In Vitro Fermentation of Porcine Milk Oligosaccharides and Galacto

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In vitro fermentation of porcine milk- and galactooligosaccharides using piglet fecal inoculum E. Difilippo, Feipeng Pan, Madelon Logtenberg, Rianne (H.A.M.) Willems, S. Braber, J. Fink-Gremmels, Henk A. Schols, and H. Gruppen J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b05384 • Publication Date (Web): 22 Feb 2016 Downloaded from http://pubs.acs.org on February 22, 2016

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Journal of Agricultural and Food Chemistry

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Abstract

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In this study, the in vitro fermentation by piglet fecal inoculum of galacto-oligosaccharides

3

(GOS) and porcine milk oligosaccharides (PMOs) was investigated in order to identify

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possible preferences for individual oligosaccharide structures by piglet microbiota. Firstly

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acidic PMOs and GOS with degree of polymerization 4-7 were depleted within 12 h of

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fermentation, whereas fucosylated and phosphorylated PMOs were partially resistant to

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fermentation. GOS structures containing β1-3 and β1-2 linkages were preferably fermented

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over GOS containing β1-4 and β1-6 linkages. Upon in vitro fermentation, acetate and butyrate

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were produced as the main organic acids. GOS fermentation by piglet inoculum showed

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unique fermentation pattern with respect to preference of GOS size and organic acids

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production.

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Keywords: human fermentation, fibers, non-digestible carbohydrates, gas chromatography,

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liquid chromatography, short chain fatty acids, sugars, organic acids

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ACS Paragon Plus Environment


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Introduction

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Milk is the first food source for infants, providing not only energy but also offering protection

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against pathogens.1 The third largest class of compounds present in human milk are

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oligosaccharides (HMOs).1 Numerous health related effects of HMOs, involving direct

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interactions with pathogen and/or indirect effects upon their fermentation by the intestinal

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microbiota have been reported.2 When human milk is lacking during the early stage of life of

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infants, cow milk based infant formula is used.3, 4 However, cow milk has a low content of

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milk oligosaccharides, therefore in many cases infant formulas are fortified with prebiotics

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such as galacto-oligosaccharides (GOS).5,

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ingredients that can influence the intestinal microbiota composition resulting in beneficial

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effects for the host.5, 7, 8 GOS are produced from lactose using β-galactosidases of microbial or

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yeast source.9 The enzymatic reaction results in a GOS mixture containing oligosaccharides

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varying in degree of polymerization, mostly from 2 to 8, and in types of glycosidic linkages

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present.5 Commonly, GOS have a glucose monomer at their reducing end to which multiple

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galactose monomers with different linkages (β1-2, β1-3, β1-4 and β1-6) are attached.10 GOS

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exhibit less structural complexity than HMOs. The major part GOS resist degradation in the

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upper intestinal tract, consequently reaching the colon, as is the case for HMOs.8, 11-13 In the

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colon, GOS enhance Bifidobacteria growth, potentially protecting the infant against infections

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and pathogen development.2,

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(SCFAs) are produced, among which butyrate.15 Butyrate have been shown to be anti-

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inflammatory and anti-carcinogenic, also enhancing (in vitro) the intestinal barrier function.5,

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16, 17

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Prebiotics are defined as non-digestible food

Moreover, upon GOS fermentation, short chain fatty acids

Recent study showed that in vivo GOS fermentation in young pigs (piglets) was

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extensive, not being conclusive on microbiota utilization towards individual GOS structures.18

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The understanding of the preference of microbiota towards specific oligosaccharide structures 3


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is important for the understanding of bacterial metabolization by the intestinal microbiota

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including enzyme activity involved and to target specific oligosaccharides to be more

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beneficial for the gut microbiota for animal health. In the present study, in vitro fermentation

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by piglet fecal inoculum was used in order to control different stages of fermentation, thereby

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focusing on the fate of individual oligosaccharides and organic acids produced.

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Oligosaccharides investigated were from porcine milk as present as primary feed source at

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early stage of piglet life and GOS.

