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.     

Nutritional quality of storage proteins during germination of Indian bean (Dolichos lablab. var. lignosus) seeds

The dry Indian bean seed composed of starch is the major component (33%) and protein accounted for 25% of dry weight. The ability of germination to increase the nutritional quality of storage proteins was studied by germinating the Indian bean seeds for 0, 8, 16, 24 and 32 h and evaluated the nutritional quality through an in vitro protein digestibility (IVPD), protein efficiency ratio (PER), apparent and true digestibility. The IVPD of raw Indian bean seeds by pepsin alone was 60.12% and the digestibility by pepsin and trypsin together improved to 64.24%. The in vitro digestibility by both processes increased appreciably with germination and marked increase was noticed in the early stage of germination. The PER values followed the same pattern as the value of weight gain of rats fed with diets containing raw and germinated Indian bean. The lowest PER values were observed with raw bean diet. However, the value increased in rats fed with diets of Indian bean germinated for different intervals of time, reaching comparable PER values with the group maintained on casein diet. The true and apparent nitrogen digestibility of raw bean low being only 82 and 72%, respectively observed with casein diet. Diets with germinated bean protein showed a marked increase in both parameters, although the values were still less than that displayed by the casein fed rats. Germination is a simple biochemical enrichment tool and significantly improves palatability, digestibility and the nutritive utilisation of proteins in Indian bean seeds.     

Compression Strength of Dairy Gels and Microstructural Interpretation

Contour plots were developed for the compression stress (at 20% deformation) of single-component, mixed and filled protein gels. Samples were made by heating and acidification from skim milk powder, SMP (0–20% TS), whey protein isolate, WPI (0–10% TS), and recombined cream, within pH 3.6–3.9, 4.6–4.8 and 5.1–5.3. At higher pH, WPI gels were stronger than SMP gels. WPI had a reinforcing effect on SMP gels, while small additions of SMP to WPI gels resulted in weaker mixed gels. Filled gels containing cream had higher compression strengths than mixed gels. Micrographs showed linking of casein chains by WPI strands in mixed gels and compatibility of fat globules with casein micelles in the protein network of filled gels.     

Gelation of casein-whey protein mixtures

Heated milk consists of a mixture of whey protein-coated casein micelles and soluble whey protein aggregates. The acid-induced gelation properties of heated milk are consistently different from those of unheated milk—i.e., a shift in gelation pH, stronger gels, and a different microstructure of the gels. In this study we investigated the role of the different fractions of denatured whey proteins on the acid-induced gelation, the gel hardness, and the microstructure. Both whey protein fractions contribute to the observed shift in gelation pH, although by a different mechanism. Obtaining gels with high gel hardness occurs most effectively when all denatured whey proteins are present as whey protein aggregates. It was observed that disulfide bridge exchange reactions during the acid-induced gelation at ambient temperature play an important role for both whey protein fractions. Additionally, disulfide interactions seem to occur between the aggregates and the casein micelles during the gel state. In this study, we show the development of a new approach for confocal scanning laser microscopy measurements—i.e., separate staining of the proteins in milk. By using this method, we were able to determine that, although whey protein aggregates are not linked to the casein micelles, they nevertheless gel at the same moment. This work adds to a better understanding of the role of denatured whey proteins during acid-induced gelation and could improve the effective use of whey proteins.     

不同分子量葡聚糖对大豆分离蛋白冷致混合凝胶的流变性质和质构性质的影响

为研究不同分子量葡聚糖对大豆分离蛋白冷致凝胶强度的影响,采用流变仪和质构仪等手段进行分析。研究结果表明:GDL诱导的大豆分离蛋白冷致凝胶是一种弱蛋白凝胶,酸化速率随着GDL含量的增加而增加。高酸化速率条件下形成的大豆分离蛋白冷致凝胶的凝胶起始点早且最快达到模量峰值;低酸化速率条件下形成的大豆分离蛋白冷致凝胶的凝胶起始点晚且最终模量较低。不同分子量和浓度的葡聚糖添加会改变大豆分离蛋白冷致凝胶的凝胶强度。同分子量的葡聚糖与大豆分离蛋白混合体系形成的冷致蛋白多糖凝胶随着葡聚糖浓度的增加其粘弹性质呈现先上升后下降的趋势;而随着葡聚糖分子量增加混合凝胶的粘弹性质变化越显著。     

Effects of disulphide bonds between added whey protein aggregates and other milk components on the rheological properties of acidified milk model systems

Micro- and nanoparticulated whey protein (MWP, NWP) were added to non-fat milk model systems and processed into chemically (glucono-delta-lactone) acidified milk gels. Model systems contained 5% protein in total and were made at two levels of casein (2.5% and 3.5%, w/w) with and without the thiol-blocking agent N-ethylmaleimide. The systems were characterised in terms of thiol groups, gel electrophoresis, particle size, and rheology during processing (homogenisation, heat treatment and acidification). The results showed that the formation of disulphide-linked structures in milk model systems was closely related to the increased particle size and rheological behaviour of the gels. MWP enriched systems produced, upon acidification, weak protein networks and required the addition of whey protein isolate (WPI) to increase gel strength. However, systems containing NWP exhibited pronounced increase in particle size and higher firmness of acidified gels through both covalent and non-covalent interactions.     

