Effect of Roasting on Oligosaccharide Abundance in Arabica Coffee

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Food and Beverage Chemistry/Biochemistry

Effect of Roasting on Oligosaccharide Abundance in Arabica Coffee Beans TIAN TIAN, Samara Freeman, Mark Corey, J. Bruce German, and Daniela Barile J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b02641 • Publication Date (Web): 03 Sep 2018 Downloaded from http://pubs.acs.org on September 4, 2018

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Journal of Agricultural and Food Chemistry

Effect of Roasting on Oligosaccharide Abundance in Arabica Coffee Beans Tian Tian,ǂ Samara Freeman,† Mark Corey,§ J. Bruce German, ǂ, † and Daniela Barile*,ǂ,† ǂDepartment of Food Science and Technology, University of California, Davis, California 95616, United States † Foods for Health Institute, University of California, Davis, One Shields Avenue, Davis, California 95616, United States § Keurig Green Mountain, Inc., Waterbury, Vermont 05676, United States * To whom correspondence should be addressed. Tel: +1-530-752-0976; Fax: +1-530-752-4759; E-mail: [email protected]

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ABSTRACT: Emerging research into the bioactivities of indigestible carbohydrates is

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illuminating the potential of various foods and food streams to serve as novel sources of

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health-promoting compounds. Oligosaccharides (OS) are widely present in milks and some

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plants. Our previous research demonstrated the presence of OS in brewed coffee and spent coffee

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grounds. Armed with this new knowledge, the next step toward improving the utilization of these

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valuable components involved investigating the effect of roasting on the formation and

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abundance of coffee OS. In the present study, we used advanced mass spectrometry to analyze a

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variety of coffee samples and demonstrated that a great structural diversity and increased

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abundance of OS is associated with higher roasting intensity. The present investigation also

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evaluated methods for OS extraction and fractionation. A preparative-scale chromatographic

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method, based on activated carbon, was developed to isolate enough amounts of OS from coffee

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to enable future confirmation of prebiotic and other in vitro activities.

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KEYWORDS: Coffea arabica, mass spectrometry, oligosaccharides, prebiotics

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INTRODUCTION

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Roasting of coffee beans is a very important step in coffee processing. It is associated with the

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development of organoleptic parameters that are important indicators of the quality of the roasted

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beans. The two major types of polysaccharides that involve structural modifications in green

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coffee beans during roasting are type II arabinogalactans and galactomannans.1 A series of

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reactions involving polysaccharides have been studied, including depolymerization, dehydration,

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cleavage of carbon-carbon linkages, their incorporation in melanoidins through transglycolation

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reactions, and some structural modifications at the sugar reducing end.2, 3 4, 5. Additionally

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previous research has indicated that the roasting process opens the cell wall matrix, enables the

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hydrolysis reactions of polysaccharides, and promotes the formation and release of

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oligosaccharides (OS).6

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OS are carbohydrates generally consisting of two to twenty monomers linked by a variety of

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O-glycosidic bonds.7 Other than sucrose, tiny amount of raffinose (0.77%) and stachyose

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(0.51%), there is no evidence for the presence of naturally occurring OS in green coffee beans. 8

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Only a few OS hypothesized to derive from the structural modifications of coffee

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polysaccharides during roasting,7, 9, 10 have been found in roasted coffee beans. Our previous

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research established an OS extraction and purification method, and elucidated the composition

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and concentration of OS in dark roasted coffee beans and spent coffee grounds.11

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OS are targets of new investigations because they exhibit highly selective prebiotic activity.

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Prebiotics are dietary ingredients that cannot be digested by human-produced digestive enzymes,

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yet they provide a health benefit to the host mediated by selectively stimulating the growth

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and/or activity of one or a limited number of host gut microbiota.13 Several food OS, including

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galactooligosaccharides and inulin, have been reported to act as prebiotics.14 Additionally, a 3 ACS Paragon Plus Environment


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recent in vitro study demonstrated that bovine milk OS can block attachment of pathogens to the

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epithelial cells and interact directly with intestinal cells. 15

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Only a few studies have explored the biological activities of coffee OS. In one study, purified

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coffee mannooligosaccharides resisted degradation when exposed to a series of digestive enzyme;

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moreover, the mannooligosaccharides were fermented by human fecal bacteria, and the products

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of fermentation included beneficial short chain fatty acids.16 In another study, consumption of

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coffee was positively associated with an increased population and metabolic activity of

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Bifidobacterium spp. in feces of healthy adult volunteers who consumed three cups of coffee

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daily for three weeks.17 In a third study, Escherichia coli and Clostridium spp. were decreased,

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whereas Bifidobacterium spp. were increased in the colons of mice that consumed coffee,

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compared with their numbers in a control group.18

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With an increasing interest in identifying sources of selective prebiotics to serve as novel

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food ingredients to improve human health, there is a considerable demand for natural,

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concentrated sources of novel oligosaccharides with improved bioactivity. However, it is

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extremely difficult to obtain functional oligosaccharides on a large scale since oligosaccharides

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have diverse sugar sequences and are found in only trace amounts in the obvious sources (e.g.

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bovine milk). Armed with this new knowledge, the next step toward improving the utilization of

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these valuable components involved investigating the effect of conventional coffee processing on

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the formation of OS. The degree of roasting is one of the key factors that could determine the

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type and amount of OS formed and hence transferred into brewed coffee. Several investigators

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discussed the effect of roasting on degradation and structural modification of carbohydrates,

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especially polysaccharides, in coffee beans.2, 12 However, the structural modifications of coffee

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carbohydrates induced by roasting remain incompletely characterized. 4 ACS Paragon Plus Environment

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Investigating the effect of roasting on the composition and abundance of coffee OS is important

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as it could lead to an improved understanding of the structures of coffee OS. In the present study,

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we used advanced mass spectrometry to analyze a variety of coffee samples and demonstrated

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that a great structural diversity and increased abundance of OS is associated with higher roasting

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intensity.