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Materials and methods

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Acetic acid, acetonitrile (ACN), ammonium formate, formic acid, chloroform and methanol

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were purchased from Biosolve BV (Valkenswaard, The Netherlands). Butyric acid, lactic

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acid, succinic acid, maltotriose, 2-(N-morpholino)ethanesulfonic acid, 2-ethylbutyric acid,

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sodium chloride, 2-picoline borane complex and sodium hydroxide were purchased from

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Sigma Aldrich (Steinheim, Germany). Propionic acid, oxalic acid and 2-aminobenzamide

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were purchased from VWR International (Amsterdam, The Netherlands). β3’-, β4’- and β6’-

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Galactosyllactose were purchased from Carbosynth (Compton, UK), while 3’- and 6’- sialyl-

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N-acetylactosamine, 3’-fucosyllactose, lacto-N-(neo)tetraose and lacto-N-(neo)hexaose were

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purchased from Dextra (Reading, UK). 3’- and 6’-Sialyllactose and lacto-N-fucosylpentaose

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were purchased from Sigma Aldrich. Water was filtered using a Milli-Q water purification

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system (Millipore, Darmstadt, Germany) and it was referred as water in the text. Vivinal GOS

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powder (FrieslandCampina Domo, Borculo, The Netherlands), dry matter (DM) 97%, with

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GOS 69%, lactose 23%, glucose and galactose together 5% on DM, is referred to in the text

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as GOS. Composition of GOS-DPs are 31% for GOS-DP2 (different from lactose), 38% for

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DP3, 18%

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manufacture.

for DP4, 8% for DP5 and 5% w/w for GOS-DP≥5, as reported by the

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Oligosaccharide extraction and separation

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Porcine milk oligosaccharides (PMOs) from porcine colostrum (37 mL) (Proefaccommodatie

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de Tolakker, Utrecht University, Utrecht, The Netherlands) were extracted, freeze-dried and

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dissolved in water (70 mg/mL), and subsequently separated using Size Exclusion

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Chromatography (SEC) as reported elsewhere.19 Collection of 103 fractions (7 mL each) was

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performed as described before.19 In total, 4 runs were performed with an injection volume of

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2.86 mL each injection. Afterwards, the fractions were pooled and freeze dried: pool 1:

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fraction 290 - 500 mL, pool 2: fraction 501 - 697 mL, pool 3: fraction 698 - 719 mL, pool 4:

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fraction 720- 741 mL, pool 5: fraction 742 - 770 mL. The freeze-dried pools were dissolved

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in 36 mL water. For each pool, 1 mL was used for oligosaccharide characterization, while the

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remaining volumes were mixed and freeze dried again to obtain porcine milk oligosaccharides

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(PMOs) with a reduced amount of lactose and PMOs with DP2.

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Fermentation of GOS and PMOs by pig fecal inoculum

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Culture medium

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The culture medium was based on a modified simulated ileal environment medium (SIEM),

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not containing carbohydrates prepared as reported elsewhere.20 The pH was set at 6.0 using

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MES buffer. All the culture medium components were provided by Tritium Microbiology

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(Veldhoven, The Netherlands).

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Fecal inoculum

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Inoculum for the in vitro fermentation was obtained from fecal material from 3 Dutch

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Landrace piglets (age of 3 weeks). The piglets were fed on porcine milk over the feeding

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period, combined with pig formula (de Heus, Ede, The Netherlands) for the 2nd day to the 8th

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day of life. From the 9th day of life until the end of the 3 weeks the piglets received pellets (de 5


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Heus). Immediately after defecation, fecal material from each piglet was collected in

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eppendorf tubes, which was immediately put into an insulated box with crushed ice and

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afterwards stored at -80 °C. The pig fecal inoculum was prepared as described elsewhere,

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with some minor modifications.21 Feces (± 100mg) of three piglets were pooled after

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defrosting in an anaerobic cabinet (gas phase: 96% N2 and 4% H2). The pooled feces were

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diluted 6 times (w/v) with sterilized 0.9% (w/v) NaCl solution and subsequently homogenized

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using a vortex. The homogenization was facilitated by the addition of sterile glass beads. The

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fecal solution was added to the culture medium in a 20 mL flasks at a 5:82 (v/v) ratio, in order

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to obtain the final inoculum. Afterwards, the bottle was closed with rubber stoppers and

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aluminum caps to maintain anaerobic conditions. Next, the inoculum was kept overnight into

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an incubator shaker (Innova 40, New Brunswick Scientific, Nijmegen, The Netherlands) (39

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°C, 100 rpm) to stabilize the bacterial population.

