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Chemical Characterization of Potentially Prebiotic Oligosaccharides in Brewed Coffee and Spent Coffee Grounds TIAN TIAN, Samara Freeman, Mark Corey, J. Bruce German, and Daniela Barile J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b04716 • Publication Date (Web): 20 Mar 2017 Downloaded from http://pubs.acs.org on March 27, 2017

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Chemical Characterization of Potentially Prebiotic Oligosaccharides in Brewed Coffee and

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Spent Coffee Grounds

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† ,† , † Tian Tian,ǂ Samara Freeman, Mark Corey,§ J. Bruce German, ǂ and Daniela Barile* ǂ,

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ǂ

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United States

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95616, United States

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§

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* To whom correspondence should be addressed:

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

Foods for Health Institute, University of California, Davis, One Shields Avenue, Davis, CA

Keurig Green Mountain, Inc., Waterbury, VT 05676, United States

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Daniela Barile; email address: [email protected]; Tel: +1-530-752-0976; Fax: +1-530-752-

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4759

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ABSTRACT: Oligosaccharides are indigestible carbohydrates widely present in mammalian

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milk and in some plants. Milk oligosaccharides are associated with positive health outcomes;

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however, oligosaccharides in coffee have not been extensively studied. We investigated the

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oligosaccharides and their monomeric composition in dark roasted coffee beans, brewed coffee

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and spent coffee grounds. Oligosaccharides with a degree of polymerization ranging from 3 to

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15, and their constituent monosaccharides were characterized and quantified. The

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oligosaccharides identified were mainly hexoses (potentially galacto-oligosaccharides and

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manno-oligosaccharides) containing a heterogeneous mixture of glucose, arabinose, xylose and

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rhamnose. The diversity of oligosaccharides composition found in these coffee samples, suggests

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that they could have selective prebiotic activity towards specific bacterial strains able to

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deconstruct the glycosidic bonds and utilize them as a carbon source.

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KEYWORDS: coffee, coffee industrial residues, mass spectrometry, oligosaccharides,

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prebiotics

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INTRODUCTION Coffee is one of the most popular beverages worldwide and is usually prepared from two

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commercially grown varieties—Coffea arabica and Coffea canephora (also known as robusta)

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or their blends. Because of the great demand for coffee, large amounts of industrial waste

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products, such as spent coffee grounds (from instant coffee production, and the direct preparation

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of beverages in cafeterias, restaurants, or homes), silver skin (from roasting), and other by-

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products of the fruit and bean processing, are generated annually.1, 2 Industrial coffee residues are

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sometimes disposed of inappropriately and, therefore, represent an environmental concern,

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especially in developing countries. New publications have shown that spent coffee grounds

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represent potent sources of functional compounds and exhibit beneficial activities such as the

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prebiotic, antimicrobial and antioxidant properties.2 Therefore, there is a pressing need to

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identify such compounds and utilize the industrial residues for the extraction of functional

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molecules, thus generating added value.3

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Several components, including caffeine, polyphenols, melanoidins, diterpenes, etc., have

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been investigated in brewed coffee and its spent coffee grounds for several functional

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properties.4 Even though carbohydrates make up 50–54% of green coffee beans and 38–42% of

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roasted coffee beans by weight (dry basis), 5 they have not been as extensively studied as a result

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of their high structural complexity. Green C. arabica beans have a content of polysaccharides as

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high as 50% (on dry basis), which include galactomannans, arabinogalactans, and cellulose.3

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Unlike coffee polysaccharides, coffee oligosaccharides are small in size and have not been

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fully isolated and characterized. Oligosaccharides are carbohydrates generally consisting of two

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to twenty monomers linked by a variety of O-glycosidic bonds.6 Other than sucrose, there is no

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evidence for the presence of naturally occurring oligosaccharides in green coffee beans.

