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Influence of degree of roasting on the antioxidant capacity and genoprotective effect of instant coffee: Contribution of the melanoidin fraction. Raquel Del Pino-García, Maria L. González-Sanjosé, Rivero-Pérez Maria Dolores, and Pilar Muñiz J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 29 Sep 2012 Downloaded from http://pubs.acs.org on September 30, 2012

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Journal of Agricultural and Food Chemistry

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Influence of Degree of Roasting on the Antioxidant Capacity and Genoprotective

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Effect of Instant Coffee: Contribution of the Melanoidin fraction.

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Raquel Del Pino-García*, María L. González-SanJosé, María D. Rivero-Pérez and Pilar

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Muñiz.

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Department of Biotechnology and Food Science, Faculty of Sciences, University of Burgos,

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Pza Misael Bañuelos, 09001, Burgos, Spain.

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*Corresponding author: Miss Raquel del Pino García, Pza Misael Bañuelos, Facultad de

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Ciencias, Departamento de Biotecnología y Ciencia de los Alimentos, 09001, Burgos, Spain.

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E-mail: [email protected]

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Phone: +34-947258800

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Fax: +34-947258831

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ABSTRACT: Roasting process induces chemical changes in coffee beans that strongly affect

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the antioxidant activity of coffee. In this study, the polyphenol and melanoidin contents and the

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antioxidant activity of three instant coffees with different roasting degrees (light, medium,

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dark) were assessed. Coffee brews were separated into fractions and the potential biological

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activity of the melanoidins was evaluated by simulating their gastrointestinal digestion. Total

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antioxidant capacity, hydroxyl radical scavenger activity, lipid peroxidation inhibition

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capacity, and protection against DNA oxidative damage (in vitro and ex vivo–genoprotective

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effect) were determined. We report that instant coffee has a high total antioxidant capacity and

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protective effect against certain oxidative stress biomarkers (lipids and DNA), although this

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capacity decreases with the roasting degree. Our study confirms the hypothesis that several of

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the polyphenols present in coffee may become part of the melanoidins generated during

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roasting. Furthermore, the elevated genoprotective effect of melanoidin-digested fractions is

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noteworthy.

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KEYWORDS:

Antioxidant

activity;

DNA

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Polyphenols; Roasting process; Soluble coffee.

protection;

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2

Lipid

peroxidation;

MRPs;

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INTRODUCTION

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Coffee, which is one of the most widely consumed beverages in the world, is appreciated

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for its pleasant flavour and aroma as well as the stimulatory properties arising from its caffeine

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content. During the past few years, evidence of the health benefits and the important

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contribution of brewed coffee to the intake of dietary antioxidants has helped to increase coffee

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consumption 1. Several factors, including the species (Arabica or Robusta) and cultivars 2, 3, the

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origin 4, the kind of roasting process (conventional or torrefacto) 5, the degree of roasting

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and the brewing procedure 1, 9 can influence the antioxidant activity of coffee beverages.

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Therefore, the antioxidant activity of coffee is known to be strongly affected by the roasting

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process. In fact, although various phenolic compounds (i.e. chlorogenic acid) originally found

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in coffee are partially lost during this process10, the antioxidant content can be maintained by

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the formation of new antioxidants, such as the melanoidins generated via the Maillard reactions

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(MR) that take place during roasting 7, 8, 11-13. Melanoidins are brown, water-soluble, nitrogen-

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containing macromolecular compounds, that are responsible for roasted coffee's colour and

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aroma. Although their structure is extremely complex and complete knowledge about it is still

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lacking, it has been proposed that the incorporation of phenolic compounds plays an important

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role in melanoidin formation 14. It has also been suggested that part of the antioxidant activity

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of the melanoidins found in coffee could be due to low-molecular-weight compounds non-

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covalently linked to them 15.

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Although the effect of coffee roasting on the antioxidant activity of coffee brews has been

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investigated previously, these studies produced contradictory results: i) an increase in the

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antioxidant capacity of brews from medium-roasted coffee and a decrease in those from dark

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coffee 6-8; ii) an increase in the antioxidant activity of brews with degree of roasting16, 17; iii) a

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decrease of antioxidant capacity of brews with the roasting degree

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activity of coffee unaffected by the degree of roasting 21. Several factors, including the use of

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different methods to determine the antioxidant capacity 19, the type of coffee employed for the ACS Paragon Plus Environment

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3, 18-20

; iiii) antioxidant


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experiments

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roasting

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literature discrepancies. Furthermore, it must be taken into account that for instant coffee, after

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the roasting process, there is an additional thermal extraction treatment at high temperature and

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a drying process that also affect the composition and antioxidant activity of coffee.

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, the use of different time-temperature profiles to reach the same degree of

, and the lack of a standard definition of roasting degree 6, could explain these

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Despite the great economic importance of instant coffee, there have been few reports

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concerning its antioxidant potential 15, 21 as most studies in the literature refer to the antioxidant

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activity of brewing roasted coffee. The objective of this study was therefore to evaluate the

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effect of the degree of roasting of instant coffee on its antioxidant activity, as well as on the

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biomarkers of oxidative stress. In addition, this study attempts to determine the contribution of

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the different compounds found in roasted coffee on its overall antioxidant capacity. To this

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end, the coffee was separated into fractions and the potential biological activity of melanoidins

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evaluated by simulating their gastrointestinal digestion. The main contribution to new

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knowledge in the field is the elevated genoprotective effect found in the instant coffee brews

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after melanoidin digestion.

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MATERIALS AND METHODS

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Chemicals. 2,2’-Azinobis 3-ethylbenzothiazoline-6-sulfonic acid (ABTS), 2,2-diphenyl-1-

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picrylhydrazyl (DPPH), 2,2’-diazobis-(2-aminodinopropane)-dihydrochloride (ABAP), 6-

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hydroxyl-2,5,7,8-tetramethyl-2-carboxylic acid (Trolox), 2,4,6-Tris (2-pyridyl)-S-triazine

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(TPTZ), thiobarbituric acid (TBA), 2-deoxy-D-ribose, gallic acid (GA), Low-Melting agarose,

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Normal-Melting agarose, menadione, Dulbecco's modified eagle medium (DMEM), phosphate

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buffered saline (PBS), fetal calf serum (FCS), Tritton X-100, bovine serum albumin (BSA),

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pancreatin, pepsin, bile salts and calf thymus DNA were purchased from Sigma–Aldrich Co

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(St. Louis, MO, USA). Butanol, ethanol, methanol, potassium persulfate (K2O8S2), ferric(III)-

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chloride acid (FeCl3), ferrous(II)-sulphate (FeSO4), hydrogen peroxide (H2O2), L-ascorbic acid, ACS Paragon Plus Environment

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citric acid, ethylenediaminetetraacetic acid (EDTA), trichloroacetic acid (TCA) and Folin-

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Ciocalteau reagent were obtained from Panreac (Barcelona, Spain). Dimethyl sulfoxide

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(DMSO) and copper(II)-sulfate pentahydrate (SO4Cu·5H2O) were purchased from Merck

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(Darmastadt, Germany).

