Ussing chamber results for amino acid absorption of protein hydrolysates in porcine jejunum must be corrected for endogenous protein

BACKGROUND: Ussing chambers have been used extensively as an ex vivo model to investigate intestinal nutrient absorption. In this study Ussing chambers were used to investigate the absorption of amino acids and peptides in pig jejunal tissue using a highly hydrolysed casein hydrolysate (PeptoPro®), casein and whey protein isolate after they had been digested with pepsin and pancreatin to simulate digestion products in the jejunum. Jejunal tissue was collected from three pigs and mounted into Ussing chambers, equilibrated, and the luminal chamber loaded with one of three test nitrogen sources. Luminal solution samples were taken every 10 min over a 90 min incubation period and the amino acid concentration determined. To determine the endogenous amino acid contribution of the tissue to the luminal solution, Ussing chambers containing no test N source (blanks) were prepared and treated similarly to the Ussing chambers containing the test N sources. RESULTS: The endogenous amino acid contribution was 0.5 mg in the luminal solution at time 0 and increased to 1 mg after 90 min. The mean amino acid absorption after 10 min incubation for the pre‐digested highly hydrolysed casein hydrolysate (16.6%) was significantly (P < 0.05) higher than for the pre‐digested casein (7.6%) and whey protein isolate (6.7%). CONCLUSION: Endogenous amino acids were a quantitatively significant portion of the luminal solution amino acids present in the Ussing chambers containing the test N sources. Ussing chambers may be a suitable tool for studying amino acid absorption of protein hydrolysates but correction for the endogenous amino acid contribution from the intestinal tissue must be made. Copyright © 2009 Society of Chemical Industry     

Combined microwave and enzymatic treatments for ��-lactoglobulin and bovine whey proteins and their effect on the IgE immunoreactivity

Effect of combined microwave (MW) and enzymatic hydrolysis on the human immunoglobulin E (IgE)-binding properties of ??-lactoglobulin (??-lg) and other whey proteins (WP) was investigated. Separated ??-lg and full whey protein isolate (WPI) were hydrolyzed with trypsin, chymotrypsin, mixture of trypsin/chymotrypsin, and pepsin at three microwave power levels: 50?W during 1 and 5?min, 100 and 200?W during 1 and 3?min. The immunoreactivity of the obtained hydrolysates resulting from combined microwave protease treatment was assessed using sera of young patients allergic to bovine whey proteins. The application of microwave treatment at 200?W enhances the hydrolysis of ??-lg by pepsin in 3?min and decreases significantly its immunoreactivity. The extensive hydrolysis of the microwave-treated ??-lg and WPI with trypsin, chymotrypsin, and the mixture of trypsin with chymotrypsin did not have an impact on the IgE binding of the products obtained in all the studied conditions.     

Whey Protein Antigenicity Reduction by Fungal Proteinases and a Pepsin/Pancreatin Combination

Whey protein components were hydrolyzed with Corolase 7092? (peptidases from Aspergillus strains), pepsin and Corolase PP? (a mixture of pancreatic enzymes), either individually or in combination, in trials to eliminate protein allergenicity. The hydrolysates were characterized by physico-chemical and by immunological techniques using sera from patients allergic to milk proteins. Enzyme specificity rather than degree of hydrolysis or molecular mass distribution of hydrolysates determined the residual antigenicity of the whey proteins. Ultrafiltration was a prerequisite for obtaining hypoallergenic whey protein hydrolysates.     

Peptic hydrolysis of ovine β-lactoglobulin and α-lactalbumin Exceptional susceptibility of native ovine β-lactoglobulin to pepsinolysis

Ovine β-lactoglobulin (BLG, a mixture of variants A and B at a ratio of 46/54) and α-lactalbumin (ALA) were subjected to pepsin activity. The degree of peptic hydrolysis of native whole BLG reached 63%, 74%, 82% and 87% after 2, 4, 8 and 20 h hydrolysis, respectively. BLG variant B was degraded completely after 2 h of pepsin digestion while variant A was degraded gradually showing 19%, 44%, 61% and 73% hydrolysis after 2, 4, 8 and 20 h, respectively. The main factors responsible for the exceptional pepsin susceptibility of ovine BLG are the slightly different tertiary structure of ovine BLG (compared with bovine BLG) as perceived from near circular dichroism spectra at pH 2, and its higher surface hydrophobicity, as demonstrated by a higher binding activity to 1-anilinonaphthalene-8-sulphonate. Reversed phase-high performance liquid chromatograms (RP-HPLC) profiles of the peptic hydrolysates of BLG showed the production of hydrophobic peptides at the early stages of hydrolysis, while more hydrophilic peptides appeared only at a later stage of hydrolysis. Mass spectroscopy analysis allowed the characterisation of 17 and 13 peptides after 2 and 20 h hydrolysis, respectively. Most of the enzyme activity was oriented first towards the N-terminal part of the molecule and later towards the C-terminal part of the protein; little or no activity was observed in the central region of the molecule even after 20 h hydrolysis. Native ovine ALA was almost completely degraded by pepsin, yielding 93%, 94%, 95% and 98% hydrolysis after 2, 4, 8 and 24 h, respectively. The RP-HPLC profile of the ALA hydrolysate showed 5 major hydrophobic peptides and 7 minor more hydrophilic peptides, which did not change with the time of hydrolysis.     

