Enzymatic Hydrolysis Of Milk Proteins Research Articles

Enzymatic hydrolysis and extensive heat treatment induce distinct modifications of cow's and camel milk proteins of relevance for production of infant formula

Camel milk is an alternative to cow's milk as protein source for infant formula. Reduced allergenicity is usually obtained through enzymatic hydrolysis of milk proteins. Heat treatment is another route to modify allergenicity. This study investigated the effect of enzyme hydrolysis and extensive heat treatment on the size, aggregation mechanisms, and modifications of camel milk proteins in comparison to the effect on cow's milk proteins. Disulphide bonds stabilized casein micelles in cow's milk but not in camel milk. The formation of large stable complexes by extensive heat treatment was prominent in cow's milk and not in camel milk. In both milks heat treatment led to equal levels of Maillard reaction, whereas sugar independent cross-linking was less pronounced in camel milk. Low sequence identity, different relative composition, and complete absence of β-lactoglobulin in camel milk are likely explanations for the different properties of cow's and camel milk. Production of infant formula based on camel milk should accordingly employ process parameters specifically adapted to this type of milk.

Modeling Tool for Studying the Influence of Operating Conditions on the Enzymatic Hydrolysis of Milk Proteins.

Systematic modeling of the enzymatic hydrolysis of milk proteins is needed to assist the study and production of partially hydrolyzed milk. The enzymatic hydrolysis of milk proteins was characterized and evaluated as a function of the temperature and protease concentration using Alcalase, Neutrase and Protamex. Modeling was based on the combination of two empirical models formed by a logarithmic and a polynomial equation to correlate the kinetic constants and the operating conditions. The logarithmic equation fitted with high accuracy to the experimental hydrolysis curves with the three proteases (R2 > 0.99). The kinetic constants were correlated with the operating conditions (R2 > 0.97) using polynomial equations. The temperature and protease concentration significantly affected the initial rate of hydrolysis, i.e., the kinetic constant a, while the kinetic constant b was not significantly affected. The values for the kinetic constant a were predicted according to the operating conditions and they were strongly correlated with the experimental data (R2 = 0.95). The model allowed for a high-quality prediction of the hydrolysis curves of milk proteins. This modeling tool can be used in future research to test the correlation between the degree of hydrolysis and the functional properties of milk hydrolysates.

Influence of high-pressure homogenization on structural properties and enzymatic hydrolysis of milk proteins

This study investigated the effects of HPH processing parameters on the conformational structure of bovine serum albumin (BSA) and whey protein isolate (WPI).BSA and WPI dispersions (1% w/v in phosphate buffer, pH = 7.5) were treated in a bench-scale HPH unit at different pressures (100, 150, 200 MPa) and number of passes (1, 2, 3, 5). The modifications of proteins' primary structure (carbonyl groups), secondary structure (α-helix, β-sheet, turn), tertiary and quaternary structure (free -SH groups) were analyzed together with their particle size distribution (PSD). HPH-assisted hydrolysis with trypsin and α-chymotrypsin was performed (200 MPa, 1 pass). Hydrolysis degree and molecular weight distribution of peptides were determined and compared with those of untreated protein dispersions.HPH treatments did not affect proteins' primary structure while slight modifications of secondary structure were detected. Interestingly, free - SH groups in BSA increased with increasing the pressure due to partial unfolding, while decreased in WPI, possibly due to disaggregation and compaction of native WPI aggregates. HPH pre-treatment allowed enhancing BSA hydrolysis reaction rate, while the compaction of WPI aggregates caused a reduced hydrolysis extent. In conclusion, HPH caused an appreciable modification of protein conformation and affected the degree of enzymatic hydrolysis.

Artificial neuronal networks (ANN) to model the hydrolysis of goat milk protein by subtilisin and trypsin.

