Spent coffee grounds extract rich in manooligosaccharides promote a

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Bioactive Constituents, Metabolites, and Functions

Spent coffee grounds extract rich in manooligosaccharides promote a healthier gut microbial community in a dose dependent manner Sergio Pérez-Burillo, Silvia Pastoriza, Alejandro Fernández-Arteaga, German Luzón, Nuria Jiménez-Hernández, Giuseppe D'Auria, Pilar Francino, and José A. Rufián-Henares J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b06604 • Publication Date (Web): 06 Feb 2019 Downloaded from http://pubs.acs.org on February 7, 2019

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Journal of Agricultural and Food Chemistry

Spent coffee grounds extract rich in manooligosaccharides promote a healthier gut microbial community in a dose dependent manner

Sergio Pérez-Burillo1, Silvia Pastoriza1, Alejandro Fernández-Arteaga2, Germán Luzón2, Nuria Jiménez-Hernández3, Giuseppe D’Auria3, M. Pilar Francino3,4 & José Ángel RufiánHenares1,5*

1Departamento

de Nutrición y Bromatología, Instituto de Nutrición y Tecnología de los

Alimentos, Centro de Investigación Biomédica, Universidad de Granada, Granada, Spain. 2Departamento

de Ingeniería Química, Facultad de Ciencias,

Universidad de Granada, Granada, Spain. 3Unitat

Mixta d’Investigació en Genòmica i Salut, Fundació per al Foment de la

Investigació Sanitària i Biomèdica de la Comunitat Valenciana (FISABIO-Salut Pública) / Institut de Biologia Integrativa de Sistemes (Universitat de València), València, Spain 4CIBER 5Instituto

en Epidemiología y Salud Pública, Madrid, Spain

de Investigación Biosanitaria ibs.GRANADA, Universidad de Granada, Granada, Spain.

* Corresponding author: Telephone: +34 958 24 28 41 E-mail: [email protected]

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ABSTRACT

2

Coffee is one of the most consumed beverages around the world and as a

3

consequence, spent coffee grounds are a massively produced residue that is causing

4

environmental problems. Reusing them is a major focus of interest nowadays. We extracted

5

mannoligosaccharides (MOS) from spent coffee grounds and submitted them to an in vitro

6

fermentation with human feces. Results obtained suggest that MOS are able to exert a

7

prebiotic effect on gut microbiota by stimulating the growth of some beneficial genera such

8

as

9

Pseudoflavonifractor, or Veillonella. Moreover, SCFA production also increased in a dose

10

dependent manner. However, we observed that 5-hydroxymethylfurfural, furfural and

11

polyphenols (which are either produced or released from spent coffee grounds matrix

12

during hydrolysis) could have an inhibitory effect on some beneficial genera such as

13

Faecalibacterium, Ruminococcus, Blautia, Butyricimonas, Dialister, Collinsella, or

14

Anaerostipes which could affect negatively to the prebiotic activity of MOS.

Barnesiella,

Odoribacter,

Coprococcus,

Butyricicoccus,

Intestinimonas,

15 16 17

KEY WORDS: Mannooligosaccharides, mannan, MOS, short chain fatty acids, gut microbiota, prebiotic activity, 5-hydroxymethylfurfural, polyphenols.

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Introduction

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Coffee brew is consumed all over the world and, in fact, is the food product most

27

consumed in the world, after water.1 Coffee has been related to health since ancient times

28

and still today is the main object of study in many research projects related to chronic

29

diseases.2 Coffee is thought to have a protective role on health, mostly due to its

30

phytochemical compounds such as chlorogenic acids and other polyphenolic substances.3

31

However, carbohydrates also account for an important part of the coffee bean. In green

32

coffee, carbohydrates account for up to 60% of the dry weight, with sucrose as the main

33

monosaccharide. Among polysaccharides, which can range from 37 to 55% of the dry

34

weight, cellulose, hemicellulose, galactomannans and arabinogalactans are found. Although

35

galactomannans are the main polysaccharide present in beans, in coffee infusion they are

36

not the main ones due to a low solubility.4 On the other hand, roasting has an important

