Emulsion properties of casein and whey protein hydrolysates and the relation with other hydrolysate characteristics

Casein and whey protein were hydrolyzed using 11 different commercially available enzyme preparations. Emulsion-forming ability and emulsion stability of the digests were measured as well as biochemical properties with the objective to study the relations between hydrolysate characteristics and emulsion properties. All whey protein hydrolysates formed emulsions with bimodal droplet size distributions, signifying poor emulsion-forming ability. Emulsion-forming ability of some casein hydrolysates was comparable to that of intact casein. Emulsion instability was caused by creaming and coalescence. Creaming occurred mainly in whey hydrolysate emulsions and in casein hydrolysate emulsions containing large emulsion droplets. Coalescence was dominant in casein emulsions with a broad particle size distribution. Emulsion instability due to coalescence was related to apparent molecular weight distribution of hydrolysates; a relative high amount of peptides larger than 2 kDa positively influences emulsion stability.     

Preparation and characterization of papain-modified sesame (Sesamum indicum L.) protein isolates

Defatted sesame meal ( approximately 40-50% protein content) is very important as a protein source for human consumption due to the presence of sulfur-containing amino acids, mainly methionine. Sesame protein isolate (SPI) is produced from dehulled, defatted sesame meal and used as a starting material to produce protein hydrolysate by papain. Protein solubility at different pH values, emulsifying properties in terms of emulsion activity index (EAI) and emulsion stability index (ESI), foaming properties in terms of foam capacity (FC) and foam stability (FS), and molecular weight distribution of the SPI hydrolysates were investigated. Within 10 min of hydrolysis, the maximum cleavage of peptide bonds occurred as observed from the degree of hydrolysis. Protein hydrolysates have better functional properties than the original SPI. Significant increase in protein solubility, EAI, and ESI were observed. The greatest increase in solubility was observed between pH 5.0 and 7.0. The molecular weight of the hydrolysates was also reduced significantly during hydrolysis. These improved functional properties of different protein hydrolysates would make them useful products, especially in the food, pharmaceutical, and related industries.     

Preparation of whey protein hydrolysates using a single- and two-stage enzymatic membrane reactor and their immunological and antioxidant properties: characterization by multivariate data analysis

An initial 5% (w/v), followed thereafter with replacement aliquots of 3% (w/v), whey protein isolate (WPI) (ca. 86.98% Kjeldahl N x 6.38), was hydrolyzed using Protease N Amano G (IUB 3.4.24.28, Bacillus subtilis) in an enzymatic membrane reactor (EMR) fitted with either a 10 or 3 kDa nominal molecular weight cutoff (NMWCO) tangential flow filter (TFF) membrane. The hydrolysates were desalted by adsorption onto a styrene-based macroporous adsorption resin (MAR) and washed with deionized water to remove the alkali, and the peptides were desorbed with 25, 50, and 95% (v/v) ethyl alcohol. The desalted hydrolysates were analyzed for antibody binding, free radical scavenging, and molecular mass analysis as well as total and free amino acids (FAA). For the first time a quantity called IC50, the concentration of peptides causing 50% inhibition of the available antibody, is introduced to quantify inhibition enzyme-linked immunosorbent assay (ELISA) properties. Principal component analysis (PCA) was used for data reduction. The hydrolysate molecular mass provided the most prominent influence (PC1 = 57.35%), followed by inhibition ELISA (PC2 = 18.90%) and the antioxidant properties (PC3 = 10.43%). Ash was significantly reduced in the desalted fractions; the protein adsorption recoveries were high, whereas desorption with alcohol was prominently influenced by the hydrophobic/ hydrophilic amino acid balance. After hydrolysis, some hydrolysates showed increased ELISA reactivity compared with the native WPI.     