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.     

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.     

A Comparison of Creep Phenomena in Food Protein Gels

Rheological properties of gels made by heating (90°C for 20 min) and subsequent cooling (5°C) dispersions of gelatin, soy protein isolate, and a mixture of whey protein and casein were determined by creep measurements at various temperatures. The elastic component of soy protein isolate gels became soft with increasing temperature but did not fully melt even at 90°C. Whey protein concentrate gels deformed continuously upon compression above 70°C, reflecting viscous flow. The gel obtained from a mixture of casein and whey protein concentrate following acidification with glucono-δ-lactone and heating revealed significant plastic deformation, indicating the network was formed by packing of small size particles.     

Gelation of casein-whey mixtures: effects of heating whey proteins alone or in the presence of casein micelles.

The aim of the present work was to investigate the role of whey protein denaturation on the acid induced gelation of casein. This was studied by determining the effect of whey protein denaturation both in the presence and absence of casein micelles. The study showed that milk gelation kinetics and gel properties are greatly influenced by the heat treatment sequence. When the whey proteins are denatured separately and subsequently added to casein micelles, acid-induced gelation occurs more rapidly and leads to gels with a more particulated microstructure than gels made from co-heated systems. The gels resulting from heat-treatment of a mixture of pre-denatured whey protein with casein micelles are heterogeneous in nature due to particulates formed from casein micelles which are complexed with denatured whey proteins and also from separate whey protein aggregates. Whey proteins thus offer an opportunity not only to control casein gelation but also to control the level of syneresis, which can occur.     

Uptake of divalent ions (Mn+2 and Ca+2) by heat-set whey protein gels

Divalent salts are used commonly for gelation of polymer molecules. Calcium, Ca(+2), is one of the most common divalent ions that is used in whey protein gels. Manganese, Mn(+2), is also divalent, but paramagnetic, enhancing relaxation decay rates in magnetic resonance imaging (MRI) and can be used as a probe to understand the behavior of Ca(+2) in whey protein gels. The objective of this study was to investigate the diffusion of Ca(+2) and Mn(+2) ions in heat-set whey protein gels by using MRI and nuclear magnetic resonance (NMR) relaxometry. Whey protein gels were immersed in solutions containing MnCl(2) and CaCl(2) at neutral pH. Images obtained with gels immersed in MnCl(2) solution revealed a relaxation sink region in the gel's surface and the thickness of the region increased with time. These "no signal" regions in the MR images were attributed to uptake of Mn(+2) by the gel. Results obtained with CaCl(2) solution indicated that since Ca(+2) did not have the paramagnetic effect, the regions where Ca(+2) diffused into the gel exhibited a slight decrease in signal intensity. The relaxation spectrums exhibited 3 populations of protons, for gels immersed in MnCl(2) solution, and 2 populations for gels in CaCl(2) solution. No significant change in T(2) distributions was observed for the gels immersed in CaCl(2) solution. The results demonstrated that MRI and NMR relaxometry can be used to understand the diffusion of ions into the whey protein gel, which is useful for designing gels of different physical properties for controlled release applications. PRACTICAL APPLICATION: Design of food systems for delivery of bioactive compounds requires knowledge of diffusion rates and structure. Utilizing magnetic resonance imaging the diffusion rates of ions can be measured. Relaxation spectra could yield information concerning molecular interactions.     

Effect of glucono-δ-lactone acidification and heat treatment on the physicochemical properties of silver carp mince

The physicochemical and rheological properties of silver carp mince with different levels of glucono-δ-lactone (GDL) with or without heat treatment at 90 °C for 20 min were investigated. Breaking force, deformation, and whiteness of GDL-induced gels increased with increasing GDL concentration. Heat treatment of GDL-induced gels led to a significant increase in breaking force and whiteness, but decreased deformation. Disulphide bond formation was observed in GDL-induced gels, which increased after heating of the acidified gels. Solubility profiles of gels in different solutions suggested that gel networks were mainly due to hydrophobic interactions, although other chemical interactions may also occur. SDS-PAGE showed that acidification resulted in a large decrease in myosin heavy chain (MHC) band intensity, suggesting the extensive formation of covalent cross-links among silver carp myosins during GDL-induced gelation.     

