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Enzymatic synthesis and purification of galactosylated chitosan oligosaccharides reducing adhesion of enterotoxigenic Escherichia coli K88 Ya Lu Yan, Ying Hu, David J Simpson, and Michael G. Gänzle J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 08 Jun 2017 Downloaded from http://pubs.acs.org on June 10, 2017
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Journal of Agricultural and Food Chemistry
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Enzymatic synthesis and purification of galactosylated chitosan oligosaccharides
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reducing adhesion of enterotoxigenic Escherichia coli K88
3
Ya Lu Yana), Ying Hua), David J. Simpsona), Michael G. Gänzlea,b)*
4
a)
5
Canada.
6
b)
7
P.R. China.
University of Alberta, Dept. of Agricultural, Food and Nutritional Science, Edmonton,
Hubei University of Technology, College of Bioengineering and Food Science, Wuhan,
8
9
*Corresponding author footnote
10
University of Alberta, Dept. of Agricultural, Food and Nutritional Science, 4-10 Ag/For
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Centre, Edmonton, AB T6E2P5, Canada
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Phone:+1 780 492 3634
13
Email:
[email protected] 14
15
16
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Abstract
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Enterotoxigenic Escherichia coli (ETEC) K88 cause diarrhea in weaned piglets, and
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represent a suitable model system for ETEC causing childhood diarrhea. This study
20
aimed to evaluate the effects of oligosaccharides against ETEC K88 adhesion to porcine
21
erythrocytes with two bioassays. Galactosylated chitosan-oligosaccharides (Gal-COS)
22
were synthesized through transgalactosylation by β-galactosidase. Fractions 2 – 5 of Gal-
23
COS were obtained through cation exchange and size exclusion chromatography.
24
Fractions 2 – 5 of acetylated Gal-COS were obtained through chemical acetylation
25
followed by size exclusion chromatography. Gal-COS F2 containing the largest
26
oligosaccharides had the highest anti-adhesion activity with the minimum inhibitory
27
concentration of 0.22 g/L, followed by F3 and F4. Acetylation of Gal-COS decreased
28
their ability to reduce ETEC K88 adhesion. The composition of active oligosaccharides
29
was determined with LC-MS. Galactosylation of COS produces oligosaccharides which
30
reduce ETEC K88 adhesion; moreover, resulting oligosaccharides match the composition
31
of human milk oligosaccharides, which prevent adhesion of multiple pathogens.
32
Keywords:
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Galacto-oligosaccharides, enterotoxigenic Escherichia coli, beta-galactosidase, pathogen
34
adhesion.
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Introduction
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Indigestible dietary oligosaccharides including galacto-oligosaccharides (GOS) have
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several biological activities that improve host health by modulating composition and
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activity of intestinal microbiota, by preventing the adhesion of pathogens to intestinal
40
tissues, and by specific interactions with the immune system.1, The conceptual template
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for the food applications of oligosaccharides with specific health benefits are human milk
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oligosaccharides (HMOs). HMO comprise over 150 different oligosaccharides and their
43
concentration in human milk ranges from 5 to 20 g/L. 2 HMOs consist of five
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monosaccharides, galactose, glucose, N-acetyl-glucosamine (GlcNAc), fucose, and N-
45
acetyl-neuraminic acid (sialic acid).2 HMOs shape the development of infant
46
microbiome, 3 block adhesion of pathogens including Escherichia coli 4 , Salmonella
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Fyris,4 and Campylobacter jejuni5 to host cells, and thus prevent infections.
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In food and feed applications, bifidogenic properties of HMO are currently substituted by
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other non-digestible oligosaccharides including galacto-oligosaccharides (GOS), fructo-
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oligosaccharides (FOS), inulin, and lactulose, which stimulate growth and activity of
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intestinal microbiota.6,7 The ability of oligosaccharides to inhibit adhesion of pathogen,
52
however, is highly specific for the target organism and the oligosaccharide structure1,8
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and HMO are thus not readily replaced by other glycans. The structural diversity of
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HMOs, a prerequisite for their interference with adhesion of multiple pathogens, also
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impedes the production at the commercial scale. A commercial GOS preparation also
56
inhibited the adherence of enteropathogenic Escherichia coli (EPEC) to Hep-2 and Caco-
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2 cells.9 However, the use of GOS to reduce adhesion of enterotoxigenic Escherichia coli,
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a major cause of childhood diarrhea, has not been reported. 3 ACS Paragon Plus Environment
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GOS are synthesized by transgalactosylation of lactose with β-galactosidase. 10 In
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response to the demand for improved HMOs substitutes, transgalactosylation with β-
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galactosidase was extended to acceptor carbohydrates other than lactose to synthesize a
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diversity of oligosaccharides.1 Fructose, mannose, sucrose, N-acetyl-glucosamine, and
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fucose have been used in transgalactosylation reactions as acceptor sugars to synthesize
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diverse galactosylated oligosaccharides.10,11,12 Of these acceptor carbohydrates, N-acetyl-
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glucosamine (GlcNAc) has received particular attention because it is a constituent of
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HMO,2 binds to fimbriae of E. coli that mediate adhesion to the intestinal mucosa,13 and
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occurs in nature as the constituent monosaccharide of chitin, an abundant polysaccharide
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occurring in the shell of arthropods and the cell wall of fungi. Chitin and chitin-
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oligosaccharides are poorly soluble in water; commercial conversion of chitin from
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shrimp generally yields the deacetylated chitosan or chitosan-oligosaccharides (COS).
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COS exhibit high solubility in water, however, they are also highly reactive as they
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contain aldehyde and amino groups as reactants in the Maillard reaction, which occurs
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even during incubation at ambient temperature.14
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Chitobiose, chitotriose, and COS are acceptor sugars for β-galactosidase to produce di-,
75
tri-, tetra-oligosaccharides with galactose and GlcNAc / glucosamine.15 GOS as well as
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COS reduce adhesion of pathogenic E. coli to intestinal mucosal cells,9,16 however, the
77
activity of galactosylated COS remains unknown. Therefore, this study aimed to
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determine whether galactosylated COS inhibit adhesion of enterotoxigenic E. coli
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expressing K88 fimbriae to porcine cells. 17 , 18 ETEC K88 is an important cause for
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diarrhea in weaning piglets;19 different from human ETEC strains, is a model system that
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readily allows validation of in vitro results in vivo.17,18 To determine whether
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galactosylation influences the biological activity, oligosaccharides were fractionated by
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cation exchange and size exclusion chromatography. The influence of acetylation was
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determined by chemical acetylation.
