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

This study aimed to sensorially optimize kombucha tea enriched with <i>Prunus spinosa</i> 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.