37

influence on carbohydrate content. Sucrose and low molecular weight oligosaccharides are

38

almost completely degraded whereas polysaccharide content is reduced to around 24-39%

39

of the dry weight. Roasting also partially hydrolyses polysaccharides to a lower degree of

40

polymerization making them more soluble.5

41

Due to its high consumption, coffee by-products are massively produced specially

42

spent coffee grounds. The main problem is that they are not properly disposed of,

43

generating a growing environmental concern.5,6 Therefore, spent coffee grounds have

44

become a major focus of interest regarding their reutilization as a source of bioactive

45

compounds.7-12

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Since hot water is not able to extract the whole carbohydrate fraction during coffee

47

brewing, spent coffee grounds are still rich in polysaccharides, with galactomannans 3 ACS Paragon Plus Environment


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representing around 50% of them and arabinogalactans and cellulose accounting for the

49

other 50%.4 Therefore, mannanoligosaccharides (MOS) are one of the bioactive compounds

50

that can be obtained from spent coffee grounds. MOS are short chain carbohydrates

51

produced from coffee galactomannans usually through acid, alkaline or enzymatic

52

hydrolysis.13 Their physiological effects arise from different functional properties: they are

53

able to enhance the growth of beneficial bacteria, decrease blood pressure, reduce fat

54

absorption, or decrease attachment of pathogenic bacteria to the intestinal mucosa.14-15

55

Finding ingredients that can promote the growth of beneficial gut bacteria is

56

becoming a major focus of interest in order to produce functional foods. That is because

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now it is very clear that the gut microbiota has important implications in different diseases

58

and is therefore closely related to health status.16 In this sense, the gut microbiota has been

59

linked to inflammatory bowel disease,17,18 obesity,19,

60

immune system disorders,22 among others. It has been reported that around 40 g of

61

polysaccharides reach the colon every day, being an important source of nutrients for gut

62

microbes and acting as prebiotic agents that enhance the growth of some of them.23 The

63

metabolization of fiber takes places specially in the proximal colon resulting in the

64

production of several metabolites such as short chain fatty acids (SCFAs), mainly acetate,

65

propionate and butyrate, which have an important role in host health, not only in the gut.24

66

Dietary fiber is the main nutrient for gut microbes and it has been proven to modulate gut

67

microbiota in vitro and in vivo with beneficial effects for host health.23

20

autism spectrum disorders,21 and

68

Taking all this information into account, the objective of this study was to evaluate

69

the effects of MOS produced from spent coffee grounds on gut microbial communities and

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also on SCFAs production, after in vitro fermentation with human fecal samples.

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Materials and methods

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Reagents

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Acetic, formic and propionic acids, 1-phenyl-3-methyl-5-pyrazolone (PMP),

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chloroform, mannose, manobiose, manotriose, manotetraose, mannopentaose and

76

mannohexaose standards, sodium di-hydrogen phosphate, tryptone, cysteine, sodium

77

sulphide, resazurin, Folin-Ciocalteu reagent, sodium carbonate, gallic acid, 5-

78

(Hydroxymethyl)furfural (HMF), furfural and acetonitrile (HPLC grade) were purchased

79

from Sigma-Aldrich (Darmstadt, Germany).

80 81

Samples

82

A mix of spent coffee grounds and water (10% w/w) was hydrolyzed in a reactor

83

(270 mL of distilled water and 30 g of spent coffee grounds) by applying high temperatures

84

and high pressure for 60 minutes and taking samples after 10 minutes, 30 minutes and 60

85

minutes. Four experiments were carried out varying only the temperature: experiment A

86

was carried out at 220 oC, experiment B was carried out at 185 oC, experiment C was

87

carried out at 170 oC and experiment D was carried out at 180 oC. Each experiment was

88

composed of the three samples taken after 10 minutes, 30 minutes and 60 minutes. Aliquots

89

were taken and stored at -80 oC for MOS quantification, HMF, furfural and total

90

polyphenols quantification and for in vitro fermentation.