Protein hydrolysates induce CCK release from enteroendocrine cells and act as partial agonists of the CCK1 receptor

Protein has been reported to be the most satiating of all macronutrients. Upon gastrointestinal digestion, peptides are generated that stimulate the release of satiety hormones such as cholecystokinin (CCK) from enteroendocrine cells. As such, bioactive peptides could be the target of Functional Food ingredients with satiating effects. We set up an in vitro assay system to investigate if different protein hydrolysates exhibit varying CCK-releasing properties. Soy, pea, potato, casein, and whey protein hydrolysates were incubated with the enteric endocrine cell line STC-1 that endogenously expresses and secretes CCK. Release of CCK was measured by ELISA. All hydrolysates induced CCK release at low concentrations (>0.1 mg.L -1)); however, no significant differences in CCK-releasing properties between the different protein hydrolysates were found, suggesting a generic, nonspecific peptide-sensing mechanism in the STC-1 cells on hydrolyzed protein. As the ELISA exhibits sensitivity to all CCK isoforms possessing the C-terminal CCK octapeptide but varying in biological activity at the CCK 1 receptor (CCK 1R), a secondary module was added to the STC-1 cell assay. Intracellular calcium measurements were performed in CHO-CCK 1R cells. Following exposure of the STC-1 cells to the protein hydrolysates, the medium was tested on the CCK 1R assay. Released CCK was measured with higher sensitivity and lower variability than in the ELISA. Surprisingly, we found that some protein hydrolysates (soy > potato > casein) also directly stimulated CCK 1R-expressing cells, while whey and pea protein hydrolysates were inactive. As CCK 1R is expressed in the GI tract, direct interaction of CCK 1R with dietary peptides may contribute to their satiety effects. Future experiments developing bioactive ingredients for Functional Foods for weight management could involve isolation of the active, CCK 1R-activating peptides in, for example, soy protein hydrolysates.     

Hydrolysis of whey protein isolate with Bacillus licheniformis protease: aggregating capacities of peptide fractions

In a previous study, peptides aggregating at pH 7.0 derived from a whey protein hydrolysate made with Bacillus licheniformis protease were fractionated and identified. The objective of the present work was to investigate the solubility of the fractionated aggregating peptides, as a function of concentration, and their aggregating capacities toward added intact proteins. The amount of aggregated material and the composition of the aggregates obtained were measured by nitrogen concentration and size exclusion chromatography, respectively. The results showed that of the four fractions obtained from the aggregating peptides, two were insoluble, while the other two consisted of 1:1 mixture of low and high solubility peptides. Therefore, insoluble peptides coaggregated, assumedly via hydrophobic interactions, other relatively more soluble peptides. It was also shown that aggregating peptides could aggregate intact protein nonspecifically since the same peptides were involved in the aggregation of whey proteins, beta-casein, and bovine serum albumin. Both insoluble and partly insoluble peptides were required for the aggregation of intact protein. These results are of interest for the applications of protein hydrolysates, as mixtures of intact protein and peptides are often present in these applications.     

Angiotensin I converting enzyme-inhibitory peptides from commercial wet- and dry-milled corn germ

Bioprocesses were developed to enhance the value of proteins from deoiled corn germ. Proteins were hydrolyzed with trypsin, thermolysin, GC 106, or Flavourzyme to generate the bioactive peptide sequences. At an enzyme to substrate ratio of 1:100, protein hydrolysis of wet-milled germ was greatest using thermolysin followed by trypsin, GC 106, and Flavourzyme. For the dry-milled corn germ, protein hydrolysis was greatest for GC 106 and least for Flavourzyme. Electrophoretic patterns indicated that the hydrolysis conditions used were adequate for generating low molecular weight peptides for both germs. Unhydrolyzed dry- and wet-milled corn germ did not appear to contain angiotensin I converting enzyme (ACE)-inhibitory peptides. After hydrolysis with trypsin, thermolysin, and GC 106 but not Flavourzyme, ACE inhibition was observed. ACE inhibition was greatest for the GC 106 hydrolysate for both wet- and dry-milled corn germ. Denaturing the protein with urea before hydrolysis, in general, increased the amount of ACE-inhibitory peptides found in the hydrolysate. Membrane fractionations of both the wet- and dry-milled hydrolysates indicated that most of the ACE-inhibitory peptides were in the <1 kDa fraction. Examination of the control total protein extracts (before treatment with proteases) from wet- and dry-milled germ revealed that neither had ACE-inhibitory properties. However, when both total corn germ control protein extracts were fractionated, the <1 kDa fraction of wet-milled corn germ proteins exhibited ACE inhibition, whereas the comparable low molecular weight fraction from dry-milled corn germ did not.     

