Whey-derived peptides interactions with ACE by molecular docking as a potential predictive tool of natural ACE inhibitors

Several milk/whey derived peptides possess high in vitro angiotensin I-converting enzyme (ACE) inhibitory activity. However, in some cases, poor correlation between the in vitro ACE inhibitory activity and the in vivo antihypertensive activity has been observed. The aim of this study is to gain insight into the structure-activity relationship of peptide sequences present in whey/milk protein hydrolysates with high ACE inhibitory activity, which could lead to a better understanding and prediction of their in vivo antihypertensive activity. The potential interactions between peptides produced from whey proteins, previously reported as high ACE inhibitors such as IPP, LIVTQ, IIAE, LVYPFP, and human ACE were assessed using a molecular docking approach. The results show that peptides IIAE, LIVTQ, and LVYPFP formed strong H bonds with the amino acids Gln 259, His 331, and Thr 358 in the active site of the human ACE. Interestingly, the same residues were found to form strong hydrogen bonds with the ACE inhibitory drug Sampatrilat. Furthermore, peptides IIAE and LVYPFP interacted with the amino acid residues Gln 259 and His 331, respectively, also in common with other ACE-inhibitory drugs such as Captopril, Lisinopril and Elanapril. Additionally, IIAE interacted with the amino acid residue Asp 140 in common with Lisinopril, and LIVTQ interacted with Ala 332 in common with both Lisinopril and Elanapril. The peptides produced naturally from whey by enzymatic hydrolysis interacted with residues of the human ACE in common with potent ACE-inhibitory drugs which suggests that these natural peptides may be potent ACE inhibitors

Protective effect of β-Lactoglobulin against heat induced loss of antioxidant activity of resveratrol

Resveratrol exhibits many health benefits; however, low water-solubility and instability under processing conditions such as heating can be some of the main challenges for its processing and formulation. Here the complexation of β-lactoglobulin (β-Lg) with resveratrol was investigated to improve its solubility and stability. The solubility of resveratrol in water was determined as 7 mg/100 ml. Resveratrol-β-Lg nanoparticles (181.8 nm) were produced at pH 6 and 75 °C for 45 min. Heating resveratrol solutions at 75 °C for 45 min resulted in isomerization of resveratrol and reduced antioxidant activity. However, resveratrol-β-Lg nanocomplexes which had undergone the same heat treatment exhibited improved antioxidant activity. Heating under pasteurisation conditions led to similar results and both native β-Lg and nanoparticles exhibited a protective effect against heat-induced chemical changes in resveratrol, resulting in enhanced antioxidant activity. Fluorescence measurements revealed strong interactions of resveratrol with both native protein and nanoparticles

Surfactant TWEEN20 provides stabilisation effect on anthocyanins extracted from red grape pomace

Red grape pomace, a wine-making by-product is rich in anthocyanins and has many applications in food and pharmaceutical industry. However, anthocyanins are unstable during processing and storage. This study aimed to investigate the stability of anthocyanins obtained by hydroalcoholic extraction (with and without sorbic acid) and colloidal gas aphrons (CGA) separation; a surfactant (TWEEN20) based separation. Anthocyanins in CGA samples showed higher stability (half-life = 55 d) than in the crude extract (half-life = 43 d) and their stability increased with the concentration of TWEEN20 in the CGA fraction (6.07–8.58 mM). The anthocyanins loss in the CGA sample (with the maximum content of surfactant, 8.58 mM) was 34.90%, comparable to that in the crude ethanolic extract with sorbic acid (EE-SA) (31.53%) and lower than in the crude extract (44%). Colour stabilisation was also observed which correlated well with the stability of individual anthocyanins in the EE and CGA samples. Malvidin-3-o-glucoside was the most stable anthocyanin over time

Chemical characterisation and determination of sensory attributes of hydrolysates produced by enzymatic hydrolysis of whey proteins following a novel integrative process

The overall aim of this work was to characterize the major angiotensin converting enzyme (ACE) inhibitory peptides produced by enzymatic hydrolysis of whey proteins, through the application of a novel integrative process. This process consisted of the combination of adsorption and microfiltration within a stirred cell unit for the selective immobilization of β-lactoglobulin and casein derived peptides (CDP) from whey. The adsorbed proteins were hydrolyzed in-situ which resulted in the separation of peptide products from the substrate and fractionation of peptides. Two different hydrolysates were produced: (i) from CDP (IC50 =287μg/mL) and (ii) from β-lactoglobulin (IC50=128μg/mL). IC50 is the concentration of inhibitor needed to inhibit ACE by half. The well known antihypertensive peptide IPP and several novel peptides that have structural similarities with reported ACE inhibitory peptides were identified and characterized in both hydrolysates. Furthermore, the hydrolysates were assessed for bitterness. No significant difference was found between the control (milk with no hydrolysate) and hydrolysate samples at different concentrations (at, below and above the IC50)