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Material and methods
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Preparation of β-galactosidase. Lactococcus lactis MG1363 expressing the LacLM type
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β-galactosidase of L. plantarum.11,15 were streaked onto modified M17 (mM17) agar
88
plates that were supplemented with 5% glucose and 5 mg/L erythromycin and incubated
89
at 30 ºC under anaerobic condition for 48 h. Crude cell extract (CCE) was prepared as
90
previously described.15 Cells were harvested by centrifugation, washed and resuspended
91
in 50 mM phosphate buffer (PB) (pH 6.5) with 10% glycerol and 1 mM magnesium
92
chloride. Cells were disrupted with a Mini Beadbeater (model 693, Biospec, Bartlesville,
93
OK) for 2 min and cell debris was removed by centrifugation at 15,300 × g for 10 min at
94
4 ºC. Protein content and β-galactosidase activity of CCE were measured by Bradford
95
protein assay and o-nitrophenyl-β-galactoside respectively. When necessary, CCE was
96
diluted with PB containing 10% glycerol and MgCl2 to standardize the activity to 25-30
97
µmol/(min × mg protein).12
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Transgalactosylation reaction of β-galactosidase with lactose and COS. COS with a
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degree of polymerisation (DP) ranging from 2 to 6 and a degree of deacetylation of > 95%
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were provided by Glycobio (Dalian, China). The size distribution of the COS was
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confirmed by size exclusion chromatography as described below (data not shown).
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Transgalactosylation reactions were conducted by the addition of 20% (v/v) of
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standardized CCE into 180 g/L of COS and 180 g/L of lactose, followed by incubation
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for 16 h at 45 ºC. Reactions were terminated by addition of perchloric acid to a
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concentration of 3.5% (v/v). Control reactions contained 360 g/L lactose with 20% CCE
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(control without COS); 360 g/L COS with 20% CCE (v/v) (control without GOS); or 180
107
g/L COS and 180 g/L lactose without CCE (control without CCE). The
108
transgalactosylation and control reactions were monitored by measuring the UV-Vis
109
absorbance in the range of 200-500 nm for 16 h with 1 h intervals. Oligosaccharide
110
samples were lyophilized prior to analyses described below.
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Preparation of acetylated COS and acetylated galactosylated COS. The acetylation of
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COS and Gal-COS was performed as described20 with some modifications. COS or Gal-
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COS were dissolved in 50:50 (v/v) methanol: water at the concentration of 40 g/L. Acetic
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anhydride (0.5 equiv/glucosamine unit of COS) was added to COS or Gal-COS with
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continuous stirring at room temperature for 4 h. The acetylation was terminated by 40-
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fold dilution with water. Oligosaccharides were lyophilized. The degree of acetylation of
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acetylated oligosaccharides was measured by titration with 0.1 M of NaOH by dissolving
118
acetylated oligosaccharides into 0.1 M
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conducted in triplicates.
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Lyophilized
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chromatography as described above. Five fractions were collected. Acetylated COS,
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acetylated Gal-COS, and five collected fractions were lyophilized and re-dissolved to a
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concentration of 10 g / L prior to analysis by HPAEC-PAD and LC-MS, and tested for
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anti-adhesive activities with the methods described below.
acetylated
Gal-COS
were
HCl as described. 21 , 22 All reactions were
further
separated
by
size
exclusion
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High Performance Anion Exchange Chromatography with Pulsed Amperometric
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Detection (HPAEC-PAD). Oligosaccharides were diluted to 1 g/L prior to analysis on a
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HPAEC-PAD ICS-3000 system (Dionex, Oakville, Canada). Samples (10 µL) were
128
separated on a CarboPac PA-20 Dionex carbohydrates column using water (A), 200 mM
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NaOH (B), and 1 M sodium acetate (C) as eluent at flow rate of 0.25 mL/min with
130
following gradient: 0 min 6% B, 20 min 100% B. GOS were further analyzed using
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gradient starting at 30.4% B, 1.3% C and increasing to 30.4% B, 11.34% C at 22 min.
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Hemagglutination assay to determine the effect of oligosaccharides on ETEC K88
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adhesion to porcine erythrocytes. All samples used in the assay were lyophilized and
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re-dissolved in phosphate–buffer saline (PBS) (pH 7.2). The hemagglutination assay was
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performed with the porcine ETEC ECL 13795 with K88 fimbriae as described18 with
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some modifications. E. coli ECL13795 were cultivated on Minca agar overnight and
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washed with 1 mL PBS. Twenty-five-microliter E. coli cell suspensions with an OD600nm
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of 5 – 10 were diluted 2-fold in V-bottom 96-well polystyrene microtiter plates (Corning).
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The same volume of PBS or tested oligosaccharides with different concentrations was
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added, and the suspensions were incubated for 5 min before the addition of 25 µL of
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erythrocyte suspension into each well. Erythrocytes were prepared by 2-fold dilution of
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10% porcine red blood cells (Innovative Research, Novi, MI, USA). The plate was
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incubated at 4 ºC overnight prior to visual inspection. Activity was noted when the
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sample carbohydrate increased the cell density of ETEC presenting hemagglutination and
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lack of a defined pellet of erythrocytes at least four fold. All samples were diluted from
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10 g/L to exhaustion of the biological activity, or to 0.1 g/L.
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Enzyme-linked immunosorbent assay (ELISA) to determine the effect of
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oligosaccharides on ETEC K88 adhesion to porcine erythrocytes. The ability of
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oligosaccharides to inhibit E. coli K88 adhesion to porcine erythrocytes was also assayed
150
by ELISA. Each step of the following assay was separated by washing the wells three
151
times with PBS. A 96-well high bind microtiter plate (Corning) was coated overnight
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with 5% porcine red blood cells (Innovative Research, Novi, MI, USA) and blocked with
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3% bovine serum albumin for 1 h at 4 ºC. Oligosaccharides were dissolved in PBS at the
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concentration of 10 g/L. ETEC were mixed with PBS or oligosaccharides and then added
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into plates with 1 h incubation at 4 ºC. Red blood cells without ETEC addition, red blood
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cells with only ETEC addition, and no red blood cells but with ETEC addition were used
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as controls. The wells were then treated with mouse anti E. coli K88A antibody (Biorad,
158
USA) at a dilution of 1:2000. After 1 h incubation, the wells were treated with a goat
159
anti-mouse IgG (H+L) secondary antibody conjugated to horseradish peroxidase
160
(Invitrogen, Fisher Scientific, CA) for 1h. After addition of TMB substrates and
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incubated for 30 min, the reaction was stopped with 2 M sulphuric acid, and the
162
absorption at 450 nm was measured on a Varioscan Flash Microplate reader (Thermo
163
Scientific, CA).
