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Characterization and quantification of oligosaccharides in human milk and infant formula Rose Nijman, Yan Liu, Apichaya Bunyatratchata, Jennifer T. Smilowitz, Bernd Stahl, and Daniela Barile J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01515 • Publication Date (Web): 25 May 2018 Downloaded from http://pubs.acs.org on May 25, 2018

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Journal of Agricultural and Food Chemistry

Characterization and quantification of oligosaccharides in human milk and infant formula

Rose M. Nijman,† Yan Liu,§ Apichaya Bunyatratchata,§ Jennifer T. Smilowitz,§ Bernd Stahl,† and Daniela Barile§,* †

Danone Nutricia Research, Utrecht, 3584 CT, the Netherlands

§

Department of Food Science and Technology, University of California, Davis, CA, 95616,

United States

* correspondence: Daniela Barile; E-mail: [email protected]

1 ACS Paragon Plus Environment


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ABSTRACT: Oligosaccharides are known to affect the health of infants. The analysis of

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these complex molecules in (human) milk samples requires state of the art techniques. This

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study analyzed the composition and concentration of oligosaccharides in early (Day 3) and

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mature (Day 42) human milk as well as in five different infant formula brands. The

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oligosaccharide content decreased in human milk from 9.15±0.25 g/L at Day 3 to 6.38±0.29

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g/L at Day 42 of lactation. All formulas resulted to be fortified with galacto-

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oligosaccharides—one was also fortified with polydextrose and another one with long-chain

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fructo-oligosaccharides. About 130 unique oligosaccharides structures were identified in the

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human milk samples, whereas infant formula contained less diversity of structures. The

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comparisons indicated that composition and abundance of oligosaccharides unique to human

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milk are not yet reproduced in infant formulas. The analytical workflow developed is suitable

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for determination of prebiotic oligosaccharides in foods that contain diverse carbohydrate

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structures.

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KEYWORDS: human milk, mass spectrometry, oligosaccharides, infant formula


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INTRODUCTION

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Human milk has undergone constant evolutionary pressure, being the sole nourishment of

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newborns. Remarkably, abundant components in human milk are indigestible, complex

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oligosaccharides—human milk oligosaccharides (HMOS); their concentration is

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approximately 5–8 g/L in mature human milk and 12–14 g/L in early human milk. 1, 2 HMOS

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are built of five monosaccharides—D-glucose, D-galactose, N-acetylglucosamine, L-fucose

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and a type of sialic acid called N-acetylneuraminic acid (NeuAc).

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HMOS support the growth of select beneficial bacteria such as infant type bifidobacteria

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within the infant intestine. 3-6 A beneficial intestinal microbiota dominated by bifidobacteria,

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supports postnatal intestinal maturation, nutrient absorption and maturation of the immune

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system in newborns. 7-11 HMOS possess anti-infective activities as they inhibit pathogens and

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toxins from binding to intestinal cells. 12-15 Monosaccharide moieties such as fucose 12, 13, 16,

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sialic acid 14 and also galactose play a role in biological recognition processes between

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eukaryotic cells of the host and also between pathogens and hosts. An important beneficial

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function of sialylated oligosaccharides is their contribution to the cognitive development of

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infants, as sialic acid is an essential nutrient for brain development and is used for sialylation

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of brain gangliosides. Sialylated oligosaccharides also play a specific role in maturation of

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the immune system 10, prevention of allergy development 10 and expression of anti-

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inflammatory properties. 17

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Because of their multi-functional, health-promoting activities, non-digestible

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oligosaccharides have been gaining immense interest by the infant formula industry. Infant

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formula is usually based on bovine milk, which contains much lower quantities of prebiotics

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than human milk; a prebiotic being a substrate that is selectively utilized by host 3 ACS Paragon Plus Environment


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microorganisms conferring a health benefit. 18 Additionally, bovine milk oligosaccharides are

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mostly sialylated, with only trace amounts of fucosylated oligosaccharides. Most infant

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formula manufacturers fortify their products with prebiotics, in an attempt of mimicking the

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composition of human milk. While galacto-oligosaccharides (GOS)-containing infant

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formulas have been found to enrich fecal bifidobacteria 19-22, there are inconsistencies in the

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literature with respect to understanding the growth rates of various bifidobacterial strains on

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GOS. For example, some adult-strains of Bifidobacterium have been found to grow more

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efficiently on GOS than several HMOS-oriented strains 23 abundant in the breastfed infant

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gut. 24, 25 On the other hand, some HMOS-oriented strains of Bifidobacterium have been

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reported to grow well on GOS. 23 Furthermore, in depth analyses on the fecal samples of

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infants on formula containing GOS have shown that the microbial composition at species-

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level within the Bifidobacterium genus was more similar to human-milk fed infants

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compared to infants fed control formula. 26, 27

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The presence of fecal short chain fatty acids in the host’s intestine has been found to exert

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benefit to the host by promoting defense functions of the host epithelial cells and protect the

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cell from enteropathogenic infection. 28 Breastfed infants have a much lower colonic pH and

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higher concentrations of fecal acetate than formula-fed infants. 29 Although statistics were

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not performed on the breastfed infant group that served as a reference, the fecal pH of infants

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who consumed infant formula containing GOS was intermediary between the control infant

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formula group (6.87) and the breastfed group (5.37). 22 In another study, the fecal pH of

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infants consuming infant formula with 8 g/l GOS/FOS (9:1) was in the range of breastfed

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infants (5.19 ± 0.40). 30 With respect to gut symptomology, compared to the control infant

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formula group, infant formulas containing GOS increased the frequency 31, 32 and softness of 4 ACS Paragon Plus Environment

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stools from infants and toddlers. 20, 21, 33 Stool consistency and frequency of preterm infants

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receiving GOS containing formula, was not significantly different from infants receiving

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human milk. 34 In other studies however, GOS resulted in lower frequencies and firmer stools

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compared with the breastfed group. 19, 21 These results are unsurprising as the intake of infant

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formula has been found to support gut dysbiosis (increased numbers of pathogens) and

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consequent metabolic effects such as higher fecal pH and lower concentrations of fecal short

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chain fatty acids compared with exclusively breastfed infants. 29 In a recent comparative

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study, the direct microbiota-independent effects of GOS on the intestinal barrier function

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were demonstrated. 68 Recent research also showed prebiotic activity for polydextrose

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(PDX). In a clinical trial in which an infant formula contained both GOS and PDX, fecal

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Bifidobacterium was similar between the breastfed and GOS/PDX-compared with the control

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infant formula group. 21 However, the mechanism by which the addition of GOS and PDX in

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infant formula affects the fecal microbial composition, growth of specific strains of

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Bifidobacterium or microbial function within the gut compared with breastfed infants is still

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poorly understood. Stool characteristics of infants fed a prebiotic mixture consisting of PDX,

