Whey Protein Denaturation Research Articles

Microstructure and rheology of acid milk gels and stirred yoghurts –quantification of process-induced changes by auto- and cross correlation image analysis

The aim of the study was to evaluate whether emerging multimodal microscopy techniques combined with quantitative image analysis were capable of detecting differences in the microstructure of model dairy systems, and to evaluate whether hydrodynamic cavitation led to differences in microstructure compared to pressure homogenisation.Hydrodynamic cavitation and pressure homogenisation were combined with severe heat treatment to obtain milk samples of different degrees of fat homogenisation and different degrees of whey protein denaturation. Glucono-δ-lactone (GDL) induced acid milk gels and stirred yoghurts were produced from the treated milks, followed by rheological analysis and evaluation of the microstructure by multimodal coherent anti-stokes raman scattering and two-photon excitation microscopy combined with auto and cross-correlation image analysis.Autocorrelation analysis of the protein network of GDL-gels could quantitatively describe differences in acid gel strength, and results indicated that differences in the degree of homogeneity and branching of the protein network were linked to differences in size and shape of the protein domains formed during acidification. However, for the stirred yoghurts, the autocorrelation analysis was limited by the heterogeneous structure of the protein network.By cross-correlation analysis, quantification of the relative distribution of fat and protein was possible for both GDL-gels and yoghurts. Results indicated that severe heat treatment of the milk base translated into differences in the interaction between fat globules and the protein network, which could be explained by a relatively higher degree of whey protein association with the milk fat globule membrane.

Influence of chaperone-like activity of caseinomacropeptide on the gelation behaviour of whey proteins at pH 6.4 and 7.2.

The effect of caseinomacropeptide (CMP) on the heat-induced denaturation and gelation of whey proteins (2.5–10%, w/v) at pH 6.4 and 7.2, at a whey protein:CMP ratio of 1:0.9 (w/w), was investigated using differential scanning calorimetry (DSC), oscillatory rheology (90 °C for 20 min) and confocal microscopy. Greater frequency-dependence in the presence of CMP suggested that the repulsive interactions between CMP and the whey proteins affected the network generated by the non-heated whey protein samples. At pH 6.4 or 7.2, CMP increased the temperature of denaturation of β-lactoglobulin by up to 3 °C and increased the gelation temperature by up to 7 °C. The inclusion of CMP strongly affected the structure of the heat-induced whey protein gels, resulting in a finer stranded structure at pH 6.4 and 7.2. The presence of CMP combined with a lower heating rate (2 °C/min) prevented the formation of a solid gel of whey proteins after heating for 20 min at 90 °C and at pH 7.2. These results show the potential of CMP for control of whey protein denaturation and gelation.

Effect of heating milk on whey protein denaturation and cheese-making properties

The cheese-making properties of milk heated at 80 °C for different times (0–8 min), and mixtures of severely-heated milk (80 °C, 30 min) with non-heated milk were evaluated in separate experiments. In both approaches, milk pH was adjusted to 6.3, 6.7 or 7.0 before heating. Despite different distributions of denatured whey protein (WP) between casein micelle surface and serum phase, heating pH had no significant effect on cheese-making properties. The two approaches were compared by reporting the results as a function of WP denaturation rate in milk. The mixed milk approach showed a higher detrimental effect on rennet-induced coagulation kinetics and curd contraction capacity during cooking than the heated milk approach. For similar WP-denaturation rate, higher yield was obtained for the mixed milk than the heated milk as a result of the better protein retention and larger increase of cheese moisture.