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Samples. To investigate the effects of roasting independently from the coffee source, three

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instant coffees produced from the same blend of coffee beans (80% Arabica and 20% Robusta)

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were supplied by the Nestlé Research Center (Lausanne, Switzerland) in three different

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roasting degrees. Green coffee beans were roasted in a pilot plant for 6 minutes at different

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levels and they were characterised by a Color-Test Neuhaus (CTn) value of 110, 85 and 60,

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corresponding to light (water loss 14.2%) medium (water loss 16.2%) and dark (water loss

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18.9%) roasted coffee, respectively. Despite differences in the roasting process, all coffees

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were given the same post-treatments in order to obtain the three final soluble powder coffees.

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Preparation of Coffee Samples (C, F, M, FMD, MD). 1.000 g of the different instant

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coffees was resuspended in 100 mL of hot milli-Q water (55 ºC) for 3 min whilst stirring

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continuously. The coffee brews (C) were then filtered and stored at 4 ºC for short time until

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analysis (C-110, C-85 and C-60 samples, respectively).

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An aliquot of each sample was subjected to ultrafiltration using an Amicon ultrafiltration-

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cell (model-8050, Amicon, Beverly, MA) equipped with a 10 kDa cutoff membrane (Millipore

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Corp., Vedford, MA, USA)

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and stored at -80 ºC until analysis. Freeze-dried filtrate samples (F) containing the low-

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molecular-weight compounds present in coffee were identified as F-110, F-85 and F-60,

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respectively. Melanoidins-containing freeze-dried retentate samples (M) were identified as M-

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110, M-85 and M-60, respectively.

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. The filtrate and retentate fractions were freeze-dried, weighed

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Simulated gastrointestinal digestion of samples containing coffee melanoidins (M) was

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performed following a slight modification of the method described by Rufián-Henares J.A. and

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Morales F.J. (2007) 15. Briefly, 0.4 g of each M sample was diluted with milli-Q water (8 mL)

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and the pH adjusted to 2.0 using 6 M HCl. A 125-μL aliquot of freshly prepared pepsin (0.16 ACS Paragon Plus Environment

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g/mL in 0.1 M HCl) was then added and the mixture incubated at 37ºC for 3 h in a shaking

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water bath. After the gastric digestion step, the pH was adjusted to 7.0 with 0.5 M NaHCO3

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and 2 mL of freshly prepared pancreatinin-bile mixture (0.004 g of pancreatinin and 0.025 g of

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bile salts in 1 mL of 0.1 M NaHCO3) was added before incubating for 1 h at 37 ºC in a shaking

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water bath. Then, enzymes were deactivated by thermal treatment at 100 ºC for 4 min in a

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water bath in order to suppress their contribution from the further studies. Finally, the digested

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solutions were ultrafiltered and freeze-dried following the same procedure as described above.

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The freeze-dried filtrate extracts containing low-molecular-weight compounds non-covalently

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linked to melanoidins were identified as FMD-110, FMD-85 and FMD-60, respectively. The

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freeze-dried retentate extracts containing digested melanoidins were identified as MD-110,

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MD-85 and MD-60, respectively.

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Determination of Total Polyphenols (TPP). The total phenol content was evaluated using 22

with GA as standard. Thus, 100 μL of diluted coffee sample

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the Folin-Ciocalteau reaction

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was added to 500 μL of Folin-Ciocalteau reagent and 400 μL of sodium carbonate solution

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(75g/L). The results were expressed in μg of GA equivalent per mg of sample (absolute

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approach) or per mg of coffee (relative approach).

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Browning measurement. The absorbance of the coffee samples at 360 and 420 nm was

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recorded using a UV-Vis spectrophotometer (U-2000 Hitachi) in order to estimate the

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intermediate and final Maillard reaction products (MRPs) content based on the browning

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associated with the formation of melanoidins 23.

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Determination of Total Antioxidant Capacity (TAC).

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ABTS method. This assay is based on the decoloration that occurs when the radical cation

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ABTS·+ is reduced to ABTS 24. The assay consisted of 980 μL of ABTS·+ solution and 20 μL

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of the sample. The absorbance at 734 nm was measured after 20 min of reaction and the results

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expressed in μg of Trolox equivalent per mg of sample (absolute approach) or per mg of coffee

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(relative approach).


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DPPH method. This method is based on reduction of the radical DPPH· 25. The reaction was

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started by adding 20 μL of each sample solution to 980 μL of DPPH· (60 μM in methanol). The

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absorbance at 517 nm was measured after 2 h and the results expressed in μg of Trolox

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equivalent per mg of sample (absolute approach) or per mg of coffee (relative approach).

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FRAP method - “Ferric Reducing/Antioxidant Power”. This method is based on the

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increase in absorbance at 593 nm due to formation of TPTZ-Fe(II) complexes in the presence

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of a reducing agent 26. The reactive mixture was prepared by mixing 25 mL of sodium acetate

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buffer solution (0.3 M, pH 3.6), 2.5 mL of TPTZ (10 mM), 2.5 mL of FeCl 3 (20 mM) and 3

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mL of milli-Q water. Then, 30 μL of each sample was added to 970 μL of the latter reactive

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mixture and incubated at 37 ºC for 30 min. The results were expressed in mM of Fe(II) per mg

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of sample (absolute approach) or per mg of coffee (relative approach).