91 92

In vitro fermentation

93

The in vitro fermentation method was carried out according to the protocol

94

described by Pérez-Burillo et al.25 A Fecal sample from four healthy donors (not taking

95

antibiotics, people with body mass index within the “normal weight range”: mean Body 5 ACS Paragon Plus Environment


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Mass Index = 21.3), was obtained in the morning, placed in sterile containers and stored at

97

4 oC until the inoculum was prepared (as an equal mixture of feces from all four donors).

98

Briefly, 0.5 mL of MOS mixture obtained from the reactor was placed in a screw-cap tube.

99

Then, 7.5 mL of fermentation medium (948.75 mL of peptone water (15 g/L), 50 mL of

100

reductive solution (312 mg of cysteine and 312 mg of sodium sulphide in 48 mL of water

101

and 2 mL of NaOH 1M), and 1.25 mL of resazurin (0.1% w/v)) and 2 mL of inoculum

102

(consisting of a solution of 32% faeces in phosphate buffer 100 mM, pH 7.0) were added,

103

to reach a final volume of 10 mL. Nitrogen was bubbled through the mix to produce an

104

anaerobic atmosphere and the mix was then incubated at 37 ºC for 24 hours under

105

oscillation. Immediately afterwards, the samples were immersed in ice, to stop microbial

106

activity. Aliquots were taken for DNA isolation and SCFAs analysis and stored at -80 ºC

107

until analysis.

108 109

Short chain fatty acids determination

110

Short chain fatty acid (SCFAs) determination was performed on the supernatant

111

obtained after in vitro fermentation. The analysis was carried out according to the

112

procedure described by Panzella et al.11 with few modifications. SCFAs determination was

113

carried out on Accela 600 HPLC (Thermo Scientific) equipped with a pump, an

114

autosampler and a UV-VIS PDA detector set at 210 nm; the mobile phase used was 0.1 M

115

phosphate buffer (pH 2.8)/acetonitrile 99:1 v/v delivered at a 1.25 mL/min flow rate; the

116

column used was an Aquasil C18 reverse phase (Thermo Scientific) (150 × 4.6 mm, 5 µm),

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with a total run-time of 30 min.

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The sample did not require any pretreatment before injecting. Briefly, the SCFA

119

standards were prepared in the mobile phase at concentrations ranging from 5 to 10000 6 ACS Paragon Plus Environment

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ppm. After the fermentation process, 1 mL of MOS fermentation supernatant was

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centrifuged to remove solid particles, filtered through a 0.22 m nylon filter and finally

122

transferred to a vial for HPLC analysis. Results are expressed as mol/g of spent coffee

123

grounds. Chromatograms are depicted in Figures S1A-S1C.

124 125

MOS HPLC determination

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Pre-column derivatization: MOS mixtures obtained from the reactor were labeled

127

with PMP following the procedure described by Zang et al.26 with few modifications.

128

Briefly, 0.1 mL of sample was mixed in a screw-cap pyrex tube with 0.1 mL of NaOH 0.3

129

M. Right after, 0.12 mL of PMP 0.5 M was added and the mixture was vortexed for 10

130

seconds and placed at 70 oC for 60 minutes. Thereafter, 0.1 mL of HCl 0.3 M was added to

131

neutralize the NaOH. The mixture was then mixed with 0.5 mL of chloroform, vortexed,

132

and the aqueous layer collected, discarding the organic one. This step was done two more

133

times. Samples were filtered through a 0.22 m filter and injected in the HPLC.

134

HPLC analysis: Determination of MOS was carried out on Accela 600 HPLC

135

(Thermo Scientific) equipped with a pump, an autosampler and a UV-VIS PDA detector set

136

at 245 nm; the mobile phase used was A) Milli-Q water with 0.1% of formic acid, and B)

137

acetonitrile with 0.1% of formic acid running with the following gradient: 2% of B to 20%

138

of B in 25 minutes (0-25 minute), 20% of B for 15 minutes (25-40 minute), 20% of B to

139

2% of B in 5 minutes (40-45 minute), and 2% of B for 15 minutes (45-60 minute). Standard

140

curves were performed for each sugar in the range of 5-0.15 ppm. The column used was an

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Accucore C18 reversed phase 150 mm x 2.1 mm and 2.6 μm of particle size. Results are



142

expressed as mg/g of spent coffee grounds. Chromatograms are depicted in Figures S1D-

143

S1F.

