Whey Protein Denaturation Research Articles

Impact of Storage Temperature on Physical Stability and Protein-Lipid Co-oxidation in Whey Protein functional Emulsions.

The aim of this study was to investigate the effect of different storage temperatures (4°C, 25°C and 45°C, denoted as T-4, T-25 and T-45, respectively) on the physical stability and protein-lipid co-oxidation of whey protein functional emulsions during the storage period at storage times of 1, 2, 3 and 6 months. At the 6th month of storage, samples stored at 45°C were less physically stable than samples stored at 4°C, as evidenced by an increase in particle size (5.50%), a decrease in zeta potential (7.93%), and a decrease in adsorbed protein concentration (36.8%). The most severe protein-lipid co-oxidation was induced in whey protein functional emulsions under 45°C temperature conditions with increasing storage time. Protein oxidation products (carbonyls, N′-formyl-L-kynurenine, and sulfhydryl groups) and lipid oxidation products (lipid hydroperoxides, thiobarbituric acid reactive substances) were at their highest levels, and significant molecular changes were observed in the molecules of adsorbed protein or unadsorbed proteins. As the storage temperature rises it exposes the active sites such as internal sulfhydryl and hydrophobic groups in the protein, promoting cross-linking and aggregation. Cluster and correlation analyses showed that protein-lipid co-oxidation of emulsions inevitably occurs during storage, but low-temperature storage can delay this process. This study is of guiding significance for the quality maintenance of functional nutritional emulsions and the selection of storage conditions. In addition, this study may provide clearer solutions to the problems of whey protein denaturation and formation of lipid oxides caused by protein-lipid co-oxidation in the food industry, as well as to the problem of increasing the shelf life of products by choosing the appropriate storage, packaging, and transportation temperatures.

Influence of β-lactoglobulin and κ-casein genotype on whey protein denaturation in heated bovine milk and rennet whey

Whey proteins provide nutritional and functional properties in many dairy products, and their denaturation induced by heat has drawn attention in dairy research and industry. Denatured whey proteins have functional properties that are beneficial for certain applications, such as yogurt, while uncontrolled denatured protein aggregation can impair certain processes, such as rennet coagulation. The possible effect of genetic polymorphism of β-lg and κ-CN on heat-induced denaturation of whey proteins in milk and in rennet whey was investigated in this study. No effect of κ-CN genotype on the denaturation of β-lg and α-lac was detected, while the A variant of β-lg was more heat-stable than the B variant. More denatured β-lg B was shown to interact with casein micelles to form insoluble aggregates compared to the A variant. These results may provide guidance for selection of specific milk genotypes for applications where whey protein denaturation needs to be minimised or else preferred, such as foaming and emulsification.

Assessment of the impact of pasteurisation on protein denaturation in sheep sweet whey by asymmetrical flow field-flow fractionation according to sampling period

Whey recovery is limited in artisanal cheese manufacture. This by-product of cheese manufacture was traditionally considered as waste and is highly polluting. However, interest in the nutritional properties of native whey and its proteins has recently increased. Pretreatment is required to preserve highly perishable whey for processing. Only a few studies have focused on sheep whey, despite its considerable nutritional and technological potential. Here, we investigated the effect of heat treatment on the denaturation and formation of protein aggregates in sweet sheep whey collected in January, April and July. Two pasteurisation protocols were studied (72 °C for 1 min and 80 °C for 15 s). Microbiological quality was assessed by checking for the presence of microbes after five days of storage at 4 °C. Asymmetrical flow field-flow fractionation (AF4) coupled to UV detector, multiangle light scattering (MALS) and refractometer (dRi) is a mild, non-naturing technique for determining the extent of denaturation of whey proteins and the size of whey protein aggregates. Greater whey protein denaturation and larger whey protein aggregates were observed for the 80 °C/15 s protocol than for the 72 °C/1 min protocol, for the January and April samples. A decrease in Immunoglobulin G (IgG) content was observed after heat treatments in the samples from July but not significantly for the other proteins. Moreover, the retention times of the monomeric whey protein peaks on the AF4 fractogram were higher for this period, indicating that the proteins were larger. Microbiological testing showed that both pasteurisation treatments were sufficiently effective to ensure good sanitary quality. The pasteurisation schedule best preserving native proteins was heating at 72 °C for 1 min.

