Whey Protein Denaturation Research Articles

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.

Whey protein mucoadhesive properties for oral drug delivery: Mucin–whey protein interaction and mucoadhesive bond strength

Whey protein is a natural polymer recently used as an excipient in buccoadhesive tablets but its mucoadhesive properties were barely studied. In this work, we characterize mucoadhesion of whey protein in order to determine the mechanisms and optimal conditions for use as excipient in oral drug delivery. Thus, native and denatured whey protein (NWP and DWP) were investigated and the effect of concentration and pH were also studied. Many methods of characterization were selected to allow the study of chemical and physical interactions with mucin and then the results were bound with an ex vivo experiments. Turbidity of WP–mucin mixture increased at acidic pH 1.2 till 4.5 indicating interaction with mucin but not at pH 6.8. No interaction with mucin was also found by ITC method at pH 6.8 for native and denatured whey protein used at 1% (w/w). Forces of bioadhesion evaluated by viscosity measurements were the best for high concentrated (10.8%) DWP solutions at pH 6.8 and were low at pH 1.2 for NWP and DWP solutions. Addition of chemical blockers indicated that hydrogen bondings and disulfide bridges were the main mechanisms of interactions with mucin. Reticulation of DWP with calcium ions to obtain microparticles (MP) did not influence the ability of interaction with mucin as shown by FTIR analysis. These results correlated with ex vivo study on rat tissue demonstrating important adhesion (75%) of WP MP on the intestine and null on the stomach after 2h of deposit.

Effect of heating strategies on whey protein denaturation—Revisited by liquid chromatography quadrupole time-of-flight mass spectrometry

Previous standards in the area of effect of heat treatment processes on milk protein denaturation were based primarily on laboratory-scale analysis and determination of denaturation degrees by, for example, electrophoresis. In this study, whey protein denaturation was revisited by pilot-scale heating strategies and liquid chromatography quadrupole time-of-flight mass spectrometer (LC/MC Q-TOF) analysis. Skim milk was heat treated by the use of 3 heating strategies, namely plate heat exchanger (PHE), tubular heat exchanger (THE), and direct steam injection (DSI), under various heating temperatures (T) and holding times. The effect of heating strategy on the degree of denaturation of β-lactoglobulin and α-lactalbumin was determined using LC/MC Q-TOF of pH 4.5–soluble whey proteins. Furthermore, effect of heating strategy on the rennet-induced coagulation properties was studied by oscillatory rheometry. In addition, rennet-induced coagulation of heat-treated micellar casein concentrate subjected to PHE was studied. For skim milk, the whey protein denaturation increased significantly as T and holding time increased, regardless of heating method. High denaturation degrees were obtained for T >100°C using PHE and THE, whereas DSI resulted in significantly lower denaturation degrees, compared with PHE and THE. Rennet coagulation properties were impaired by increased T and holding time regardless of heating method, although DSI resulted in less impairment compared with PHE and THE. No significant difference was found between THE and PHE for effect on rennet coagulation time, whereas the curd firming rate was significantly larger for THE compared with PHE. Micellar casein concentrate possessed improved rennet coagulation properties compared with skim milk receiving equal heat treatment.

