In vitro infant fecal fermentation characteristics of bovine milk osteopontin and 2′-fucosyllactose combination

Abstract

Breast milk is rich in bioactive proteins and oligosaccharides, including osteopontin (OPN) and 2′-fucosyllactose (2’-FL), which are believed to promote the growth of beneficial microbiota and regulate intestinal barrier function. In this study, fermentation substrates including DOPN (digested OPN fragment), 2’-FL and their combinations in varying proportions, were prepared through in vitro gastrointestinal digestion, dialysis and freeze-drying. Changes in gas production, organic acid levels, ammonia N concentration and bacterial population abundance were studied using an in vitro batch fermentation model, with feces inocula from healthy infants. Results showed that DOPN fermentation by colonic microorganisms produced approximately 1.7 mL of total gas, with acetate (28.6 mM) as the predominant metabolite, along with lower levels of propionate (3.9 mM) and branched chain fatty acids (2.7 mM), accompanied by a substantial accumulation of ammonia N (504.6 mg/L). When the DOPN and D2’-FL ratio ranged from 1:36 to 1:3, the mixture exhibited a pronounced bifidogenic effect, with the relative abundance of B. longum reaching 42–56 %. This was accompanied by sustained high production of acetate (55.0–59.9 mM), lactate (8.5–11.1 mM) and succinate (3.2–3.8 mM), while protein fermentation by-products, such as ammonia N, remained at low levels (8.3–104.6 mg/L). Taken together, the combination of OPN and 2’-FL established a synergistic microenvironment that enhanced both microbial diversity and metabolite functionality, providing a biochemical basis for their potential application in infant formula products.