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Separation of charged oligosaccharides by cation exchange chromatography and
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size exclusion chromatography. To separate charged oligosaccharides from lactose and
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GOS, reaction mixtures obtained with COS, GOS and galactosylated COS (Gal-COS)
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were separated through solid phase extraction method with cation exchange column
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Strata SCX (55 µm, 70A, Phenomenex, USA). The SCX column was conditioned with 3
169
mL of methanol prior to extraction. Column was equilibrated with 0.1% trifluoroacetic
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acid (TFA). Sugar samples were diluted with 0.1% (TFA) to a concentration of 10 g/L
171
and loaded onto the column. Charged oligosaccharides, containing COS and Gal-COS,
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were eluted with 0.2% triethylamine. Fractions collected during loading of the column
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were referred as Gal-COS flow through (Gal-COS FT) and fractions collected after
174
elution were referred to as Gal-COS. The collected fractions were lyophilized and re-
175
dissolved to a concentration of 10 g/L. All fractions were analysed by HPAEC-PAD and
176
hemagglutination assay.
177
Lyophilized COS, GOS, and Gal-COS fractions obtained by cation exchange
178
chromatography were further separated by size exclusion chromatography on a Superdex
179
peptide 10/300 GL column (10x240 mm, 13 µm, GE healthcare life sciences, USA)
180
eluted with 0.2M ammonium acetate at 0.3 mL/min. Separations were carried out on a
181
Agilent 1200 HPLC system coupled to refractive index (RI) and multiple wavelength
182
detector. Glucose (Fisher), lactose (Sigma), raffinose (Sigma), stachyose (Sigma), inulin
183
from chicory (Sigma), and dextran (100,000-200,000 Da, Sigma) were used as external
184
standards for molecular weight calibration. Five fractions were collected for each sample.
185
All collected fractions were lyophilized and re-dissolved to a concentration of 10 g/L
186
prior to analysis by HPAEC-PAD, LC/MS and anti-adhesive activities.
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Liquid chromatography/electrospray ionization mass spectrometry (LC-ESI-MS).
188
LC-ESI-MS analysis of oligosaccharides was conducted by Mass Spectrometry Facility
189
of University of Alberta. LC-MS was performed using an Agilent 1200 SL HPLC system
190
with GlycanPac AXH-1 column (2.1x150 mm, 1.9 µm, Thermo Scientific, Sunnyvale,
191
USA), thermostated at 40 ºC, with a buffer gradient system composed of 96:4 (v/v)
192
acetonitrile (ACN): water as mobile phase A and 80 mM ammonium formate in water, 9 ACS Paragon Plus Environment
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pH 4.4 as mobile phase B. Oligosaccharides were separated using the following gradient:
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10% B for 2 min, 10% to 30% B over a period of 10 min, 30% to 75% B over of 9 min,
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75% to 80% B over a period of 1 min, 80% to 10% B over a period of 2 min and held at
196
10% B for 3 min.
197
Mass spectra were acquired in positive mode ionization using an Agilent 6220 Accurate-
198
Mass TOF HPLC/MS system (Santa Clara, CA, USA) equipped with a dual sprayer
199
electrospray ionization source with the second sprayer providing a reference mass
200
solution. Mass spectrometric conditions were drying gas 10 L/min at 300 ºC, nebulizer 30
201
psi, and mass range 100-3200 Da. Analysis of the HPLC-MS data was done using the
202
Agilent Mass Hunter Qualitative Analysis software (ver.B.07.00).
203
Stability of COS, acetylated-COS, Gal-COS, and acetylated Gal-COS during
204
storage. Freeze-dried COS, acetylated-COS, Gal-COS, and acetylated Gal-COS (0.01-
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0.05g/ vial) were placed into individual HPLC vials and stored in closed containers at 37
206
ºC and 6 ºC for 7 d. The water activity (aW) in the containers was maintained by addition
207
of saturated sodium chloride, saturated sodium bromide, and silica gel beads to achieve a
208
constant aW of 0.75, 0.55, and 0.1, respectively. UV-vis absorbance spectra of tested
209
oligosaccharides were measured before and after 7 d storage. The absorbance was
210
recorded in the range of 200-600 nm. Tested sugars were dissolved to a concentration of
211
10 g/L prior to analysis. The compositions of tested sugar before and after storage were
212
analyzed by HPAEC-PAD with the method described above.
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Statistical analysis. Purification and fractionation was performed from three independent
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enzymatic reactions. Bioassays were performed in triplicate technical repeats. Results of
215
the bioassay-guided fractionation are presented as means ± standard error of the mean.
216
Results
217
Transgalactosylation and separation of COS. Transgalactosylation of COS and lactose
218
with β-galactosidase were analyzed by HPAEC-PAD (Fig. 1) Transgalactosylation
219
activity of β-galactosidase was apparent but galactosylated oligosaccharides were
220
partially obscured by the presence of glucose, galactose, COS, and galacto-
221
oligosaccharides (Fig 1A and 1B). Previously, Gal-COS were synthesized via
222
transgalactosylation of galactose from lactose to the non-reducing end of COS by β-
223
galactosidase.15 Charged oligosaccharides including COS and Gal-COS were separated
224
from the reaction mixture by cation exchange chromatography. In the separation of
225
transgalactosylation reactions with COS as acceptor, COS and Gal-COS were bound to
226
the column and eluted with triethylamine (Fig. 1B); glucose, galactose, lactose and
227
galacto-oligosaccharides did not bind to the columns (data not shown).
228
The enzymatic reaction mixture and all fractions obtained by separation with cation
229
exchange chromatography were tested for anti-adhesion activity against ETEC K88. All
230
samples were adjusted to 10 g/L and diluted to exhaustion of the biological activity.
231
Enzymatic reactions without further purification had the strongest anti-adhesive activities,
232
followed by Gal-COS purified by cation exchange columns (Table 1). Glucose, galactose,
233
lactose, COS and GOS had no effect on agglutination of ETEC to porcine erythrocytes.
234
Monitoring the transgalactosylation reactions with lactose or lactose and COS by
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measuring UV absorbance and comparison to reactions with COS and CCE suggested
236
that the presence of COS leads to formation of Maillard compounds. Oligosaccharide
237
mixtures generated by enzymatic synthesis in presence of COS thus require further
238
separation before conclusions on their biological activity can be drawn. COS reduced
239
K88 adhesion after separation on cation exchange columns, however, the effective
240
concentration was twice as high as the effective concentration of Gal-COS (Table 1). The
241
results indicate that Gal-COS have higher anti-adhesion activity than COS or GOS.
242
Fractionation of oligosaccharides by SEC. In order to determine the effect of molecular
243
weight of oligosaccharides on anti-adhesion activities against ETEC K88, COS, GOS and
244
Gal-COS were separated based on their molecular weight. Five different fractions were
245
collected for each sample and designated F1 to F5 in order of decreasing molecular
246
weight. Most Gal-COS eluted in F4 and F3, followed by F2 and F5 (Fig 2, upper trace).