AVALIAÇÃO DO ÍNDICE DE SOROPROTEÍNA NÃO DESNATURADA COMO UM INDICADOR DE TRATAMENTO TÉRMICO PARA LEITE UHT E LEITE EM PÓ

Durante o processamento térmico de produtos lácteos ocorrem diversas modificações químicas dentre as quais a desnaturação de soroproteínas é bastante evidente e estudada. Este fenômeno pode gerar gelificação em leite UHT e problemas de solubilização em leite em pó, dois importantes produtos lácteos amplamente difundidos no mercado nacional. O índice de soroproteína não-desnaturada (WPNI) pode ser uma ferramenta útil para avaliar o grau de severidade ao qual o leite está sendo submetido e, portanto, permite inferir sobre a ocorrência dos possíveis defeitos associados à desnaturação proteica. Neste estudo, verificou-se o teor de soroproteína não-desnaturada como indicador de tratamento térmico em leite submetido aos processamentos de ultrapasteurização e de secagem. As amostras comerciais de leite UHT e leite em pó analisadas apresentaram um intervalo de confiança de WPNI (2,39 ± 1,41) mg WPN.mL-1 e (2,63 ± 1,38) mg WPN.mL-1, respectivamente. De acordo com a análise estatística de variância, todas as amostras avaliadas diferiram entre si com um p-valor < 0,001; sendo que 90% das amostras de leite UHT e 67% para as amostras de leite em pó foram classificadas como submetidas a médio tratamento térmico. Os dados obtidos permitiram inferir sobre a severidade do tratamento térmico associado a cada tipo de produto analisado e foram capazes de demonstrar a falta de padronização dos processos de tratamento térmico nas amostras estudadas, fato este que pode ter como consequência o desenvolvimento de características indesejáveis nos produtos ao longo do período de estocagem e comercialização.

Yogurt made from milk heated at different pH values

Milk for yogurt manufacture is subjected to high heat treatment to denature whey proteins. Low milk pH values (≤6.5) at heating result in most denatured whey proteins becoming associated with casein micelles, whereas high milk pH values (≥7.0) at heating result in the formation of mostly soluble (nonmicellar) denatured whey protein complexes. There are conflicting reports on the relative importance of soluble and casein-bound whey protein aggregates on the properties of acid gels. Prior studies investigating the effect of pH of milk at heating used model gels in which milk was acidified by glucono-δ-lactone; in this study, we prepared yogurt gels using commercial starter cultures. Model acid gels can have very different texture and physical properties from those made by fermentation with starter cultures. In this study, we investigated the effects of different pH values of milk at heating on the rheological, light backscatter, and microstructural properties of yogurt gels. Reconstituted skim milk was adjusted to pH values 6.2, 6.7, and 7.2 and heated at 85°C for 30 min. A portion of the heated milk samples was readjusted back to pH 6.7 after heating. Milks were inoculated with 3% (wt/wt) yogurt starter culture and incubated at 40°C until pH 4.6. Gel formation was monitored using dynamic oscillatory rheology, and parameters measured included the storage modulus (G′) and loss tangent (LT) values. Light-backscattering properties, such as the backscatter ratio (R) and the first derivative of light backscatter ratio (R′), were also monitored during fermentation. Fluorescence microscopy was used to observe gel microstructure. The G′ values at pH 4.6 were highest in gels made from milk heated at pH 6.7 and lowest in milk heated at pH 6.2, with or without pH adjustment after heating. The G′ values at pH 4.6 were lower in samples after adjustment back to pH 6.7 after heating. No maximum in the LT parameter was observed during gelation for yogurts made from milk heated at pH 6.2; a maximum in LT was observed at pH ~4.8 for samples heated at pH 6.7 or 7.2, with or without pH adjustment after heating. Higher R-values were observed with an increase in pH of heating, with or without pH adjustment after heating. The sample heated at pH 6.2 had only one major peak in its R′ profile during acidification, whereas samples heated at pH 6.7 and 7.2 had 2 large peaks. The lack of a maximum in LT parameter and the presence of a single peak in the R′ profile for the samples heated at pH 6.2 were likely due to the partial solubilization of insoluble calcium phosphate when milk was acidified to this lower pH value. No clear differences were observed in the microstructures of gels between the different treatments. This study indicates that heating milk at the natural pH (~6.7) created an optimum balance of casein-bound and soluble denatured whey proteins, which resulted in yogurt with the highest gel stiffness.