beta-lactoglobulin hydrolysis. 1. Peptide composition and functional properties of hydrolysates obtained by the action of plasmin, trypsin, and Staphylococcus aureus V8 protease.

beta-Lactoglobulin (betaLg) was subjected to limited hydrolysis by trypsin, plasmin, and endoproteinase from Staphylococcus aureus V8 (S.aur.V8) to degrees of hydrolysis (DH) of 1, 2, and 4%. The several hydrolysates had different peptide compositions (determined by reversed-phase HPLC and gel-permeation chromatography [GPC]). GPC under nondenaturing, denaturing, and denaturing plus reducing conditions showed that the peptides formed were linked by hydrophobic interactions or by disulfide bonds or were not linked at all. At very low protein concentration, some differences in emulsion-forming properties were observed: only the plasmin hydrolysates could form emulsions with a uniform particle-size distribution. The emulsions formed with S.aur.V8 hydrolysates had poor emulsion-stabilizing properties. Some hydrolysates showed increased foam-forming properties in comparison with the intact protein. All foams formed were stable. Overall, the plasmin hydrolysate (DH4) contained relatively much larger molecules and/or hydrophobic molecules. Many molecules were disulfide-linked peptides. This hydrolysate also had the best functional properties.     

FTIR spectra of whey and casein hydrolysates in relation to their functional properties

Mid-infrared spectra of whey and casein hydrolysates were recorded using Fourier transform infrared (FTIR) spectroscopy. Multivariate data analysis techniques were used to investigate the capacity of FTIR spectra to classify hydrolysates and to study the ability of the spectra to predict bitterness, solubility, emulsifying, and foaming properties of hydrolysates. Principal component analysis revealed that hydrolysates prepared from different protein sources or with different classes of proteolytic enzymes are distinguished effectively on basis of their FTIR spectra. Moreover, multivariate regression analysis showed satisfactory to good prediction of functional parameters; the coefficient of determination (R(2)) varied from 0.60 to 0.92. The accurate prediction of bitterness and emulsion forming ability of hydrolysates by using only one uncomplicated and rapid analytical method has not been reported before. FTIR spectra in combination with multivariate data analysis proved to be valuable in protein hydrolysate fingerprinting and can be used as an alternative for laborious functionality measurements.     

Production of cod (Gadus morhua) muscle hydrolysates. Influence of combinations of commercial enzyme preparations on hydrolysate peptide size range

The complement of enzyme activities of a selection of commercial protease preparations were determined using fluorogenic substrates. Alcalase was used in combination with other commercial enzyme preparations to produce cod muscle (Gadus morhua) hydrolysates. Each muscle hydrolysate was characterized with respect to the percentage degree of hydrolysis (DH %), peptide molecular weight range, and free amino acid content. The enzyme preparations containing predominantly protease or endopeptidase activities achieved high DH % and produced significant amounts of peptides below a molecular weight of 3000. Alcalase combined with exopeptidase-rich preparations produced hydrolysates rich in low-molecular-weight peptides. Selecting combinations of enzyme preparations with complementary activity profiles could be used to manipulate the peptide molecular weight profile of hydrolysates.     

Angiotensin I converting enzyme inhibitory peptides purified from bovine skin gelatin hydrolysate.

