A proteomic approach to detect lactosylation and other chemical changes in stored milk protein concentrate

Milk proteins undergo chemical changes such as lactosylation, deamidation and protein cross-linking during processing and storage of milk products. A proteomic technique combining two-dimensional gel electrophoresis and mass spectrometry was used to investigate chemical modifications to proteins, in milk protein concentrate (MPC80), during storage. Lactosylation, deamidation and protein cross-linking were observed on 2-DE gels. They were storage temperature-, humidity- and time-dependent. Lactosylated whey proteins were well separated on 2-DE in vertical stacks of spots. The masses of the spots varied by multiples of 324, indicating the attachment of lactose to lysine residues in the proteins. The trypsin-digested spots of α-lactalbumin were analysed by MALDI-TOF mass spectrometry, which indicated multiple lactosylation sites. The lactose adducts on gels were quantified by image analysis, allowing development of adducts over time to be monitored. The results show that proteomics can be used for the detection and quantification of chemical modifications to proteins in stored MPC80.     

Structural changes induced by high-pressure processing in micellar casein and milk protein concentrates

Reconstituted micellar casein concentrates and milk protein concentrates of 2.5 and 10% (wt/vol) protein concentration were subjected to high-pressure processing at pressures from 150 to 450 MPa, for 15 min, at ambient temperature. The structural changes induced in milk proteins by high-pressure processing were investigated using a range of physical, physicochemical, and chemical methods, including dynamic light scattering, rheology, mid-infrared spectroscopy, scanning electron microscopy, proteomics, and soluble mineral analyses. The experimental data clearly indicate pressure-induced changes of casein micelles, as well as denaturation of serum proteins. Calcium-binding α_S1- and α_S2-casein levels increased in the soluble phase after all pressure treatments. Pressurization up to 350 MPa also increased levels of soluble calcium and phosphorus, in all samples and concentrations, whereas treatment at 450 MPa reduced the levels of soluble Ca and P. Experimental data suggest dissociation of calcium phosphate and subsequent casein micelle destabilization as a result of pressure treatment. Treatment of 10% micellar casein concentrate and 10% milk protein concentrate samples at 450 MPa resulted in weak, physical gels, which featured aggregates of uniformly distributed, casein substructures of 15 to 20 nm in diameter. Serum proteins were significantly denatured by pressures above 250 MPa. These results provide information on pressure-induced changes in high-concentration protein systems, and may inform the development on new milk protein-based foods with novel textures and potentially high nutritional quality, of particular interest being the soft gel structures formed at high pressure levels.     

Effect of storage time and temperature of milk protein concentrate (MPC85) on the renneting properties of skim milk fortified with MPC85

This study investigated the effect of storage temperature (20–50 °C) and time (0–60 days) on the renneting properties of milk protein concentrate with 85% protein (MPC85). Reconstituted skim milk was fortified with the MPC85 (2.5% w/w) and the renneting properties of the skim milk/MPC85 systems were investigated using rheology. It was found that the final complex modulus (final G∗) and the yield stress of the rennet-induced skim milk/MPC85 gels decreased exponentially with storage time of the MPC85 for storage temperatures greater than 20 °C, with a greater effect at the higher storage temperatures. Changes in the solubility of MPC85 with storage time were correlated with the rheological properties. The primary phase of renneting (cleavage of κ-casein) was not affected by the storage of the MPC85; hence the effect was related to the secondary stage of renneting (aggregation/coagulation of rennet-treated casein micelles). Using a temperature–time superposition method, a master curve was formed from the final G∗, yield stress and solubility results. This suggested that the same physical processes affected the solubility and rennet gelation properties of the milks. It is proposed that the MPC85 protein in rennet-treated skim milk/MPC85 solutions may transform from an interacting material, when solubility is high, to an inert or weakly interacting material, when solubility is low, and that this results in the reduced final G∗ and yield stress of the rennet gels when MPC85 is stored at elevated temperatures for long periods.     