Amaltheys: A fluorescence-based analyzer to assess cheese milk denatured whey proteins

The cheese industry faces many challenges to optimize cheese yield and quality. A very precise standardization of the cheese milk is needed, which is achieved by a fine control of the process and milk composition. Thorough analysis of protein composition is important to determine the amount of protein that will be retained in the curd or lost in the whey. The fluorescence-based Amaltheys analyzer (Spectralys Innovation, Romainville, France) was developed to assess pH 4.6-soluble heat-sensitive whey proteins (sWP*) in 5 min. These proteins are those that can be denatured upon heat-treatment and further retained in the curd after coagulation. Monitoring of sWP* in milk and subsequent adaptation of the process is a reliable solution to achieve stable cheese yield and quality. Performance of the method was evaluated by an accredited laboratory on a 0 to 7 g/L range. Accuracy compared with the reference Kjeldahl method is also provided with a standard error of 0.25 g/L. Finally, a 4-mo industrial trial in a cheese plant is described, where Amaltheys was used as a process analytical technology to monitor sWP* content in ingredients and final cheese milk. Calibration models over quality parameters of final cheese were also built from near-infrared and fluorescence spectroscopic data. The Amaltheys analyzer was found to be a rapid, compact, and accurate device to help implementation of standardization procedures in the dairy industry.

Enhanced stability of anthocyanin-based color in model beverage systems through whey protein isolate complexation

Anthocyanins are often used in functional foods and beverages as colorants and nutraceuticals. However, these natural compounds may undergo chemical degradation during storage leading to color fading and loss of bioactivity. In particular, vitamin C (l-ascorbic acid) is known to accelerate anthocyanin degradation. In this study, the influence of various food-grade biopolymers on the physical and chemical stability of model beverages containing anthocyanin (0.025%), ascorbic acid (0 or 0.05%), and calcium salt (0 or 0.01%) was examined under accelerated conditions (40°C for 7days). Four biopolymers (1%) were examined for their potential to inhibit anthocyanin degradation: native whey protein; denatured whey protein; citrus pectin; and beet pectin. The physical stability was determined by measuring changes in absorbance, color, and visual appearance. Solutions containing anthocyanin and calcium salt (0 or 0.01%) were stable throughout storage, while those with added ascorbic acid were the least stable. The addition of biopolymers, particularly heat denatured whey protein, significantly enhanced the stability of the anthocyanin during storage. Fluorescence quenching studies showed that the anthocyanin may have formed complexes with the whey protein through hydrogen bonding that resulted in their enhanced stability in the presence of ascorbic acid. This study provides information that may improve the stability of anthocyanins in food and beverage systems.

Properties of partially denatured whey protein products: Formation and characterisation of structure

Partially denatured whey protein (PDWPC) products have been manufactured using a controlled heating process that allows control of the degree of denaturation of the whey proteins. This is assessed by following the change in free sulphydryl content of the protein as heating progresses. This allows the formation of soluble whey protein aggregates of diverse particle size and morphology. The PDWPC's have been made using different manufacturing conditions (temperature, pH, degree of denaturation) to give aggregated PDWPC powders with a degree of denaturation in the range 45–98% and particle size 3–17 μm. Particle size analysis, scanning electron microscopy and density analysis show that the particles have aggregated structures that range from compact, particulate gel-like to fibrillar phase-separated structures, with intermediate structures formed under some conditions. These structures are consistent with the known gel structures formed in whey protein concentrate gels. The structure of the PDWPC particles differs from that of microparticulated whey proteins. The possibility of using PDWPC's as ingredients tailored to the needs of food manufacturers is discussed.

Influence of heating treatment and membrane concentration on the formation of soluble aggregates

This study investigated the effect of thermal denaturation on milk concentrated by ultrafiltration (UF) with or without diafiltration (DF) at various time–temperature combinations. The non-sedimentable protein fractions present in milk after heating were analyzed using size exclusion chromatography and their composition was determined. The extent of whey protein denaturation and the composition of the soluble complexes depended not only on heating temperature and time, but also on the protein concentration. There was a greater extent of protein aggregation in concentrated milk compared to 1× milk, and there was a significant difference in the size and composition of the aggregates present in the non-sedimentable fraction depending on whether the milk was concentrated only by UF or if DF was also used. DF milk contained a higher concentration of non-sedimentable protein compared to UF milk. In all samples, κ-casein and β-lactoglobulin were the main proteins present in the soluble aggregates, but higher temperatures and longer heating times resulted in greater extents of denaturation of α-lactalbumin. This work clearly identified differences in the extent of aggregation between milk concentrated by UF or UF and DF, and unconcentrated milk, with important consequences on the processing functionality of caseins.

