Limits...
A versatile and scalable strategy for glycoprofiling bifidobacterial consumption of human milk oligosaccharides.

Locascio RG, Niñonuevo MR, Kronewitter SR, Freeman SL, German JB, Lebrilla CB, Mills DA - Microb Biotechnol (2008)

Bottom Line: For quantitative consumption, deuterated and reduced human milk oligosaccharide (HMO) standards were used.A detailed analysis of consumption glycoprofiles indicated strain-specific capabilities towards differential metabolism of milk oligosaccharides.This method overcomes previous limitations in the quantitative, multi-strain analysis of bacterial metabolism of HMOs and represents a novel approach towards understanding bacterial consumption of complex prebiotic oligosaccharides.

View Article: PubMed Central - PubMed

Affiliation: Departments of Viticulture & Enology, Chemistry and Food Science & Technology, and Microbiology Graduate Group, University of California, Davis, CA 95616, USA.

Show MeSH

Related in: MedlinePlus

Schematic of experimental procedure. Schematic illustration for the workflow utilized for the real‐time monitoring of bacterial growth, sample preparation, MS analysis, data collection and analysis. (a and b) Clonal expansion and real‐time monitoring of bacterial growth performed in anaerobic chamber. (c) Sample collection. (d) Sample inactivation, sterilization and clean‐up. (e) Internal standard addition. (f) TipTop PGC oligosaccharide isolation. (g) Sample MS analysis, data acquisition and processing. Time gains in sample processing as compared with a previously developed method (LoCascio et al., 2007).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3815754&req=5

f1: Schematic of experimental procedure. Schematic illustration for the workflow utilized for the real‐time monitoring of bacterial growth, sample preparation, MS analysis, data collection and analysis. (a and b) Clonal expansion and real‐time monitoring of bacterial growth performed in anaerobic chamber. (c) Sample collection. (d) Sample inactivation, sterilization and clean‐up. (e) Internal standard addition. (f) TipTop PGC oligosaccharide isolation. (g) Sample MS analysis, data acquisition and processing. Time gains in sample processing as compared with a previously developed method (LoCascio et al., 2007).

Mentions: A plate reader‐based bacterial growth assay was coupled with MS analysis of the spent cultures, and a custom‐developed data analysis software to obtain detailed HMO consumption patterns for 12 bifidobacterial strains. For every strain, the following growth kinetics parameters were calculated: growth rate, apparent lag time, generation time and maximum optical density (OD) reached. These were analysed in combination with the HMO consumption glycoprofiling data to obtain a detailed description of each strain's ability to metabolize specific HMOs. In the automated MS analysis error bars were calculated from at least six relative standard deviations of both the control and samples. Computing the propagation of error was necessary because the per cent HMO consumption was computed by normalizing the MS intensity of the samples to that of the control. Per cent relative standard deviation widely ranged from 1% to 18% and an average of less than 10% for each oligosaccharide peak. The combined use of 96‐well plates and TipTop cartridges reduced the amount of HMO needed for bacterial growth by fivefold, and has reduced the time to perform the oligosaccharide extraction process of a given sample from 41.5 to 3.5 h (Fig. 1). This method enabled monitoring bacterial consumption of the most abundant fucosylated and neutral oligosaccharide isomers in human breast milk, accounting by number for 54% of the total number of individual oligosaccharides from a pooled human milk sample. The HMO masses monitored comprise several isomers representing the highest abundance milk oligosaccharides, and accounting for approximately 70% of all HMOs in milk (Ninonuevo et al., 2006) (Table S1).


A versatile and scalable strategy for glycoprofiling bifidobacterial consumption of human milk oligosaccharides.

Locascio RG, Niñonuevo MR, Kronewitter SR, Freeman SL, German JB, Lebrilla CB, Mills DA - Microb Biotechnol (2008)

Schematic of experimental procedure. Schematic illustration for the workflow utilized for the real‐time monitoring of bacterial growth, sample preparation, MS analysis, data collection and analysis. (a and b) Clonal expansion and real‐time monitoring of bacterial growth performed in anaerobic chamber. (c) Sample collection. (d) Sample inactivation, sterilization and clean‐up. (e) Internal standard addition. (f) TipTop PGC oligosaccharide isolation. (g) Sample MS analysis, data acquisition and processing. Time gains in sample processing as compared with a previously developed method (LoCascio et al., 2007).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3815754&req=5

f1: Schematic of experimental procedure. Schematic illustration for the workflow utilized for the real‐time monitoring of bacterial growth, sample preparation, MS analysis, data collection and analysis. (a and b) Clonal expansion and real‐time monitoring of bacterial growth performed in anaerobic chamber. (c) Sample collection. (d) Sample inactivation, sterilization and clean‐up. (e) Internal standard addition. (f) TipTop PGC oligosaccharide isolation. (g) Sample MS analysis, data acquisition and processing. Time gains in sample processing as compared with a previously developed method (LoCascio et al., 2007).
Mentions: A plate reader‐based bacterial growth assay was coupled with MS analysis of the spent cultures, and a custom‐developed data analysis software to obtain detailed HMO consumption patterns for 12 bifidobacterial strains. For every strain, the following growth kinetics parameters were calculated: growth rate, apparent lag time, generation time and maximum optical density (OD) reached. These were analysed in combination with the HMO consumption glycoprofiling data to obtain a detailed description of each strain's ability to metabolize specific HMOs. In the automated MS analysis error bars were calculated from at least six relative standard deviations of both the control and samples. Computing the propagation of error was necessary because the per cent HMO consumption was computed by normalizing the MS intensity of the samples to that of the control. Per cent relative standard deviation widely ranged from 1% to 18% and an average of less than 10% for each oligosaccharide peak. The combined use of 96‐well plates and TipTop cartridges reduced the amount of HMO needed for bacterial growth by fivefold, and has reduced the time to perform the oligosaccharide extraction process of a given sample from 41.5 to 3.5 h (Fig. 1). This method enabled monitoring bacterial consumption of the most abundant fucosylated and neutral oligosaccharide isomers in human breast milk, accounting by number for 54% of the total number of individual oligosaccharides from a pooled human milk sample. The HMO masses monitored comprise several isomers representing the highest abundance milk oligosaccharides, and accounting for approximately 70% of all HMOs in milk (Ninonuevo et al., 2006) (Table S1).

Bottom Line: For quantitative consumption, deuterated and reduced human milk oligosaccharide (HMO) standards were used.A detailed analysis of consumption glycoprofiles indicated strain-specific capabilities towards differential metabolism of milk oligosaccharides.This method overcomes previous limitations in the quantitative, multi-strain analysis of bacterial metabolism of HMOs and represents a novel approach towards understanding bacterial consumption of complex prebiotic oligosaccharides.

View Article: PubMed Central - PubMed

Affiliation: Departments of Viticulture & Enology, Chemistry and Food Science & Technology, and Microbiology Graduate Group, University of California, Davis, CA 95616, USA.

Show MeSH
Related in: MedlinePlus