Enzymatic modification of isolated bovine milk oligosaccharides to mimic human milk oligosaccharides with increased antimicrobial activity

 描述（由申请方提供）：母乳摄入与肠道感染的发生率和严重程度降低相关，导致婴儿发病率和死亡率降低。已知人乳低聚糖（HMO）通过各种机制（包括诱饵机制和增加有益微生物的生长）促成这一结果。特定细菌的生长增加改善了肠上皮的屏障功能，并提供了空间竞争，减少了病原体的定植。虽然已经成功地合成了几种HMO，但大多数已知的HMO都没有大规模的来源。大量的HMO样寡糖（OS）对于其抗菌功能的临床前测试至关重要。牛乳含有HMO样OS（BMO），但它们通常岩藻糖基化程度较低。HMO的岩藻糖基化和唾液酸化对于通过诱饵效应的直接抗微生物作用和通过促进细菌生长的间接抗微生物作用都是重要的。低水平的岩藻糖基化和唾液酸化导致OS对生长促进作用的特异性较差。在奶酪生产过程中，乳清渗透物作为废物副产品产生，并且它包含所有的BMO。我们可以从工业奶酪制造商那里获得大量乳清渗透物并分离BMO。然而，为了增加它们的直接和间接抗微生物效力，这些OS必须被修饰以增加它们的唾液酸化和岩藻糖基化。通过酶法修饰，我们将创建一类新的BMO为基础的OS具有增加的抗菌活性。首先，我们将从牛乳乳清废物流中分离出克至千克数量的OS。接下来，我们将用岩藻糖基化、唾液酸化或两者的组合来修饰它们的结构。最后，我们将检查原始和修改后的OS对几种病原体的诱饵作用和几种益生元特异性的抗生素的抗菌作用。第一阶段将证明这种方法的可行性，以产生公斤数量的HMO样抗菌OS的临床前测试。

 DESCRIPTION (provided by applicant): Consumption of human milk is associated with reduced incidence and severity of enteric infections, leading to reduced morbidity and mortality in infants. Human milk oligosaccharides (HMO) are known to contribute to this outcome through various mechanisms, including a decoy mechanism and by increasing the growth of beneficial microbes. Increased growth of specific commensals improves the barrier function of the intestinal epithelium and provides spatial competition, reducing pathogen colonization. While several HMO have been successfully synthesized, no large-scale source for the majority of known HMO exists. Large quantities of HMO-like oligosaccharide (OS) are vital for preclinical testing of their antimicrobial functions. Bovine milk contains HMO-like OS (BMO), but they are typically less fucosylated. The fucosylation and sialylation of HMO is important for both direct antimicrobial action via the decoy effect and indirect antimicrobial action via commensal growth enhancement. Low levels of fucosylation and sialylation result in OS with poor specificity of action for growth promotion. During cheese production, whey permeate is produced as a waste by-product, and it contains all of the BMO. We can acquire large quantities whey permeate from industrial cheese manufacturers and isolate the BMO. However, to increase their direct and indirect antimicrobial potency, these OS must be modified to increase their sialylation and fucosylation. By enzymatic modification, we will create a novel class of BMO-based OS with increased antimicrobial activities. First, we will isolate gram to kilogram quantities of OS from bovine milk whey waste streams. Next, we will modify their structures with fucosylation, sialylation, or a combination of both. Finally, we will examine the original and modified OS for antimicrobial actions against several pathogens for decoy action and several commensals for prebiotic specificity. Phase I will demonstrate the feasibility of this approach to generate kilogram quantities of HMO-like antimicrobial OS for preclinical testing.