Effects of microwave and ultrasound pretreatments on enzymolysis of milk protein concentrate with different enzymes

Milk protein concentrate was pretreated either by microwave irradiation or by ultrasound before initiation of 3‐h enzymatic hydrolysis. The duration of pretreatment ranged from 1 to 8 min at a power level of 800 W, with the control not being subjected to any pretreatment, and five enzymes (Alcalase, Trypsin, Neutrase, Alkaline Protease and Flavourzyme) were employed. The effects of microwave and ultrasound pretreatments on the kinetics and degree of hydrolysis, protein solubility, bitterness and angiotensin‐converting enzyme inhibitory activity were evaluated. Pretreatments increased the degree of hydrolysis and stabilised the solubility of the hydrolysates but could not significantly reduce bitterness of the hydrolysates The angiotensin‐converting enzyme inhibitory activity of the hydrolysates were improved with 5‐min ultrasound‐pretreated Neutrase hydrolysates giving IC₅₀ value of 0.23 mg mL^?1. Kinetic parameters showed improved catalytic efficiencies. Pretreatments of milk protein concentrates with either microwave or ultrasound significantly improve the bioactivity and functional characteristics of the resulting hydrolysates.     

Antibody binding and functional properties of whey protein hydrolysates obtained under high pressure

This paper examines the potential of high hydrostatic pressure to produce whey protein hydrolysates that combine low immunoglobulin (Ig)G- and IgE-binding with acceptable functional properties, with the aim to produce milk-based ingredients with reduced potential allergenicity that could be used in hypoallergenic foods. Treatment with pepsin and chymotrypsin under high pressure produced, in minutes, hydrolysates in which α-lactalbumin and β-lactoglobulin were totally proteolysed, giving rise to large and hydrophobic peptides. Such hydrolysates presented reduced antigenicity and human IgE-binding properties. The hydrolysates obtained with pepsin at 400 MPa showed improved heat stability, particularly at a pH, close to the isoelectric point of the whey proteins, and their emulsion activity indexes at pH 7.0 were superior to those of the untreated whey proteins. These results suggest that the peptides present retained low antigenicity together with sufficient capacity to form emulsions.     

Microwave irradiation under different pH conditions induced a decrease in β-lactoglobulin antigenicity

Whey proteins adjusted at pH values 2, 4.6, 9 and at the natural milk pH (pH 6.8) were subjected to microwave irradiation at 300 W for 20 min or 700 W for 10 min. The protein composition of treated and native whey proteins were evaluated by Lowry’s method and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The ability of treated whey to bind IgG polyclonal antibody was determined by an enzyme-linked immunosorbent assay (ELISA) using sera obtained from rabbit immunized to β-Lactoglobulin (β-Lg). Significantly higher losses in soluble protein concentrations were observed for microwave irradiated whey proteins at pH value 4.6 (35.6% at 300 W and 44.33% at 700 W) (P < 0.0001) compared with those irradiated at the natural milk pH (26% at 300 or 700 W). The electrophoretic patterns of these proteins revealed a considerable decrease in intensity of the band corresponding to α-lactalbumin but only a slight modification was observed for the electrophoretic profiles of β-lactoglobulin. The data obtained with a rabbit anti-β-lactoglobulin immunoglobulin indicated a low antigenic response for microwave-irradiated whey proteins at the natural milk pH (up to 29.32% as well as 300–700 W) (P < 0.001). The lowest antigenicity was observed for samples adjusted to pH 4.6 followed by microwave irradiation at 300 or 700 W (46.99% at 300 W and 41.16% at 700 W) (P < 0.0001).     