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GOS and lactulose were found to be more similar to those of breastfed infants in comparison

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with infants fed control infant formula. 35 A specific mixture of scGOS and lcFOS in a ratio

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of 9:1 has been developed to closely resemble the molecular size distribution of

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oligosaccharides in human milk and a wealth of literature has assessed its usefulness. In a

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recent review by Scholtens et al. 36, ten studies were reported that showed increases in fecal

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bifidobacteria and lactobacilli by infant formula containing scGOS/lcFOS. The review

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furthermore summarized seven studies that found positive effects of this mixture on stool

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consistency and stool frequency. The World Allergy Organization suggests using prebiotic



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supplementation in infants that are not-exclusively breastfed; 12 out of the 19 publications

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included in a systematic review from 2016 had tested the prebiotic mixture scGOS/lcFOS

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(9:1). 37 The addition of a 9:1 scGOS/lcFOS mixture in a concentration of 0.8 g/100 mL to

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infant formula was approved in the EU and USA. 38, 39 Moro and Boehm 40 reviewed 39

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clinical studies, of which 24 contained the prebiotic mixture of scGOS/lcFOS (9:1), and

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found that the benefits of various prebiotics are structure-dependent and cannot be

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extrapolated from one to the other.

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No comprehensive research has yet characterized and quantified the oligosaccharide

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profile in several commercially available infant formulas directly related to known changes

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of oligosaccharide profile in human milk at various lactation stages.

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The aim of the present work was to compare the oligosaccharide profile of human milk,

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including neutral and acidic oligosaccharides, at Day 3 with that of Day 42 of lactation.

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Additionally, the oligosaccharide profile of infant formulas aimed for 0-12 month old infants

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was compared between several brands. A nano liquid chromatography (LC) chip system with

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graphitized carbon was employed for separation of extracted HMOS into individual isomeric

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forms prior to detection by a quadrupole time-of-flight (Q-TOF) mass analyzer. This

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technique has been shown to be ideal for oligosaccharide characterization thanks to its ability

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to resolve tens of isomers, high sensitivity and reproducibility, while requiring only minute

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amounts of sample. 41, 42

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Quantification of the major oligosaccharides (selected based on abundance for secretor

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status; intact or after hydrolysis) was achieved by employing high-performance anion-

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exchange chromatography (HPAEC) with pulsed amperometric detection (PAD) and a UV

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enzymatic D-Glucose/D-Fructose kit. HPAEC-PAD has already been successfully used for


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oligosaccharide quantification in whey permeate 43 which, however has a simpler

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composition compared with infant formula, therefore additional purification strategies were

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employed in the present study.

110 111

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MATERIALS AND METHODS

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Samples and Chemicals. Twenty breast milk samples were obtained from ten women who

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delivered term infants (>37 weeks gestation) and were enrolled in the UC Davis Lactation

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Study in Davis, California. Breast milk was collected from each mother at two lactation

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stages, Day 3 and Day 42 postnatal. Milk samples were collected in the morning using a

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modified published method

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Harmony Manual Breast pump by the mother 2-4 h after feeding her infant. Subjects

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collected the milk into a bottle, inverted 6 times, aliquoted 12 mL into a 15 mL

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polypropylene tube, and subsequently froze the sample in the kitchen freezer (-20 ºC).

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Samples were picked up, transported to the lab on dry ice, and stored at -80 ºC until

122

processing.

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The UC Davis Institutional Review Board approved all aspects of the study and informed

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consent was obtained from all mothers (protocol #: 216198).

125 126 127

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involving milk collection from one breast using a Medela

Five infant formula brands, labelled as A, B, C, D, E and commercially available in the United States, were obtained from a local supermarket or directly from the manufacturers. All infant formulas studied were intended for 0-12 month old infants. Infant formulas

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were prepared according to the instructions on the packages. The analytical grade standards

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for glucose, galactose and lactose were purchased from Sigma-Aldrich (St. Louis, MO, 7 ACS Paragon Plus Environment


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USA). Long-chain fructose-oligosaccharide (lcFOS) standard (Inulin Orafti) was provided by

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Beneo (Mannheim, Germany). Short-chain galacto-oligosaccharide (scGOS) standard

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(Vivinal GOS powder) was received from Friesland Campina Domo (Borculo, the

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Netherlands). Polydextrose (PDX) standard (Litesse® two powder) was provided by Danisco

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(Reigate, United Kingdom). All other oligosaccharide standards were purchased from V-

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LABS, INC. (Covington, LA, USA). Carbonate-free NaOH solution (50% w/w) was

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purchased from Fisher Scientific (Fair Lawn, NJ, USA). NaOAc and the reduction reagent

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NaBH4 were from Sigma–Aldrich (St. Louis, MO, USA). Carrez solution I and II were

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purchased from Fisher Scientific (Fair Lawn, NJ, USA). A food grade lactase, separated from

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Aspergillus oryzae, was obtained from BIO-CAT (Troy, VA, USA). Endo and exo-inulinase

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from Aspergillus niger were purchased from Megazyme (Bray Co., Wicklow, Ireland).

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Enzymatic D-Glucose/D-Fructose kit was obtained from R-Biopharm (Darmstadt, Germany).

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Bio-Gel P2 extra fine powder was purchased from Bio-Rad (Hercules, CA, USA). Dionex

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OnGuard A II cartridges were obtained from Thermo Fisher (Sunnyvale, CA, USA). All

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solvents used in sample preparation and elution were optima grade from Fisher Scientific

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(Fair Lawn, NJ, USA). 18.2 MΩ.cm nanopure water at 25 °C was used throughout the

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experiment and is referred to as water.

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Oligosaccharide Purification Prior to Analysis by Mass Spectrometry. Milk and

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infant formula samples were purified according to a previously described method. 45 All

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samples were prepared in duplicate. Fifty microliters of each sample were diluted with an

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equal volume of water. After briefly mixing with a vortex mixer, diluted samples were

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centrifuged at 4 °C for 30 min at 14000 × g to remove the lower density milk fat globules.

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The skim milk was collected and treated with four volumes of Folch solution (2:1 (v/v)


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chloroform:methanol) to remove residual lipid and some protein. The solution was

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centrifuged at 4 °C for 30 min at 14000 × g. The upper methanol layer containing

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carbohydrates was collected. Two volumes of –30 °C ethanol were added and the solution

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was incubated at –30 °C for 1 h to precipitate the remaining proteins. Proteins were

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separated by centrifuging at 4 °C for 30 min at 14000 × g. The supernatant was collected and

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dried by vacuum centrifugation at 35 °C. The dried samples containing oligosaccharides

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were rehydrated in 50 µl of water. All rehydrated samples were mixed with a vortex mixer

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and sonicated well to obtain a homogeneous solution.