247
The carbohydrate concentration in Gal-COS F1 was below the detection limit of the RI
248
detector. The molecular weight range of fractions was estimated as follows: F2, MW
249
1500-2500 Da, corresponding to DP8 and higher; F3, MW 900-1500 Da, corresponding to
250
DP6 – 8; F4, MW 500-1200 Da, corresponding to DP 3 – 6, F5, less than 500 Da and DP
251
of less than 3. All fractions were tested for anti-adhesive activities against ETEC K88
252
(Table 2). Fractions containing Gal-COS were two to 8 times more active when
253
compared to the corresponding factions containing COS only. The activity of Gal-COS
254
decreased with decreasing molecular weight (Table 2).
255
In addition to carbohydrates, F5 contained compounds with UV absorbance at 254 and
256
280 nm. This confirmed that Maillard reaction products influenced the results of the
257
hemagglutination assay as Gal-COS elution fraction 5 (Table 2) and COS-containing 12 ACS Paragon Plus Environment
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control reactions (data not shown) inhibited E. coli adhesion to porcine red blood cells at
259
10 g/L.
260
F1 and F5 were excluded from further analysis owing to the low yield, the low activity,
261
and the presence of UV-absorbing compounds in F5. F2, F3 and F4 of Gal-COS were
262
analyzed by HPAEC-PAD. Different patterns of oligosaccharides were observed in each
263
fractions, however, the lack of galactosylated COS standards prevented identification
264
(data not shown).
265
The effect of Gal-COS on adhesion of E. coli was confirmed with an additional bioassay
266
based on ELISA detection of K88 fimbriae (Fig. 4). Consistent with results obtained with
267
the hemagglutination assay, the highest anti-adhesive activity was observed with Gal-
268
COS after purification with cation exchange columns; Gal-COS F2 and Gal-COS F3 also
269
exhibited strong anti-adhesion effects (Fig. 4). Lactose and COS did not reduce ETEC
270
K88 attachment onto porcine red blood cells. Different from the hemagglutination assay,
271
however, the Gal-COS reaction mixture had no anti-adhesive activity (Fig. 4), further
272
indicating that Maillard reaction products interfere with the hemagglutination assay.
273
Taken together, ELISA confirmed that the Gal-COS and the Gal-COS fractions F2, F3
274
and F4 inhibit adherence of ETEC K88 to porcine red blood cells, and that Maillard
275
reaction products interfere with the hemagglutination assay.
276
Identification of oligosaccharides by LC-ESI-MS. Active fractions were analyzed by
277
LC-MS to achieve an accurate determination of Gal-COS. The high resolution MS
278
provided information on the chemical formula; the presence of Gal-COS was deduced
279
from the enzymatic method of synthesis and the absence of the corresponding products in
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the control reactions. Galactosylated COS were present in all active fractions but absent
281
in the corresponding fractions containing fractionated COS (Table 3 and data not shown).
282
The predominant degree of polymerization in the respective fractions corresponded to the
283
separation range of the SEC column used for fractionation (Table 3 and Figure 2).
284
Galactosylated COS were the major compounds in each of the fractions tested (Table 3);
285
Gal-GOS were composed of glucosamine and GlcNAc with one to four galactosyl-
286
residues.
287
Preparation of acetylated COS and acetylated galactosylated COS. Gal-COS
288
contained both glucosamine and GlcNAc as constituting monosaccharides. To determine
289
whether the degree of acetylation influences activity, Gal-COS were separated by cation
290
exchange chromatography and acetylated chemically. Titration of Gal-COS before and
291
after acetylation estimated the degree of acetylation as 50%. Analysis of acetylated Gal-
292
COS by LC-MS confirmed the synthesis of N-acetyl-glucosamine containing
293
oligosaccharides. LC-MS results (Table 4) showed galactosylated oligosaccharides with
294
different degree of acetylation. Acetylated Gal-COS fractions differed in their anti-
295
adhesive activity (Table 5). The largest fraction (Fig. 4) with molecular weight from 1700
296
to 2500 Da exhibited the strongest activity, followed by the fractions with molecular
297
range of 1000 to 2000 Da and 700 to 1300 Da. However, acetylation decreased the anti-
298
adhesive activity 3 – 10 fold when compared to the corresponding fractions containing
299
Gal-COS (Table 2 and 5).
300
Stability of Gal-COS and acetylated Gal-COS during storage. Past studies and
301
observations during purification of Gal-COS in this study indicated that handling of COS
302
at ambient temperature may result in formation of Maillard products which affect their 14 ACS Paragon Plus Environment
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activity.14 To determine whether acetylation or galactosylation influences the reactivity
304
during dry storage, the degradation of Gal-COS was monitored after lyophilisation. Gal-
305
COS were compared to COS, acetylated COS and acetylated Gal-COS. Samples were
306
stored at different temperatures and water activities. The change in UV-Vis absorbance
307
after 7 days of storage was used as a simple tool to monitor major changes. The
308
differences in the UV-Vis spectra are plotted in a heat map to monitor the stability over
309
time. An increased absorption was observed for COS storage under 37 ºC for all three
310
water activities conditions as indicated with lighter colour area in heat map (Figure 5). A
311
minor increase in absorbance was observed in acetylated COS, Gal-COS and acetylated
312
Gal-COS under all conditions. No changes were observed in COS stored at 6 ºC with all
313
tested water activities. An increased UV absorption between 340-360 nm was observed
314
for Gal-COS maybe because the Mallard reaction products formed during enzymatic
315
reaction. COS were not stable when stored at 37ºC, however, galactosylation, acetylation,
316
or both would increase their stability. The lower UV absorption of acetylated Gal-COS
317
after 7 d storage when compared to Gal-COS indicated a lower reactivity.
318
Discussion
319
This study demonstrates that Gal-COS inhibit adhesion of ETEC K88 to porcine blood
320
cells, and may thus be used to inhibit pathogen adhesion in vivo. The activity was
321
confirmed by two bioassays, a hemagglutination assay, and an ELISA assay targeting
322
K88 fimbriae. Transgalactosylation strongly increased the anti-adhesive activity of COS;
323
moreover, high molecular weight oligosaccharides had higher biological activity while
324
acetylation decreased the effect on pathogen adhesion.
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325
Transgalactosylation of COS with β-galactosidase as catalyst and lactose as galactosyl-
326
donor provides an inexpensive approach to synthesize oligosaccharides that contain
327
GlcNAc- or glucosamine and exhibit biological activity.1 The production of glucosamine
328
or COS currently employs acid hydrolysis of chitin from crab and shrimp shell 23 or
329
enzymatic hydrolysis of chitosan or chitin using chitosanase24 or chitinase,25 respectively.
330
COS inhibited adhesion of EPEC16 and ETEC (this study) to eukaryotic cells, however,
331
COS are highly reactive and form Maillard products during storage or in enzymatic
332
reactions. These products interfere with their biological activity. Cation exchange and
333
size exclusion purification excluded the effects of Maillard products on hemagglutination
334
assay. ELISA assay confirmed the inhibition of ETEC adhesion by Gal-COS. Moreover,
335
Gal-COS with a DP ranging from 8 – 12 exhibited the highest activity; these compounds
336
have a lower reactivity in the Maillard reaction when compared to short-chain COS.
