An egg yolk protein by-product following ethanol extraction of phospholipids (YP) was hydrolyzed with pepsin to produce and identify novel peptides that revealed antioxidant, ACE inhibitory and antidiabetic (α-glucosidase and DPP-IV inhibitory) activities. The peptic hydrolysate of YP was fractionated by ion-exchange chromatography and reversed-phase high-pressure liquid chromatography. Isolated peptides were identified using mass spectrometry (MALDI-ToF) and the Mascot Search Results database. Four peptides of MW ranging from 1,210.62 to 1,677.88 Da corresponded to the fragments of Apolipoprotein B (YINQMPQKSRE; YINQMPQKSREA), Vitellogenin-2 (VTGRFAGHPAAQ) and Apovitellenin-1 (YIEAVNKVSPRAGQF). These peptides were chemically synthesized and showed antioxidant, ACE inhibitory or/and antidiabetic activities. Peptide YIEAVNKVSPRAGQF exerted the strongest ACE inhibitory activity, with IC50 = 9.4 µg/mL. The peptide YINQMPQKSRE showed the strongest DPPH free radical scavenging and DPP-IV inhibitory activities and its ACE inhibitory activity (IC50) reached 10.1 µg/mL. The peptide VTGRFAGHPAAQ revealed the highest α-glucosidase inhibitory activity (IC50 = 365.4 µg/mL). A novel nutraceutical effect for peptides from an egg yolk hydrolysate was shown.

In the present study, whey protein concentrate (WPC-80) and β-lactoglobulin were hydrolyzed with a noncommercial serine protease isolated from Asian pumpkin (Cucurbita ficifolia). Hydrolysates were further fractionated by ultrafiltration using membranes with cut-offs equal 3 and 10 kDa. Peptide fractions of molecular weight lower than 3 and 3–10 kDa were further subjected to the RP-HPLC. Separated preparations were investigated for their potential as the natural inhibitors of dipeptidyl peptidase (DPP-IV), α-glucosidase and angiotensin converting enzyme (ACE). WPC-80 hydrolysate showed higher inhibitory activities against the three tested enzymes than β-lactoglobulin hydrolysate. Especially high biological activities were exhibited by peptide fractions of molecular weight lower than 3 kDa, with ACE IC50 <0.64 mg/mL and DPP-IV IC50 <0.55 mg/mL. This study suggests that peptides generated from whey proteins may support postprandial glycemia regulation and blood pressure maintenance, and could be used as functional food ingredients in the diet of patients with type 2 diabetes.

Deriving non-conventional enzymes from cheaper sources than those used for commercially available enzymes may result in the production of hydrolysates with beneficial features, while drastically reducing the cost of hydrolysis. This is especially significant for enzymatic hydrolysis as a method of protein waste utilization. We have previously described the ability of non-commercial serine protease from Yarrowia lipolytica yeast to produce/release bioactive peptides from egg white protein by-products (EP). The enzymatic hydrolysis of EP was carried out for 24 h using the serine protease at an enzyme: substrate ratio of 1:30 (w/w). The obtained hydrolysate was characterized by protein degradation of 38% and also exhibited an antioxidant and cytokine-inducing activity. The isolation procedure (ultrafiltration and RP-HPLC) of bioactive peptides from the EP hydrolysate provided peptide fractions with significant antioxidant and ACE inhibitory activities. Three homogeneous and three heterogeneous peptide fractions were identified using MALDI-TOF/MS and the Mascot Search Results database. The peptides, mainly derived from ovalbumin, were composed of 2-19 amino-acid residues. We have thus demonstrated a novel ability of serine protease from Y. lipolytica to release biopeptides from an EP by-product.

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The main objective of this study was to determine potential application of a serine proteinase derived from Asian pumpkin for obtaining biologically active peptides from casein. The course of casein hydrolysis by three doses of the enzyme (50, 150, 300 U/mg of protein) was monitored for 24 hours by the determinations of: hydrolysis degree DH (%), free amino group content (μmole Gly/g), RP HPLC peptide profiles and by polyacrylamide gel electrophoresis. In all hydrolyzates analyzed antioxidant activities were determined using three tests: the ability to reduce iron ions in FRAP test, the ability to scavenge free radicals in DPPH test, and Fe(2+) chelating activity. The antimicrobial activity of obtained peptide fractions was determined as the ability to inhibit the growth of Escherichia coli, Bacillus cereus and Pseudomonas fluorescens in a diffusion plate test. The deepest degradation, expressed as the DH [%] and the free amino group content (67% and 7528 µmole Gly/mg, respectively), was noted in samples hydrolyzed with 300 U/ml of enzyme for 24 hours, while in other samples the determined values were about three and two times lower. The results were in agreement with the peptide profiles obtained by RP HPLC. The highest antioxidative activities determined in all tests were seen for the casein hydrolysate obtained with 300 U/mg protein of serine proteinase after 24 h of reaction (2.15 µM Trolox/mg, 96.15 µg Fe(3+)/mg, 814.97 µg Fe(2+)/mg). Antimicrobial activity was presented in three preparations. In other samples no antimicrobial activity was detected.

The effect of whey protein hydrolysate (WPH) addition on growth of standard yoghurt cultures and Bifidobacterium adolescentis during co-fermentation and its viability during storage at 4ºC in yoghurts has been evaluated. WPH was obtained with the use of serine protease from Y. lipolytica yeast. Stirred probiotic yoghurts were prepared by using whole milk standardized to 16% of dry matter with the addition of either whey protein concentrate, skim milk powder (SMP), WPH-SMP (ratio 1:1), WPH. The hydrolysate increased the yoghurt culture counts at the initial stage of fermentation and significantly inhibited the decrease in population viability throughout the storage at 4ºC in comparison to the control. The post-fermentation acidification was also retarded by the addition of WPH. The hydrolysate did not increase the Bifidobacterium adolescentis counts at the initial stage. However, the WPH significantly improved its viability. After 21 days of storage, in the yogurts supplemented with WPH, the population of these bacteria oscillated around 3.04 log10 CFU/g, while in samples where SMP or whey protein concentrate was used, the bacteria were no longer detected.

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In the present study the effect of hydrolysis with non-commercial Cucurbita ficifolia serine protease on a reduction of the IgE and IgG binding capacity of whey protein concentrate and αs-casein was investigated. The intensity of the protein degradation was analyzed by the degree of hydrolysis, the free amino groups content and RP-HPLC. The ability to bind the antibodies by native proteins and their hydrolysates was determined using a competitive ELISA test. Deep hydrolysis contributed to a significant reduction of immunoreactive epitopes present in WPC. In the case of IgE and IgG present in the serum pool of children with CMA, the lowest binding capacity was detected in the 24 h WPC hydrolysate, where the inhibition of the reaction with native WPC was ≤23 and ≤60 %, respectively. The analysis of the IgG reactivity in the antiserum of the immunized goat showed that the lowest antibody binding capacity was exhibited also by 24 h WPC hydrolysate at a concentration of 1000 μg/ml where the inhibition of the reaction with nWPC was ≤47 %. One-hour hydrolysis of α-casein was sufficient to significant reduction of the protein antigenicity, while the longer time (5 h) of hydrolysis probably lead to the appearance of new epitopes reactive with polyclonal.