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Determination of hydroxyl radical scavenger activity (HRSA). Deoxyribose (2-deoxy27

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D-ribose) decays when exposed to hydroxyl radicals generated by the Fenton reaction

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reaction mixture containing 1 mM deoxyribose, 5 mM phosphate buffer (pH 7.4), 0.1 mM

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ascorbic acid, 0.1 mM FeCl3, 1 mM H2O2, 0.1 mM EDTA and 100 μL of the samples, in a total

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volume of 1 mL, was incubated for 60 min at 37 ºC. The samples were then mixed with 1.5 mL

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of TCA (28% w/v) and 1 mL of TBA (1% w/v) and heated at 100ºC for 15 min. The

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absorbance was recorded at 532 nm and the results expressed as % oxidation inhibition with

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respect to a control test following an absolute (per mg of sample) or a relative approach (per

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mg of coffee).

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. The

Lipid peroxidation inhibition. The experiments were carried out in rat liver microsomal 28

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preparations

using peroxyl radicals as oxidant. The total microsomal protein content was

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determined using the Bradford method

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incubated with a solution of 10 mM ABAP in the presence of 20 μL of sample (diluted to 0.5

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mg/mL in milli-Q water). The incubation temperature was set at 37ºC for a period of 90 min.

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Lipid peroxidation was evaluated using the thiobarbituric acid reactive substances (TBARS)

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assay to quantify malondialdehyde. The absorbance measured at 532 nm was proportional to

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. The microsomal fraction (1 mg/mL of protein) was


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the quantity of peroxyl radicals generated and the results were expressed as % oxidation

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inhibition with respect to a control test following an absolute (per mg of sample) or a relative

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approach (per mg of coffee).

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DNA oxidative damage. Agarose gels. Oxidative DNA damage was measured following 30

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the method described by Rivero D. et al. (2005)

with a reaction mixture containing calf

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thymus DNA (100 μg), ascorbic acid (10 mM), copper(II) sulfate (0.1 mM) and 300 μL of the

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coffee samples (diluted to 10 mg/mL in milli-Q water) in a total volume of 500 μL. The

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mixture was incubated in a shaking water bath at 37ºC for 60 min. The resulting fragments

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were separated by electrophoresis on 1% agarose gels using an electrophoresis system (Hoefer-

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PS500XT, San Francisco, CA, USA). The gel was run at 100V, 400 mA and 100W for 45 min.

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Genoprotective effect. Comet assay. This assay, also known as single-cell gel

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electrophoresis under alkaline conditions, was performed in the cell line HT-29 (Human colon

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carcinoma cell line), according to a previously reported method

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DMEM supplemented with 10% FCS at 37 ºC and 5% CO2. After incubation with 50 μL of

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coffee samples (diluted to 1 mg/mL in DMSO) and 15 μL of catalase (100 U/mL) for 24 h at

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37ºC and 5% CO2, oxidation was induced with 50 μL of menadione (20 μM, diluted in

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DMEM). The resulting cell suspensions were collected, centrifuged, resuspended in preheated

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1% low-melting-point agarose and added to frosted microscope slides precoated with 1%

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normal-melting-point agarose, which were then immersed in cold lysing solution (2.5 M NaCl,

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100 mM EDTA, 100 mM Tris, 1% sodium-lauryl-sarcosineate, 1% Tritton X-100, 10%

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DMSO, pH 10) overnight at 4 ºC. The microscope slides were then placed in an electrophoresis

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tank and the DNA allowed to uncoil for 40 min in alkaline electrophoresis buffer (1 mM

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EDTA, 0.3 N NaOH, pH 10). Electrophoresis was conducted at 4 ºC, 25 V, 500 mA and 150

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W. The slides were subsequently neutralized with Tris buffer (0.4 M, pH 7.5) and stained with

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ethidium bromide, then observed using a fluorescent microscope and photographed with a

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digital camera. The photographs were analyzed using the CometScoreTM Freeware, Version

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1.5.2.6. software (TriTek Corp., Sumerduck, VA, USA). For each analysis, 60 individual cells ACS Paragon Plus Environment

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. Cells were grown in

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were calculated and their tail length evaluated. DNA damage was expressed as % of relative

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tail length in relation to the oxidized control ([T/C2]%) using an absolute approach (per 50 μg

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of sample).

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Statistical procedure. Statistical analysis of the data was carried out using one-way

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analysis of variance (ANOVA). The least significant difference (LSD) test was applied in order

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to determine the statistical significance between various groups. A minimum significance level

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of p < 0.05 was considered. Linear regression was used to study the possible correlations

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between the parameters studied. The Statgraphics® Centurion, Version 16.1.11.0 software

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(Statpoint Technologies, Icn., Warrenton, VA, USA) was used.

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RESULTS AND DISCUSSION

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Coffee brews (C) and derived ultrafiltrated fractions (F, M, FMD and MD) obtained from

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three instant coffees that differed in their degree of roasting (light: CTn-110, medium: CTn-85,

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dark: CTn-60) were characterised and their antioxidant activity assessed using different

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methodologies. Data from several of these assays were analyzed in two different ways,

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following an absolute or a relative approach. The absolute approach gives information

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regarding the net activity of each coffee sample, irrespective of its concentration, as the

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experiments were carried out with the same amount of each sample resuspended in milli-Q

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water and the results are expressed per mg of sample. A prior weighing of the amount of

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ultrafiltrated fractions obtained from each sample also allowed to follow a relative approach

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(results expressed per mg of coffee), which describes the contribution of each fraction to the

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overall antioxidant activity of the parent coffee. In the Table 1 it is specified the amount of

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freeze-dried fraction of each ultrafiltrated sample (F, M, FMD and MD) obtained per gram of

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coffee. For samples FMD and MD it is also indicated the recovery for this fractions expressed

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per gram of M sample. The amount in the filtrate samples (F and FMD) decreased with degree

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of roasting, whereas an increase in the amount of sample weighed with the roasting degree was ACS Paragon Plus Environment

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observed for the retentate samples (M and MD). These results are in line with those reported by

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other studies, which found that melanoidins represented around the 38-40% of the compounds

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in roasted coffee 15.

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Characterization of instant coffee samples: Total polyphenol (TPP) content and

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browning measurement. The total polyphenol (TPP) content and the browning related to the

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presence of melanoidins were determined in the coffee samples (Table 2) in order to attempt to

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elucidate the important changes that the roasting process induces in the chemical composition

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of coffee beans.

