Cranberry Xyloglucan Structure and Inhibition of Escherichia coli

May 14, 2015 - Cranberry juice has been recognized as a treatment for urinary tract infections on the basis of scientific reports of proanthocyanidin ...
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Cranberry Xyloglucan Structure and Inhibition of Escherichia coli Adhesion to Epithelial Cells Arland T. Hotchkiss, Alberto Nunez, Gary D Strahan, Hoa Chau, Andre White, Jannie Marais, Kellie Hom, Malathi Srilakshmi Vakkalanka, Rong Di, Kit L. Yam, and Christina Khoo J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b00730 • Publication Date (Web): 14 May 2015 Downloaded from http://pubs.acs.org on May 18, 2015

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Journal of Agricultural and Food Chemistry

Cranberry Xyloglucan Structure and Inhibition of Escherichia coli Adhesion to Epithelial Cells Arland T. Hotchkiss, Jr. †*, Alberto Nuñez†, Gary D. Strahan†, Hoa K. Chau†, André K. White†, Jannie P.J. Marais‡, Kellie Hom§, Malathi S. Vakkalanka#, Rong Diζ, Kit L. Yam# and Christina Khoo‡



U.S. Department of Agriculture, Agricultural Research Service, Eastern Regional Research

Center, 600 E. Mermaid Lane, Wyndmoor, PA 19038, United States ‡

Ocean Spray Cranberries, Inc., One Ocean Spray Drive, Lakeville-Middleboro, MA 02349

United States §

Department of Pharmaceutical Sciences, University of Maryland, School of Pharmacy, 20 North

Pine Street, Baltimore Maryland 21201, United States #

Department of Food Science, Rutgers University, 65 Dudley Road, New Brunswick, NJ 08901,

United States ζ

Department of Plant Biology and Pathology, Rutgers University, 59 Dudley Road, New

Brunswick, NJ 08901, United States

*Corresponding author (Tel: 215-233-6448; Fax: 215-233-6795; E-mail: [email protected])

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ABSTRACT: Cranberry juice has been recognized as a treatment for urinary tract infections

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based on scientific reports of proanthocyanidin anti-adhesion activity against Escherichia coli as

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well as from folklore. Xyloglucan oligosaccharides were detected in cranberry juice and the

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residue remaining following commercial juice extraction that included pectinase-maceration of

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the pulp. A novel xyloglucan was detected based on tandem mass spectrometry analysis of an ion

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at m/z 1055 that was determined to be a branched, three hexose, four pentose oligosaccharide

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consistent with an arabino-xyloglucan structure. Two-dimensional nuclear magnetic resonance

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spectroscopy analysis provided through-bond correlations for the α-L-Araf (1→2) α-D-Xylp

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(1→6) β-D-Glcp sequence proving the S-type cranberry xyloglucan structure. Cranberry

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xyloglucan-rich fractions inhibited the adhesion of E. coli CFT073 and UTI89 strains to T24

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human bladder epithelial cells and E. coli O157:H7 to HT29 human colonic epithelial cells.

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SSGG xyloglucan oligosaccharides represent a new cranberry bioactive component with E. coli

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anti-adhesion activity and high affinity for type 1 fimbriae.

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KEYWORDS: Cranberry, Arabino-Xyloglucan, Escherichia coli, Anti-Adhesive, Urinary Tract

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Infection

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INTRODUCTION Current therapies for bacterial infections in humans and animals are based largely on the

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use of antibiotics. Since their introduction in the 1940s, antibiotic drugs have proven an effective

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treatment in many bacterial illnesses; however, their frequent and long-term use has given rise to

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antibiotic-resistant bacterial strains that have necessitated the development and implementation

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of increasingly more powerful drugs. Sub-therapeutic use of antibiotics as growth promoters in

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the livestock animal industry has contributed to a reservoir of antibiotic-resistant bacteria in the

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environment. Hospital admissions of patients with serious, and in some cases life-threatening

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bacterial infections, are on the rise. Urinary tract infections (UTIs), for example, have been a

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pervasive health care problem resulting in millions of doctor office visits per year. UTIs are

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generally defined as the presence of bacteria (>100,000 cells/mL) in the urine. These infections

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are commonly caused by Gram-negative bacteria, particularly E. coli,1 and occur primarily in

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women. UTIs begin with adherence to, then colonization of bacteria on urinary tract epithelial

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cells. Adherence by E. coli is performed by proteinaceous fimbriae on the bacterial cell surface,

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which attach to specific oligosaccharide receptors on epithelial cells. One in four women with

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UTIs will experience a recurrence of the infection and are often found to be more prone to these

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infections. Natural substances which could treat and/or prevent UTIs could be beneficial for

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women suffering from this condition since antibiotic treatment in many cases causes a secondary

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vaginal yeast infection requiring subsequent treatment with antifungal agents.

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Cranberry juice is acidic (≤ pH 2.6), and rich in anthocyanins and tannins giving it an

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astringent taste.2 The juice is prepared by milling and pressing after a hot (50 °C, 1 h)

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commercial pectinase maceration of the berries. Cranberry pectin has very high-methoxy group

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content, which requires a second hot commercial pectinase treatment following pressing and

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prior to juice filtration and concentration. Cranberry juice is considered a healthy juice with

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prebiotic properties,3 and there is a large volume of literature on the role of cranberry

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phytochemicals in preventing or mitigating UTIs.4-6 Cranberry proanthocyanidins have

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antioxidant properties7,8 and were reported to inhibit adhesion of p-fimbriated E. coli to

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uroepithelial cells.9 The same α-Gal-(1-4)-ß-Gal receptor is required for red blood cell

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agglutination and p-fimbriated E. coli adhesion to uroepithelial cells.9 Cranberry juice plus D-

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mannose prevented bacterial adherence, colonization and ultimately prevented uncontrollable

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UTIs due to E. coli type 1 fimbriae specifically binding to D-mannose, rather than sucrose or

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fructose.10 Previously, pectic oligosaccharides were reported to inhibit the adhesion of

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enterohemorrhagic and enteropathogenic strains of E. coli to HT29 cells.11 This establishes a

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precedent for plant cell wall oligosaccharides that have anti-adhesive properties for E. coli

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binding to epithelial cell receptors. The cranberry plant cell wall structure consists of

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parenchyma cellulose, hemicellulose and pectin rich in arabinan.12

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Xyloglucan is a well-known hemicellulose that cross-links type 1 plant cell walls found

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in dicotyledonous and non-commelinoid monocotyledonous plants.13 Xyloglucan, which has a

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ß-(1-4)-glucan backbone, is able to hydrogen bond to the surface of cellulose microfibrils

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forming a network that connects adjacent microfibrils in cell walls. The xyloglucan network is

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intermeshed with the pectin network of cell wall matrix polysaccharides.14 This makes

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xyloglucan an important polysaccharide in the growth and development of primary cell walls.14

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There is a block-like structure in xyloglucan in which a 6-11 sugar sequence is repeated

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throughout the polysaccharide. Carbohydrate characterization of xyloglucan oligosaccharide

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fragments has been used to describe polysaccharide structures specific for plant taxonomic

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groups.15-18 Three types of xyloglucan structure have been described with fucogalacto-

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xyloglucan the most commonly distributed in about half of the monocot taxonomic orders and all

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dicot orders except for the Solanales, Laminales, Gentianales and Ericales.14,18 Xyloglucan from

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these later orders contains arabino-xyloglucan structure. Small amounts of a third xyloglucan

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structure is also present in commelinoid monocots (grasses, bromeliads, palms and cypresses) as

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randomly distributed single xylose substituents on a cellulosic backbone.14 A single letter

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nomenclature was developed to describe xyloglucan substituent sequences.19 We investigated

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cranberry xyloglucan structure and detected arabino-xyloglucan (SSGG) oligosaccharide

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fractions with anti-E. coli adhesion activity for epithelial cell receptors.