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However, a few oligosaccharides have been found in roasted coffee beans, and they seem to 3 ACS Paragon Plus Environment

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derive from two polysaccharides, arabinogalactans and galactomannans, through several

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chemical reactions during the roasting process.7-9

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The amount of oligosaccharides that are transferred into brewed coffee depends on factors

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such as the variety and origin of green beans, and the degree of roasting, as well as the brewing

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conditions used. Structural elucidation and quantification of oligosaccharide profile in coffee will

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aid the understanding for their functional properties. Oligosaccharides are targets of new

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investigations because they exhibit highly specific functions such as acting as prebiotics by

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feeding beneficial bacteria, blocking attachment of pathogens in the gut, and interacting directly

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with intestinal cells. Prebiotics are dietary ingredients that cannot be digested by human-

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produced digestive enzymes, yet they provide a health benefit to the host mediated by selectively

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stimulating the growth and/or activity of one or a limited number of host gut microbiota.10 Some

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abundant food oligosaccharides, including galactooligosaccharides and inulin, have prebiotic

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activity.11

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The prebiotic properties of oligosaccharides highly depend on structural conformation,

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including their degree of polymerization, monosaccharide composition, and their glycosidic

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linkages. Identification of novel oligosaccharide structures can be challenging because of a range

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of molecular diversity and complexity of molecular structure, e.g., the heterogeneous

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compositions and stereochemistry of each monosaccharide unit, the possibility of branching, and

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different linkages. As a matter of fact, arabinogalactooligosaccharides and

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galactooligosaccharides—which may be composed of the same group of constituent

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monosaccharides and linked by the same glycosidic linkages as coffee oligosaccharides, possess

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biological activity on several bacteria known as probiotic in in vitro studies.12-17

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Especially, mannooligosaccharides derived from mannan in spent coffee grounds have been

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isolated and their beneficial effect on large bowel function was documented through a series of

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studies. Mannooligosaccharides were shown to promote growth of bifidobacteria as well as

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increasing the production of short chain fatty acids by in vitro studies,18, 19 animal models,20 and

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in the human studies.21, 22 In addition, previous publications have also discussed their beneficial

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effect on reduction of fat absorption on animal and humans.23-25 However, no results have been

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published yet to discuss the structure and potential prebiotic effect of intact oligosaccharides in

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brewed coffee or spent coffee grounds.

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High-resolution mass spectrometry and tandem mass spectrometry offer the ability to

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comprehensively characterize oligosaccharide structures; these spectral technologies have

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already been successfully used to provide detailed information about the consumption of human

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milk oligosaccharides by probiotic bacteria in in vitro studies.26 Here, we present the isolation,

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identification and characterization of oligosaccharides from roasted coffee beans, brewed coffee

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and its spent coffee grounds. Multiple chromatographic and spectroscopic analytical tools were

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used to identify and quantify the purified oligosaccharides.

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MATERIALS AND METHODS

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Samples, Reagents and Instruments. Dark roasted ground C. arabica and liquid coffee

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brew were provided by Keurig Green Mountain. A blend of coffee beans sourced from

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throughout the world was used. The blend contained green coffee components from Southeast

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Asia, East Africa, and South America. Roasted beans were ground using No.5 grinder (see Table

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S1 for particle size information). All the samples were stored at –20 oC until ready to use.

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To study the effect of brewing conditions, coffee samples were brewed using four brewing

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appliances, including a French press, a Bunn brewer, a K-cup coffee maker, and Espresso

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machine (hereafter referred to as French press, Bunn, K-cup, and Espresso). All the beans were

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roasted to espresso roast (25 Agtron units color), and ground with Ditting® Grinder (Ditting

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Maschinen AG, Bachenbülach, Switzerland) to different sizes (described in Table S1). French

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press method used beans ground with No. 9.0 grinder. Coffee was left to steep for 4 min in a

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bodum® Chambord model French Press, before depressing plunger and decanting coffee extract.

