Changes in Caprine Milk Oligosaccharides at Different Lactation

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Changes in caprine milk oligosaccharides at different lactation stages analyzed by high performance liquid chromatography coupled to mass spectrometry Andrea Martin-Ortiz, Daniela Barile, Jaime Salcedo, F Javier Moreno, Alfonso Clemente, ANA ISABEL RUIZ MATUTE, and Maria Luz Sanz J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b05104 • Publication Date (Web): 10 Apr 2017 Downloaded from http://pubs.acs.org on April 11, 2017

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Journal of Agricultural and Food Chemistry

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Changes in caprine milk oligosaccharides at different lactation stages analyzed by

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high performance liquid chromatography coupled to mass spectrometry

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Andrea Martín-Ortiz1, Daniela Barile2, Jaime Salcedo2, F. Javier Moreno3, Alfonso

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Clemente4, Ana I. Ruiz-Matute1*, María L. Sanz1

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(Spain)

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Shields Avenue, Davis, CA 95616, USA

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Instituto de Química Orgánica General (CSIC). Juan de la Cierva, 3 28006 Madrid,

Department of Food Science and Technology, University of California Davis, One

Instituto de Investigación en Ciencias de la Alimentación, CIAL (CSIC-UAM), C/

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Nicolás Cabrera, 9, Campus de Cantoblanco - Universidad Autónoma de Madrid, 28049

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Madrid, Spain

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(CSIC), Profesor Albareda 1, 18008 Granada, Spain

Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas

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*Corresponding author:

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Email: [email protected]

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Tel. +34915622900

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Fax: +34925644853

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Abstract

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Changes of the abundance of caprine milk oligosaccharides (CMO) at different lactation

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stages has been evaluated by hydrophilic interaction liquid chromatography coupled to

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mass spectrometry (HILIC-Q MS) and nano-flow liquid chromatography-quadrupole-

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time of flight mass spectrometry (nano-LC-Chip-QTOF MS). Eight major

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oligosaccharides (OS) were quantified at different lactation stages by HILIC-Q MS,

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whilst the use of nano-LC-Chip-QToF MS allowed expanding the study to forty-nine

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different OS by monitoring neutral non- and fucosylated species, as well as acidic

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species containing not only N-acetyl-neuraminic acid or N-glycolyl-neuraminic acid

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residues but also the combination of both sialic acids. Overall, the most abundant OS

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decreased with lactation time, whereas different trends were observed for minor

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oligosaccharides. 6’-Sialyl-lactose was the most abundant acidic OS whilst galactosyl-

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lactose isomers were identified as the most abundant neutral OS. This is the first time

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that a comprehensive study regarding the changes of the abundance of CMO, both

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neutral and acidic, at different lactation stages is carried out.

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Keywords: goat milk oligosaccharides, lactation stages, hydrophilic interaction liquid

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chromatography, nanoflow liquid chromatography-quadrupole-time of flight mass

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spectrometry

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Introduction

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Human milk (HM) is considered the ideal food for neonates, not only because of its

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complex nutrients mixture fulfilling infant’s requirements but also because of the

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presence of bioactive components exerting important physiological effects for the

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neonate. 1 Among those, human milk oligosaccharides (HMOs) have been described to

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act as prebiotics and as potent inhibitors of bacterial adhesion to intestinal epithelial

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receptors.

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proportion in the gastrointestinal tract, they may play a role in the local intestinal

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immune system of the breast-fed infants, thus, acting in the prevention of infectious

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diseases. 2-8

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HMOs are complex structures with different glycosidic linkages, degrees of

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polymerization and monomeric composition including hexoses (Hex, D-glucose and D-

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galactose), N-acetyl-glucosamine (GlcNAc), L-fucose (Fuc), and sialic acid [N-acetyl-

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neuraminic acid (Neu5Ac)].

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present in HM,

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to acidic ones (10-25%).

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diversity, at present, it is not feasible to totally replicate HMOs profile by synthetic

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methods; only HMOs of lower molecular weight have been successfully produced by

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molecular engineering approaches.