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MATERIALS AND METHODS

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Materials. The following were obtained from Sigma-Aldrich (St. Louis, MO):

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phosphate-buffered saline tablets (PBS; pH 7.4 ± 0.2, 0.1 mol/L), non-essential amino acid

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solution, trypsin-EDTA solution, Dulbecco’s modified Eagle medium with GlutaMAX-1

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(DMEM), tryptic soy agar and 2,5-dihydroxybenzoic acid. Fetal Bovine Serum was obtained

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from the American Type Culture Collection (ATCC, Manassas, VA). PBS was filter sterilized

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with a 0.2µm syringe filter prior to use.

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Xyloglucan-oligosaccharides with a degree of polymerization (DP) of 7 to 9 were

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purchased from Megazyme (Bray, Ireland). Commercial juices were purchased from a local

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grocery store.

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Oligosaccharide isolation. A cranberry (Vaccinium macrocarpon, mainly Stevens

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variety harvested in Massachusetts during 2009) hull enzyme-treated concentrate fraction (A1)

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was produced using Klerzyme 150 pectinase (DSM Food Specialties, South Bend, IN) during

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cranberry depectinization. Fractionation of A1 was accomplished by utilizing a FLASH-40

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system (Biotage, Charlotte, NC), converted to accept Biotage SNAP KP-C18-HS 120 g

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cartridges. A1 (20 g) was dissolved in 200 mL of deionized (DI) water and 50 mL of the solution

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was loaded onto the pre-conditioned (300 mL methanol, 300 mL DI water) C18 column

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cartridge. The C18 column cartridge was eluted first with 500 mL of DI water, followed by 500

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mL of a 15% methanol/water mixture (35 mL/min flow rate). The remaining phenolic content

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was washed from the column with 500 mL of methanol. The column was reconditioned before

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loading it again with the next 50 mL of A1 solution. After the C18 separation, the 15%

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methanol/water fractions were combined and dried to produce the A2 fraction (4.97 g of a pink

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colored powder). An unknown peak was observed in A2 chromatograms at approximately 6.7

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min during high pressure liquid chromatography (HPLC) analysis of fraction A2 using an 1100

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series (Agilent Technologies, Santa Clara, CA) system equipped with a refractive index detector

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and HPX-87C HPLC column (Bio-Rad Laboratories, Inc., Hercules, CA). Fraction A2 was

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further purified by Sephadex LH20 (Sigma-Aldrich, St. Louis, MO) chromatography to eliminate

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phenolic pigments. Fraction A2 (4.8 g) was dissolved in 60 mL of DI water and the mixture was

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loaded on a 300 x 45 mm Sephadex LH20 column (pre-conditioned with 500 mL of DI water).

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The column was eluted with 500 mL of DI water using a Masterflex L/S pump (Cole-Parmer,

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Vernon Hill, IL) at a flow rate of 2.5 mL/min, followed by lyophilization to produce 4.32 g of a

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slightly pink, off-white powder A6 fraction.

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Carbohydrate analysis. The neutral sugar content (NS), uronic acid content (UA),

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degree of methyl esterification (DE) and degree of acetylation (DA) were determined as reported

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previously20 except that the NS (glucose standard) was used as the basis for DE and DA instead

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of the UA. Monosaccharide analysis by high-performance anion-exchange chromatography with

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pulsed amperometric detection (HPAEC-PAD) following methanolysis was according to the

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procedure reported previously.21 Unhydrolysed oligosaccharides were also separated by HPAEC-

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PAD according to the procedure reported previously.11

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High Performance Size Exclusion Chromatography. Cranberry samples (10-20

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mg/mL) were dissolved in 0.05 M NaNO3 and 0.01% NaN3 mobile phase by stirring at room

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temperature, centrifuged at 50,000 g for 10 min and filtered through a 0.22 or 0.45 µm Millex

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HV filter (Millipore Corp., Bedford, MA). The model 1200 series solvent delivery system,

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included a degasser, pump and auto sampler (Agilent Corp.) with flow rate set at 0.7

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mL/min. The injection volume was 200 µL. Samples were run in triplicate. The column set

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consisted of three 300 mm x 7.8 mm i.d., 13 µm, TSK GMPWXL size exclusion columns in

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series and two 40 mm x 6.0 mm i.d., 12 µm, guard columns of the same material (Tosoh

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Bioscience, Tokyo, Japan) with one guard column placed before the separatory columns and the

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other prior to the detectors. The column temperature was controlled in a water bath set at 35

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°C. Column effluent was detected with a HELEOS II multi-angle laser light scattering

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photometer (Wyatt Technology, Santa Barbara, CA), in series with a model Viscostar

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II differential pressure viscometer (Wyatt Technology) and an differential refractive index

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interferometer (Wyatt Technology). Electronic outputs from these three detectors were processed

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with ASTRA software version 6.1.1.17 (Wyatt Technology).

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Mass Spectrometry. Matrix-assisted laser desorption ionization mass spectrometry with

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tandem time of flight mass spectrometry (MALDI-TOF/TOF MS/MS) utilized a 4700

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Proteomics Analyzer mass spectrometer (Applied Biosystems, Framingham, MA) in the positive

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reflectron mode. Spectra were obtained by averaging 1000 and 2500 acquired spectra in the MS

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and MS/MS modes, respectively. Collision induced dissociation (CID) with air at approximately

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1 x 10-6 Torr and 1 keV acceleration voltage was used for obtaining the MS/MS spectra for

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selected oligosaccharides. Conversion of TOF to mass (Da) for the monoisotopic ions, [M+Na]+,

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was based on calibration of the instrument with a peptide standard calibration kit (Applied

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Biosystems). Oligosaccharide samples (3-5 mg) were dissolved in 1 mL of water and cleaned

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with CarboPrep 90 graphitized carbon cartridges, 3 mL, 250 mg (Restek, Bellefonte, PA). The

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cartridges were first conditioned by passing 3 mL of acetonitrile:water (50:50) and then washed

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4 times with 3 mL of water. After conditioning, the oligosaccharide solution was passed through

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the graphitized carbon cartridge, washed 3 times with 3 mL of water and the water wash was

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discarded. The oligosaccharides were eluted with 1 mL of acetonitrile:water (30:70, v/v)

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containing 0.1% TFA. From this solution, 2 µL were mixed with 10 µL of a solution of 2,5-

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dihydroxy benzoic acid (10 mg/mL in acetonitrile:water (50:50), 0.1% TFA), and spotted onto a

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MALDI plate for analysis.