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Controlling roasting parameters, and optimizing both the extraction in a preparative purification

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methods will lead to a more effective and targeted extraction of OS. Here, we studied the effect

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of roasting intensity on the composition and abundance of coffee OS and selected the optimum

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roasting conditions for the production of OS. The present investigation also evaluated methods

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for OS extraction and fractionation. A preparative-scale chromatographic method, based on

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activated carbon, was developed to isolate enough amounts of OS from coffee to enable future

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confirmation of prebiotic and other in vitro activities.

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MATERIALS AND METHODS Chemicals and Reagents. D-galactose, D-glucose, D-mannose, L-arabinose, D-xylose,

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L-rhamnose, and L-allose were from Sigma-Aldrich (St. Louis, MO, USA). Analytical grade

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standard 2’-fucosyllactose (2’-FL) was purchased from V-Laboratories (Covington, LA, USA).

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A commercial sucrose/D-glucose/D-fructose kit was from R-biopharm AG (Darmstadt,

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Germany). The total carbohydrate colorimetric assay kit was purchased from BioVision,

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Incorporated (Milpitas, CA, USA). The Tri-Sil HTP (HDMS:TMCS:Pyridine) reagent was

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purchased from Thermo Fisher Scientific (Waltham, MA, USA). The Discovery® DSC-C8

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1-mL solid-phase extraction columns and the Discovery DSC-C8 column packing material

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were purchased from SUPELCO (Bellefonte, PA, USA). The Extract-Clean TM Carbo porous 5 ACS Paragon Plus Environment


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graphitized carbon cartridges (150 mg) were purchased from GRACE Davison Discovery

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Science (Deerfield, IL, USA). The activated carbon charcoal (50–200 mesh) was purchased from

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FisherbrandTM (Columbia, MD, USA).

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The 60-mL filtration tubes with a Teflon Frits were purchased from SUPELCO (Bellefonte,

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PA, USA). The 0.22-µm MILLEX GP filter units were purchased from Merck Millipore Ltd.

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(Tullagreen, Carrigtwohill, Co. Cork, IRL). All solvents used were HPLC-MS grade (Fisher

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Scientific, Fiar Lawn, NJ).

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Processing and Characterization of Coffee Beans. Raw or roasted ground Coffea arabica

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were provided by Keurig® Green Mountain, Inc. (Waterbury, VT, USA). A blend of coffee beans

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sourced from Southeast Asia, East Africa, and South America was used. Beans were ground

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using a No. 5 Ditting® grinder. The coffee bean samples were stored at –20 oC until ready to use.

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To study the effect of roasting intensity on the composition and abundance of OS, seven

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samples of the blended beans were roasted and coded from R1 to R7, where R1 was the least

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roasted and R7 was the most heavily roasted. Green beans from the same blend were also

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analyzed. The roasting levels of beans were indicated by the color characteristics of the ground

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beans, including the Agtron/SCAA color measurement and CIE L*a*b*. The Agtron numbers

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were obtained using a roast classification system (the Specialty Coffee Association of America

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and Agtron Inc. (Reno, NV, USA)). Twenty grams of ground beans were transferred to a plastic

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Petri dish and compared to the color disk. Both samples and the color disk were viewed and

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compared against a black neutral density background provided in the kit. The colorimetric

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measurement was performed using a portable Chromameter CR-410 (Konica Minolta, Ramsey,

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NJ, USA) on the ground coffee beans directly. The chromameter was calibrated with a standard

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white plate (D65 illuminant, Y=93.4, x = 0.3162, y = 0.3342). Values for L* (lightness), a* 6 ACS Paragon Plus Environment

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(red-green), and b* (yellow-blue) were obtained. The chroma [c = (a2+b2)1/2] and the hue [arctan

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(b/a)] were calculated accordingly.19-21

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Small Scale OS Isolation and Purification for Characterization and Quantification. OS

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from ground C. arabica beans were isolated and purified as previously described.11 Briefly, a 5-g

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sample of ground coffee beans was boiled with 100 mL of nanopure water (Direct-Q® 5 UV

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Remote Water Purification System, Millipore Sigma, Burlington, MA, USA) for 20 min under

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constant stirring, resulting in a liquid coffee extract. The liquid coffee extract was prepared in

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triplicate. Total dissolved solids (Table 1) were measured (Ultramete II™ 4P, Myron L®

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Company, Carlsbad, CA, USA) to check efficiency of extraction. The pH values (Table 1) of the

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resulting liquid coffee extract were also measured with SevenCompact S220-Basic, pH/Ion

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benchtop meter (Mettler-Toledo, LLC, Columbus, OH, USA). The majority of lipids and some

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proteins in the coffee extract were removed using the Folch method by mixing one volume of

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coffee extract with four volumes of a mixture of chloroform and methanol (2:1, vol:vol). The

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mixture was mixed vigorously with a vortex mixer, and then was centrifuged at 4,000 ×g for 30

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min at 4 oC. The upper aqueous layer containing OS was vacuum dried, re-suspended in

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nanopure water, and subjected to stepwise solid-phase extraction. The OS-containing solution

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was first loaded to a Discovery® DSC C8 column to remove residual lipids, small peptides, and

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polysaccharides. The entire eluate was then loaded onto the porous graphitized carbon column.

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The hydrophilic OS were bonded by porous graphitized carbon cartridge. The bonded OS

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fraction was eluted stepwise with 20% acetonitrile, then 40% acetonitrile/0.05% trifluoacetic

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acid. Those OS-rich eluate fractions were combined, was vacuum dried and suspended in

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nanopure water for LC-MS and GC-FID analysis.



