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<meta name="robots" content="INDEX,FOLLOW,NOARCHIVE" /><meta name="citation_inbook_title" content="Glycoscience Protocols (GlycoPODv2) [Internet]" /><meta name="citation_title" content="Preparation of oligosaccharides from human milk" /><meta name="citation_publisher" content="Japan Consortium for Glycobiology and Glycotechnology" /><meta name="citation_date" content="2022/03/17" /><meta name="citation_author" content="Junko Amano" /><meta name="citation_pmid" content="37590643" /><meta name="citation_fulltext_html_url" content="https://www.ncbi.nlm.nih.gov/books/NBK593903/" /><meta name="citation_keywords" content="human milk oligosaccharides (HMOs)" /><meta name="citation_keywords" content="diversity" /><meta name="citation_keywords" content="prebiotics" /><meta name="citation_keywords" content="anti-infections" /><link rel="schema.DC" href="http://purl.org/DC/elements/1.0/" /><meta name="DC.Title" content="Preparation of oligosaccharides from human milk" /><meta name="DC.Type" content="Text" /><meta name="DC.Publisher" content="Japan Consortium for Glycobiology and Glycotechnology" /><meta name="DC.Contributor" content="Junko Amano" /><meta name="DC.Date" content="2022/03/17" /><meta name="DC.Identifier" content="https://www.ncbi.nlm.nih.gov/books/NBK593903/" /><meta name="description" content="Human milk contains over 200 kinds of oligosaccharides with larger size and greater diversity compared to bovine milk, which mainly contains small oligosaccharides. The amounts of oligosaccharides, excluding lactose, is 5–15 g/L in human mature milk and 20 g/L in colostrum, although the bovine colostrum contains only 1 g/L of oligosaccharides, 70% of which is 3’-sialyllactose. Human milk oligosaccharides (HMOs) have various core oligosaccharides, which are diversified by sialylation via α2,3 or α2,6 linkages and fucosylation via α1,2, α1,3, or α1,4 linkages and express various antigens, such as blood-type antigens (1–5). The variation of fucosylation depends on the mother’s genotypes: the combination of Secretor (Se) gene (fucosyltransferase-2, FUT2) and Lewis (Le) gene (FUT3), Se+/Le+, Se+/Le-, Se-/Le+, and Se-/Le-. Milk from nonsecretors (Se-/Le+ and Se-/Le-) does not contain 2’-fucosyllactose and α1,2-fucosylated HMOs. HMOs act as prebiotics, favoring the growth of beneficial bacteria, such as Bifidobacteria and Bacteroides (6,7). There is considerable evidence that virulent enteric bacteria and viruses initiate infection by binding to particular sugar chains of glycolipids and glycoproteins on the surface of their target cells. Due to their structural mimicry of the glycans of glycoproteins on the mucous membrane, HMOs are considered to protect breast-fed infants against infections by blocking the adhesion of pathogens (8,9). In the EU, infant formulas supplemented with 2’-fucosyllactose and lacto-N-neotetraose (10) for enhanced function are already available. Along with being important for infant nutrition, HMOs are a precious natural material as a tool of glycobiological research and drug development." /><meta name="og:title" content="Preparation of oligosaccharides from human milk" /><meta name="og:type" content="book" /><meta name="og:description" content="Human milk contains over 200 kinds of oligosaccharides with larger size and greater diversity compared to bovine milk, which mainly contains small oligosaccharides. The amounts of oligosaccharides, excluding lactose, is 5–15 g/L in human mature milk and 20 g/L in colostrum, although the bovine colostrum contains only 1 g/L of oligosaccharides, 70% of which is 3’-sialyllactose. Human milk oligosaccharides (HMOs) have various core oligosaccharides, which are diversified by sialylation via α2,3 or α2,6 linkages and fucosylation via α1,2, α1,3, or α1,4 linkages and express various antigens, such as blood-type antigens (1–5). The variation of fucosylation depends on the mother’s genotypes: the