(HSCG) in a simulated digestion-fermentation

Subscriber access provided by Uppsala universitetsbibliotek

Article

High antioxidant action and prebiotic activity of hydrolyzed spent coffee grounds (HSCG) in a simulated digestion-fermentation model: toward the development of a novel food supplement Lucia Panzella, Sergio Pérez-Burillo, Silvia Pastoriza, María Angeles Martín, Pierfrancesco Cerruti, Luis Goya, Sonia Ramos, José A. Rufián-Henares, Alessandra Napolitano, and Marco d'Ischia J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b02302 • Publication Date (Web): 10 Jul 2017 Downloaded from http://pubs.acs.org on July 10, 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 31

Journal of Agricultural and Food Chemistry

1 High antioxidant action and prebiotic activity of hydrolyzed spent coffee grounds (HSCG) in a simulated digestion-fermentation model: toward the development of a novel food supplement

Lucia Panzella,*,† Sergio Pérez-Burillo,∞ Silvia Pastoriza,∞ María Ángeles Martín,‡ Pierfrancesco Cerruti,§ Luis Goya,‡ Sonia Ramos,‡ José Ángel Rufián-Henares,# Alessandra Napolitano,† and Marco d’Ischia†

†

Department of Chemical Sciences, University of Naples “Federico II”, Via Cintia 4, I-80126,

Naples, Italy ∞

Departmento de Nutrición y Bromatología, Facultad de Farmacia, Universidad de Granada,

Campus Universitario de Cartuja, 18071 Granada, Spain ‡

Department of Metabolism and Nutrition, ICTAN, CSIC, José Antonio Novais 10, 28040 Madrid,

Spain §

Institute for Polymers, Composites and Biomaterials (IPCB-CNR), Via Campi Flegrei 34, I-80078

Pozzuoli, Italy #

Departamento de Nutrición y Bromatología, Instituto de Investigación Biosanitaria

ibs.GRANADA, Universidad de Granada, Granada, Spain

*Corresponding author (Tel: +39081674131; Fax: +39081674393; E-mail: [email protected])

ACS Paragon Plus Environment


Page 2 of 31

2 1

Abstract

2

Spent coffee grounds are a by-product with a large production all over the world. The aim of this

3

study was to explore the effects of a simulated digestion-fermentation treatment on hydrolyzed

4

spent coffee grounds (HSCG) and to investigate the antioxidant properties of the digestion and

5

fermentation products in human hepatocellular carcinoma HepG2 cell line. The potentially

6

bioaccessible (soluble) fractions exhibited high chemoprotective activity in HepG2 cells against

7

oxidative stress. Structural analysis of both the indigestible (insoluble) and soluble material

8

revealed partial hydrolysis and release of the lignin components in the potentially bioaccessible

9

fraction following simulated digestion-fermentation. A high prebiotic activity as determined from

10

the increase in Lactobacillus spp. and Bifidobacterium spp. as well as the production of short chain

11

fatty acids (SCFAs) following microbial fermentation of HSCG was also observed. These results

12

pave the way toward the use of HSCG as a food supplement.

13 14

Keywords: spent coffee grounds; antioxidant; reactive oxygen species; simulated digestion-

15

fermentation; HepG2 cells; short chain fatty acids

16


Page 3 of 31


3 17

Introduction

18

Coffee is by far one of the most consumed beverages in the world and the resulting spent coffee

19

grounds (SCG), because of the high content in caffeine, tannins, and polyphenols, can have harmful

20

effects on the environment, requiring proper management and disposal.1 Proposed use of SCG

21

includes production of biofuel, removal of pollutants from water and as a source of natural phenolic

22

antioxidants for use as nutritional supplements, foods, or cosmetic additives.1-9

23

SCG contain mainly carbohydrates (38–42%), proteins (8%), and chlorogenic acids (3–4%).10,11 As

24

a major outcome of the roasting process, SCG contain also melanoidins, which are usually

25

quantified to account for ca. 25% w/w of the dry weight of roasted coffee beans.12,13

26

We recently developed an expedient chemical procedure to convert SCG into an all-natural

27

biocompatible material, termed hydrolyzed spent coffee grounds (HSCG), involving an hydrolytic

