# From Chemistry to Functionality: HPLC–DAD/LC–MS/MS Characterization of Bee Product-Enriched Prunus spinosa L. Kombucha with In Vitro Antidiabetic Activity and Bioaccessibility

## TL;DR

This study explores how adding bee products to blackthorn kombucha improves its health benefits, particularly for diabetes, through detailed chemical and sensory analysis.

## Contribution

The paper introduces a novel kombucha formulation enriched with bee products and blackthorn juice, demonstrating its antidiabetic potential and bioaccessibility.

## Key findings

- Bee bread-enriched kombucha had the highest total phenolic and flavonoid content, along with strong antioxidant activity.
- Propolis-enriched kombucha showed the highest antidiabetic activity, inhibiting key digestive enzymes.
- Phenolic compounds like chlorogenic acid and catechin hydrate were prominent in enriched samples.

## Abstract

This study aimed to sensorially optimize kombucha tea
enriched
with Prunus spinosa L. (blackthorn)
juice and bee products (propolis, pollen, and bee bread), and to elucidate
the product’s bioactive profile and functional potential (antidiabetic
activity and in vitro bioaccessibility) using advanced analytical
approaches. The formulations were optimized using response surface
methodology (RSM). Alongside the evaluation of total phenolic content
(TPC), total flavonoid content (TFC), DPPH• radical scavenging
activity, and total antioxidant capacity (TAC), detailed compositional
analyses were conducted. The phenolic composition was quantitatively
characterized by HPLC–DAD, organic acid profiles were determined
using HPLC, and free amino acids were quantified by triple quadrupole
LC–MS/MS. In sensory evaluation, the bee bread-enriched kombucha
(BB-BKT) received the highest general acceptability score (8.32),
with a taste score of 8.25. At the same time, the pollen-enriched
sample (PL-BKT) stood out for its smell (8.21). Regarding bioactive
content, BB-BKT exhibited the highest values, with TPC of 257.10 mg
GAE/100 mL, TFC of 36.16 mg CE/100 mL, and DPPH• activity of
57.05 μmol TE/mL. According to HPLC–DAD results, total
phenolics increased from 106.35 μg/mL in the control sample
to 159.23–186.15 μg/mL after enrichment. Chlorogenic
acid (79.04 μg/mL) was prominent in BB-BKT, catechin hydrate
(121.2 μg/mL) in PL-BKT, and quercetin (47.32 μg/mL) and
naringenin (17.59 μg/mL) in the propolis-enriched sample (PB-BKT).
LC–MS/MS analysis revealed that PL-BKT was the richest sample
in free amino acids, with significant increases in proline (47.37
mg/100 mL), leucine (4.10 mg/100 mL), and lysine (4.54 mg/100 mL).
In antidiabetic activity assays, the propolis-enriched sample (PB-BKT)
showed the highest activity, with 40.7% inhibition of α-glucosidase
and 42.02% inhibition of α-amylase. Although a gradual decrease
in bioactives was observed during in vitro digestion, the preservation
of phenolic and flavonoid recovery generally within the ∼21–30%
range suggests that optimized enrichment may support postdigestive
functional effects.