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Oligosaccharides were reduced from the aldehyde to alditol form to eliminate anomers,

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thus simplifying analysis of chromatograms. Oligosaccharide reduction conditions were as

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described by Wu et al. 46 with some modifications. An equal volume (50 µl) of 1.0 M NaBH4

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was added to each sample and the sample was incubated at 65 °C for 1 h. To remove borates,

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salts, monosaccharides and disaccharides, 96-well plates with porous graphitized carbon

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solid-phase extraction columns (PGC-SPE) (40-µl media bed volume, 2000 µg binding

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capacity, Glygen Corp., Columbia, MD, USA) were employed. After activating the column,

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samples were loaded onto the plate. Undesired compounds were thoroughly removed by

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washing with 200 µl water six times and spinning at room temperature for 3 min at 277 × g.

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Total oligosaccharides, bound to the porous graphitized carbon, were eluted by 600 µl of

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40% acetonitrile with 1% trifluoracetic acid in water (v/v). Purified oligosaccharides were

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dried by speed vacuum centrifugation at 35 °C.

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Oligosaccharide analysis by nanoLC-chip/Q-TOF. Purified reduced milk

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oligosaccharides were analyzed by nano-LC-chip/Q-TOF mass spectrometry. Samples were

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rehydrated in 50 µl of water and diluted 100 times for mass spectrometry analysis. Diluted



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samples were injected into the Agilent 6520 (Santa Clara, CA, USA) chip/Q-TOF with a

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porous graphitized carbon (PGC) glycan micro-chip (Santa Clara, CA, USA). PGC was used

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as a separating medium because it effectively separates oligosaccharide isomers. The

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conditions reported by Wu et al. 42 were used with the following modifications. All data were

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collected in positive mode with 450–2500 mass/charge (m/z) range. The Q-TOF was

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calibrated by a dual nebulizer electrospray source with a wide mass range of calibrant ions

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(ESI-L, low concentration tuning mix, Agilent Technology). The m/z = 922.010 and m/z =

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1221.991 were chosen as internal calibrants with the reference mass parameter of detection

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window 100 ppm and minimum height 500 counts. The peak collection thresholds were set at

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200 ion counts or 0.01% relative intensity for mass spectrometry spectra. Data analysis was

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performed on Agilent Mass Hunter Quantitative Analysis and Agilent Mass Hunter Profinder

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version B.06.00.

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Data Analysis. The “Find by molecular feature” algorithm of Mass Hunter Quantitative

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Analysis was employed to extract peaks that matched an in-house HMOS database. The

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extraction was performed with an absolute peak height cut-off of 5000 ion counts and a

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quality score over 60. The isotope type used was “glycan.” Isotopes were grouped by peak

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spacing 0.025 m/z + 7.0 ppm. All extracted peaks were manually checked. The secondary

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extraction was performed in Mass Hunter Profinder. “Targeted feature extraction” was

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applied to all samples against the human milk library (database) compiled from the analyses

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of oligosaccharides in the ten milk samples. The extraction was required to match the

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compounds in the library with both accurate mass and retention time. All the extracted peaks

197

were checked and inaccurate integrations were manually corrected.


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For data analysis of infant formula, a new library containing all possible bovine milk

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oligosaccharides and GOS was assembled. The “Find by molecular feature” algorithm was

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performed to extract peaks matching the mass of the compounds in this new library. The

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extraction parameters were similar to those for human milk data analysis except that the

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absolute height filter was set to 1000 in order to extract more low-abundant compounds. A

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secondary extraction in Profinder was applied as described for the human milk samples.

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Compound exchange format files with compound name, formula, retention time and mass

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were generated.

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Quantification of Monosaccharides and Disaccharides by High-Performance Anion-

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Exchange Chromatography with Pulsed Amperometric Detection (HPAEC-PAD). Two

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volumes of samples (human milk and infant formula) were treated with one volume “Carrez

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solution I” followed by one volume of “Carrez solution II” to precipitate proteins and

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eliminate impurities that would interfere with the quantification of galactose, glucose and

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lactose. 47 The mixture was centrifuged at 4255 x g, 4 ºC for 30 min. The supernatant was

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diluted and filtered with 0.22-µm filter. For method validation and study of recovery, a

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randomly selected infant formula sample was spiked with five concentrations of glucose,

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galactose and lactose standards (Table S1). External calibration curves of glucose, galactose

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and lactose were constructed. The concentration of each spiked sample was measured using

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these external calibration curves. Recovery was calculated by the difference between the

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experimentally measured amount and original amount added in a blank sample divided by

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the spiked amount.

219 220

Quantification of monosaccharides was performed by a Thermo Scientific Dionex ICS5000+ HPAEC-PAD (Sunnyvale, CA, USA). The quantification parameters and



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conditions reported by Lee 43 were used. Glucose, galactose and lactose were separated on a

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CarboPac PA 10 column (4 x 250 mm) with a CarboPac PA 10 guard column (4 x 50 mm)

223

and detected by a disposable gold working electrode and a quadruple potential waveform.

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Elution was performed at room temperature at a flow rate of 1.2 mL/min. Glucose, galactose

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and lactose were simultaneously analyzed in the same run. A 15-min washing with 200 mM

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and re-equilibration with 10 mM NaOH was performed followed by an isocratic condition of

227

10 mM NaOH for 12 min. A gradient from 10 mM to 100 mM NaOH was used from 12 min

228

to 25 min to elute lactose.

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Quantification of bound sialic acid in human milk (Day 42) and infant formula.

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Human milk and infant formula samples were filtered by 10 kDa Amicon ultrafiltration

231

filters. A 100-µL aliquot of permeate was dried and hydrolyzed with 50 mM sulfuric acid at

232

80 °C for 1 h. The hydrolyzed permeate, containing the released sialic acid, was purified by

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Dionex–OnGuard A II cartridges. Released sialic acid was eluted by 8 mL of 50 mM NaCl.

234

48

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HPAEC-PAD on a CarboPac PA 10. After re-equilibration with 10 mM NaOAc and 100 mM

236

NaOH, the NaOAc concentration was increased from 10 mM to 100 mM from 0 min to 20

237

min with the same concentrations of NaOH.

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After bringing the volume to 10 mL, samples were filtered and analyzed by Dionex

Quantification of Oligosaccharides in Human Milk by HPAEC-PAD. HMOS were

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quantified by a standard addition method to eliminate the matrix effect (Table S3).