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Coffee is one of the beverages with the highest content in phenolic compounds (between

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200-550 mg per cup of coffee) 4. Indeed, they constitute approximately 16 % of total solids in

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instant coffee, although this content varies with degree of roasting 32. As shown in Table 2, the

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TPP content in C samples decreased significantly as the degree of roasting increased (a 20.7%

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decrease of μg of gallic acid (GA) equivalent per mg of sample from light- (C-110) to dark-

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roasted (C-60) coffee), thereby supporting the theory that the roasting process results in a

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decrease in the polyphenol content of coffee

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maintained for the remaining samples (F, M, FMD and MD), especially in the filtrate ones. In

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the relative approach, the decrease as a function of degree of roasting was similar in all

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samples except for the MD samples. Chlorogenic acids are the most important polyphenols in

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coffee, but the high temperatures used during the roasting process result in their lactonization

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and polymerization, either becoming new structures (some of which may participate in the

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formation of melanoidins) or leading to their degradation

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filtrate samples (F) contributed more than retentate samples (M) to the TPP of coffees.

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However, the values obtained in the absolute approach for TPP in M were higher than those

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obtained in the respective F. This could support the hypothesis proposed by several authors

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whereby phenolic compounds are incorporated into the structure of coffee melanoidins

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generated during the roasting process

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could be measured by the TPP approach.

15, 33, 34

6, 16

. In the absolute approach, this trend was

32

. In the relative approach, the

, so the compounds in the melanoidin structure


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Browning is considered a non-specific marker of MR development and has usually been 23, 35-37

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measured spectrophotometrically and expressed in absorbance units

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markers like high-performance liquid chromatography (HPLC) determination of furosine,

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hydroxymethylfurfural (HMF) and furfural, or liquid chromatography-electrospray ionization

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single quadrupole mass spectrometry (LC-ESI-MS) ) determination of acrylamide 23, as well as

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techniques like high-performance gel-permeation chromatography (HPGPC) and capillary

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zone electrophoresis (CZE) 38 have been used for the determination of the MR development or

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melanoidin content in food analysis. However, previous studies have shown that colour can be

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directly related to the concentration of melanoidins using an external standard method, as the

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relationship between browning and the formation of melanoidins is linear between a limited

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range of absorbance, both at 405 nm

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extinction coefficient of coffee melanoidins constant over the roasting process. However, in

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this study we have not calculated concentrations, but we have just focused on the brown colour

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of coffee samples as an indirect parameter of their amount of melanoidins. In the first stages of

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the MR, reducing sugars react with amino acids, giving rise to non-colour compounds which

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do not absorb in the visible spectra. The formation of these early MR compounds can be

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monitored at 280 nm, and a pool of more advanced ones (intermediate MRPs) at 360 nm. The

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progress of the reaction involves the production of high molecular weight compounds (final

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MRPs) that contains chromophore groups with a characteristic absorbance maximum at 420

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nm

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presented in Table 2. The amount of compounds formed during intermediate steps of MR was

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higher in light coffee (CTn-110) for all samples (C, F, M, FMD and MD) and decreased with

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degree of roasting. However, roasting induced an increase in the amount of final MRPs. This

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finding confirms that melanoidins are formed continuously upon the coffee roasting process

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and is in accordance with previous reports where it has been proposed that initial roasting

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process predominantly leads to the formation of intermediate molecular weight melanoidins

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when compared to high molecular weight melanoidins, while prolonged roasting especially

23

39

and 420 nm

37

. Some specific

, remaining the value of the molar

. The results of the absorbance values of the coffee samples at 360 and 420 nm are


11


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leads to the formation of high molecular weight melanoidins

. The results of this study

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also showed that, for the three instant coffees, the highest amounts of both intermediate and

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final MRPs were obtained for the MD samples while the F samples presented the lowest

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values.

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Total antioxidant capacity (TAC) of instant coffee samples. Initially we assessed the

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total antioxidant capacity (TAC) of samples using three methods - ABTS, DPPH and FRAP. It

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can be seen from Figure 1 that all samples behave similarly in these three assays, as confirmed

282

by a study of the correlations between them: ABTS / DPPH (r = 0.8388); ABTS / FRAP (r =

283

0.9311); DPPH / FRAP (r = 0.9219). All C samples presented an elevated antioxidant capacity,

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with the TAC decreasing significantly with increased degree of roasting. These findings are

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consistent with those reported in other studies involving the ABTS

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In contrast, previous results from other groups in the field, who found a higher antioxidant

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capacity for the medium-roasted coffee using the ABTS method6, 7, for the dark-roasted coffee

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using the DPPH assay

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ABTS, FRAP and DPPH methods

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antioxidant potential of coffee is mainly attributed to the presence of phenolic compounds and

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melanoidins. However, depending on the intensity of the roasting process, the contribution of

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each group of compounds to the TAC of the resulting coffee varies markedly and there are

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broad discrepancies in the results obtained by different authors concerning the effects of

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roasting on the antioxidant activity of coffee. Thus, Del Castillo M.D. et al. (2002) 7 suggested

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that, under mild roasting conditions, polyphenols are mainly responsible for the free radical

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scavenging activity of coffee brews whereas MRPs are more important for this activity in more

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strongly roasted coffees. In contrast, Delgado-Andrade C. and Morales F.J. (2005)

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that the contribution of the melanoidin fraction to the TAC of coffee was very modest, finding

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that is consistent with the study of Sacchetti G. et al. (2009) 10 and with our research. Vignoli

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J.A. et al. (2011)

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contributed equally to the antioxidant activity of soluble coffee, since the degradation of one

21

16

3, 19, 20

and FRAP assays 18.

, or similar TAC values for light- and dark-roasted coffees using 21

, differ notably from those obtained in this study. The

10

reported

found that both compounds, chlorogenic acids and melanoidins,


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was balanced by the formation of the other during the roasting process. Therefore, they

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concluded that the antioxidant activity depends more on the initial coffee composition

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(concentration of phenolic compounds, caffeine and melanoidins in the raw materials) than on

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the roasting degree. In our study, the antioxidant potential obtained for the F and M samples

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(Figure 1) and the study of correlations between TAC assays and the composition of coffee,

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where we found high positive correlations between TPP / ABTS (r = 0.9465), TPP / DPPH (r =

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0.9335) and TPP / FRAP (r = 0.9553), lead us to conclude that the decrease in the TAC of

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coffee upon roasting may be related to the contribution of the low-molecular-weight

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compounds present in coffee, as F samples always present a lower antioxidant capacity and

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TPP content as the degree of roasting increases. Moreover, F samples makes a much more

312

important contribution to the overall antioxidant activity of coffee (relative approach) than the

313

M samples, especially in light- (CTn-110) and medium-roasted (CTn-85) coffee. The

314

differences resulting from the degree of roasting in samples enriched in melanoidins (M) were

315

lower than those for the F samples. However, after the gastrointestinal digestion of M samples

316

and their separation into fractions, the results from the absolute approach showed that the

317

antioxidant capacity of those samples corresponding to the low molecular weight compounds

318

non-covalently linked to melanoidins (FMD) decreased with the degree of roasting, whereas

319

there were no significant differences for those samples enriched in purified melanoidins (MD).