Structural properties of stirred yoghurt as influenced by whey proteins

The effect of whey protein addition on structural properties of stirred yoghurt systems at different protein and fat content was studied using laser diffraction spectroscopy, rheology and confocal laser scanning microscopy (CLSM). The composition of heated milk systems affected micro- and macroscopic properties of yoghurt gels. Particle size increased as a function of increasing whey protein content and decreased as a function of increased fat level. Firmness (elastic modulus) and apparent viscosity of manufactured yoghurt samples increased as a function of increased interparticle interactions, mainly caused by self-aggregation of whey proteins or aggregated whey protein-coated fat globules, respectively. The resistance towards shear-induced disruption of yoghurt gels increased with an increasing proportion of casein protein in the protein mixture, whereas products with high whey protein level revealed lower resistance behaviour towards shear-forces. CLSM images illustrated that the presence of large whey protein aggregates and lower number of fat globules lead to the formation of an interrupted and coarse gel microstructure characterised by large interstitial spaces. The higher the casein fraction and/or the fat level, the less interspaced voids in the network were observed. However, it is evident that the addition of whey proteins reinforces firmness properties of low-fat yoghurts comparable to characteristics of full-fat yoghurt.     

Microstructural features of composite whey protein/polysaccharide gels characterized at different length scales

Mixed biopolymer gels are often used to model semi-solid food products. Understanding of their functional properties requires knowledge about structural elements composing these systems at various length scales. This study has been focused on investigating the structural features of mixed cold-set gels consisting of whey protein isolate and different polysaccharides at different length scales by using confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). Whey protein cold-set gels were prepared at different concentrations to emulate stiffness of various semi-solid foods. Mixed gels contained different concentrations of gellan gum, high methyl pectin or locust bean gum. Results obtained with CLSM, at the micrometer length scale, indicated the homogeneous nature of the investigated gels. Results obtained with SEM, at the sub-micron length scale, indicated the presence of spherical protein aggregates. During the gel preparation (acidification), the presence of polysaccharides in the whey protein gels led to on initially segragative phase separation into a gelled protein phase and a polysaccharide/serum phase at a micrometer length scale. At the final pH of the gels (pH 4.8, i.e. below the pI of whey proteins), the negatively charged polysaccharides interacted with the protein phase and their spatial distribution was effected by charge density. Polysaccharides with a higher charge density were more homogeneously distributed within the protein phase. Neutral polysaccharide, locust bean gum, did not interact with the protein aggregates but was present in the serum phase. Using SEM, a new type of microstructure formed in the whey protein/polysaccharide gels was characterized. It composed of a protein continuous, porous network at the length scale of 100 μm, coexisting next to the pools of serum which contained spherical protein-rich domains. Heterogeneity of the structure strongly related to the macroscopic behavior of the gels under large deformation. Upon uniaxial compression these heterogeneous gels releases a large amount of serum. Combination of the results of two microscopic techniques, CLSM and SEM, appeared to offer unique possibilities to characterize the structural elements of whey protein/polysaccharide cold-set gels over a wide range of length scales.     

pH值对罗非鱼蛋白-大豆蛋白混合热凝胶特性及体外消化性的影响

以罗非鱼和大豆为原料分别提取鱼分离蛋白(fish protein isolate,FPI)和大豆分离蛋白(soy protein isolate,SPI),制备罗非鱼蛋白-大豆蛋白(FPI∶SPI=1∶1,质量比)热诱导凝胶,探讨pH值(6.0、6.5、7.0、7.5)对混合蛋白热凝胶特性和体外消化性的影响。结果表明:pH 6.5时罗非鱼蛋白-大豆蛋白混合蛋白热凝胶储能模量G′值大于单一蛋白,凝胶特性最好,硬度和胶黏性相比FPI都得到提升。pH值会影响蛋白质与水分子的结合能力,混合蛋白热凝胶的持水性在pH 6.5时比其它pH值条件下强(P<0.05),达到最大值84.02%,与扫描电镜结果一致,网络结构致密且光滑平整。pH 6.5时,混合蛋白热凝胶的消化率达91.57%,高于其它pH值下的消化率(P<0.05)。因此,通过调节pH值可以改善FPI和SPI混合蛋白热凝胶的凝胶特性和消化性。     

Gelation of Mixed Gels Containing κ-Carrageenan and Skim Milk Components

ABSTRACT: The gelation characteristics of mixed gels containing κ-carrageenan and skim milk or milk fractions (skim milk permeate or retentate) obtained by ultrafiltration were examined. Increasing the skim milk solids content of mixtures containing carrageenan increased setting temperatures and gel strength. The milk proteins contributed to gel strength but did not influence the setting temperature of mixtures. The binding of denatured whey proteins to casein micelles affected gel network formation of milk-carrageenan mixtures containing 10% milk solids. Network formation in mixed gels containing carrageenan and milk or milk fractions was initiated by the carrageenan component and dictated primarily by the ionic content of the mixtures.     