Partial substitution of sheep and goat milks of various fat contents by the respective sweet buttermilks: Effect of cream heat treatment

The potential of sweet sheep and goat buttermilks (BMs) to partially replace sheep and goat milks was investigated. The experimental factors were: the heat treatment of cream - 66 °C/30 min or pasteurized 85 °C/20 s - the fat content of milks, i.e., standardized- or full-, reduced-, low-fat, and the level of substitution, i.e., 5, 10, 15% BM. Milks, BMs and their mixtures were analyzed for composition, rennet clotting behavior - i.e., clotting time (RCT), curd firming rate (K20), curd firmness (A30) – and syneresis of the rennet-gels. The pasteurization-induced denaturation of whey proteins decreased significantly (P < 0.05) the syneresis of BM rennet-gels but it did not affect significantly the composition, the extended RCT and low A30 of BMs. The composition and behavior of milk/BM mixtures were not affected by the cream treatment. The increase of %BM level decreased significantly (P < 0.05) the A30 and syneresis although not in a dose-dependent manner. In conclusion, the BMs from sheep or goat dairy cream - safely handled due to cream pasteurization - could be used in the manufacture of reduced- or low-fat cheeses without pre-fractionation or concentration. These findings could be the first step for the development of novel circular dairy foods.

New experimental set-up for testing microwave technology to continuously heat fouling-sensitive food products like milk concentrates

Microwave heating is an emerging alternative direct heating method to face deposit and particle formation upon heating fouling-sensitive food products. This study successfully demonstrated the implementation of a new set-up for continuous microwave heating at pilot scale (f = 2450 MHz, P = 0.6–6 kW). Reconstituted skim milk concentrate (DM = 27, 31.5 and 36 g/100 g) was heated (110–125 °C, 5 s holding time) through microwave technology and with a tubular heat exchanger in order to examine heat-related product changes and compare both technologies.The results showed significantly reduced whey protein denaturation (35–40% less β-lactoglobulin denaturation) and deposit formation (90% less fouling) of microwave heated reconstituted skim milk concentrate compared to tubular heating, whereas colour changes were insignificant for all experiments. Both methods resulted in comparable particle sizes at equal flow profiles, however, turbulent flow conditions were necessary in order to prevent elevated particle formation. Industrial relevanceMicrowave heating is a promising alternative heating method for fouling-sensitive dairy products. This study demonstrates that through short heat-uptake times gentle product handling is ensured, resulting in retained native product properties. Reduced fouling leads to extended running times, implying less and eventually shorter cleaning intervals. Moreover, compared to other direct heating methods like steam injection, no additional water/steam is added to the product. With its space-saving design and easy handling, continuous microwave heating is a rising technology with high industrial relevance.

Hydrodynamic cavitation of raw milk: Effects on microbial inactivation, physical and functional properties

The effects of hydrodynamic cavitation treatment of raw milk at different temperatures (outlet temperatures of 40, 50, 60 or 72 °C) were evaluated in relation to corresponding reference heat treatments. Physical and functional changes were studied and microbial inactivation was evaluated by the total bacterial count. Hydrodynamic cavitation reduced the mean milk fat globule size to 3.29–1.40 μm, depending on the treatment temperature. Treatment did not result in a significant increase in whey protein denaturation and did not affect acid gel formation. A reduction of >1 log unit in the total bacterial count was obtained, which was primarily ascribed to the temperature increase during treatment. Thus, this study illustrates that HC treatment can provide a heating unit allowing for simultaneous pasteurisation and reduction in MFG size, with minimal changes to the physical and functional properties of the milk.

Incorporating whey protein aggregates produced with heat and ultrasound treatment into rennet gels and model non-fat cheese systems

Native whey proteins (WP) are expulsed from cheese coagulation during syneresis. Although incorporating denatured WP aggregates into cheese gels has been previously proposed to improve the overall cheese yield, the effects of WP aggregate properties on gelation kinetics and protein retention are not yet fully understood. In this study, heat and power ultrasound were used to produce denatured whey protein aggregates with a wide range of sizes. The effects of size and hydrophobicity differences in WP aggregates produced by heat and heat coupled with ultrasound were investigated in relation to the kinetics of rennet gelation and protein retention in model non-fat cheddar cheeses. Rheological measurements showed that sufficiently large, denatured WP aggregates could avoid impairment of rennet gelation caused by native WP, irrespective of changes in the soluble calcium concentration or the surface hydrophobicity of the aggregates. WP aggregates formed by the combined heat and ultrasound treatment were more hydrophobic than the larger heat-treated aggregates and were better retained in the cheese. However, inclusion of sufficiently large aggregates in cheese milk conferred an openness to the cheese microstructure and showed promise in improving the otherwise rigid non-fat cheese.