240

Homogeneously pooled human milk samples of Day 3 and Day 42 were obtained by mixing

241

equal volumes of each donor’s milk. Samples were diluted five-fold prior to use. A stock

242

solution of nine standards—lacto-N-difucohexaose I (LNDFH I ), lactodifucotetraose

243

(LDFT), 2'-fucosyllactose (2’-FL), lacto-N-fucopentaose I (LNFP I), lacto-N-tetraose (LNT),


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lacto-N-hexaose (LNH), monofucosyllacto-N-hexaose I (MFLNH I), 6'-sialyllactose sodium

245

salt (6'-SL) and 3'-sialyllactose sodium salt (3'-SL)—was used to spike the human milk

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samples (see Table S2 and Table S3). Spiked human milk samples were filtered through 10

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kDa Amicon ultrafiltration filters (Billerica, MA, USA) at 14000 x g at 4 °C for 30 min. The

248

collected permeate was diluted and filtered through a 0.2-µm membrane for HPAEC-PAD

249

analysis. Quantification of oligosaccharides was achieved by a CarboPac PA 200 column (3

250

x 250 mm) equipped with a CarboPac PA 200 guard column (3 x 50 mm) with a 0.5 mL/min

251

flow rate. An isocratic condition of 100 mM NaOH was used to separate neutral

252

oligosaccharides. Acidic oligosaccharides were eluted with isocratic 100 mM NaOH and 10

253

mM NaOAc. Peak areas were plotted against spiked amounts of oligosaccharide standards.

254

The interception of the x axis was the amount of the corresponding oligosaccharide in human

255

milk.

256

Quantification of GOS in Infant Formula. A 2-cm wide x 100-cm long size-exclusion

257

column (SEC) was packed with pre-wetted Bio-Gel P2 extra fine powder (Bio-Rad,

258

Hercules, CA, USA). Two column volumes of degassed water were passed through the

259

column prior to use. The void volume was determined using blue dextran with a flow rate of

260

15 mL/h. Water was used as eluent. One milliliter of skimmed infant formula was loaded

261

onto the size-exclusion column. After discarding the void volume, 1-ml fractions were

262

collected and analyzed by matrix-assisted laser desorption/ionization –time of flight mass

263

spectrometry (MALDI-TOF, Microflex, Bruker, Billerica, MA, USA). Fractions containing

264

oligosaccharides with a degree of polymerization (DP) between 3 and 9 were pooled and

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dried for further hydrolysis experiments. Quantification of DP2 was not included in this

266

work, since all samples had a matrix containing high levels of lactose, which would interfere



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with the measurement accuracy of the DP2 of GOS. Pooled GOS samples were re-suspended

268

in 5 mL of water. After adjustment to pH 4.5 with hydrochloric acid, purified infant formula

269

was hydrolyzed with 0.25% lactase at 50 °C for 45 min. The enzyme was recovered using a

270

10 kDa Amicon ultrafiltration filter. Purified samples were diluted and filtered through a

271

0.22-µm polyethersulfone filter (Washington, NY, USA). Analysis of monosaccharides and

272

lactose before and after hydrolysis was done by Dionex HPAEC–PAD with a CarboPac PA

273

10 column. External calibration curves were built with commercial analytical standards to

274

calculate monosaccharides and lactose concentrations. The GOS concentration was

275

calculated based on the following equation:

276

a)

277

GOS. Gt is the total concentration of galactose after enzyme treatment. Gb is the measured

278

initial free galactose. Gl means the galactose content released from lactose.

279

b) GOSt = k * Gg

280

where k = (180 + 162n)/(180n) and n is the average number of galactose moieties in the GOS

281

molecules. For example, if n=2, k is 1.4. 49 According to the specification of the GOS

282

ingredient used in the infant formula production, the average number of galactose units was

283

estimated to be 2.7.

Gg = Gt – Gb – Gl, where Gg is the concentration (g/L) of galactose released from

284

Quantification of FOS in Infant Formulas. The AOAC method to determine total

285

fructan in food was used. 50 The stock solutions of endo- and exo-inulinase were mixed as a

286

1:10 solution (Table S4). The mixture was diluted to 400 u/mL exo-inulinase and 40 u/mL

287

endo-inulinase by using a pH 4.5 100 mM NaOAc buffer containing 1 mg/ml bovine serum

288

albumin. One-hundred microliters of enzyme mixture was added to 200 µL of each sample.

289

After adjusting the sample to pH 4.5 with glacier acetic acid, the mixture was incubated at 60


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°C and kept under agitation in a thermo mixer for 30 min. After the reaction was completed,

291

enzyme was separated by a 10 kDa Amicon ultrafiltration filter. The total released fructose

292

(Fg) was measured by an UV enzymatic D-Glucose / D-Fructose kit. FOS was calculated

293

according to the following equation:

294

FOSt=k*Fg

295

where k=(180 + 162n)/(180n) and n is the average number of fructose moieties in the FOS

296

molecules. In the infant formula, the average DP of FOS was calculated as 25. Hence, n=24

297

was the estimated factor used in the total FOS calculation.

298

Quantification of PDX in Infant Formula. Infant formula samples were mixed with a

299

vortex for 1 min and incubated at 80 ˚C for 5 min. The solution was cooled at room

300

temperature before centrifugation at 16162 x g, at 4˚C for 30 min. The top fat layer and

301

precipitate were removed and the solution was diluted 500 fold, filtered and analyzed by

302

HPAEC-PAD. The 10 g/L of standard PDX was treated the same as the infant formula

303

sample and finally diluted to 0.06, 0.03, 0.01, 0.006, 0.003 and 0.001 g/L to generate the

304

standard curve. After 5min of prewashing with 200mM and 10 min of re-equilibration with

305

10mM NaOH, a 45 min gradient from 10mM to 80mM NaOH was used to elute PDX. The

306

PDX peak eluting at 2.23 min that did not coelute with GOS, nor with other compounds in

307

the infant formula sample, was used to quantify the total concentration of intact PDX by

308

comparing the area of the 2.23 min peak in the infant formula with the same peak in the PDX

309

standard curve.

310

RESULTS AND DISCUSSION

311 312

Comparison of oligosaccharides, bound sialic acid, monosaccharides and lactose in human milk and infant formula. The oligosaccharides in human milk and commercial



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infant formulas were measured by nano LC-chip/Q-TOF MS for comparison of their content

314

and composition. Samples from ten human milk donors (collected at Day 3 and Day 42 of

315

lactation) and five infant formula brands were measured in duplicate and the nano LC-

316

chip/Q-TOF MS peak areas were averaged. The complete list of identified oligosaccharides

317

for human milk and infant formula is presented in the supplementary material (Tables S5-

318

S8). Human milk comprised about 130 individual structures, whereas infant formula

319

contained less diversity of structures. In early milk, 76 fucosylated structures, 13 sialylated, 9

320

fucosylated and sialylated and 35 neutral unfucosylated molecules were identified. In mature

321

milk, 73 fucosylated structures, 12 sialylated, 9 fucosylated and sialylated and 34 neutral

322

unfucosylated molecules were detected. Infant formula contained only 2 sialylated and 44

323

neutral unfucosylated oligosaccharides. The quantity of NeuAc, monosaccharides and lactose

324

in human milk and infant formula were also measured by HPAEC-PAD (triplicate

325

measurements). All values in the text and in table 1 till 4 are presented as mean of samples ±

326

mean of SD.