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In vitro fermentation

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GOS and PMOs, obtained as described above, were used as substrates for the fermentation by

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pig fecal inoculum. The in vitro fermentation experiment was performed as described

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elsewhere with minor modifications.21 GOS or PMOs were added to the culture medium with

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a final substrate concentration of 11.1 mg/mL. The fecal inoculum was added to the solution

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containing substrate and medium in a 1:10 ratio (v/v) and a final volume of 10 mL for the

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fermentation flasks. Control samples consisting of culture medium without substrate were

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used to monitor the background fermentation. All procedures were performed in an anaerobic

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cabinet. Flasks were closed with rubber stoppers and aluminum caps, and they were put into

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an incubator shaker (Innova 40) (100 rpm, 39 °C). After 9, 10, 12, 24 and 48 hours of the

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fermentation, two times 70 µL were taken from the fermentation bottles with a syringe and

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transferred to eppendorf tubes. In order to inactivate fecal enzymes, eppendorf tubes were put

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into a boiling water bath for 5 min and stored afterwards at -20oC.

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Characterization and quantification of GOS and PMOs

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Samples after fermentation were cleaned prior to analysis by hydrophilic interaction liquid

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chromatography with mass spectrometry (HILIC-ESI-MSn). The samples after fermentation

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were diluted 10 times in water and 150 µL were placed into a polyvinylidene fluoride

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centrifugal filter unit (0.22 µm, Merck Millipore, Amsterdam, The Netherlands) and

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centrifuged (5 min, 20000g, 20oC). Afterwards, ACN was added to the filtrate in a ratio of 1:1

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(v/v) and the samples were centrifuged (5 min, 20000g, 20oC). Supernatants were analyzed on

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a Thermo Accela Ultra High Liquid Chromatography (UHPLC) system (Thermo Scientific,

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Waltham, MA, USA.) as described elsewhere.22 Briefly, The mobile phases used were: A =

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water + 1% (v/v) acetonitrile (ACN), B = ACN, and C = 200 mM ammonium formate buffer

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(pH 4.5) with a flow rate of 300 µL/min. The elution profile was: isocratic 85% B (0-1 min);

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85-60% B (1-31 min); from 60-40% B (31-35 min); isocratic 40% B (35-36 min); from 40-

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85% B (36-36.1 min); isocratic 85% B (36.1-45 min). The mobile phase C was kept at 5%

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during the entire gradient, with auto-sampler and column oven temperature of 15 and 35 °C,

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respectively.

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In order to recognize GOS linkage type, HPAEC-PAD was used and the HPAEC profiles

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were compared with profiles as reported elsewhere.10, 15 Oligosaccharides standards were used

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for PMO characterization and quantification by HILIC-MSn.. Calibration curves of the

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corresponding milk oligosaccharide standards and GOS (0.002 to 0.5 mg/mL respectively)

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were used in order to quantify PMOs and GOS by mass spectrometry. Area and concentration

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of individual GOS-DP were obtained by selecting the mass range for each GOS-DP and

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taking into account the relative amount of a given GOS-DP in the total GOS sample. If no 7


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corresponding standards for neutral PMOs were available, GOS with a comparable DP was

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used for the calibration curve. The curves fitting the PMO standards showed a linear

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correlation with R2 of 0.90-0.99, while the curves fitting the GOS-DPs showed a linear

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correlations with R2 of 0.98-0.99.

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Organic acid analysis

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In order to quantify acetate, propionate and butyrate produced during the fermentation, GC

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analysis was performed as described elsewhere with minor modifications.15 Samples after

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fermentation were diluted 10 times with water. Aliquots (50 µL) and SCFAs standards (0.01-

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3 mg/mL) were mixed with 0.15 M oxalic acid (50 µL). The solutions were kept for 30 min

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before addition of 0.025 mg/mL 2-ethyl butyric acid as internal standard in water (143 µL). In

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order to analyze succinic and lactic acid, high performance liquid chromatography with

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refractive index detection was performed as described elsewhere.15 Briefly, Dionex system

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(Ultimate 3000 HPLC) equipped with Aminex HPX-87H column with a guard column (Bio-

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Rad, Hercules, CA, USA) was used, with a H2SO4 (5 mM) mobile phase with a flow rate of

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0.6 mL/min at 65oC.