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Multiple extractions were performed and decanted extracts were pooled together to ensure the

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maximum extraction. The Bunn extract was made by using beans ground with No.3.5 grinder,

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Bunn® Axiom Brewer, with brewing temperature set at 93.3 oC. K-Cup® brew was made by

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using beans ground with No.3.5 grinder, on a B70 Keurig® brewer at an 8 oz setting

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(approximately 236 mL). Espresso was prepared with beans ground with No. 3.0 grinder, in a La

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Marzocco espresso machine (Florence, Italy) with water pressurized to 130 psi. The spent coffee

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grounds were also analyzed. The values of total dissolved solid of the industrial samples are

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summarized in Table 1.The detailed parameters for all brewing techniques are summarized in

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Table S2.

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D-galactose, D-glucose, D-mannose, L-arabinose, D-xylose, L-rhamnose, and L-allose were

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purchased from Sigma-Aldrich (St. Louis, MO, United States). The LC-MS system used for

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analysis was Agilent 6520 Nano LC Chip-QToF (Agilent Technology, Santa Clara, CA, United

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States). The gas chromatograph with an FID (GC-FID) was from Hewlett Packard HP-6890

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(Wilmington, DE, United States). A dryer was used for evaporation of solvent with nitrogen

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(Reacti-Vap III™, Pierce, Rockford, IL, United States).

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Oligosaccharides Extraction and Purification. Oligosaccharides from ground C. arabica

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beans were extracted with hot water according to a published method27 with some modifications.

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A 5 g sample of ground coffee beans was extracted with 100 mL of water at 100 oC for 20 min

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under constant stirring. The extract was centrifuged at 4, 225 ×g for 5 min to obtain the

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supernatant. Total dissolved solids (ppm) were measured at 18–23 oC depending on the room

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temperature (Ultramete II™ 4P, Myron L® Company, Carlsbad, CA, United States). For liquid

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coffee samples, 5 mL of each coffee sample was used for further purification. The liquid coffee

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was defatted using the Folch method.28 Briefly, 1 volume of liquid coffee was mixed with 4

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volumes of 2:1 chloroform: methanol, mixed with a vortex mixer, and centrifuged to obtain the

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aqueous layer that contained the hydrophilic oligosaccharides. The aqueous layer was vacuum

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dried and re-suspended in water prior to purification by solid phase extraction using reverse

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phase C8 cartridges, followed by extraction on porous graphitized carbon cartridges.29 Residual

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hydrophobic lipids, small peptide, and polysaccharides were bound to the C8 bonded silica and

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the hydrophilic oligosaccharides were captured by a subsequent purification with porous

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graphitized carbon cartridges and stepwise elution with 20% and 40% acetonitrile/ 0.05%

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trifluoroacetic acid. The acetonitrile was removed with vacuum and the dry oligosaccharides

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were resuspended in water for further analysis by LC-MS or GC-FID.

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Characterization of Oligosaccharides by Chip-Quadrupole-Time-of-Flight Mass

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Spectrometry. An Agilent 6520 NanoChip-LC-QToF was used for compositional analysis of

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intact coffee oligosaccharides. Dried samples were suspended in 200 µL nano-pure water and

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analyzed using Nano-LC Chip QToF according a published method.30 Oligosaccharide

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separation was achieved with the micro fluidic HPLC-Chip consisting of an enrichment column

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(4 mm; 40 nL) and an analytical column (75 µm × 43 mm) with a nano-electrospray tip. Both

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columns were packed with 5 µm 250 Å porous graphitized carbon material. Binary solvent

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gradients were applied for oligosaccharides separation. Elution solvent A contained 97% nano-

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pure water, 3% acetonitrile, and 0.1% formic acid; solvent B contained 90% acetonitrile, 10%

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nano-pure water, and 0.1% formic acid. The columns were equilibrated with 100% solvent A.

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The 65-min gradient was programmed as follows: from 0 to 16% B from 2.5 to 20 min; from 16

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to 40 % B from 20 to 30 min; from 40% to 100% B from 30 to 40 min, followed by 100% B for

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10 min; from 100 to 0 % B from 50 to 55.01 min, and re-equilibrium at 0% B for another 10 min.

