Seaweed (G. gracilis) Protein Hydrolyzates: A Valuable Source of Short- and Medium-Chain Peptides with Multifunctional Properties

Abstract

The sustainable valorization of infesting marine biomass offers opportunities to address environmental challenges and emerging nutritional needs. This study investigated the invasive red alga Gracilaria gracilis as a potential source of bioactive peptides with antihypertensive and antidiabetic properties. Protein hydrolyzates were generated via enzymatic digestion and fractionated by size exclusion chromatography. Peptidomics analysis using liquid chromatography coupled with high-resolution mass spectrometry identified 362 short-chain and 97 medium-chain peptides. Antioxidant effects were confirmed via diphenyl-2-picrylhydrazyl radical (DPPH), trolox equivalent antioxidant capacity (TEAC), and ferric reducing antioxidant power (FRAP) assays: at 20 mg/mL, short-chain peptides showed a TEAC of 60.8 ± 0.7% and a FRAP activity of 4638.7 ± 87.8%, significantly higher than the medium-chain fraction (36.1 ± 3.6% and 2180.6 ± 25.8%, respectively). Short-chain peptides also demonstrated stronger angiotensin-converting enzyme inhibition (19.53 ± 0.64% at 2.07 mg/mL) compared to medium-chain peptides (12.5 ± 0.42%). Conversely, medium-chain peptides exhibited superior dipeptidyl peptidase IV inhibition. Trans-epithelial transport experiments confirmed bioavailability, with 40 short peptides and 65 medium peptides detected in the basolateral compartment. These findings demonstrate the potential of converting invasive seaweeds into multifunctional ingredients for functional foods or nutraceuticals, supporting marine biotechnology and circular bioeconomy strategies for preventive healthcare and metabolic disease management.

Keywords: Caco-2 cell; algae; angiotensin-converting enzyme inhibitory properties; antioxidant properties; bioactive peptides; dipeptidyl-peptidase IV; intestinal trans-epithelial transport; liquid chromatography coupled to high-resolution mass spectrometry; peptidomics.

1

Effects of short- and medium-chain peptides on human intestinal Caco-2 cells (mean of six determinations performed in triplicate). C: control, ns: not significant.

2

In vitro ABTS radical scavenging activity (A) and ferric-reducing antioxidant power (FRAP) activity (B) of short- and medium-chain peptides­(mean of six determinations performed in triplicate).

3

In vitro DPP-IV activity assay (A) and in situ DPP-IV activity evaluations on Caco-2 cells after 6 h of short- and medium-chain peptides treatments (B). Sitagliptin represents the positive control. Data are the means ± SD of three experiments performed in triplicate. C: control sample, ns: not significant. (*) p < 0.05, (**) p < 0.01, (***) p < 0.001, (****) p < 0.0001.

4

Percentage of in vitro ACE inhibition of short- and medium-chain peptides at different concentrations (0.09, 0.17, 0.35, 0.69, 1.04, 1.38, 2.07 mg/mL, mean of three determinations performed in duplicate).

5

GRAVY index score (grand average of hydrophobicity) of putatively identified short-chain peptides. A score below 0 indicates hydrophilic properties, while a score above 0 indicates hydrophobic properties.

6

Distribution of the identified amino acid sequences among the three conditions (T0, AP, and BL) is reported in a Venn diagram.