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Characterization of OS by Chip-Quadrupole-Time-of-Flight Mass Spectrometry. Prior

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to LC-MS analysis, dried OS samples were reconstituted in 200 µL of nanopure water, with

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2’-FL being added as internal standard at the final concentration of 0.005 g/L. Mass spectrometry

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analysis was performed with an Agilent 6520 NanoChip-LC-Quadrupole-Time-of-Flight (QToF)

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with a microfluidic nanoelectrospray PGC-Chip (II) (Agilent Technologies, Santa Clara, CA,

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USA) according to the previously published method, with minor modification.22 The

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micro-fluidic PGC-Chip (II) (Agilent Technologies, Santa Clara, CA, USA) contained an

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enrichment column (4 mm; 40 nL) and an analytical column (43 mm × 75 µm), both packed with

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porous graphitized carbon. Chromatographic separation was performed by binary solvent

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gradients of 3% acetonitrile/0.1% formic acid (solvent A) in water and 90% acetonitrile/0.1%

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formic acid in water (solvent B). The columns were initially equilibrated with 100% solvent A

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with a flow rate of 0.3 µL/min for the nano pump and 4 µL/min for the capillary pump. The

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

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to 30 min, 16 to 40% B; 30 to 40 min, 40% to 100% B, followed by 100% B for 10 min; 50 to

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55.01 min, 100 to 0% B, and re-equilibrium at 0% B for10 min.

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Mass spectra were acquired in positive mode within a m/z range of 450–2,500. OS were

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detected in their protonated form ([M+H]+). Internal mass calibration was conducted using two

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reference masses (m/z 922.009798, 1221.990637, ESI-ToF tuning mix G1969-85000, Agilent

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Technologies). Automated precursor selection was employed based on ion abundance for tandem

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MS analysis, performing up to 6 MS/MS spectra per individual MS when precursor ions were

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above ion abundance threshold. The threshold for peak selection was set at 200 ion counts for

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


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tandem MS was set on medium (~ 4 m/z). The collision energy was set at 1.8V/100 Da with an

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offset of –3.6V.

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MS data were analyzed using the molecular feature extraction function of Mass Hunter

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Qualitative Analysis software version B.06.00 and Mass Hunter Profinder B.06.00 (Agilent

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Technologies). The putative OS structures were extracted through the “find compound by

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formula” algorithm. The compounds were filtered with mass range from 450 to 2500 m/z and

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retention time between 5 and 30 min. Target compounds had an ion count higher than 1,000,

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either singly or doubly charged, and a typical isotopic distribution of small biological molecules.

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OS molecular formulas were determined from the deconvoluted mass list with in-house software,

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with an error as low as 10 ppm. All the OS compositions were confirmed by tandem MS analysis.

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The abundance of each OS was calculated as (Area os/ Area i.s. ×average Area i.s.). Average area

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of the internal standard was calculated over all roasting levels and replicates.

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Characterization and Quantification of Constituent Monosaccharides by GC-FID. A

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gas chromatograph with a flame-ionization detector (GC-FID) was used to characterize the

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constituent monosaccharides and their absolute amounts by employing methanolysis and

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trimethylsilylation derivatization following published procedure.23 Purified coffee OS (0.5 mL)

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were dried, dissolved in 0.5 mL MeOH/0.5 M HCl, and incubated at 80 oC for 16 h. The reaction

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mixture was cooled to room temperature and dried under a stream of nitrogen. The dried sample

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was mixed with 250 µL methanol and dried under a stream of nitrogen. The mixture was

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trimethylsilylated by incubating with TriSil® reagent (300 µL) for 1 h at 80 oC. The reaction

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mixture was cooled to room temperature and excess solvent was removed under a stream of

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nitrogen. A 1-mL aliquot of hexane was added to the dried sample and centrifuged at 10,000 rpm

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for 10 min to separate salts. The solution was dried under a stream of nitrogen and suspended in 9 ACS Paragon Plus Environment


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150 µL of hexane prior to injection (1 µL) into a GC coupled to an FID controlled by a

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Hewlett-Packard ChemStation. Separation was carried out using a DB-1 fused-silica capillary

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column (30 m × 250 µm i.d., 0.25 µm film thickness, J&W Scientific, Folsom, CA, USA).

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Samples were injected in the pulsed split mode with a split ratio of 5:1. The injector and the FID

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temperature was 280 oC. The GC oven temperature was programmed from 120 to 200 oC at 1.5

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o

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carrier gas at a flow rate of 2.5 mL/min in constant flow mode. Calibration curves were built for

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the following monosaccharide standards: glucose, galactose, mannose, arabinose, xylose, and

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rhamnose. Allose was used as internal standard.

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C/min, 200 oC for 5 min isothermal, and a post run of 2 min at 250 oC. Hydrogen was used as

Statistical Analysis. The differences in constituent monosaccharides and the extrapolated

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amount of OS 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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Preparative-scale Purification of Coffee OS. The entire workflow for the preparative scale purifications is depicted in Figure 1 Five grams of the dark roasted coffee beans were subjected to hot water extraction as

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previously described. The resulting extract was mixed with cold ethanol (1:2 vol:vol) and stored

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at –30 oC for 1 h to precipitate the majority of proteins, polysaccharides, and melanoidins. The

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mixture was centrifuged at 4,225 ×g for 30 min. The supernatant was defatted using the Folch

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method as previously described for the small scale. The aqueous layer was vacuum dried and

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re-suspended in water prior to solid-phase extraction by an in-house packed column.

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The C8 column was packed by dissolving 15 g of the Discovery DSC-C8 column packing material in 60 mL acetonitrile. The slurry was transferred to a 60 mL filtration tube with a 10 ACS Paragon Plus Environment

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Teflon Frit on the bottom. The column elution time was set for 30 min and the remaining

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acetonitrile was eluted slowly at ambient atmosphere pressure. The column was sealed with a

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Teflon Frit on the top. The packed column was washed three times with 100 mL of water to

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remove the residual acetonitrile and condition the column at ambient atmosphere pressure.

