Hydrolysis of whey protein isolate with Bacillus licheniformis protease: fractionation and identification of aggregating peptides

The objective of this work was to identify the dominant aggregating peptides from a whey protein hydrolysate (degree of hydrolysis of 6.8%) obtained with Bacillus licheniformis protease. The aggregating peptides were fractionated with preparative reversed-phase chromatography and identified with liquid chromatography-mass spectrometry. The results showed that the dominant aggregating peptide, at pH 7.0, was beta-lg AB [f1-45]. In addition, the peptides beta-lg AB [f90-108]-S-S-alpha-la [f50-113], alpha-la [f12-49]-S-S-alpha-la [f50-113], beta-lg AB [f90-108]-S-S-beta-lg AB [f90-108], beta-lg A [f90-157], and beta-lg AB [f135-157/158] were also identified as main aggregating peptides. The results further showed that aggregation, via hydrophobic interactions, prevented further digestion (at pH 8.0), thereby explaining the large size of the aggregating peptides. It is hypothesized that B. licheniformis protease breaks down hydrophilic segments in the substrate and, therefore, preserves hydrophobic segments that aggregate once exposed to the solvent.     

Moisture-induced aggregation of whey proteins in a protein/buffer model system

Moisture-induced protein aggregation in a dry or intermediate-moisture food matrix can contribute to the loss of product acceptability. The present study evaluated the molecular mechanisms and controlling factors for moisture-induced whey protein aggregation in a premixed protein/buffer model system. Insoluble aggregates rapidly formed during the first 3 days of storage at 35 degrees C with a slower rate afterward. Evaluation of the insoluble aggregates by solubility tests in solutions containing SDS/urea/guanidine HCl/dithiothreitol and gel electrophoresis showed that the formation of intermolecular disulfide bonds was the main mechanism for protein aggregation, and all major whey proteins were involved in the formation of insoluble aggregates. Effects of various factors on aggregation were also investigated, including moisture content, medium pH, and the addition of NaCl. The dependence of aggregation on moisture content was bell-shaped, and the maximal extent of aggregation was achieved at a moisture content of around 70-80% on a dry weight basis.     

Development of a large-scale (50 L) apparatus for ampholyte-free isoelectric focusing (autofocusing) of peptides in enzymatic hydrolysates of food proteins

It has been demonstrated that peptides in enzymatic hydrolysates of proteins can be fractionated on the basis of the amphoteric nature of the sample peptides, by a laboratory-scale isoelectric focusing apparatus, without adding a chemically synthesized carrier ampholyte. This approach is referred to as autofocusing. In the present study, a large-scale (up to 50 L) autofocusing apparatus was developed and tested. A tank (125 cm x 25 cm x 20 cm) was divided into 12 compartments by 11 plates, each with a window covered in a thin agarose gel layer supported by a nylon screen (100 mesh). The compartments at both ends were filled with 0.1 N phosphoric acid (anode) and 0.1 N NaOH (cathode), respectively, functioning as electrode compartments. The remaining compartments were used for sample compartments. Autofocusing was carried out at constant voltage according to two different methods. In method 1, all sample compartments were filled with a 1% water solution of casein or milk whey protein hydrolysates. In method 2, two compartments located in the center of the tank were filled with 5% sample solution and the others were filled with deionized water. Compositional and sequence analyses of the autofocusing fractions revealed that peptides in the two hydrolysates can be fractionated within 24 h by the present apparatus. Better fractionation was obtained by method 2, whereas enrichment of some peptides occurred by using method 1.     

