Karav Sercan, Le Parc Annabelle, Leite Nobrega de Moura Bell Juliana Maria, Frese Steven A, Kirmiz Nina, Block David E, Barile Daniela, Mills David A
Department of Food Science and Technology, University of California, Davis, California, USA.
Foods for Health Institute, University of California, Davis, California, USA.
Appl Environ Microbiol. 2016 May 31;82(12):3622-3630. doi: 10.1128/AEM.00547-16. Print 2016 Jun 15.
Milk, in addition to nourishing the neonate, provides a range of complex glycans whose construction ensures a specific enrichment of key members of the gut microbiota in the nursing infant, a consortium known as the milk-oriented microbiome. Milk glycoproteins are thought to function similarly, as specific growth substrates for bifidobacteria common to the breast-fed infant gut. Recently, a cell wall-associated endo-β-N-acetylglucosaminidase (EndoBI-1) found in various infant-borne bifidobacteria was shown to remove a range of intact N-linked glycans. We hypothesized that these released oligosaccharide structures can serve as a sole source for the selective growth of bifidobacteria. We demonstrated that EndoBI-1 released N-glycans from concentrated bovine colostrum at the pilot scale. EndoBI-1-released N-glycans supported the rapid growth of Bifidobacterium longum subsp. infantis (B. infantis), a species that grows well on human milk oligosaccharides, but did not support growth of Bifidobacterium animalis subsp. lactis (B. lactis), a species which does not. Conversely, B. infantis ATCC 15697 did not grow on the deglycosylated milk protein fraction, clearly demonstrating that the glycan portion of milk glycoproteins provided the key substrate for growth. Mass spectrometry-based profiling revealed that B. infantis consumed 73% of neutral and 92% of sialylated N-glycans, while B. lactis degraded only 11% of neutral and virtually no (<1%) sialylated N-glycans. These results provide mechanistic support that N-linked glycoproteins from milk serve as selective substrates for the enrichment of infant-associated bifidobacteria capable of carrying out the initial deglycosylation. Moreover, released N-glycans were better growth substrates than the intact milk glycoproteins, suggesting that EndoBI-1 cleavage is a key initial step in consumption of glycoproteins. Finally, the variety of N-glycans released from bovine milk glycoproteins suggests that they may serve as novel prebiotic substrates with selective properties similar to those of human milk oligosaccharides.
It has been previously shown that glycoproteins serve as growth substrates for bifidobacteria. However, which part of a glycoprotein (glycans or polypeptides) is responsible for this function was not known. In this study, we used a novel enzyme to cleave conjugated N-glycans from milk glycoproteins and tested their consumption by various bifidobacteria. The results showed that the glycans selectively stimulated the growth of B. infantis, which is a key infant gut microbe. The selectivity of consumption of individual N-glycans was determined using advanced mass spectrometry (nano-liquid chromatography chip-quadrupole time of flight mass spectrometry [nano-LC-Chip-Q-TOF MS]) to reveal that B. infantis can consume the range of glycan structures released from whey protein concentrate.
牛奶除了滋养新生儿外，还提供一系列复杂的聚糖，其结构确保了哺乳期婴儿肠道微生物群关键成员的特定富集，这一群落被称为以牛奶为导向的微生物群。牛奶糖蛋白被认为具有类似的功能，作为母乳喂养婴儿肠道中常见双歧杆菌的特定生长底物。最近，在各种婴儿携带的双歧杆菌中发现的一种细胞壁相关的内切β-N-乙酰氨基葡萄糖苷酶（EndoBI-1）被证明可以去除一系列完整的N-连接聚糖。我们假设这些释放的寡糖结构可以作为双歧杆菌选择性生长的唯一来源。我们证明了EndoBI-1在中试规模下从浓缩牛初乳中释放N-聚糖。EndoBI-1释放的N-聚糖支持婴儿双歧杆菌亚种（婴儿双歧杆菌）的快速生长，该菌种在人乳寡糖上生长良好，但不支持动物双歧杆菌亚种（乳酸双歧杆菌）的生长，后者则不能。相反，婴儿双歧杆菌ATCC 15697在去糖基化的乳蛋白部分上不生长，清楚地表明牛奶糖蛋白的聚糖部分提供了生长的关键底物。基于质谱的分析表明，婴儿双歧杆菌消耗了73%的中性N-聚糖和92%的唾液酸化N-聚糖，而乳酸双歧杆菌仅降解了11%的中性N-聚糖，几乎没有（<1%）唾液酸化N-聚糖。这些结果提供了机制支持，即牛奶中的N-连接糖蛋白作为选择性底物，用于富集能够进行初始去糖基化的婴儿相关双歧杆菌。此外，释放的N-聚糖比完整的牛奶糖蛋白是更好的生长底物，这表明EndoBI-1切割是糖蛋白消耗的关键初始步骤。最后，从牛乳糖蛋白中释放的多种N-聚糖表明，它们可能作为新型益生元底物，具有与人乳寡糖类似的选择性特性。
先前已经表明糖蛋白作为双歧杆菌的生长底物。然而，糖蛋白的哪一部分（聚糖或多肽）负责这一功能尚不清楚。在本研究中，我们使用一种新型酶从牛奶糖蛋白中切割共轭N-聚糖，并测试了各种双歧杆菌对它们的消耗情况。结果表明，聚糖选择性地刺激了婴儿双歧杆菌的生长，婴儿双歧杆菌是婴儿肠道中的关键微生物。使用先进的质谱（纳升液相色谱芯片-四极杆飞行时间质谱[nano-LC-Chip-Q-TOF MS]）确定了单个N-聚糖消耗的选择性，以揭示婴儿双歧杆菌可以消耗从乳清蛋白浓缩物中释放的一系列聚糖结构。