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Nuclear Magnetic Resonance Spectroscopy. Cranberry fraction A6 was dissolved in

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H2O (99.6% 2H, Cambridge Isotope Laboratories, Tewksbury, MA), lyophilized and redissolved

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in 0.6 mL of 99.96% enriched 2H2O and transferred to a 5 mm NMR tube. Spectra were recorded

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at 40 oC on either a Bruker Avance-II 700 MHz spectrometer (Billerica, MA) using a 5 mm xyz-

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PFG QXI HCNP probe or a Varian INOVA 500 MHz spectrometer (Santa Clara, CA) equipped

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with a z-PFG PentaProbe. Data processing was performed using NMR-Pipe,22 and analyzed

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using SPARKY.23 All spectra were referenced to the internal 1H and 13C resonances of either

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4,4-dimethyl-4-silapentane-1-sulfonic acid (DSS) or 3-(trimethylsilyl)-2,2',3,3'-

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tetradeuteropropionic acid (TMSP-d4). One dimensional 1H NMR spectra were acquired at 700

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MHz using a spectral width of 5,000 Hz, 32,768 points, 70o pulse width, and a recycle time of

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2.5 s. One dimensional 13C NMR spectra were acquired at 176 MHz using 65,536 data points, a

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70o pulse width, and a recycle delay of 2.5 s. Gradient enhanced versions of the following

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experiments were run: Three 2D TOCSY (10 ms, 120 ms and 300 ms using the DIPSI-2

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sequence24 for a spin-lock), 2D ROESY (200 ms mixing time), two 1H-13C-HSQC, long-range

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1

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ms and 300 ms spin-lock). The 2D homonuclear experiments were recorded with spectral widths

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of 5K – 7K Hz in both dimensions, using 8192 - 16384 points in the directly-detected dimension,

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512 increments in the second dimension, 32 or 64 transients per acquisition depending on signal

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intensity and a 2.5 s delay between scans. The heteronuclear experiments were acquired with a

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range of parameter values depending on the specific experiment, the instrument and the digital

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resolution needed. These parameter values were 2,097 or 7000 Hz spectral width in the 1H

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dimension, and 10K or 19K Hz in the 13C dimension (or in the HMBC 25K Hz), using 1024-

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8192 points in the directly-detected dimension, 512 increments in the second dimension, 32 or 64

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transients per acquisition depending on signal intensity and a 2.5 s delay between scans. These

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experiments were run on several batches of the sample and enabled the assignment of resonances

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and measurement of coupling constants to determine the sugar residue identities, their anomeric

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configuration and connectivity as well as the local structure.

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H-13C HSQC, JH-C-coupled HSQC, 1H-13C-HMBC (JnC-H = 6 Hz) and two HMQC TOCSY (50

Anti-adhesive Activity. A bacterial adhesion assay was carried out with human bladder

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epithelial cells T24 (ATCC #HTB-4 UEC) and E. coli CFT073 (ATCC # 700928) and UTI89

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strains.25, 26 Cranberry extracts A1 and A6 were diluted in PBS to concentrations of 0 – 5 mg/mL.

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A cranberry enriched proanthocyanidin (PAC) fraction (55% total PAC analyzed by 4-

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dimethylaminocinnamaldehyde colorimetric method with purified cranberry PACs as a reference

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standard)27 was used as positive control in the CFT073 assay and was diluted in PBS to

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concentrations of 0 – 0.5 mg/mL (pH 7). α-D-Mannose was used as positive control in the UTI89

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assay and was diluted in PBS to concentrations of 0 – 2 mg/mL. PBS was used as a negative

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control. Briefly, fluorescent-labeled uropathogenic E. coli strains CFT073 and UT189 were

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subcultured using colonizing factor agar (CFA) and Luria-Bertani (LB) broth, respectively. CFA

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was used to promote p-fimbriae growth in CFT073, while UTI89 only express type 1 fimbriae.

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Bacteria were pre-incubated with A1 and A6 fractions and control samples for 30 min at 35 °C

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before being added to T24 cells in 96-well plates at an E. coli:T24 ratio of 400:1. E. coli and T24

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co-cultures were incubated in a 5% CO2 humidified atmosphere for 1 h at 37 °C. Following the

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incubation, bacteria not adhering to the T24 cells were removed by washing with PBS. The

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fluorescence intensity (FI) was measured in a microplate reader at 480 nm excitation/516 nm

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emission (Synergy H1 Hybrid, Biotek Instruments, Winooski, VT). All samples were tested in

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triplicate.

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The effect of A6 on bacterial adhesion was also determined with E. coli O157:H7 (ATCC

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#BAA-1883) and human colonic epithelial cells HT29 (ATCC #HTB-38) using a standard assay

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reported earlier.11 Working cultures of E. coli were prepared by inoculating slopes of agar in

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universal bottles and incubating the agar for 18-24 h at 37 ºC. E. coli broth cultures for adhesion

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assays were grown in DMEM supplemented with 5% (v/v) fetal bovine serum and 1% (v/v) non-

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essential amino acid solution (SDMEM). The broth was inoculated from a slope culture and

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incubated anaerobically at 37 ºC for 18-24 h. The overnight culture was then inoculated 1% (v/v)

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into fresh SDMEM and incubated for a further 18 to 24 h under the same conditions. This was

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repeated. On the day of the assay, a 10% (v/v) inoculum was again inoculated into prewarmed

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SDMEM and incubated for 4 h aerobically at 37 ºC.

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HT29 human intestinal epithelial cells were grown in 25-cm2 tissue culture flasks in

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SDMEM at 37 ºC in 5% CO2 until approximately 90% confluent, split according to the method

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recommended by the European Collection of Cell Cultures, and stored in aliquots over liquid

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nitrogen. These aliquots were used to seed 25 cm2 flasks, which after growth were split into 12-

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well tissue culture plates. The 12 well plates were grown to approximately 95% confluence

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before being used for the adhesion assays.

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A culture of the E. coli strain was prepared as described above, then diluted 1:500 in

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PBS. The viable count of the diluted suspension was determined by spread plating onto plate

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count agar, with decimal dilution being carried out in PBS buffer as appropriate. A6 was

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dissolved in PBS (5 mg/mL) and sterilized by passing through a 0.2 µm syringe filter. The

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carbohydrate solution was further diluted in sterile PBS as required. The SDMEM was aspirated

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into a 12-well tissue culture plate with near confluent monolayers of HT29 cells, prepared as

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described above. The monolayers were washed by pipetting in 1ml of sterile PBS per well,

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swirling by hand, and then aspirating. A 0.5 mL aliquot of the A6 solution was added to the well,

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followed by 0.5 mL of bacterial suspension in PBS. Control wells contained unsupplemented

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PBS substituted for the A6 solution. All assays were performed in triplicates. The plates were

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swirled by hand to mix and then incubated at 37 ºC aerobically for 2 h.