combination of Secretor (Se) gene (fucosyltransferase-2, FUT2) and Lewis (Le) gene (FUT3), Se+/Le+, Se+/Le-, Se-/Le+, and Se-/Le-. Milk from nonsecretors (Se-/Le+ and Se-/Le-) does not contain 2’-fucosyllactose and α1,2-fucosylated HMOs. HMOs act as prebiotics, favoring the growth of beneficial bacteria, such as Bifidobacteria and Bacteroides (6,7). There is considerable evidence that virulent enteric bacteria and viruses initiate infection by binding to particular sugar chains of glycolipids and glycoproteins on the surface of their target cells. Due to their structural mimicry of the glycans of glycoproteins on the mucous membrane, HMOs are considered to protect breast-fed infants against infections by blocking the adhesion of pathogens (8,9). In the EU, infant formulas supplemented with 2’-fucosyllactose and lacto-N-neotetraose (10) for enhanced function are already available. 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<div class="pre-content"><div><div class="bk_prnt"><p class="small">NCBI Bookshelf. A service of the National Library of Medicine, National Institutes of Health.</p><p>Nishihara S, Angata K, Aoki-Kinoshita KF, et al., editors. Glycoscience Protocols (GlycoPODv2) [Internet]. Saitama (JP): Japan Consortium for Glycobiology and Glycotechnology; 2021-. </p></div></div></div>
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<div class="main-content lit-style" itemscope="itemscope" itemtype="http://schema.org/CreativeWork"><div class="meta-content fm-sec"><h1 id="_NBK593903_"><span class="title" itemprop="name">Preparation of oligosaccharides from human milk</span></h1><div class="contrib half_rhythm"><span itemprop="author">Junko Amano</span>, Ph.D. in Pharmaceutical sciences<div class="affiliation small">The Noguchi Institute<div><span class="email-label">Email: </span><a href="mailto:dev@null" data-email="pj.ro.ihcugon@onama" class="oemail">pj.ro.ihcugon@onama</a></div></div><div class="small">Corresponding author.</div></div><p class="small">Created: <span itemprop="datePublished">October 4, 2021</span>; Last Revision: <span itemprop="dateModified">March 17, 2022</span>.</p></div><div class="body-content whole_rhythm" itemprop="text"><div id="g18-prephumanmilk.Introduction"><h2 id="_g18-prephumanmilk_Introduction_">Introduction</h2><p>Human milk contains over 200 kinds of oligosaccharides with larger size and greater diversity compared to bovine milk, which mainly contains small oligosaccharides. The amounts of oligosaccharides, excluding lactose, is 5–15 g/L in human mature milk and 20 g/L in colostrum, although the bovine colostrum contains only 1 g/L of oligosaccharides, 70% of which is 3’-sialyllactose. Human milk oligosaccharides (HMOs) have various core oligosaccharides, which are diversified by sialylation via α2,3 or α2,6 linkages and fucosylation via α1,2, α1,3, or α1,4 linkages and express various antigens, such as blood-type antigens (<a class="bk_pop" href="#g18-prephumanmilk.REF.1">1</a>–<a class="bk_pop" href="#g18-prephumanmilk.REF.5">5</a>). The variation of fucosylation depends on the mother’s genotypes: the combination of Secretor (<i>Se</i>) gene (fucosyltransferase-2, FUT2) and Lewis (<i>Le</i>) gene (FUT3), <i>Se<sup>+</sup>/Le<sup>+</sup></i>, <i>Se<sup>+</sup>/Le<sup>-</sup></i>, <i>Se<sup>-</sup>/Le<sup>+</sup></i>, and <i>Se<sup>-</sup>/Le<sup>-</sup></i>. Milk from nonsecretors (<i>Se<sup>-</sup>/Le<sup>+</sup></i> and <i>Se<sup>-</sup>/Le<sup>-</sup></i>) does not contain 2’-fucosyllactose and α1,2-fucosylated HMOs. HMOs act as prebiotics, favoring the growth of beneficial bacteria, such as <i>Bifidobacteria</i> and <i>Bacteroides</i> (<a class="bk_pop" href="#g18-prephumanmilk.REF.6">6</a>,<a class="bk_pop" href="#g18-prephumanmilk.REF.7">7</a>). There is considerable evidence that virulent enteric bacteria and viruses initiate infection by binding to particular sugar chains of glycolipids and glycoproteins on the surface of their