Proteolysis and antioxidant activity of peptic,tryptic and chymotryptic hydrolysates of cow,buffalo, goat and camel caseins

Caseins of cow, buffalo, goat and camel milks were hydrolysed using pepsin, trypsin and chymotrypsin. The rate and degree of casein hydrolysis and the antioxidant activity (AA) of the casein hydrolysates (CH) were followed. Camel casein showed the highest rate and degree of hydrolysis with pepsin and trypsin, while cow casein was more rapidly hydrolysed with chymotrypsin than other caseins studied. The AA of all CH increased to a maximum after 24 h of hydrolysis. The AA of tryptic hydrolysates was higher (P < 0.002) than that of peptic hydrolysates. Camel CH exhibited higher AA than hydrolysates of other caseins.     

Dual function peptides from pepsin hydrolysates of whey protein isolate

The aim of this study was to investigate the effect of pepsin hydrolysates of whey protein isolate (WPI) on vascular relaxation and emulsifying capacity. WPI was subjected to pepsin hydrolysis for 5 h. The chromatographic profiles of the samples showed the formation of a wide variety of peptides. Addition of WPI hydrolysates in phenylephrine-contracted rat aortic rings induced a similar concentration-dependent relaxation in both endothelium-intact and endothelium-denuded preparations. In endothelium-denuded vessels the maximum relaxation induced by WPI fractions increased along the time, reaching over 70% after 3 h-hydrolysis on. In addition, the vascular relaxation was not associated with an inhibition of the angiotensin-converting enzyme or activation of K⁺ channels. Hydrolysed fractions were further evaluated for the emulsifying capacity (EC) and all tested fractions were able to keep an EC over 60%. These results reinforce the potential of WPI pepsin-hydrolysates as an option in the search for dual function peptides from whey proteins.     

Sweet potato protein hydrolysates: antioxidant activity and protective effects on oxidative DNA damage

In this study, sweet potato protein (SPP) hydrolysates were prepared by six enzymes (alcalase, proleather FG‐F, AS1.398, neutrase, papain and pepsin). The antioxidant activities and protective effect against oxidative DNA damage of SPP hydrolysates were investigated. Alcalase hydrolysates exhibited the highest hydroxyl radical‐scavenging activity (IC₅₀ 1.74 mg mL^?1) and Fe²⁺‐chelating ability (IC₅₀ 1.54 mg mL^?1) (P < 0.05). Compared with other five hydrolysates, the hydrolysates obtained by alcalase had the most abundant <3‐kDa fractions. In addition, below 3‐kDa fractions of alcalase hydrolysates showed the highest antioxidant activities and protective effects against DNA damage through both scavenging hydroxyl radicals and chelating Fe²⁺, which was probably because of the increase in several antioxidant amino acids, such as His, Met, Cys, Tyr and Phe, as well as the hydrophobic amino acids. The results suggested that enzymatic hydrolysis could be used as an effective technique to produce high value‐added peptides products from SPP.     

Rheological and structural characterization of gels from whey protein hydrolysates/locust bean gum mixed systems

The gelling ability of whey proteins can be changed by limited hydrolysis and by the addition of other components such as polysaccharides. In this work the effect of the concentration of locust bean gum (LBG) on the heat-set gelation of aqueous whey protein hydrolysates (10% w/w) from pepsin and trypsin was assessed at pH 7.0. Whey protein concentrate (WPC) mild hydrolysis (up to 2.5% in the case of pepsin and 1.0% in the case of trypsin) ameliorates the gelling ability. The WPC synergism with LBG is affected by the protein hydrolysis. For a WPC concentration of 10% (w/w), no maximum value was found in the G′ dependence on LBG content in the case of the hydrolysates, unlike the intact WPC. However, for higher protein concentrations, the behaviour of gels from whey proteins or whey protein hydrolysates towards the presence of LBG becomes very similar. In this case, a small amount of LBG in the presence of salt leads to a big enhancement in the gel strength. Further increases in the LBG concentration led to a decrease in the gel strength.     

Antioxidant activities of chicken liver hydrolysates by pepsin treatment

This study was divided into two parts: (i) an optimal hydrolysing procedure of chicken liver hydrolysates (CLHs) and (ii) the in vivo antioxidant properties of CLHs via a D‐galactose‐induced mouse model. A pepsin‐to‐raw chicken liver mass ratio (1:400, w:w) and 2‐h hydrolysing period were chosen to manufacture CLHs based on yield, peptide level and antioxidant effect. Molecular masses of CLHs were lower than 10 kDa. CLH was rich in aspartic acid and glutamic acid, and also contained both manganese and selenium, which are essential cofactors of superoxide dismutase and glutathione peroxidase, respectively. The contents of cadmium, mercury, tin, and arsenic in CLHs were very low and even no detectible. Regarding the in vivo antioxidant activity of CLHs, a dosage of 1.2 g D‐galactose kg^?1 body weight increased (P < 0.05) 2‐thiobarbituric acid reactive substances values and decreased (P < 0.05) glutathione and Trolox equivalent antioxidant capacity values, as well as superoxide dismutase, catalase, and glutathione peroxidase activities in serum and organs of mice. However, the in vivo antioxidant capacities were improved (P < 0.05) by supplementing CLHs.     