28

protocol with 6 M HCl, at 100 °C, overnight. The black powder thus obtained displayed potent

29

antioxidant properties for diverse applications, including e.g. cell protection, food lipid preservation

30

and thermal and photo-oxidative stabilization of polymers.14

31

Yet, simple chemical assays are inadequate to evaluate the actual antioxidant activity of food, and

32

the importance of in vitro digestion combined with cellular assays to determine the antioxidant

33

activity has been recently emphasized.15-20

34

SCG have been found to be good sources of prebiotic compounds following in vitro digestion.12

35

The chemical characterization of potentially prebiotic oligosaccharides in SCG have been also

36

recently reported.21

37

The anti-inflammatory potential of the metabolites produced by colonic fermentation of SCG has

38

also been described, supporting the use of SGC in the food industry as dietary fiber source with

39

health benefits.12,22 However, the physiological potential and health benefits of HSCG have not yet

40

been adequately investigated.

41

We report herein the modifications induced by in vitro gastrointestinal digestion followed by a

42

fermentation step on the antioxidant activity of HSCG. The potential antioxidant and oxidative ACS Paragon Plus Environment


Page 4 of 31

4 43

stress protection of both the potentially bioaccessible digestion and fermentation products and the

44

residual, undigested fraction of HSCG were investigated by validated cellular assays using human

45

liver HepG2 cell line. This is generally held as a sensitive model for the determination of the

46

chemoprotective potential of antioxidant compounds.23 Preliminary structural investigation on both

47

the soluble, potentially bioaccesible, fractions and the residual, indigestible, solid fraction was

48

carried out. The prebiotic activity of HSCG was also determined.

49

Materials and Methods

50

Chemicals. HSCG were prepared from espresso SCG collected from a local coffee shop as

51

previously described.14 Salivary α-amylase, pepsin from porcine, bile acids (bile extract porcine),

52

tryptone, cysteine, sodium sulphide, resazurin, inulin, o-phthaldialdehyde (OPT), glutathione

53

(GSH), and 2,7-dichlorofluorescin diacetate (DCFH-DA) were from Sigma-Aldrich. Pancreatine

54

from porcine pancreas was purchased from Alpha Aesar, tert-butylhydroperoxide (t-BOOH) from

55

Panreac, Bradford reagent from BioRad.

56

General Experimental Methods. FTIR analysis was performed using a Perkin Elmer Spectrum

57

100 spectrometer in attenuated total reflectance (ATR) mode, with an average of 32 scans and

58

resolution of 4 cm−1, in the range 4000-400 cm−1. UV-vis spectra were recorded on a Agilent/HP

59

8453 spectrophotometer. NMR spectra were recorded in D2O or CD3OD on a Bruker 400 MHz

60

NMR spectrometer. HPLC analysis was performed with an instrument equipped with a UV-vis

61

detector (Agilent, G1314A); a Phenomenex Sphereclone ODS column (250 × 4.60 mm, 5 µm) was

62

used, at a flow rate of 0.7 mL/min; a 1% formic acid (solvent A)/methanol (solvent B) gradient

63

elution was performed as follows: from 5 to 90% B, 0-45 min; the detection wavelength was 254

64

nm. Short chain fatty acid (SCFAs) determination was carried out on Accela 600 HPLC (Thermo

65

Scientific) equipped with a pump, an autosampler and a UV-VIS PDA detector set at 210 nm; the

66

mobile phase used was 0.1 M phosphate buffer (pH 2.8)/acetonitrile 99:1 v/v delivered at a 1

67

mL/min flow rate; the column used was an Aquasil C18 reverse phase (Thermo Scientific) (150 ×

68

4.6 mm, 5 µm), with a total run-time of 20 min. ACS Paragon Plus Environment

Page 5 of 31


5 69

In vitro Digestion. The in vitro digestion method followed was an adaptation to the method

70

previously described,17,24 composed of an oral phase, a gastric phase and an intestinal one. Briefly,

71

for the oral phase, 5 mL of simulated salivary fluid containing α-amylase were added to 0.5 g of

72

grinded HSCG. Such mix was incubated at 37 oC for 2 min. Then, 10 mL of simulated gastric fluid

73

containing pepsin were added and the pH lowered to 3.0 by adding 1 N HCl. The mixture was

74

incubated at 37 oC for 2 h, after that 20 mL of simulated intestinal fluid containing pancreatin and

75

bile salts were added and the pH increased to 7.0 with 1 N NaOH. The mixture was then incubated

76

at 37 oC for 2 h. In order to stop the enzymatic reactions, the tube was buried in iced water,

77

centrifuged at 14000 g for 10 min at 4 oC and the supernatant stored at -80 oC until further analysis.

