Ultrasound-Assisted NADES-Based Extraction of Spirulina Bioactives: Bioaccessibility and Intestinal Permeability Assessment Using an In Vitro Digestion/Caco-2 Cell Model and Food Application

In this study, ultrasound-assisted extraction was applied using eight NADES formulations to recover Spirulina antioxidants, and the bioaccessibility and apparent basolateral recovery of phenolics in the most potent extracts were evaluated, both in the original extracts and after their incorporation into a kefir matrix, through an in vitro digestion/Caco-2 cell model. The extracts obtained with glucose:glycerol exhibited the highest pigment and total phenolic content (TPC), while betaine:glycerol and choline chloride:citric acid extracts exhibited the highest antioxidant capacity. Although Spirulina-fortified kefir samples had significantly lower (> 90% reduction) TPCs than individual Spirulina samples, they exerted increased apparent in vitro bioaccessibility of phenolics during digestion, with the highest value reaching 355% for kefir incorporated with the glucose:glycerol extract. This elevated response is likely associated with matrix-related release, dissociation, and transformation of Folin-reactive compounds during digestion rather than reflecting actual intestinal absorption of phenolic compounds. Relatively higher apparent basolateral recovery of antioxidants in the Caco-2 cell model was also achieved in some Spirulina-fortified kefir samples. These findings highlight the potential use of NADES with ultrasonication for the enhanced recovery of Spirulina antioxidants and also revealed that kefir can serve as an effective carrier matrix for improved gastrointestinal utilization of Spirulina-derived bioactives. Practical Applications: This study shows that kefir can be considered a promising carrier matrix for Spirulina antioxidants. Since Spirulina is widely used as a functional food ingredient, its incorporation into kefir may offer a commercially attractive strategy for producing probiotic beverages with added antioxidant and natural pigment value. The present findings may assist the food industry in designing more sustainable and value-added products using microalgae-based ingredients and alternative extraction systems.