The enzymatic hydrolysis of milk proteins yield final products with improved properties and reduced allergenicity. The degree of hydrolysis (DH) influences both technological (e.g., solubility, water binding capacity) and biological (e.g., angiotensin-converting enzyme (ACE) inhibition, antioxidation) properties of the resulting hydrolysate. Phenomenological models are unable to reproduce the complexity of enzymatic reactions in dairy systems. However, empirical approaches offer high predictability and can be easily transposed to different substrates and enzymes. In this work, the DH of goat milk protein by subtilisin and trypsin was modelled by feedforward artificial neural networks (ANN). To this end, we produced a set of protein hydrolysates, employing various reaction temperatures and enzyme/substrate ratios, based on an experimental design. The time evolution of the DH was monitored and processed to generate the ANN models. Extensive hydrolysis is desirable because a high DH enhances some bioactivities in the final hydrolysate, such as antioxidant or antihypertensive. The optimization of both ANN models led to a maximal DH of 23·47% at 56·4 °C and enzyme-substrate ratio of 5% for subtilisin, while hydrolysis with trypsin reached a maximum of 21·3% at 35 °C and an enzyme-substrate ratio of 4%.

Enhancing bioactive peptide release and identification using targeted enzymatic hydrolysis of milk proteins.

Milk proteins have been extensively studied for their ability to yield a range of bioactive peptides following enzymatic hydrolysis/digestion. However, many hurdles still exist regarding the widespread utilization of milk protein-derived bioactive peptides as health enhancing agents for humans. These mostly arise from the fact that most milk protein-derived bioactive peptides are not highly potent. In addition, they may be degraded during gastrointestinal digestion and/or have a low intestinal permeability. The targeted release of bioactive peptides during the enzymatic hydrolysis of milk proteins may allow the generation of particularly potent bioactive hydrolysates and peptides. Therefore, the development of milk protein hydrolysates capable of improving human health requires, in the first instance, optimized targeted release of specific bioactive peptides. The targeted hydrolysis of milk proteins has been aided by a range of in silico tools. These include peptide cutters and predictive modeling linking bioactivity to peptide structure [i.e., molecular docking, quantitative structure activity relationship (QSAR)], or hydrolysis parameters [design of experiments (DOE)]. Different targeted enzymatic release strategies employed during the generation of milk protein hydrolysates are reviewed herein and their limitations are outlined. In addition, specific examples are provided to demonstrate how in silico tools may help in the identification and discovery of potent milk protein-derived peptides. It is anticipated that the development of novel strategies employing a range of in silico tools may help in the generation of milk protein hydrolysates containing potent and bioavailable peptides, which in turn may be used to validate their health promoting effects in humans. Graphical abstract The targeted enzymatic hydrolysis of milk proteins may allow the generation of highly potent and bioavailable bioactive peptides.

Selective enrichment of dairy phospholipids in a buttermilk substrate through investigation of enzymatic hydrolysis of milk proteins in conjunction with ultrafiltration

Extensive enzymatic hydrolysis of milk proteins in reconstituted buttermilk powder was combined with ultrafiltration to generate a phospholipid (PL) enriched fraction with maximum permeation of hydrolysed peptides. Buttermilk, naturally high in PLs, is the ideal substrate for enrichment of these bio- and techno-functionally active compounds. A 7.8 fold increase in PL was achieved in the 50 kDa retentate; 6.16±0.02% total PL compared with 0.79±0.01% in the starting substrate, an increase considerably greater than previously reported. Total lipid content (% dry matter) increased 6.3 fold in the retentate, 43.43±0.61%, from the starting substrate, 6.84±0.17%. This combined strategic approach enabled maximum enrichment of PLs with no transmission of lipid material into the permeate, 0.09±0.02% total lipid, and non-detectable levels of PLs recovered in the permeate, 0.00±0.01% total PL.

Preparation, properties, and uses of enzymatic milk protein hydrolysates

ABSTRACTEnzymatic hydrolysis of milk proteins has been a subject of numerous research studies and patents. The driving force for these studies has been the increased utilization of milk proteins. The industrial uses of milk proteins are based on their unique composition, functionality, and nutritive values. The diversity of milk protein fraction, the large number of proteinases, and controlled hydrolysis conditions used resulted in the preparation of hydrolysates suitable for several purposes. Enzymatic hydrolysis of milk proteins modifies the techno-functional and biofunctional properties of hydrolysates depending on the enzyme(s) and hydrolysis conditions used. Milk protein hydrolysates (MPH) are used commonly in normal and clinical nutrition and as a functional food ingredient. In the present review, emphasis has been made to highlight methods applied for the preparation of MPH, and the functional properties and utilization of the obtained hydrolysates.