78

A 10% of the liquid fraction was added to the solid residue in order to mimic the fraction that is not

79

readily absorbed after digestion.25 Then, the mixed fractions were frozen for further lyophilization.

80

When required, the supernatant from the digestion mixture was lyophilized, too, and then subjected

81

to chemical extraction: in brief, 300 mg of material were dissolved in 120 mL of water, then the

82

solution was taken to pH 1 with 3 M HCl and extracted with ethyl acetate (3 × 100 mL). The

83

combined organic layers were dried over Na2SO4 and taken to dryness to give a dark yellow oily

84

residue (ca. 30 mg). For comparison ethyl acetate extraction was performed also on the supernatant

85

form a control mixture containing enzymes and other ingredients but no HSCG.

86

In vitro Fermentation. The method used was adapted from a previously described protocol.26 In

87

brief, to 300 mg of lyophilized digestion solid residue, 200 µL of distilled water were added into a

88

screw-cap tube to make up the volume up to 0.5 mL. Then, 7.5 mL of fermentation final solution

89

(peptone water + resazurine) was added. Finally, 2 mL of inoculum was added being the final

90

volume 10 mL. The inoculum consisted of a solution of 32% feces in phosphate buffer 100 mM, pH

91

7.0 (fecal content composed of a mixture of equal weight of fresh morning feces of three healthy

92

adult human donors, mean body mass index = 21.3). Nitrogen was bubbled in order to reach an

93

anaerobic atmosphere and the mixture was incubated at 37 oC for 20 h under oscillation. Right after,

94

the sample was buried in ice to stop microbial activity and centrifuged. Supernatant was collected ACS Paragon Plus Environment


Page 6 of 31

6 95

and stored at -80 oC. The solid residue was also stored for direct antioxidant activity measure. Both

96

digestion and fermentation were performed in triplicate. When required, the supernatant was

97

lyophilized and subjected to chemical extraction: in brief, 100 mg of material were dissolved in 40

98

mL of water, then the solution was taken to pH 1 with 3 M HCl and extracted with ethyl acetate (3

99

× 30 mL). The combined organic layers were dried over Na2SO4 and taken to dryness to give a dark

100

yellow oily residue (ca. 4 mg). For comparison ethyl acetate extraction was performed also on the

101

supernatant of a control mixture containing all the ingredients but no HSCG.

102

SCFAs Assay. SCFAs determination (acetic, propionic and butyric acids) was carried out by HPLC

103

as described in the General Experimental Methods. The sample did not require any pretreatment

104

before injection. Briefly, the SCFA standards were prepared in the mobile phase at concentrations

105

ranging from 5 to 10000 ppm. After the fermentation process, 1 mL of supernatant was centrifuged

106

to remove solid particles, filtered through a 0.22 µm nylon filter and finally transferred to a vial for

107

HPLC analysis. A probiotic commercial milk beverage was also analyzed for comparison.

108

Prebiotic activity. The ability of bacteria to utilize HSCG as carbon source was performed as

109

previously described.12 After the digestion-fermentation step, qRTi-PCR was performed as reported

110

previously27 to assess the growth of different bacterial strains. The QIAamp DNA Stool Mini Kit

111

(Qiagen) was used for DNA extraction, after diluting the stool contents 1:10 (w/v) in phosphate

112

buffer saline (PBS). DNA was eluted in the provided buffer, and purified DNA extracts were stored

113

at −20 °C. A series of genus-specific primer pairs were used.27 PCR amplification and detection was

114

performed in an Eco Illumina thermocycler as follows. Each reaction mixture (10 µL) was

