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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.

This work proposes a method to separate proteins and polyphenols in a food byproduct with high bioactive properties and demonstrate by high performance liquid chromatography tandem mass spectrometry its efficiency. Bioactive substances were extracted using high intensity focused ultrasounds. Resulting extract (SR) was submitted to a step for the purification of proteins and to obtain a protein isolate (PI). Both extracts (SR and PI) were digested using two different enzymes (alcalase and thermolysin). Antioxidant, hypocholesterolemic, and antihypertensive properties of hydrolysates were explored. High antioxidant capacity, mainly due to the presence of polyphenols, was observed in SR and its hydrolysates. Hydrolysates obtained from PI using the alcalase enzyme showed simultaneously a high capacity to inhibit cholesterol esterase and to reduce micellar cholesterol solubility. Hydrolysate obtained from PI using the thermolysin enzyme showed a high antihypertensive capacity. Peptides and polyphenols in hydrolysates were identified by RP-HPLC-ESI-Q-TOF. Hydrolysates obtained from PI showed a high amount of peptides and a negligible amount of polyphenols while polyphenols were mainly present in hydrolysates from SR.

Pomegranate peel is a source of proteins, bioactive peptides, and phenolic compounds. The simultaneous extraction of these compounds required the use of polluting solvents and reagents that are non-suitable. This work targets the development of green methodologies based on pressurized liquids (PLE) or deep eutectic solvents (DES) for the extraction of these compounds. Extracts were digested with different proteolytic enzymes and different functionalities (antioxidant, hypocholesterolemia, and antihypertensive capacities) were evaluated. Highly antioxidant and hypocholesterolemic extracts and hydrolysates were obtained using PLE while high antihypertensive capacity was observed in the hydrolysates from proteins extracted using DES. Peptides and polyphenols were identified by HPLC-ESI-Q-TOF/MS. Higher amounts of peptides were shown in hydrolysates from DES extracts while hydrolysates from PLE extracts presented higher amounts of phenolic compounds. Some peptides were assigned to proteins from Punica granatum. Both green methods improved the extraction of bioactive compounds from pomegranate peel compared to the non-sustainable method. Industrial relevance The development of green methodologies which employ sustainable solvents such as pressurized liquids (PLE) and deep eutectic solvents (DES) allows extracting proteins and bioactive compounds from pomegranate peel. In addition, these solvents improve the extraction of health beneficial compounds compared to the non-sustainable and polluting solvents. Therefore, they could be used for the development of nutraceuticals and functional foods or even in medicinal, cosmetic, and pharmaceutical preparations.

The long exposition to reactive species results in oxidative stress which has been related with the development of cancer and other serious diseases. Olea europaea and Prunus persica seeds present a high protein content and preliminary results demonstrated their high potency to obtain bioactive peptides. The protective effect against oxidative damage exerted by peptides released from Olea europaea and Prunus persica seeds has been evaluated in this work. Seed hydrolysates showed protection against oxidation through four different mechanisms: inhibition of the formation of hydroxyl radicals, scavenging of free radicals, reduction of oxidizing compounds, and inhibition of lipid peroxidation. Moreover, seed hydrolysates also reduced the oxidative stress induced by an oxidizing agent on human cancer cells. Despite protection evaluated by individual mechanisms seemed to be significantly affected by the seed genotype, overall protection of seed hydrolysates was not so different. Seeds hydrolysates were not cytotoxic on normal cells but they demonstrated antiproliferative effect on human cancer cells (HeLa, PC-3, and HT-29). Peptides in all seed hydrolysates were sequenced by RP-HPLC-ESI-Q-TOF. Eighteen common peptides were observed among olive seed hydrolysates while a wider variability was observed among Prunus seed hydrolysates.