Reducing the oil content in coated and deep‐fried chicken using whey protein

Whey protein has been investigated for its oil barrier properties in fried foods. Whey protein has been implemented successfully non‐film forming applications to reduce the fat content of deep‐fried chicken. As a 10% postbreading dip, whey protein solutions significantly lowered oil absorption of boneless patties by 37% compared to undipped controls. In bone‐in chicken thighs, 10% whey protein postbreading dip caused a 15% reduction in oil absorption during frying which was doubled by 10% β‐lactoglobulin postbreading dip at pH 2. A 30% lipid reduction was observed in deep‐fried battered and floured chicken strips when the strips were dipped in a 10% solution of denatured whey protein prior to frying compared to control chicken strips. When added to the batter of deep‐fried chicken nuggets, a significant fat reduction was observed for nuggets to which 3% whey protein was added to the batter compared to the control. In chicken strips that were marinated in a 15% solution of whey protein, a 35% fat reduction was observed. In spite of these results, questions that still remain. These studies have been undertaken in fully cooked products, not in raw coated or par‐fried products. The impact of rethermalization method is not known. Only preliminary research on the impact of WPI on the frying oil with respect to the formation of polar materials has been undertaken. On the positive side, the processes for introducing whey protein as an oil inhibition agent utilize processing steps that are already very common, e.g. incorporation into the batter or a marinade. Whey protein has the potential to be employed as an oil inhibiting agent in fried foods especially as it is relatively inexpensive and easy to come by.

Membrane fouling during ultra- and microfiltration of whey and whey proteins at different environmental conditions: The role of aggregated whey proteins as fouling initiators

For the concentration of whey proteins from whey by ultrafiltration (UF) or fractionation by microfiltration (MF), mass transfer through the membrane is limited by deposit formation which is variable and not well understood, yet. There is little knowledge on the predominant molecular mechanism responsible for membrane fouling during whey micro- and ultrafiltration. Some works indicate that whey protein aggregates induce or enhance membrane fouling. The impact of whey protein aggregates as well as the interaction of native whey proteins and whey protein aggregates during membrane fouling was studied in both dead-end lab scale and cross-flow pilot scale. By means of targeted heat-treatment, a defined amount of protein aggregates was formed in β-Lactoglobulin (β-Lg) model suspensions as well as in sweet whey. β-Lg aggregates were found to accelerate membrane fouling during MF and UF due to covalent thiol/disulfide reactions. For sweet whey cross-flow filtration, membrane fouling was accelerated by whey protein aggregates up to a certain degree of whey protein denaturation. Above a critical value of 30% protein denaturation, flux increased again. This effect is explained by an improved erosion of larger whey protein aggregates as well as a reduced reactivity of heat-aged whey protein aggregates.

Denaturation and Physical Characteristics of Spray-Dried Whey Protein Isolate Powders Produced in the Presence and Absence of Lactose, Trehalose, and Polysorbate-80