115

composed of 5 µL of KAPA SYBR Fast Master Mix (Kapa Biosystems), 0.25 µL of each specific

116

primer (at a concentration of 10 µM) and 2 µL of template DNA. Standard curves were created

117

using serial 10-fold dilutions of bacterial DNA extracted from pure cultures with a bacterial

118

population ranging from 2 to 9 log10 CFUs, as determined by plate counts. One strain belonging to

119

each of the bacterial genera or groups targeted in this study was used to construct the standard

120

curve. More specifically, DNA was extracted from the following strains: Bifidobacterium longum ACS Paragon Plus Environment

Page 7 of 31


7 121

CECT 4551, Clostridium coccoides DSMZ 935, Bacteroides fragilis DSMZ 2151, Lactobacillus

122

salivarius CECT 2197, obtained from the Spanish Collection of Type Cultures (CECT) or the

123

German Collection of Microorganisms and Cell Cultures (DSMZ).

124

Cellular Assays. Human hepatoma HepG2 cells were maintained in a humidified incubator

125

containing 5% CO2 and 95% air at 37 ºC. They were grown in Dulbecco’s Modified Eagle

126

Medium/Nutrient Mixture F-12 (DMEM F-12) from Biowhitaker, supplemented with 2.5%

127

Biowhitaker foetal bovine serum (FBS) and 50 mg/L of each of the following antibiotics:

128

gentamicin, penicillin and streptomycin.

129

To assay direct effect, cells were incubated with doses ranging from 1 to 100 µg/mL (depending of

130

the assay) of HSCG, digested HSCG, fermented HSCG or residual fraction from fermented HSCG

131

for 20 h. To assay for a protective effect, cells were pre-treated with the noted doses (see Table 1

132

and Figures 1 and 2) of the four samples for 20 h, then the medium was removed, cells were washed

133

with PBS and 400 µM t-BOOH (dissolved in the medium) was added for 2 h, after which the cell

134

cultures were processed as detailed below for each assay.

135

Cell viability was measured by the crystal violet assay as previously reported.28 Briefly, HepG2

136

cells were seeded at low density (10000 cells per well) in 96-well plates, grown for 20 h under the

137

different conditions and incubated with crystal violet (0.2% in ethanol) for 20 min. Plates were

138

rinsed with water, allowed to dry, and 1% sodium dodecylsulfate added. The absorbance of each

139

well was measured using a microplate reader at 570 nm.

140

Cellular reactive oxygen species (ROS) were quantified by the dichlorofluorescein assay as

141

previously described.15,29 Briefly, the cells were seeded in 24-well plates (200000 cells per well) in

142

medium containing FBS, which was replaced with the FBS-free medium the next day. After 20 h, 5

143

µM DCFH-DA was added to the wells which were incubated at 37 ºC for 30 min, after that cells

144

were washed with PBS, treated with fresh FBS-free medium with the different concentrations of the

145

four HSCG samples and ROS production was monitored for 120 min. For the protection assay, cells

146

were seeded and left overnight before treating them with the HSCG samples for 20 h. Then DCFHACS Paragon Plus Environment


Page 8 of 31

8 147

DA was added for 30 min and cells were washed with PBS prior to the addition of 400 µM t-BOOH

148

to every well but controls with further incubation for 2 h. Control cells without t-BOOH treatment

149

were used as negative control. Multiwell plates were measured in a fluorescent microplate reader at

150

excitation wavelength of 485 nm and emission wavelength of 528 nm. Results are expressed as

151

percent of fluorescence units.

152

GSH content was quantitated by a fluorometric assay as previously described.15 Briefly, HepG2

153

cells were plated in 60 mm diameter plates at a concentration of 1.5 × 106 cells/plate. Cells were

154

treated with the different quantities of the samples for 20 h, collected by scraping in 1.5 mL of PBS

155

and centrifuged (1500 rpm, 4 ºC, 5 min), and lysed by adding 110 µL of 5% w/v trichloroacetic acid

156

containing 2 mM EDTA. Protein was measured by the Bradford reagent. After centrifugation of the

157

cells (7500 rpm, 4 ºC, 30 min), 50 µL of supernatant were transferred to wells in a 96-well plate.