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Mass spectrometry was set in the positive mode. Mass calibration was performed using

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internal reference masses (m/z 922.009798, 1221.990637). Automated precursor selection was

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applied for selecting peaks for tandem fragmentation. The threshold for peak selection was set at

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200 ion counts for MS and 5 ion counts for MS/MS. The acquisition rate was 0.63 spectra/s. The

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isolation width for tandem MS was medium (~ 4 m/z). The collision energy was set at 1.8V/100

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Da with an offset of -3.6V.

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Mass spectra were analyzed using the molecular feature extraction in Mass Hunter

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Qualitative Analysis software version B.06.00. Oligosaccharide molecular formulas were

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determined for masses with an error as low as 10 ppm.

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Characterization and quantification of monosaccharides fingerprint by GC-FID. A gas

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chromatograph with an FID (GC-FID) was used to characterize the composition of

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monosaccharides and their absolute amounts. Carbohydrates have low volatility; therefore,

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preparation of derivatives was necessary for GC analysis. Methanolysis and trimethylsilylation

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(TMS) of purified coffee oligosaccharides was performed following a published procedure.31, 32

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Methanol containing 0.5 M HCl was prepared by adding 2.8 mL of acetyl chloride to 20 mL

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anhydrous methanol. Purified coffee oligosaccharides (0.5 mL) were dried, suspended in 0.5 mL

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MeOH/HCl, and incubated at 80 oC for 16h. The solutions were cooled to room temperature and

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dried under a stream of nitrogen; 250 µL methanol was added and then dried under a stream of

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nitrogen. TriSil® reagent (300 µL) was added to each sample and the vials were incubated at 80

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o

C for 1 h. The residues were cooled at room temperature and excess solvent was removed under

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a stream of nitrogen. The TMS derivatives were extracted by adding 1 mL of hexane and

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centrifuging at 10,000 rpm for 10 min. The solution was dried and suspended in 150 µL of

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hexane prior injection (1 µL) into GC-FID. The GC-FID was equipped with a capillary

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split/splitless inlet, coupled to an FID controlled by an HP ChemStation. A DB-1 fused-silica

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capillary column (30 m × 0.25 mm i.d., 0.25 µm film thickness, J&W Scientific, Folsom, CA

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United States) was used for separation of monosaccharides. Hydrogen was used as carrier gas

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with the flow rate of 2.5 mL/min. Samples were injected in the pulsed split mode with the split

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ratio of 5:1. The injector and the FID temperature was 280 oC. The GC temperature program was

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as follows: 120–200 oC by 1.5 oC /min, 200 oC for 5 min, and a post run of 2 min at 250 oC.

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Calibration curves were built for the following monosaccharide standards: glucose, galactose,

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mannose, arabinose, xylose, and rhamnose. Allose was used as internal standard. The detector

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response factors for individual monosaccharides were calculated to quantify each

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monosaccharide in oligosaccharide chains.

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Statistical Analysis. The differences in constituent monosaccharides and total amount of

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oligosaccharides among different samples were analyzed using ANOVA followed by Tukey’s

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post-hoc test. All statistical analyses were conducted in R software, version 3.1.2. The threshold

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of significant difference was set at p ≤ 0.05.

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RESULTS AND DISCUSSION

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Structural elucidation of coffee oligosaccharides is the first step to understanding their biological

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activity; at the same time, achieving high sample purity is critical for successful oligosaccharide

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analysis. Additionally, the method for extracting oligosaccharides from the cell wall matrix must

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be chosen with careful consideration, based on the particular properties of the compounds (such

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as polarity and solubility) and keeping in mind the goal of minimizing degradation or structural

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alterations of the oligosaccharides. Hot water extraction, a well-known method for plant

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oligosaccharide extraction, is often followed by protein precipitation with ethanol, dialysis and

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ultrafiltration.27, 33, 34 However, in our work, solid-phase extraction was a more efficient way to

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obtain high purity oligosaccharides, which was a crucial requirement to accomplish

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characterization by mass spectrometry.