Bovine skin gelatin was hydrolyzed with sequential protease treatments in the order of Alcalase, Pronase E, and collagenase using a three-step ultrafiltration membrane reactor. The molecular weight distributions of the first, second, and third hydrolysates were 4.8-6.6, 3.4-6.6, and 0.9-1.9 kDa, respectively. The angiotensin I converting enzyme (ACE) inhibitory activity of the third hydrolysate (IC(50) = 0.689 mg/mL) was higher than that of the first and second hydrolysates. Two different peptides showing strong ACE inhibitory activity were isolated from the hydrolysate using consecutive chromatographic methods including gel filtration chromatography, ion-exchange chromatography, and reversed-phase high-performance liquid chromatography. The isolated peptides were composed of Gly-Pro-Leu and Gly-Pro-Val and showed IC(50) values of 2.55 and 4.67 microM, respectively.     

Improved storage stability of model infant formula by whey peptides fractions

The purpose of this study was to evaluate the shelf-life stability (6 months) of model infant formula with whey protein hydrolysates or peptidic fractions as carrageenan replacers. Whey protein hydrolysates were prepared with trypsin and followed by ultrafiltration of the hydrolyzed mixture, and peptidic fractions were isolated from the ultrafiltered tryptic hydrolysate by anion- or cation-exchange chromatography. The stability of the model infant formula was evaluated using a stratification method based on fat content differences between the top and bottom strata of the samples. With protein hydrolysate-based formulations, the creaming rate of the fat in the product was slightly higher than in the standard formulation (with carrageenan), which is indicative of lower storage stability. The addition of cationic fractions to model infant formula also resulted in lower product stability, whereas the fat creaming rate was retarded in anionic fraction based formulations. The physicochemical characteristics of certain peptides combined with the reported high emulsifying properties of peptidic sequences found within these fractions may account for their ability to act as carrageenan replacers.     

Investigations into inhibitor type and mode, simulated gastrointestinal digestion, and cell transport of the angiotensin I-converting enzyme-inhibitory peptides in Pacific hake (Merluccius productus) fillet hydrolysate

Fish protein hydrolysate (FPH) produced by incubation of Pacific hake fillet with 3.00% Protamex at pH 6.5 and 40 degrees C for 125 min demonstrated in vitro ACE-inhibitory activity (IC50 = 165 microg/mL), which was enhanced by ultrafiltration through a 10 kDa molecular weight cutoff membrane (IC50 = 44 microg/mL). However, after simulated gastrointestinal digestion, FPH and ultrafiltrate had similar ACE-inhibitory activity (IC 50 = 90 microg/mL), indicating that FPH peptides act as "pro-drug type" inhibitors and that enrichment by ultrafiltration may be unnecessary. Matrix-assisted laser desorption/ionization-time of flight mass spectrometry confirmed that the molecular weights of major peaks were <1 kDa regardless of ultrafiltration. ACE-inhibitory activities of digested hydrolysates were not significantly affected by preincubation with ACE ( P > 0.05) and exhibited a competitive inhibitory mode. A permeability assay using fully differentiated colorectal adenocarcinoma (Caco-2) cells showed an apical to basolateral transport of peptides that ranged from approximately 2 to 20% after 2 h at 37 degrees C. Pacific hake fillet hydrolysates are a potentially bioavailable source of ACE-inhibitory peptides awaiting further in vivo study.     

Fractionation and enzymatic hydrolysis of soluble protein present in waste liquors from soy processing

A waste effluent from a soymeal concentrates plant was centrifuged and ultrafiltered by successive processing in membranes of 10, 30, and 50 kDa, with further concentration of the resulting stream using a 5 kDa membrane. The separated fractions (5-10, 10-30, 30-50, and >50 kDa) were subjected to chemical, nutritional, and functional characterization. Resuspension of the retentates in salt-containing systems improved the protein solubility, the emulsifying capacity, and the gelation capacity, whereas the emulsion stability and the foam capacity and stability decreased with respect to the values obtained using distilled water, and the oil absorption capacity was not affected. The digestibility of the fraction >50 kDa was comparable to that of casein. The fractions of higher molecular mass (30-50 and >50 kDa) were subjected to enzymatic hydrolysis with a commercial protease, to give products with improved emulsifying activity and stability, particularly for hydrolysates with a degree of hydrolysis between 20 and 30%.     