target cells. Due to their structural mimicry of the glycans of glycoproteins on the mucous membrane, HMOs are considered to protect breast-fed infants against infections by blocking the adhesion of pathogens (<a class="bk_pop" href="#g18-prephumanmilk.REF.8">8</a>,<a class="bk_pop" href="#g18-prephumanmilk.REF.9">9</a>). In the EU, infant formulas supplemented with 2’-fucosyllactose and lacto-<i>N</i>-neotetraose (<a class="bk_pop" href="#g18-prephumanmilk.REF.10">10</a>) for enhanced function are already available. Along with being important for infant nutrition, HMOs are a precious natural material as a tool of glycobiological research and drug development.</p><p>
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<b>Protocol</b>
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</p><p>The following protocol has been revised based on “Milk oligosaccharides ~Preparation of oligosaccharides from human milk” in GlycoPOD ver.1 (https://jcggdb.jp/GlycoPOD/protocolShow.action?nodeId=t114) (<a href="https://jcggdb.jp/GlycoPOD/protocolShow.action?nodeId=t114).%20(Note" ref="pagearea=body&targetsite=external&targetcat=link&targettype=uri"><b>Note</b></a>
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<b>1</b>).</p><div id="g18-prephumanmilk.Materials"><h3>Materials</h3><p>1. Column for size-exclusion chromatography filled with gel, such as Sephadex G-25 (medium grade)</p><p>2. Anion-exchange column filled with gel, such as TOYOPEARL Super Q-650M (Tosoh Bioscience)</p><p>3. 5% (w/v) aqueous phenol</p><p>4. H<sub>2</sub>SO<sub>4</sub></p><p>5. 100, 200, 300, 400, and 500 mM pyridinium acetate buffer (pH 5.0)</p><p>6. 10 mg/mL 2,5-dihydroxybenzoic acid in 40% (v/v) acetonitrile-water if using matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS)</p><p>7. Millipore ultrapure water or redistilled water is recommended as water</p></div><div id="g18-prephumanmilk.Instruments"><h3>Instruments</h3><p>1. Photometer for measuring visible light and plastic disposable cuvette, if possible.</p><p>2. Fraction collector</p><p>3. Cooling centrifuge with swing bucket</p><p>4. Evaporator or Speed Vac</p><p>5. MALDI-TOF MS</p></div><div id="g18-prephumanmilk.Methods"><h3>Methods</h3><p>For a better understanding, <a class="figpopup" href="/books/NBK593903/figure/g18-prephumanmilk.F1/?report=objectonly" target="object" rid-figpopup="figg18prephumanmilkF1" rid-ob="figobg18prephumanmilkF1">Figure 1</a> shows the entire process described below and the yield obtained at each step.</p><dl class="temp-labeled-list"><dt>1.</dt><dd><p class="no_top_margin">Removal of fats and proteins</p><dl class="temp-labeled-list"><dt>a.</dt><dd><p class="no_top_margin">Centrifuge milk (10–20 mL per tube) for 15 min at 5,000 ×<i>g</i> at 4°C.</p></dd><dt>b.</dt><dd><p class="no_top_margin">Remove the creamy fat layer with a spatula, and transfer the water layer (each ~5 mL) to a new tube.</p></dd><dt>c.</dt><dd><p class="no_top_margin">Add 10 mL of methanol and 15 mL of chloroform.</p></dd><dt>d.</dt><dd><p class="no_top_margin">Shake well and leave at 4°C.</p></dd><dt>e.</dt><dd><p class="no_top_margin">Centrifuge for 5 min at 5,000 ×<i>g</i> at 4°C.</p></dd><dt>f.</dt><dd><p class="no_top_margin">Collect upper layer from all tubes.</p></dd><dt>g.</dt><dd><p class="no_top_margin">Evaporate the solvent to dry and dissolve with 20 mL of water (the crude oligosaccharide fraction)</p></dd></dl></dd><dt>2.</dt><dd><p class="no_top_margin">Gel filtration</p><dl class="temp-labeled-list"><dt>a.</dt><dd><p class="no_top_margin">Apply the crude oligosaccharide fraction to a Sephadex G-25 column (5 cm of ID × 67 cm) equilibrated with water.</p></dd><dt>b.</dt><dd><p class="no_top_margin">Elute with water under hydrostatic pressure and collect in 20 mL fractions.</p></dd><dt>c.</dt><dd><p class="no_top_margin">Take aliquots and dilute with water to 100 μL.</p></dd><dt>d.</dt><dd><p class="no_top_margin">Add 100 μL of 5% aqueous phenol and 0.5 mL of H<sub>2</sub>SO<sub>4</sub>.