# Comprehensive LC-ESI-HRMS/MS Profiling and Assessment of Texture, Predicted Glycaemic Index, Antioxidant Activity and Digestive Enzyme Inhibition of Gluten- and Lactose-Free Cookies Enriched with Pomegranate By-Products

## TL;DR

This study shows that adding pomegranate seed flour to gluten- and lactose-free cookies improves their antioxidant activity and reduces their predicted glycaemic index.

## Contribution

The novel use of pomegranate seed flour as a functional ingredient in gluten- and lactose-free cookies is demonstrated.

## Key findings

- Pomegranate seed flour addition increased antioxidant activity and reduced predicted glycaemic index in cookies.
- PSF-enriched cookies inhibited digestive enzymes like pancreatic lipase and α-amylase in a concentration-dependent manner.
- LC-ESI-HRMS/MS identified 36 bioactive compounds in pomegranate seed flour, including flavonoids and phenolic acids.

## Abstract

This study investigated gluten- and lactose-free cookies enriched with pomegranate seed flour (PSF, 5 and 10% w/w), a sustainable by-product of juice processing. LC-ESI/HRMS/MS analysis of PSF identified 36 bioactive compounds, mainly flavonoids, phenolic acids, hydrolysable tannins, and polar lipids. PSF incorporation significantly affected colour and texture, increasing friability, as evidenced by a reduction in breaking force from 35.37 N in the control cookie to 21.72 N in cookies enriched with 10% PSF, while maintaining good sensory acceptability. Total phenol (≈1.60–1.82 mg GAE/g) and flavonoid contents were only slightly affected by PSF addition; however, antioxidant activity markedly increased, with FRAP values rising from 55.8 to 67.82 μM Fe (II)/g and DPPH IC50 values decreasing from 31.38 to 12.72 μg/mL in the 10% PSF-enriched cookies. The enriched cookies inhibited pancreatic lipase, α-amylase, and α-glucosidase in a clear concentration-dependent manner and showed a reduced predicted glycaemic index (pGI 46.80 vs. 50.08 in the control). Multivariate analysis confirmed a clear dose-dependent effect of PSF on functional, textural, and sensory properties. Overall, pomegranate seed flour proved to be an effective upcycled ingredient for enhancing the functional profile of gluten- and lactose-free bakery products. Further studies using digestion models and in vivo or clinical approaches are needed to clarify the nutritional relevance and health effects of PSF-enriched foods.