The denaturation (loss of protein through aggregation and/or change in secondary structure) and physical characteristics such as powder morphology, particle size and size distribution, amorphous/crystalline behavior, and solubility of whey protein isolate (WPI) were investigated in a spray-drying process. The protective efficacy of sugars (lactose and trehalose) and low-molecular-weight surfactant polysorbate-80 (Tween-80) on the secondary structure (β-turn, β-sheet and α-helix) and physical characteristics of spray-dried WPI was quantified. The WPI, WPI+sugar, and WPI+Tween-80 formulations were spray dried maintaining the total solids at 10% (w/w). The inlet and outlet temperatures were maintained at 180 and 80°C, respectively. The results showed that the loss of protein through denaturation and aggregation was not significant (p > 0.05). However, a significant (p < 0.05) alteration of the secondary structural elements was observed. Due to spray drying of WPI without protectants, the β-sheet and β-turn were decreased by 4.4 and 14.5%, respectively, and the random coil increased by 20.7%. The α-helix of WPI remained unaltered during the spray-drying process. The presence of Tween-80 effectively protected the α-helix and β-sheet but the β-turn remained vulnerable and was decreased. No significant (p > 0.05) change in the solubility of WPI was observed due to spray drying. Spray drying of WPI+sugar produced essentially amorphous particles. The dried powder particles were spherical with wrinkled or folded surface.

Effect of high pressure treatment on inactivation of vegetative pathogens and on denaturation of whey proteins in different media

Cell suspensions of Listeria monocytogenes 5366, Staphylococcus aureus 976 and Pseudomonas aeruginosa 110T were exposed to high pressure treatment from 200 to 600 MPa for 5, 10 and 15 min at an initial temperature of 20 °C in raw skimmed bovine milk, whey and phosphate buffer, pH 7.0 in order to determine their degree of inactivation. S. aureus was the most baroresistant bacteria followed by L. monocytogenes and P. aeruginosa. A treatment at 600 MPa for 15 min was needed to obtain a reduction of 7 log10 cycles of S. aureus in all media. This treatment was selected to determine the degree of denaturation of the main whey proteins. Lactoperoxidase activity was not affected by pressure treatment applied to milk, whereas a denaturation of 80%, 70%, 44% and 7% was obtained for β-lactoglobulin, lactoferrin, immunoglobulin G (IgG) and α-lactalbumin, respectively. The effect of the treatment media on denaturation was not noticeable for lactoperoxidase, α-lactalbumin and IgG whereas β-lactoglobulin was more sensitive when treated in milk and lactoferrin in whey. Treatment of milk at 75 °C for 15 s induced a lower denaturation of β-lactoglobulin, lactoferrin and IgG compared to pressure treatment, whereas produced a higher decrease of enzymatic activity of lactoperoxidase.

Pilot-scale purification of α-lactalbumin from enriched whey protein concentrate by anion-exchange chromatography and ultrafiltration

A method based on anion exchange chromatography (AIEX) in combination with ultrafiltration (UF) was developed to obtain kilogram-scale amounts of bovine α-lactalbumin (α-La) of high purity from α-La-enriched whey protein concentrate (αWPC). Initially, α-La was successfully purified, at laboratory scale, from a 10% solution of αWPC. Removal of casein and denatured whey protein by acid precipitation resulted in a α-La purity of 78%. A further purification by AIEX eluting with sodium acetate (NaAc) buffer led to a final purity and recovery of 100 and 85%, respectively. Based on this, the process was simplified and optimized for pilot-scale purification by selective binding of β-lactoglobulin (β-Lg). Batchwise expanded bed AIEX using a column with 5 L of Q Sepharose matrix was performed. Pure α-La was obtained in the run through buffer. In the end, 1100 g of holo form α-La was obtained with a purity of 97.4% in protein and a recovery of 80%, calculated based on the protein detectable in reversed phase high-performance liquid chromatography (RP-HPLC). Thus, by combining AIEX and UF, heating was avoided allowing production of α-La in kilo-scale with high purity and recovery from αWPC with a minimum volume of buffer solutions.

Rennet Coagulation and Cheesemaking Properties of Thermally Processed Milk: Overview and Recent Developments.

Thermally induced changes in milk proteins and minerals, particularly interactions among caseins and denatured whey proteins, influence important properties of dairy products in both positive and negative ways. Whereas the extensive protein connectivity and increased water-holding capacity resulting from such heat-induced protein modification account for the much desired firmness of acid gels of yogurt, thermal processing, on the other hand, severely impairs clotting and adversely affects the cheesemaking properties of rennet-coagulated cheeses. In technological terms, the principal ongoing challenge in the cheese industry is to take advantage of the water-holding capacity of thermally aggregated whey proteins without compromising the rennetability of cheese milk or the textural and functional attributes of cheese. Including some recent data from the authors' laboratory, this paper will discuss important aspects and current literature on the use of thermally processed milk in the production of rennet-coagulated cheeses and also some of the potential alternatives available for inclusion of whey proteins in cheese, such as the addition of microparticulated whey proteins to cheese milk.