</p></dd><dt>e.</dt><dd><p class="no_top_margin">Keep for 20 min at room temperature.</p></dd><dt>f.</dt><dd><p class="no_top_margin">Measure OD490 nm (detection of hexoses)</p></dd><dt>g.</dt><dd><p class="no_top_margin">Draw a chromatogram plotting the absorbance against the fraction number on the horizontal axis, and find the elution of lactose with the highest peak near the column volume.</p></dd><dt>h.</dt><dd><p class="no_top_margin">Pool the fractions containing oligosaccharides larger than lactose.</p></dd></dl></dd><dt>3.</dt><dd><p class="no_top_margin">Separation of neutral and acidic oligosaccharides</p><dl class="temp-labeled-list"><dt>a.</dt><dd><p class="no_top_margin">Apply a part of the pooled fraction to a TOYOPEARL Super Q-650M column (1.6 cm of ID × 10 cm) equilibrated with water.</p></dd><dt>b.</dt><dd><p class="no_top_margin">Elute with 40 mL of water under hydrostatic pressure, and collect this pass-through fraction (that is unbound to the column) as a neutral oligosaccharide fraction, N.</p></dd><dt>c.</dt><dd><p class="no_top_margin">Elute with 40 mL of 100, 200, 300, 400 and 500 mM pyridinium acetate buffer (pH 5.0) stepwise under hydrostatic pressure, store the five fractions, and name as acidic oligosaccharide fractions, A1, A2, A3, A4, and A5, respectively (<b>Note 2</b>).</p></dd><dt>d.</dt><dd><p class="no_top_margin">Perform Steps 2c–f to determine the amounts of hexose in the fractions, if necessary.</p></dd></dl></dd><dt>4.</dt><dd><p class="no_top_margin">Size fractionation by gel filtration</p><dl class="temp-labeled-list"><dt>a.</dt><dd><p class="no_top_margin">Apply the neutral oligosaccharide fraction, N to a Sephadex G-25 column (5 cm of ID × 67 cm)</p></dd><dt>b.</dt><dd><p class="no_top_margin">Elute with water under hydrostatic pressure and collect in 20 mL fractions.</p><p>Perform Steps 2c–f to determine the amounts of hexose in the fractions, if necessary.</p></dd><dt>c.</dt><dd><p class="no_top_margin">Take an aliquot of each fraction to detect oligosaccharides and put on a MALDI plate.</p></dd><dt>d.</dt><dd><p class="no_top_margin">Add 2,5-dihydroxybenzoic acid solution as a matrix to the sample and mix and dry the mixture.</p></dd><dt>e.</dt><dd><p class="no_top_margin">Measure using MALDI-TOF MS</p></dd><dt>f.</dt><dd><p class="no_top_margin">Pool fractions depending on molecular sizes, for example, N-1, N-2 (mainly contains oligosaccharides with decaose cores), N-3 (mainly contains oligosaccharides with octaose cores), N-4 (mainly contains oligosaccharides with hexose cores), N-5 (mainly contains oligosaccharides with tetraose cores), and N-6 (mainly contains lactose) (<b>Note 3</b>).</p></dd></dl></dd><dt>5.</dt><dd><p class="no_top_margin">Labeling with pyrene butanoic acid hydrazide (PBH) for further detailed analysis of the structures (<a class="figpopup" href="/books/NBK593903/figure/g18-prephumanmilk.F2/?report=objectonly" target="object" rid-figpopup="figg18prephumanmilkF2" rid-ob="figobg18prephumanmilkF2">Figure 2</a>, <b>Note 4</b>)</p><dl class="temp-labeled-list"><dt>a.</dt><dd><p class="no_top_margin">Heat oligosaccharides (approximately 1 nmol) and PBH (100 nmol) in 20 μL of methanol in the presence of 0.1 N acetic acid in a glass vial tightly sealed with a screw cap at 80°C for 20 min.</p></dd><dt>b.</dt><dd><p class="no_top_margin">Neutralize by adding dilute NaOH solution.</p></dd><dt>c.</dt><dd><p class="no_top_margin">Add 30 μL of 1.7 M NaBH<sub>4</sub> solution and incubated at 40°C for 20 min.</p></dd><dt>d.</dt><dd><p class="no_top_margin">Neutralize by adding dilute acetic acid solution.</p></dd><dt>e.</dt><dd><p class="no_top_margin">Add 400 μL of water and 500 μL of chloroform.</p></dd><dt>f.</dt><dd><p class="no_top_margin">Shake well and centrifuge for 5 min.</p></dd><dt>g.</dt><dd><p class="no_top_margin">Discard lower chloroform layer.</p></dd><dt>h.