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.MS Analysis of Roasted Coffee Beans. Oligosaccharides were analyzed by nanoLC-Chip-

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QToF to obtain compositional information, including the size of individual oligosaccharides

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(degree of polymerization) and type of constituent monosaccharides. A typical chromatogram for

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oligosaccharides in dark roast coffee is shown in Figure 1. The oligosaccharides were eluted with

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the retention times between 5 and 30 minutes of the solvent gradient. Because single stage MS

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only reveals information about size of molecules, to determine the individual monosaccharides

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and their size (degree of polymerization), the purified coffee oligosaccharides were also analyzed

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by tandem MS, which provided high resolution and accuracy for compositional identification. An

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example of a tandem spectrum for one oligosaccharide in dark roasted coffee (m/z = 991.3382)

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and its fragments is shown in Figure 2. The loss of 162.05 Da corresponded to the loss of a

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hexose residue due to the cleavage of glyosidic bonds. Overall, the fragment ions allowed

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identification of the composition as six hexose residues and one free reducing end.

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Table 2 presents the details of oligosaccharide composition obtained by tandem MS and

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Mass Hunter Quantitative analysis (using a mass error as low as 10 ppm). Oligosaccharides

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identified in the roasted coffee samples were mainly of the hexose type, with different degrees of

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polymerization. Several ions corresponded to oligosaccharides made of pentose residues linked

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to hexose. Overall, the oligosaccharides in roasted coffee were composed of from 3 to 15 hexose

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or hexose-pentose combinations.

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Understanding the precursor polysaccharide structures will enable prediction of some coffee

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oligosaccharide structural information. Structurally, type II arabinogalactans in coffee beans have

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a backbone composed of β-(1→3)-linked galactopyranosyl residues frequently substituted at O-6

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position by side chains formed by 1→6-and 1→3-linked β-galactosyl units and α-arabinosyl

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residues.33 The 1→6 galactosyl side chains can be terminated by glucuronic acid residues.35 The

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occurrence of rhamnoarabinosyl and rhamnoarabinoarabinosyl side chains was also reported as a

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structural feature of coffee arabinogalactans.36 As far as galactomannans are concerned, the

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structure has been described as linear polysaccharides with a main backbone of β-(1→4)-linked

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D-mannopyranose residues, 4-5% of which were substituted at O-6 with side chains of single α-

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(1→6) linked D-galactopyranose residues.9 The mannan backbones were sometimes interspersed

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with β-(1→4)-linked D-glucopyranose and a side chain of single arabinose was also reported.37

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Previous research also reported the presence of singly, doubly and consecutively acetylated

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mannose residues in coffee galactomannan.37

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As degradation products from the two polysaccharides described above, we hypothesize that

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the hexose oligosaccharides would also be mainly β-(1→3)-linked galactopyranosyl residues or

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β-(1→4)-linked D-mannose residues with β-(1→6)-galactosyl/glucosyl units. The hexose-

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pentose oligosaccharides in coffee were possibly β-(1→3)-linked galactopyranosyl residues or β-

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(1→4)-linked D-mannose residues with an α-(1→3)-arabinosyl/xylosyl residue. The

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oligosaccharide profiles described in the present work are in partial agreement with the structural

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characterization of galactomannan derivatives published by Nunes and Coimbra.7 These authors

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performed ESI-MS analysis of galactomannan derived oligosaccharide in roasted coffee and also

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found the major DP3 fractions were hexose oligosaccharides and pentosyl-containing

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trisaccharide Pent-Hex2. Besides, they demonstrated the occurrence of oligosaccharide with

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acetylated species, oligosaccharide derivatives containing acid residues, modified residues of

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caramelization reactions, and Amadori compounds. The prediction of monosaccharide

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composition based on both mass spectrum and precursor information enabled the selection of

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appropriate monosaccharide standards for quantification (see GC analysis of constituent

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monosaccharides of coffee oligosaccharides). However, analysis and enzymatic assays beyond

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the scope of this work would be required to fully illustrate the oligosaccharide structures,

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including all linkages.