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Results and discussion

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Fermentation of porcine milk oligosaccharides (PMOs)

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As a result of rapid colonic bacterial colonization, piglets can extensively ferment dietary

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fibers.22-26 GOS and PMOs present in the diet of piglets are almost completely fermented in

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the colon, resulting in trace amounts of oligosaccharides present in the feces other than intact

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GOS or PMO structures.18,

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fermentation of GOS is suggested to start already in the small intestine and to continue

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extensively in cecum and colon.24, 25 In order to study any preference and differentiation in

22

Similarly as found for dietary fibers, piglets intestinal

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rate of fermentation among GOS and PMO structures, in vitro fermentation was performed

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using pig fecal inocula. Prior to monitor their degradation profiles, the main PMOs were

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identified and quantified by HILIC-MSn.19 Through single ion monitoring, in total thirteen

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main PMOs representing about 77% of the total peak area, were identified and monitored

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during the in vitro fermentation. Using previously gained knowledge,22 PMOs identified were

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the neutral β3’- and β6’- galactosyllactose (β3’- and β6’-GL), lacto-N-neotetraose (LNnT),

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lacto-N-pentaose-I (LNP-I), lacto-N-neohexaose (LNnH), 2’- and 3’- fucosyllactose (2’- and

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3’-FL); and the acidic 3’- and 6’- sialyllactose (3’- and 6’-SL), 3’- and 6’- sialyl-N-

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acetyllactosamine (3’- and 6’-SLN), phosphorylated lactose (P+ lactose) and Neu5Ac(α2-

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6)GlcNAc(β1-3)Gal(β1-4)Glc. The degradation of individual PMOs during fermentation was

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indirectly expressed via the abundance of remaining oligosaccharides present (Figure 1).

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Overall, a decrease in concentration upon fermentation was observed for each PMO, with

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individual differences in degradation rate (Figure 1). One exception was phosphorylated

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lactose, which increased in concentration during the first 12 h of fermentation, while being

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partially depleted afterwards (Figure 1 C). The lactose present was attributed partially to the

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small amount of lactose still present after purification with SEC and partially to lactose

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released by bacterial degradation of PMOs. Lactose was the first carbohydrate to be fully

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fermented by the porcine microbiota, decreasing approximately 98% during the first 9 h of

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fermentation. Trimers, such as β3’-GL, β6’-GL, 2’-FL and 3’-FL were all fermented with a

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different rate.

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The trimers β3’-GL and β6’-GL diminished about 50% of their initial concentrations in the

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first 9 h of fermentation, whereby β3’-GL is more quickly fermented than β6’-GL (Figure 1

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B). LNnH is fermented faster than LNnT and LNnP-I, decreasing 80% of its original 9


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concentration during 12 h of fermentation. All the neutral PMOs are almost completely

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fermented within 24 h, whereas LNnT still was not consumed completely. Most probably,

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part of the LNnT present at 24 h is released by the degradation of larger PMOs.27 LNnT is

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totally consumed in the subsequent 24 h by the piglet colonic microbiota. Acidic PMOs are

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fermented for 50% during the first 12 h and are fermented faster than neutral PMOs (Figure 1,

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C). Surprisingly, the phosphorylated lactose (P+lactose) more than doubled in concentration

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during the first 12 h of fermentation, with a subsequent decrease to about 50% of its starting

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concentration. Hypothetically, the increase of phosphorylated lactose could be derived from

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fermentation of larger phosphorylated PMOs present, although they were not reported so far

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to be present in domestic animal milk samples.22, 27 Overall, fermentation of acidic and neutral

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PMOs was almost complete in 48 h, with the exception of fucosylated PMOs and

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phosphorylated lactose. The resistance of fucosylated structures during the in vitro

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fermentation might be an indication of a relevant physiological role by preventing microbial

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pathogens for binding to intestinal cells in the piglets, as suggested for human studies.28, 29 In

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conclusion, in vitro fermentation with piglet microbiota showed a rapid consumption of

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PMOs, with different fermentation rates detected for individual PMOs. Piglet microbiota

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firstly ferment small PMOs, such as neutral and acidic trimers, and afterwards neutral larger

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PMOs, each with its own specific rate.