</dt><dd><p class="no_top_margin">Add 500 μL of new chloroform and repeat extraction procedure.</p></dd><dt>i.</dt><dd><p class="no_top_margin">Take upper aqueous phase including the PBH-labeled oligosaccharides.</p></dd><dt>j.</dt><dd><p class="no_top_margin">Dry using a vacuum centrifuge.</p></dd><dt>k.</dt><dd><p class="no_top_margin">Dissolve in water and apply onto a Sep-Pak C18 cartridge.</p></dd><dt>l.</dt><dd><p class="no_top_margin">Wash with five-bed volume of water</p></dd><dt>m.</dt><dd><p class="no_top_margin">Elute with five-bed volume of water-acetonitrile (6:4, v/v)</p></dd><dt>n.</dt><dd><p class="no_top_margin">Evaporate solvent and store at −30°C in the dark until use (<b>Note 5</b>).</p></dd></dl></dd></dl></div><div id="g18-prephumanmilk.Notes"><h3>Notes</h3><p>1. This protocol is for relatively large-scale preparation of intact oligosaccharides from human milk (use up to 250 mL). An example of the preparation results is shown in <a class="figpopup" href="/books/NBK593903/figure/g18-prephumanmilk.F1/?report=objectonly" target="object" rid-figpopup="figg18prephumanmilkF1" rid-ob="figobg18prephumanmilkF1">Figure 1</a>. When using a bigger quantity, such as one liter, the method by Kobata (<a class="bk_pop" href="#g18-prephumanmilk.REF.11">11</a>) is recommended.</p><p>2. Most monosialylated oligosaccharides are recovered in A1 fraction, and some monosulfated oligosaccharides are found in A2 although detailed structural study is not done.</p><p>3. Each fraction by gel filtration contains several oligosaccharides. Separation and detection of closely related or isomeric structures is difficult with using intact oligosaccharides. It is necessary to derivatize oligosaccharides and use a method with better resolution. Lectin column chromatography will be valuable for further separation. <i>Aleuria aurantia</i> lectin-immobilized column chromatography is effective for fucosylated oligosaccharides.</p><p>4. Labeling of oligosaccharides with a sensitive fluorescent derivative allows for further separation and structural analysis. There are many labeling reagents, such as 2-aminopyridine and 2-aminobenzamide. Among them, PBH-labeled oligosaccharides (<a class="figpopup" href="/books/NBK593903/figure/g18-prephumanmilk.F2/?report=objectonly" target="object" rid-figpopup="figg18prephumanmilkF2" rid-ob="figobg18prephumanmilkF2">Figure 2</a>) have strong hydrophobicity, and therefore, it can separate isomers on high-performance liquid chromatography (HPLC). The detection sensitivity of PBH-oligosaccharides on MALDI-MS is 1000 times that of intact oligosaccharides. Since many negative ions by MALDI are also generated and can be applied to MS<sup>2</sup>, MS<sup>3</sup>, and MS<sup>4</sup>, isomers even in the mixture can be identified using diagnostic fragment ions, which are produced from particular structures. The author determined many kinds of fucosylated isomers containing Type 1H, Le<sup>a</sup>, and Le<sup>x</sup> using this method (<a class="bk_pop" href="#g18-prephumanmilk.REF.2">2</a>).</p><p>5. For example, PBH-labeled oligosaccharides can be analyzed on HPLC with Inertsil WP300 C<sub>18</sub> column (GL Sciences) (4.6 × 500 mm) using water-acetonitrile eluent system for neutral oligosaccharides and 50 mM acetic acid-triethylamine (pH 5.0)-acetonitrile eluent system for acidic oligosaccharides. PBH-oligosaccharides are detected at Ex 341 nm/Em 376 nm.</p></div></div><div id="g18-prephumanmilk.References"><h2 id="_g18-prephumanmilk_References_">References</h2><dl class="temp-labeled-list"><dt>1.</dt><dd><div class="bk_ref" id="g18-prephumanmilk.REF.1">Kobata A. Structures and application of oligosaccharides in human milk. Proc Jpn Acad Ser B Phys Biol Sci. 2010;86(7):731-47. doi: <a href="https://doi.org/10.2183/pjab.86.731" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">10<wbr style="display:inline-block"></wbr>.2183/pjab.86.731</a>. PMID: 20689231. [<a href="/pmc/articles/PMC3066539/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC3066539</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/20689231" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 20689231</span></a>]</div></dd><dt>2.