158

Then, 15 µL of 1 M NaOH were added, followed by 175 µL of 0.1 M sodium phosphate buffer (pH

159

8.0) containing 5 mM EDTA. 10 µL per well of a 10 mg/mL methanolic solution of OPT were

160

finally added. After 15 min at room temperature in the dark, fluorescence was measured (emission

161

wavelength: 460 nm; excitation wavelength: 340 nm). The results were expressed as percent related

162

to untreated cells.

163

Statistical Analysis. Statistical significance of the data was tested by one-way analysis of variance

164

(ANOVA), followed by the Duncan test to compare the means that showed significant variation (p

165

< 0.05); all the statistical analyses were performed using Statgraphics Plus software, version 5.1.

166

For the cellular assays, prior to analysis data were tested for homogeneity of variances by the test of

167

Levene; for multiple comparisons, one-way ANOVA was followed by a Bonferroni test when

168

variances were homogeneous or by Tamhane test when variances were not homogeneous. The level

169

of significance was p < 0.05. A SPSS version 23.0 program was used.

170 171 172 ACS Paragon Plus Environment

Page 9 of 31


9 173

Results and Discussion

174

Determination of Antioxidant Properties of HSCG Following Simulated Digestion and

175

Fermentation in HepG2 cells.

176

Evaluation of the actual bioavailability of polyphenols in food and food supplements based on data

177

concerning their absorption, metabolism, tissue and organ distribution is crucial to establish their

178

effects on human body.18,30,31 Drug bioavailability depends on several factors such as administration

179

route, phenotype, age, gender, and food interaction,31 however studies carried out on animals or

180

human subjects are necessarily complex, expensive, and lengthy. This is the reason why different in

181

vitro procedures that mimic the physiochemical and biochemical conditions encountered in the

182

gastrointestinal tract have been actively developed and tested, providing preliminary data on the

183

potential bioavailability of different components of the food under evaluation.32-37 The importance

184

of in vitro digestion combined with cellular assays to determine the antioxidant activity has been

185

recently emphasized,15-20 and the HepG2 cell line is widely used to study the biotransformation and

186

the chemopreventive potential of different compounds as a model system of the human liver.23,38

187

We are aware that i) phase-II metabolism of the phenolic compounds produced after the simulated

188

gastrointestinal digestion should be duly considered since it can limit their bioavailability and

189

reduce their biological activity,39 and that ii) expression of drug-metabolizing enzymes and drug

190

transporters in transformed cell lines is often low and variable. Notwithstanding that, genotyping of

191

phase I and phase II enzymes and drug transporter polymorphisms in these cells confirmed HepG2

192

as a suitable model for metabolic studies, also because the low levels of sulfotransferase and N-

193

acetyltransferase reported in these cells are still high enough to allow metabolism.38

194

To test the biocompatibility and cytoprotective properties of both the liquid (potentially

195

bioaccessible) fractions from digestion (HSCG-dig) and fermentation (HSCG-ferm) treatment and

196

the solid residue after fermentation (HSCG-res), together with raw HSCG for comparison, doses of

197

1-20 µg/mL were considered physiological and realistic,14 but higher concentrations were also

198

tested in order to ensure that no cytotoxic effect induced by the samples was observed in the cell ACS Paragon Plus Environment


Page 10 of 31

10 199

culture. Thus, crystal violet assay (Table 1) shows that doses up to 100 µg/mL of any of the four

200

products did not affect HepG2 cell viability after 20 h.

201

Despite a slight increase in ROS observed for HSCG-dig at 1 and 5 µg/mL, no physiologically

202

relevant changes in ROS production (Figure 1A) and GSH concentration (Figure 2A) were

203

observed after plain treatment of cells with all four samples, indicating no harmful alteration of the

204

redox status. Thus, direct treatment with the HSCG products did not induce cellular stress or

205

oxidative damage which could impair cell functionality. In order to test the cytoprotective effect of

206

the products in a stressful condition, a model of oxidative stress induced by a potent pro-oxidant,

207

tert-butylhydroperoxide (t-BOOH) was established.15 In agreement with this model, 400 µM t-

208

BOOH evoked a condition of oxidative stress exhibited by decreased cell viability (Table 1) and

