Peptides derived from dolicholin, a phaseolin-like protein in country beans (Dolichos lablab), potently stimulate cholecystokinin secretion from enteroendocrine STC-1 cells

Peptides derived from soybean beta-conglycinin and pork protein stimulate cholecystokinin (CCK) secretion from the enteroendocrine cells (EECs) and suppress food intake. Here we examined CCK-releasing activities from the enteroendocrine cell line STC-1, in peptides derived from underutilized legumes, and found much higher activity in the peptic hydrolysate of Country beans (CBP) compared to that from other legume-derived peptides including beta-conglycinin peptone. Active components in CBP were separated into acetonitrile-soluble fractions, but the activities were abolished after pronase treatment. To identify the Country bean protein containing the active peptides, Country bean protein extracts in an alkaline solution (CBE) were fractionated based on isoelectric point or molecular weight. Peptones prepared from CBE fractions containing a 51 kDa major protein stimulated CCK release, but other fractions did not. N-Terminal sequence analysis indicated that the 51 kDa protein is a phaseolin-like globular protein, and we designated this protein dolicholin. These results indicate that Country bean-derived peptides are very potent legume peptides in stimulating CCK secretion from EECs and that the stimulant peptides originate from dolicholin, a newly identified phaseolin-like globular protein in Country beans.     

Potato extract (Potein) suppresses food intake in rats through inhibition of luminal trypsin activity and direct stimulation of cholecystokinin secretion from enteroendocrine cells

Dietary proteins and trypsin inhibitors are known to stimulate the secretion of the satiety hormone cholecystokinin (CCK). A potato extract (Potein) contains 60% carbohydrate and 20% protein including trypsin inhibitory proteins. In this study, we examined whether Potein suppresses food intake in rats and whether it directly stimulates CCK secretion in enteroendocrine cells. In fasted rats, food consumption was measured up to 6 h after the oral administration of Potein or soybean trypsin inhibitor (SBTI). CCK-releasing activities of Potein and SBTI were examined in the murine CCK-producing cell line STC-1. Potein inhibited the trypsin activity in vitro with a potency 20-fold lower than that of SBTI. Oral administration of Potein dose-dependently suppressed food intake for 1-6 h. Potein, but not the SBTI, dose-dependently induced CCK secretion in STC-1 cells. These results suggest that Potein suppresses food intake through the CCK secretion induced by direct stimulation on enteroendocrine cells and through inhibition of luminal trypsin.     

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.     

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.     

Release of angiotensin I converting enzyme (ACE) inhibitory activity during in vitro gastrointestinal digestion: from batch experiment to semicontinuous model

Gastrointestinal digestion is of major importance in the bioavailability of angiotensin I converting enzyme (ACE) inhibitory peptides, bioactive peptides with possible antihypertensive effects. In this study, the conditions of in vitro gastrointestinal digestion leading to the formation and degradation of ACE inhibitory peptides were investigated for pea and whey protein. In batch experiments, the digestion simulating the physiological conditions sufficed to achieve the highest ACE inhibitory activity, with IC(50) values of 0.076 mg/mL for pea and 0.048 mg/mL for whey protein. The degree of proteolysis did not correlate with the ACE inhibitory activity and was always higher for pea than whey. In a semicontinuous model of gastrointestinal digestion, response surface methodology studied the influence of temperature and incubation time in both the stomach and small intestine phases on the ACE inhibitory activity and degree of proteolysis. For pea protein, a linear model for the degree of proteolysis and a quadratic model for the ACE inhibitory activity could be constituted. Within the model, a maximal degree of proteolysis was observed at the highest temperature and the longest incubation time in the small intestine phase, while maximal ACE inhibitory activity was obtained at the longest incubation times in the stomach and small intestine phase. These results show that ACE inhibitory activity of pea and whey hydrolysates can be controlled by the conditions of in vitro gastrointestinal digestion.     

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.     