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The coffee OS extract was loaded on the manually packed C8 column and washed with 150

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mL of water. The eluate was collected in three 50-mL fractions (C8 eluate fraction 1, 2 and 3).

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To further purify the OS in this eluate, each fraction was incubated with 2 g of activated carbon

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for 3 h. The activated carbon was packed into a12 mL filter tubes with a Teflon Frit on the

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bottom. The remaining solution was eluted slowly and collected (AC eluate fraction 1). The

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column was sealed with a Teflon Frit on the top and washed three times with 10 mL water to

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remove residual monosaccharides and sucrose (AC eluate fraction 2). The OS were eluted with

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30 mL of 20% acetonitrile (AC eluate fraction 3, target fraction containing OS). All eluate

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fractions were dried under vacuum, combined, resuspended in water, and passed through a 0.22

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µm MILLEX GP filter unit (Merck Millipore Ltd., Tullagreen, Carrigtwohill, Co. Cork, IRL) to

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remove residual activated carbon powder before further analysis.

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A microplate colorimetric carbohydrate assay (Biovision) was used to quantify the purified

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coffee OS. A commercial 2 mg/mL glucose standard (0, 2, 4, 6, 8, and 10 µL) was used to

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establish a calibration curve. The final volume of each calibration point was adjusted to 30 µL

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with nanopure water. A 30 µL aliquot of purified coffee OS was used at adjusted dilution.

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Glucose standards and samples were incubated with 150 µL of sulfuric acid (98%) at 85 oC for

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15 min. After incubation, 30 µL of developer (provided by the manufacturer) was added to each

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well. Samples were mixed for 5 min and absorbance was measured at 490 nm by a SpectraMax



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190 Microplate Reader (Molecular Devices, San Jose, CA, USA). The quantity of total

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carbohydrates was calculated based on the calibration curve.

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RESULTS AND DISCUSSION

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Characteristic Parameters of the Coffee Beans and the Brewed Extract. The

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Agtron/SCAA roast measurement and CIE L*a*b* color characteristics of the ground coffee

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beans from the different roasting levels are indicated in Table 1. The Agtron number of samples

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varied between 90 and 25, which corresponds, respectively, to the degrees of roasting intensity

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varying from the lightest roast with distinguishable coffee flavor characteristics (90) to the

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espresso roast (25). L* and b* values, and calculated chroma and hue values of roasted beans

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were significantly lower (p < 0.001) than that of the green beans, and also decreased as the

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roasting intensity increased. The a* value increased from green beans to light roasted beans, and

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then decreased along the increased roasting intensity.

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The significant inverse correlation between L*, b* values and intensity of roasting were well

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discussed in previous publications.19, 20 However, the discussions about the change in a* value

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during roasting were controversial. Moss and Otten,24 and Somporn et al. 20reported an elevated

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a* value with increased levels of roasting. Bicho et al.25 reported a sharp increase of a* values in

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roasted beans as compared to green beans, followed by a decreased trend with a continued

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roasting process, which was also observed in this study.

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The change in pH and total dissolved solids in brewed extract as a result of roasting level are

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also shown in Table 1. Prior to roasting, the pH of green coffee brew was 5.73, close to the value

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of 5.8 reported by Ramalakshmi et al.26 The pH first decreased to 4.82 in the brew of the lightest

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roasted beans, and increased up to 5.32 with elevated roasting intensity. A previous study also

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observed the same trend.27 The decrease in pH was attributed to the formation of formic, acetic, 12 ACS Paragon Plus Environment

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glycolic, and lactic acids as the coffee beans were roasted to medium level. The increase in pH in

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the dark roasted beans are possibly caused by the destruction of organic acids formed during

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roasting and those present initially (citric acid, malic acid, and chlorogenic acids).28

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An increased total dissolved solids as a result of heavier roasting intensity was also noticed.

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This result can be partially due to the effect of roasting on opening the cell wall structures and

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facilitating the release of polysaccharides and OS. 6

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Effects of Roasting on the Formation and Abundance of OS. The diversity in OS

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structures, including monosaccharide compositions, linkages, and isomers and the range of sizes

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provides the basis for a selective support of probiotic growth. Only bacteria equipped with a

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specific set of glycoside hydrolases, transporters and other molecules contributed to the OS

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degradation would be able to cleave the various monosaccharides and utilize them as a carbon

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source.29 Therefore, even different isomers with the same composition are not equivalent in

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biological function and separation and differentiation achieved at structural isomer level is crucial

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for the structure-function analysis of OS.

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The effect of roasting intensity on the formation and abundance of OS was studied by

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analyzing the green beans and seven samples roasted to different intensities (Table 2). Using

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tandem MS/MS, we identified and confirmed a total of 26 OS structures, including isomers

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corresponding to 16 unique OS compositions, via analysis of characteristic fragments. The OS

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compositions, retention time, and accurate mass for all samples are displayed in Table 2. The

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identified OS ranged from 3 to 15 hexose (hex) or hexose-pentose (pent) combinations. The

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relative abundances, monitored as peak areas, of 26 OS were averaged for each set of replicates.