Microstructure of kajmak skin layer made during kajmak production

Kajmak is a unique dairy product with a long tradition in Balkan countries and the Middle East. Kajmak skin layers are formed during 60 minutes of kajmak production, influencing the quality and determining the structure of the final product. The influence of milk composition and heat treatments (75-95 °C/5min) on the composition, microstructure and protein distribution of kajmak skin layers was investigated in this study. Microstructure was determined using scanning electron microscopy (SEM), while the distribution of protein fractions was studied by SDS PAGE in reducing and non-reducing conditions. Kajmak skin layers obtained from milk treated at common intense heat regime were characterized with β-LG and α-LA mainly as disulfide-linked complexes, while lower temperature (75 °C) caused the presence of whey proteins in dominantly individual form. More severe treatments (85 °C, 90 °C and 95 °C) caused the full involvement of β-LG in the complexes, since a certain amount of α-LA was found as a monomer. The kajmak skin layer composition, severity of the applied milk heat treatment and the linked whey proteins exhibited a very strong influence on kajmak skin layer microstructure. The microstructure of kajmak skin layer with a low fat content and fat/protein (F/P) ratio was characterized by a compact structure with a dense protein matrix. High fat content and F/P ratio resulted in a dispersive form of the stretched protein network. Kajmak skin layers produced from milk with added whey proteins were characterized by fibrous, thread-like segments, due to the incorporated denatured whey proteins, especially linked β-LG.

Infant milk formulae processing: Effect of wet-mix total solids and heat treatment temperature on rheological, emulsifying and nutritional properties

This study investigated the effects of total solids (TS) in the wet-mix and pasteurisation temperature on the whey protein (WP) denaturation, rheological behaviour, emulsifying properties and available lysine content of a model infant milk formula (IMF). IMF with 50% or 60% (w/w) TS were produced by wet-mix process and pasteurised at 75 °C or 100 °C for 18 s. Measurements were taken after dispersion, pasteurisation, homogenisation and spray-drying of wet-mix. Pasteurisation at 100 °C caused extensive WP denaturation (above 80%) and increased the viscosity. Available lysine content was 52% higher in powdered IMF obtained from wet-mixes pasteurised at 75 °C compared to 100 °C. A significant reduction in energy consumption could be achieved by producing IMF from 60% TS wet-mixes pasteurised at 75 °C, while obtaining powders with less than 7% WP denaturation, no loss of available lysine during processing and post-reconstitution stability for 2 h.

The effect of heat treatment on the front-face fluorescence spectrum of tryptophan in skim milk

The effect of heat treatment on tryptophan front-face fluorescence emission spectra of reconstituted skim milk was assessed. Samples were heat-treated from 70 to 100 °C at six holding times. Treating milk at 90 and 100 °C induced a decrease in fluorescence intensity (∼37 and ∼47 %) and a red shift (12 and 14 nm), respectively, with both parameters significantly affected at 99.9 % by temperature, time, and the interaction of both factors. Red shift, which could be attributed to whey protein denaturation and tryptophan exposure to increasing solvent polarity, was found to follow a quadratic trend as a function of holding time (from 0 to 60 min). The decrease of fluorescence intensity observed at those two temperatures and in the mentioned holding time range was also a quadratic function of the associated red shift. Emission intensity change at a single wavelength (∼340 nm) was partly caused by direct intensity decrease and indirectly due to the emission maximum shift. Tryptophan fluorescence allowed sample classification by PCA analysis according to heat load, i.e., temperature and time. These results established the bases for development of a cost-effective sensor based on simple, economic optical components for measurement of a few well-selected emission wavelengths, which associated with data processing, should allow inline control of milk heat treatment.

