Accepted Manuscript

Application of the QUENCHER methodology to the food industry

Muriel Henrion, Mathieu Servaes, Frank Thielecke, Vincenzo Fogliano

PII: S0308-8146(17)31272-4
DOI: http://dx.doi.org/10.1016/j.foodchem.2017.07.119
Reference: FOCH 21503

To appear in: Food Chemistry

Received Date: 25 April 2017

Revised Date: 17 July 2017
Accepted Date: 24 July 2017

Please cite this article as: Henrion, M., Servaes, M., Thielecke, F., Fogliano, V., Application of the QUENCHER
methodology to the food industry, Food Chemistry (2017), doi: http://dx.doi.org/10.1016/j.foodchem.2017.07.119

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Application of the QUENCHER methodology to the food industry

Muriel Henrion a, *, Mathieu Servaes a, Frank Thielecke b, c, Vincenzo Fogliano d

a
Department of Science and Nutrition, Nestlé NPTC Beverage Center, Orbe, Switzerland
b
Cereal Partners Worldwide, I-Center, Orbe, Swizerland
c
Present address: Thielecke Consultancy, Bettenstrasse 60a, 4123 Allschwill, Switzerland
d
Food Quality & Design group Wageningen University, The Netherlands

* Corresponding author. E-mail address: muriel.henrion@rdor.nestle.com

Highlights

• Measuring antioxidant capacity without extraction is suitable for food industries

• The QUENCHER method can be optimized considering grinding and weighting steps

• Adaptation of ABTS solution for high TEAC materials prevents for dilution necessity

Abstract

The QUENCHER method is a time and cost-saving extraction-free procedure measuring in vitro

antioxidant capacity which appears highly relevant from an industrial perspective. However,

grinding and exact weighting of material may be considered as critical points and were

addressed in the present paper. Increasing sample weight at constant ABTS volume reduced

TEAC values up to 50 %. Working at higher ABTS radical concentration than recommended

furthermore increased the TEAC values by 30 %. Both weight and ABTS concentration effect

could yet be predicted using a general model built on refined wheat (adjusted R2: 0.9986). Only

cryo-milling enabled to reduce granulometry of bran-rich samples in recommended range.

Consequent size reduction increased TEAC values up to 90 %. Impact of ultrafine jet-milling did

however not systemically impact more TEAC values than cryo-milling. The proposed model

1
approach allowed taking the best advantages of QUENCHER and confirmed this method as

ideal for industrial applications.

Keywords: antioxidant capacity, whole grain, ABTS, factory monitoring, breakfast cereals

1. Introduction

Consumer’s expectations regarding nutrition and health have increasingly grown these past

decades. As a result, the offer in manufactured food products has taken a sharp turn to more

nutritionally relevant alternatives. Whole grain enrichment is amongst the key initiatives

supported by the food industry to meet these requirements. Furthermore, the regulatory frame in

Europe has greatly strengthen with the application in 2007 of the EC Regulation 1924/2006 on

Nutrition and Health Claims ensuring that “any claim made on a food’s labelling, presentation or

advertising in the European Union is clear, accurate and based on scientific evidence”. Along

with this regulation come guidelines regarding i) sufficient characterization of the

food/constituent for which the claim is made, ii) demonstration of beneficial effects and iii)

establishment of cause and effect relationship. Part of this regulation thus drives for solid,

reproducible and relevant methodologies for food characterization.

Assessment of food antioxidant capacity is a common approach in food science research to

address health potential or functional properties of foodstuffs. Antioxidant capacity as measured

in vitro is not relevant to address the redox balance in vivo (Gökmen, Serpen & Fogliano, 2009),

with the exception of what happens within the gastrointestinal tract, where “simple” quenching of

free radicals might occur inside the intestinal lumen (Vitaglione, Napolitano & Fogliano, 2008,

Nikki, 2010, Fraga, Oteiza & Galleano, 2014). However, as it encompasses additive and

synergistic effects of all antioxidant compounds in a product, measurement of total antioxidant

capacity still provides a more realistic picture than the separate effect of individual components

(Floegel, Kim, Chung, Koo & Chun, 2011). When it comes to the examination of food products,

2
antioxidant capacity testing is thus popular to build databases and calculate antioxidant dietary

intake (Wu et al., 2004, Carlsen et al., 2010). This parameter may also be of use when it comes

to comparing raw materials (Mille, Rigelhof, Marquart, Prakash & Kanter, 2000, Martínez-Tomé

et al., 2004), or giving insight on processing impact (Dewanto, Xianzhong & Rui, 2002, Zhang &

Hamauzu, 2004, Finocchiaro, et al., 2007, Han & Koh, 2011). Beyond the health beneficial

image of antioxidant compounds also lies their preservative effect during shelf-life, which is of

key importance for manufactured food products (Shahidi & Zhong, 2010). The increasing

demand of consumers for clean labelling and natural antioxidants reinforces the need for rapid

evaluation and screening of antioxidant capacity of raw materials and their sensitivity to the

oxidative damage.

