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

小型企业创新研究(SBIR)第一阶段项目的更广泛的商业影响是评估一种负担得起的酶法生产通常存在于母乳中的适销对路的天然化合物。一旦这一战略得到验证，它就可以在国内和工业用户中实施。这项技术能够将牛奶中的乳糖转化为非常理想的化合物。这些稀有而有价值的化合物可以帮助促进有益肠道细菌的生长，是人类母乳的组成部分。这些化合物广泛用于强化婴儿食品，它们在肠道健康、支持平衡的肠道微生物区系、有益于免疫力和改善认知大脑健康方面发挥作用。此外，新的研究表明，包含稳定的生物活性天然化合物的负担得起的食品强化技术将有利于婴儿期和整个生命阶段的健康肠道。因此，这项技术为食品和膳食补充剂制造商打开了新的商机，旨在开发独特的增强肠道的营养解决方案。拟议的项目将通过酶作用产生在母乳中发现的化合物，包括N-乙酰氨基葡萄糖(LacNAc)。研究计划包括测试β-己糖基转移酶生产的可扩展性，该酶将由Kphaffi异源生产。为了验证100个L工作容量生物处理反应器的工业可扩展性、产品生成量和产率，生成的BHT将被用于催化半乳糖与N-乙酰氨基葡萄糖(GlcNAc)的重复加成。除了低聚半乳糖外，这些产品还将包括由顺序的转半乳糖化反应产生的LacNAc二糖。回收的产品将在临床前安全性/毒性研究中进行测试。在这项研究期间收集的数据将允许进行更精确的成本效益分析，其中将包括产品产量、碳平衡、微生物组效益和代谢数据。由于BHT的特异性、一步反应合成、低成本底物、可持续性和总体低环境影响，生产LacNAc的更高效的酶生物合成方法有望节省成本。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

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.