Physiochemical properties, volatile compounds and consumer acceptance of novel ultra-high temperature milk during storage

Ultra-high temperature (UHT) treatment, achieved through direct heating (D-UHT) and indirect heating (ID-UHT), imparts a long shelf-life to milk, allowing for non-refrigerated distribution. This study investigates the impact of a novel D-UHT method (155 °C, 0.5 s) on milk properties, volatile compounds, and consumer acceptance, contrasting it with traditional ID-UHT. Compared to ID-UHT, D-UHT milk showed smaller particle size (0.63–0.65 μm) and reduced whey protein denaturation (43.11–61.54 %). No statistically significant difference in consumer overall liking scores was observed between D-UHT and ID-UHT milk samples (from 5.3 to 5.9; P > 0.05). Emoji-check-all-that-apply analysis revealed a more positive attitude towards D-UHT milk before 1-month storage. Identification of 36 volatile compounds, including 18 aroma-active compounds, in the milk samples facilitated characterization through principal component analysis. The profile of D-UHT milk after a 1-month storage period closely resembled that of raw milk. Our findings suggest that novel D-UHT milk may offer enhanced nutrition and consumer acceptance within the first month, attributed to lower heat exposure and a volatile compound profile resembling raw milk.

Comparing physicochemical properties related to thermal stability of caprine and bovine milk protein concentrate dispersions

Thermal stability and physicochemical properties as a function of heating temperature, pH and salt addition was compared between caprine and bovine milk protein concentrate dispersions. Caprine milk protein concentrate (MPC) had a lower amount of heat coagulation time and a higher percentage of sediments than bovine MPC. Calcium ion activity of caprine MPC was higher than that of bovine. The amount of κ-casein in the serum of caprine MPC dispersions was higher than that of bovine, and increased with increasing temperatures, pH and the addition of NaCl and citrate. The amount of whey protein in serum was also higher in heated caprine MPC compared with bovine. Particle size of caprine MPC was larger than bovine MPC and was more susceptible to induced aggregation. The lower thermal stability of caprine MPC might be associated with the higher calcium ion activity and the higher amount of κ-casein dissociation promoted by whey protein denaturation and complexation in serum.

The denaturation of whey proteins in a processed cheese environment

The kinetics of whey protein denaturation at three pH values (pH 5.4, 5.7 and 6.0) was determined in processed cheese made from rennet casein/whey protein concentrate (RC-PC), or milk protein concentrate (MPC-PC). Samples were heated from 75 to 100 °C for up to 5400 s. Denaturation of both proteins was faster with increasing pH. The reaction order for β-lactoglobulin and α-lactalbumin denaturation was 1.5 and 1.0 respectively. There was a change in temperature dependence of the rate constants at about 85 °C for both proteins. Kinetic parameters were consistent with unfolding as rate-determining at low temperatures and aggregation as rate-determining at higher temperatures. β-Lactoglobulin denaturation was slower at low temperatures and faster at high temperatures for the MPC-PC when compared with RC-PC with a similar effect at all pH. α-Lactalbumin denaturation was more rapid for MPC-PC than RC-PC at all temperatures, with a greater difference at lower pH.

Prior denaturation and aggregation of whey proteins: is this a useful strategy for increasing the content of these proteins in UHT high-protein dairy beverages?

UHT treatments applied to dairy products have been associated with modifications on the proteins’ structure that may trigger the occurrence of fouling, sedimentation and/or gelation. These issues are particularly relevant regarding formulated whey protein-rich dairy beverages. In this context, this study aimed to evaluate the impact of a two steps preheating treatment coupled with homogenization (pretreatment) prior to UHT treatment to allow an increase of whey protein concentration in UHT high-protein dairy beverage. The pretreatment was applied to 10% w/w protein formulated beverages displaying casein (CN) to whey protein (WP) ratios of 40:60 and 30:70. The pretreated beverages were compared with non-pretreated beverage containing 50CN:50WP ratio. The applied pretreatment induced the denaturation of around 97.8–99.4% of WP. After UHT treatment, despite the higher concentration of WP, pretreated beverages displayed similar particle size distribution and equivalent or lower sedimentation level than the non-pretreated samples. The present study reveals the potential of the pretreatment for allowing the increase of WP content in UHT treated high-protein beverages. The results reinforce the rationalization of process development as a useful strategy for producing stable high-protein products.

Gastric clot formation and digestion of milk proteins in static in vitro infant gastric digestion models representing different ages

Gastric digestion conditions change during infancy from newborn towards more adult digestion conditions, which can change gastric digestion kinetics. However, how these changes in gastric digestion conditions during infancy affect milk protein digestion has not been investigated. Therefore, we aimed to investigate milk protein digestion with static in vitro gastric digestion models representing one-, three- and six-month-old infants. With increasing age, gastric clots and soluble proteins were digested more extensively, which may partly be attributed to the looser gastric clot structure. Larger differences with increasing age were found for heated than unheated milk proteins, which might be caused by the presence of denatured whey proteins. Taken together, these findings show that gastric milk protein digestion increases during infancy. These in vitro gastric digestion models could be used to study how milk protein digestion changes with infant age, which may aid in developing infant formulas for different age stages.