Inhibition of dipeptidyl peptidase IV by enzymatic hydrolysates derived from primary and secondary whey of fresh and Oaxaca cheeses

Currently, there is an interest in studying natural inhibitors of dipeptidyl peptidase IV (DPP‐IV) for the treatment of type II diabetes. To look for novel inhibitors, the primary and secondary whey of fresh and Oaxaca cheeses was obtained, and the electrophoretic patterns were determined. Hydrolysis with pepsin and trypsin of primary and secondary whey from fresh and Oaxaca cheeses, respectively, showed DPP‐IV inhibition with values of ~51 and 55%, and also an IC50 of 92.86 μg/mL for Oaxaca cheese‐derived peptides. Data show the potential of whey enzymatic hydrolysates from fresh and Oaxaca cheeses as antidiabetic agents.     

Thermal denaturation of bovine β-lactoglobulin in different protein mixtures in relation to antigenicity

Denaturation of β-lactoglobulin (BLG) was studied in relation to its antigenicity at two heat treatments in several native protein mixtures; allergenicity was determined by enzyme-linked immunosorbent assay based on BLG capacity to bind with immunoglobulin G (IgG) antibodies. The influence of other proteins on BLG denaturation correlated with altered antigenicity. Treatment at 72 °C/15 s enhanced antigenicity in a BLG+α-lactalbumin (ALA) mixture, possibly due to exposed epitopes in the unfolded structure. Treatment at 100 °C/30 s mostly resulted in BLG-led protein aggregation through thiol/disulphide interactions and decreased antigenicity by fragmentation and masking of epitopes, the extent of which was mixture-dependent. The presence of IgG resulted in diminished antigenicity in BLG + ALA + IgG at 100 °C/30 s in comparison with BLG + ALA. ALA governed whey protein denaturation over BLG in BLG + ALA + IgG + bovine serum albumin (BSA), possibly catalysed by BSA at 100 °C/30 s, resulting in a higher retention of antigenicity than in other mixtures.     

Effects of pepsin hydrolysis on the soy β‐conglycinin aggregates formed by heat treatment at different pH

The effects of pepsin hydrolysis on the β‐conglycinin aggregates formed by heat treatment at different pH were investigated. Results showed that fibrils were still observed, whereas the random aggregates were easily to be digested in the simulated gastric fluid. Electrophoresis and molecular weight analysis indicated that large aggregates still existed after pepsin treatment for fibrils. Hydrolysis resulted in changes in the apparent viscosity (ηapp) of 6% fibril solutions. The ηapp at the shear rate range (0–30 s^?1) increased in the order of fibrils < fibrils with pepsin for 60 min < fibrils with pepsin for 30 min. Smaller peptide/fibril fragments were generated, and additional aggregates were reformed during the hydrolysis process, as evidenced by thioflavin T and atomic force microscopy images. The native β‐conglycinin hydrolysates comprised a mixture of polypeptides enriched in about 47 kDa. These findings would provide valuable information about effects of enzymatic hydrolysis on plant oligometric globulin aggregates.     

Effects of In Vitro Proteolysis on the Allergenicity of Major Whey Proteins

The effects of in vitro proteolysis on the allergenicity of major whey proteins (α-lactalbumin and β-lactoglobulin) by simulating the human gastrointestinal conditions were evaluated. The proteolysis of demineralized whey with pepsin (pH 2.0 for 30 min) and/or various pancreatic enzymes (pH 7.5 for 60 and 240 min) was performed by the pH-stat technique at 37°C. The enzyme inactivation was performed by heating at 80°C for 20 min. Allergenicity of the hydrolyzates was evaluated by RAST inhibition using sera obtained from children allergic to whey proteins. Selective proteolysis of whey by pepsin and α-chymotrypsin was the most efficient combination of enzymes to reduce the allergenicity of both α-lactalbumin and β-lactoglobulin. The above hydrolyzate could be used to develop an ingredient for infant milk formula with a lower allergenicity.     