Influence of Fatty acids on foaming properties of cider

Seventy-seven ciders from four consecutive harvests, which were produced at industrial scale by cider-makers from the region of Asturias (northern Spain), were analyzed to evaluate their foam capacity. The Bikerman method for the evaluation of foaming characteristics was adapted to ciders. In foaming, there are two parameters, foam formation and foam stability, which are found to be related to each other. To determine the relationship between fatty acid content and foaming properties of cider, the multivariate analysis technique of canonical correlation analysis was applied. Foam stability is positively related to the content of caprylic acid. Foam height is positively related to linolenic, pentadecanoic, and palmitic acid and negatively related to stearic and linoleic acid.     

Synergistic effect of high and low molecular weight molecules in the foamability and foam stability of sparkling wines

The foam of sparkling wines is a key parameter of their quality. However, the compounds that are directly involved in foam formation and stabilization are not yet completely established. In this work, seven sparkling wines were produced in Bairrada appellation (Portugal) under different conditions and their foaming properties evaluated using a Mosalux-based device. Fractionation of the sparkling wines into four independent fractions, (1) high molecular weight material, with molecular weight higher than 12 kDa (HMW), (2) hydrophilic material with molecular weigh between 1 and 12 kDa (AqIMW), (3) hydrophobic material with molecular weigh between 1 and 12 kDa (MeIMW), and (4) hydrophobic material with a molecular weight lower than 1 kDa (MeLMW), allowed the observation that the wines presenting the lower foam stability were those that presented lower amounts of the MeLMW fraction. The fraction that presented the best foam stability was HMW. When HMW is combined with MeLMW fraction, the foam stability largely increased. This increase was even larger, approaching the foam stability of the sparkling wine, when HMW was combined with the less hydrophobic subfraction of MeLMW (fraction 3). Electrospray tandem mass spectrometry (ESI-MS/MS) of fraction 3 allowed the assignment of polyethylene glycol oligomers (n = 5-11) and diethylene glycol 8-hydroxytridecanoate glyceryl acetate. To observe if these molecules occur in sparkling wine foam, the MeLMW was recovered directly from the sparkling wine foam and was also analyzed by ESI-MS/MS. The presence of monoacylglycerols of palmitic and stearic acids, as well as four glycerylethylene glycol fatty acid derivatives, was observed. These surface active compounds are preferentially partitioned by the sparkling wine foam rather than the liquid phase, allowing the inference of their role as key components in the promotion and stabilization of sparkling wine foam.     

Mechanical and structural properties of milk protein edible films cross-linked by heating and gamma-irradiation

The mechanical properties of cross-linked edible films based on calcium caseinate and two type of whey proteins (commercial and isolate) were investigated. Cross-linking of the proteins was carried out using thermal and radiative treatments. Size-exclusion chromatography performed on the cross-linked proteins showed that gamma-irradiation increased the molecular weight of calcium caseinate, while it changed little for the whey proteins. However, heating of the whey protein solution induced cross-linking. For both cross-linked proteins, the molecular weight distribution was >/=2 x 10(3) kDa. Combined thermal and radiative treatments were applied to protein formulations with various ratios of calcium caseinate and whey proteins. Whey protein isolate could replace up to 50% of calcium caseinate without decreasing the puncture strength of the films. Films based on commercial whey protein and calcium caseinate were weaker than those containing whey protein isolate. Electron microscopy showed that the mechanical characteristics of these films are closely related to their microstructures.     

Hydrophobicity of bitter peptides from soy protein hydrolysates

Soy peptides were characterized for flavor, chemical properties, and hydrophobicity to investigate their relationships with bitterness. Five peptide fractions ranging in average molecular mass from 580 to 11300 Da were fractionated by ultrafiltration from two commercial soy protein hydrolysates. The bitterness of fractionated peptides was related to molecular mass, with maximum bitterness observed at approximately 4000 Da for one hydrolysate and 2000 Da for the other. The bitterness increased as the peptide M(w) decreased to 3000 Da for the first hydrolysate and to 2000 Da for the second one and then decreased as the peptide M(w) decreased below 1000 Da. The peptide fraction with molecular mass of <1000 Da showed the lowest bitterness for both. The hydrophobicity data based on Q values do not support Ney's Q rule as a predictor of bitterness for soy peptides.     