320

However, in the relative approach, the contribution to the overall TAC of coffee increased with

321

degree of roasting for MD samples due to the increase in purified melanoidin content with the

322

increase in roasting degree.

323

It must be mentioned that phenolic compounds and melanoidins are not the only

324

components that affect the antioxidant activity of instant coffee or coffee brews. Non-phenolic

325

compounds as caffeine and derivatives, trigonelline or hydroxymethylfurfural (HMF) are also

326

of interest due to their antioxidant properties and present different mechanism of antioxidant

327

activity 36. The content of trigonelline and HMF in coffee is dependent on the roasting degree,

328

whereas caffeine remains stable

40

. Therefore, these compounds can influence the results of


13


Page 14 of 34

329

antioxidant activity observed, but their contribution has not been extensively discussed in this

330

study.

331

Hydroxyl radical scavenger activity (HRSA) of instant coffee samples. Hydroxyl

332

radicals are extremely reactive and may be generated under physiological conditions in the

333

human body, where they can react with proteins, unsaturated fatty acids, DNA, etc. 4. We used

334

the deoxyribose assay to detect possible scavengers of hydroxyl radicals. This assay had

335

already been employed in a study which showed that coffee was able to inhibit hydroxyl

336

radicals-induced oxidation by acting as a secondary antioxidant that prevent the generation of

337

these radicals 4. Other authors 2 studied the HRSA in green and roasted coffee and reported that

338

the dialysates (MW < 3500 Da) either from green or roasted coffee showed antiradical activity,

339

while only the retentates (MW > 3500 Da) from the roasted coffee samples were active.

340

However, to the best of our knowledge, there are no previous studies regarding the effect of the

341

degree of roasting or the gastrointestinal digestion of coffee on its HRSA. Our results, which

342

are presented in Figure 2 as % oxidation inhibition, show a decrease in the scavenger activity

343

of C samples with an increase in the degree of roasting, specifically a 17.4% decrease of

344

HRSA from CTn-110 to CTn-60. A similar behaviour was also observed for filtrate samples (F

345

and FMD). The M samples did not present significant differences between the three coffees,

346

whereas an increase of HRSA from light (CTn-110) to dark (CTn-60) coffee was obtained for

347

MD samples. It should be noted that, in contrast to the TAC results, the samples obtained after

348

the gastrointestinal digestion of M samples (FMD and MD) present high HRSA values.

349

Furthermore, the absolute approach shows that MD samples have the highest HRSA for all

350

three coffees. The contribution of FMD and MD samples to the overall HRSA of coffee

351

(relative approach) was dependent on the degree of roasting. Thus, the FMD was more

352

important in light-roasted coffee, both fractions made a similar contribution in medium-roasted

353

coffee and MD presented a higher inhibition value in dark-roasted coffee. It should also be

354

noted that a positive correlation was found between HRSA / intermediate MRPs content (r =

355

0.8107) and HRSA / final MRPs content (r = 0.5923). ACS Paragon Plus Environment

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356

Lipid peroxidation inhibition of instant coffee samples. Lipid peroxidation inhibition

357

was evaluated as a biomarker of oxidative lipid stress by determining the ability of samples to

358

scavenge peroxyl radicals generated in the chain reactions that lead to microsome oxidation.

359

As shown in Figure 3, C-110 sample presented a high lipid peroxidation inhibition of 81.9%

360

per 10 μg of coffee (with respect to a control oxidized test), inhibition that decreased from

361

light- to dark-roasted coffee until a 57.9%. This trend was maintained for the remaining

362

samples (F, M and FMD), except for the MD ones, in which no significant differences were

363

observed. F samples contributed in a higher extend than the M samples to the protection

364

against lipid oxidation (relative approach), whereas we found rather slight differences between

365

the lipid peroxidation inhibition of FMD and MD samples. This results were rather similar to

366

those obtained for the total antioxidant capacity (TAC), especially as regards the DPPH assay

367

(correlation between lipid peroxidation inhibition / DPPH, r = 0.9528). Taking into account the

368

total polyphenol, intermediate and final MRPs composition of the samples, these results agree

369

with previous papers which have shown the importance of polyphenols in peroxidation

370

inhibition 4 and the greater influence of compounds formed in the intermediate steps of the MR

371

than the final products of these reactions on the peroxyl radical scavenging capacity 7, 8.

372

Protection of instant coffee samples against DNA oxidation (in vitro and ex vivo-

373

genoprotective effect). DNA protection against oxidative damage, which may lead to single-

374

and double-strand breaks of DNA 30, was the last oxidative stress biomarker to be assessed in

375

this study. In order to evaluate the protection of DNA against oxidation, we carried out two

376

determinations - the first one in vitro (on calf thymus DNA) in agarose gels, and the other one

377

ex vivo (on cell cultures) in order to assess the genoprotective effect of coffee.

378

Hydroxyl radicals induce DNA chain-breakages, thereby generating smaller DNA

379

fragments that can be separated by electrophoresis in agarose gels. Samples may avoid DNA

380

damage as a result of their free radical scavenging properties, their copper (II) chelating ability,

381

thereby avoiding the generation of hydroxyl radicals in the presence of ascorbic acid, or direct

382

DNA protection due to the interaction between DNA and the compounds present in the ACS Paragon Plus Environment

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383

samples

. Figure 4 shows a photograph of one of the agarose gels obtained. The same

384

amount of each sample (300 μL of a 10 mg/mL dilution) was used for incubation with DNA

385

and the degree of DNA protection was determined by comparing the DNA bands obtained in

386

the gel for the different samples with those for a non-oxidized (C) and an oxidized control (O).