337
Fimbriae mediated pathogen-host interactions are highly specific for host species, body
338
site, and in several cases, also the age of the animals.8 For example, host adaptation of
339
Salmonella enterica Typhimurium was related to allelic variations in the glycan binding
340
domain of FimH. 26 Likewise, host specificity of ETEC relates to the fimbriae that
341
mediate adhesion. ETEC expressing class 1b and class 5 fimbria cause childhood
342
diarrhea, 27 while ETEC with fimbria F4 (K88) cause disease in animals.19 Porcine
343
aminopeptidase N, a membrane bound glycoprotein, was recently identified as receptor
344
for F4 fimbriae. Binding of F4 fimbriae to aminopeptidase N was dependent on
345
decoration of the protein with sialic acid. 28 ETEC K88 fimbriae bind to GlcNAc, N-
346
acetyl-galactosamine, N-acetylmannosamine, and D-galactosamine
347
oligosaccharides consisting of GlcNAc and galactose residues at the reducing end were
29
and β-linked
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348
suggested to be essential for adherence of K88 fimbriae to host cells.29, 30 , 31 , 32 In
349
agreement with the GlcNAc and galactose-containing target glycans of K88 fimbriae, the
350
effect of Gal-COS containing β-linked galactosyl and GlcNAc or glucosamine residues
351
on ETEC K88 attachment to porcine red blood cells was stronger when compared to non-
352
galactosylated COS (Table 2). However, acetylation to match the acetylation in porcine
353
glycans recognized by K88 fimbriae reduced activity.
354
ETEC induced diarrhea in suckling and post-weaning piglets cause significant economic
355
losses in swine production. ETEC initiate host colonization through receptor-fimbriae
356
interaction; diarrhea is caused by heat-labile or heat-stable enterotoxins secreted after
357
colonization29. ETEC with fimbriae K88 (F4) and F18 infect suckling and post-weaning
358
piglets, respectively.19,
359
processing, reduced K88 attachment to swine epithelia in vivo and in vitro. 33
360
Exopolysaccharides extracted form Lactobacillus reuteri also reduced ETEC K88
361
adherence in vitro18 and in vivo.17 Other food- or feed-derived glycans including wheat
362
bran, galactomannan or phenolic compounds from locust bean, or glycopeptides derived
363
from ovomucin interfered with adhesion of ETEC K88,34 however, the active compounds
364
have not been identified. Bioassay-guided separation and fractionation of Gal-COS as
365
performed in this study allowed to relate molecular weight and degree of acetylation of
366
Gal-COS to their anti-adhesion activity. Moreover, the use of two complementary
367
bioassays confirmed that the oligosaccharides target K8 fimbriae (Table 2 and Figure 4).
368
The activity of Gal-COS decreased with decreasing molecular weight and acetylation
369
decreased activity. This result matches observations on the anti-adhesive activity of COS
370
against EPEC, which also decreased with reduced DP and increased acetylation.16
30
Casein glycomacropetide, a glycoprotein obtained from cheese
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371
Remarkably, the galactosylation of COS increased anti-adhesion activity when compared
372
to COS. Gal-GOS are promising alternatives to antibiotic therapy for the swine industry.
373
Bacterial pathogens other than ETEC K88 also recognize β-linked galactosylated
374
oligosaccharides,8 and Gal-COS may thus target additional human or animal pathogens.
375
The concept of using oligosaccharides for therapeutic applications is increasingly
376
validated by in vivo studies.17 Oligosaccharide synthesis with glycosyl hydrolases
377
requires high concentrations of both donor and acceptor sugars, and generates
378
oligosaccharide mixtures differing in the site of glycosylation, the linkage type, and the
379
degree
380
oligosaccharides were produced with other retaining glycosyl hydrolases including ɑ-
381
galactosidase, 35 fucosidase, trans-sialidase 36 and N-acetyl-glucosaminidase.1 Metabolic
382
engineering of microorganisms to synthesise oligosaccharides with glycosyl transferases
383
provides an alternative approach for oligosaccharide production. Fucosyllactose has
384
recently become commercially available through fermentation of fucose and lactose with
385
E. coli harbouring fucosyltransferase. 37 Oligosaccharide synthesis with intracellular
386
glycosyl transferases generates lower yields when compared to glycosyl hydrolases but
387
produces defined oligosaccharides.
388
transferase activity and the preference for the linkage type of retaining glycosyl
389
hydrolases.39,40,41 Relating the selectivity of oligosaccharide synthesis to the specificity of
390
glycan recognition of pathogens, however, makes the use of oligosaccharide mixtures
391
preferential as these may target multiple pathogens. Human milk oligosaccharides are a
392
mixture of more than 150 oligosaccharides targeting adhesion of multiple pathogens.4
of
polymerisation15,17
(this
38
paper).
In
addition
to
β-galactosidases,
Site-directed mutagenesis can improve the
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Journal of Agricultural and Food Chemistry
393
In conclusion, this study demonstrated that partially purified galactosylated chitosan-
394
oligosaccharides strongly interfered with adhesion of ETEC K88 to porcine erythrocytes.
395
In addition, the influence of the molecular weight and degree of acetylation was
396
elucidated, which is necessary to address the low solubility of chitin oligosaccharides,
397
and the high reactivity of chitosan oligosaccharides. Acetylation of COS or Gal-COS
398
increases stability but decreases anti-adhesive activity.
399
Acknowledgements
400
The Alberta Livestock and Meat Agency is acknowledged for funding (Grant No.
401
2015B009R).