Protein-peptide interactions in mixtures of whey peptides and whey proteins

The effects of several conditions on the amounts and compositions of aggregates formed in mixtures of whey protein hydrolysate, made with Bacillus licheniformis protease, and whey protein isolate were investigated using response surface methodology. Next, the peptides present in the aggregates were separated from the intact protein and identified with liquid chromatography-mass spectrometry. Increasing both temperature and ionic strength increased the amounts of both intact protein and peptides in the aggregates. There was an optimal amount of added intact WPI that could aggregate with peptides, yielding a maximal amount of aggregated material in which the peptide/protein molar ratio was around 6. Under all conditions applied, the same peptides were observed in the protein-peptide aggregates formed. The dominant peptides were beta-lg AB [f1-45], beta-lg AB [f90-108], and alpha-la [f50-113]. It was hypothesized that peptides could form a kind of glue network that can include beta-lactoglobulin via hydrophobic interactions at the hydrophobic binding sites at the surface of the protein.     

Functional region identification in proteins by accumulative-quantitative peptide mapping using RP-HPLC-MS

A new method was developed to identify regions in proteins from which peptides are derived with specific functional properties. This method is applicable for systems in which peptides of a hydrolyzed protein possess specific functional properties, but are too large to be sequenced directly and/or the peptide mixture is too complex to purify and characterize each peptide individually. In the present work, aggregating peptides obtained by proteolytic hydrolysis of soy glycinin were used as a case study. The aggregating peptides are isolated and subsequently further degraded with trypsin to result in peptides with a mass <5000 Da to enable sequence identification using RP-HPLC-MS in combination with MS/MS. Prior to RP-HPLC the peptides are fractionated using anion and cation exchange chromatography. The fractions obtained are analyzed with RP-HPLC-MS. The peptides, with identified sequences, were quantified using the peak areas of the RP-HPLC chromatograms measured at 214 nm. Next, the peak areas were corrected for the molar extinction coefficient of the individual peptides, followed by accumulative-quantitative peptide mapping. The results show that in complex systems, based on the method described, the regions in the parental protein from which the functional peptides originate can be properly identified.     

Correlations between biochemical characteristics and foam-forming and -stabilizing ability of whey and casein hydrolysates

Whey protein and casein were hydrolyzed with 11 commercially available enzymes. Foam properties of 44 samples were measured and were related to biochemical properties of the hydrolysates using statistical data analysis. All casein hydrolysates formed high initial foam levels, whereas whey hydrolysates differed in their foam-forming abilities. Regression analysis using the molecular weight distribution of whey hydrolysates as predictors showed that the hydrolysate fraction containing peptides of 3-5 kDa was most strongly related to foam formation. Foam stability of whey hydrolysates and of most casein hydrolysates was inferior to that of the intact proteins. The foam stability of casein hydrolysate foams was correlated to the molecular weight distribution of the hydrolysates; a high proportion of peptides >7 kDa, composed of both intact casein and high molecular weight peptides, was positively related to foam stability.     

Effects of moisture-induced whey protein aggregation on protein conformation, the state of water molecules, and the microstructure and texture of high-protein-containing matrix

Moisture-induced protein aggregation through intermolecular interactions such as disulfide bonding can occur in a high-protein-containing food matrix during nonthermal processing and storage. The present study investigated the effect of moisture-induced whey protein aggregation on the structure and texture of such high-protein-containing matrices using a protein/buffer model system. Whey proteins in the protein/buffer model systems formed insoluble aggregates during 3 months' storage at temperatures varying from 4 to 45 degrees C, resulting in changes in microstructure and texture. The level of aggregation that began to cause significant texture change was an inverse function of storage temperature. The protein conformation and the state of water molecules in the model system also changed during storage, as measured by differential scanning calorimetry and Fourier transform infrared spectroscopy. During storage, the model system that had an initially smooth structure formed aggregated particles (100-200 nm) as measured by scanning electron microscopy, which lead to an aggregation network in the high-protein-containing matrix and caused a harder texture.     