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).

The effect of whey protein denaturation on light backscatter and particle size of the casein micelle as a function of pH and heat-treatment temperature

Reconstituted low-heat skim milk with pH adjusted to 6.3, 6.7, and 7.1 was heated at 80 and 90 °C for 10 min to evaluate the relationship between the attachment of denatured whey proteins to the surface of the casein micelle and the intensity of optical light backscatter response. Both optical light backscatter intensity and casein particle size showed a significant negative correlation with pH and positive correlation with heat treatment temperature. When samples were analysed independently by respective pH, there was a significant correlation of both light backscatter and particle size with temperature for samples at a pH of 6.3, and between light backscatter and temperature at pH 7.1. These results show the potential for the use of optical light backscatter techniques as a measurement of changes in casein micelle particle size induced by heat treatment.

Controlling heat induced aggregation of whey proteins by casein inclusion in concentrated protein dispersions

Formation of soluble, heat stable whey protein (WP) particles may be achieved via caseins and their chaperone like activity. The aim of this study was to establish effects of caseins on the properties of heat induced WP aggregates. Concentrated dairy protein mixtures (10% total-solids) were prepared with various casein:WP ratios (5:95–30:70) at adjusted pH (5.7–7.5) and heated to 80–120 °C, under a constant shear (500 s−1). Increasing casein fraction at higher pH reduced the average size of WP aggregates and the surface hydrophobicity with simultaneous reduction in apparent viscosity at pH 6.7. WP denaturation was minimal when 30% casein was included in the dispersions. Inhibition of WP denaturation at elevated pH with inclusion of caseins may be attributed to a chaperon like activity of the casein. Thus, by increasing pH and the casein content in the system it was possible to form nano-size whey protein particles.

Reassociation of dissociated caseins upon acidification of heated pH-adjusted skim milk

Milk was heated at different pH (pH 6.5–7.1) and temperatures (20–120°C/10min). This resulted in different levels of casein and denatured whey proteins to be distributed between the colloidal and serum phases. The milks were subsequently acidified and the distribution of protein between colloidal and serum was monitored at different pH. On acidification to pH 5.4, the serum phase caseins and denatured whey proteins partially reassociated with the caseins, although a complex behaviour was observed at ∼pH 5.4 where additional casein dissociation occurred in some samples. At pH below 5.4 the caseins and denatured whey proteins rapidly aggregated. No separate aggregation of κ-casein/denatured whey protein complexes or κ-casein depleted micelles was observed. The earlier gelation of milks heated at higher pH was likely to be due to the destabilisation of the entire milk protein system rather than a preferential aggregation of the serum phase proteins.

Influence of protein content and storage conditions on the solubility of caseins and whey proteins in milk protein concentrates

Milk protein concentrate (MPC) powders with protein content ranging from 35% (MPC35) to 87% (MPC90) (w/w) protein in dry matter were prepared on a pilot scale. The solubility of the MPCs and the individual caseins and whey protein therein was determined as a function of storage time and temperature. Insolubility developed more rapidly in high protein MPCs and at higher storage temperatures and was primarily related to insolubility of the micellar caseins. Non-micellar caseins retained solubility throughout storage, whereas development of insolubility of whey proteins was less extensive than that of caseins, but was promoted in MPC80 prepared from milk with significant amounts of denatured whey proteins. It is hypothesised that the development of insolubility in MPCs arises due to aggregation of casein micelles at high temperature during drying. High calcium ion activity and the gradual conversion from an aqueous to a non-aqueous surrounding contribute to this instability.