402
Supporting Information Available: Figure S1. HPAEC-PAD chromatography of COS
403
before and after storage at 37 ºC;
404
Reference
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oligosaccharides are associated with protection against diarrhea in breast-fed infants. J. Pediatr. 2004, 145, 297-303. 6 Ackerman, D. L.; Craft, K. M.; Townsend, S. D. Infant food applications of complex carbohydrates: Structure, synthesis, and function. Carbohydr. Res. 2017, 437, 16-27. 7 Davis, L. M. G.; Martinez, I.; Walter, J.; Goin, C.; Hutkins, R. W. Barcoded Pyrosequencing reveals that consumption of galactooligosaccharides results in a highly specific bifidogenic response in humans. Plos One 2011, 6, e25200. 8 Shoaf-Sweeney, K. D.; Hutkins, R. W. Adherence, anti-adherence, and oligosaccharides preventing pathogens from sticking to the host. Adv. Food Nutr. Res. 2009, 55, 101e161. 9 Shoaf, K.; Mulvey, G. L.; Armstrong, G. D.; Hutkins, R. W. Prebiotic galactooligosaccharides reduce adherence of enteropathogenic Escherichia coli to tissue culture cells. Infect. Immun. 2006, 74, 6920-6928. 10 Gänzle, M. G. Enzymatic synthesis of galacto-oligosaccharides and other lactose derivatives (hetero-oligosaccharides) from lactose. Int. Dairy J. 2012, 22, 116-122. 11 Schwab, C.; Sorensen, K. I.; Gänzle, M. G. Heterologous expression of glycoside hydrolase family 2 and 42 βgalactosidases of lactic acid bacteria in Lactococcus lactis. Syst. Appl. Microbiol. 2010, 33, 300-307. 12 Schwab, C.; Lee, V.; Sorensen, K. I.; Gänzle, M. G. Production of galactooligosaccharides and heterooligosaccharides with disrupted cell extracts and whole cells of lactic acid bacteria and bifidobacteria. Int. Dairy J. 2011, 21, 748-754. 13 Rhen, M., Klemm, P., Korhonen, T.K. Identification of two new hemagglutinins of Escherichia coli, N-acetyl-D-glucosamine-specific fimbriae and a blood group Mspecific agglutinin, by cloning the corresponding genes in Escherichia coli K-12. J. Bacteriol. 1986, 168, 1234-1242. 14 Hrynets, Y.; Ndagijimana, M.; Betti, M. Studies on the formation of Maillard and caramelization products from glucosamine incubated at 37 °C. J. Agric. Food Chem. 2015, 63, 6249-6261. 15 Black, B. A.; Yan, Y.; Galle, S.; Hu, Y.; Curtis, J. M.; Gänzle, M. G. Characterization of novel galactosylated chitin-oligosaccharides and chitosan-oligosaccharides. Int. Dairy J. 2014, 39, 330-335. 16 Quintero-Villegas, M. I.; Aam, B. B.; Rupnow, J.; Sorlie, M.; Eijsink, V. G.; Hutkins, R. W. Adherence inhibition of enteropathogenic Escherichia coli by
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chitooligosaccharides with specific degrees of acetylation and polymerization. J. Agric. Food Chem. 2013, 61, 2748-2754. 17 Chen, X. Y.; Woodward, A.; Zijlstra, R. T.; Gänzle, M. G. Exopolysaccharides synthesized by Lactobacillus reuteri protect against enterotoxigenic Escherichia coli in piglets. Appl. Environ. Microbiol. 2014, 80, 5752-5760. 18 Wang, Y.; Gänzle, M. G.; Schwab, C. Exopolysaccharide synthesized by Lactobacillus reuteri decreases the ability of enterotoxigenic Escherichia coli to bind to porcine erythrocytes. Appl. Environ. Microbiol. 2010, 76, 4863-4866. 19 Xia, P.; Zou, Y.; Wang, Y.; Song, Y.; Liu, W.; Francis, D. H.; Zhu, G. Receptor for the F4 fimbriae of enterotoxigenic Escherichia coli (ETEC). Appl. Microbiol. Biotechnol. 2015, 99, 4953-4959. 20 Hu, Y.; Du, Y.; Wang, X.; Feng, T. Self-aggregation of water-soluble chitosan and solubilization of thymol as an antimicrobial agent. Journal of Biomedical Materials Research Part a 2009, 90A, 874-881. 21 Liu, D.; Wei, Y.; Yao, P.; Jiang, L. Determination of the degree of acetylation of chitosan by UV spectrophotometry using dual standards. Carbohydr. Res. 2006, 341, 782-785. 22 Zhang, Y. Q.; Xue, C. H.; Xue, Y.; Gao, R. C.; Zhang, X. L. Determination of the degree of deacetylation of chitin and chitosan by X-ray powder diffraction. Carbohydr. Res. 2005, 340, 1914-1917. 23 Hossain, G. S.; Shin, H. D.; Li, J.; Wang, M.; Du, G.; Chen, J.; Liu, L. Metabolic engineering for amino-, oligo-, and polysugar production in microbes. Appl. Microbiol. Biotechnol. 2016, 100, 2523-2533. 24 Thadathil, N.; Velappan, S. P. Recent developments in chitosanase research and its biotechnological applications: A review. Food Chem. 2014, 150, 392-399. 25 Liu, L.; Liu, Y.; Shin, H.; Chen, R.; Li, J.; Du, G.; Chen, J. Microbial production of glucosamine and N-acetylglucosamine: advances and perspectives. Appl. Microbiol. Biotechnol. 2013, 97, 6149-6158. 26 Yue, M.; Han, X.; De Masi, L.; Zhu, C.; Ma, X.; Zhang, J.; Wu, R.; Schmieder, R.; Kaushik, R. S.; Fraser, G. P.; Zhao, S.; McDermott, P. F.; Weill, F. X.; Mainil, J. G.; Arze, C.; Fricke, W. F.; Edwards, R. A.; Brisson, D.; Zhang, N. R.; Rankin, S. C.; Schifferli, D. M. Allelic variation contributes to bacterial host specificity. Nat. Commun. 2015, 6, 8754