144 145

Folin-Ciocalteu total phenolics assay

146

Total phenolic content was estimated following the procedure described by

147

Singleton and Rossi27 with few modifications and adapted to a microplate reader. The assay

148

was performed on the samples obtained from the reactor. Briefly, sodium carbonate 10%

149

was prepared and then the needed volume was diluted to 2.35%. 30 µL of sample were

150

placed by triplicate in the wells of a transparent 96-well polystyrene microplate (Biogen

151

Científica, Spain), followed by 255 µL of the sodium carbonate and finally 15 µL of the

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Folin-Ciocalteu reagent. Absorbance lectures at 725 nm were taken for 60 minutes at 37 oC.

153

The equipment used was a FLUOStar Omega microplate reader (BMG Labtech, Germany).

154

A calibration curve was performed using gallic acid as the standard with concentrations

155

ranging from 0.01 to 1.00 mg/mL. The results obtained are expressed as mol gallic acid

156

equivalents (GAE) per g of spent coffee grounds.

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HMF and furfural assay

159

HMF and furfural determination was performed according to the method described

160

by Rufián-Henares et al.28 One mL of the MOS mixture taken from the reactor was

161

centrifuged at 10000 rpm for 5 minutes. Then, it was filtered through a 0.22 m filter. The

162

sample was ready for HPLC injection. Determination was carried out on Accela 600 HPLC

163

(Thermo Scientific) equipped with a pump, an autosampler and a UV-VIS PDA detector set

164

at 284 nm. The mobile phase was made up of a mixture of acetonitrile in water (5% v/v)


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delivered at the flow rate of 1 mL/min under isocratic conditions through C18 reverse phase

166

column. 20 L of sample were injected. HMF and furfural was quantified by the external

167

standard method within the range 0.01–50.00 mg/L. Results are expressed as mg/g of spent

168

coffee grounds.

169 170

DNA extraction and sequencing

171

DNA extraction was performed using a NucliSENSEasyMAG platform

172

(Biomérieux) following the standard protocol. Microbial genomic DNA was used at a

173

concentration of 5 ng/μL in 10 mMTris (pH 8.5) for the Illumina protocol for 16S rRNA

174

gene Metagenomic Sequencing Library Preparation (Cod. 15044223 Rev. A). PCR primers

175

targeting the 16S rRNA gene V3 and V4 regions were designed as in Klindworth et al.29

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Primer

177

TGTATAAGAGACAGCCTACGGGNGGCWGCA-G3’

178

CGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC3’.

179

Primers contained adapter overhang sequences added to the gene-specific sequences,

180

making them compatible with the Illumina Nextera XT Index kit (FC-131-1096). After 16S

181

rRNA gene amplification, amplicons were multiplexed and 1 mL of amplicon pool was run

182

on a Bioanalyzer DNA 1000 chip to verify amplicon size (~550 bp). After size verification,

183

libraries were sequenced in an Illumina MiSeq sequencer according to manufacturer’s

184

instructions in a 2x300 cycles paired-end run (MiSeq Reagent kit v3 MS-102-3001).

sequences

are:

Forward

5’TCGTCGGCAGCGTCAGATG-

185 186

Bioinformatic analysis


and

Reverse

5’GTCT-


187

Quality assessment of sequencing reads was performed with the prinseq-lite

188

program applying the following parameters:30 a minimal length (min_length) of 50 nt and a

189

quality score threshold of 30 from the 3’-end (trim_qual_right), using a mean quality score

190

(trim_qual_type) calculated with a sliding window of 10 nucleotides (trim_qual_window).

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Read 1 and read 2 from Illumina sequencing where joined using fastq-join from the ea-tools

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suite.31 Taxonomic affiliations were assigned using the RDP_classifier from the Ribosomal

193

Database Project (RDP).32 Reads that had an RDP score below 0.8 were assigned to the

194

next higher taxonomic rank, leaving the last rank as unidentified. We assigned 6 taxonomic

195

levels, which were kingdom, phylum, class, order, family and genus.