Effect of casein/whey ratio on the thermal denaturation of whey proteins and subsequent fouling in a plate heat exchanger

Dairy fouling is a ubiquitous problem in food processing, however, the fouling mechanism is not fully understood and investigations arose mainly from experiments with model systems that contained only whey proteins, typically reconstituted from whey protein isolate powder (WPI). The effect of casein on fouling has been rarely considered despite it is the major component of milk proteins. To fill this gap, whey protein-based model fluids containing different casein concentrations and fixed content of added calcium were prepared, leading to various Casein/WPI mass ratios. The effect of Casein/WPI on β-lactoglobulin (BLG) denaturation at molecular level and subsequent fouling behavior in the pilot-scale plate heat exchanger during pasteurization treatment was investigated. It was shown that Casein/WPI significantly affects the fouling behavior: at low Casein/WPI, fouling mass dropped dramatically until a minimum value was reached located at Casein/WPI of 0.2. While at higher Casein/WPI, fouling mass increased with elevated Casein/WPI. Element mapping of the fouling layer also reveals that different structures and fouling mechanisms occur depending on Casein/WPI ratio. Finally, it was established that contrary to WPI solutions, BLG thermal denaturation is poorly correlated to decrease/extent of fouling for casein protein-based solutions showing that the presence of casein deeply modifies mineral and protein interactions and fouling build-up.

Interactions of Native and Denatured Whey Proteins with Caseins and Polysaccharides

In this review, interactions of native or denatured whey proteins with other proteins and polysaccharides were addressed. Chemical structures of whey proteins and caseins as representatives of proteins and of gum Arabic and dextran as representatives of polysaccharides were explained. Whey protein, as a mixture of different proteins, such as beta-lactoglobulin, alpha-lactalbumin, or bovine serum albumin, has a highly complex nature, and therefore, the main interaction occurs within these proteins upon processing. Structu-re of whey protein includes hydrogen bonds, disulfide bridges and free thiol group, all of which allows whey proteins highly reactive with other polymers. With these properties, whey proteins can be denatured via heating or acidification in a controlled way; and therefore, several functional particles with different sizes and shapes could be obtained. Here we explained the interactions of native and denatured whey proteins with caseins, gum Arabic and dextran in terms of their behaviuor in solutions or dispersions, their functional and rheological properties. Denaturation process includes mainly hydrophobic interacti-ons and is most of the time irreversible, whereas the complex formation of proteins with polysaccharides includes electrostatic and/or steric interactions and complex formation could be reversible or irreversible depending on the type of application. Such interactions are important for the stability of food materials especially during processing and storage, therefore, a deep insight on this subject is important.

Influence of heat treatment of goat milk on casein micelle size, rheological and textural properties of acid gels and set type yoghurts.

Acid gels and yoghurts were made from goat milk that was heated at 72°C/30 s, 85°C/5 min, and 95°C/5 min, followed by acidification with starter culture at 43C until pH 4.6. The rheological and textural properties of acid gels and yoghurts were analyzed using dynamic low amplitude oscillatory rheology and back extrusion texture analysis, respectively. The effect of goat milk heat treatment on the mean casein micelle diameter and protein profile was also determined by dynamic light scattering and SDS PAGE electrophoresis, respectively. The shortest gelation and fermentation time was recorded for yoghurt prepared from milk heated at 85°C/5 min. Also, the pH of gelation, the storage moduli (G') and yield stress were higher for this yoghurt, compared with the other two. Textural properties of goat milk yoghurts such as firmness and consistency were strongly affected by milk heat treatment, and the highest values were recorded for yoghurt produced from milk preheated at 85°C/5 min, as well. The largest casein micelles were measured after 85°C/5 min treatment and their size decreased at higher temperature, despite higher denaturation of whey proteins at the most intense heat regime, indicating the structure changes that influence on the acid gelation.

Structural characterization of heat-induced protein aggregates in model infant milk formulas