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The peak areas of selected OS are shown in Figure 2 and Figure 3, and the data for all the OS in

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coffee beans are shown in Figures S1–S3. In general, hexose OS exhibited a greater level of 13 ACS Paragon Plus Environment


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structural diversity with a wider degree of polymerization (DP) range and more isomeric

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structures with higher abundance, compared with hexose-pentose OS. A recent model study also

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reported the presence of hybrid hexose-pentose OS and suggested that the production of hybrid

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structures was due to non-enzymatic transglycosylation reactions, which may involve the

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arabinose side chains of coffee arabinogalactan.5

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The OS profiles of the coffee samples varied among different roasting levels. Green bean

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samples only contained simple OS of (Hex)3 and (Hex)4, whereas OS with up to 15

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monosaccharide units were found in roasted coffee samples (Table 2). Figure 2 presents the

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relative abundance of selected small hexose OS (DP 3 to 4). These small OS were more often

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present in green beans, and light to light medium roasted beans (Table 2). Three OS, i.e. (Hex)3

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isomer1&3, and (Hex)4 isomer1, were found both in green and roasted beans, and their relative

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abundances were higher in green beans (Fig. 2). Some unique structures, i.e. (Hex)3 isomer 2,

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and (Hex)4 isomer 3 &4, were only found in green beans and were in moderate abundance.

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(Hex)4 isomer 2 and (Hex)3–Pent were only present in light to light medium samples (R1, R2 and

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R3). Arabinogalactans are known to be more susceptible to thermal degradation during roasting,

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indeed, a 10 to 20-fold decrease in the molecular weight of arabinogalactans was noticed even

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after a light roast.2 The small OS found in roasted coffee beans, particularly those

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pentose-containing structures, are possibly derived from the removal of arabinose side chains,

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the fission of the galactose backbone, followed up by the non-enzymatic transglycosylation

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reactions.5 These small OS can be rapidly degraded during the roasting process, resulting in their

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low abundance in green or light roasted beans.1, 10

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Roasting levels appeared to have a direct effect on the OS of DP 5-15. Except for the (Hex)5 isomer 1, only present in very dark roasted beans (R7), and the (Hex)4-Pent only present in 14 ACS Paragon Plus Environment

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R5-R7, the OS made of 5 to 13 monomers were present in all roasted coffee beans. However, a

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higher number of larger OS structures was associated with more intense roasting. (Hex)14 was

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found in light medium to very dark roasted beans (R3–R7) and (Hex)15 was only found in

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medium-very dark beans (R4–R7) (Table 2). A greater number of isomeric structures was found

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for (Hex)5–(Hex)7, whereas only a single isomer was found for larger compounds with DP 8-15.

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The changes in relative abundance of the selected OS made of 5–15 monomers in roasted beans

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are shown in Figure 3. Overall, the relative abundance of individual OS increased with elevated

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intensity of roasting, with (Hex)6 isomer 2 being the only exception. Moreover, an interesting

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trend was noticed between the relative abundance and the degree of polymerization. The relative

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abundance decreased from (Hex)5 (relatively high) to (Hex)9, and increased again from (Hex)10 to

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(Hex)15..

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Structurally, coffee arabinogalactans have a backbone of β-(1→3)-linked galactopyranosyl

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residues frequently substituted at the O-6 position by side chains formed by 1→6-linked

301

β-galactosyl, or at O-3 position by α-arabinosyl, rhamnoarabinosyl and rhamnoarabinoarabinosyl,

302

and glucuronic acid residues.3, 30, 31 Coffee galactomannans have been described as linear

303

polysaccharides with a main backbone composed of β-(1→4)-linked mannopyranose residues,

304

which are sometimes interspersed with β -(1→4)-linked glucopyranose. Some acetylated

305

mannopyrannose residues in the backbones are also present in O-2 and or O-3 position. The side

306

chains usually occur at O-6, with side chains of a single α-(1→6)-linked D-galactose residue or a

307

single arabinose residue. 32, 33

308

polysaccharide structures during roasting.1, 2, 9, 34, 35 Nunes and Coimbra35 observed the

309

de-polymerization and de-branching of galactomannans and arabinogalactans, as well as

310

decreased size of the arabinosyl side chains in arabinogalactans with an increase in degree of

A series of publications reported changes in coffee



Page 16 of 37

311

roasting. They subsequently identified structural modifications on galactomannan, including

312

those that involved in Maillard reactions, caramelization, isomerization, oxidation, and

313

decarboxylation, that were due to roasting process.9 Redgewell et al.2 investigated the degree and

314

nature of polysaccharide degradation at different roasting levels; they reported that

315

arabinogalactans and galactomannans were degraded up to 60% and 36%, respectively, after a

316

dark roasting. Although those studies only demonstrated the changes in polysaccharides, both the

317

fission of the backbone and cleavage of the side chains can lead to the generation of OS.

318

In the absence of commercial standards for the novel oligosaccharides identified in coffee, it

319

is difficult to achieve the quantification purpose by mass spectrometry. Gas chromatography

320

therefore was used to obtain the total oligosaccharides amount extrapolated from the total

321

monosaccharides amount, also reveal the presence of the individual constituent monosaccharides.

322

Table 3 illustrate the absolute amount of constituent monosaccharides and the extrapolated total

323

amount of OS (calculated as the sum of constituent monosaccharides amounts measured by GC)

324

in raw and roasted beans.

325

All six constituent monosaccharides were present in roasted beans (R1–R7) in various

326

amounts, as shown in Table 3. Although the amount of constituent monosaccharides varied,

327

galactose and mannose consistently dominated the compositions, with lower amounts of

328

arabinose and glucose, and trace amounts of rhamnose and xylose. Galactose and mannose were

329

possibly units of OS derived from the backbone of arabinogalactans and galactomannans.

330

Arabinose was possibly a monomer of OS derived from the arabinogalactans side chain. Glucose

331

may have derived either from the galactomannans or arabinogalactans backbone. Xylose was

332

also identified in another study and proposed to derive from mannose through isomerization and

333

oxidative decarboxylation during the roasting process.36 Nunes et al.31 reported the presence of 16 ACS Paragon Plus Environment

Page 17 of 37


334

rhamnoarabinosyl and rhamnoarabinoarabinosyl side chains in coffee arabinogalactan-proteins,

335

which could explain the low level of rhamnose found in the present study. There was a positive

336

correlation between the total amounts of OS and degree of roasting. ANOVA indicated a

337

significant difference among the total amount (mg/g coffee beans) of OS in beans roasted at

338

different levels (p = 3.37 × 10-6). This trend corresponded well with the individual OS identified

339

through mass spectrometry as their relative abundance increased with elevated degree of

340

roasting.