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Fermentation of galacto-oligosaccharides (GOS)

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Although GOS are not naturally occurring in the piglet diet, the in vitro fermentation of GOS

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by piglet fecal microbiota showed that GOS is quite an easy fermentable substrate, as, on

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average, 36% of residual GOS were present after 12 h of fermentation. However, a difference

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in fermentation rate was noticed concerning the different GOS-DPs, being present for the 59,

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42, 29, 27 and 25% for DP3, 4, 5, 6 and 7, respectively, after 12 h of fermentation. GOS-DP2 10



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were fermented first, as expected from a previous investigation on GOS fermented by human

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fecal inoculum.15 After 24h of fermentation, almost all GOS were metabolized. Lactose as

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present in the PMOs mixture was totally consumed within the first 12 h of fermentation

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(Figure 1, A), whereas GOS-DP2 including lactose, was still present for 12% after 12 h of

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fermentation. Comparing DP2 oligosaccharides in PMOs and GOS (at time point 12 h) it can

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be concluded that lactose was fermented much faster than non-lactose GOS-DP2 structures.

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Lactose was introduced in the pig diet not only by nursing, but also by the cow milk based pig

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formula, presumably leading to adaptation of the pig microbiota to lactose. On the contrary,

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GOS-DP2 were not present at any phase of the pig diet. Hence, a slower adaptation of the

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microbiota than for lactose was expected. For GOS-DP3-7 (Figure 2), longer fermentation

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times compared with fermentation times found for dimers were observed (9-12 h), with

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different utilization rates depending on the DP. In the first 12 h of in vitro fermentation by

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piglet fecal inoculum, GOS-DP5-7 were mostly degraded (about 30% remaining

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oligosaccharides), while GOS-DP3 was still present in relatively high abundance (60%

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remaining oligosaccharides) (Figure 2). This was not observed during 12 h of in vitro

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fermentation of GOS by human fecal inoculum, where about 70% of remaining GOS-DP4-6

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and about 50% of remaining GOS-DP3 were observed.15 After 24h of fermentation, all GOS-

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DPs showed to be fully fermented without DP discrimination. As expected from GOS

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fermentation by human fecal inoculum, also piglet fecal microbiota showed preference

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regarding specific GOS structures.15 Using HPAEC-PAD analysis, not only the decrease

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during fermentation time of GOS-DP, but also the decrease of individual GOS isomers was

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monitored. In Figure 3, GOS peaks are assigned with numbers as reported previously.30

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Isomeric GOS structures were degraded differently depending on their structures as can be

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seen for lactose and for Gfadapta 11


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OS-DP2 such as Gal(1-3)Gal, Gal(1-3)Glc and Gal(1-2)Glc (Figure 3, numbers 2.3, 2.5 and

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2.6, respectively). This observation was similar to that observed in a previous study, reporting

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that β1-2 and β1-3 linkages were more easily degradable than β1-4 linkages during GOS-

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DP2 fermentation by human fecal inoculum.15 As expected from the HILIC-MSn profile,

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GOS-DP3 were harder to degrade than GOS-DP2. The trimers indicated by numbers 3.8 and

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3.9 were easier to ferment by pig inoculum than trimers numbers 3.4, 3.5, 3.6 and 3.7 (Figure

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3), as expected from GOS fermentation by human fecal inoculum.30

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A relevant difference between piglet and human inocula, was noticed for Gal(β1-4)Gal(β1-

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4)Glc and Gal(β1-4)Gal(β1-4)Fru (numbers 3.6 + 3.7, Figure 3), which accumulated with an

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increase of 17% during the first 12 h of fermentation by piglet fecal inocula, while they were

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easily fermentable substrates for the human microbiota.15 A similar preference towards the

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degradation of specific linkages was noticed when comparing piglet and human in vitro

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fermentation: β1-3/β1-2 linkages were preferred over β1-4/β1-6 linkages for GOS-DP2-3.

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Literature describes human and pig microbiota composition to be similar, mainly consisting

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of Firmicutes and Bacteriodes.31 On the other hand, differences were reported to occur in

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Bifidobacteria species, relevant for GOS degradation.31 Studies reported on extremely low

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amounts of Bifidobacteria species present in the gastro-intestinal tracts of piglets (< 1% of the

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total bacteria present).