Beta-conglycinin embeds active peptides that inhibit lipid accumulation in 3T3-L1 adipocytes in vitro

Obesity is a worldwide health concern because it is a well-recognized predictor of premature mortality. The objective was to identify soybean varieties that have improved potential to inhibit fat accumulation in adipocytes by testing the effects of soy hydrolysates having a range of protein subunit compositions on lipid accumulation and adiponectin expression in 3T3-L1 adipocytes. The results showed that differences in the protein distribution of 15 soy genotypes led to different potentials for the reduction of fat accumulation. The inhibition of lipid accumulation of soy alcalase hydrolysates in 3T3-L1 adipocytes ranged from 29 to 46%. Soy hydrolysates made from genotypes with 45.3 +/- 3.3% of total protein as beta-conglycinin, on average, showed significantly higher inhibition of lipid accumulation compared to those with 24.7 +/- 1.5% of extracted total protein as beta-conglycinin. Moreover, after in vitro simulated digestion with pepsin-pancreatin of the soy alcalase hydrolysates, 86% of the original activity remained. Adiponectin expression was induced in 3T3-L1 adipocytes treated with 15 soy hydrolysates up to 2.49- and 2.63-fold for high and low molecular weight adiponectin, respectively. The inhibition of lipid accumulation calculated from a partial least squares (PLS) analysis model correlated well with experimental data (R(2) = 0.91). In conclusion, it was feasible to differentiate soy varieties on the basis of the potential of their proteins to reduce fat accumulation using a statistical model and a cell-based assay in vitro. Furthermore, beta-conglycinin embeds more peptides than glycinin subunits that inhibit lipid accumulation and induce adiponectin in 3T3-L1 adipocytes. Therefore, soy ingredients containing beta-conglycinin may be important food components for the control of lipid accumulation in adipose tissue.     

beta-Conglycinins among sources of bioactives in hydrolysates of different soybean varieties that inhibit leukemia cells in vitro

Soybean is a complex matrix containing several potentially bioactive components. The objective was to develop a statistical model to predict the in vitro anticancer potential of soybean varieties based on the correlation between protein composition and bioactive components after simulated gastrointestinal enzyme digestion with their effect on leukemia mouse cells. The IC 50 values of the hydrolysates of soy genotypes (NB1-NB7) on L1210 leukemia cells ranged from 3.5 to 6.2 mg/mL. Depending on genotype, each gram of soy hydrolysates contained 2.7-6.6 micromol of total daidzein, 3.0-4.7 micromol of total genistein, 0.5-1.3 micromol of glycitein, 2.1-2.8 micromol of total saponins, 0.1-0.2 micromol of lunasin, and 0.1-0.6 micromol of Bowman-Birk inhibitor (BBI). The IC 50 values calculated from a partial least-squares (PLS) analysis model correlated well with experimental data ( R (2) = 0.99). Isoflavones and beta-conglycinin positively contributed to the cytotoxicity of soy on L1210 leukemia cells. Lunasin and BBI were potent L1210 cell inhibitors (IC 50 = 13.9 and 22.5 microM, respectively), but made modest contributions to the activity of defatted soy flour hydrolysates due to their relatively low concentrations. In conclusion, the data demonstrated that beta-conglycinins are among the major protein components that inhibit leukemia cell growth in vitro. Furthermore, it was feasible to differentiate soybean varieties on the basis of the biological effect of their components using a statistical model and a cell-based assay.     

Study of the effect of soy proteins on the acid-induced gelation of casein micelles

The objective of this research was to understand whether addition of soy protein to milk protein affects the properties of acid-induced casein gels. Different samples were prepared by suspending casein micelles pellets in milk serum containing soy proteins or whey proteins as well as mixtures of the two proteins. Glucono-delta-lactone was added, and the changes in apparent size (in diluted systems) as well as the viscoelastic properties of the mixtures were measured. Size exclusion chromatography was also carried out to characterize the soluble phase of the various mixtures before and after heating. Soy protein affected the gelation of the mixtures; however, not to the same extent as whey proteins, which dominated formation of the network in soy-whey-casein systems. It was concluded that, up to a critical ratio of soy/whey proteins, soy proteins can be incorporated in the mix without a significant change in structure of the casein gels.     

