Chemical Characterization of Potentially Prebiotic Oligosaccharides in

Subscriber access provided by University of Newcastle, Australia

Article

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

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 33

Journal of Agricultural and Food Chemistry

1

Chemical Characterization of Potentially Prebiotic Oligosaccharides in Brewed Coffee and

2

Spent Coffee Grounds

3

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

4

ǂ

5

United States

6

†

7

95616, United States

8

§

9

* 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

10

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

11

4759

1 ACS Paragon Plus Environment


12

ABSTRACT: Oligosaccharides are indigestible carbohydrates widely present in mammalian

13

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

14

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

15

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

16

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

17

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

18

oligosaccharides identified were mainly hexoses (potentially galacto-oligosaccharides and

19

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

20

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

21

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

22

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

23 24

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

25

prebiotics

26


Page 2 of 33

Page 3 of 33


27 28

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

29

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

30

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

31

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

32

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

33

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

34

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

35

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

36

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

37

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

38

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

39

molecules, thus generating added value.3

40

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

41

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

42

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

43

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

44

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

45

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

46

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

47

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

48

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

49

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

50

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


51

derive from two polysaccharides, arabinogalactans and galactomannans, through several

52

chemical reactions during the roasting process.7-9

53

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

54

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

55

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

56

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

57

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

58

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

59

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

60

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

61

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

62

abundant food oligosaccharides, including galactooligosaccharides and inulin, have prebiotic

63

activity.11

64

The prebiotic properties of oligosaccharides highly depend on structural conformation,

65

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

66

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

67

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

68

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

69

different linkages. As a matter of fact, arabinogalactooligosaccharides and

70

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

71

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

72

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

73

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


Page 4 of 33

Page 5 of 33


74

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

75

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

76

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

77

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

78

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

79

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

80

brewed coffee or spent coffee grounds.

81

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

82

comprehensively characterize oligosaccharide structures; these spectral technologies have

83

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

84

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

85

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

86

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

87

used to identify and quantify the purified oligosaccharides.

88

MATERIALS AND METHODS

89

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

90

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

91

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

92

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

93

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

94

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

95

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

96

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



97

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

98

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

99

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

100

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

101

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

102

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

103

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

104

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

105

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

106

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

107

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

108

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

109

Table S2.

110

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

111

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

112

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

113

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

114

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

115

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

116

Oligosaccharides Extraction and Purification. Oligosaccharides from ground C. arabica

117

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

118

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

119

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


Page 6 of 33

Page 7 of 33


120

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

121

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

122

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

123

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

124

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

125

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

126

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

127

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

128

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

129

the hydrophilic oligosaccharides were captured by a subsequent purification with porous

130

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

131

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

132

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

133

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

134

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

135

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

136

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

137

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

138

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

139

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

140

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

141

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

142

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



143

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

144

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

145

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

146

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

147

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

148

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

149

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

150

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

151

Da with an offset of -3.6V.

152

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

153

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

154

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

155

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

156

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

157

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

158

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

159

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

160

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

161

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

162

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

163

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

164

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

165

o

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


Page 8 of 33

Page 9 of 33


166

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

167

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

168

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

169

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

170

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

171

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

172

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

173

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

174

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.

175

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

176

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

177

response factors for individual monosaccharides were calculated to quantify each

178

monosaccharide in oligosaccharide chains.

179

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

180

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

181

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

182

of significant difference was set at p ≤ 0.05.

183

RESULTS AND DISCUSSION

184

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

185

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

186

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

187

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

188

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



189

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

190

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

191

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

192

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

193

characterization by mass spectrometry.

194

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

195

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

196

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

197

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

198

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

199

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

200

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

201

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

202

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

203

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

204

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

205

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

206

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

207

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

208

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

209

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

210

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

211

or hexose-pentose combinations.


Page 10 of 33

Page 11 of 33

212


Understanding the precursor polysaccharide structures will enable prediction of some coffee

213

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

214

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

215

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

216

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

217

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

218

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

219

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

220

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

221

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

222

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

223

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

224

mannose residues in coffee galactomannan.37

225

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

226

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

227

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

228

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

229

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

230

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

231

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

232

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

233

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



234

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

235

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

236

caramelization reactions, and Amadori compounds. The prediction of monosaccharide

237

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

238

appropriate monosaccharide standards for quantification (see GC analysis of constituent

239

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

240

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

241

including all linkages.

242

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

243

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

244

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

245

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

246

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

247

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

248

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

249

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

250

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

251

extrapolated via quantification of the constituent monosaccharides after methanolytic cleavage

252

and derivatization.

253

GC Analysis of Constituent Monosaccharides of Coffee Oligosaccharides. The GC

254

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

255

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

256

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


Page 12 of 33

Page 13 of 33


257

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

258

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

259

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

260

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

261

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

262

amounts (2.1% and 1.2%, respectively).

263

Partial structural information on coffee oligosaccharides was inferred by combining the

264

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

265

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

266

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

267

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

268

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

269

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

270

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

271

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

272

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

273

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

274

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

275

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

276

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

277

indicated the possibilities that rhamnoarbinosyl and rhamnoarabinoarabinosyl side chains of type

278

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

279

oligosaccharides.



280

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

281

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

282

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

283

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

284

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

285

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

286

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

287

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

288

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

289

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

290

oligosaccharides with select probiotic strains.

291

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

292

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

293

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

294

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

295

and also analyzed for oligosaccharides.

296

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

297

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

298

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

299

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

300

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

301

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

302

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


Page 14 of 33

Page 15 of 33


303

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

304

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

305

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

306

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

307

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

308

other brewing methods.

309

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

310

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

311

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

312

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

313

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

314

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.



Page 28 of 33

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


Page 29 of 33


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



Page 30 of 33

Figure 3.

Allose (IS)

Mannose

Response

Galactose Arabinose Allose (IS) Xylose Allose (IS)

Glucose

Rhamnose

Retention Time (Minutes)


Page 31 of 33


Figure 4.



Figure 5


Page 32 of 33

Page 33 of 33


For Table of Contents Only


Chemical Characterization of Potentially Prebiotic Oligosaccharides in

Recommend Documents