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After incubation, the bacterial suspension was aspirated from the wells. A 1 mL aliquot

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of PBS was added to each well, the plate swirled briefly by hand, and the PBS removed. The

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washing step was repeated two more times. A 70 µL aliquot of trypsin-EDTA solution was

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added to each well, the plate was rocked to ensure even coverage, and then it was incubated at 37

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ºC for 5 min. A 1 mL aliquot of PBS was then pipetted into each well and pipette-mixed until the

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monolayer was completely dislodged and clumps dissolved (as determined visually). Bacteria in

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cell suspension were then enumerated by plate counting on plate count agar plates with decimal

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dilutions performed in PBS as required. All plates were incubated at 37 ºC for 18-24 h before

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colonies were enumerated. Viable counts were calculated for all wells and the inoculum and are

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expressed as CFU/mL. For each test the mean and the standard error of the triplicate wells were

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calculated. Statistical significance was determined by one-way analysis of variance, using

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ANOVA software.

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RESULTS AND DISCUSSION

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Carbohydrate Analysis. The A1 fraction produced by commercial pectinase treatment

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of cranberry hulls contained pectin fragments and significant amounts of neutral sugar-rich

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material (Table 1). The A2 fraction was neutral sugar-rich and had relatively low amounts of

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uronic acid indicating little pectin was present. The most purified of the cranberry fractions (A6)

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consisted of a neutral sugar-rich polysaccharide with a weight-average molar mass of 2,750 Da

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compared to 3,410 Da for A2 and 5,510 Da for A1. The z-average hydrodynamic radius and

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weight-average intrinsic viscosity were consistent with low molecular weight and low viscosity

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polysaccharides. Fraction A6 had no absorption at 230 nm and 280 nm indicating the lack of the

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aromatic structures present in proanthocyanidins (PACs).

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The monosaccharide compositions of the A2 and A6 cranberry fractions were dominated

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by glucose, arabinose and xylose with very little galacturonic acid and rhamnose present (Table

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2). This indicated that the A2 and A6 polysaccharide fragments were possibly a xyloglucan. The

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A1 fraction contained a significant amount of galacturonic acid, but the low levels of rhamnose

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detected indicated that little rhamnogalacturonan was present. Therefore, A1 contained

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homogalacturonan-rich pectin and other polysaccharides released from the pectinase treatment of

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cranberry hulls.

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A series of oligosaccharide peaks were present in A2 and A6 cranberry fraction HPAECPAD chromatograms that were consistent with the retention times of xyloglucan standard

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oligosaccharides (Figure 1). However, the structure of the Megazyme standard xyloglucan

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oligosaccharides was galacto-xyloglucan. For example, the DP 7 xyloglucan oligosaccharide

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consisted of a cellotetraose backbone and three single xylose substituents attached to three of the

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glucose residues. The DP 8 and DP 9 xyloglucan standards had one or two of the xylose residues

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substituted with a galactose residue, respectively. This xyloglucan structure is typical of

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fucogalacto-xyloglucan without fucose. However, relatively low amounts of galactose were

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detected in the A2 and A6 monosaccharide composition, which indicated that the cranberry

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xyloglucan structure might differ from fucogalacto-xyloglucan.

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Mass Spectrometry. MALDI-TOF mass spectrometry of the A6 fraction produced a

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series of [M+Na]+ ions (Figure 2) that were consistent with xyloglucan structure (Table 3). No

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[M+Na]+ ions were observed corresponding to PACs.9 The ions at m/z 1085, 1217, 1379 and

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1525 were reported for argan tree (Sapotaceae) xyloglucan with Hex4Pent3, Hex4Pent4,

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Hex5Pent4, and Hex5Pent4dHex composition, respectively, and described as fucogalacto-

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xyloglucan structure.17 Ions at m/z 791 and 923 were previously reported in tobacco suspension

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culture xyloglucan with SGG and/or XXG, and SXG and/or XSG structure, respectively.15 The

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very abundant m/z 1217 ion in cranberry A6 (Figure 2) was previously reported to have XXSG

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structure in oleander and olive fruit xyloglucan.16,18 However, the diagnostic XXSG MS/MS

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fragments16 were not present in the cranberry MALDI-TOF/TOF MS/MS spectrum for the m/z

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1217.37 ion shown in Figure 3B. Therefore, the SSGG xyloglucan structure appeared to be the

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predominant cranberry xyloglucan structure as was reported for m/z 1217 in tobacco.15 Ions at

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m/z 1127, 1259, 1289, 1331 and 1421 ions were previously reported as acetylated xyloglucan

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oligosaccharides in tobacco.15 The m/z 1289 and 1331 ions were assigned previously as XLXG

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and/or XXLG, and UXGGG and/or XUGGG structure, respectively.15 However, in cranberry

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xyloglucan, these ions are more likely to represent an alternative structure due to the relatively

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low amount of galactose present in the monosaccharide composition. Therefore, XSGGG-Ac and

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XSGGG-Ac2 cranberry xyloglucan structures appear to be more appropriate. The

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Hex4PentndHexHexA2Me3 xyloglucan composition series has never been reported before but

271

these structures are similar to the m/z 1525 XUFG structure reported for the Argan tree (Ericales)

272

xyloglucan and the xylan oligosaccharides containing two 4-O-methyl-glucuronic acids from the

273

same source.17 Another very abundant cranberry ion, m/z 1055, also has never been reported

274

previously for a xyloglucan structure and was used for further MS/MS structural elucidation. The

275

cranberry m/z 1055 and 1217 ions also were detected as their potassiated forms ([M+K]+) at m/z

276

1071 and 1233, respectively. The most abundant cranberry MALDI-TOF MS ions were m/z

277

1055, 1085 and 1217 depending on batch to batch variation in A6.

278

The MS/MS spectra of oligosaccharides by MALDI-TOF/TOF produced a set of ions that

279

provides the essential information for carbohydrate structural characterization.28 The MS/MS

280

spectrum represents two types of ions corresponding to cross-ring fragmentation between two

281

bonds on the same sugar residue and glycosidic bond cleavage between two sugar residues.

282

When the charge of the resulting fragment is located toward the reducing end of the

283

oligosaccharide, then the ions are designated as X ions for cross-ring fragmentation, and Y and Z

284

ions for the glycosidic bond fragments. If the charge is located at the nonreducing end, then the

285

ions are designed as A for cross-ring fragmentation, and B and C for the glycosidic bond

286

fragments. The fragmented bond site is indicated with the corresponding letter, a subscript

287

number, and a Greek letter to designate the branched chain involved. Superscript numbers

288

preceding the ions X or A indicates the cleaved bonds in the sugar ring.