</dt><dd><div class="bk_ref" id="g18-prephumanmilk.REF.2">Amano J, Osanai M, Orita T, Sugahara D, Osumi K. Structural determination by negative-ion MALDI-QIT-TOFMS<sup>n</sup> after pyrene derivatization of variously fucosylated oligosaccharides with branched decaose cores from human milk. <span><span class="ref-journal">Glycobiology. </span>2009 Jun;<span class="ref-vol">19</span>(6):601–14.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/19240274" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 19240274</span></a>] [<a href="http://dx.crossref.org/10.1093/glycob/cwp026" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>3.</dt><dd><div class="bk_ref" id="g18-prephumanmilk.REF.3">Ruhaak LR, Lebrilla CB. Advances in analysis of human milk oligosaccharides. Adv Nutr. 2012 May;3(3):406-14. doi: <a href="https://doi.org/10.3945/an.112.001883" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">10<wbr style="display:inline-block"></wbr>.3945/an.112.001883</a>. PMID: 22585919. [<a href="/pmc/articles/PMC3649477/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC3649477</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/22585919" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 22585919</span></a>]</div></dd><dt>4.</dt><dd><div class="bk_ref" id="g18-prephumanmilk.REF.4">Bode L. Human milk oligosaccharides: every baby needs a sugar mama. Glycobiology. 2012 Sep;22(9):1147-62. doi: <a href="https://doi.org/10.1093/glycob/cws074" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">10<wbr style="display:inline-block"></wbr>.1093/glycob/cws074</a>. PMID: 22513036. [<a href="/pmc/articles/PMC3406618/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC3406618</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/22513036" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 22513036</span></a>]</div></dd><dt>5.</dt><dd><div class="bk_ref" id="g18-prephumanmilk.REF.5">Corona L, Lussu A, Bosco A, Pintus R, Marincola F C, Fanos V, Dessi A. Human milk oligosaccharides: A comprehensive review towards metabolomics. <span><span class="ref-journal">Children. </span>2021 Sep;<span class="ref-vol">8</span>(9):804.</span> doiPMID: . [<a href="/pmc/articles/PMC8465502/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC8465502</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/34572236" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 34572236</span></a>] [<a href="http://dx.crossref.org/10.3390/children8090804" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>6.</dt><dd><div class="bk_ref" id="g18-prephumanmilk.REF.6">Yu ZT, Chen C, Newburg DS. Utilization of major fucosylated and sialylated human milk oligosaccharides by isolated human gut microbes. <span><span class="ref-journal">Glycobiology. </span>2013 Nov;<span class="ref-vol">23</span>(11):1281–92.</span> [<a href="/pmc/articles/PMC3796377/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC3796377</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/24013960" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 24013960</span></a>] [<a href="http://dx.crossref.org/10.1093/glycob/cwt065" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>7.</dt><dd><div class="bk_ref" id="g18-prephumanmilk.REF.7">Marcobal A, Barboza M, Sonnenburg ED, Pudlo N, Martens EC, Desai P, Lebrilla CB, Weimer BC, Mills DA, German JB, Sonnenburg JL. Bacteroides in the infant gut consume milk oligosaccharides via mucus-utilization pathways. <span><span class="ref-journal">Cell Host Microbe. </span>2011 Oct;<span class="ref-vol">10</span>(5):507–14.</span> [<a href="/pmc/articles/PMC3227561/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC3227561</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/22036470" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 22036470</span></a>] [<a href="http://dx.crossref.org/10.1016/j.chom.2011.10.007" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>8.