341

Galactose and mannose were consistently the dominant sugars throughout roasting.

342

Galactose concentrations remained relative high throughout all the roasting levels, whereas the

343

increase of mannose was more significant (Table 3) with the elevated roasting intensities. Recent

344

studies showed that coffee arabinogalactans and galactomannans undergo remarkably different

345

structural changes during roasting.2, 37 Arabinogalactans are particularly labile to thermal

346

degradation, and in our study they began to depolymerize after a light roasting. The OS derived

347

through the fission and debranching of arabinogalactans were generated after light roasting and

348

remained in high concentration with elevated roasting intensities. Considering that galactose

349

residues are the building blocks of coffee arabinogalactans, the high concentration of galactose at

350

all roasting levels can be explained. On the other hand, galactomannans were moderately

351

degraded even in dark roasted beans, and their extractability increased probably due to changes

352

in cell wall structures.2 The mannose-containing OS derived from galactomannan may slowly

353

start to appear under elevated level of roasting, resulting in a more significant increase in

354

absolute concentration.

355 356

Interestingly, the amount of arabinose in OS increased until light-medium roasting (R3) and decreased with elevated intensity of roasting. Arabinose residues in arabinogalactans are 17 ACS Paragon Plus Environment


Page 18 of 37

357

sensitive to thermal degradation,12 and these residues are involved in the formation of

358

melanoidins38-40 and acids.28 Moreira et al.41 further demonstrated structural modifications of

359

α-(1→5)-l-arabinotriose induced by roasting, including dehydration, oxidation, and cleavage of a

360

carbon-carbon bond at the reducing end of OS, and reported the products of these modifications,

361

e.g. aldehydes, dialdehydes, and acids. Accordingly, the decreased amount of arabinose may

362

have resulted from the further structural modification caused by higher intensity of roasting.

363

The concentration of glucose in coffee OS was stable in beans roasted at all degrees, which is

364

in agreement with previous published results.2 OS of green beans did not contain arabinose,

365

rhamnose, or xylose; however, they contained high amounts of glucose.

366

Previous work reported high amounts of sucrose, up to 90 mg/g, in green coffee beans

367

(especially in Coffea arabica species).3, 42 Although porous graphitized carbon cartridges were

368

used to purify OS and remove residual sucrose and glucose, a small amount may have remained.

369

A commercial sucrose/D-glucose/D-fructose kit was used to quantify free glucose, fructose, and

370

sucrose in the purified green coffee bean extract, and only 0.47 mg/g sucrose and 0.15 mg/g free

371

D-glucose were present. This amount could not explain the high amount (2.039±0.042 mg/g

372

coffee bean) of glucose in green coffee bean samples. In the present work, we found green beans

373

contained two unique hexose OS isomers, (Hex)4 isomer 3 and 4, with moderate relative

374

abundance. The beans also contained a high abundance of (Hex)3 isomer 3. The higher

375

concentration of glucose may be due to the presence of those short chain hexose OS.

376

Preparative-scale Purification of Coffee OS. The discovery of potential prebiotic activity

377

requires generation of an adequate amount of OS of interest in high purity in order to test the

378

activity. Several methods, such as liquid-liquid extraction, ion-exchange resin and size-exclusion

379

chromatography, activated carbon chromatography, and nano filtration have been developed to 18 ACS Paragon Plus Environment

Page 19 of 37


380

purify and enrich OS from food mixtures. 43-45 Among those, activated carbon chromatography

381

was the most efficient purification method and has been used in a wide spectrum of applications

382

to remove impurities and concentrate OS. For example, it has been tested for separating

383

maltopentaose from mixtures of simple sugars and other maltooligosaccharides,46 isolating

384

hexose OS from honey,47 purifying fructooligosaccharides from a fermentative broth,45 and etc.

385

The purification consists of three steps: absorption of OS onto the activated carbon, washing the

386

column with water, and desorption of OS by solvents, e.g. ethanol or acetonitrile in water

387

solution.

388

Following characterization of the OS present in roasted coffee, we performed a preparative

389

scale purification to obtain adequate pure OS to perform in vitro tests of probiotic activity.

390

Because coffee roasted to the highest intensity (R7, Agtron # 25) was the most abundant source

391

of OS, the R7 sample was selected for extraction and purification of OS in preparative scale for

392

testing. In-house columns packed with C8-silica gel base octyl bonding material successfully

393

removed proteins, peptides, and some browning pigments and activated carbon treatment of

394

samples successfully concentrated the coffee OS. C8 column fractionation yielded three 50-mL

395

fractions. The first 50-mL fraction was completely transparent, whereas the second and third

396

fractions were slightly brown. The LC-MS results revealed that OS were primarily in the first

397

two fractions, whereas trace levels were found in the third fraction. However, all fractions after

398

purification on the C8 column still contained some impurities and therefore were subject to

399

additional activated carbon absorption of OS for further purification.

400

After incubating the purified OS samples with activated carbon, there was no detectible OS

401

remaining in AC eluate fraction 1 and 2. After desorption of the OS with acetonitrile/water

402

solution (resulting in AC eluate fraction 3), the final solution contained 7.807±0.376 mg OS as 19 ACS Paragon Plus Environment


403

measured by a total carbohydrate assay kit conducted according to the instructions. No glucose

404

or sucrose was detected in the final OS-containing solution as confirmed by a commercial

405

sucrose/D-glucose/D-fructose assay kit.