Influence of partially demineralized milk proteins on rheological properties and microstructure of acid gels

Innovative clean label processes employed in the manufacture of acid gels are targeted to modify the structure of proteins that contribute to rheological properties. In the present study, CO₂-treated milk protein concentrate powder with 80% protein in dry matter (TMPC80) was mixed with nonfat dry milk (NDM) in different ratios for the manufacture of acid gels. Dispersions of NDM and TMPC80 that provided 100, 90, 70, and 40% of protein from NDM were reconstituted to 4.0% (wt/wt) protein and 12.0% (wt/wt) total solids. Dispersions were adjusted to pH 6.5, followed by heat treatment at 90°C for 10 min. Glucono-δ-lactone was added and samples were incubated at 30°C, reaching pH 4.5 ± 0.05 after 4 h of incubation. Glucono-δ-lactone levels were adjusted to compensate for the lower buffering capacity of samples with higher proportions of TMPC80, which is attributable to the depletion of buffering minerals from both the serum and micellar phase during preparation of TMPC80. Sodium dodecyl sulfate-PAGE analysis indicated a higher amount of caseins in the supernatant of unheated suspensions with increasing proportions of CO₂-treated TMPC80, attributable to the partial disruption of casein micelles in TMPC80. Heat treatment reduced the level of whey proteins in the supernatant due to the heat-induced association of whey proteins with casein micelles, the extent of which was larger in samples containing more micellar casein (i.e., samples with a lower proportion of TMPC80). Particle size analysis showed only small differences between nonheated and heated dispersions. Gelation pH increased from ?5.1 to ?5.3, and the storage modulus of the gels at pH 4.5 increased from ?300 to ?420 Pa when the proportion of protein contributed by TMPC80 increased from 0 to 60%. Water-holding capacity also increased and gel porosity decreased with increasing proportion of protein contributed by TMPC80. The observed gel properties were in line with microstructural observations by confocal microscopy, wherein sample gels containing increasing levels of TMPC80 exhibited smaller, well-connected aggregates with uniform, homogeneous pore sizes. We concluded that TMPC80 can be used to partially replace NDM as a protein source to improve rheological and water-holding properties in acid gels. The resultant gels also exhibited decreased buffering, which can improve the productive capacity of yogurt manufacturing plants. Overall, the process can be leveraged to reduce the amount of hydrocolloids added to improve yogurt consistency and water-holding capacity, thus providing a path to meet consumer expectations of clean label products.     

Effect of milk supplementation and culture composition on acidification, textural properties and microbiological stability of fermented milks containing probiotic bacteria

In the present work, the combined effect of milk supplementation and culture composition on acidification, textural properties, and microbiological stability of fermented milks containing probiotic bacteria, was studied. Three powders (whey, casein hydrolysate, and milk proteins) were tested as supplementation. Two strains of probiotic bacteria, Lactobacillus acidophilus (LA5) and Lactobacillus rhamnosus (LC35), were used in pure culture, and in mixed culture with Streptococcus thermophilus (ST7). Acidifying activity was enhanced with mixed cultures, compared to pure cultures resulting in a shorter time to reach pH 4.5. Acidifying activity was greatly improved with casein hydrolysate, with a reduction of the fermentation time by about 55% by comparison with the other supplementations. The stability of probiotic bacteria was weakly affected by milk supplementation and culture composition. However, pure cultures were more stable than mixed cultures. The texture of the fermented products was not dependent on culture composition, but strongly dependent on milk supplementation. Sweet whey supplementation gave products with lower firmness and viscoelasticity than products supplemented with casein hydrolysate or milk proteins (decrease by 70%). It was observed that all products containing probiotic counts over 2.2×10⁷ CFU mL⁻¹ are suitable for the development of a lactic beverage containing probiotics.     

Rheological and structural characterization of gels from whey protein hydrolysates/locust bean gum mixed systems

The gelling ability of whey proteins can be changed by limited hydrolysis and by the addition of other components such as polysaccharides. In this work the effect of the concentration of locust bean gum (LBG) on the heat-set gelation of aqueous whey protein hydrolysates (10% w/w) from pepsin and trypsin was assessed at pH 7.0. Whey protein concentrate (WPC) mild hydrolysis (up to 2.5% in the case of pepsin and 1.0% in the case of trypsin) ameliorates the gelling ability. The WPC synergism with LBG is affected by the protein hydrolysis. For a WPC concentration of 10% (w/w), no maximum value was found in the G′ dependence on LBG content in the case of the hydrolysates, unlike the intact WPC. However, for higher protein concentrations, the behaviour of gels from whey proteins or whey protein hydrolysates towards the presence of LBG becomes very similar. In this case, a small amount of LBG in the presence of salt leads to a big enhancement in the gel strength. Further increases in the LBG concentration led to a decrease in the gel strength.     

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.