Research and Development of a Peptide Complex Technology

Results of biotechnological research on controlled hydrolysis of casein and production of peptide complexes are summarized in the present article. Selection of processing parameters and optimization of the conditions of enzymatic hydrolysis of milk proteins allowed for the development of a resource-efficient technology for the production of peptide complexes. Enzymatic hydrolysis of peptide bonds R1-CONH-R2 + H2O  R1COOH + NH2R2 was considered using the example of casein and a range of proteolytic enzymes (trypsin and chymotrypsin) belonging to the hydrolase class. Enzyme solution (0.1% m/v) was added to each protein solution so that the final enzymesubstrate ratio was 1:25, 1:50, or 1:100. The enzyme-substrate ratio of 1:50 was shown to be optimal, and the recommended temperature and pH values were 50±1°С and 7.50±0.01, respectively. The degree of hydrolysis is one of the parameters characterizing the overall changes in the amino acid composition of proteins. Therefore, a time period of 12.00±0.05 h was chosen as the optimal duration of the hydrolysis process. Further research was focused on the analysis of peptide profiles using MALDI-TOF MS based on identification of peptide sequences. The studies have shown that casein hydrolysates are rich in biologically active peptide complexes. The detection of such complexes in hydrolysates of casein was the main result of the study. For example, the peptide β-casokinin (amino acid sequence Ala-Val-Pro-Tyr-Pro-Gln-Arg) is an inhibitor of angiotensin-converting enzyme.

Enzymatic Hydrolysis of Milk Proteins as a Tool for Modification of Functional Properties at Interfaces of Emulsions and Foams - A Review

Enzymatic Hydrolysis of Milk Proteins as a Tool for Modification of Functional Properties at Interfaces of Emulsions and Foams - A Review

Proteins Profile in Milk from Three Species of Ruminants

Milk proteins, caseins and whey proteins, are very important nutritionally, as they contain all essential aminoacids in optimal proportions and are the most important source of bioactive peptides. These peptides are protein fragments resulting from enzymatic hydrolysis of milk proteins, which carry numerous beneficial effects on the cardiovascular, nervous, gastrointestinal and immune systems. In this research, total proteins, caseins and whey proteins respectively, were dosed in milk from three species of ruminants-cow, goat and sheep, using a very sensitive method, Bradford photometric method. The highest content of total proteins was obtained in sheeps’ milk (65.92 mg/ml) and the lowest in cows’ milk (40.03 mg/ml), intermediate values occurring in goats’ milk (46.79 mg/ml). The lowest amount of caseins was found in cow milk (28.26 mg/ml), followed by sheep milk (42.55 mg/ml) and goat milk (44.03 mg/ml). When the case of whey proteins, the highest values occur in sheep milk (23.36 mg/ml) and the lowest in cow milk (11.79 mg/ml), goat milk having intermediate values (17.7 mg/ml). The results obtained indicate the dependence of protein concentration in milk of the studied ruminant species and stresses the importance of including goat and sheep milk in daily diet, along with cow milk, for an optimal intake of protein.

Quantitative studies on the enzymatic hydrolysis of milk proteins brought about by cardosins precipitated by ammonium sulfate

Hydrolysis of whey proteins may produce peptide mixtures with better functional properties than the original protein mixture, viz. higher solubilites and lower allergenic effects. Cynara cardunculus is a wild plant that possesses (aspartic) proteases in its flower cells; those enzymes exhibit general proteolytic and specific milk clotting activities, which are rather useful in traditional cheesemaking. This study was thus aimed at characterizing the enzymatic action of crude extracts of said plant after preliminary purification by salting out with ammonium sulfate at two different concentration levels, viz. 30% and 70% saturation. The coagulant activity on milk, and the proteolytic activity using casein and azocasein as substrates, of the crude extract and of each precipitated fraction were measured at 37°C and pH 5.2. The profile of hydrolysis of the major whey proteins, i.e. α-lactalbumin (α-La), β-lactoglobulin (β-Lg) and bovine serum albumin (BSA) was characterized by gel permeation chromatography and polyacrylamide gel electrophoresis (PAGE) in the presence of sodium dodecyl sulfate. The 30% and 70% saturation fractions exhibited lower coagulant and proteolytic specific activities than the crude extract. However, the relative ratio of coagulant to proteolytic activity, which is a useful indicator of appropriateness for cheesemaking, was higher for the partially purified fractions. The extents of hydrolysis of whey proteins brought about by the partially purified extracts were above those by their crude counterpart, but qualitative hydrolysis patterns were essentially identical to each other; by 24 h, α-La was substantially depleted, whereas β-Lg was very poorly hydrolyzed and BSA was only slightly hydrolyzed. The native proteins were converted to lower and lower molecular weight peptides.