Effects of high hydrostatic pressure on antimicrobial protein stability and the rheological and shelf-life properties of donkey milk.

The effects of high hydrostatic pressure (HHP) and heat treatments on antimicrobial protein stability and on the physico-chemical, microbiological, rheological and shelf-life properties of donkey milk were investigated. Although heat treatment at 75°C for 2 min resulted in 1.50 log CFU ml-1 microbial inactivation, losses in activities of lysozyme (58%) and lactoferrin (82%) were observed due to whey protein denaturation. By contrast, HHP application at 400 MPa caused lower enzyme activity losses (22 and 37% respectively) whilst maintaining a significant reduction of microbial load (1.80 log CFU ml-1). Color analyses showed that the lightness values of all samples decreased during storage. Higher flow consistency (viscosity) and lower flow behavior indexes were observed in heat-treated samples compared to untreated and HHP-treated ones, which can be explained by advanced protein denaturation during heat-treatment. The results suggest that HHP is a more suitable process than heat treatment for preservation of donkey milk within the conditions studied.

Towards creating sustainable foods from side streams: Heat-induced structure formation in blends of sunflower seed press cakes and cheese whey under moderate shear

Processing of oilseeds generates low value by-products, which still contain valuable components. Sustainable and circular food chains require valorising the entire stream or producing less refined fractions of it. One approach could be blending with other protein-containing side streams to obtain novel, nutritionally valuable and techno-functional food ingredients. In this study, sunflower press cake was co-processed with components from whey, a cheese-making by-product. Blends with constant dry matter and protein content but different press cake to whey protein ratios (0–225 g/kg press cake) were used to investigate the contributions of both side streams to structure formation during heating (80–140 °C) under moderate shear using a Rapid Visco Analyser. The denaturation of whey proteins contributed to an increased viscosity, but the highest viscosities were still achieved at high ratios of press cake, underlining the importance of the fibre fraction for structure formation. Treatments at 120 and 140 °C increased the amount of insoluble material and water holding capacity of the blends, and analyses of the serum phase and curd showed that sunflower and whey proteins formed heat-induced, insoluble aggregates. Confocal laser scanning microscopy confirmed the presence of large protein particles dispersed in the matrix rather than the presence of a continuous network, as was the case for heating without shear. Furthermore, the protein particles were more defined and showed a smoother appearance with increasing press cake concentration. This research provides fundamental insights in the colloidal interactions between biomacromolecule blends during processing (e.g., protein cross-linking, microphase separation), and demonstrates the importance of understanding the critical process parameters (e.g., heat, shear) leading to structure formation, facilitating the successful integration of complex materials such as press cakes and setting the basis for further processing of the blends and their utilisation as ingredients, for instance in functional drinks, snacks, or semi-solid spreads.

Chemical composition and amino acids profile of Gouda cheese supplemented with denatured whey protein paste

Chemical composition and amino acids profile of Gouda cheese supplemented with denatured whey protein paste

Rheological, Physicochemical, Microbiological, and Aroma Characteristics of Sour Creams Supplemented with Milk Protein Concentrate.

Milk protein concentrate (MPC) is widely used to enhance the stability and texture of fermented dairy products. However, most research has focused on yogurt products, and the effects of MPC on sour cream characteristics remain unknown. Therefore, we investigated the effects of different MPC levels (0%, 1%, 2%, and 3% w/w) on the rheological, physicochemical, microbiological, and aroma characteristics of sour creams in this study. We found that MPC supplementation stimulated the growth of lactic acid bacteria (LAB) in sour creams, resulting in higher acidity than that in the control sample due to the lactic acid produced by LAB. Three aroma compounds, acetaldehyde, diacetyl, and acetoin, were detected in all sour cream samples. All sour creams showed shear-thinning behavior (n=0.41-0.50), and the addition of MPC led to an increase in the rheological parameters (ηa,50, K, G', and G″). In particular, sour cream with 3% MPC showed the best elastic property owing to the interaction between denatured whey protein and caseins. In addition, these protein interactions resulted in the formation of a gel network, which enhanced the water-holding capacity and improved the whey separation. These findings revealed that MPC can be used as a supplementary protein to improve the rheological and physicochemical characteristics of sour cream.

