Bioaccessibility and Cellular Uptake of Lutein, Zeaxanthin and Ferulic Acid from Muffins and Breads Made from Hairless Canary Seed, Wheat and Corn Blends

**Background:** Hairless canary seed (HCS) is a novel whole grain rich in carotenoids (lutein, zeaxanthin) and phenolic acids (ferulic acid), which are associated with eye health, cognitive function, and reduced risk of chronic diseases. However, the bioaccessibility and intestinal absorption of these compounds from HCS-based baked products had not been characterized. This study aimed to evaluate the in vitro bioaccessibility and cellular uptake of lutein, zeaxanthin, and ferulic acid from muffins and breads made with HCS, wheat, and corn blends.

**Methods:** Three low-fat muffins were prepared: 100% HCS, HCS/corn 1:1, and HCS/corn 1:2 (w/w). Four breads were made: 100% wheat (control), wheat/HCS 85/15, 75/25, and 50/50 (w/w). Samples underwent simulated oral (α-amylase, 30 s), gastric (pepsin, pH 2, 2 h), and intestinal (pancreatin/bile, pH 7.5, 3 h) digestion. The aqueous phase (micellar fraction) was collected. Because untreated digesta damaged Caco-2 cells, a cleanup procedure was developed: addition of AEBSF protease inhibitor followed by C18 SampliQ solid-phase extraction (SPE) column. Cleaned digesta were filtered (0.45 µm then 0.2 µm) and incubated with differentiated Caco-2 cells (11–14 days post-confluency) for 4 h. Lutein, zeaxanthin, and ferulic acid were quantified by HPLC/UPLC in muffins, breads, digesta (before and after cleanup), and cell harvests. Bioaccessibility was defined as the percentage of the original compound in the aqueous digesta; cellular uptake as the percentage retained by cells. One-way ANOVA with Tukey’s test (p < 0.05) was used.

**Key Results:** - **Muffins:** Lutein bioaccessibility was 92–94% before cleanup and 81–86% after cleanup (no significant differences among muffins). Zeaxanthin bioaccessibility ranged from 63–79% (untreated) to 38–48% (cleaned). Ferulic acid bioaccessibility was 105–229% (untreated) and 53–133% (cleaned), with values >100% indicating release of bound ferulic acid during digestion. Cellular uptake was low: lutein 7–9%, zeaxanthin 4–11%, ferulic acid 0.5–1.8%. - **Breads:** Lutein bioaccessibility was highest in wheat/HCS 50/50 bread (79.7% untreated, 59.7% cleaned) and lowest in control (41.9% untreated, 33.9% cleaned). Zeaxanthin bioaccessibility was 27.4–61.1% (untreated) and 17.7–32.2% (cleaned). Ferulic acid bioaccessibility was 98–103% (untreated) and 81–102% (cleaned). Cellular uptake of lutein was 9.2% (50/50) and 4.3% (75/25); no lutein was detected in cells from control or 85/15 breads. Zeaxanthin was not detected in any bread cell harvest. Ferulic acid uptake was 0.2%. - **Cleanup:** The C18+AEBSF treatment maintained Caco-2 monolayer integrity over 4 h, but reduced lutein, zeaxanthin, and ferulic acid concentrations in digesta by approximately 10–30%.

**Clinical Implications:** HCS-based muffins and breads provide a means to increase dietary intake of lutein, zeaxanthin, and ferulic acid. Although bioaccessibility is high, cellular uptake is low, suggesting that only a small fraction of these compounds is absorbed in the small intestine. Enrichment of products with higher amounts of these bioactives may be necessary to achieve physiologically relevant absorption. The study also highlights the need for improved in vitro models (e.g., co-culture with HT29-MTX mucus-producing cells) to better mimic in vivo conditions and avoid artifacts from digesta cleanup. Overall, HCS is a promising functional food ingredient for boosting carotenoid and polyphenol intake, potentially contributing to eye and cardiovascular health.