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alternative sources with similar bioactive properties to HMOs that can be exploited by

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the food or pharmaceutical industry on a large scale, either through direct consumption

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or as a functional ingredient in infant formulas, mimicking biological functions of

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oligosaccharides present in HM. 15, 16 In this sense, naturally-occurring OS isolated from

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milk of animal species could be considered. 17, 18

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Moreover, as they are not digested and absorbed only in a small

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More than 200 different OS have been described to be

with predominance of neutral fucosylated OS (50-70%) compared 12, 13

However, due to their specific structural composition

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Therefore, there is great interest in finding



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Goat milk is known to contain oligosaccharides structurally similar to those present in

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HM

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Recently, in vitro studies have revealed the potential prebiotic activity of

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oligosaccharides from caprine whey.

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oligosaccharides isolated from goat milk reduce intestinal inflammation in rats with

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induced colitis

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Therefore, goat milk could be a potentially promising functional food ingredient for

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improving intestinal health, 24 especially if supplemented in infant formulae.

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Liquid chromatography (LC) is the most widespread technique for the analysis of

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oligosaccharides from mammal milks. Although several LC operation modes such as

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normal phase (NP) 25 and high performance anion exchange chromatography (HPAEC)

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4, 14, 22, 23, 26, 27

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interaction liquid chromatography (HILIC) is considered an efficient operation mode for

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the analysis of complex OS mixtures

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isomeric oligosaccharide and good peak shapes. In a recent study from our group, 20 the

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main OS in different goat colostra have been quantified by HILIC-Q MS. Additionally,

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up to 78 caprine milk oligosaccharides (CMO) structures were identified by nanoflow

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liquid chromatography-quadrupole-time of flight mass spectrometry (Nano-LC-Chip–

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Q-TOF MS). Combination of information provided by both techniques allowed

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obtaining comprehensive information, not provided in previous studies, about goat

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colostra oligosaccharide composition.

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Changes of the oligosaccharide abundance of human and bovine milks at different

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lactation stages have been widely studied, observing a decrease in their total content

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during lactation and a modification in the OS profile.

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about the changes of CMO composition during lactation. A transcriptomic study of

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and in higher concentrations than milks from other farm mammals.

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Moreover, it has been proved that

and they can improve barrier function of epithelial cell co-cultures.

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have been widely used for the analysis of these carbohydrates, hydrophilic

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providing appropriate resolution between

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However, little is known


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glycosylation-related genes in goat colostrum and milk obtained after 120 days of

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lactation has recently shown that most of them were more expressed in the former,

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which might be related to the higher oligosaccharides concentration found in colostrum.

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(6’-SL) and disialyl-lactose in colostra and milks of two goat breeds sampled at

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different stages of lactation (up to 90 days) was reported. 27, 35 In general, a decrease of

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OS content was observed during lactation, although 3’-SL contents increased in

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colostrum from 0 h to 24 h. Formiga de Sousa et al. 36 also detected a decrease in sialic

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acid monomers, obtained after hydrolysis of CMO, at different lactation stages (85, 105,

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125 and 145 days). However, to the best of our knowledge, a more comprehensive study

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of the evolution of both neutral and acidic CMO during lactation has not yet been

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carried out.

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Therefore, the present work investigated the changes of the abundance of CMO during

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the first months of lactation in individual and pooled milks from goats of the same breed

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(Murciano-Granadina) by HILIC-Q MS and Nano-LC-Chip–Q-TOF MS.

Recently, the content of three OS, namely 3’-sialyl-lactose (3’-SL), 6’-sialyl-lactose

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Materials and Methods

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Chemicals and reagents

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Acetic acid was from Normasolv (Barcelona, Spain); ammonium acetate and

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ammonium hydroxide were from Panreac (Barcelona, Spain), and ethanol of analytical

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grade was acquired from Lab-Scan (Gliwice, Poland). Acetonitrile (ACN) and formic

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acid (HPLC-MS grade) were obtained from Fisher-Scientific (Fair Lawn, NJ, USA).

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ESI-TOF Low concentration Tuning Mix G1969–85000 was acquired from Agilent

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Technologies (Santa Clara, CA, USA). Nanopure water (18.2 MΩ.cm, 25 °C) was used

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throughout all experiments.



