Immobilization of thermolysin enzyme on dendronized silica supports. Evaluation of its feasibility on multiple protein hydrolysis cycles.

This work evaluates different dendrimer-silica supports for the immobilization of enzymes by multipoint covalent binding. Thermolysin was immobilized on two dendrimers (PAMAM and carbosilane) with two different generations (zero (G0) and first (G1)). Results were compared with a control, a silica support functionalized with a monofunctional molecule. Dendrimers increased the number of available sites to bind the enzyme. Despite the enzyme was immobilized on all supports, G0 dendrimers immobilized a 30% more enzyme than G1. Thermolysin immobilized on G0 dendrimer supports showed the highest activity and could be employed in three consecutive hydrolysis cycles. Optimal immobilization time was 1 h while optimal protein loading was 25 mg enzyme/100 mg support. Enzyme activity was promoted when using 5 mg of immobilized enzyme at 750 rpm, 60 °C, and 2 h of hydrolysis. Under these conditions, the activity of thermolysin increased up to the 78% of the free enzyme activity. Kinetics of the hydrolysis reaction using the immobilized thermolysin was also studied and compared with the obtained using the free thermolysin. The addition of ZnCl2 and NaCl during the immobilization procedure increased thermolysin activity in the second (22% more) and in the third (14% more) hydrolysis clycles.

Evaluation of the relationship between the peptide profiles and the lipid-lowering properties of olive seed hydrolysates as a tool for tuning hypocholesterolemic functionality.

Olive processing generates large amounts of stones with high protein contents. Previous studies have demonstrated that Manzanilla variety olive seed proteins release peptides with lipid-lowering capacity. However, no work has demonstrated their roles in the overall hypolipidemic activity. Moreover, further studies using different olive varieties are required to propose a solid method for the exploitation of olive seeds. Twenty different olive varieties were employed in this work. Proteins were extracted using high-intensity focused ultrasound and digested with Alcalase. The released peptides were identified using proteomic techniques, and their capabilities to reduce the absorption of dietary cholesterol (by inhibiting cholesterol esterase enzyme, binding bile acids, and reducing micellar cholesterol solubility) or the biosynthesis of endogenous cholesterol were evaluated. Peptides with different lipid lowering capacities were obtained from all varieties although the genotype significantly affected the hypolipidemic characteristics. Univariate and multivariate statistical analyses showed strong correlations, positive and negative, between the presence of certain peptides in the hydrolysates and their capacity to reduce exogenous cholesterol absorption and endogenous cholesterol synthesis. Therefore, the selection of the olive seed genotype can direct its lipid-lowering properties, e.g., by promoting the reduction of dietary cholesterol absorption or the inhibition of cholesterol biosynthesis.

Identification of Peptides Potentially Responsible for In Vivo Hypolipidemic Activity of a Hydrolysate from Olive Seeds.

Previous studies demonstrated that peptides produced by the hydrolysis of olive seed proteins using Alcalase enzyme showed in vitro multifunctional lipid-lowering capability. This work presents a deeper insight into the hypolipidemic effect of olive seed peptides. The capability of olive seed peptides to inhibit endogenous cholesterol biosynthesis through the inhibition of HMG-CoA reductase enzyme was evaluated observing a 38 ± 7% of inhibition. Two in vivo assays using different peptides concentrations (200 and 400 mg/kg/day) were designed to evaluate the hypolipidemic effect of olive seed peptides in male and female mice. A low concentration of hydrolysate reduced total cholesterol in male mice in a 20% after 11 weeks compared to the mice feeding with hypercholesterolemic diet. A higher hydrolysate concentration showed a greater reduction in total cholesterol (25%). The analysis of the olive seed hydrolysate by reverse phase high-performance liquid chromatography mass spectrometry (RP-HPLC-MS) enabled the identification of peptides that could be responsible for this hypolipidemic effect.

Isolation and identification by high resolution liquid chromatography tandem mass spectrometry of novel peptides with multifunctional lipid-lowering capacity.