Intercultivar Variation in the Quantity of Monomeric Proteins,Soluble and Insoluble Glutenin,and Residue Protein in Wheat Flour and Relationships to Breadmaking Quality

A new fractionation procedure based on differential solubility was applied to wheat flour proteins to evaluate the relationship between protein fractions and functionality for breadmaking. Flour was initially extracted with 50% 1-propanol. Monomeric proteins (mainly gliadins) and soluble glutenin contained in the 50% propanol soluble extract were fractionated by selective precipitation of the glutenin by increasing the concentration of 1-propanol to 70%; monomeric proteins remain in the supernatant. Insoluble glutenin in the 50% propanol insoluble residue was extracted using 50% 1-propanol containing 1% dithiothreitol (DTT) at 60°C. Protein in the final residue was extracted using SDS with or without DTT. It comprised mainly Glu-1D high molecular weight glutenin subunits and nongluten polypeptides. For seven Canadian cultivars of diverse breadmaking quality, there was relatively little variation in the percentage of flour protein corresponding to monomeric proteins (48–52%) and residue protein (14–18%). In contrast, intercultivar variation in soluble and insoluble glutenin was substantial, with contents of 10–20% and 12–28% of flour protein, respectively. Soluble and insoluble glutenin were also highly correlated with physical dough properties, accounting for 83–95% of the variation of individual dough rheological parameters (except dough extensibility), and ≈ 74% of the variation in loaf volume. In contrast, monomeric and residue protein fractions were poorly associated with breadmaking quality. However, among the four protein fractions, only residue protein was significantly correlated (r = -0.79) with dough extensibility. The flour sample with the highest and lowest concentrations of insoluble and soluble glutenin, respectively, as well as marginally the lowest concentrations of monomeric and residue proteins was Glenlea, a cultivar of the Canada Western Extra Strong Red Spring wheat class which characteristically possesses distinctly strong dough mixing properties.     

Heat-induced aggregation of whey proteins: comparison of cheese WPC with acid WPC and relevance of mineral composition

Heat-induced aggregation of whey proteins in solutions made from two commercial whey protein concentrates (WPCs), one derived from mineral acid whey (acid WPC) and the other from cheese whey (cheese WPC), was studied using polyacrylamide gel electrophoresis (PAGE), size exclusion chromatography (SEC), and transmission electron microscopy (TEM). Heat treatment (75 degrees C) of acid WPC solutions (12.0%, w/w, pH 6.9) resulted in formation of relatively small "soluble" aggregates that were predominantly disulfide-linked. By contrast, heat treatment of the cheese WPC solutions (under the same conditions) caused formation of relatively large aggregates, containing high proportions of aggregates linked by noncovalent associations. The rate of aggregation of both beta-lactoglobulin and alpha-lactalbumin at 75 degrees C, measured as the loss of native proteins by PAGE, was higher in the cheese WPC solution than in the acid WPC solution. Cross dialysis of the two WPC solutions resulted in alteration of the mineral composition of each WPC solution and reversing their heat-induced aggregation behavior. The results demonstrated that the mineral composition is very important in controlling the aggregation behavior of WPC products.     

Effects of ionic strength on the enzymatic hydrolysis of diluted and concentrated whey protein isolate

To identify the parameters that affect enzymatic hydrolysis at high substrate concentrations, whey protein isolate (1-30% w/v) was hydrolyzed by Alcalase and Neutrase at constant enzyme-to-substrate ratio. No changes were observed in the solubility and the aggregation state of the proteins. With increasing concentration, both the hydrolysis rate and the final DH decreased, from 0.14 to 0.015 s(-1) and from 24 to 15%, respectively. The presence of 0.5 M NaCl decreased the rate of hydrolysis for low concentrations (to 0.018 s(-1) for 1% WPI), resulting in similar rates of hydrolysis for all substrate concentrations. The conductivity increase (by increasing the protein concentration, or by addition of NaCl) has significant effects on the hydrolysis kinetics, but the reason for this is not yet well understood. The results show the importance of conductivity as a factor that influences the kinetics of the hydrolysis, as well as the composition of the hydrolysates.     