387

As far as the influence of the roasting degree of coffee on DNA protection is concerned, a

388

slightly higher protection of CTn-110 coffee in samples C, F and FMD was observed, whereas

389

M samples protected DNA in a similar way, and DNA protection increased with degree of

390

roasting for the MD samples. The high protection provided by retentate fractions (M and MD),

391

which was much higher than that provided by C samples and filtrates (F and especially FMD),

392

was surprising. We have been unable to find any similar assay in coffee in the literature to

393

make a comparison. However, as the HRSA assay did not provide as many differences

394

between samples, it is possible that DNA protection is due to both the ability of coffee samples

395

to scavenge hydroxyl radicals and the ability of melanoidins and other compounds with a high

396

molecular weight present in coffee to prevent hydroxyl radical generation (by acting as

397

efficient copper (II) chelating agents), as well as their direct interaction with DNA to protect

398

against oxidation.

399

Finally, we researched the ex vivo genoprotective effect of instant coffee samples in the HT-

400

29 cell line (Human colon carcinoma cell line). DNA damage was evaluated using the “comet

401

assay” or single-cell gel electrophoresis under alkaline conditions, where DNA fragments form

402

the comet's tail, as can be seen in the photographs of Figure 5. The comet assay had been

403

previously used to study the ex vivo protective effects of coffee on the DNA of human

404

lymphocytes after H2O2 oxidation, observing pronounced protective effects

405

tried to determine whether the degree of roasting or the gastrointestinal digestion of

406

melanoidins could influence the genoprotective effect of instant coffee. The results of this

407

study are presented in Figure 5 (D) and expressed as % of relative tail length with respect to

408

the oxidized control ([T/C2] %) using the absolute approach. Every sample was found to

409

protect DNA against oxidation, although a higher degree of DNA protection was found for the ACS Paragon Plus Environment

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41

. We therefore

Page 17 of 34


410

C and F samples of light-roasted coffee (CTn-110), with M samples showing no significant

411

differences in terms of degree of roasting. However, the most remarkable finding of the comet

412

assay was that the samples obtained after melanoidin digestion (FMD and MD) provided the

413

highest degree of DNA protection, thereby suggesting that digestion of the original

414

melanoidins (M) induces modifications in their structures that lead to an increased

415

genoprotective effect. After gastrointestinal digestion of M samples, FMD was found to

416

present a higher genoprotective effect than MD for CTn-110 coffee, whereas MD provided a

417

greater degree of inhibition of DNA oxidation than FMD for CTn-60 coffee. No significant

418

differences were found between FMD and MD fractions for CTn-85 coffee, although both

419

fractions presented a higher genoprotective effect than the original melanoidins of this instant

420

coffee. Therefore, the digestion of the melanoidins from light-roasted instant coffee seems to

421

result in the release of compounds from the melanoidin structure that present a higher

422

protection against oxidation of DNA in vivo than the melanoidin core. Conversely, in dark-

423

roasted coffee the melanoidin core retained a higher genoprotective effect than the bounded

424

compounds released during the digestion of the original melanoidins. A high positive

425

correlation was observed between this assay and the HRSA assay (r = 0.8218), where FMD

426

and MD fractions also presented a high activity.

427

In conclusion, instant coffee has a high total antioxidant capacity and protective effect

428

against certain oxidative stress biomarkers (lipids and DNA), although this capacity decreases

429

with the roasting degree due to the lower antioxidant activity and lesser contribution of the low

430

molecular weight compounds present in coffee as the degree of roasting increases. However,

431

melanoidin-enriched fractions contribute considerably, and in a similar manner, to the overall

432

antioxidant activity of coffee irrespective of the degree of roasting. After gastrointestinal

433

digestion of melanoidins, the compounds non-covalently linked to melanoidins in light-roasted

434

coffee are the main contributors to the antioxidant activity, whereas in dark-roasted coffee the

435

purified melanoidin fractions contribute to a greater extent. Likewise, whereas phenolic

436

compounds contribute more than melanoidins to the TAC and lipid peroxidation inhibition ACS Paragon Plus Environment

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437

ability of coffee, HRSA and DNA protection against oxidative damage are mainly determined

438

by its intermediate and final MRPs content. The main contribution to new knowledge in the

439

field is the elevated genoprotective effect found in the fractions obtained after the digestion of

440

original melanoidins present in instant coffee. In addition, our study confirms the hypothesis

441

that several of the phenolic compounds originally present in coffee may form part of the

442

melanoidins generated during the roasting process.

443


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Page 19 of 34

444


ABBREVIATIONS AND NOMENCLATURE

445 446

C, coffee brews; CTn, color-test-number; F, filtrate samples from coffee brews; FMD, filtrate

447

samples from digested melanoidins; FRAP, ferric reducing-antioxidant power; HRSA,

448

hydroxyl radical scavenger activity; M, melanoidins retentated samples from coffee brews;

449

MD, melanoidins retentated samples from digested melanoidins; MR, Maillard reactions;

450

MRPs, Maillard reaction products; TPP, total polyphenols; TAC, total antioxidant capacity.

451 452

Notes

453 454

The authors declare no competing financial interest

455


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456

Page 20 of 34

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457 458

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Muñiz, P. Inhibition of induced DNA oxidative damage by beers: Correlation with the

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content of polyphenols and melanoidins. J. Agric. Food Chem. 2005, 53, 3637-3642.

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source of antioxidant polyphenols in the Japanese population. J. Agric. Food Chem. 2009,

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57, 1253-1259.

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Smit, G. Electron spin resonance (ESR) studies on the formation of roasting-induced

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antioxidative structures in coffee brews at different degrees of roast. J. Agric. Food Chem.

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2008, 56, 4597-4604.

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activity of melanoidins from coffee brews by different antioxidant methods. J. Agric.

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Food Chem. 2005, 53, 7832-7836.

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(35) Adams, A.; Borrelli, R. C.; Fogliano, V.; De Kimpe, N. Thermal degradation studies of food melanoidins. J. Agric. Food Chem. 2005, 53, 4136-4142. (36) Yen, W. J.; Wang, B. S.; Chang, L. W.; Duh, P. D. Antioxidant properties of roasted coffee residues. J. Agric. Food Chem. 2005, 53, 2658-2663.

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(37) Brands, C. M. J.; Wedzicha, B. L.; Van Boekel, M. A. J. S. Quantification of melanoidin

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concentration in sugar - casein systems. J. Agric. Food Chem. 2002, 50, 1178-1183.