327

As shown in Figure 1, fucosylated oligosaccharides comprised 58.2±7.4% of total

328

oligosaccharides in human milk (Day 42), a value that is in agreement with the previously

329

reported content (between 46% and 70%). 45 Oligosaccharides with up to four fucose

330

residues were detected. The oligosaccharide LNT, known for its prebiotic potential,

331

constituted a large part (17.0±6.6%) of total oligosaccharides. HMOS’ sialylated fraction of

332

the Day 42 milk samples constituted 8.3±2.0% of total oligosaccharides, based on

333

abundance, and 3.4±0.9% of these oligosaccharides contained both sialic acid and fucose

334

residues. Ninonuevo et al. 45 previously found sialylated oligosaccharides to fall between 3%

335

and 36% of the total abundance of oligosaccharides in human milk. Neutral unfucosylated


Page 17 of 36


336

oligosaccharides represented the remainder of the pool. The relative abundances of the

337

oligosaccharides in the Day 3 samples were very similar to those in the Day 42 samples,

338

except for a slightly higher relative abundance of fucosylated oligosaccharides in the Day 3

339

samples, and a lower abundance of LNT. It is clear from Figure 1 that the oligosaccharides in

340

the infant formulas differed from those in human milk. The sialylated fraction of infant

341

formula contained only 2.8±1.5% of total oligosaccharides and 4.7±2.3% of the

342

oligosaccharides was neutral non-fucosylated. The majority (92.4±3.7%) of the

343

oligosaccharides consisted of hexose oligomers.

344

The content of bound NeuAc in a pooled sample of human milk from the ten donors was

345

0.344±0.008 g/L (Table 1); this form of sialic acid was previously reported as being between

346

0.26 and 0.70 g/L at four to eight weeks of lactation. 51, 52 Oligosaccharide-bound NeuAc in

347

the infant formulas, was on average 0.043±0.001 g/L. NeuAc was previously reported as

348

being between 0.014 and 0.072 g/L in infant and follow-on formulas recommended for

349

different age groups (up to 12 months). 51, 52 The lower sialic acid content in formula results

350

in significantly lower levels of sialic acid in brain and saliva of formula-fed infants than in

351

breastfed infants. 53

352

Free monosaccharides were also found to be quite low in both human milk and in infant

353

formula (Table 2), with concentrations lower than 0.4 g/L in all samples, except for glucose

354

in infant formula, which was on average >1.5 g/L. Lactose was highly abundant in all

355

samples, with levels in infant formula (56.8±1.83) comparable with those in mature human

356

milk at Day 42 (56.7±0.92) and higher than those in human milk at Day 3 of lactation

357

(45.2±1.31).



358

Page 18 of 36

As described above, the oligosaccharides of special health interest—sialylated

359

oligosaccharides, α1,2-fucosylated oligosaccharides and LNT—were abundant in the Day 3

360

and Day 42 human milk samples. The infant formulas were all supplemented with hexose

361

oligomers, and neutral non-fucosylated milk oligosaccharides and sialylated oligosaccharides

362

derived from bovine milk used as base for the formulation were identified only at low

363

abundance. Fucosylated oligosaccharides were absent from the five formula brands assayed,

364

and the prebiotic oligosaccharide LNT also was not detected. The content of bound sialic

365

acid was about eight times lower in infant formula compared to human milk. Human milk

366

contains a complex mixture of oligosaccharides, whereas infant formula contains mainly

367

single components. The overall composition of the five different infant formula brands was

368

similar; consisting mainly of hexose oligomers (Figure 1 displays an average). However,

369

infant formula brands may differ in the number, quantity and types of hexose oligomers

370

added, with emerging science showing different effects on i.e. the infant’s gut health. It is

371

therefore worth to look at the particular differences among infant formula brands in more

372

detail, which will be discussed below.

373 374

HMOS variation during lactation measured by HPAEC-PAD. The amounts of

375

oligosaccharides in human milk were compared between early and mature milk to investigate

376

changes in composition occurring over time. Table 3 displays the concentration of nine

377

oligosaccharides in Day 3 and Day 42 pooled human milk as determined by HPAEC-PAD

378

(triplicate measurement). The total amount of oligosaccharides in human milk decreased

379

significantly remarkably from Day 3 by Day 42, which is consistent with previous

380

observations.

1, 51, 54, 55

The total concentration of the 9 HMOS measured was 6.38±0.29 g/L


Page 19 of 36


381

at Day 42 milk, which is in agreement with the concentration range (5 to 8 g/L) previously

382

reported by Kunz et al. 2 A total of 9.15±0.25 g/L was measured in early milk (Day 3), which

383

is lower than the 12–14 g/L value (depending on the milk type) recently reported by Thurl et

384

al.

385

quantified in the two studies. The total oligosaccharide content in human milk was 30.3%

386

lower at Day 42 compared to Day 3 of lactation; this decrease in total oligosaccharides over

387

time might be partially compensated by the increase in volume ingested by the baby over

388

time. 56 Especially, the α1,2-linked fucosylated oligosaccharides decreased (approximately by

389

35%) at Day 42 of lactation (Table 3), consistently with findings reported by others.

390

Three of the nine analysed oligosaccharides increased over time: LNH (+94.6%), 3’-SL

391

(+9.0%), and LNT (+6.2%).

1

This difference is likely attributable to the different individual oligosaccharides

1, 57, 58

392 393

Oligosaccharides in infant formula. The types and quantities of prebiotic oligosaccharides

394

added to five infant formula brands, labelled as A, B, C, D, E and commercially available in

395

the United States, were measured. Table 4 displays the quantities of the oligosaccharides

396

with a DP≥3, according to the official fiber regulation, detected in each infant formula as

397

measured by HPAED-PAD. The five formulas analyzed resulted to contain non-digestible

398

oligosaccharides in the form of GOS. There were some differences in GOS content among

399

the formulas, with formulas A, B and C containing between 3.5 and 4.0 g/L GOS, and

400

formula D containing the highest quantity of 4.45±0.01 g/L. Only formula E contained a

401

much lower amount of GOS (1.99±0.04 g/L). Nano LC-chip/Q-TOF MS chromatograms

402

revealed that all of the infant formula tested presented a predominant degree of

403

polymerization (DP) of 3,4 and 5 (see table 4). Products B and E also contained higher DP



404

isomers which were derived from the addition of polydextrose and maltodextrins in the

405

formulations.