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Statistical analysis

198

Pearson correlations were carried out through multivariate analysis with

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Statgraphics Centurion XVI.I. Bacterial abundances between groups, SCFA, HMF and

200

polyphenols were compared by analysis of variance (ANOVA) at p < 0.05 confidence level

201

with Statgraphics Centurion XVI.I and R software.

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Results and discussion

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MOS quantification

205

MOS quantification was carried out after 10, 30 and 60 minutes of hydrolysis in

206

every experiment. For each sample, mannose, mannobiose, mannotriose, mannotetraose,

207

mannopentaose and mannohexaose were determined. Table 1 shows the MOS content in

208

each sample. All carbohydrates were detected except mannotetraose. After 10 minutes,

209

temperature was not stable yet and MOS values are similar among the different

210

experiments. However, experiment B had significantly lower (p < 0.05) amounts of total 10 ACS Paragon Plus Environment

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MOS than the other experiments. The highest levels of mannose, mannobiose and

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mannopentaose were obtained in experiment A, whereas mannotriose and manohexaose

213

were significantly higher in experiment C.

214

After 30 minutes of hydrolysis, experiment A showed the highest MOS content

215

with statistical significance (p < 0.05), followed by experiments B and D, with no

216

significant differences among the two of them. Finally, experiment C showed significantly

217

lower (p < 0.05) amounts than the other three experiments. This makes sense since

218

experiment A was carried out at a higher temperature, whereas in experiment C the lowest

219

temperature was used. On the other hand, experiments B and D showed very similar

220

amounts of MOS, probably due to the very close working temperature. Individually, each

221

sugar followed the tendency described above. Finally, at 60 minutes experiment A

222

decreased in all six sugars, probably due to a thermal degradation related with the high

223

temperatures applied; experiment B had significantly higher values than the other three

224

experiments, whereas experiment C had significantly lower values. Individually, each sugar

225

followed the tendency described above.

226

As a result of these trends, the overall highest amounts of individual and total

227

MOS were obtained after 60 minutes in experiment B, as at this point MOS concentrations

228

reached values beyond those that had been obtained at 30 minutes in experiment A.

229 230

Microbial composition

231

As the objective of this project was to study the possible use of spent coffee

232

grounds rich in MOS (after hydrolysis) as potential prebiotic, an initial in vitro fermentation

233

of a pure MOS mix before starting with spent coffee grounds hydrolysis was performed.

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Accordingly, 30 mg of MOS/mL (5 mg/mL of each compound) were fermented. Significant 11 ACS Paragon Plus Environment


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stimulation in the growth of some genera, in comparison with basal situation of the

236

microbial community, were found. Therefore, Bifidobacterium, Barnesiella, Anaerostipes,

237

Blautia, Clostridium XVIb, Coprococcus, Butyricicoccus, Intestinimonas, Ruminococcus

238

and Bilophila showed significantly higher abundances when MOS where used (Figure

239

S2A).

240

Once the prebiotic effect of pure MOS could have on some bacteria was

241

confirmed, and since MOS have been described as prebiotic compounds, the next step was

242

to unravel whether higher amounts of MOS would lead to a more beneficial microbial

243

community structure. For this purpose, the samples corresponding to 30 minutes were used

244

for several reasons: a) after 10 minutes the temperature was not stable and thus, MOS

245

production was very variable. In this sense, MOS production was not sufficiently different

246

among studies as to be able to make comparisons and therefore to establish clear links to

247

certain microorganisms. b) After 60 minutes, experiment A suffered a decrease in

248

oligosaccharide content (probably due to a thermal degradation) which would yield new

249

compounds with potential effect on microbial growth, making this experiment useless for

250

comparisons. c) Related to the previous reason, during carbohydrate heating there is a

251

production of furanic compounds (such as HMF and furfural) from non-enzymatic

252

browning.28 In this sense, we observed an increase in the levels of furanic compounds with

253

hydrolysis time and temperature (HMF and furfural, Table 2), which have been reported to

254

inhibit some bacterial growth.33 Therefore we decided to avoid samples obtained after 60

255

minutes due to the excessive accumulation of furanic compounds; in fact, we detected that

256

some bacteria stopped growing or even decreased in abundance after 30 minutes (described

257

in section 3.2.1.). In addition, comparisons were made between experiments and not


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between samples from the same experiment, using only the sample obtained after 30

259

minutes of hydrolysis.