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(38) Morales, F. J. Application of capillary zone electrophoresis to the study of food and food-

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model melanoidins. Food Chem. 2002, 76, 363-369. (39) Silván, J. M.; Morales, F. J.; Saura-Calixto, F. Conceptual study on maillardized dietary fiber in coffee. J. Agric. Food Chem. 2010, 58, 12244-12249. ACS Paragon Plus Environment

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(40) Alves, R. C.; Costa, A. S. G.; Jerez, M.; Casal, S.; Sineiro, J.; Núñez, M. J.; Oliveira, B.

561

Antiradical activity, phenolics profile, and hydroxymethylfurfural in espresso coffee:

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Influence of technological factors. J. Agric. Food Chem. 2010, 58, 12221-12229.

563

(41) Bichler, J.; Cavin, C.; Simic, T.; Chakraborty, A.; Ferk, F.; Hoelzl, C.; Schulte-Hermann,

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R.; Kundi, M.; Haidinger, G.; Angelis, K.; Knasmüller, S. Coffee consumption protects

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human lymphocytes against oxidative and 3-amino-1-methyl-5H-pyrido[4,3-b]indole

566

acetate (Trp-P-2) induced DNA-damage: Results of an experimental study with human

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volunteers. Food Chem. Toxicol. 2007, 45, 1428-1436.

568 569

FUNDING

570 571

This research forms part of the project BU004A08 supported by “Consejería de Educación,

572

Junta de Castilla y León”. The PhD grant of R. Del Pino-García is funded by the PIRTU

573

programme “Consejería de Educación de la Junta de Castilla y León” and the “European Social

574

Fund”.

575


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576

FIGURE CAPTIONS

577

Figure 1. Total antioxidant capacity (TAC) determined using the ABTS (A), DPPH (B) and

578

FRAP (C) assays. The ABTS and DPPH values are expressed in μg of Trolox equivalent per

579

mg of sample (absolute approach) or per mg of coffee (relative approach). The FRAP results

580

are expressed in mM of Fe(II) per mg of sample (absolute approach) or per mg of coffee

581

(relative approach). Data expressed as mean values ± standard error (n = 3). Latin letters

582

indicate significant differences as a function of the roasting degree (CTn-110, CTn-85, CTn-

583

60). Greek letters refer to significant differences between the samples (C, F, M, FMD, MD)

584

obtained from each coffee.

585 586

Figure 2. Determination of hydroxyl radical scavenger activity (HRSA) using the deoxyribose

587

assay. The results are expressed as % oxidation inhibition with respect to a control oxidation

588

test using an absolute (per mg of sample) or a relative approach (per mg of coffee). Data

589

expressed as mean values ± standard error (n = 3). Latin letters indicate significant differences

590

as a function of the roasting degree (CTn-110, CTn-85, CTn-60). Greek letters refer to

591

significant differences between the samples (C, F, M, FMD, MD) obtained from each coffee.

592 593

Figure 3. Determination of lipid peroxidation inhibition. The results are expressed as %

594

oxidation inhibition with respect to a control oxidation test using an absolute (per 10 μg of

595

sample) or a relative approach (per 10 μg of coffee). Data expressed as mean values ± standard

596

deviation (n = 3). Latin letters indicate significant differences as a function of the roasting

597

degree (CTn-110, CTn-85, CTn-60). Greek letters refer to significant differences between the

598

samples (C, F, M, FMD, MD) obtained from each coffee.

599 600

Figure 4. Agarose gel electrophoresis separation of DNA (protection of coffee samples against

601

DNA oxidation). P: Molecular Weight Standard from 23.1 K to 125 pb; C: non-oxidized DNA

602

control; O: oxidized DNA control. ACS Paragon Plus Environment

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Page 26 of 34

603 604

Figure 5. Comet assay in HT-29 cell line. Photographs analyzed using the CometScoreTM

605

programme: A) non-oxidized control, B) oxidized control (cells treated with menadione), C)

606

cells incubated in presence of 50 μg of sample C 110 and treated with menadione. D) DNA

607

migration evaluated by the comet tail length (n = 60 cells for each sample). The results are

608

expressed as [T/C2] % = % of relative tail length with respect to the oxidized control,

609

following an absolute approach (per 50 μg of sample). Data expressed as mean values ±

610

standard error (n = 3). Latin letters indicate significant differences as a function of the roasting

611

degree (CTn-110, CTn-85, CTn-60). Greek letters refer to significant differences between the

612

samples (C, F, M, FMD, MD) obtained from each coffee.


26

Page 27 of 34


TABLES Table 1. Amount of freeze-dried fraction of each ultrafiltrated samples (F, M, FMD, MD) obtained per gram of coffee or gram of M sample. Samples

g sample / g coffee

Samples

g sample / g coffee

g sample / g M sample

F-110

0.560 ± 0.005 c

FMD-110

0.217 ± 0.005 c

0.567 ± 0.012 c

F-85

0.553 ± 0.003 b

FMD-85

0.199 ± 0.006 b

0.504 ± 0.013 b

F-60

0.545 ± 0.004 a

FMD-60

0.180 ± 0.005 a

0.446 ± 0.011 a

M-110

0.383 ± 0.003 a

MD-110

0.157 ± 0.006 a

0.409 ± 0.013 a

M-85

0.394 ± 0.003 b

MD-85

0.182 ± 0.007 b

0.461 ± 0.015 b

M-60

0.404 ± 0.004 c

MD-60

0.206 ± 0.004 c

0.510 ± 0.010 c

Data expressed as mean values ± standard error (n = 3). Latin letters indicate significant differences as a function of the degree of roasting (CTn-110, CTn-85, CTn-60) between the values obtained for each sample (F, M, FMD, MD).