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27 Mortezaei, N.; Epler, C. R.; Shao, P. P.; Shirdel, M.; Singh, B.; McVeigh, A.; Uhlin, B. E.; Savarino, S. J.; Andersson, M.; Bullitt, E. Structure and function of enterotoxigenic Escherichia coli fimbriae from differing assembly pathways. Mol. Microbiol. 2015, 95, 116-126. 28 Melkebeek, V.; Rasschaert, K.; Bellot, P.; Tilleman, K.; Favoreel, H.; Deforce, D.; De Geest, B. G.; Goddeeris, B. M.; Cox, E. Targeting aminopeptidase N, a newly identified receptor for F4ac fimbriae, enhances the intestinal mucosal immune response. Mucosal Immunology 2012, 5, 635-645. 29 Jin, L. Z.; Zhao, X. Intestinal receptors for adhesive fimbriae of enterotoxigenic Escherichia coli (ETEC) K88 in swine - a review. Appl. Microbiol. Biotechnol. 2000, 54, 311-318. 30 Moonens, K.; Van den Broeck, I.; De Kerpel, M.; Deboeck, F.; Raymaekers, H.; Remaut, H.; De Greve, H. Structural and functional insight into the carbohydrate receptor binding of F4 fimbriae-producing enterotoxigenic Escherichia coli. J. Biol. Chem. 2015, 290, 8409-8419. 31 Grange, P. A.; Mouricout, M. A.; Levery, S. B.; Francis, D. H.; Erickson, A. K. Evaluation of receptor binding specificity of Escherichia coli K88 (F4) fimbrial adhesin variants using porcine serum transferrin and glycosphingolipids as model receptors. Infect. Immun. 2002, 70, 2336-2343. 32 Sarabia-Sainz, A.; Ramos-Clamont, G.; del Carmen Candia-Plata, Ma Maria; Vazquez-Moreno, L. Biorecognition of Escherichia coli K88 adhesin for glycated porcine albumin. Int. J. Biol. Macromol. 2009, 44, 175-181. 33 Gustavo Hermes, R.; Molist, F.; Francisco Perez, J.; Gomez de Segura, A.; Ywazaki, M.; Davin, R.; Nofrarias, M.; Korhonen, T. K.; Virkola, R.; Martin-Orue, S. M. Casein glycomacropeptide in the diet may reduce Escherichia coli attachment to the intestinal mucosa and increase the intestinal lactobacilli of early weaned piglets after an enterotoxigenic E. coli K88 challenge. Br. J. Nutr. 2013, 109, 1001-1012. 34 Gonzalez-Ortiz, G.; Francisco Perez, J.; Gustavo Hermes, R.; Molist, F.; JimenezDiaz, R.; Maria Martin-Orue, S. Screening the ability of natural feed ingredients to interfere with the adherence of enterotoxigenic Escherichia coli ( ETEC) K88 to the porcine intestinal mucus. Br. J. Nutr. 2014, 111, 633-642. 35 Wang, Y.; Black, B. A.; Curtis, J. M.; Gänzle, M. G. Characterization of alphagalacto-oligosaccharides formed via heterologous expression of alpha-galactosidases from Lactobacillus reuteri in Lactococcus lactis. Appl. Microbiol. Biotechnol. 2014, 98, 2507-2517
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36 Holck, J.; Larsen, D. M.; Michalak, M.; Li, H.; Kjaerulff, L.; Kirpekar, F.; Gotfredsen, C. H.; Forssten, S.; Ouwehand, A. C.; Mikkelsen, J. D.; Meyer, A. S. Enzyme catalysed production of sialylated human milk oligosaccharides and galactooligosaccharides by Trypanosoma cruzi trans-sialidase. New Biotechnology 2014, 31, 156-165 37 Weichert, S.; Jennewein, S.; Huefner, E.; Weiss, C.; Borkowski, J.; Putze, J.; Schroten, H. Bioengineered 2 '-fucosyllactose and 3-fucosyllactose inhibit the adhesion of Pseudomonas aeruginosa and enteric pathogens to human intestinal and respiratory cell lines. Nutr. Res. 2013, 33, 831-838. 38 Crout, D. H.; Vic, G. Glycosidases and glycosyl transferases in glycoside and oligosaccharide synthesis. Current Opinion in Chemical Biology 1998, 2, 98-111 39 Zakariassen, H.; Hansen, M. C.; Joranli, M.; Eijsink, V. G. H.; Sorlie, M. Mutational Effects on Transglycosylating Activity of Family 18 Chitinases and Construction of a Hypertransglycosylating Mutant. Biochemistry (N. Y. ) 2011, 50, 5693-5703 40 Jorgensen, F.; Hansen, O. C.; Stougaard, P. High-efficiency synthesis of oligosaccharides with a truncated beta-galactosidase from Bifidobacterium bifidum. Appl. Microbiol. Biotechnol. 2001, 57, 647-652 41 Meng, X.; Dobruchowska, J. M.; Pijning, T.; Gerwig, G. J.; Kamerling, J. P.; Dijkhuizen, L. Truncation of domain V of the multidomain glucansucrase GTF180 of Lactobacillus reuteri 180 heavily impairs its polysaccharide-synthesizing ability. Appl. Microbiol. Biotechnol. 2015, 99, 5885-5894 405
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Table 1. Inhibition of erythrocyte agglutination by ETEC K88 strain with oligosaccharides at various concentrations. Hemagglutination was determined with E. coli K88 ECL13795 (O149, virotype STb:LT:EAST1:F4), and results were based on three independent assays. Different letters indicate statistical differences (P 10
COS elutiona)
6.67 ± 0.96a
COS FTb)
9.44 ± 0.00b
Glucose
> 10
Galactose
> 10
Glucosamine
> 10
Sample
Lactose Gal-COS mixture
a)
> 10 c)
0.69 ± 0.00c
Gal-COS FT b)
5.00 ± 0.00a
Gal-COSa)
2.92 ± 0.48d
GOS > 10 charged oligosaccharide fraction after purification by cation exchange chromatography.
b)
uncharged fraction of enzymatic reaction products that did not bind to cation exchange column.
c)
unpurified enzymatic reaction products or equivalent incubation of enzyme and COS
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Table 2. Inhibition of erythrocyte agglutination by ETEC K88 strain with Gal-COS purified by cation exchange chromatography at various concentrations. The corresponding COS fractions were used for comparison. Hemagglutination was determined with E. coli K88 ECL13795 (O149, virotype STb:LT:EAST1:F4), and results were based on three independent assays. Values in the same column that do not share a common superscript are significantly different (P 10
COS F2
8.33 ± 0.96a
COS F3
2.78 ± 0.48b
COS F4
6.11 ± 0.96a
COS F5
> 10
Gal-COS F2
0.22 ± 0.00c
Gal-COS F3
1.88 ± 0.24d
Gal-COS F4
3.06 ± 0.48b
Gal-COS F5
> 10
GOS fractions
> 10 in all tested fractions
Sample
a)
Fractions numbers 1 – 5 correspond to fractions with decreasing molecular weight after separation with size exclusion chromatography.