Influence of milk protein concentrates with modified calcium content on enteral dairy beverage formulations: Physicochemical properties

Because of their high protein and low lactose content, milk protein concentrates (MPC) are typically used in the formulation of ready-to-drink beverages. Calcium-mediated aggregation of proteins during storage is one of the main reasons for loss of storage stability of these beverages. Control and calcium-reduced MPC [20% calcium-reduced (MPC-20) and 30% calcium-reduced (MPC-30)] were used to evaluate the physicochemical properties in this study. This study was conducted in 2 phases. In phase I, 8% protein solutions were prepared by reconstituting the 3 MPC and adjusting the pH to 7. These solutions were divided into 3 equal parts, 0, 0.15, or 0.25% sodium hexametaphosphate (SHMP) was added, and the solutions were homogenized. In phase II, enteral dairy beverage formulations containing MPC and a mixture of gums, maltodextrin, and sugar were evaluated following the same procedure used in phase I. In both phases, heat stability, apparent viscosity, and particle size were compared before and after heat treatment at 140°C for 15 s. In the absence of SHMP, MPC-20 and MPC-30 exhibited the highest heat coagulation time at 30.9 and 32.8 min, respectively, compared with the control (20.9 min). In phase II, without any addition of SHMP, MPC-20 exhibited the highest heat coagulation time of 9.3 min compared with 7.1 min for control and 6.2 min for MPC-30. An increase in apparent viscosity and a decrease in particle size of reconstituted MPC solutions in phases I and II with an increase in SHMP concentration was attributed to casein micelle dissociation caused by calcium chelation. This study highlights the potential for application of calcium-reduced MPC in dairy-based ready-to-drink and enteral nutrition beverage formulations to improve their heat stability.     

Effects of standardization of whole milk with dry milk protein concentrate on the yield and ripening of reduced-fat cheddar cheese

Commercial milk protein concentrate (MPC) was used to standardize whole milk for reduced-fat Cheddar cheesemaking. Four replicate cheesemaking trials of three treatments (control, MPC1, and MPC2) were conducted. The control cheese (CC) was made from standardized milk (casein-to-fat ratio, C/F approximately 1.7) obtained by mixing skim milk and whole milk (WM); MPC1 and MPC2 cheeses were made from standardized milk (C/F approximately 1.8) obtained from mixing WM and MPC, except that commercial mesophilic starter was added at the rate of 1% to the CC and MPC1 and 2% to MPC2 vats. The addition of MPC doubled cheese yields and had insignificant effects on fat recoveries (approximately 94% in MPC1 and MPC2 vs. approximately 92% in CC) but increased significantly total solids recoveries (approximately 63% in CC vs. 63% in MPC1 and MPC2). Although minor differences were noted in the gross composition of the cheeses, both MPC1 and MPC2 cheeses had lower lactose contents (0.25 or 0.32%, respectively) than in CC (0.60%) 7 d post manufacture. Cheeses from all three treatments had approximately 10(9) cfu/g initial starter bacteria count. The nonstarter lactic acid bacteria (NSLAB) grew slowly in MPC1 and MPC2 cheeses during ripening compared to CC, and at the end of 6 mo of ripening, numbers of NSLAB in the CC were 1 to 2 log cycles higher than in MPC1 and MPC2 cheeses. Primary proteolysis, as noted by water-soluble N contents, was markedly slower in MPC1 and MPC2 cheeses compared to CC. The concentrations of total free amino acids were in decreasing order CC > MPC2 > MPC1 cheeses, suggesting slower secondary proteolysis in the MPC cheeses than in CC. Sensory analysis showed that MPC cheeses had lower brothy and bitter scores than CC. Increasing the amount of starter bacteria improved maturity in MPC cheese.     

Physico-chemical factors controlling the foamability and foam stability of milk proteins: Sodium caseinate and whey protein concentrates

We explored the foaming behavior of the two main types of milk proteins: flexible caseins and globular whey proteins. Direct foam comparison was complemented with measurements in model experiments such as thin foam films, dynamic surface tension, and protein adsorption. Foaming was studied as a function of pH (from below to above isoelectric point, pI) and range of ionic strengths. Maximum foamability was observed near pI ≈ 4.2 for WPC in contrast to sodium caseinate which had minimum foaming near pI = 4.6. Good foamability behavior correlated well with an increased adsorption, faster dynamic surface tension decrease and increased film lifetime. Differences in the stability of the foams and foam were explained with the different molecular structure and different aggregation behavior of the two protein types. Far from its isoelectric pI, casein adsorption layers are denser and thicker thus ensuring better stabilization. Added electrolyte increased further the adsorption and the repulsion between the surfaces (probably by steric and/or osmotic mechanism). In contrast the globular molecules of WPC probably could not compact well to ensure the necessary films and foams stabilization far from pI, even after electrolyte addition.     