Identification of strong aggregating regions in soy glycinin upon enzymatic hydrolysis

Upon hydrolysis with chymotrypsin, soy glycinin has a strong tendency to aggregate. The regions of glycinin from which the aggregating peptides originate were identified by accumulative-quantitative peptide mapping. To this end, the aggregating peptides were further hydrolyzed with trypsin to obtain peptides of which the sequence can be identified using RP-HPLC-MS/MS. This resulted in a hydrolysate in which 90% of the proteinaceous material was dissolved. The soluble fraction was analyzed using the method of accumulative-quantitative peptide mapping: fractionation using ion exchange chromatography, followed by identification of peptides by RP-HPLC-MS/MS, quantification based on the absorbance at 214 nm, and finally peptide mapping. For the peptide mapping the proportions in which each of the five glycinin subunits are present, as determined by Edman degradation, were taken into account. The results showed that mainly the basic polypeptide and a part of the acidic polypeptide, close to the location of the disulfide bridge between the basic and acidic polypeptides, are present in the aggregating peptide fraction. On the basis of the results obtained, an aggregation mechanism was proposed. The hydrophilic acidic polypeptides shield the hydrophobic basic polypeptides, and the former are preferentially degraded upon hydrolysis. This results in a net increase in hydrophobicity of the remaining material, which mainly consists of the basic polypeptide fragments. This increase in hydrophobicity is proposed to be the driving force in the aggregation of chymotrypsin-derived peptides of glycinin.     

Rheological properties and characterization of polymerized whey protein isolates.

Whey protein polymers were formed by heating whey protein isolate solutions at 80 degrees C. Flow behaviors of whey protein polymers produced from different protein concentrations and heating times were comparable to various flow behaviors of hydrocolloids. Polymer formation was found to be a two-phase process. The initial protein concentration was a significant factor that determines the size and/or shape of the primary polymer in the first phase as shown by intrinsic viscosity. Heating time was a factor in determining the aggregation in the second phase as shown by apparent viscosity. Intrinsic viscosity of whey protein polymers was as high as 141.7 +/- 7.30 mL/g, compared to 5.04 +/- 0.20 mL/g for native whey proteins. The intrinsic viscosity and gel electrophoresis data suggested that disulfide bonds played an important role in whey polymer formation.     

NMR relaxation and water self-diffusion studies in whey protein solutions and gels

The changes in water proton transverse relaxation behavior induced by aggregation of whey proteins are explained in terms of the simple molecular processes of diffusion and chemical exchange. The water self-diffusion coefficient was measured in whey protein solutions and gels by the pulsed field gradient NMR method. As expected, water self-diffusion was reduced with increased protein concentrations. Whatever the concentration, the water molecules were free to diffuse over distances varying from 15 to 47 mum. Water diffusion was constant over these distances, demonstrating that no restrictions were found to explain the water hindrance. The modification in protein structure by gelation induced a decrease in water diffusion. The effects of protein concentration on water diffusion are discussed and modeled. Two approaches were compared, the obstruction effect induced by a spherical particle and the cell model, which considered two water compartments with specific self-diffusion coefficients.     

Rennet-induced aggregation of heated pH-adjusted skim milk

Heated (20-100 °C/0-30 min) skim milks (pH 6.5-7.1) were diluted in buffer (pH 7.0). Rennet was added, and the particle size with time was measured. For all samples, the size initially decreased (lag phase) and then increased (aggregation phase). Milks heated at ≤60 °C had short lag phases and rapid aggregation phases regardless of pH. Milks heated at >60 °C at pH 6.5 had long lag phases and slow aggregation phases. As the pH increased, the lag phase shortened and the aggregation phase accelerated. The aggregation time was correlated with the level of whey protein associated with the casein micelles and with the level of κ-casein dissociated from the micelles. Heated milks formed weak gels when renneted. It is proposed that the milks heated at low pH have whey proteins associated with the casein micelles and that these denatured whey proteins stabilize the micelles to aggregation by rennet and therefore inhibit gelation. In the milks heated at higher pH, the whey proteins associate with κ-casein in the serum and, on rennet treatment, the κ-casein-depleted micelles and the serum-phase whey protein/κ-casein complexes aggregate; however, the denatured whey proteins stabilize the aggregates so that gelation is still inhibited.     