27


Page 28 of 34

Table 2. Total polyphenol (TPP) content and browning measurement of coffee samples. TPP

A

A

Browning measurement

B

Samples

Absolute approach

Relative approach

(Equiv μg GA/ mg sample)

(Equiv μg GA/ mg coffee)

Absorbance 360 nm (Intermediate MRPs)

Absorbance 420 nm (Final MRPs)

C-110

222 ± 3 c / ε

222 ± 3 c / δ

0.793 ± 0.033 c / βγ

0.169 ± 0.018 a / β

C-85

203 ± 3 b / ε

203 ± 3 b / ε

0.708 ± 0.015 b / β

0.205 ± 0.009 b / γ

C-60

176 ± 4 a / ε

176 ± 4 a / ε

0.664 ± 0.011 a / γ

0.247 ± 0.013 c / γ

F-110

174 ± 3 c / γ

97.2 ± 1.9 c / γ

0.633 ± 0.014 c / α

0.086 ± 0.015 a / α

F-85

148 ± 4 b / γ

81.8 ± 2.3 b / δ

0.574 ± 0.026 b / α

0.119 ± 0.009 b / α

F-60

132 ± 2 a / γ

72.0 ± 0.9 a / δ

0.509 ± 0.019 a / α

0.143 ± 0.009 c / α

M-110

183 ± 2 b / δ

70.2 ± 0.8 b / β

0.761 ± 0.013 c / β

0.202 ± 0.011 a / γ

M-85

166 ± 5 a / δ

65.4 ± 1.9 a / γ

0.723± 0.016 b / β

0.234 ± 0.008 b / δ

M-60

159 ± 7 a / δ

64.4 ± 2.7 a / γ

0.693 ± 0.005 a / γ

0.281 ± 0.024 c / δ

FMD-110

100 ± 3 c / α

21.7 ± 0.6 c / α

0.835 ± 0.048 c / γ

0.159 ± 0.007 a / β

b/α

b/β

0.177 ± 0.009 a / β

b/α

FMD-85

85.9 ± 4.7

17.1 ± 0.9

0.689 ± 0.019

FMD-60

76.2 ± 3.1 a / α

13.7 ± 0.6 a / α

0.585 ± 0.042 a / β

0.202 ± 0.011 b / β

MD-110

120 ± 2 a / β

18.8 ± 0.4 a / α

0.987 ± 0.019 c / δ

0.396 ± 0.023 a / δ

MD-85

116 ± 5 a / β

21.1 ± 1.0 b / β

0.918 ± 0.036 b / γ

0.445 ± 0.010 b / ε

MD-60

114 ± 4 a / β

23.5 ± 0.9 c / β

0.849 ± 0.025 a / δ

0.477 ± 0.007 c / ε

TPP values are expressed in μg of gallic acid (GA) equivalent per mg of sample (absolute

approach) or per mg of coffee (relative approach).

B

Browning is expressed as the absorbance

values obtained at 360 nm and 420 nm. Data expressed as mean values ± standard error (n = 3). Latin letters indicate significant differences as a function of the degree of roasting (CTn-110, CTn-85, CTn-60) between the values obtained for each sample (F, M, FMD, MD). Greek letters refer to significant differences between the samples (C, F, M, FMD, MD) obtained from each coffee with a determined degree of roasting (CTn-110, CTn-85, CTn-60).


28

Page 29 of 34


Figure 1. (Two-column figure) ABTS·+ Assay

A

* of sample (Absolute)

350

Equiv μg Trolox/mg *

300

δ c δ b γ a

δ c ε

CTn 110

b δ a

γ

γ

c β

a

250

b

CTn 85

γ γ

CTn 60

a a

β

β β β

a

a a a

200

CTn 110 CTn 85

α

γ

c δ γ b a

150

* of coffee (Relative)

β γ γ

a a a

100

CTn 60

c α b α a α α

50

β β

α

α

a b b

c b a

0 C

F

M

FMD

MD

Samples

DPPH · Assay

B

* of sample (Absolute) CTn 110

600

Equiv μg Trolox/mg *

500

ε

ε

c

c

400

CTn 85 CTn 60

* of coffee (Relative) ksd

γ

δ

b ε a

b δ a

δ c β

γ

b

b γ a

300

CTn 110

β

ba

CTn 85

δ a

200

CTn 60

β

δ c γ b γ a

c α

α α β

γ β β

b α

a a a

a

a a a

100

β α

α α α

α

c b a

a a a

0 C

F

M

FMD

MD

Samples

FRAP Assay

C

* of sample (Absolute)

3

ε

c Equiv mM Fe(II)/mg *

2,5 2

CTn 110

δ c

CTn 85 CTn 60

ε

δ

b ε

b δ a

a

δ c

γ

CTn 110

c γ γ b a

γ

b

1,5

* of coffee (Relative)ksd

β

γ

a

c δ b δ a

1

c

γ γ

b a

CTn 60

a a a

c α β

CTn 85

β β β

α

b α a

0,5

α α

c b α a

α β β

a b c

0 C

F

M

FMD

Samples


29

MD


Page 30 of 34

Figure 2. (Two-column figure)

Absolute

Hydroxyl Radical Scavenger Activity (HRSA) Assay

CTn 110

100

CTn 85

oxidation inhibition %

90 80

β

70

c

60

ε

β

c δ b ε a

b β a

αβ

c α b α

αβ

a a a

c

β

β

a

β

CTn 60

γ

Relative

a b

CTn 110

b a

CTn 85 CTn 60

a

50

α αβ β

γ

δ γ c b δ a

40 30

γ β γ

a a a

β α

20

α

c b a

10

α

c a b

0 C

F

M

FMD

Samples


30

α β

MD

Page 31 of 34



Lipid Peroxidation Inhibition Assay

oxidation inhibition %

Absoluta

100 90 80 70 60 50 40 30 20 10 0

CTn 110

δ c

ε

c

γ

b

δ b

γ

a

CTn 85

γ β

c βγ δ

b γ

a

a

b

CTn 60

β

Relative Relativa

γ

ab a

CTn 110 CTn 85

α

δ γ c b γ a

α α β

c α γ β β α α α

β α α

c b a

C

F

M

FMD

Samples


31

CTn 60

a a a

b α a

α α α

a b c

MD


Figure 4. (One-column figure)


32

Page 32 of 34

Page 33 of 34



A

B

C

relative tail length %- [T/C2]%

D

Comet Assay

100 90 80 70 60 50 40 30 20 10 0

Negative control

Oxidized control CTn 110 CTn 85

δ

αβ a

controls

C

γδ β c b

γ b γ ab a

CTn 60

γ γ γ a a a

F

M

β α b α ab a

FMD

Samples


33

β b α α ab a

MD


Maillard reactions (MR) Melanoidins

Phenolic compounds

Roasting Antioxidant activity

Grinding & Extraction

C.

MW

Phenolic compounds

Instant coffee

or

??

C.

MW

Melanoidins Potential bioactivity ??

In vitro Gastrointestinal digestion

Genoprotective effect – Comet assay

TOC GRAPHIC A

Non-oxidized control

B

Oxidized control

C

C110 sample protection


34

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Influence of the Degree of Roasting on the ... - ACS Publications

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