Biological transformation of chlorophyll-rich spinach (Spinacia oleracea L.) extracts under in vitro gastrointestinal digestion and colonic fermentation

Chlorophyll can be obtained from a variety of green vegetables. In this study, chlorophyll-rich spinach (Spinacia oleracea L.) extracts were subjected to early-life and adult-life gastrointestinal digestion and colonic fermentation in a murine model in vitro to investigate the biological transformation of the chlorophyll. Chlorophylls a and b were the main compounds present in the extracts. Furthermore, some other compounds were also confirmed, such as 15¹-hydroxy-lactone chlorophyll a, 13²-hydroxy chlorophyll a, and 15¹-hydroxy-lactone chlorophyll b. Chlorophylls favored pheophytinization and oxidative reactions under in vitro early-life and adult-life gastrointestinal digestion, leading to the formation of pheophytin a, pheophytin b, 13²-hydroxy pheophytin a, and 15¹-hydroxy-lactone pheophytin a. 16S rRNA gene sequencing conveyed that pheophytins modulated the gut microbiota composition during in vitro colonic fermentation. Notably, Blautia in the gut microbiota of 3-week-old mice (early life) and unclassified Lachnospiraceae in 8-week-old mice (adult life) were advantageous for transforming the pheophytins to pheophorbide a, pheophorbide b, 15¹-hydroxy-lactone pheophorbide a, and 13²-hydroxy pheophorbide a, thereby demonstrating the loss of the phytol chain in the pheophytins. Meanwhile, total short-chain fatty acids, as well as acetic, propionic, and butyric acids, were increased by the process of microbial fermentation in the presence of pheophytins. Our study provides fundamental insight into the contribution of diverse gut microbiota functions toward the biological transformation of pheophytins.