260

At phylum level, nearly 100% of the bacterial populations belonged to the phyla

261

Actinobacteria, Firmicutes, Bacteroidetes or Proteobacteria. Experiment A (with the

262

highest MOS content) had a significant (p < 0.05) enrichment in phylum Firmicutes

263

compared with the other three experiments. Moreover, Bacteroidetes showed a tendency to

264

become more abundant in experiments with higher amounts of MOS. In addition,

265

Bacteroidetes abundance was significantly higher (p < 0.05) in experiments A, B and D

266

compared with experiment C (the one with the lowest amounts of MOS). These results are

267

in accordance with previously published literature stating that fiber-rich diets increase

268

Firmicutes and Bacteroidetes phyla.34 The same authors described that an increase in

269

Firmicutes and Bacteroidetes is accompanied by a decrease in Proteobacteria. Our results

270

also show how Proteobacteria abundance is significantly higher (p < 0.05) in the

271

experiment with the lowest amounts of MOS (Figure 1A). On the other hand, no

272

significant differences in the Firmicutes:Bacteroidetes ratio were observed.

273

The observed increase in Firmicutes could be explained by the higher abundance

274

of some specific genera: Clostridium XIVb, Coprococcus, Butyricicoccus, Intestinimonas,

275

Pseudoflavonifractor and Veillonella. In addition, statistically significant differences

276

related with MOS content were found in the Bacteroidetes genera Barnesiella and

277

Odoribacter and in the Proteobacteria ud-Burkholderiales and Bilophila. Experiment A

278

(with the highest MOS levels) showed significantly higher (p < 0.05) abundances of the

279

above mentioned taxa than the other three experiments. On the other hand, experiment C

280

(with the lowest MOS amounts) generally showed significantly lower abundances of such

281

bacteria than the other experiments (Figure 1B). These results indicate that a higher supply 13 ACS Paragon Plus Environment


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of MOS translates into a higher abundance of these bacteria. Moreover, these assumptions

283

are supported by significantly positive correlations between each bacterial abundance and

284

every MOS analyzed (Figure 2). Except for Bilophila, all of the bacterial taxa associated

285

with MOS have been linked mainly to positive effects on health, summarized in Table 3.

286

According to these results, MOS could have a prebiotic effect over gut microbial

287

communities, promoting a healthier microbial structure.

288

The analysis of microbial -diversity in terms of the Shannon and Simpson

289

diversity indexes was also performed. In experiment A, the Shannon diversity index was

290

significantly higher than in the other experiments. Moreover, the Simpson diversity index

291

followed the same behavior, being significantly higher in A than in the other three

292

experiments (where no significant differences were found). These results are in accordance

293

with published literature about the effect of fiber-rich diets on the increase of gut microbial

294

diversity.34 Additionally, statistically significant correlations between Shannon diversity

295

index and mannose, mannobiose, mannotriose, mannopentaose, mannohexaose, and total

296

MOS were found (Figure 2). Finally, the Simpson diversity index was also positively

297

correlated with mannose, mannobiose, mannopentaose and total MOS (Figure 2).

298

According to these results, we observed that a higher MOS concentration led to a richer

299

microbial community in terms of diversity.