Contribution of whey protein denaturation to the in vitro digestibility, biological activity and peptide profile of milk protein concentrate

The impact of whey protein (WP) denaturation on the in vitro digestibility and biological activity of milk protein concentrate-85 (MPC85) was investigated. MPC85S1 and MPC85S2 having undenatured WP levels equal to 16.6 and 6.0 g/100 g overall protein, respectively, had similar in vitro protein digestibility corrected amino acid scores equal to 1.14. The samples were subjected to in vitro simulated gastrointestinal digestion while sampling was performed every 30 min during gastric (GD) followed by intestinal (GID) digestion. Liquid chromatography–mass spectroscopy showed that MPC85S1-GD, MPC85S2-GD, MPC85S1-GID and MPC85S2-GID had 50, 38, 47 and 66 unique peptides, respectively. The degree of hydrolysis, molecular mass distribution, dipeptidyl peptidase-IV inhibition, oxygen radical absorbance capacity and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) radical scavenging activity of the digests were compared. Overall, the results showed higher digestibility and bioactivities for low-denatured MPC85 compared to high-denatured MPC85 upon GD, however, following GID, both samples were digested to a similar extent.

Effect of Direct Steam Injection and Instantaneous Ultra-High-Temperature (DSI-IUHT) Sterilization on the Physicochemical Quality and Volatile Flavor Components of Milk.

The effects of variations in the heat treatment process of milk on its quality and flavor are inevitable. This study investigated the effect of direct steam injection and instantaneous ultra-high-temperature (DSI-IUHT, 143 °C, 1-2 s) sterilization on the physicochemical properties, whey protein denaturation (WPD) rate, and volatile compounds (VCs) of milk. The experiment compared raw milk as a control with high-temperature short-time (HTST, 75 °C 15 s and 85 °C 15 s) pasteurization and indirect ultra-high-temperature (IND-UHT, 143 °C, 3-4 s) sterilization. The results showed no significant differences (p > 0.05) in physical stability between milk samples with different heat treatments. The DSI-IUHT and IND-UHT milks presented smaller particle sizes (p < 0.05) and more concentrated distributions than the HTST milk. The apparent viscosity of the DSI-IUHT milk was significantly higher than the other samples (p < 0.05) and is consistent with the microrheological results. The WPD of DSI-IUHT milk was 27.52% lower than that of IND-UHT milk. Solid-phase microextraction (SPME) and solvent-assisted flavor evaporation (SAFE) were combined with the WPD rates to analyze the VCs, which were positively correlated with ketones, acids, and esters and negatively associated with alcohols, heterocycles, sulfur, and aldehydes. The DSI-IUHT samples exhibited a higher similarity to raw and HTST milk than the IND-UHT samples. In summary, DSI-IUHT was more successful in preserving the milk's quality due to its milder sterilization conditions compared to IND-UHT. This study provides excellent reference data for the application of DSI-IUHT treatment in milk processing.

Sustainable Approaches in Whey Cheese Production: A Review

Whey cheeses have been produced from the very early steps of cheesemaking practices as a sustainable way to utilize whey, which is the main by-product of cheesemaking. Traditional whey cheeses, manufactured with similar processes, are Ricotta, Ricotta salata or Ricottone, and Ricotta fresca in Italy; Anthotyros, Myzithra, Manouri, Xynomyzithra, and Urda in Greece; Urda in Serbia and Romania as well as in other countries such as Israel; Lor in Turkey; Anari in Cyprus; Skuta in Croatia and Serbia; Gjetost and Brunost in Norway; Mesost and Messmör in Sweden; Mysuostur in Iceland; Myseost in Denmark; Requeijão in Portugal; and Requesón in Spain and Mexico. The production of whey cheese is based on the denaturation of whey proteins by heating to 88–92 °C. The specific processing conditions and aspects of the microbiology of whey cheeses are discussed. The special characteristics of whey cheeses, which are high pH and high moisture content, make them susceptible to microbial growth. Due to the limited shelf life of these products, extended research has been carried out to extend the shelf life of whey cheese. The sustainable preservation approaches, such as modified atmosphere packaging, addition of herbs and/or plant extracts, and bio-preservation methods are reviewed. Moreover, novel whey cheeses focused on functional properties have developed during the last 10 years.