Page 20 of 36

406

If compared with human milk concentration, the added amount of oligosaccharides to

407

infant formula appeared adequate. With average daily intakes of human colostrum (day 3)

408

and mature human milk (Day 42) as 371 ml/L and 711 ml/L, respectively, based on our data

409

59

410

the analyzed formulas contained only one type of added prebiotic oligosaccharide (GOS),

411

whereas two resulted to contain an additional oligosaccharide class. As shown in table 4,

412

formula E contained Polydextrose (PDX) whereas formula D contained long-chain Fructose-

413

oligosaccharide (lcFOS), the high-molecular weight fraction of inulin extracted from chicory

414

plants. Formula B was fortified with GOS and it also contained maltodextrin. However,

415

maltodextrin is a digestible carbohydrate (e.g. digested by salivary and pancreatic amylases

416

in the gastrointestinal tract of infants) and, therefore, does not belong to the health-promoting

417

group of prebiotic oligosaccharides that are utilized by intestinal microbiota. For this reason,

418

maltodextrin was not measured in this study.

, infants would consume about 3.4 g (Day 3) and 4.5 g (Day 42) HMOS per day. Three of

419

Although the DP2 fraction of GOS was not measured in this study, it is scientifically

420

relevant and, therefore, is part of the total GOS content. The total short-chain GOS: long-

421

chain FOS ratio in formula D was 9:1, calculated referring to the detailed GOS analyses by

422

Coulier et al. 60 The health of an infant, especially in terms of intestinal and immune health is

423

strongly influenced by diet during the first few months of life. 40 The present work

424

demonstrates that there are substantial differences in the types and amounts of

425

oligosaccharides among infant formula brands available in the USA, all with their inherent


Page 21 of 36


426

health benefits. At the same time, it must be noted that these prebiotics here measured in

427

infant formula, compared with HMOS, differ substantially in structure, hence in the

428

microbial function and intestinal functions as well. This study showed that the quantity and

429

composition of oligosaccharides in human milk, unlike infant formula, changes over the

430

course of lactation. It is clear from the results that the oligosaccharide composition of human

431

milk is highly complex and dynamic, unlike that of infant formula. Compositional

432

differences between infant formulas exist and are dependent on the number and type of

433

oligosaccharides present. A distinction in expected health outcomes can therefore be made

434

not only between human milk and infant formula, but also among various infant formula

435

brands. The results suggest that the infant formula industry can benefit from supplementing a

436

complex mixture of HMOS to infant formula. As a matter of fact, production of a handful of

437

HMOS is now possible at the large scale, and supplementation of the synthetically produced

438

oligosaccharides 2’-FL and Lacto-N-neotetraose (LNnT) to infant formula was recently

439

approved by the EFSA Panel on Dietetic Products. 61 The latter was not measured in the

440

current study due to its low abundance in human milk. 1 Several in vitro and in vivo tests

441

have already been carried out to assess the potential for health claims. Steenhout et al.

442

demonstrated that the addition of both 2’-FL and LNnT to starter formula shifted the fecal

443

microbiota closer to that observed in infants fed human milk. 62 Results from Goehring et al.

444

63

445

and adaptive immune profiles more similar to those of breastfed infants. Although 2’-FL

446

seems to be a more prominent candidate based on (amongst others) its abundance in human

447

milk 1, more research is needed to investigate the health benefits of both HMOS before a

448

recommendation can be made about the preferred molecule for infant formula fortification.

suggest that feeding infant formula with fortified 2’-FL (0.2 and 1.0 g/L) modifies innate



Page 22 of 36

449

Currently, no health claims exist for HMOs in the EU or USA and the maximum amount

450

allowed in infant formula (2 g/L) is well below the overall HMOS concentration in milk. 64

451

Experimental evidence on HMOS characterization of different population groups, suggests

452

that LNT would be a better candidate for supplementation than LnNT to represent the core

453

HMOS structure, due to their different structure and abundance in human milk. The

454

comprehensive work by Thurl et al. 1 demonstrated that LNT and other neutral

455

oligosaccharides based on the core type 1 structure, are significantly more represented in

456

human milk compared to LnNT, which is a type 2 structure typically found in ruminant milks

457

rather than human. Based on a recent review more systematic studies on sample collection

458

and comparable analytics are needed to determine the concentrations of HMOS. 65

459

Additionally, more research is needed to determine the effect of the addition of single HMOS

460

structures to infant formula, keeping in mind the complex mixture and size distribution of

461

oligosaccharides present in human milk.

462 463

ACKNOWLEDGMENTS

464

The authors value the scientific discussions with D. Klaassen - van de Beek, Danone Nutricia

465

Early Life Nutrition US and thank Cora Dillard for editing this manuscript.

466

AUTHOR INFORMATION

467

Corresponding Author

468

*(D.B.) Phone: 530-752-0976. Fax: 530-752-0976. E-mail: [email protected]

469

Funding sources

470

D.B. received funding from USDA NIFA (Hatch project 232719) and Danone Nutricia.

471

Notes 22 ACS Paragon Plus Environment

Page 23 of 36


472

RMN and BS are employees of Danone Nutricia Research. YL, AB, JTS and DB declare that

473

the research was conducted in the absence of any commercial or financial relationships that

474

could be construed as a potential conflict of interest.

475 476 477

ABBREVIATIONS USED

478

oligosaccharides; 2'-FL, 2'-fucosyllactose; 3'-SL, 3'-sialyllactose; 6'-SL, 6'-sialyllactose; DP,

479

degree of polymerization; HMOS, human milk oligosaccharides; HPAEC, high-performance

480

anion-exchange chromatography; LC, liquid chromatography; LDFT, lactodifucotetraose;

481

LNDFH I, lacto-N-difucohexaose I; LNFP I, lacto-N-fucopentaose I; LNH, lacto-N-hexaose;

482

LNnT, Lacto-N-neotetraose; LNT, lacto-N-tetraose; MFLNH I, monofucosyllacto-N-hexaose

483

I; NeuAc, N-acetylneuraminic acid; PAD, pulsed amperometric detection; PDX,

484

polydextrose; PGC, porous graphitized carbon; PGC-SPE, porous graphitized carbon solid-

485

phase extraction columns; SEC, size exclusion column.

(lc)FOS, (long-chain) fructo-oligosaccharides; (sc)GOS, (short-chain) galacto-



Page 24 of 36

ASSOCIATED CONTENT Supporting information Supporting information is available: Details on methods used. This material is available free of charge via the Internet at http://pubs.acs.org.