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.     

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.     

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.     

Antioxidant activity of some protein hydrolysates and their fractions with different isoelectric points

Antioxidant activities of commercially available enzymatic hydrolysates of milk and plant proteins were examined. Among them, soy protein and wheat gluten hydrolysates showed strong 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity and antioxidation activity against linoleic acid oxidation in emulsion systems. Peptide fractions with higher antioxidant activities than crude enzymatic hydrolysates of gluten and soy protein were prepared without toxic solvents and reagents. Peptides in these plant protein hydrolysates were fractionated on the basis of the amphoteric nature of sample peptides by preparative isoelectric focusing without adding chemically synthesized carrier ampholytes, which is termed autofocusing. The acidic fractions from both protein hydrolysates showed stronger DPPH radical scavenging activities than the basic fractions, while the basic fractions strongly suppressed 2,2'-azobis (2-amidinopropane) dihydrochloride-induced oxidation of linoleic acid in an emulsion system. These acidic and basic peptide fractions would be useful to examine the mechanism underlying the antioxidant activities of peptides in food.     

Lower weight gain and hepatic lipid content in hamsters fed high fat diets supplemented with white rice protein, brown rice protein, soy protein, and their hydrolysates

The physiological effects of the hydrolysates of white rice protein (WRP), brown rice protein (BRP), and soy protein (SP) hydrolyzed by the food grade enzyme, alcalase2.4 L, were compared to the original protein source. Male Syrian Golden hamsters were fed high-fat diets containing either 20% casein (control) or 20% extracted proteins or their hydrolysates as the protein source for 3 weeks. The brown rice protein hydrolysate (BRPH) diet group reduced weight gain 76% compared with the control. Animals fed the BRPH supplemented diet also had lower final body weight, liver weight, very low density lipoprotein cholesterol (VLDL-C), and liver cholesterol, and higher fecal fat and bile acid excretion than the control. Expression levels of hepatic genes for lipid oxidation, PPARα, ACOX1, and CPT1, were highest for hamsters fed the BRPH supplemented diet. Expression of CYP7A1, the gene regulating bile acid synthesis, was higher in all test groups. Expression of CYP51, a gene coding for an enzyme involved in cholesterol synthesis, was highest in the BRPH diet group. The results suggest that BRPH includes unique peptides that reduce weight gain and hepatic cholesterol synthesis.     

Bioactivity of antioxidants in extruded products prepared from purple potato and dry pea flours

Measuring antioxidant activity using a biologically relevant assay adds important evidence to aid in understanding the role of phytochemicals based on data from in vivo and chemical assays of extrusion processed purple potato and pea flours. A cellular antioxidant activity assay could provide biologically relevant information on bioactive compounds in raw as well as processed food products. The objective of this study was to investigate the complete phytochemical profiles, antioxidant activity, cellular antioxidant activity, and their contribution to bioactivity in purple potato flour, dry pea flour, raw formulations, and extrusion cooked products prepared with the above ingredients. The free fraction of extracts contributed 68, 64, and 88% to total phenolics, total antioxidant activity (ORAC value), and total flavonoids, respectively, in purple potato flour (PPF). Similarly, extracts in the free fraction contributed 87, 86, and 64% to total phenolics, total antioxidant activity (ORAC value), and total flavonoids, respectively, in dry pea flour (DPF). The amount of total phenolics and total flavonoids in purple potato flour and the antioxidant activity of PPF and DPF were comparable to published data. However, a higher amount in the total flavonoids and lower in the total phenolics of DPF were observed. Caffeic, p-coumaric, and ferulic acids were mostly observed in the bound extracts of raw formulations as in the extrudates, whereas chlorogenic acid was predominant in the free extracts. The extruded products had significantly higher (p < 0.05) content of total phenolics, ORAC antioxidant activity, and flavonoids, compared to the raw formulations. Extrusion processing increased the cellular antioxidant activity of the extrudates prepared from 35:65 and 50:50 PPF/DPF (w/w) of ingredients compared with control raw formulations in a dose-dependent manner. Increase of PPF significantly increased (p < 0.05) the cellular antioxidant activity of 35-50% PPF formulations.     