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Analytical grade standards of 6’-sialyllactose (6’-SL) sodium salt, 3’-sialyllactose (3’-

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SL) sodium salt, 3’-sialyl-N-acetyllactosamine, 3’-galactosyl-lactose, 4’-galactosyl-

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lactose and 6’-galactosyl-lactose were purchased from Carbosynth (Berkshire, UK),

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whereas 3α,4β-galactotriose was obtained from Dextra Laboratories (Reading, UK) and

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2’-fucosyllactose (2’-FL) was from V-Labs (Covington, LA, USA). Nylon FH

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membranes (0.22 µm; Millipore, Bedford, MA, USA) were used for filtering

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ACN:water (50:50, v:v) standard solutions before injection.

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Milk samples

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Individual milk samples from one Murciano-Granadina goat (GM) were collected at an

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experimental farm at Estación Experimental del Zaidín (Granada, Spain). Samples were

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collected at days 2, 10, 20, 30 and 40 of lactation (GM2, GM10, GM20, GM30 and

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GM40, respectively). Moreover, milks kindly provided by Hermanos Archiduque farm

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(Granada, Spain) from twelve individual Murciano-Granadina goats (GP) were

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collected at days 1, 7, 30 and 120 and pooled (GP1, GP7, GP30 and GP120,

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respectively). GM2 and GP1 samples were considered colostrum. Samples were

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immediately frozen at -80 °C until their analysis. The Spanish guidelines for

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experimental animal protection (Royal Decree 53/2013) in line with the corresponding

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European Directive (2010/63/EU) were followed animals care and handle. The Ethics

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Committee for Animal Research from the Animal Nutrition Unit approved the

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experimental protocol.

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Fat and protein removal

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Samples were defatted by centrifugation at 6500 × g for 15 min at 5 ºC and kept in an

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ice bath for 30 min. Whatman Nº 1 filter paper was used for the removal of lipid layer

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supernatant.4


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The removal of the protein fraction was carried out by the addition of two volumes of

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cold ethanol to the skimmed colostrum and milk samples. After shaking for 2 h in an ice

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bath, solutions were centrifuged at 6500 × g for 30 min at 5 ºC. Supernatants were

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collected and ethanol was removed ple in a rotary evaporator (Büchi Labortechnik AG,

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Flawil, Switzerland) at 37 ºC. The carbohydrate fraction present in the aqueous solution

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was frozen and freeze-dried.

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Chromatographic analyses

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HILIC-Q MS

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CMO quantification was performed on an Agilent 1200 series HPLC system (Hewlett-

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Packard, Palo Alto, CA, USA) equipped with an oven (Kariba Instruments, UK) and

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coupled to a quadrupole HP-1100 mass detector (Hewlett-Packard) with an electrospray

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ionization (ESI) source using the method previously optimized by Martín-Ortiz et al. 20.

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Samples were diluted with 50:50 (v:v) acetonitrile (ACN):water, filtered through a 0.22

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µm filter (Millipore, Madrid , Spain) and 5 µL were injected.

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An ethylene bridge hybrid with trifunctionally-bonded amide phase (BEH X-Bridge

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column; 150 x 4.6 mm; 3.5 m particle size, 135 Å pore size, Waters, Hertfordshire, UK)

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at a flow rate of 0.4 mL min-1 was used for LC experiments. For these assays, a gradient

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of ACN:water with addition of 0.1% ammonium hydroxide was used as follows: from 0

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to 46 min a linear gradient from 15 to 50% aqueous phase, kept 10 min at these

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conditions, then ramped to original composition in 1 min and finally equilibrated for 15

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min. The ESI source was operated under positive polarity using 4 kV capillary voltage

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at 300 ºC with a nitrogen drying gas flow of 12 L min−1 and a nebulizer (N2, 99.5%

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purity) pressure of 276 kPa; fragmentor voltage was varied from 80 to110 V. Adducts

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formed under optimal conditions were evaluated. Ions corresponding to [M+Na]+ of the

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oligosaccharides under analysis were monitored in SIM mode using default variable



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fragmentor voltages. HPChem Station software version 10.02 (Hewlett-Packard) was

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used for data processing.