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Table 3. List of compounds identified in Gal-COS elution fractions and their mass accuracy Oligosaccharides fractions
Gal-COS F2
Gal-COS F3
Gal-Gal-(GlcN)4(GlcNAc)6 Gal-(GlcN)6(GlcNAc)5 Gal-(GlcN)7(GlcNAc)4 Gal-(GlcN)8(GlcNAc)3 Gal-(GlcN)5(GlcNAc)5 Gal-Gal-(GlcN)6(GlcNAc)3 Gal-(GlcN)8(GlcNAc)2 Gal-Gal-(GlcN)6(GlcNAc)2 Gal-Gal-(GlcN)7(GlcNAc) Gal-Gal-Gal-Gal-(GlcN)5 Gal-Gal-Gal-(GlcN)6 Gal-Gal-(GlcN)7 Gal-Gal-Gal-Gal-(GlcN)4 Gal-Gal-Gal-(GlcN)5 Gal-Gal-(GlcN)6 Gal-Gal-Gal-(GlcN)4 Gal-(GlcN)6 (GlcN)7 Gal-Gal-(GlcN)4 Gal-(GlcN)5 (GlcN)6 Gal-Gal-Gal-(GlcN)4 Gal-Gal-(GlcN)5 Gal-Gal-Gal-(GlcN)
Measured Mass (Da) 2204.8849 2161.8854 2119.8750 2077.8624 2000.8215 1917.7775 1874.7822 1714.6989 1672.6779 1471.5760 1470.5855 1469.5893 1310.5067 1309.5378 1308.5296 1148.4479 1146.4698 1145.4932 986.3933 985.4063 984.4209 1148.4457 1147.4640 987.3800
Error (Da) 0.0172 0.0123 0.0125 0.0104 0.0172 0.0104 0.0095 0.0111 0.0007 0.0101 0.0036 -0.0086 0.0096 0.0005 0.0005 0.0036 -0.0064 0.0009 0.0018 -0.0011 0.0000 0.0015 0.0037 0.0046
Gal-Gal-(GlcN)4
986.3921
0.0007
Gal-(GlcN)5
985.4026
-0.0048
Gal-Gal-(GlcN)3
825.3326
0.0001
Compounds1, 2)
3
Gal-COS F4
Gal-(GlcN)4
824.3348 -0.0038 (GlcN)5 823.3563 0.0017 Gal-Gal-(GlcN)2 664.2489 -0.0049 (GlcN)4 662.2852 -0.0006 (GlcN)3 501.2179 0.0009 1) The position of N-acetyl-glucosamine groups does not correspond to its position in different oligosaccharides; however, as a result of enzymatic catalysis, galactose units are at the non-reducing end. 2) Nomenclature: Gal, Galactose; GlcN, N-glusosamine; GlcNAc, N-acetyl-glucosamine 26 ACS Paragon Plus Environment
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Table 4. List of compounds identified in acetylated Gal-COS elution fractions and their mass accuracy Oligosaccharides fractions
Acetylated Gal-COS F2
Acetylated Gal-COS F3
Acetylated Gal-COS F4
Compounds
Measured mass (Da)
Error (Da)
Compounds
Measured mass (Da)
Error
Gal-(GlcN)(GlcNAc)10 Gal-Gal-Gal-(GlcN)(GlcNAc)8 Gal-(GlcNAc)10 Gal-Gal-(GlcNAc)9 Gal-(GlcN)(GlcNAc)9 Gal-Gal-Gal-(GlcNAc)8 Gal-Gal-(GlcN)(GlcNAc)8
2371.9303 2289.8933 2210.8655 2169.8457 2168.8497 2128.8214 2127.8300
0.0044 0.0204 0.0084 0.0151 0.0031 0.0173 0.0010
Gal-Gal-Gal-(GlcN)-(GlcNAc)7 Gal-(GlcNAc)9 Gal-(GlcN)(GlcNAc)8 Gal-Gal-Gal-(GlcN)(GlcNAc)6 Gal-(GlcN)(GlcNAc)7 Gal-Gal-(GlcN)(GlcNAc)6
2086.8016 2007.7821 1965.7672 1883.7253 1762.6841 1721.6646
0.0081 0.0044 0.0000 0.0113 -0.0037 0.0033
Gal-Gal-(GlcN)(GlcNAc)8 Gal-Gal-GlcN-(GlcNAc)7 Gal-Gal-Gal-(GlcN)(GlcNAc)4 (GlcNAc)7 Gal-(GlcNAc)6 Gal-Gal-(GlcNAc)5 Gal-Gal-(GlcN)(GlcNAc)4 Gal-Gal-(GlcN)(GlcNAc)4 Gal-Gal-(GlcN)(GlcNAc)3 Gal-Gal-Gal-(GlcN)(GlcNAc)2 Gal-(GlcN)(GlcNAc)3
2127.8303 1924.7427 1477.5656 1439.5653 1398.5427 1357.5210 1315.5033
0.0103 0.0020 0.0103 -0.0009 0.0030 -0.0017 0.0008
Gal-Gal-Gal-(GlcN)(GlcNAc)3 (GlcNAc)6 Gal-(GlcNAc)5 Gal-(GlcN)(GlcNAc)4 Gal-Gal-(GlcN)2(GlcNAc)2 Gal-Gal-(GlcN)(GlcNAc)3
1274.4845 1236.4851 1195.4652 1153.4464 1070.4117 1112.4238
0.0085 -0.0017 0.0050 -0.0033 -0.0009 0.0007
1315.5069 1112.4237 1071.4047 950.3716
0.0044 0.0006 0.0081 0.0012
Gal-Gal-(GlcN)(GlcNAc)2 Gal-Gal-Gal-(GlcN)(GlcNAc) (GlcNAc)4 Gal-Gal-(GlcN)(GlcNAc)
909.3452 868.3246 830.3283 706.2670
0.0015 0.0074 0.0002 0.0026
1) The position of N-acetyl-glucosamine groups does not correspond to its position in different oligosaccharides; however, as a result of enzymatic catalysis, galactose units are at the non-reducing end. 2) Nomenclature: Gal, Galactose; GlcN, N-glusosamine; GlcNAc, N-acetyl-glucosamine
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Table 5. Inhibition of erythrocyte agglutination by ETEC K88 strain with acetylated GalCOS elution fractions at various concentrations. Hemagglutination was determined with E. coli K88 ECL13795 (O149, virotype STb:LT:EAST1:F4), and results were based on three independent assays. Different letters indicate statistical differences (P 10
Acetylated Gal-COS F2
5.83 ± 0.96a
Acetylated Gal-COS F3
6.11 ± 0.96a
Acetylated Gal-COS F4
> 10
Acetylated Gal-COS F5
> 10
Sample
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Figure legends. Figure 1. HPAEC-PAD results of transgalactosylation reaction with lactose and COS and purification of the active oligosaccharide fractions. Panel A, Chromatographic trace of unpurified enzymatic reactions; chromatograms of lactose and COS are shown for comparison. Panel B. Chromatographic traces of charged oligosaccharides purified by cation exchange chromatography. The corresponding fraction obtained with separation of COS is shown for comparison. Chromatograms are representative of three independent reactions. Galactose, glucose and lactose elute at 11.2, 11.6 15.9 min, respectively. Figure 2. Size exclusion results for different fractions of Gal-COS elution. Fraction 2 is the largest followed by fraction 3 and followed by fraction 4 and fraction 5 was the smallest fraction collected. LMW dextran (450,000-650,000), inulin, stachyose (DP 4), raffinose (DP3), and lactose (DP 2), were used as molecular weight standards. Chromatography was based on three independent reactions. Figure 3. Size exclusion results for different fractions of acetylated Gal-COS elution. Fraction 2 is the largest followed by fraction 3 and followed by fraction 4 and fraction 5 was the smallest fraction collected. LMW dextran (450,000-650,000), inulin, stachyose (DP 4), raffinose (DP3), and lactose (DP 2), were used as molecular weight standards. Chromatography was based on three independent reactions. Figure 4. Quantification of E. coli K88 ECL13795 binding to porcine erythrocytes with ELISA targeting K88 antibodies. ETEC were incubated with erythrocytes without addition of glycans (no glycan control), or with addition of 10 g/L of the carbohydrates as indicated on the x-axis. Results are reported as means ± standard deviation of three independent assays. The values of columns that do not share a common superscript differ significantly (P