Identification and characterization of topoisomerase II inhibitory peptides from soy protein hydrolysates

Topoisomerases are targets of several anticancer agents because their inhibition impedes the processes of cell proliferation and differentiation in carcinogenesis. With very limited information available on the inhibitory activities of peptides derived from dietary proteins, the objectives of this study were to employ co-immunoprecipitation to identify inhibitory peptides in soy protein hydrolysates in a single step and to investigate their molecular interactions with topoisomerase II. For this, soy protein isolates were subjected to simulated gastrointestinal digestion with pepsin and pancreatin, and the human topoisomerase II inhibitory peptides were co-immunoprecipitated and identified on a CapLC- Micromass Q-TOF Ultima API system. The inhibitory activity of these peptides from soy isolates toward topoisomerase II was confirmed using three synthetic peptides, FEITPEKNPQ, IETWNPNNKP,and VFDGEL, which have IC 50 values of 2.4, 4.0, and 7.9 mM, respectively. The molecular interactions of these peptides evaluated by molecular docking revealed interaction energies with the topoisomerase II C-terminal domain (CTD) (-186 to -398 kcal/mol) that were smaller than for the ATPase domain (-169 to -357 kcal/mol) and that correlated well with our experimental IC 50 values ( R (2) = 0.99). In conclusion, three peptides released from in vitro gastrointestinal enzyme digestion of soy proteins inhibited human topoisomerase II activity through binding to the active site of the CTD domain.     

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.     

Use of caseinophosphopeptides as natural antioxidants in oil-in-water emulsions

Chelators are valuable ingredients used to improve the oxidative stability of food emulsions. Caseins and casein peptides have phosphoseryl residues capable of binding transition metals. Thus, the ability of enriched caseinophosphopeptides to inhibit lipid oxidation in corn oil-in-water emulsions was investigated. Enriched caseinophosphopeptides (25 microM) inhibited the formation of lipid oxidation at both pH 3.0 and 7.0 as determined by lipid hydroperoxides and hexanal. Calcium (0-100 mM) had no influence on the antioxidant activity of the enriched caseinophosphopeptides. Casein hydrolysates were more effective inhibitors of lipid oxidation than the enriched caseinophosphopeptides at equal phosphorus content. Thus, antioxidant properties might not be uniquely attributed to chelating metals by phosphoseryl residues but also by scavenging free radicals. Overall, the observed antioxidant activity of casein hydrolysates means they could be utilized to decrease oxidative rancidity in foods.     

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.     

Heat-induced soy-whey proteins interactions: formation of soluble and insoluble protein complexes

The aggregation behavior during heating of a solution containing soy protein and whey protein isolate (WPI) was studied using rheology, confocal microscopy, gel filtration, and electrophoresis. Soy/WPI mixtures formed gels at 6% total protein concentration with a high elastic modulus (G') and no apparent phase separation. The ratio of soy to WPI was fundamental in determining the type of network formed. Systems containing a high soy to WPI ratio (>70% soy protein) showed a different evolution of the elastic modulus during heat treatment, with two apparent stages of network development. Whey proteins formed disulfide bridges with soy proteins during heating, and at low ratios of soy/WPI, the aggregates seemed to be predominantly formed by 7S, the basic subunits of 11S and beta-lactoglobulin. Size exclusion chromatography indicated the presence of high molecular weight soluble complexes in mixtures containing high soy/WPI ratios. Results presented are the first evidence of interactions between soy proteins and whey proteins and show the potential for the creation of a new group of functional ingredients.