Heat treatments induce structural modifications of bovine milk proteins. In this study, we aimed to investigate how these structural modifications are affected by the whey protein profile of infant milk formulas (IMFs) and heat treatment parameters. Three model IMFs (1.3% of proteins; casein:whey protein ratio 40:60) differing by the whey protein profile (1:3.37 α-lactalbumin (α-LA):β-lactoglobulin (β-LG), 1:3.26:1.80 α-LA:β-LG:lactoferrin (LF) or 1:0.13:0.40 α-LA:β-LG:LF), were heated at 67.5 °C or 80 °C to reach an akin whey protein denaturation extent of 65%. Protein structures were analyzed by asymmetrical flow field-flow fractionation coupled with multiangle light scattering and differential refractometer, transmission electron microscopy and electrophoresis. The unheated IMFs were used as reference. The results showed that LF addition in IMFs induced partial casein micelle disintegration before heating. In the absence of added LF, the heat-denatured whey proteins either formed soluble whey protein aggregates or casein micelle-bound whey protein aggregates. The latter were favored at the expense of soluble aggregates in the heated IMFs with the LF content increase and the concomitant β-LG content decrease. Consequently, the casein micelle structure was strongly dependent on the β-LG and LF amounts in IMFs and on the heating temperature. In the IMFs containing β-LG and LF and at temperature greater than the β-LG denaturation temperature, the casein micelles exhibited filamentous appendages on its surface after heating. Below the denaturation temperature of β-LG and in IMFs containing trace β-LG amount, the heated casein micelles were perfectly spherical with a smooth surface.

Influence of desialylation of caseinomacropeptide on the denaturation and aggregation of whey proteins

The effect of the addition of caseinomacropeptide (CMP) or desialylated CMP on the heat-induced denaturation and aggregation of whey proteins was investigated in the pH range 3 to 7 after heating at 80°C for 30 min. The rate and temperature of denaturation, the extent of aggregation, and the changes in secondary structure of the whey proteins heated in presence of CMP or desialylated CMP were measured. The sialic acid bound to CMP favored the denaturation and aggregation of whey proteins when the whey proteins were oppositely charged to CMP at pH 4. A transition occurred at pH 6, below which the removal of sialic acid enhanced the stabilizing properties of CMP against the denaturation and aggregation of the whey proteins. At pH >6, the interactions between desialylated CMP and the whey proteins led to more extensive denaturation and aggregation. Sialic acid bound to CMP influenced the denaturation and aggregation behavior of whey proteins in a pH-dependent manner, and this should be considered in future studies on the heat stability of such systems containing CMP.

Design and characterization of casein–whey protein suspensions via the pH–temperature-route for application in extrusion-based 3D-Printing

The current interest in individualized food through additive manufacturing has identified a need for more information on the formulation and printability of potential ingredients. Here, the effect of formulation parameters of casein–whey protein suspensions like the pH (4.8–5.4) as well as the casein content (8.0–12.0% (w/w)) mixed with whey protein (2.0–3.0% (w/w)) and the effect of pre-processing parameters including the denaturation of whey proteins (80 °C, 10 min; adjusted pH 6.55, 6.9 and 7.1) on the gel formation via the pH–temperature (T)-route was studied. Rheological measurements showed that the sol–gel transition temperature (G’ = 1 Pa) decreased and the aggregation rate of the casein–whey protein suspensions increased with increasing heating pH value. The aggregation rate was considered to be a key parameter predicting the printability of formulations. By exceeding a certain aggregation rate (250 Pa/10 K), casein–whey protein suspensions were found to be printable resulting in firm and stable gels.

Heat induced conformational changes of whey proteins in model infant formulae: Effect of casein and inulin

The effect of casein (CAS) and inulin (INUL), at ratios commonly found during infant formula processing, on the conformational changes of whey proteins (WP) was investigated. Model systems were prepared as aqueous dispersions of WP isolate alone or with calcium caseinate and/or INUL, and heated for 30 min at 66, 70 or 75 °C. Heat treatment of WP induced denaturation and aggregation. Solubility of WP system decreased by 25% when heated at 75 °C. Upon heating, the presence of CAS did not prevent WP denaturation, but increased protein solubility up to 100% in WP–CAS system and prevented aggregation. INUL affected the secondary structure of WP and increased the denaturation temperature, although it did not prevent aggregation nor solubility loss at 75 °C. In conclusion, presence of CAS prevented aggregation and solubility loss of WP during heating while INUL induced WP conformational changes impacting the denaturation stage.