Preparation of antioxidant enzymatic hydrolysates from alpha-lactalbumin and beta-lactoglobulin. Identification of active peptides by HPLC-MS/MS

We have investigated the antioxidant activity of hydrolysates from whey proteins bovine alpha-lactalbumin (alpha-La) and beta-lactoglobulin A (beta-Lg A) by commercial proteases (pepsin, trypsin, chymotrypsin, thermolysin, and Corolase PP). Corolase PP was the most appropriate enzyme to obtain antioxidant hydrolysates from alpha-La and beta-Lg A (ORAC-FL values of 2.315 and 2.151 micromol of Trolox equivalent/mg of protein, respectively). A total of 42 peptide fragments were identified by HPLC-MS/MS in the beta-Lg A hydrolysate by Corolase PP. One of the sequences (Trp-Tyr-Ser-Leu-Ala-Met-Ala-Ala-Ser-Asp-Ile) possessed radical scavenging (ORAC-FL value of 2.621 micromol of Trolox equivalent/micromol of peptide) higher than that of butylated hydroxyanisole (BHA). Our results suggest that whey protein hydrolysates could be suitable as natural ingredients in enhancing antioxidant properties of functional foods and in preventing oxidation reaction in food processing.     

Antioxidant properties of casein calcium peptides and their effects on lipid oxidation in beef homogenates

The antioxidant activity of casein calcium peptides in several in vitro assay systems was investigated. Casein calcium peptides were prepared by the microbial enzymic hydrolysis of casein calcium. The main peak of the molecular mass distribution of the peptides was about 3 kDa. Casein calcium peptides showed strong antioxidant activity with the beta-carotene bleaching method, and they also showed scavenging activity against radicals such as superoxide radicals, 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals, and hydroxyl radicals. Antioxidant activity was increased with an increasing peptide concentration. Casein calcium peptides also showed strong antioxidant activity against lipid oxidation in ground beef homogenates. These results suggest that casein calcium peptides are a suitable natural antioxidant that prevents the lipid oxidation of meat and related food ingredients.     

Preparation and characterization of beta-carotene nanodispersions prepared by solvent displacement technique

This work demonstrated the preparation of protein-stabilized beta-carotene nanodispersions using the solvent displacement technique. The emulsifying performance of sodium caseinate (SC), whey protein concentrate (WPC), whey protein isolate (WPI), and a whey protein hydrolysate (WPH, 18% degree of hydrolysis) was compared in terms of particle size and zeta-potential of the nanodispersions. SC-stabilized nanodispersions exhibited a bimodal particle size distribution: large particles (stabilized by casein micelles) with a mean particle size of 171 nm and small particles (stabilized by casein submicelles) of 13 nm. This was confirmed with transmission electron microscopy analysis. Most of the beta-carotene precipitated (87.6%) was stabilized in the small particles. On the other hand, the nanodispersions stabilized by the whey proteins were polydispersed with larger mean particle sizes. The mean particle size of WPC and WPI was 1730 and 201 nm, respectively. The SC-stabilized nanodispersion was expected to be more stable as indicated by its higher absolute zeta-potential value (-31 mV) compared to that of WPC (-15 mV) and WPI (-16 mV). Partially hydrolyzed whey protein possessed improved emulsifying properties as shown by WPH-stabilized samples. It was interesting to note that increasing the SC concentration from 0.05 to 0.5 wt % increased the particle size of beta-carotene stabilized by casein micelles, while the reverse was true for those stabilized by SC submicelles. Microfluidization at 100 MPa of SC solution dissociated the casein micelles, resulting in a decrease in mean particle size of the casein micelle-stabilized particles when the SC solution was used to prepare nanodispersions. The results from this work showed that protein-stabilized beta-carotene nanodispersions could be prepared using the solvent displacement technique.