Enzymatic Hydrolysis of Milk Proteins Under Alkaline and Acidic Conditions

ABSTRACTThe pH‐stat and osmometric methods were adopted for control of hydrolysis of casein and whey proteins by chymotrypsin, trypsin and pepsin. Experiments in alkaline medium showed that the mean pK values determined for amino groups in milk protein hydrolysates at 52°C were 7.11 for casein hydrolysates and 7.18 for whey protein hydrolysates. During hydrolysis in acidic medium the osmotic coefficients determined for casein and whey proteins enabled calculation of the calibration factor for the osmometer (1.05) which could be assumed as constant. Results will enable monitoring of DH during hydrolysis of milk proteins in such systems.

Enzymatic Hydrolysis of Milk Proteins Used for Emulsion Formation. 2. Effects of Calcium, pH, and Ethanol on the Stability of the Emulsions

Oil-in-water emulsions were prepared with native milk proteins (caseinate and β-lactoglobulin) or the same proteins after different extents of breakdown by treatment with trypsin. The effect of trypsin treatment of the emulsions after formation was also investigated. The stability of emulsions was measured by determining the particle sizes with light scattering. Different destabilization conditions were used; namely, decreasing pH, adding calcium ions, or incorporation of ethanol. Comparisons were made between emulsions containing modified and unmodified proteins. Trypsinolysis rendered caseinate emulsions less stable to decreasing pH and increasing concentrations of ethanol, but the stability to calcium increased and correlated with the surface composition of individual types of casein molecules and their calcium sensitivity. Emulsions containing β-lactoglobulin, on the other hand, showed improved stabilities with hydrolysis under all conditions compared with emulsions containing unhydrolyzed protein. Ke...

Enzymatic Hydrolysis of Milk Proteins Used for Emulsion Formation. 1. Kinetics of Protein Breakdown and Storage Stability of the Emulsions

Hydrolysis of β-lactoglobulin and sodium caseinate in solution or while adsorbed at oil-water interfaces in emulsions formed from unmodified proteins was carried out with trypsin. The hydrolysis was monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and dynamic light scattering. The stability during storage of the emulsions containing hydrolyzed proteins was also investigated by small-angle laser light scattering. The order of the hydrolysis of the individual caseins at the oil surface was β > αs >>> κ compared with αs > β > κ in solution. The overall rate of hydrolysis of sodium caseinate was the same whether adsorbed to the interface or in solution. Conversely, the rate of hydrolysis of β-lactoglobulin was much higher on the surface of emulsion than in solution. Emulsions formed from hydrolyzed protein solutions were less stable during storage than those formed from unmodified proteins and subsequently hydrolyzed or those formed with unmodified protein solutions. Keywords: Emulsions; casein; β-lactoglobulin; trypsin; peptides; emulsion stability

Use of the o-Phthalaldehyde and N-Acetyl-L-Cysteine the Evaluation of Milk Proteins

o-Phthalaldehyde and N-acetyl-Lcysteine are used in the determination of milk proteins. Three procedures are proposed and compared. One of them is based on reaction of o-phthalaldehyde and N-acetyl-Lcysteine with the intact proteins and the two others on reaction of the reagents with the released amino acids after total acid hydrolysis of the protein samples. When the protein sample is hydrolyzed, calibration is performed either with a hydrolyzed protein standard or with isoleucine. A procedure for the measurement of the degree of enzymatic hydrolysis of milk proteins without separation of the unhydrolyzed protein, which makes use of the same reagents, is also described. In all cases absorbance is measured at 335nm.