Control of heat-induced aggregation of whey proteins using casein

The ability of alphas1/beta-casein and micellar casein to protect whey proteins from heat-induced aggregation/precipitation reactions and therefore control their functional behavior was examined. Complete suppression (>99%) of heat-induced aggregation of 0.5% (w/w) whey protein isolate (pH 6.0, 85 degrees C, 10 min) was achieved at a ratio of 1:0.1 (w/w) of whey protein isolate (WPI) to alphas1/beta-casein, giving an effective molar ratio of 1:0.15, at 50% whey protein denaturation. However, in the presence of 100 mM NaCl, heating of the WPI/alphas1/beta-casein dispersions to 85 degrees C for 10 min resulted in precipitation between pH 6 and 5.35. WPI heated with micellar casein in simulated milk ultrafiltrate was stable to precipitation at pH>5.4. Protein particle size and turbidity significantly (P相似文献   

Physical properties, molecular structures, and protein quality of texturized whey protein isolate: effect of extrusion temperature

Although extrusion technology has contributed much to increasing the effective utilization of whey, the effect of extrusion conditions on the functional properties of the proteins is not well understood. In this work, the impact of extrusion temperature on the physical and chemical properties, molecular structures, and protein quality of texturized whey protein isolate (WPI) was investigated at a constant moisture content and compared with WPI treated with simple heat only. The Bradford assay methods, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and reversed-phase high-performance liquid chromatography techniques were used to determine protein solubility and to analyze compositional changes in the two major whey proteins, α-lactalbumin and β-lactoglobulin. Circular dichroism and intrinsic tryptophan fluorescence spectroscopic techniques were applied to study the secondary and tertiary structures of the proteins. This study demonstrated that extrusion temperature is a critical but not the sole determining factor in affecting the functional properties of extruded WPI.     

beta-lactoglobulin hydrolysis. 2. Peptide identification, SH/SS exchange, and functional properties of hydrolysate fractions formed by the action of plasmin.

beta-Lactoglobulin (betaLg) was hydrolyzed by plasmin to a degree of hydrolysis of 4%. The hydrolysate was fractionated by ion-exchange chromatography and subsequent hydrophobic-interaction chromatography. The betaLg peptide fraction consisting of smaller peptides (mostly <2 kDa) had poor foam- and emulsion-forming and -stabilizing properties. Most of the betaLg peptides were identified (in either the nonreduced or reduced form) by mass spectrometry on the basis of the known primary structure of the intact protein and the specificity of the enzyme. The peptides formed during betaLg/plasmin-hydrolysis were (1) peptides lacking a cysteyl residue, (2) peptides composed of a single amino acid chain containing intramolecular disulfide bonds, and (3) peptides composed of two amino acid chains linked by an intermolecular disulfide bond. It appeared that significant SH/SS-exchange had taken place during hydrolysis. Many of the peptides present in the peptide fraction that exhibited good functional properties were disulfide-linked fragments.     

Differences in Alcohol‐Soluble Protein from Genetically Altered Wheat Using Capillary Zone Electrophoresis,One‐ and Two‐Dimensional Electrophoresis,and a Novel Gluten Matrix Association Factor Analysis