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Limitations of MS. The Nano LC-Chip-QToF provided the necessary high resolution and

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high mass accuracy for the identification of oligosaccharide compositions. However, it must be

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noted that all hexose residues have the same molecular formula (C6H10O5) and thus the same

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mass (162.0528 Da); therefore, MS, by virtue of measuring solely accurate mass, cannot

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discriminate among hexose isomers, nor pentose isomers. Moreover, MS can only provide

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oligosaccharide relative abundance in part due to changes in ionization. The accurate

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quantification of coffee oligosaccharides could not be achieved without appropriate standards to

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obtain the unique response factors for each oligosaccharide, yet no commercial standards are

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available for coffee oligosaccharides. Therefore, the total amount of oligosaccharides was

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extrapolated via quantification of the constituent monosaccharides after methanolytic cleavage

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and derivatization.

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GC Analysis of Constituent Monosaccharides of Coffee Oligosaccharides. The GC

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profile of the TMS derivatives of coffee oligosaccharides after methanolic HCl treatment is

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shown in Figure 3. Pentose (arabinose, and xylose) and a deoxyhexose (rhamnose) were in lower

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abundance in general and eluted earlier than hexose (galactose, mannose, and glucose)

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molecules. Figure 4 shows an example of a typical monosaccharide composition of

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oligosaccharides in dark roasted coffee, including the response factors of each monosaccharide

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standard. Mannose and galactose, respectively at 37.1% and 42.2%, were the predominant

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constituents of oligosaccharides. Arabinose (7.8%) and glucose (9.5%) were also detected as

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major monosaccharide constituents, whereas, xylose and rhamnose were found only in trace

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amounts (2.1% and 1.2%, respectively).

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Partial structural information on coffee oligosaccharides was inferred by combining the

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results from MS and GC analysis. The oligosaccharides in brewed coffee mainly belonged to

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galactomannans and type II arabinogalactan, with galactose and mannose being the most

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represented in the backbone and other lower abundant monosaccharides (arabinose, xylose, and

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rhamnose) either mainly located in the side chain or interspersed in the main backbones.

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Mannose and galactose were found in approximately similar amounts. This fact is in agreement

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with the previously published results that soluble coffee fiber contained almost equal proportions

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of arabinogalactans and galactomannans.17 Also, about 9.5% glucose was detected in our

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oligosaccharide extracts. Similar results were reported by Redgwell et al, who found more than

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10% of glucose residues as non-reducing terminal units on β-(1→6)-galactosyl side chains of

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type II arabinogalactan.35 Arabinose was found to constitute about 7.8% (weight%) or 9%

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(mol%) of the total oligosaccharides, which is similar to the results (8 mol%) reported by

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Gniechwitz et al.17 Trace amounts of xylose were also reported in previous research35 possibly

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deriving from the side chain type II arabinogalactan, too. The presence of rhamnose residues

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indicated the possibilities that rhamnoarbinosyl and rhamnoarabinoarabinosyl side chains of type

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II arabinogalactan were cleaved off during the roasting process hence generating smaller

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oligosaccharides.

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The structure-function properties of oligosaccharides in many foods have been studied and

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one of the well-demonstrated activities is their prebiotic function. However, the efficacy of

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prebiotics may vary due to the specificity of their interactions with target commensal bacteria.

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The oligosaccharides in coffee are, in principle, non-digestible, thus making them possible

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candidates for prebiotic activity. Unlike commercially available prebiotics, which consist of

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repetition of the same monomer (fructose for inulin and galactose for galactooligosaccharides),

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the coffee oligosaccharides identified exhibited higher diversity in both size and composition.

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Their sizes ranged from those of 3 hexoses to 15 hexoses or hexose-pentose combination, and

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their constituent monosaccharides included 6 different monosaccharide types. The diversity in

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both the size and monosaccharide composition provides the basis for future matching of coffee

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oligosaccharides with select probiotic strains.