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differ from the species reported in human intestinal tract.31, 34 Our results indicate a difference

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in microbiota composition based on the fermentation characteristics of oligosaccharides.

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Organic acid production upon PMOs and GOS fermentation by piglet fecal inoculum

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The degradation of PMOs and GOS by piglet fecal inoculum resulted in the production of

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organic acids: acetate (A), succinate (S), butyrate (B), lactate (L) and propionate (P) as shown

31-33

Certain Bifidobacteria species present in piglet intestine could

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in Figure 4. The molar ratios A:S:B:L:P observed after 24 h of fermentation were of

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49:1:20:2:27 and 45:1:21:4:29 for PMOs and GOS, respectively. Acetate was the most

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abundant organic acid detected after 24 h of PMOs and GOS fermentation by piglet fecal

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inoculum. Similarity acetate was the most abundant organic acid produced for GOS in vitro

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fermentation by human fecal inoculum.35 Next to acetate, also propionate and butyrate (Figure

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4, A and B) were produced for both GOS and PMOs fermentation by piglet fecal inoculum,

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whereas mainly succinate and propionate were co-produced for GOS fermentation by human

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fecal inoculum.15,

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human inoculum showed differences in A:S:B:L:P molar ratios: 45:1:21:4:29 and 75:10:6:2:8,

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for piglet and human inoculum, respectively.15, 16 Overall, after fermentation for 24 h, butyrate

261

was produced in higher abundance upon GOS fermentation by piglet microbiota than by

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human microbiota.

263

16

For 24 h of in vitro fermentation, GOS fermentation with piglet and

The absolute amount of butyrate was of 1.4 and 0.7 µmol/mg substrate for GOS fermented by

264

piglet and human inoculum, respectively.15,

16

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inoculum, about 7 and 4 µmol organic acids per mg of substrate were produced for GOS and

266

PMOs, respectively. Fermentation of GOS by human fecal inocula resulted in about 12

267

µmol/mg organic acids production in 24 h of fermentation.15 Overall, in the intestinal tract,

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1010-11 and 1014 bacteria per gram of intestinal content for piglets and human are present,

269

respectively.31, 36

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In summary, differently from in vivo piglet fermentation, in vitro fermentation provide the

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opportunity to observe the preference of piglet microbiota on fermentation of individual

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oligosaccharides. During in vitro fermentation, acidic PMOs and neutral PMO trimers were

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the first to be degraded followed by larger neutral PMOs. Fucosylated and phosphorylated

During fermentation for 24 h by piglet

13


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PMO were the most resistant to fermentation. Interestingly, while PMO trimers were quickly

275

degraded, GOS trimers showed a slow apparent degradation rate for the first 12 h of

276

fermentation. GOS trimers degradation rate was hypothesized to be influenced not only by

277

utilization by the microbiota, but also by the release of trimeric degradation products upon

278

fermentation of larger GOS. In addition, GOS containing β1-3 and β1-2 linkages were faster

279

fermented than GOS containing β1-4 and β1-6 linkages. Major products upon PMOs and

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GOS fermentation by piglet microbiota were acetate, propionate and butyrate.

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glycoscience. From chemistry to system biology, Kamerling, J. P.; Boons, G. J.; Lee, Y. C.;

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397

Figure Captions

398

Figure 1. Proportion (%) of remaining PMOs during in vitro batch fermentation by piglet

399

fecal inocula. Concentration per individual PMOs at t = 0 was set to 100%. In A) lactose and

400

2’-, and 3’- fucosyllactose (2’-, and 3’-FL) are shown; in B) β 3’-, and 6’- galactosyllactose (β

401

3’-, and 6’-GL), lacto-N-neotetraose (LNnT), lacto-N-pentaose-I (LNP-I), and lacto-N-

402

neohexaose (LNnH) are shown; while in C) 3’-, and 6’- sialyllactose (3’-, and 6’-SL), 3’-, and

403

6’- sialyl-N-acetyllactosamine (3’-, and 6’-SLN), phosphorylated lactose (P+ lactose), and

404

Neu5Ac(α2-6)GlcNAc(β1-3)Gal(β1-4)Glc are shown.

405

Figure 2. Proportion (area %) of remaining GOS during in vitro fermentation by piglet fecal

406

inoculum as measured by HILIC-MSn. Concentrations per PMOs at t=0 were set to 100%. DP

407

= degree of polymerization.