Wheat protein composition and organization play interrelated roles in determining physical properties for technological purposes. In prior research, a number of isogenic wheat lines of Bobwhite that have high levels of expression of the native Dx5 and/or Dy10 high‐molecular‐weight glutenin subunits (HMW‐GS) were examined vis‐à‐vis physical properties related to separation. In particular, these altered lines were characterized by poor mixing properties, the formation of mixtures in water that could not be separated by conventional mechanical methods, reduced water absorption, unique milling properties, and severely limited development of microscopic fibrils. These attributes suggested inherent organizational differences at submicroscopic and molecular levels among the various lines. Therefore, proteins were fractionated from whole meals using 70% ethanol to elucidate solubility characteristics and compositions and to infer structural properties. Capillary zone electrophoresis and one‐ and two‐dimensional SDS‐PAGE (2DE) revealed striking differences in the protein composition and solubility among these new lines and the Bobwhite from which they were derived. Generally, Bobwhite yielded soluble protein that included not only what would be considered as classical gliadins but also some of each of the HMW‐GS as monomers or polymers with low degrees of polymerization, whereas the genetically altered lines produced far less total soluble protein and very limited amounts of HMW‐GS. In the extreme, high levels of expression of Dx5 subunit not only led to reduced solubility of the HMW‐GS but also limited the solubility of the many other proteins that are normally soluble. In addition, a matrix association factor similar to the classical separation factor of analytical chemistry and chemical engineering was introduced and applied to 2DE data for insoluble and soluble protein to summarize and index relative involvement of specifically enhanced proteins in the insoluble gluten matrix after equilibration with ethanol. The highest relative association was determined for the HMW‐GS lines enriched in Dx5 or Dy10 protein and the lowest for Bobwhite. Greater association was indicated for Dx5 than for Dy10 protein in these lines. The value of the association factor was likely influenced by differences in glutamine‐to‐cysteine ratios and differences in altered glutenin chain configurations stemming from high levels of expression of a single or limited number of HMW‐GS.     

Screening for nutritive peptides that modify cholesterol 7alpha-hydroxylase expression

Bioactive peptides with a variety of effects have been described from several nutritive proteins. They exhibit antimicrobial, blood-pressure lowering, antithrombotic, immunomodulatory, and cholesterol-modulating effects. In this study, we have examined whether peptides derived from food proteins might influence bile acid synthesis. A reporter gene cell line that carries a cholesterol 7alpha-hydroxylase promoter fragment fused to firefly luciferase ( cyp7a-luc) was used to screen for nutritive peptides affecting cyp7a expression, the enzyme catalyzing the rate-limiting step in bile acid synthesis. Proteolytic hydrolysates were prepared from soy protein and bovine casein with pepsin, trypsin, chymotrypsin, and elastase and size fractionated using ultrafiltration. Several bioactive hydrolysates could be identified that inhibited luciferase expression. Also, an activation of kinase (AKT, ERK, p38-MAPK) signaling could be observed. Selected hydrolysates were further fractionated by reversed-phase HPLC. Bioactive HPLC-fractions were obtained from casein but not from soy hydrolysates; however, activity could not be recovered in single peak fractions. Peptides in such fractions were identified by mass spectrometry. Five selected peptides from alpha S1-casein present in active fractions were synthesized, but none of these showed activity in the cyp7a-luc screening system. However, two of them activated MAP-kinase signaling similar to the hydrolysates, which suggests, that these peptides are involved in cyp7a regulation by the casein hydrolysates.     

Heat-induced phenomena in soy protein suspensions. Rheometric data and theoretical interpretation.

Heat-induced aggregation of soy proteins in aqueous suspensions was studied through cone and plate rheometry for two different heating conditions. The rheometric data obtained covered the temperature range from 20 degrees C (stable colloidal suspension) to approximately 90 degrees C (onset of network formation). Calorimetric data for the soy protein samples were also obtained to evaluate the degree of protein denaturation in the rheometric cell. Heat-induced transitions in soy globulins, such as dissociation, denaturation, and aggregation, were analyzed in relation to the rheological response of the suspension. The viscosity of the stable colloidal suspension satisfies the Cross model. A viscosity equation for the aggregating suspension was also derived by considering the fractal structure of the particle clusters and the Brownian aggregation mechanism. This equation is suitable to describe the experimental viscosity data.