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The MS/MS spectrum in Figure 3A corresponds to the sodiated oligosaccharide,

290

[M+Na]+, with m/z 1055.32 (Figure 2). This precursor ion is consistent with an oligosaccharide

291

structure formed by 4 pentoses and 3 hexoses. The ion at m/z 305.3 in the MS/MS spectrum

292

(Figure 3A) suggests that two pentoses are present as a side chains, labeled as fragments C2α and

293

C2β in structure 1 (Figure 4). No ion corresponding to a side antenna with 1, 3 or 4 pentoses is

294

observed in the in the spectrum. The ion at m/z 893 indicates the loss of a hexose from the

295

precursor ion (1055-162) (cleavage C4), (Figure 4). This suggests that one of the hexoses is not

296

linked to a side chain. The spectrum is not dominated by Y or B ions but with ions that are

297

consistent with cross-ring fragmentation as indicated in 1. As the spectrum shows, the cross-ring

298

cleavage (loss 104 Da) at the pentose-end produces the ion at 951 (1,4X4α or 1,4X3β fragment) that

299

further loses a pentose to generate the ion at 819 and a hexose to generate the ion at m/z 657.

300

However, these two ions can also correspond to the 1,5X3α and 1,2X2 fragments, respectively.

301

Some of the ions are generated from subsequent fragmentation of the 951 fragment, such as the

302

ion at m/z 891 (951-60). The significant ion at m/z 465 could also be explained by subsequent

303

cleavages of the ion at 951 after losing 1 hexose, 2 pentoses and a cross-ring fragment (951-162-

304

132-132-60 = 465). Therefore, several ions in Figure 3A could be produced by multiple

305

fragmentation patterns. Further analysis of the fragmentation pattern suggests that the isobaric

306

structure 2 (Figure 4) is also present. The evidence supporting structure 2 is found in the ion at

307

m/z 921, which corresponds to the cross-ring cleavage 1,5X*2 of the hexose at the nonreducing

308

terminus. Therefore, both SSG and GSS structures were present and assigned (Table 3). The

309

spectrum in Figure 3B is consistent with the structures in Figure 4 but with an extra hexose. The

310

cross-ring cleavage of the end terminus pentose produces the ion at m/z 1113 (1217-104), but

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also the loss of a hexose from the precursor ion generates the ion at m/z 1055, and that fragment

312

is consistent with the spectrum and structures in Figure 3A.

313

Nuclear Magnetic Resonance Spectroscopy. NMR analysis of the anomeric region in

314

the 1D-1H spectrum (Figure 5) reveals that the resonances near 4.5 ppm have H1-H2 coupling

315

constants of JH1-H2 ~ 7-9 Hz, indicating that the H1 and H2 are axial-axial orientation. Therefore,

316

they were assigned as β-D-Glc and β-D-Gal. The resonances at 4.94, 5.24, 5.21, 5.15, 5.31, 5.28

317

ppm have JH1-H2 ~3.8-4.7 Hz and denote that the H1 and H2 have an axial-equatorial orientation.

318

Likewise, the peak at 5.08 ppm has a shoulder that is also 3.5 Hz from the main peak. These

319

chemical shifts and coupling constants are characteristic of pyranoses, such as rhamnose, fucose

320

and xylose. Several peaks in the region of ~5.1 – 5.2 ppm have very small or indiscernible

321

coupling in the 1D-1H spectra. These attributes support an assignment of α-L-Araf and there is

322

no evidence for β-L-Araf in the sample. Furanoses such as these undergo rapid re-puckering of

323

their rings, resulting in an averaging of the coupling constants. The absence of aromatic

324

resonances in the 1D-1H NMR spectra supported the absence of PACs and other phenolic

325

compounds in cranberry fraction A6.

326

Analysis of the 2D HSQC (Figure 6) confirms β-Glc, α-Xylp, and α-Araf are present, as

327

well as other residues expected from the monosaccharide composition and 1D-1H spectrum. The

328

HSQC reveals that the proton resonances at 4.48-4.57 ppm are separated into two main clusters

329

based on their 13C resonances. One is centered at a 13C frequency of ~105 ppm, and corresponds

330

to β-D-Glcp, whereas the other cluster is at ~107 ppm and corresponds to β-D-Galp. Such β-D-

331

Glcp resonances are commonly found in S and G structures in SG or GS environments.29 While

332

the β-D-Galp resonances can be found in xyloglucan F and L structures,29 it is also present in

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rhamnogalacturonan pectin and this monosaccharide is less abundant in the cranberry fractions.

334

Therefore, it is unclear what structure includes the galactose in cranberry A6.

335

The HMBC and long range HSQC experiments provide evidence for α-D-Xylp -C1 → β-

336

D-Glcp-H6 correlations, confirming xyloglucan structure. In addition, there are two sets of

337

through-bond α-L-Araf-C1→ α-D-Xylp-H2 correlations, which are consistent with S-type

338

structures (Figure 7). One of these xylose residues also has an HMBC correlation to a glucose

339

H6, and thus proves an entire S-type structure of α-L-Araf (1→2) α-D-Xylp (1→6) β-D-Glcp.

340

The fact that there are two sets of α-L-Araf (1→2) α-D-Xylp correlations, suggests that the S

341

structures are in slightly different environments, as would be expected from GSS and SSG

342

sequences. Although additional arabinose and xylose residues were observed in the NMR

343

spectra, their sequential connectivity could not be determined due to spectral congestion and

344

overlapping resonances.

345

There is also evidence of acetylation of some of the sugars in the fraction. Analysis of the

346

HSQC, HMBC, long-range HSQC and HMQC-TOCSY experiments revealed the presence of

347

methyl groups (~2.2 ppm in 1H and ~20 ppm in 13C) that have HMBC correlations to carbonyl

348

carbons (~175 ppm), and these in-turn have correlations for the H2 resonances (~4.3 ppm) of

349

several α-L-Araf residues. While each sample batch demonstrated acetylation correlations, we

350

noted that there was batch-to-batch variation in the specific arabinose residue involved in these

351

correlations. However, acetylation was never observed for those arabinose residues identified as

352

being in the S-type xyloglucan structure.

353

The NMR data also indicate the presence of secondary components consisting of β-D-

354

GalpA, α-L-Rhap, and α-L-Fucp in addition to the β-D-Galp discussed above. The β-D-GalpA is

355

indicated by through-bond correlations in the HMBC spectrum between the C6 in the carboxylic

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acid group and the H5 in its sugar ring. The presence of α-L-Fucp is indicated by H6 methyl

357

resonances (1.26 ppm) that have TOCSY correlations to H5 protons on the directly attached

358

sugar rings (3.44 - 4.74 ppm and have JH5-H6 = 6-7 Hz). These α-L-Fuc correlations can be

359

followed in the HMBC, long-range HSQC and HMQC-TOCSY experiments from the methyl

360

resonances to corresponding anomeric resonances (data not shown). Additional methyl

361

resonances exist (1.10 – 1.20 ppm) which are consistent with α-L-Rhap. Spectral congestion

362

does not allow through-bond correlations of these α-L-Rhap methyl protons to their anomeric

363

resonances. Therefore, the α-L-Rhap assignment is likely but cannot be verified due to the lack

364

of through-bond correlations.