</dt><dd><div class="bk_ref" id="g18-prephumanmilk.REF.8">Newburg DS, Walker WA. Protection of the neonate by the innate immune system of developing gut and of human milk. <span><span class="ref-journal">Pediatr Res. </span>2007 Jan;<span class="ref-vol">61</span>(1):2–8.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/17211132" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 17211132</span></a>] [<a href="http://dx.crossref.org/10.1203/01.pdr.0000250274.68571.18" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>9.</dt><dd><div class="bk_ref" id="g18-prephumanmilk.REF.9">Moore RE, Xu LL, Townsend SD. Prospecting human milk oligosaccharides as a defense against viral infections. <span><span class="ref-journal">ACS Infect Dis. </span>2021 Feb 12;<span class="ref-vol">7</span>(2):254–263.</span> [<a href="/pmc/articles/PMC7890562/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC7890562</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/33470804" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 33470804</span></a>] [<a href="http://dx.crossref.org/10.1021/acsinfecdis.0c00807" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>10.</dt><dd><div class="bk_ref" id="g18-prephumanmilk.REF.10">Vandenplas Y, Berger B, Carnielli VP, Ksiazyk J, Lagstrom H, Luna MS, Migacheva N, Mosselmans J-M, Pecaud J-C, Possner M, Singhal A, Wabitsch M. Human milk oligosaccharides: 2’-Fucosyllactose and lacto-<em>N</em>-neotetraose in infant formula. <span><span class="ref-journal">Nutrients. </span>2018 Sep;<span class="ref-vol">10</span>(9):1161.</span> doiPMID: . [<a href="/pmc/articles/PMC6164445/" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pmc">PMC free article<span class="bk_prnt">: PMC6164445</span></a>] [<a href="https://pubmed.ncbi.nlm.nih.gov/30149573" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 30149573</span></a>] [<a href="http://dx.crossref.org/10.3390/nu10091161" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>11.</dt><dd><div class="bk_ref" id="g18-prephumanmilk.REF.11">Kobata A. Isolation of oligosaccharides from human milk. <span><span class="ref-journal">Methods Enzymol. </span>1972;<span class="ref-vol">28</span>:262–271.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/661578" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 661578</span></a>] [<a href="http://dx.crossref.org/10.1016/0076-6879(72)28026-0" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd></dl></div><h2 id="NBK593903_footnotes">Footnotes</h2><dl class="temp-labeled-list small"><dt></dt><dd><div id="g18-prephumanmilk.FN1"><p class="no_top_margin">The authors declare no competing or financial interests.</p></div></dd></dl><div class="bk_prnt_sctn"><h2>Figures</h2><div class="whole_rhythm bk_prnt_obj bk_first_prnt_obj"><div id="g18-prephumanmilk.F1" class="figure bk_fig"><div class="graphic"><img src="/books/NBK593903/bin/g18-prephumanmilk-Image001.jpg" alt="Figure 1: . Preparation scheme for one human milk sample and the amount of hexose in each fraction." /></div><h3><span class="label">Figure 1: </span></h3><div class="caption"><p>Preparation scheme for one human milk sample and the amount of hexose in each fraction.</p></div></div></div><div class="whole_rhythm bk_prnt_obj"><div id="g18-prephumanmilk.F2" class="figure bk_fig"><div class="graphic"><img src="/books/NBK593903/bin/g18-prephumanmilk-Image002.jpg" alt="Figure 2: . Labeling of oligosaccharides with pyrene butanoic acid hydrazide." /></div><h3><span class="label">Figure 2: </span></h3><div class="caption"><p>Labeling of oligosaccharides with pyrene butanoic acid hydrazide. The reduction shown in Steps 5b–d makes it more stable.</p></div></div></div></div><div id="bk_toc_contnr"></div></div></div>
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