209

GSH (Figure 2B), as well as overproduction of ROS (Figure 1B). Interestingly, data in Table 1

210

show that pre-treatment with 1-10 µg/mL of HSCG-dig and HSCG-ferm and 5-10 µg/mL HSCG

211

completely protected HepG2 cell viability from stress-induced death, indicating a clear defense of

212

cell integrity by the spent coffee products against a stressful challenge. The antioxidant activity

213

observed for HSCG-ferm would indicate that the gut microbiota has a strong metabolic activity

214

against HSCG, releasing antioxidant compounds that could be absorbed through the colonic

215

intestinal tract.

216

The ROS-scavenging effect of HSCG has been recently reported.14 In the present study, Figure 1B

217

shows that 1-20 µg/mL of HSCG-dig, HSCG-ferm and raw HSCG significantly reduced ROS

218

overproduction evoked by t-BOOH, suggesting a quenching ability of reactive species in a cellular

219

environment, which could explain the observed reduced oxidative stress and cell protection. We

220

have previously reported a similar ROS reducing effect in this same cell line treated with coffee

221

melanoidin15 and with a green coffee bean extract or pure chlorogenic acid.40

222

Comparable to what previously reported with HSCG,14 a dose-dependent recovery of the depleted

223

GSH was observed with 5-10 µg/mL of HSCG-dig and a complete rescue was confirmed with 5-10


Page 11 of 31


11 224

µg/mL HSCG, whereas the other conditions tested showed no significant recovery of the decreased

225

GSH (Figure 2B). GSH is the main non-enzymatic cellular antioxidant attenuating oxidative stress

226

by acting as both a reducing agent and a substrate of glutathione peroxidase. Maintaining GSH

227

levels above a critical threshold is therefore a crucial issue to guarantee cell survival under

228

oxidative stress conditions.

229

The present GSH data agree with previous results obtained in the same cell line with other coffee

230

constituents,15,40 and clearly indicates that cells exposed to HSCG and HSCG-dig showed a

231

protected redox status in a situation of oxidative stress. Thus, the protective mechanism of HepG2

232

cell integrity and functionality by HSCG samples can be described in terms of regulation of the

233

cellular redox status, as a consequence of the scavenging of ROS by HSCG which would result in

234

the recovery of GSH and reduced oxidative damage and cell death.

235

Notably, in all the cellular assays performed no significant protective effect was observed for the

236

HSCG-res sample, suggesting that most part of the beneficial antioxidant activity exhibited by

237

HSCG is transferred to the soluble, potentially bioaccessible fraction. Although much of the

238

evidence for bioactivity of polyphenols evaluated on cell lines may be of little significance in vivo,41

239

HSCG-ferm containing human gut microbiota derived metabolites may be considered much more

240

relevant in this regard.

241 242

Structural Transformations of HSCG Following Simulated Digestion and Fermentation.

243

To gain an insight into the structural transformations that occur following simulated digestion-

244

fermentation of HSCG, the ATR-FTIR spectra of HSCG and HSCG-res were recorded and are

245

shown in Figure 3, together with the subtracted spectrum (HSCGS-res – HSCG). In the high

246

frequency region, HSCG and HSCG-res showed similar spectral profiles, with a broad band located

247

at 3300 cm-1 and a pattern of signals in the 2900-2800 cm-1 range. These features are due to

248

hydroxyl groups of cellulose and hemicellulose10 and to the hydrocarbon moieties of lignins,42

249

respectively. A decrease in these latter signals is observed in the subtracted spectrum, indicating ACS Paragon Plus Environment


Page 12 of 31

12 250

partial hydrolysis of lignins following the simulated digestion-fermentation, which could in part

251

account for the higher antioxidant activity observed for the soluble, potentially bioaccessible

252

fractions compared to HSCG-res. At lower wavenumbers, HSCG shows two absorption bands in

253

the carbonyl stretching region, located at 1740 and 1705 cm-1, likely due to acetyl groups of

254

hemicellulose and carboxylic acids, respectively.43 The subtracted spectrum shows a remarkable

255

reduction of these signals in HSCG-res, likely due to hydrolysis and removal of hemicellulose and

