SBIR Phase I: Development of an enzymatic method to produce compounds found in human milk at commercial scale

The broader/commercial impact of this Small Business Innovation Research (SBIR) Phase I project is to assess an affordable enzymatic method for producing marketable natural compounds usually found in human milk. Once this strategy is validated, it could be implemented by domestic and industrial users. The technology is capable of transforming the lactose in milk to highly desirable compounds. These rare and valuable compounds can help enhance growth of beneficial gut bacteria and are building blocks of human breast milk. These compounds are widely desirable to fortify infant food, where they play a role in intestinal health, supporting balanced gut microbiota, benefiting immunity, and improving cognitive brain health. Furthermore, new research suggests that affordable technologies for food fortification containing stable bioactive natural compounds will benefit the healthy gut beyond infancy and across life stages. As such, this technology opens new business opportunities for food and dietary supplement manufacturers aiming to develop unique gut-strengthening nutrition solutions. The proposed project will enzymatically generate compounds found in human milk, including N-acetylglucosamine (LacNAc). The research plan consists of testing the scalability of enzyme β-hexosyltransferase (BHT) production, which will be heterologously produced by K. phaffi. To validate industrial scalability, product generation, and yields from 100 L working volume bioprocessing reactors, the BHT generated will be utilized to catalyze the repeated addition of galactose to N-acetylglucosamine (GlcNAc). The products, in addition to galactooligosaccharides, will include LacNAc disaccharides, generated by sequential transgalactosylation reactions. The recovered products will be tested in preclinical safety/toxicity studies. Data collected during this study will allow for a more precise cost-benefit analysis, which will include product yields, carbon balances, microbiome benefits, and metabolic data. Cost savings are expected from a more efficient enzymatic biosynthesis method for producing LacNAc due to BHT specificity, synthesis in one-step reactions, low-cost substrates, sustainability, and overall low environmental impact. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.