300 301

Microbial growth inhibitors

302

The appearance of inhibitors of microbial growth during biotransformation

303

processes is a main issue that has been widely reported.33 Such potential inhibitors include

304

furanic compounds (like HMF and furfural) and some polyphenols. HMF and furfural are


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metabolites produced during non-enzymatic browning and therefore are already present in

306

roasted coffee beans35 and spent coffee grounds.36 The inhibition caused by furanic

307

compounds has been widely reported, mostly in relation to the fermentation of

308

lignocellulosic residues.33 In the case of polyphenols, it is widely known that they are

309

strong antioxidants and therefore play a protective role in health. Some polyphenols, mostly

310

complex ones such as flavonoids, chlorogenic acids, tannins, ellagitannins, etc., can be

311

metabolized by some gut microbial communities, yielding simpler phenolics. These

312

metabolites can be absorbed and exert a beneficial effect either systemically or locally in

313

the colon.37 However, some other polyphenols have also been described as possible growth

314

inhibitors for some microorganisms.33,38 In this sense, it has been reported that ferulic acid

315

and quercetin can inhibit or reduce the growth of some fiber degrading bacterial

316

communities in rats, leading to a lower SCFA production and an increased excretion of

317

oligosaccharides in feces.39

318

One of the objectives of the project was to study the potential use of hydrolyzed

319

spent coffee ground extracts without further purification, since they still have some

320

beneficial compounds, such as polyphenols. However, it was hypothesized that along

321

hydrolysis (due to high temperatures) some Maillard compounds (such as HMF and

322

furfural) would appear. Therefore, the possible inhibitory effect of HMF and furfural on

323

microbial growth we tested, by in vitro fermentation of a MOS mixture with and without

324

addition of HMF-furfural mix at two concentrations (10 and 1 mg/mL). Growth inhibition

325

of Barnesiella, Coprobacter, Butyricimonas, Odoribacter, Anaerostipes, Blautia,

326

Clostridium XVIb, Ruminococcus, Clostridium IV, Rumiococcus2, Flavonifractor,

327

Intestinimonas, Oscillibacter, Clostridium XVIII and Bilophila (Figure S2B) were observed



328

under this in vitro fermentation conditions. Therefore, the inhibitory activity of HMF and

329

furfural was demonstrated.

330

The analysis of bacterial communities showed that the relative abundances of

331

some genera were lower in the fermentations employing coffee grounds hydrolyzed for

332

longer than 30 minutes (Figure 3). These bacteria included some SCFA producers

333

Collinsella, Ruminococcus and Blautia (acetate producers), Blautia and Dialister

334

(propionate producers), and Anaerostipes, Faecalibacterium, and Butyricimonas (butyrate

335

producers).16 In view of this pattern, it could be hypothesized that some microbial inhibitors

336

that accumulate during hydrolysis, such as furanic compounds and/or polyphenols, can

337

negatively affect such microbial genera. The relative abundance of several of these taxa had

338

increased with coffee grounds hydrolysed for 30 minutes in comparison to those

339

hydrolysed for 10, whereas the relative abundance of Faecalibacterium, Ruminococcus and

340

Blautia already decreased with coffee grounds hydrolysed for 30 minutes, suggesting a

341

more harmful effect of such inhibitors towards them. However, in experiment C, the

342

relative abundance of these three genera increased between 10 minutes and 30 minutes of

343

coffee grounds hydrolysis. This could be related with the lower HMF and furfural values

344

found in experiment C.

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Taking all this information into account, the analysis of HMF and furfural

346

concentrations in the samples collected after 10, 30 and 60 minutes of hydrolysis was

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performed. As expected, HMF and furfural concentrations increased with hydrolysis time

348

due to longer exposure to high temperature (Table 2). HMF and furfural production also

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depend on the temperature applied, and thus, in those experiments in which the applied

350

temperature was higher, HMF and furfural concentrations were also higher. We found

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statistically significant negative correlations (p < 0.05) between HMF and furfural and 16 ACS Paragon Plus Environment

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Blautia, Dialister, Faecalibacterium, Ruminococcus, Butyricimonas, and Anaerostipes

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(Figure 2). Correlation coefficients ranged from -0.3325 (in the case of Ruminococcus) to

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-0.8312 (for Butyricimonas). Therefore, HMF and furfural seem to inhibit or decrease the

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growth of these microorganisms, and accordingly reduce the prebiotic effect of MOS.