Characterisation of β-lactoglobulin nanoparticles and their binding to caffeine

The production of β-Lg nanoparticles by a simple heat-induced denaturation method without the need to add chemicals was performed at different conditions of pH, and temperature of denaturation. Optimum conditions were set as 0.2% β-Lg, pH 6 and simply heating at 75 °C for 45 min. At these conditions, a monodisperse solution with colloidal stability was obtained and the yield of aggregation was over 90%. Shape and size of nanoparticles were determined by Dynamic Light Scattering and by electron microscopy. A monodisperse particle size distribution of spherical shape particles (200 nm-300nmin diameter) was obtained. The stability of the aggregates towards various types of dissociating buffers was studied. Sodium dodecyl sulphate (SDS) and urea had a strong effect on the size of the nanoparticles, while 2-Mercaptoethanol and Dithiothreitol (DTT) had no significant effect. Therefore hydrogen bonding and hydrophobic interactions were the predominant interactions responsible for the microstructure. Maximum yield of caffeine encapsulation of 13.54% was obtained at caffeine to β-Lg molar ratio of 50:1. Rapid nanoparticle degradation and increase in polydispersity during the incubation of β-Lg nanoparticles at simulating stomach conditions was observed due to enzymatic attack. Nevertheless, little release of entrapped caffeine was noted. Total release was achieved at intestinal conditions. Finally, the adsorption of caffeine to both native and denatured β-Lg followed a Langmuir adsorption isotherm model and caffeine had three times more affinity for partially denatured β-Lg in nanoparticles than for native protein

Protein hydrolysate from Pterygoplichthys disjunctivus, armoured catfish, with high antioxidant activity

Pterygoplichthys disjunctivus locally named as armored catfish ” is a by-catch of tilapia fishing which accounts for up to 80% of total captured fish in the Adolfo Lopez Mateos dam, in Michoacán, México affecting the economy of its surrounding communities. This invasive fish is discarded by the fishermen since native people do not consume it; partly due to its appearance yet is rich in protein. The aim of this study was to produce hydrolysates from armoured catfish using food grade proteases (neutrases HT and PF and alcalase PAL) and investigate process conditions (pH and temperature) that lead to high degree of hydrolysis, antioxidant activity, and Angiotensin I-Converting Enzyme (ACE) Inhibitory activity. No other similar research has been reported on this underutilized fish. The antioxidant activity was measured by three different methods, ABTS, FRAP, ORAC with relevance to food and biological systems in order to obtain a more comprehensive assessment of the activity. In addition, main peptide sequences were identified. All enzymes produced hydrolysates with high antioxidant activity. In particular, the protease HT led to the highest antioxidant activity according to ABTS (174.68 mol Trolox equivalent/g fish) and FRAP (7.59 mg ascorbic acid equivalent/ g fish) methods and almost the same as PAL according to the ORAC method (51.43 mol Trolox equivalent/g fish). Moreover, maximum activities were obtained at mild pH and temperature conditions (7.5; 50 °C). Interestingly, the ORAC values obtained here were higher than others previously reported for fish hydrolysates and similar to those reported for fruits such as, blueberries, apples and oranges. The peptide sequence IEE(E) was present in several peptides in both hydrolysates; this sequence may be partly responsible for the high antioxidant activity particularly for the one based on the iron reducing power. These findings will be relevant to the valorization of other fish/fish muscle discards and could contribute to the production of food supplements and nutraceuticals

Colloidal gas aphrons separation to obtain polyphenol rich fractions from artichoke agro-industrial discards

Artichoke agro-industrial discards have proved an excellent source of polyphenolic compounds with high antioxidant capacity. The aim of this study was to further separate these bioactive molecules from an ethanolic extract by using Colloidal Gas Aphrons (CGAs) generated from a cationic surfactant, Cetyl trimethylammonium bromide, CTAB (1 mM) and a nonionic one, Tween 20 (10 mM). Total proteins, total sugars, total phenolic content (TPC), antioxidant capacity (AOC) and polyphenolic profile were determined in the crude extracts and CGA fractions. Highest recovery was obtained with the nonionic Tween 20 at CGA to feed volumetric ratio 5 (RE% = 64) whereas the cationic CTAB proved more efficient (RE% = 58) at the highest ratio studied here (ratio 11). Antioxidant capacity followed the same trend as the recovery of polyphenols and highest enrichment in antioxidant activity for each of the surfactants (1.5 for Tween 20 and 1.4 for CTAB) was obtained at those conditions that led to maximum recovery. Highest selectivity was obtained with CTAB. Electrostatic interactions drove the separation with the cationic surfactant whereas with Tween 20 hydrophobic interactions were predominant. Polyphenolic profile of feed and CGAs fractions comprised mainly mono and dicaffeoylquinic acids and a small proportion of flavones. In most of the cases no enrichment of any particular polyphenol was found in CGAs samples. However, with Tween and at ratios 5 and 11 CGAs were enriched in dicaffeoylquinic acid. Future research should focus on assessing if the surfactant could also offer any advantage to the formulation of these polyphenols