Page 25 of 36


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30. Moro, G.; Minoli, I.; Mosca, M.; Fanaro, S.; Jelinek, J.; Stahl, B.; Boehm, G., Dosage-related bifidogenic effects of galacto- and fructooligosaccharides in formula-fed term infants. Journal of pediatric gastroenterology and nutrition 2002, 34, 291-295. 31. Ben, X. M.; Zhou, X. Y.; Zhao, W. H.; Yu, W. L.; Pan, W.; Zhang, W. L.; Wu, S. M.; Van Beusekom, C. M.; Schaafsma, A., Supplementation of milk formula with galacto-oligosaccharides improves intestinal micro-flora and fermentation in term infants. Chin. Med. J. (Engl.) 2004, 117, 927931. 32. Ben, X. M.; Li, J.; Feng, Z. T.; Shi, S. Y.; Lu, Y. D.; Chen, R.; Zhou, X. Y., Low level of galacto-oligosaccharide in infant formula stimulates growth of intestinal Bifidobacteria and Lactobacilli. World J. Gastroenterol. 2008, 14, 6564-6568. 33. Ribeiro, T. C.; Costa, R.; Almeida, P. S.; Pontes, M. V.; Leite, M. E.; Filadelfo, L. R.; Khoury, J. C.; Bean, J. A.; Mitmesser, S. H.; Vanderhoof, J. A.; Scalabrin, D. M., Stool pattern changes in toddlers consuming a follow-on formula supplemented with polydextrose and galactooligosaccharides. Journal of pediatric gastroenterology and nutrition 2012, 54, 288-290. 34. Boehm, G.; Lidestri, M.; Casetta, P.; Jelinek, J.; Negretti, F.; Stahl, B.; Marini, A., Supplementation of a bovine milk formula with an oligosaccharide mixture increases counts of faecal bifidobacteria in preterm infants. Archives of disease in childhood. Fetal and neonatal edition 2002, 86, F178-181. 35. Ziegler, E.; Vanderhoof, J. A.; Petschow, B.; Mitmesser, S. H.; Stolz, S. I.; Harris, C. L., Term infants fed formula supplemented with selected blends of prebiotics grow normally and have soft stools similar to those reported for breast-fed infants. J Pediatr Gastroenterol Nutr 2007, 44, 359-364. 36. Scholtens, P. A.; Goossens, D. A.; Staiano, A., Stool characteristics of infants receiving short-chain galacto-oligosaccharides and longchain fructo-oligosaccharides: A review. World J. Gastroenterol. 2014, 20, 13446-13452. 37. Cuello-Garcia, C. A.; Fiocchi, A.; Pawankar, R.; Yepes-Nuñez, J. J.; Morgano, G. P.; Zhang, Y.; Ahn, K.; Al-Hammadi, S.; Agarwal, A.; Gandhi, S.; Beyer, K.; Burks, W.; Canonica, G. W.; Ebisawa, M.; Kamenwa, R.; Lee, B. W.; Li, H.; Prescott, S.; Riva, J. J.; Rosenwasser, L.; Sampson, H.; Spigler, M.; Terracciano, L.; Vereda, A.; Waserman, S.; Schünemann, H. J.; Brożek, J. L., World Allergy Organization - McMaster university guidelines for allergic disease prevention (GLAD-P): prebiotics. World Allergy Organ. J. 2016, 9, 1-10. 38. U.S. Food and Drug Administration, GRAS Notice 477: Long-chain inulin. In 2013. 39. EFSA NDA Panel (EFSA Panel on Dietetic Products Nutrition and Allergies), Scientific Opinion on the essential composition of infant and follow-on formulae. EFSA Journal 2014 2014, 12, 3760, 106 pp. 40. Moro, E. G.; Boehm, G., Clinical outcomes of prebiotic intervention trials during infancy: A review. Func. Food Rev. 2012, 4, 101-113. 41. Niñonuevo, M. R.; Lebrilla, C. B., Mass spectrometric methods for analysis of oligosaccharides in human milk. Nutr. Rev. 2009, 67, S216S226. 42. Wu, S.; Grimm, R.; German, J. B.; Lebrilla, C. B., Annotation and structural analysis of sialylated human milk oligosaccharides. J. Proteome Res. 2010, 10, 856-868. 43. Lee, H.; de MeloSilva, V.; Liu, Y.; Barile, D., Short communication: Quantification of carbohydrates in whey permeate products using highperformance anion-exchange chromatography with pulsed amperometric detection. J. Dairy Sci. 2015, 98, 7644-7649.



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44. Ferris, A. M.; Jensen, R. G., Lipids in human milk: a review. 1: Sampling, determination, and content. Journal of pediatric gastroenterology and nutrition 1984, 3, 108-122. 45. Ninonuevo, M. R.; Park, Y.; Yin, H.; Zhang, J.; Ward, R. E.; Clowers, B. H.; German, J. B.; Freeman, S. L.; Killeen, K.; Grimm, R.; Lebrilla, C. B., A strategy for annotating the human milk glycome. J. Agric. Food Chem. 2006, 54, 7471-7480. 46. Wu, S.; Tao, N.; German, J. B.; Grimm, R.; Lebrilla, C. B., Development of an annotated library of neutral human milk. J. Proteome Res. 2010, 9, 4138–4151. 47. Cataldi, T. R. I.; Angelotti, M.; Bianco, G., Determination of mono- and disaccharides in milk and milk products by high-performance anion-exchange chromatography with pulsed amperometric detection. Anal. Chim. Acta 2003, 485, 43-49. 48. Hurum, D. C.; Rohrer, J. S., Determination of sialic acids in infant formula by chromatographic methods: a comparison of highperformance anion-exchange chromatography with pulsed amperometric detection and ultra-high-performance liquid chromatography methods. J. Dairy Sci. 2012, 95, 1152-1161. 49. Slegte, J., Determination of trans galactooligosaccharides in selected food products by ion exchange chromatography collaborative study. J. AOAC Int. 2002, 85, 417-423. 50. McCleary, B. V.; Murphy, A.; Mugford, D. C., Measurement of total fructan in foods by enzymatic/spectrophotometric method: collaborative study. J AOAC Int. 2000, 83, 356-364. 51. Martín-Sosa, S.; Martín, M. J.; García-Pardo, L. A.; Hueso, P., Sialyloligosaccharides in human and bovine milk and in infant formulas: variations with the progression of lactation. J. Dairy Sci. 2003, 86, 52-59. 52. Wang, B.; Brand-Miller, J.; McVeagh, P.; Petocz, P., Concentration and distribution of sialic acid in human milk and infant formulas. Am. J. Clin. Nutr. 2001, 74, 510-515. 53. Tram, T. H.; Miller, J. C. B.; McNeil, Y.; McVeagh, P., Sialic acid content of infant saliva: comparison of breast fed with formula fed infants. Arch. Dis. Child. 1997, 77, 315-318. 54. Coppa, G. V.; Gabrielli, O.; Pierani, P.; Catassi, C.; Carlucci, A.; Giorgi, P. L., Changes in carbohydrate composition in human milk over 4 months of lactation. Pediatrics 1993, 91, 637-641. 55. Newburg, D. S.; Neubauer, S. H., Chapter 4 - Carbohydrates in milks: analysis, quantities, and significance. In Handbook of Milk Composition, Robert, G. J., Ed. Academic Press: San Diego, 1995; pp 273-349. 56. Choua, G.; El Kari, K.; El Haloui, N.; Slater, C.; Aguenaou, H.; Mokhtar, N., Quantitative assessment of breastfeeding practices and maternal body composition in Moroccan lactating women during six months after birth using stable isotopic dilution technique. Int. J. Matern. Child Health 2013, 1, 45-50. 57. Asakuma, S.; Urashima, T.; Akahori, M.; Obayashi, H.; Nakamura, T.; Kimura, K.; Watanabe, Y.; Arai, I.; Sanai, Y., Variation of major neutral oligosaccharides levels in human colostrum. Eur. J. Clin. Nutr. 2007, 62, 488-494. 58. Chaturvedi, P.; Warren, C. D.; Altaye, M.; Morrow, A. L.; Ruiz-Palacios, G.; Pickering, L. K.; Newburg, D. S., Fucosylated human milk oligosaccharides vary between individuals and over the course of lactation. Glycobiology 2001, 11, 365-372.