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The levels of total polyphenols in the samples collected after 10, 30 and 60

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minutes of hydrolysis were also analyzed. Experiment A showed, in average, lower

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amounts of polyphenols than the other three experiments. This could be related with the

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higher hydrolysis temperature, which could degrade some polyphenols. Experiments B, C

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and D showed similar polyphenol content in average, with no significant differences (Table

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2). However, polyphenol content increased along hydrolysis time, which could be related

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with the constant release of polyphenols as the degree of hydrolysis of the spent coffee

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grounds increases. As previously stated, polyphenols could also act as microbial inhibitors.

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In this sense, statistically significant negative correlations (p < 0.05) for Blautia,

365

Faecalibacterium, Ruminococcus and Collinsella (Figure 2) were found. Correlation

366

coefficients ranged from -0.3228 (for Collinsella) to -0.7334 (for Faecalibacterium). In this

367

sense, polyphenols released by spent coffee grounds could reduce the growth of some gut

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bacteria and therefore reduce fiber degradation and SCFA production. Blautia,

369

Faecalibacterium and Ruminococcus showed significant negative correlations with HMF,

370

furfural and total polyphenols. This could mean that these bacteria are negatively affected

371

by both kinds of inhibitors. Accordingly, as depicted in Figure 3, these bacteria seem to be

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more aggressively affected and their abundances already start decreasing with coffee

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grounds hydrolyzed for 30 minutes.

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Short chain fatty acids production 17 ACS Paragon Plus Environment


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Several health effects are attributed to SCFAs. Decreasing the luminal pH is one of

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the most obvious, which can counter the growth of pathogenic bacteria. On the other hand,

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butyrate is used as substrate by the epithelial cells, being important for their functionality

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and, overall, the three main SCFAs (acetate, propionate and butyrate) are important for the

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maintenance of the gut barrier.

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colonocytes, acetate and propionate are mostly absorbed and incorporated to different

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metabolic routes related to energy balance. Thus, these SCFAs have a protective role in

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diet-induced obesity.40 On the other hand, butyrate and propionate have been related to the

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production of gut hormones and therefore in reducing food intake.40 Moreover, it is thought

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that SCFAs could have an important role in colorectal cancer protection (mostly butyrate)

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by increasing motility, irrigation, reducing inflammation and increasing apoptosis. In fact,

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it has been suggested that the protective effect of dietary fiber over colorectal cancer

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depends upon the production of butyrate.41 Propionate along with butyrate has also been

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associated with regulation of intestinal inflammation through induction of T-regulatory cell

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differentiation.24

24

Moreover, while butyric acid is mostly metabolized by

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SCFAs production is summarized in Table 2. SCFA production showed a similar

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pattern across experiments as, generally, higher amounts of MOS led to higher SCFAs

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production though for 60 minutes of coffee grounds hydrolysis the levels of SCFAs

394

decreased (Table 2). In experiment A this makes sense since MOS production was lower at

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60 minutes. However, this was found even in experiments were MOS production increased

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until the end. The decrease in SCFAs production with coffee grounds that have undergone

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more than 30 minutes of hydrolysis could be attributed to the inhibitory effect exerted by

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HMF, furfural and/or polyphenols. As described in section 3.2.1, some SCFAs producers

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are inversely correlated with HMF, furfural and polyphenol concentrations, which could 18 ACS Paragon Plus Environment

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mean that they are negatively affected by those substances. As a consequence, SCFAs

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production is reduced. Moreover, supporting these findings, SCFAs producers

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Anaerostipes, Collinsella, Coprococcus, Faecalibacterium, Bididobacterium, Veillonella,

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Dialister, Butyricimonas, Butyricicoccus and Intestinimonas correlated positively with the

404

corresponding SCFAs (Figure 2). Therefore, this could explain, at least in part, SCFAs

405

production.

406

When comparing experiments, a higher MOS production usually translated into a

407

higher SCFAs production (Table 2). Mean acetate values over the three measured time

408

points ranged from 75.05 mol/g of spent coffee grounds for experiment A to 130.79 mol/

409

g for experiment D. Experiment A showed significantly lower production of acetate (p

Spent coffee grounds extract rich in manooligosaccharides promote a

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