Effect of pH on heat-induced interactions in high-protein milk dispersions and application of fluorescence spectroscopy in characterizing these changes

This study investigated casein-whey protein interactions in high-protein milk dispersions (5% protein wt/wt) during heating at 90°C for 1.5 to 7.5 min at 3 different pH of 6.5, 6.8, and 7.0, using both conventional methods (gel electrophoresis, physicochemical properties) and fluorescence spectroscopy. Conventional methods confirmed the presence of milk protein aggregates during heating, similar to skim milk. These methods were able to help in understanding the denaturation and aggregation of milk proteins as a function of heat treatment. However, the results from the conventional methods were greatly affected by batch-to-batch variations and, therefore, differentiation could be drawn only in nonheated samples and samples heated for a longer duration. The front-face fluorescence spectroscopy was found to be a useful tool that provided additional information to conventional methods and helped in understanding differences between nonheated, low-, and high-heated samples, along with the type of sample used (derived from liquid or powder milk protein concentrates). At all pH values, tryptophan maxima in nonheated samples derived from powdered milk protein concentrates presented a blue shift in comparison to samples derived from liquid milk protein concentrates, and tryptophan maxima in heated samples presented a red shift. With the heating of the sample, Maillard emission and excitation spectra also showed increases in the peak intensities from 408 to 432 and 260 to 290 nm, respectively. As the level of denaturation increased with heating, a marked differentiation can be seen in the principal component analysis plots of tryptophan, Maillard emission, and excitation spectra, indicating that the front-face fluorescence technique has a potential to monitor and classify samples according to milk protein interactions as a function of pH and heat exposure. Overall, it can be said that the pattern of protein-protein interactions in high-protein dispersions was similar to the observation reported in skim milk systems, and fluorescence spectroscopy with chemometrics can be used as a rapid, nondestructive, and complementary method to conventional methods for following heat-induced changes.     

Functionality of milk protein concentrate 80 with emulsifying salts and its applications in analogue cheeses

Milk protein concentrate 80 (MPC80) was prepared with different emulsifying salts (ES). The effects on particle size (D50), solubility, and surface hydrophobicity (H₀) of MPC80 were then observed after production. The molecular weight and secondary structure of MPC80 protein were also investigated through sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Fourier Transform Infrared (FTIR) spectrometry. Particle size (D50) was reduced from 31.37 to 20.67 μm following the addition of sodium phosphate (SPS). The solubility of MPC80 fortified with sodium citrate salt (SCS), SPS, and sodium pyrophosphate (SPP) was improved by 80.32, 78.23, and 55.80%, respectively. The SDS-PAGE pattern showed no significant difference between the control and single-ES-fortified samples, but major protein bands (α_s-CN, β-CN, κ-CN, BSA, and β-LG) had a stronger intensity than binary-ES-fortified MPC80. The FTIR results showed that the β-sheet content of ES-fortified MPC80 was relatively higher than that in the control. Compared with analogue cheeses made by MPC80 with and without ES, SCS–SPP (92:8)-modified MPC80 presented a better fluid mass and homogeneous colour.     

The ultrafiltration molecular weight cut-off has a limited effect on the concentration and protein profile during preparation of human milk protein concentrates

Optimizing protein intake for very low birth weight (<1,500 g) infants is fundamental to prevent faltering postnatal growth with the potential association of impaired neurodevelopment. The protein content of human milk is not sufficient to support the growth of very low birth weight infants. To meet their elevated protein requirements, human milk is currently fortified using typically bovine milk-based protein isolates (>85% on a dry basis). However, these products have several limitations for use in this vulnerable population. To overcome the shortcomings of bovine milk-based protein supplement, a human milk protein concentrate (HMPC) was developed. In preliminary attempts using 10 kDa ultrafiltration (UF) membranes, it was not possible to reach the protein content of commercial protein isolates, presumably due to the retention of human milk oligosaccharides (HMO). Consequently, it was hypothesized that the use of a UF membrane with a higher molecular weight cut-off (50 kDa rather than 10 kDa) could improve the transmission of carbohydrates, including HMO, in the permeate, thus increasing the protein purity of the subsequent HMPC. The results showed that permeate fluxes during the concentration step were similar to either UF molecular weight cut-off, but the 50-kDa membrane had a higher permeate flux during the diafiltration sequence. However, it was not sufficient to increase the protein purity of the human milk retentate, as both membranes generated HMPC with similar protein contents of 48.8% (10 kDa) and 50% (50 kDa) on a dry basis. This result was related to the high retention of HMO, mainly during the concentration step, although the diafiltration step was efficient to decrease their content in the HMPC. As the major bioactive proteins (lactoferrin, lysozyme, bile salt stimulated lipase, and α1-antitrypsin) in human milk were detected in both HMPC, the 50-kDa membrane seems the most appropriate to the preparation of HMPC in terms of permeation flux values. However, improving the separation of HMO from proteins is essential to increase the protein purity of HMPC.     