Physicochemical properties of fluid milk with different heat treatments and HS-GC-IMS identification of volatile organic compounds

To improve understanding of the effects of industrial heating processes on its physiochemical and flavour properties, fluid milk was evaluated for protein denaturation and flavour volatiles after high-temperature short-time (HTST), indirect ultra-high temperature (IND-UHT) and direct ultra-high temperature with direct steam infusion (INF-dUHT) treatments. Protein denaturation was evaluated by particle size, free sulphydryl content, fluorescence spectra, native whey protein retention rate and microstructure. Volatile organic compounds (VOCs) were compared by headspace-gas chromatography-ion mobility spectrometry (HS-GC-IMS). HTST treatment exerted the lowest negative effect on the aggregation and denaturation of native whey proteins including α-lactalbumin, β-lactoglobulin and lactoferrin, followed by INF-dUHT. IND-UHT caused the most severe denaturation of native whey proteins. HTST-treated milk was characterised by a high level of low molecular weight ketones and INF-dUHT milk possessed the greatest abundance of aldehydes, whereas IND-UHT milk exhibited the highest contents of high molecular weight methyl ketones, which contributed to an unpleasant aroma.

Influence of the biologically active supplement "Immunocort" on the production and quality of the "Mozzarella Ukrainian" cheese

Experimental studies have proven the effectiveness of the use of the herbal dietary supplement "Immunocort" in the technology of rennet cheese "Mozzarella Ukrainian". "Immunocort" is an herbal preparation that contains a balanced complex of immunoactive plants aimed at strengthening the immune system and eliminating immunodeficiency. The composition of the dietary supplement includes astragalus herb, echinacea root, Damascus blackberry seeds, currant leaf, string grass, Uncaria bark, and ant tree bark. Based on organoleptic and physical-chemical studies, it was established that the best option in the manufacture of "Mozzarella Ukrainian" cheese is to add to the normalized mixture before its pasteurization of the "Immunocort" supplement in the amount of 3 % by weight of the normalized mixture. A study was conducted on the effect of the coagulation temperature and pasteurization temperature on the synergistic properties of the cheese clot. These indicators confirmed the significant importance for the transition of solids into whey in the process of making rennet cheese "Mozzarella Ukrainian" with the bio additive "Immunocort". It was found that with an increase in temperature, there was a decrease in the syneretic ability of cheese clots. The efficiency of using solids increased with increasing pasteurization temperature. The relatively low content of dry substances in whey at high pasteurization temperatures can be explained by the denaturation of whey proteins and their transition to a cheese clot. The best results were achieved at the pasteurization temperature (86±2) °C and the coagulation temperature (32±2) °C. The data obtained could make it possible to manage the process of making new types of cheeses with vegetable additives, depending on the specific conditions and requirements for the product

Heat-induced changes in blends of skimmed buffalo and bovine milk

This study investigated the physical, chemical, and structural changes in mixtures of buffalo and bovine milk (0:100, 25:75, 50:50, 75:25, 100:0) induced by heating at 80, 85, 90, and 95 °C for 5 min. No significant changes in particle size, zeta potential, and calcium activity were observed in heated buffalo milk and its mixtures with bovine milk, irrespective of the heating temperature, but heating at ≥ 85 °C induced a significant decrease in pH. The increase in viscosity with heating was dependent on the ratio of buffalo to bovine milk and the heating temperature. The variation in casein dissociation, whey proteins denaturation and their association with themselves and casein micelles, and the alteration in salt balance were key factors that contribute to significant heat-induced changes in pH and viscosity of milk blends.

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.

Microencapsulation of Lactobacillus plantarum 299v Strain with Whey Proteins by Lyophilization and Its Application in Production of Probiotic Apple Juices

The viability of probiotics is strictly influenced by the production, storage, and digestion, while microencapsulation is a technology that can protect them against harsh environments. In this study, the impact of different core-to-wall ratios and wall material formulations on physical properties and the cell number of the microcapsules were investigated. The samples with core-to-wall ratio 1:1 have a significantly higher cell number, encapsulation efficiency, and bulk density than samples with core-to-wall ratio 1:1.5. The yields of the encapsulation method were changes in the opposite direction. Meanwhile, core-to-wall ratios and formulation have a significant effect on the cell number of the microcapsules during the in vitro SGJ test, whereas time, core-to-wall ratios, and formulation have a similar influence in the in vitro SIJ test. Moreover, probiotic apple juices stored at 4 °C for 6 weeks kept the highest cell number at the end. Furthermore, probiotic apple juices fortified by microcapsules coated with WP:DWP 1:1 in core-to-wall ratio 1:1 and stored at 4 °C for 4–8 weeks exhibited a significantly lower pH value. In summary, both whey proteins and denatured whey proteins are as good as coating material for microencapsulation of probiotic bacteria Lactobacillus plantarum 299v strains. These microcapsules have high potential in the production of probiotic apple juice even by fermentation or fortification methods.