406

Page 20 of 37

Activated carbon successfully removed impurities while absorbing the OS. The brown color

407

of the final OS-containing solution possibly derived from a small amount of either polyphenols

408

or melanoidins that remained even after two stages of solid-phase extraction. Melanoidins are the

409

final products of the Maillard browning reactions that occur during roasting.39 Melanoidins

410

account for up to 29% (w/w) of dry matter in brewed coffee.39 The chemical structures of

411

melanoidins are largely unknown, yet several researchers proposed that melanoidins mainly

412

consist of the degradation products of carbohydrates formed in the early stages of the Maillard

413

reaction; these degradation products are polymerized through aldol-type condensation and may

414

also be linked by amino compounds.39, 48-50 The incorporation of OS into melanoidin without

415

degradation of the glycosidic bonds during the Maillard reaction has been reported.48 A few

416

attempts have been performed to evaluate the implication of melanoidins on gut microbiota.

417

Ames et al. demonstrated that model melanoidins increased the counts of bacteroides and

418

clostridia, and bifidobacteria from 6 h to 24 h incubation through the in vitro study.51 Jaquet et al.

419

reported the increase in the population and the metabolic activity of Bifidobacterium spp. in a

420

3-week coffee consumption clinical trial on healthy adult volunteers, which may result from the

421

implication of melanoidins metabolism by the gut microbiota. 17 However, melanoidins in coffee

422

were possibly present with OS or polysaccharides and therefore the clinical study result cannot

423

be directly related to the effect of melanoidins.

424 425

The characterization of OS in roasted coffee beans in this study highlighted coffee as a putative source of functional OS. This study explored the difference in OS profiles among beans 20 ACS Paragon Plus Environment

Page 21 of 37


426

roasted to different levels. The size, numbers of structural isomers, and abundance of OS were all

427

elevated with increased intensity of roasting. Coffee OS were characterized with a wide range of

428

DP with various constituent monosaccharides and possibilities of branching, all of which

429

provided the basis for selective simulation of a specific gut microbiota species. Furthermore, our

430

results suggest that the formation and extraction of diverse OS structures can be optimized by

431

manipulating the degree of roasting. OS purification using an in-house packing column allowed

432

us to isolate sufficient amounts for future testing of bioactivity. The application of active carbon

433

OS adsorption especially aided removal of impurities and concentration of target OS. Other

434

techniques, such as membrane filtration, could also be applied or combined with activated

435

carbon in future studies to further investigate coffee OS and their potential prebiotic activities.

436

ACKNOWLEDGEMENTS

437

The authors acknowledge financial support from the Keurig Green Mountain, Inc., Waterbury,

438

Vermont USA, and thank C. J. Dillard for editing this manuscript.

439

ABBREVIATIONS USED

440

OS, oligosaccharides; DP, degree of polymerization; LC, liquid chromatography; 2’-FL,

441

2’-fucosyllactose; QToF, quadrupole time-of-fight; GC, gas chromatography; FID, flame

442

ionization detector; MRS, De Man-Rogose-Sharp.

443

CONFLICT OF INTEREST

444

MC was an employee of Keurig Green Mountain, Inc. at the time the work was performed. The

445

remaining authors declare that the research was conducted in the absence of any commercial

446

relationships that could be construed as a potential conflict of interest.

447



Page 22 of 37

448

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449

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29. Goh, Y. J.; Klaenhammer, T. R. Genetic mechanisms of prebiotic oligosaccharide

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32. Nunes, F. M.; Domingues, M. R.; Coimbra, M. A. Arabinosyl and glucosyl residues as

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33. Navarini, L.; Gilli, R.; Gombac, V.; Abatangelo, A.; Bosco, M.; Toffanin, R. Polysaccharides

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41. Moreira, A. S.; Coimbra, M. A.; Nunes, F. M.; Domingues, M. R. M. Roasting-induced

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43. Hernández, O.; Ruiz-Matute, A. I.; Olano, A.; Moreno, F. J.; Sanz, M. L. Comparison of

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29-36.

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46. Lee, J.-W.; Kwon, T.-O.; Moon, I.-S. Adsorption of monosaccharides, disaccharides, and

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49. Kato, H.; Tsuchida, H. Estimation of melanoidin structure by pyrolysis and oxidation. Prog.

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489-495.

580 27 ACS Paragon Plus Environment


Page 28 of 37

581 Figure Legends Figure 1. The workflow for preparative scale production of coffee OS. Figure 2. Relative abundances, expressed as peak areas, of small hexose OS (DP3~4) present in green beans and/or roasted beans, as analyzed by LC-NanoChip-QToF. Figure 3. Relative abundances, expressed as peak areas, of selected medium-large hexose OS (DP5~15) present in roasted beans, as analyzed by LC-NanoChip -QToF.


Page 29 of 37


Table 1. CIE L*a*b*, Chroma, and Hue Color Characteristics of the Green Beans and Ground Roasted Coffee Samples (R1–R7), and pH and total dissolved of Their Liquid Coffee Extracts brewed extract dry ground bean characteristics characteristics Sample1