408

Figure 3. HPAEC-PAD chromatograms of GOS fermentation with piglet fecal inoculum,

409

with structures of oligosaccharides as present in the GOS mixture (2.1-3.9) (according to

410

Ladirat et al.).30 DP= degree of polymerization

411

Figure 4. Organic acid amount and relative concentration during in vitro fermentation of

412

GOS (A) and PMOs (B) by piglet fecal inoculum and of GOS by human fecal inocula. DP =

413

degree of polymerization. Peak numbers correspond to structures in the insert.

414

20



Page 20 of 25

Figures Figure 1.

21


Page 21 of 25


Figure 2.

Remaining oligosaccharides (% area)

120 100

DP 2 , including lactose

80

DP 3 60

DP 4 DP 5

40

DP 6 20

DP 7

0 0

12

24

36

48

Hours

22



Page 22 of 25

Figure 3. Peak

Compound

Glc/Gal Glucose/Galactose 2.1

β-D-Gal-(1↔1)-α-D-Glc + β-D-Gal-(1↔1)-β-D-Glc

2.2 (L)

β-D-Gal-(1→4)-D-Glc (Lactose)

2.3

β-D-Gal-(1→3)-D-Gal

2.4


2.5

β-D-Gal-(1→3)-D-Glc

2.6

β-D-Gal-(1→2)-D-Glc β-D-Gal-(1→4)-β-D-Gal-(1→6)-D-Glc

3.4

or β-D-Gal-(1→6)-β-D-Gal-(1→4/6)-D-Glc β-D-Gal-(1→4)-β-D-Gal-(1→6)-D-Glc

3.5

or β-D-Gal-(1→6)-β-D-Gal-(1→4/6)-D-Glc

3.6 + 3.7

β-D-Gal-(1→4)-β-D-Gal-(1→4)-D-Glc + β-D-Gal-(1→4)-β-D-Gal-(1→4)-Fru β-D-Gal-(1→4)-β-D-Gal-(1→3)-D-Glc

3.9

β-D-Gal-(1→4)-β-D-Gal-(1→2)-D-Glc 2.2 (L)

3.6 + 3.7

3.8


2.4


2.5


2.6


DP4-c

β-D-Gal-(1→4)-β-D-Gal-(1→6)-D

or β-D-Gal-(1→6)-β-D-Gal-(1→4/

β-D-Gal-(1→4)-β-D-Gal-(1→6)-D

or β-D-Gal-(1→6)-β-D-Gal-(1→4/

β-D-Gal-(1→4)-β-D-Gal-(1→4)-D

+ β-D-Gal-(1→4)-β-D-Gal-(1→4)-

3.8

β-D-Gal-(1→4)-β-D-Gal-(1→3)-D

3.9

β-D-Gal-(1→4)-β-D-Gal-(1→2)-D

DP6 DP7

DP5 DP6

DP5

3.6 + 3.7 DP4-d

3.9 DP4-b

DP4-a

3.5

2.3

DP3

2.1

3.4 2.4 2.5 2.6 3.5

2.2 (L)

Glc/Gal

3.8

3.4

Response(nC)

β-D-Gal-(1→4)-D-Glc (Lactose)

2.3

t0 t12 t24

2.0

4.0

6.0

8.0

10.0

12.0

14.0

16.0

Time (min)


18.0

20.0

t48 22.0 23

Page 23 of 25


µmol/mg substrate

Figure 4.

A) 15.0

lactate 10.0

succinate butyrate

5.0

propionate acetate

0.0 0

10

12

24

48

24

48

Hours

B) II) µmol/mg substrate

15.0

10.0

5.0

0.0 0

10

12

415

Hours

24



Page 24 of 25

Table of Contents (TOC) Graphic

Oligosaccharide fermentation with piglet inocula Porcine milk oligosaccharides

Galacto-oligosaccharides

100

µmol/mg substrate

6

5

4 50 3

2

1

0

Remaining oligosaccharides (% )

7

0 0

10

12

24

48

0

10

12

24

48

Fermentation time (hours) Lactate

Succinate

Butyrate

Propionate

Acetate

pmo%

25


In Vitro Fermentation of Porcine Milk Oligosaccharides and Galacto

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