Short communication: Effects of nanofiltration and evaporation on the gel properties of milk protein concentrates with different preheat treatments

This study aimed to evaluate the effects of different concentration methods (nanofiltration and evaporation) and heat treatments on the gel properties of milk protein concentrate (MPC). The MPC gels were produced using glucono-δ-lactone (GDL) as an acidifier with different preheat treatments (30 min at 80°C and 5 min at 92°C). We then evaluated the effect of preheat treatments on MPC gel properties, including storage modulus (G′), loss tangent (tan δ), firmness, whey separation, and microstructure. The results indicated that without preheating, evaporation (EP)-MPC had higher G′ and firmness, and lower tan δ and whey separation than nanofiltration (NF)-MPC. These results suggest that EP-MPC produced a better acid-induced gel than NF-MPC when no preheat treatments were performed. After preheating, however, except for a very small difference in the final G′ (EP-MPC was higher), the 2 MPC did not differ significantly in firmness, final tan δ, or whey separation. Additionally, compared with the gel of unheated MPC, both preheat-treated gels (NF-MPC and EP-MPC) achieved increased G′ and firmness and decreased tan δ and whey separation. The preheat-treated MPC also displayed a more flexible-stranded network. These findings demonstrate that, given a suitable heating treatment, NF-MPC compares favorably with EP-MPC in achieving desired gel properties.     

Changes in the physical properties,solubility, and heat stability of milk protein concentrates prepared from partially acidified milk

A limiting factor in using milk protein concentrates (MPC) as a high-quality protein source for different food applications is their poor reconstitutability. Solubilization of colloidal calcium phosphate (CCP) from casein micelles during membrane filtration (e.g., through acidification) may affect the structural organization of these protein particles and consequently the rehydration and functional properties of the resulting MPC powder. The main objective of this study was to investigate the effects of acidification of milk by glucono-δ-lactone (GDL) before ultrafiltration (UF) on the composition, physical properties, solubility, and thermal stability (after reconstitution) of MPC powders. The MPC samples were manufactured in duplicate, either by UF (65% protein, MPC65) or by UF followed by diafiltration (80% protein, MPC80), using pasteurized skim milk, at either the native milk pH (~pH 6.6) or at pH 6.0 after addition of GDL, followed by spray drying. Samples of different treatments were reconstituted at 5% (wt/wt) protein to compare their solubility and thermal stability. Powders were tested in duplicate for basic composition, calcium content, reconstitutability, particle size, particle density, and microstructure. Acidification of milk did not have any significant effect on the proximate composition, particle size, particle density, or surface morphology of the MPC powders; however, the total calcium content of MPC80 decreased significantly with acidification (from 1.84 ± 0.03 to 1.59 ± 0.03 g/100 g of powder). Calcium-depleted MPC80 powders were also more soluble than the control powders. Diafiltered dispersions were significantly less heat stable (at 120°C) than UF samples when dissolved at 5% solids. The present work contributes to a better understanding of the differences in MPC commonly observed during processing.     

Lipid and protein structure analysis of frankfurters formulated with olive oil-in-water emulsion as animal fat replacer

Lipid and protein structural characteristics of frankfurter formulated with olive oil-in-water emulsion stabilized with soy protein isolate (SPI) as pork backfat replacer were investigated using Fourier transform infrared spectroscopy (FT-IR). Proximate composition and textural properties were also evaluated. Different frankfurters were reformulated: F/PF with pork backfat, F/SPI with oil-in-water emulsion stabilized with SPI and F/SPI + SC + MTG with emulsion stabilized with a combination of SPI, sodium caseinate (SC) and microbial transglutaminase (MTG). Replacement of pork backfat with these emulsions produced an increase (P < 0.05) of hardness, springiness, cohesiveness and chewiness but a reduction (P < 0.05) of adhesiveness. F/SPI and F/SPI + SC + MTG frankfurters showed the lowest (P < 0.05) half-bandwidth in the 2922 cm⁻¹ band, which could be related to lipid chains were more ordered than in F/PF. Modifications in the amide I band profile revealed a higher concentration of aggregated intermolecular β-sheets in F/SPI + SC + MTG samples. Lipid and protein structural characteristics could be associated with specific textural properties of healthier frankfurters.     