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59. Neville, M. C.; Keller, R.; Seacat, J.; Lutes, V.; Neifert, M.; Casey, C.; Allen, J.; Archer, P., Studies in human lactation: milk volumes in lactating women during the onset of lactation and full lactation. Am. J. Clin. Nutr. 1988, 48, 1375-1386. 60. Coulier, L.; Timmermans, J.; Bas, R.; Van Den Dool, R.; Haaksman, I.; Klarenbeek, B.; Slaghek, T.; Van Dongen, W., In-depth characterization of prebiotic galacto-oligosaccharides by a combination of analytical techniques. J. Agric. Food Chem. 2009, 57, 8488-8495. 61. EFSA NDA Panel (EFSA Panel on Dietetic Products Nutrition and Allergies), Statement on the safety of lacto-N-neotetraose and 2′-Ofucosyllactose as novel food ingredients in food supplements for children. EFSA Journal 2015, 13. 62. Steenhout, P.; Sperisen, P.; Martin, F.-P.; Sprenger, N.; Wernimont, S.; Pecquet, S.; Berger, B., Term infant formula supplemented with human milk oligosaccharides (2′Fucosyllactose and Lacto-N-neotetraose) shifts stool microbiota and metabolic signatures closer to that of breastfed Infants. The FASEB Journal 2016, 30, 275.7. 63. Goehring, K. C.; Marriage, B. J.; Oliver, J. S.; Wilder, J. A.; Barrett, E. G.; Buck, R. H., Similar to those who are breastfed, infants fed a formula containing 2′-Fucosyllactose have lower inflammatory cytokines in a randomized controlled trial. The Journal of Nutrition 2016, 146, 2559-2566. 64. Salminen, S., Regulatory Aspects of Human Milk Oligosaccharides. Nestle Nutrition Institute workshop series 2017, 88, 161-170. 65. Thurl, S.; Munzert, M.; Boehm, G.; Matthews, C.; Stahl, B., Systematic review of the concentrations of oligosaccharides in human milk. Nutr. Rev. 2017, 75, 920-933.

FIGURE CAPTIONS Figure 1. Schematic representation of the relative abundance of major oligosaccharide-groups in human milk at Day 3 and Day 42 of lactation (average of ten donors), and in infant formula (average of five brands) as analyzed by nano LC-chip/Q-TOF MS.



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TABLES

Table 1. Concentrations of bound sialic acid in mature human milk (Day 42) a and infant formula b Sialic acid (g/L)c Human milk (Day 42)

0.344±0.008

Infant formula

0.043±0.001

a

Average of ten donors.

b

Average of five infant formulas

c

Values are average ± SD of triplicate measurements

Table 2. Free monosaccharide and lactose concentrations in early human milk (Day 3)a, mature human milk (Day 42)a and infant formulab Galactose

Glucose (g/L) c

Lactose (g/L) c

(g/L) c Early milk (Day 3)

-

0.156±0.004

45.2±1.31

Mature human milk (Day 42)

Trace

0.236±0.009

56.7±0.92 30

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Infant formula

0.373±0.012

1.538±0.043

a


b

Average of five infant formulas

c

Values are average ± SD of triplicate measurements

56.8±1.83



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Table 3. Concentrations of oligosaccharides in early human milk (Day 3) a and mature human milk (Day 42)a Day 3 human

Day 42 human Change in concentration at Day

Oligosaccharide

milk (g/L)b

milk (g/L)b

42 compared to Day 3 (%)

LNDFHI

2.10±0.06

1.93±0.05

-8.1

LDFT

0.36±0.01

0.24±0.01

-33.8

2'-FL

3.75±0.10

2.48±0.13

-33.9

LNT

0.48±0.00

0.51±0.03

+6.2

LNFP I

1.81±0.03

0.58±0.03

-67.7

MFLNHI

0.11±0.01

0.11±0.01

0.0

LNH

0.08±0.01

0.16±0.01

+94.6

6'-SL

0.34±0.03

0.25±0.02

-27.1


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a

3'-SL

0.11±0.01

0.12±0.00

+9.0


b

Total

9.15±0.25

6.38±0.29

Values are average ± SD of triplicate

-30.3 measurements

Table 4. Contents of prebiotics (DP≥3) in five different US infant formula brands as obtained by HPAED-PAD. The Predominant Degree of polymerization (DP), evaluated by nano LC Q ToF, is also reported for GOS in all formula studied. Values are presented as average ± SD (between triplicates).

US formula formula A formula B formula C formula D formula E

Short-chain Galactooligosaccharides* of DP≥3 (g/L) 3.74±0.05 4.00±0.06 3.50±0.04 4.45±0.01 1.99±0.04

Long-chain Fructooligosaccharides (g/L)

Polydextrose Predominant (g/L) Degree of Polymerization (GOS)

n.a. n.a. n.a. 0.80±0.012 n.a.

n.a. n.a. n.a. n.a. 2.01±0.01

DP 3,4 and 5 DP 3,4 and 5 DP 3,4 and 5 DP 3,4 and 5 DP 3,4 and 5

* Although present 60, prebiotic DP2 is not considered in this table.



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Figure 1

100%

Neutral 5%

Fucosylated and sialylated 3%

Fucosylated and sialylated 3% Sialylated 5%

Sialylated 5%

Fucosylated 57%

Fucosylated 55%

Sialylated 3%

90% 80% 70%

Hexose oligomers 92%

60% 50% 40% 30%

[SERIES NAME] [VALUE]

20%

LNT 17%

Neutral 20%

10%

Neutral 20%

0% Human milk D3

Human milk D42

Infant formula


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