365

The anomeric proton orientations (α or β) were confirmed by means of their C-H

366

coupling constants, JC1-H1, as derived from the C-H coupled HSQC and HMBC experiments.

367

This determination helps to confirm resonance assignments and utilizes the fact that axially

368

oriented H-1 have JC1-H1~160 Hz, and those with an equatorial H-1 have values ~170 Hz.28 A

369

summary of the anomeric chemical shifts, their assignments, and likely substructures can be

370

found in Table 4 and are annotated in Figure 6. These assignments are based on coupling

371

constants and through-bond correlations, and are consistent with literature values of both the 1H

372

and 13C chemical shifts.16,18,30-35 The same xyloglucan oligosaccharides with SSGG structure

373

(m/z 1217 most abundant ion) were also detected in commercial cranberry juice with the

374

MALDI-TOF MS ion intensity directly proportional to the amount of cranberry juice present

375

(data not shown). No xyloglucan oligosaccharides were detected in commercial apple, grape or

376

pomegranate juices using the same graphitized carbon extraction method detailed above for

377

cranberry fractions.

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Anti-adhesive Activity of Cranberry Fractions. The anti-adhesive activity of cranberry

379

xyloglucan-rich fractions was determined by bacterial adhesion to epithelial cell assays using

380

both uropathogenic and enterohemorrhagic E. coli strains in order to identify binding to type 1

381

(UTI89) or P- (CFT073) fimbriae and those produced by enterohemorrhagic E. coli (O157:H7).

382

Fimbriae are important bacterial virulence factors required for binding to epithelial cells in the

383

urinary tract; type 1 fimbriae are typically associated with cystitis and p-fimbriae with

384

pyelonephritis infections. Specific binding of p-fimbriated E. coli to uroepithelial cells decreased

385

with increasing concentrations (0 – 5 mg/mL) of cranberry extract A6 in a dose-dependent

386

manner (Figure 8). The half-maximal inhibitory concentration of A6 was 0.82 mg/mL. As

387

previously reported by Kimble et al.,25 a cranberry polyphenol extract enriched in

388

anthocyanidins, polyphenolic acids, flavonols and PACs was used as a positive control in the p-

389

fimbriated E. coli assay, and exhibited a specific binding reduction between 0 – 1.25 mg/mL

390

with a half-maximal inhibitory concentration of 0.12 mg/mL. Therefore, the oligosaccharide

391

enriched extract A6 showed 6.8 times lower binding affinity to the p-fimbriated E. coli compared

392

to the cranberry polyphenolic enriched standard. Cranberry extract A1 showed a slightly higher

393

inhibitory effect compared to A6 (0 – 30 mg/mL A1) with a half-maximal inhibitory

394

concentration of 0.64 mg/mL (data not shown), possibly due to presence of phenolic compounds,

395

especially PACs in extract A1.

396

A concentration-dependent (0 – 5 mg/mL A6) reduction of binding was also observed in

397

the type 1 E. coli assay (Figure 9). The half-maximal inhibitory concentration of A6 was 0.66

398

mg/mL. These results are comparable to the half-maximal inhibitory concentration of α-D-

399

Mannose (0.9 mg/mL) standard, a natural ligand for type 1 fimbriae. Cranberry A1 exhibited a

400

half-maximal inhibitory concentration of 4.3 mg/mL, and therefore was less effective at

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preventing bacterial adhesion compared to A6 for type 1 fimbriated E. coli. The cranberry extract

402

A1 was enriched in pectic oligosaccharides and contained polyphenolic compounds compared to

403

the xyloglucan oligosaccharide-enriched A6 fraction. Thus, the higher levels of xyloglucan

404

oligosaccharides correlated with the higher anti-adhesion activity for type 1 fimbriated E. coli.

405

From these results, purified cranberry A6 xyloglucan oligosaccharides have a higher binding

406

affinity toward type 1 fimbriated E. coli compared to p-fimbriated strains.

407

A non-dose-dependent response was observed for the anti-adhesive activity of A6 in the

408

bacterial adhesion assay reported earlier.11 The adhesion of E.coli O157:H7 to human colonic

409

epithelial HT29 cells was blocked by A6 at low concentrations (0.001-0.1 mg/mL) relative to the

410

unsupplemented PBS control (Figure 10). At higher concentrations, the cranberry A6 fraction

411

was not able to inhibit E. coli O157:H7 adhesion to HT29 cells. Enterohemorrhagic E. coli

412

produces several adhesive structures including the E. coli common pilus and type IV pilli.36,37

413

Prebiotic oligosaccharides (galacto-oligosaccharides and pectic-oligosaccharides) were also

414

reported to have dose-dependent anti-adhesive properties against enterohemorrhagic and

415

enteropathogenic E. coli strains,11,38 P-fimbriated E. coli and verocytotoxins produced by

416

enterohemorrhagic strains of E. coli utilize the same α-Gal-(1-4)-ß-Gal terminal oligosaccharide

417

receptor for adhesion to epithelial cells, while type 1 fimbriae utilize a α-Man-(1-2)-Man

418

receptor.39 It is unlikely that xyloglucan, pectic- and galacto-oligosaccharides prevent E. coli

419

adhesion through receptor mimicry since α-Man-(1-2)-Man or α-Gal-(1-4)-ß-Gal have not been

420

detected in cranberry or citrus fractions or prebiotic products. However, this data indicates that

421

cranberry xyloglucan oligosaccharides have a higher affinity for type 1 fimbriae, while pectic-

422

and galacto-oligosaccharides have higher affinity for P-fimbriae and those produced by

423

enterohemorrhagic and enteropathogenic E. coli strains.

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In conclusion, the cranberry xyloglucan structure was characterized for the first time as

425

an arabino-xyloglucan with SSGG structure. This is the first member of the Ericales with this

426

type of xyloglucan structure, but only the second plant in the order to have the xyloglucan

427

characterized. A new xyloglucan heptasaccharide from cranberry was characterized by MALDI-

428

TOF/TOF MS/MS and NMR with SSG and GSS oligosaccharide structure. NMR data, including

429

through-bond correlations, coupling constants and chemical shift values, enabled the assignment

430

of α and β linkages, the sugar residues and confirmed the structural sequence. NMR provided

431

direct through-bond correlation evidence for α-L-Araf (1→2) α-D-Xylp linkages and α-D-Xylp

432

(1 → 6) β-D-Glcp linkages, as found in xyloglucans with S-type structures. There appear to be

433

two cranberry xyloglucan S structures with slightly different physical environments, which can

434

be attributed to SSG and GSS sequences. The cranberry A6 fraction that included SSGG-type

435

xyloglucan oligosaccharides prevented adhesion of both p-fimbriated and type 1 uropathogenic

436

E. coli strains to human uroepithelial cells and veritoxigenic E. coli to colonic epithelial cells in

437

vitro using three independent assays. In addition to proanthocyanidins, SSGG-type xyloglucan

438

oligosaccharides are new bioactive cranberry juice ingredients with potential to control urinary

439

tract infections. However, more research is needed to determine the exact mechanism of

440

xyloglucan oligosaccharide anti-adhesion activity against pathogenic E. coli.