In vitro antioxidant activities of hydrolysates obtained from Iranian wild almond (Amygdalus scoparia) protein by several enzymes

A protein extract from wild almond was hydrolysed using five different enzymes (pepsin, trypsin, chymotrypsin, alcalase and flavourzyme). The hydrolysates were then assayed for their antioxidant activities. The highest extent of proteolysis was obtained with alcalase (0.35; determined as the change in the absorbance at 340 nm, ΔA₃₄₀) and the lowest was with pepsin (ΔA₃₄₀ = 0.12). Radical scavenging activities obtained by 2, 2′‐azino‐bis (3‐ethylbenzothiazoline‐6‐sulphonic acid) and ferric‐reducing abilities of the hydrolysates demonstrated that the hydrolysate from alcalase had significantly (P < 0.05) greater antioxidant activity. Analysis of the molecular weight distributions showed that peptides produced by alcalase were smaller than those produced by chymotrypsin, trypsin and flavourzyme. Based on the current study, the hydrolysates produced by alcalase can be suggested as potential antioxidant agents in food industry and for use in functional foods.     

Antioxidant and nitric oxide inhibitory activities of tilapia (Oreochromis niloticus) protein hydrolysate: effect of ultrasonic pretreatment and ultrasonic‐assisted enzymatic hydrolysis

Ultrasound was incorporated to processing of fish protein hydrolysate to facilitate homogenate pretreatment and enzymatic hydrolysis of tilapia (Oreochromis niloticus) muscle protein. Their effects on Flavourzyme hydrolysis and biological activities of the tilapia hydrolysate were examined. The ultrasound‐assisted hydrolysis caused reduction in degree of hydrolysis ranging from 23% to 35% relative to that of the conventional process. The 70 W ultrasound‐assisted hydrolysis process increased DPPH radical‐scavenging activity and reducing power of tilapia hydrolysate prepared from the non‐pretreatment homogenate by 33% and 45%, respectively. All hydrolysates have no cytotoxicity on RAW264.7 cell lines at the maximum concentration of 20 mg protein mL^?1. The 70 W ultrasound pretreatment at 30 and 45 min combined with conventional hydrolysis is the suitable condition for producing tilapia hydrolysate with nitric oxide inhibitory and antioxidative activities on RAW264.7 cell lines, respectively. As a result, ultrasound could be applied to enzymatic protein hydrolysis either as pretreatment or during the hydrolysis.     

Optimisation of microwave‐assisted extraction of prune (Prunus domestica) antioxidants by response surface methodology

Optimal conditions for microwave‐assisted extraction (MAE) of total phenols (TP), epicatechin gallate and antioxidant activity from prune (Prunus domestica), rejected in transformation process of plum to prune, were determined by response surface methodology. The central composite design was used to study the effects of three independent variables: microwave power, irradiation time and solvent polarity on the TP, epicatechin gallate and antioxidant activity. Epicatechin gallate was identified as a major phenolic compound in prune extract by RP‐HPLC. Microwave power and irradiation time significantly affected all responses (P < 0.01). The highest TP (598.89 mg GAE/100 g prune) was obtained using water as an extraction solvent at 500 W, during 115 s. However, the optimal conditions for epicatechin gallate extraction were ethanol 80%, 435 W and 120 s. MAE is more efficient than conventional extraction method to obtain TP from prune. The experimental values were reasonably close to the predicted values confirming the validity of the predicted models.     

Improving antioxidant activities of whey protein hydrolysates obtained by thermal preheat treatment of pepsin,trypsin, alcalase and flavourzyme

Antioxidant activity of whey protein concentrate (WPC) hydrolysates was evaluated. Hydrolysates were obtained by pepsin, trypsin, alcalase and flavourzyme enzymatic reaction and preheat treatment of 95 °C for 5 or 10 min. The degree of hydrolysis (DH) was determined by 2,4,6‐trinitrobenzene sulphonic acid method, and antioxidant properties were determined by three spectrophotometric methods: ferricyanide method, ferric reducing/antioxidant power assay and diphenyl‐picryl hydrazinyl radical‐scavenging activity. For all the enzymes, briefly preheat treatment (95 °C/5 min) increased DH of WPC. Alcalase hydrolysates showed the highest antioxidant activity by three methods. The changes in antioxidant activity was coincidental with the changes in DH (R² = 0.988). Hydrolysates analysed by polyacrylamide gel electrophoresis and high performance liquid chromatography indicated that the α‐La was hydrolysed completely by pepsin, trypsin and alcalase and was resistant to flavourzyme to some extent; β‐lactoglobulin was only completely hydrolysed by trypsin and alcalase. Results indicated that antioxidant activity of hydrolysates was greatly related to the exposure of amino acid residues.