# In Vitro Evaluation of ACE and DPP-IV Inhibitory, and GLP-1 Stimulation Activities of Collagen Hydrolysate Enriched in Tripeptides

## TL;DR

A collagen hydrolysate enriched in tripeptides shows better enzyme inhibition and GLP-1 stimulation than a standard collagen hydrolysate, suggesting potential health benefits.

## Contribution

A tripeptide-enriched collagen hydrolysate was shown to have enhanced ACE/DPP-IV inhibition and GLP-1 stimulation compared to a benchmark formulation.

## Key findings

- The tripeptide-enriched collagen hydrolysate (CH) showed significantly higher ACE and DPP-IV inhibitory activity than the benchmark collagen hydrolysate (BCH).
- CH reduced cellular DPP-IV activity in Caco-2 cells and stimulated GLP-1 secretion in STC-1 cells, unlike BCH.
- Peptidomic analysis showed CH had more short- and medium-length peptides and a 1.7-fold higher degree of hydrolysis than BCH.

## Abstract

Background/Objectives: Collagen hydrolysates are widely used as nutritional ingredients for skin and joint health; however, growing evidence indicates that collagen may also exert beneficial effects on cardiometabolic pathways. Short peptides have been shown to modulate angiotensin-converting enzyme (ACE) and dipeptidyl peptidase IV (DPP-IV), key regulators of blood pressure and glucose homeostasis. This study aimed to assess the dual ACE- and DPP-IV inhibitory and GLP-1 stimulation activities, respectively of a tripeptide-enriched formulation (CH). The study was performed using a benchmark collagen hydrolysate (BCH) as reference. Methods: ACE and DPP-IV inhibitory activities were evaluated using in vitro enzymatic assays. Cellular compatibility and in situ DPP-IV inhibition were assessed in Caco-2 intestinal cells, while glucagon-like peptide-1 (GLP-1) secretion was measured in STC-1 enteroendocrine cells. The degree of hydrolysis was determined by OPA assay, and nanoLC–HRMS was used to characterize and compare the proteomic profiles of the samples. Results: Both hydrolysates exhibited dose-dependent ACE and DPP-IV inhibition; however, CH showed significantly higher inhibitory activity at comparable concentrations. CH also reduced cellular DPP-IV activity in Caco-2 cells and stimulated GLP-1 secretion in STC-1 cells, whereas BCH showed limited or non-significant cellular effects. Peptidomic analysis revealed an enrichment of short- and medium-length peptides in CH, while BCH contained a higher proportion of long peptides (>2000 Da). Consistently, CH exhibited a 1.7-fold higher degree of hydrolysis than BCH. Conclusions: The tripeptide-enriched collagen hydrolysate demonstrated superior enzymatic and cellular bioactivity compared with the benchmark formulation, supporting its potential as a multifunctional bioactive ingredient for health applications.