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Effects of Brewing on Oligosaccharide Distribution in Liquid Coffee and Spent Coffee

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Grounds. The effects of brewing techniques on the presence and abundance of oligosaccharides

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were investigated by analyzing coffee brewed using the following methods: French press, Bunn,

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K-cup, and Espresso machine. The spent coffee grounds were subjected to hot water extraction

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and also analyzed for oligosaccharides.

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Oligosaccharide compositions reported in table 2 were found in all four liquid coffee

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samples. The chromatograms of French press, Bunn and K-cup were similar, whereas espresso

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coffee had a rather distinct oligosaccharide profile with more abundant molecules larger than DP

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10. Analysis by MS of the spent coffee grounds indicated the presence of oligosaccharides

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similar to what observed in the corresponding liquid coffee samples, albeit in lower relative

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abundance. The only exception was that the espresso coffee extract contains more abundant

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oligosaccharides with DP above 10 while oligosaccharides with DP below 10 in lower relative

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abundance in the espresso coffee extract than in the extract of the spent grounds. The

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oligosaccharide profile of extract and spent coffee grounds of espresso appears to well correlate

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and provide complementary oligosaccharides profiles. This could be explained by the finer bean

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size (No.3.0 grinder), and the pressure applied during espresso brewing, which might have

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accelerated the release of oligosaccharides with higher DP in the liquid phase compared to the

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other brewing methods.

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Figure 5 and Table 3 present the total amount of oligosaccharides (Figure 5a) and amount of

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constituent monosaccharides (Figure 5b) in liquid coffee and spent grounds obtained by different

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brewing techniques. ANOVA was conducted to compare the amount of oligosaccharides in liquid

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coffee and in hot water extract of spent coffee grounds. The result indicated the significant

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difference with p value equal to 2.06×10-7. Tukey’s method for post hoc test indicated significant

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higher level of oligosaccharides in espresso coffee than the rest of three brewing techniques and

315

all spent coffee grounds.

316

Comparing the amount of oligosaccharides between liquid coffee and their corresponding

317

spent coffee grounds in each technique: comparable amount of oligosaccharides were found in

318

the residues compared to their corresponding liquid coffee, but espresso coffee contained a

319

significantly higher amount of oligosaccharides than its spent coffee grounds. The presence of

320

oligosaccharides in spent coffee grounds provided another possible source of bioactive

321

oligosaccharides. Moreover, the presence of significant amounts of DP 0.05) Different letters in the same column indicates the statistical difference.

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Relative Abundance (Counts)

Figure 1. 5 Hex m/z 829.2826

7 Hex m/z 1153.3857

8 Hex m/z 1315.4398 9 Hex m/z 1477.4916

3 Hex m/z 505.1781 2 Hexa + 1 Pentb m/z 457.1539

4 Hex m/z 649.2189 3 Hex + 1Pent m/z 637.2193

4 Hex + 2 Pent m/z 931.3129 4 Hex + 1Pent m/z 799.2724

6 Hex m/z 991.3256 6 Hex+ 1 Pen m/z 961.324

10 Hex m/z 1477.4916

Hex: Hexose Unit

b

Pent: Pentose Unit

12 Hex m/z 1964.6648 13 Hex m/z 1063.3613 Z=2

14 Hex m/z 1144.3846 Z=2 15 Hex m/z 1225.4033 Z=2

Retention Time (Minutes) a

11 Hex m/z 1802.6006

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Journal of Agricultural and Food Chemistry

Figure 2.

Relative Abundance (Counts)

(Hex)2

Hex: Hexose Unit (Hex)3 (Hex)4 a

Hex

(Hex)5

(Hex)6

Free reducing end

Mass-to-Charge (m/z)

a

Hex: Hexose Unit

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Figure 3.

Allose (IS)

Mannose

Response

Galactose Arabinose Allose (IS) Xylose Allose (IS)

Glucose

Rhamnose

Retention Time (Minutes)

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Journal of Agricultural and Food Chemistry

Figure 4.

31 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Figure 5

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Journal of Agricultural and Food Chemistry

For Table of Contents Only

33 ACS Paragon Plus Environment