total roast

Agtron

classification2

number

L*

a*

b*

Chroma3

Hue4

pH

solids5

raw

NA

56.41±0.90 h

1.69±0.06 h

17.30±0.21 h

17.38±0.21 h

1.47±0.00 h

5.73±0.00 h

1978±101 h

R1

light

90

25.36±0.11 i

9.79±0.11 i

13.21±0.17 i

16.44±0.20 i

0.93±0.00 i

4.82±0.04 i

2187±31 hi

R2

moderately light

75

25.20±0.16 i

9.27±0.04 j

12.49±0.22 j

15.55±0.20 j

0.93±0.01 i

4.86±0.01 i

2158±41 hi

R3

light medium

65

23.01±0.15 j

8.63±0.06 k

9.87±0.11 k

13.11±0.12 k

0.85±0.00 j

4.99±0.01 j

2380±10 i

R4

medium

50

19.27±0.18 k

5.95±0.09 l

5.02±0.12 l

7.78±0.14 l

0.70±0.01 k

5.08±0.00 k

2337±46 i

R5

moderately dark

40

18.76±0.03 l

5.34±0.25 m

4.26±0.06 m

6.84±0.16 m

0.67±0.03 l

5.16±0.03 l

2201±3 i

R6

dark

35

18.19±0.07 kl

5.01±0.11 n

3.70±0.18 n

6.23±0.19 n

0.64±0.01 k

5.20±0.01 l

2338±96 hi

R7

very dark

25

17.05±0.04 m

3.68±0.04 o

2.04±0.02 o

4.21±0.04 o

0.51±0.00 m

5.32±0.00 m

2352±201 i

green

dissolved

beans

1

Samples were ground beans; green beans were from the same batch as roasted beans. Data represents the mean of n = 3.

2

Bulk roast classification is based on the Agtron Scores. 29 ACS Paragon Plus Environment


3

Chroma = (a*2+b*2)1/2

4

Hue = arctan (b*/a*).

5

Total dissolved solids (ppm) were measured at 18 °C.

Page 30 of 37

Numbers followed by the same letter, within a column, are not significantly different (p > 0.05).


Page 31 of 37


Table 2. OS found in green beans and seven roasted beans. retention

accurate

exact

mass

time

mass

mass

error2

green

(minutes)

(g/mol)

(g/mol)

(ppm)

beans

(Hex)2Pent

4.39

474.158

474.158

0.84

(Hex)3 isomer1

6.81

504.169

504.169

0.00

(Hex)3 isomer2

11.24

504.171

504.169

3.97

(Hex)3 isomer3

14.42

504.180

504.169

21.82

(Hex)3Pent

15.28

636.217

636.211

9.43

(Hex)4 isomer1

11.23

666.223

666.222

1.50

(Hex)4 isomer2

12.49

666.223

666.222

1.50

(Hex)4 isomer3

12.34

666.222

666.222

0.00

(Hex)4 isomer4

14.83

666.222

666.222

0.00

(Hex)4Pent

14.08

798.266

798.264

2.51

(Hex)5 isomer1

13.42

828.275

828.275

0.00

(Hex)5 isomer2

15.17

828.275

828.275

0.00

OS compositions1

Presence in individual samples

R1

R2

R3

R4

R5

R6

R7



Page 32 of 37

1.52

1476.483 1476.485

1.35

21.73

1638.538 1638.539

0.61

(Hex)11

22.61

1800.593 1800.592

0.56

(Hex)12

23.5

1962.645 1962.644

0.51

(Hex)13

24.11

2124.700 2124.697

1.41

(Hex)14

24.85

2286.749 2286.750

0.44

(Hex)15

25.28

2448.795 2448.803

3.27

(Hex)5 isomer3

16.74

828.275

828.275

0.00

(Hex)5Pent

17.42

960.317

960.316

1.04

(Hex)6 isomer1

16.36

990.327

990.328

1.01

(Hex)6 isomer2

17.97

990.327

990.328

1.01

(Hex)7 isomer1

18.14

1152.379 1152.380

0.87

(Hex)7 isomer2

21.43

1152.378 1152.380

1.74

(Hex)8

19.52

1314.431 1314.433

(Hex)9

20.66

(Hex)10

1

Hex: hexose; Pent: Pentose; (Hex)n: OS composed of n hexose units.

2

mass error = (accurate mass-exact mass)/exact mass ×106.


Page 33 of 37


Table 3. Constituent Monosaccharides1 and Extrapolated Total OS in Beans Roasted at Different Intensities as Analyzed by Gas Chromatography2 Extrapolated sample

arabinose

rhamnose

xylose

mannose

galactose

glucose total OS

green

ND

ND

ND

0.249±0.013 f

0.443±0.041 e

2.039±0.042 a

2.730±0.013 d

R1

0.496±0.016 b 0.059±0.004 b

0.085±0.004 b

0.514±0.019 ef

1.380±0.086 cd 0.557±0.051 b

3.090±0.101 d

R2

0.450±0.082 b 0.058±0.008 b 0.115±0.036 ab

0.538±0.073 e

1.355±0.228 d

2.994±0.472 d

R3

0.658±0.012 a 0.107±0.018 a

0.998±0.020 d

2.059±0.055 ab 0.658±0.137 b 4.653±0.058 bc

R4

0.460±0.032 b 0.060±0.003 b 0.126±0.006 ab 1.214±0.015 cd

1.891±0.119 b

R5

0.458±0.023 b 0.053±0.002 b 0.121±0.003 ab 1.335±0.073 bc

1.812±0.091 bc 0.526±0.074 b

4.306±0.118 c

R6

0.547±0.007 ab 0.066±0.002 b

0.152±0.012 a

1.572±0.129 b

2.400±0.130 a

0.569±0.028 b

5.305±0.248 b

R7

0.482±0.026 b 0.063±0.001 b 0.124±0.007 ab

2.597±0.093 a

2.492±0.016 a

0.671±0.126 b

6.429±0.014 a

0.172±0.010 a

0.477±0.045 b

0.680±0.198 b 4.432±0.375 bc

1

Monosaccharides are expressed as mean mg/g coffee bean ± SD (n = 3); ND, not detected.

2

Numbers followed by the same letter, within a column, are not significantly different (p > 0.05).



Page 34 of 37

Figure 1.


Page 35 of 37


Figure 2.

green beans



Page 36 of 37

Figure 3


Page 37 of 37


Table of content graph


Effect of Roasting on Oligosaccharide Abundance in Arabica Coffee

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