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Quantitative analysis was performed in triplicate by the external standard method, using

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calibration curves within the range 5-100 mg L-1 for 3’-SL, 6’-SL, 3’-sialyl-N-acetyl-

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lactosamine and 3’-galactosyllactose. Considering the absence of glycoly-neuraminyl-

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lactose (Neu5Gc-lactose) as commercial standard, the responses of N-acetyl-neuraminic

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and N-glycolyl-neuraminic acids were evaluated. Negligible differences were observed

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between the responses of these monosaccharides; therefore, Neu5Gc-lactose isomers

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were quantified using 3’-SL calibration curves. Reproducibility of the method was

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estimated on the basis of the intra-day and inter-day precision, calculated as the relative

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standard deviation (RSD) of retention times and concentrations of oligosaccharide

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standards obtained in five independent measurements. Good precision values were

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obtained (RSD ranging 6–8%). Limit of detection (LOD) and limit of quantitation

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(LOQ) were calculated as three and ten times, respectively, the signal to standard

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deviation of the noise ratio (S/σΝ). Values ranging from 0.03 - 0.16 mg mL−1 and 0.1 -

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0.5 mg mL−1 were obtained for LOD and LOQ, respectively.

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Nano-LC-Chip–Q-TOF MS -

Sample preparation

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Considering that the high content of lactose present in goat milk would cause

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interference with the analysis of lower abundance oligosaccharides, this disaccharide

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was removed from milk samples by size exclusion chromatography (SEC) using a Bio-

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Gel P2 (Bio-Rad, Hercules, CA, USA) column (90 × 5 cm). Water was selected as the

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mobile phase at a flow of 1.5 mL min-1 at 4 ºC. Oligosaccharide fraction was dissolved

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in water (20% wt:v) and 25 mL was injected into the column. Fractions of 10 mL were


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collected. The degree of polymerization (DP) of collected fractions was determined by

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ESI-MS (Agilent 1200 series HPLC system, Hewlett-Packard) at positive polarity

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selecting the corresponding m/z values. Fractions with DP ≥3 were pooled and freeze-

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dried.

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-

MS analysis

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Prior to MS analysis, purified and dried OS of GM and GP samples were reconstituted

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to a final concentration of 0.1 mg mL-1 with nanopure water, 2’-FL being added as

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internal standard (final concentration of 0.001g L-1). MS analysis was carried out using

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an Agilent 6520 accurate-mass Quadrupole-Time-of-Flight (Q-TOF) LC/MS with a

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microfluidic nano-electrospray chip containing a graphitized carbon enrichment and

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analytical columns (Agilent Technologies, Santa Clara, CA, USA) as previously

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described.

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90% ACN/0.1% formic acid in water (solvent B) at a flow rate of 0.3 µL min-1 for the

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nano pump and an isocratic method (100% A) at a flow rate of 4 µL min-1 for the

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capillary pump were applied. Positive ionization mode with a mass/charge (m/z) range

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of 450–2500 was selected for data acquisition. Automated precursor selection was

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employed based on abundance, with up to 6 MS/MS per MS. A precursor isolation

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window of 1.3 m/z and a fragmentation energy set at 1.8 V/100 Da was used. Reference

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masses of m/z 922.009 and 1221.991 were selected for internal calibration (ESI-TOF

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Low concentration Tuning Mix G1969–85000, Agilent Technologies).

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OS identification was performed using the Find Compounds by Formula function of

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Mass Hunter Qualitative Analysis Version B.06.00 (Agilent Technologies) which

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allowed matching the masses of oligosaccharides with the caprine milk oligosaccharide

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databases, OS composition confirmed by MS/MS as described in literature.

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A binary gradient of 3% ACN/0.1% formic acid in water (solvent A), and

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Abundances of each OS relative to the internal standard (Area OS/Area i.s.) were obtained

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in triplicate.

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Statistical analysis

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Statistica version 7.1 (2005) by Statsoft Inc. (Tulsa, USA) was used for statistical

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analyses. One-way ANOVA test followed by a Fisher test as a post hoc comparison of

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means were used to determine significant differences (P

Changes in Caprine Milk Oligosaccharides at Different Lactation

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