Interactions between milk whey protein and polysaccharide in solution

In this work we have studied the interactions between a commercial whey protein concentrate (WPC) and two anionic polysaccharides (sodium alginate, SA, and λ-carrageenan, λ-C) in the aqueous phase. The concentration of WPC at 1.0% and the pH 7.0 of the aqueous phase were maintained constant, while polysaccharides (PS) were evaluated within a 0.0–1.0% concentration range. Interactions between WPC and PS in the aqueous phase were analysed by fluorescence spectroscopy, absorption spectroscopy in the presence of methylene blue (MB), and confocal laser scanning microscopy. The results from these methodologies revealed differences in the molecular dynamics of mixed systems. The nature of the interactions between WPC and PS depended on the PS type, its relative concentration in the aqueous phase and also on the two WPC fractions. Whey protein concentrate/sodium alginate (WPC/SA) mixed systems were distinguished by a tendency to protein aggregation in the aqueous phase and their segregation into separated microdomains. On the other hand, WPC/λ-carrageenan (WPC/λ-C) mixed systems showed a high degree of attractive interactions over the whole range of concentrations. The ultrastructure revealed the existence of hybrid macromolecular entities (biopolymer network). Interaction of WPC and polysaccharide in the aqueous phase has an effect on the adsorption of mixed systems at the air–water interface and on their foaming characteristics.     

Changes in the surface protein of the fat globules during ultra-high pressure homogenisation and conventional treatments of milk

Disruption of fat globules upon homogenisation provokes a reduction of their size and a concomitant increase in their specific surface area. In order to overcome this phenomenon, the milk fat globule membrane (MFGM) adsorbs non-native MFGM proteins. The aim of the present study was to examine the effects of UHPH conditions (temperature and pressure) on the milk fat globule and the surface proteins by comparison with conventional treatments applied in the dairy industry. Transmission electron microscopy and SDS-PAGE revealed major differences. In UHPH-treated milk, casein micelles were found to be adsorbed on the MFGM in a lesser extent than in conventional homogenisation–pasteurisation. However, greater adsorption of directly bonded casein molecules, released by UHPH through the partial disruption of casein micelles, was observed especially at high UHPH pressures. Adsorption of whey proteins on the MFGM of conventionally homogenised–pasteurised milk was mainly through intermolecular disulfide bonds with MFGM material, whereas in UHPH-treated milk, disulfide bonding with both indirectly and directly adsorbed caseins was also involved.     

Changes in protein compositions and their effects on physical changes of shrimp during boiling in salt solution

Dried shrimp is a high-value fishery product of Thailand. Boiling shrimp in salt solution is an important step during the production of dried shrimp and affects significantly the quality of dried shrimp. However, not much information is so far available on the effects of various boiling parameters on the quality changes of shrimp, especially in terms of the changes of shrimp protein compositions and their consequences on microstructural and physical changes of shrimp. The present work was thus aimed at studying the effects of boiling time and concentration of salt solution on the protein fractions, microstructural and physical changes of boiled shrimp. In addition, the relationships between protein compositions, cooking loss, texture and microstructure of shrimp were established using coupled image and fractal analysis. Boiling was investigated at various concentrations of salt solution and boiling times. Boiled shrimp was then evaluated in terms of its protein fractions, microstructure in terms of fractal dimension and physical changes (cooking loss and hardness). The relationships between all studied parameters were monitored and simple correlations between them were determined. The changes of cooking loss, hardness as well as the normalized changes of fractal dimension (ΔFD/FD₀) highly correlated with the changes of protein compositions of shrimp.     

The combined effect of antioxidants and modified atmosphere packaging on protein and lipid oxidation in beef patties during chill storage

Effect of rosemary extract and ascorbate/citrate (1:1) in combination with modified atmosphere packaging (100% N(2), 80% O(2)/20% N(2)) on protein and lipid oxidation in minced beef patties during storage in the dark for up to 6 days at 4°C was investigated. A high level of oxygen in the packaging atmosphere was found to increase both lipid and protein oxidation during storage as evaluated by TBARS analysis of secondary lipid oxidation products and by 2,4-dinitrophenylhydrazine derivatization of protein carbonyls. Both antioxidant systems tested were found to inhibit lipid oxidation but not protein oxidation. In contrast, ascorbate/citrate was found to promote protein oxidation. Rosemary extract was found to regenerate or protect α-tocopherol whereas the packaging atmospheres had no effect on α-tocopherol stability. In high oxygen atmospheres both antioxidants protected the fresh red meat colour with ascorbate/citrate being more efficient than the rosemary extract, whereas no effect of antioxidant on meat colour was found in beef patties stored in 100% nitrogen.