Survivability of alginate-microencapsulated Lactobacillus plantarum during storage, simulated food processing and gastrointestinal conditions

A comparison between the most investigated alginate-based encapsulating agents was performed in the current study. Here, the survivability of Lactobacillus plantarum microencapsulated with alginate (Alg) combined with skim milk (Sm), dextrin (Dex), denatured whey protein (DWP) or coated with chitosan (Ch) was evaluated after exposure to different heat treatments and in presence of some food additives, during storage and under simulated gastrointestinal condition. In addition, the encapsulated cells were evaluated for production of different bioactive compounds such as exopolysacchar.ides and antimicrobial substances compared with the unencapsulated cells. The results showed that only Alg-Sm maintained the viability of the cells >106 cfu/g at the pasteurization temperature (65 °C for 30 min). Interestingly, storage under refrigeration conditions increased the viability of L. plantarum entrapped within all the tested encapsulating agents for 4 weeks. However, under freezing condition, only Alg-DWP and Alg-Sm enhanced the survival of the entrapped cells for 3 months. All the microencapsulated cells were capable of growing at the different NaCl concentrations (1%–5%) except for cells encapsulated with Alg-Dex, showed viability loss at 3% and 5% NaCl concentrations. Tolerance of the microencapsulated cells toward organic acids was varied depending on the type of organic acid. Alg-Ch and Alg-Sm provide better survival for the cells under simulated gastric juice; however, all offer a good survival for the cells under simulated intestinal condition. Our findings indicated that Alg-Sm proved to be the most promising encapsulating combination that maintains the survivability of L. plantarum to the recommended dose level under almost all the stress conditions adopted in the current study. Interestingly, the results also revealed that microencapsulation does not affect the metabolic activity of the entrapped cells and there was no significant difference in production of bioactive compounds between the encapsulated and the unencapsulated cells.

Optimation of microorganism level of semi-solid emergency food based on denaturated whey protein concentrate (WPC) with different mineral concentration and sterilization levels

When post-disaster conditions occur, the problem of food procurement becomes a critical factor which able to disrupt the fulfillment of nutrition for the society, especially children. Procurement of emergency food products is one solution that can be offered to meet daily energy for the victims in need. The raw material for making emergency food products that can be used is denatured Whey Protein Concentrate (WPC), as a source of protein, sweet potato flour as a source of carbohydrates and mineral mix as a determinant of the texture and power of product acceptance. Emergency food products are also incidental, so they require a long shelf life, and sterilization is one way that can be done to extend the shelf life of food products. The factors tested in this study included protein levels, mineral levels, and the level of sterilization with the number of microorganisms as the response variable. The optimal combination of factors tested will produce the minimum number of microorganisms. This study aims to analyze and evaluate the combination of material formulations and sterilization rates using the analytical data from Response Surface Methode (RSM) with a validation of single objective optimization method in order to have a minimum number of microorganisms to meet the nutritional needs that are incidental. Based on the response surface analysis, the optimum formula of semi-emergency food was 11.6% protein content; added 4% mineral mix; and sterilized at 8.09 D log cycle.

A numerical model for studying the thermal denaturation-aggregation of whey proteins under continuous thermal processing

A computational fluid dynamics model was designed to study the problem of thermal processing of a liquid food product containing whey proteins within a heat exchanger consisting of heating, holding and cooling tubular sections. This physical problem is associated with strong coupling between the phenomena of fluid flow, heat transfer, and thermal denaturation-aggregation of whey proteins. Our primary objective was to investigate the two-way coupling between these phenomena within the heat exchanger. This was carried out by analyzing the model predictions of velocity, temperature and product properties at both axial and radial directions. Attention was focussed on the whey proteins present in a cream cheese formulation. The thermal denaturation-aggregation kinetics was supposed to follow that of the beta-lacto-globulin, which plays a major role in fouling when milk derivatives are submitted to thermal processing in heat exchangers. Model predictions demonstrated that the apparent viscosity of the liquid product exhibited a complex behavior along the processing unit: in addition to its dependence on local temperature, it was affected by the local degree of denaturation of whey proteins – and hence on the product history previous to this position in the heat exchanger. The numerical model was structured into a sequence of computational domains; its versatility was illustrated by changing the length of the holding section and then assessing the impact on the final degree of denaturation of the whey proteins present in the liquid product.