Effect of β-lactoglobulin on perception of astringency in red wine as measured by sequential profiling

Astringency is a predominant sensory attribute that influences the overall quality of red wine. The application of whey proteins as functional and nutritional food additives is popular but their use is uncommon to enology. Here whey proteins as a suitable food component to improve the sensory quality of red wine were investigated. This work focused on the sensory perception of astringency in red wine treated with β-lactoglobulin and gelatin. Ovalbumin precipitation method was used to assess astringency pre- and post-treatment and compared to the perceived astringency. A sequential profiling sensory technique was used to evaluate astringency in relation to other attributes over repeated consumption of red wine. The intensity of astringency increased insignificantly over repeated sips at 60 s intervals for the treated and untreated red wine. The difference in astringency perception (p < 0.05) between the wine samples was shown at 30 s after swallowing. Wines treated with β-lactoglobulin and gelatin significantly reduced astringency and the total polyphenol content. The reduction in astringency indicates that these proteins actively bind and precipitate polyphenols which are known to contribute to perception of astringency. Furthermore, the good agreement between the chemical and sensory methods supports this mechanism for reduction of astringency

Highly proteolytic bacteria from semi-ripened Chiapas cheese elicit angiotensin-I converting enzyme inhibition and antioxidant activity

Chiapas cream cheese (CCH) manufacturing process involves a long acid-enzymatic coagulation period of full-fat cow raw milk to achieve an acid and crumbly cheese. These sensorial aspects are related to lactic acid bacteria activity during ripening. Our main objective was to test the hypothesis that CCH contained highly proteolytic strains able to release bioactive compounds upon milk-protein hydrolysis. First, the proteolysis of CCH was evaluated considering the peptide and amino acid profiles of cheese samples collected from Veracruz (AVCH) and Tabasco (HTCH). The angiotensin-converting-enzyme (ACE) inhibitory activity in cheese water-soluble fractions was evaluated. Thereafter, strains from both CCH samples were isolated and selected based on their proteolytic capability, genetic fingerprint differentiation and growth conditions. Finally, a range of activities in vitro were tested in milk fractions fermented with selected strains. CCH showed ACE inhibitory activity: IC50=1.75-2.75 mg/mL. Interestingly, AVCH contains 0.78 g/kg of the antihypertensive γ-aminobutyric acid. Three highly proteolytic strains showed ACE and high antioxidant activities upon milk fermentation. In conclusion, CCH contain proteolytic strains able to release bioactive compounds from milk proteins and potentially useful to produce functional ingredients and foods

Bioaccessible peptides released by in vitro gastrointestinal digestion of fermented goat milks

In this study, ultrafiltered goat milks fermented with the classical starter bacteria Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus salivarus subsp. thermophilus or with the classical starter plus the Lactobacillus plantarum C4 probiotic strain were analyzed using ultra-high performance liquid chromatography-quadrupole-time-of-flight tandem mass spectrometry (UPLC-Q-TOF-MS/MS) and/or liquid chromatography-ion trap (LC-IT-MS/MS). Partial overlapping of the identified sequences with regard to fermentation culture was observed. Evaluation of the cleavage specificity suggested a lower proteolytic activity of the probiotic strain. Some of the potentially identified peptides had been previously reported as angiotensin converting enzyme (ACE)-inhibitory, antioxidant and antibacterial and might account for the in vitro activity previously reported for these fermented milks. Simulated digestion of the products was conducted in presence of a dialysis membrane to retrieve the bioaccessible peptide fraction. Some sequences with reported physiological activity resisted digestion but were found in the non-dialyzable fraction. However, non-previously detected sequences such as the antioxidant αs1-casein 144YFYPQL149, the antihypertensive αs2-casein 90YQKFPQY96 and the antibacterial αs2-casein 165LKKISQ170 were found in the dialyzable fraction of both fermented milks. Moreover, in the fermented milk including the probiotic strain, the k-casein dipeptidyl peptidase IV inhibitor (DPP-IV) 51INNQFLPYPY60 as well as additional ACE-inhibitory or antioxidant sequences could be identified. With the aim to anticipate further biological outcomes, quantitative structure activity relationship (QSAR) analysis was applied to the bioaccessible fragments and led to propose potential ACE inhibitory sequences