256

carboxylic acid constituting HSCG following the enzymatic treatment. No other significant

257

differences could be observed among the two samples, either in the cellulose/hemicellulose alcohol

258

C-O stretching region (bands at 1160, 1060 and 1034 cm-1)44 or in the aromatic skeleton vibrations

259

(1615 ant 1515 cm-1) and C-H bending of aromatic methoxy groups (1461 and 1110 cm-1).45 The

260

increase in absorption in the 1600-1500 cm-1 range in the HSCG-res spectrum can be attributed to

261

the amide groups of some residual enzymes used for the simulated digestion-fermentation

262

treatment.

263

To gain information about the nature of the components released from HSCG following simulated

264

digestion and fermentation, the soluble fractions HSCG-dig and HSCG-ferm were preliminarily

265

analyzed by both HPLC and 1H NMR, but no significant differences were observed compared to

266

control mixtures containing enzymes and other ingredients but no HSCG. Accordingly, to avoid

267

interferences from these components, in other experiments the HSCG-dig and the HSCG-ferm

268

samples were taken to pH 1 and repeatedly extracted with ethyl acetate. HPLC analysis with UV

269

detection at 254 nm of the organic extractable fraction from HSCG-dig showed the presence of

270

minute amounts of chromatographically defined compounds but significant quantities of organic

271

polymeric material eluted as a broad peak at the end of the gradient program. The presence of

272

conjugated, aromatic polymer was also well apparent from the UV-vis spectrum of the organic

273

extract of HSCG-dig reported in Figure 4, featuring a broadband absorption in the range 200-600

274

nm, with maxima at around 280 and 310 nm, typical of lignin moieties with hydroxycinnamic acid

275

type structures;46-48 for comparison the UV-vis spectrum of the organic extractable fraction from the ACS Paragon Plus Environment

Page 13 of 31


13 276

control, simulated digestion mixture not containing HSCG is also shown at the same concentration,

277

displaying no significant absorption above 300 nm. Unfortunately, all attempts to further

278

characterize the main extractable components of HSCG-dig by NMR failed, likely due to the great

279

chemical heterogeneity of lignin polymers.

280

Very little information could be obtained on the ethyl acetate extractable fraction of HSCG-ferm:

281

apparently no polymeric material was present in this case, as highlighed by both HPLC and UV-vis

282

analysis, suggesting efficient metabolization of the lignin moieties by the colonic microflora;

283

however, not very significant differences could be observed either in the elutoghraphic profiles or in

284

the UV-vis or NMR spectra when the extractable fraction of HSCG-ferm was compared with that

285

obtained form a control fermentation mixture not containing HSCG.

286

Prebiotic Activity of HSCG.

287

Given the nutritional composition of SCG,12 and the presence of insoluble components, these by-

288

products should have an interesting activity on the gut microbiota. Since it was found that SCG

289

increase the levels of Lactobacillus spp. and Bifidobacterium spp. after in vitro fermentation,12 we

290

decided to analyze the effect of microbial fermentation over HSCG by determination of changes in

291

microbial composition and production of SCFAs, which are known to have healthy properties such

292

as immunomodulation through their attachment to the GPR43 receptor.49 Table 2 shows the changes

293

in microbial composition after fermentation of HSCG, compared to those produced by a

294

commercial milk beverage enriched with a probiotic strain known to produce large amount of

295

SCFAs after fermentation, a positive control made of inulin (a commercial prebiotic) and a control

296

sample (fermentative medium without any nutrient). HSCG showed a clear prebiotic activity since

297

its fermentation increased the population of Bifidobacterium spp. and Lactobacillum spp.

298

(compared with the control), which are known microbial species with healthy properties. At the

299

same time, a decrease (compared with the control) was observed on Clostridium spp. and

300

Bacteroides spp., which are microorganisms related with different pathologies. The same effect was

301

also obtained for the milk beverage and inulin (probiotic and prebiotic controls, respectively). ACS Paragon Plus Environment


Page 14 of 31

14 302

However, the amount of Bifidobacterium spp. and Lactobacillum spp. was statistically higher (P

(HSCG) in a simulated digestion-fermentation

Recommend Documents