This work describes the isolation, characterization, and identification by RP-HPLC-ESI-Q-TOF of novel peptides that interfere in the fat digestion and absorption mechanisms by multiple pathways. Peptides were ultrafiltrated and peptides in the most active fraction were further separated by semipreparative RP-HPLC. Nine different subfractions were obtained observing a high amount of peptides in subfraction F3. Peptides in subfraction F3 could simultaneously reduce the solubility of cholesterol in micelles and inhibit pancreatic cholesterol esterase and pancreatic lipase, even after a simulated gastrointestinal digestion. The identification of lipid-lowering peptides has been scarcely performed and when done, low selectivity or sensitivity of employed identification techniques or conditions did not yield reliable results. Separation and detection of peptides by RP-HPLC-ESI-Q-TOF-MS was optimized and most favorable conditions were employed for the identification of peptides using de novo sequencing. Ten different peptides with 4-9 amino acids were identified. Main feature of identified peptides was the high acidity derived from a high presence of amino acids glutamic acid and aspartic acid in their sequences.

Identification by hydrophilic interaction and reversed-phase liquid chromatography-tandem mass spectrometry of peptides with antioxidant capacity in food residues.

HILIC- and RP-HPLC-ESI-Q-TOF identification of bioactive peptides with antioxidant capacity in peach by-products was carried out. Peach seeds contain more than 40% of proteins (as dried and defatted basis) and could constitute a cheap source of bioactive peptides. Extracted proteins were digested using four different commercial enzymes. Five assays based on different antioxidant mechanisms were employed for a reliable evaluation of the antioxidant capacity of the extracts. Thermolysin enzyme originated the extract with the most favorable antioxidant capacity. Probably due to a synergic effect among antioxidant peptides, it was not possible to find a peptide fraction with a higher antioxidant capacity than the whole extract. Eighteen peptides were identified in the whole hydrolysate when combining HILIC- and RP-HPLC-ESI-Q-TOF. A high percentage of hydrophobic amino acids were observed within their sequences which is a characteristic feature of the antioxidant nature of peptides.

Development of a capillary high performance liquid chromatography-ion trap-mass spectrometry method for the determination of VLIVP antihypertensive peptide in soybean crops.

Soybean peptide VLIVP presents a very high antihypertensive activity (IC50 value 1.69µM), even higher than extensively studied IPP and VPP peptides from milk. Nevertheless, no much attention has been paid to this peptide and there is no method enabling its determination in soybeans. The aim of this work was the development of an analytical methodology for this purpose. A methodology consisting of the extraction of soybean proteins, their digestion with Protease P enzyme, their chromatographic separation using capillary-HPLC, and IT-MS detection was optimized. Protein extraction was performed by the use of high intensity focused ultrasounds to obtain a reduced extraction time. Optimization of chromatographic and mass spectrometry parameters enabled the separation of VLIVP peptide within just 7min and its sensitive detection. The analytical characteristics of the capillary-HPLC-IT-MS method were evaluated through the study of linearity, LOD, LOQ, study of the presence of matrix interferences, precision, and recovery. The method enabled to detect as low as 3.6ng of peptide and to determine as low as 12ng of peptide in 1g of soybean (as dry basis). Finally, the developed method was applied to the determination of the antihypertensive peptide VLIVP in different soybean varieties. The results showed the highest yield of VLIVP peptide in variety Mazowiecka II from Poland.

Isolation and identification of antioxidant peptides from commercial soybean-based infant formulas.

Soybean-based infant formulas (SBIFs) based on soybean protein isolate (90% of proteins) are an interesting alternative to cow's milk infant formulas. Different works have demonstrated the presence of bioactive peptides in different soybean-based foodstuffs. The aim of this work was the evaluation, for the first time, of antioxidant peptides in five different commercially available SBIFs. Ultrafiltration through 10 kDa molecular weight cut-off filters was the most suitable extraction method. Despite peptide concentrations ranging between 1.19 and 2.27 mg/mL, similar antioxidant capacities were detected in all SBIF extracts. Extracts were further fractionated according to their molecular weight by ultrafiltration, and fractions from 5 to 10 kDa, 3 to 5 kDa, and below 3 kDa were obtained. The most active fraction was further fractionated by off-gel isoelectrofocusing and reversed-phase chromatography. Antioxidant fractions were also submitted to simulated gastrointestinal digestion (GI) with pepsin and pancreatin to evaluate their antioxidant capacity after digestion. Peptides were identified by HPLC-ESI-Q-ToF-MS/MS. At least 120 peptides were identified in every antioxidant fraction, with 42 peptides common to all SBIFs.