441

ASSOCIATED CONTENT

442

Supporting Information

443

SI 1: Molar mass, intrinsic viscosity and hydrodynamic radius of cranberry fractions.

444

2: NMR data for α-D-Xylp -C1 → β-D-Glcp-H6 correlations.

445

3: 2D NMR data for acetylation of Araf residues.

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4: 2D NMR data for GalAp residues.

447

This material is available free of charge via the Internet at http://pubs.acs.org.

448

AUTHOR INFORMATION

449

Corresponding Author

450

*E-mail: [email protected], Phone: (215) 233-6448, Fax: (215) 233-6795.

451

Funding

452

This work was supported by a Cooperative Research and Development Agreement with Ocean

453

Spray Cranberries, Inc. (58-3K95-1-1503).

454

Notes

455

The authors declare no competing financial interest.

456

Mention of brand or firm name does not constitute an endorsement of the U.S. Department of

457

Agriculture above others of a similar nature not mentioned.

458

ACKNOWLEDGEMENTS

459

The authors acknowledge helpful discussions with Drs. Darón Freedberg (FDA), Eugene

460

Mazzola (FDA) and Bruce Coxon (The Eunice Kennedy Shriver National Institute of Child

461

Health and Human Development, NIH) for interpretation of NMR data.

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26 553

(34) Jansson P.E.; Stenutz, R.; Widmalm G. Sequence determination of oligosaccharides and

554

regular polysaccharides using NMR spectroscopy and a novel Web-based version of the

555

computer program casper. Carbohydr. Res. 2006, 341, 1003-1010.

556

(35) Mazzola, E.P.; Parkinson, A.; Kennelly, E.J.; Coxon, B.; Einbond, L.S.; Freedberg, D.I.

557

Utility of coupled-HSQC experiments in the intact structural elucidation of three complex

558

saponins from Blighiasapida. Carbohydr. Res. 2011, 346, 759-768.

559

(36) Rendon, M.A.; Saldana, Z.; Erdem, A.L.; Monteiro-Neto, V.; Vazquez, A.; Kaper, J.B.

560

Commensal and pathogenic Escherichia coli use a common pilus adherence factor for epithelial

561

cell colonization. Proc. Natl. Acad. Sci. USA 2007, 104, 10637-10642.

562

(37) Xicohtencati-Cortes, J; Monteiro-Neto, V; Ledesma, M.A.; Jordan, D.M.; Francetic, O.;

563

Kaper, J.B.; Puente, J.L.; Giron, J.A. Intestinal adherence associated with type IV pili of

564

enterohemorrhagic Escherichia coli O157:H7. J. Clin. Invest. 2007, 117, 3519-3129.

565

(38) Shoaf, K.; Mulvey, G.L.; Armstrong, G.D.; Hutkins, R.W. Prebiotic galactooligosaccharides

566

reduce adherence of enteropathogenic Escherichia coli to tissue culture cells. Infect. Immun.

567

2006, 74, 6920-6928.

568

(39) Hotchkiss, A.T.; Buddington, R.K. Intestinal infections and prebiotics: The role of

569

oligosaccharides in promoting health. Functional Food Reviews 2011, 3, 119-134.

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27 Table 1. Carbohydrate Analysis of Cranberry Fractions. UA

DE

DA

NS

(mole%

(mole%

(mole%

(mole%

(SD))

(SD))

(SD))

(SD))

A1

35.6 (3.5)

7.0 (0.9)

1.7 (0.3)

53.8 (2.7)

A2

5.3 (0.1)

4.9 (0.2)

1.5 (0.0)

86.0 (2.2)

A6

13.0 (0.6)

7.5 (0.1)

1.7 (0.0)

81.3 (0.5)

UA = Uronic Acid, DE = Degree of Methyl Esterification, DA = Degree of Acetylation, NS = Neutral Sugar

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28 Table 2. Monosaccharide Composition of Cranberry Fractions. Glc

Ara

Gal

Xyl

Rha

Fuc

GalA

(mole%)

(mole%)

(mole%)

(mole%)

(mole%)

(mole%)

(mole%) (mole%)

A1

25.4

10.7

7.4

6.6

0.3

0.2

49.4

0.1

A2

46.0

28.5

4.6

17.0

0.5

0.4

3.2

0.0

A6

50.3

25.7

4.4

14.8

0.5

0.4

3.7

0.2

Glc = glucose, Ara = arabinose, Gal = galactose, Xyl = xylose, Rha = rhamnose, Fuc = fucose, GalA = galacturonic acid, GlcA = glucuronic acid

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GlcA

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Page 30 of 46

29 Table 3. MALDI-TOF Mass Spectrometry of A6. [M+Na]+

Composition

Assigned

[M+Na]+

Composition

Structure 791.24

Hex3Pent2

SGG, XXG

Assigned Structure

1421.44

Hex5Pent4-Ac

GSSGGAc

821.26

Hex4Pent

XGGG

1493.47

Hex2Pent6dHexHexAMe

833.25

Hex2PentdHexHexAMe

1511.48

Hex5Pent5

SSXGG

923.28

Hex3Pent3

SXG, XSG

1525.48

Hex5Pent4dHex

XUFG

953.30

Hex4Pent2

SGGG

1625.49

Hex2Pent7dHexHexAMe

965.29

Hex2Pent2dHexHexAMe

1757.55

Hex2Pent8dHexHexAMe

1055.32

Hex3Pent4

SSG, GSS

1889.58

Hex2Pent9dHexHexAMe

1085.33

Hex4Pent3

GSXG

2021.64

Hex2Pent10dHexHexAMe

1097.34

Hex2Pent3dHexHexAMe

2153.68

Hex2Pent11dHexHexAMe

1127.34

Hex4Pent3-Ac

XSGG-Ac

2285.74

Hex2Pent12dHexHexAMe

1217.37

Hex4Pent4

SSGG

2417.77

Hex2Pent13dHexHexAMe

1229.38

Hex2Pent4dHexHexAMe

1259.36

Hex4Pent4-Ac

SSGG-Ac

1289.39

Hex5Pent3-Ac

XSGGG-Ac

1331.37

Hex5Pent3-Ac2

XSGGGAc2

1379.43

Hex5Pent4

SSGGG

Hex = hexose, Pent = pentose, dHex = deoxyhexose (rhamnose or fucose), Ac = O-acetyl, G = ß-D-glucose, X = ß-D-glucose with a terminal α-D-xylose substituent at the O-6 position,

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30 S = ß-D-glucose with α-L-Araf-(1-2)-α-D-Xylp at the O-6 position, L = ß-D-glucose with ß-DGalp-(1-2)-α-D-Xylp at the O-6 position, F = ß-D-glucose with α- L Fucp-(1-2)-ß-D-Galp-(1-2)α-D-Xylp at the O-6 position, U = ß-D-glucose with ß-D-Xylp-(1-2)-Xylp with the O-6 position.

