The OS used in this study are from pooled human milk provided by milk banks in San Jose, CA and Austin, TX. HMOs were isolated from the milk using a previously described procedure involving defatting, chloroform/methanol extraction, ethanol precipitation, and evaporation.6 (link) A sample enrichment step with solid phase extraction (SPE) employing graphitized carbon cartridge (GCC) was used before the analysis. GCC (150 mg bed weight, 4mL volume) were purchased from Alltech (Deerfield, IL). Sodium borohydride (98%) and 2, 5-dihydroxybenzoic acid (DHB) were obtained from Sigma–Aldrich (St. Louis, MO). Standard HMOs were purchased from Dextra Laboratories (Earley Gate, UK). α(1–2)-Fucosidase was obtained from EMD CALBIOCHEM (La Jolla, CA), β(1–3)-galactosidase and α(2–3)-neuraminidase from New England Biolab (Beverly, MA), β(1–4)-galactosidase from ProZyme (San Leandro, CA), and α(1–3,4)-fucosidase from Sigma–Aldrich (St. Louis, MO). The non-selective sialidase was purified and provided by Prof. David Mills from the Department of Viticulture and Enology in UC Davis. All reagents are of analytical or HPLC grade.
Protocol detail
Isolation and Analysis of Human Milk Oligosaccharides
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Austin
Carbon
Chloroform
Dihydroxybenzoic acid
Ethanol
Fucosidase
Galactosidase
Hplc
Human milk
Methanol
Milk
Sialidase
Sodium borohydride
Solid phase extraction
Corresponding Organization :
Other organizations : University of California, Davis, Agilent Technologies (United States)
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Variable analysis
independent variables
- Milk source location (San Jose, CA and Austin, TX)
- Isolated human milk oligosaccharides (HMOs)
- Defatting, chloroform/methanol extraction, ethanol precipitation, and evaporation procedure for isolating HMOs
- Solid phase extraction (SPE) with graphitized carbon cartridge (GCC) for sample enrichment
- Reagents (sodium borohydride, 2,5-dihydroxybenzoic acid, standard HMOs, enzymes, etc.) are of analytical or HPLC grade
- Positive control: Standard HMOs purchased from Dextra Laboratories
- Negative control: Not explicitly mentioned
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