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31 Table 4. Chemical Shift Assignments of Identified Sugar Resonances.

Possible C1

C2

C3

C4

C5

C6

C6(Me)

C=O

C(Acyl-Me)

(ppm)

(ppm)

(ppm)

(ppm)

(ppm)

(ppm)

(ppm)

(ppm)

(ppm)

H1

H2

H3

H4

H5

H5e

H6

H6e

H6(Me)

H(Acyl-Me)

Possible

(ppm)

(ppm)

(ppm)

Structure

Residue

Structure (ppm) (ppm) (ppm) (ppm) (ppm) (ppm) (ppm)

Environment a-L-Fucp

94.53

74.00

74.75

---

72.73

---

19.34

---

---

5.21

3.58

3.84

3.85

3.94

---

---

---

1.29

---

α-D-Galp

98.10

---

---

---

---

---

---

---

---

5.28

3.76

3.84

3.94

4.08

---

---

---

---

---

β-D-Galp

107.40

78.37

---

---

---

---

---

---

---

4.53

3.68

---

3.94

3.60

---

---

3.80

---

---

β-D-Galp

107.30

---

---

---

---

---

---

---

---

4.53

3.66

3.83

3.94

3.64

---

---

3.80

---

---

β-D-Galp

107.20

---

---

---

---

---

---

---

---

4.54

3.40

---

---

3.61

---

---

---

---

---

β-D-Galp

107.10

---

---

---

---

---

---

---

---

4.53

3.66

3.67

3.93

3.65

---

3.91

3.92

---

---

β-D-GalpA

97.7

77.05

---

---

---

62.77

---

173.7

---

4.65

3.28

3.62

3.94

3.81

---

---

---

---

---

β-D-Glcp

105.10

75.49

---

---

---

---

---

---

---

4.53

3.40

---

3.68

---

---

---

---

---

---

β-D-Glcp

105.00

75.54

---

---

---

---

---

---

---

4.52

3.38

---

3.65

---

---

---

---

---

---

β-D-Glcp

105.20

75.48

---

---

---

---

---

---

---

4.51

3.35

3.50

3.67

3.83

---

3.93

3.93

---

---

β-D-Glcp

105.40

75.78

---

---

---

---

---

---

---

4.51

3.32

---

---

---

---

---

---

---

---

α-L-Araf

110.23

85.21

79.48

---

69.58

---

---

174.13

19.51

5.07

4.12

4.00

4.20

3.79

3.88

---

---

---

2.07

α-L-Araf

110.20

---

---

---

---

---

---

---

---

5.07

4.12

4.03

3.95

3.71

3.89

---

---

---

---

α-L-Araf

110.20

81.95

---

---

69.53

---

---

176.70

23.03

5.10

4.27

3.92

4.10

3.72

3.82

---

---

---

2.12

α-L-Araf

109.90

86.51

---

86.68

---

---

---

---

---

5.14

4.11

3.97

4.13

3.72

---

---

---

---

---

α-L-Araf

109.80

82.43

86.25

85.28

---

---

---

176.70

22.90

5.17

4.35

3.96

4.14

3.73

3.82

---

---

---

2.20

α-L-Araf

111.91

83.85

---

86.52

---

---

---

---

---

5.16

4.19

3.93

4.07

3.71

3.83

---

---

---

G, S or X

XSG, GSG, S

α-L-Araf

111.92

---

---

---

---

---

---

---

---

5.19

4.19

3.92

4.08

3.71

3.8

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---

---

---

SSG,GSS

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Journal of Agricultural and Food Chemistry

32 74.62 / 74.70a

---

---

---

---

---

5.11

3.61

---

---

---

3.87

---

---

---

---

---

---

---

---

---

5.13

3.68

---

---

3.53

---

---

---

---

---

---

---

---

---

---

5.07

3.58

3.77

3.72

3.66

3.70

---

---

---

---

---

---

---

---

---

---

5.12

3.83

---

---

---

---

---

---

---

---

75.80

---

---

---

---

---

---

4.94

3.53

3.71

3.72

3.58

3.70

---

---

---

---

81.47

75.75

---

64.49

---

---

---

---

4.93

3.54

3.69

3.66

3.70

3.86

---

---

---

---

83.06

72.24

---

---

---

---

---

---

4.95

3.58

3.65

3.72

3.56

3.88

---

---

---

---

α-L-Rhap

101.20

α-L-Rhap

101.07

α-L-Rhap

100.80

α-L-Rhap

101.60

---

---

α-D-Xylp

101.56

77.88

α-D-Xylp

101.10

α-D-Xylp

101.40

a

---

---

---

74.64 / 72.21a

Two resonances were observed, but assignment as C2, C3 and/or C4 could not be determined

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S or X

GSS, SSG

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33 FIGURE CAPTIONS

Figure 1. HPAEC-PAD oligosaccharide analysis of cranberry fractions A6 (A) and A2 (B) compared with xyloglucan oligosaccharide standards (C, degree of polymerization indicated above each peak).

Figure 2. MALDI-TOF MS of fraction A6.

Figure 3. MALDI-TOF/TOF MS/MS of A. m/z 1055.32 and B. m/z 1217.37.

Figure 4. Xyloglucan structure (1) based on MALDI-TOF/TOF MS/MS of m/z 1055.32. An alternate structure (2) is also consistent with MALDI-TOF/TOF MS/MS xyloglucan fragmentation.

Figure 5. Anomeric region of 1D 1H-NMR spectrum of fraction A6.

Figure 6. NMR HSQC of anomeric region showing primary assignments.

Figure 7. Correlations between α-L-Araf-C1→ α-D-Xylp-H2 were observed consistent with S structures.

Figure 8. Adhesion of E. coli CFT073 to uroepithelial T24 cells in the presence of cranberry A6 fraction.

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34

Figure 9: Adhesion of E. coli UTI89 to T24 cells in the presence of cranberry A6 fraction.

Figure 10. Adhesion of E.coli O157:H7 to HT29 cells in the presence of cranberry A6 fraction.

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35 Figure 1

300

1 2 3

250

Response[nC]

200

A

150

3

100

B 8

2

50

7

9 C

0

1

-50 0.0

10.0

20.0

30.0 Tim e [m in]

40.0

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60.0

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36 Figure 2

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37 Figure 3

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38 Figure 4

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39 Figure 5

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40 Figure 6

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41 Figure 7

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42 Figure 8.

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43 Figure 9.

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44

Adhesion relative to control (%)

Figure 10. 100 90 80 70 60 50 40 30 20 10 0 0.001 0.005 0.01

0.05

0.1

0.5

0.8

1

2.5

A6 (mg/ml)

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45 Table Of Contents Graphic

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