During the past decades a large number of antioxidant methodologies have been developed

and/or updated. Most commonly used assays include TEAC (Miller, Rice-Evans, Davies,

Gopinathan & Milner, 1993), FRAP (Benzie & Strain, 1996), DPPH (Brand-Williams, Cuvelier &

Berset, 1995) and ORAC (Prior, Wu & Schaich, 2005). The main drawback of these popular

methodologies is that they are all extraction-dependent. This not only introduces a high bias

when comparing results from one study to another, but, does also not allow considering the

contribution of antioxidant compounds that are not soluble, being either chemically bound,

physically trapped, or part of insoluble antioxidant material (Gökmen, Serpen & Fogliano, 2009).

The QUENCHER methodology, standing for QUick, Easy, New, CHEap and Reproducible, was

the first to address this issue by proposing a direct measurement of antioxidant activity

considering also insoluble material (Serpen, Capuano, Fogliano & Gökmen, 2007). Briefly, the

method relies on the surface reaction phenomenon between solid (bound or trapped antioxidant

compounds) and liquid (soluble free radicals) materials. Reaction between a radical and an

antioxidant may thus occur at the interface when they come into contact, regardless of the

hydrophobicity of the compound of interest. This method, first based on the generation of ABTS

free radicals has been largely adopted and tested on various food matrices (Serpen, Gökmen,

3
Pellegrini & Fogliano, 2008, Açar, Gökmen, Pellegrini & Fogliano, 2009, Serpen, Gökmen &

Fogliano, 2012a, Del Pino-García, García-Lomillo, Rivero-Pérez, González-SanJosé & Muñiz.,

2015) and also on other insoluble innovative materials for various applications (Cusola, Valls,

Vidal & Roncero, 2015, Mata, Nakkala & Sadras, 2016). The procedure can be adapted to

different colored probe such as DPPH, FRAP, CUPRAC and ORAC (Amigo-Benavent, del

Castillo & Fogliano, 2010, Serpen, Gökmen & Fogliano, 2012b, Tufan, Çelik, Özyürek, Güçlü &

Apak, 2013, Kraujalis, Venskutonis, Kraujalienė & Pukalskas, 2013, Del Pino-García, García-

Lomillo, Rivero-Pérez, González-San José & Muñiz, 2015). Furthermore, the QUENCHER

method has even been evaluated in combination with a conventional procedure to evaluate the

Global Antioxidant Response (GAR) of various foods under physiological conditions, assessing

antioxidant capacity from both low molecular weight compounds potentially bioaccessible and

resulting undigested solid material (Delgado-Andrade, Conde-Aguilera, Haro, Pastoriza &

Rufián-Henares, 2010, Pastoriza, Delgado-Andrade, Haro & Rufián-Henares, 2011, Álvarez,

Pastoriza, Alonso-Olalla, Delgado-Andrade & Rufián-Henares, 2014, Arques, Pastoriza,

Delgado-Andrade, Clemente & Rufián-Henares, 2016)

Industries are seeking for reliable and quick analytical methodologies that can be easily adapted

to large scale and possible to automate. The QUENCHER procedure appears as highly relevant

from an industrial perspective for both the absence of an extraction procedure (saving time and

use of solvents) and the operational simplicity (spectrophotometric procedure easily adaptable to

any laboratory). This method is particularly relevant when dealing with cereal-based foods,

where the major part of antioxidants are bound (Serpen, Gökmen, Pellegrini & Fogliano, 2008).

In this paper the QUENCHER procedure was critically revised considering some aspects of the

procedure that could be potential drawbacks for intensive testing in cereal industry. In particular

the grinding (especially for bran-rich matrixes) and the exact weighting of material were

considered as critical points to address.

4
2. Material and Methods

2.1 Chemicals and Reagents

ABTS (2,2’-Azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium and Trolox (6-hydroxy-

2,5,7,8-tertramethyl-chroman-2-carboxylic acid) were supplied by SIGMA-ALDRICH (St-Louis,

USA). Ethanol and Methanol analytical grade and potassium peroxodisulfate were supplied by

MERCK (Darmstadt, Germany). Ultrapure water was used for all experiments and solvent

preparations (MilliQ System, Millipore, Bedford, MA)

2.2 Foods

A total of 31 different cereal raw materials entering in the composition of breakfast cereals

worldwide were analyzed. Cereals were supplied either as grains or as raw flours, as they were

used in the factories. Table 1 summarizes the material investigated in this study. Whole grain

wheat flakes and whole grain wheat extruded pellets were selected as examples of bran-rich

products challenging to mill below the 300 µm recommended particle size and were shipped

from one of the factories providing the raw materials. Finally, refined wheat and whole grain

wheat flours used as reference and/or testing material were supplied by Grand Moulins de

Cossonay (Groupe Minoterie SA, Switzerland).

2.3 Sample milling

Pre-milling of cereal grains was performed using a Retsch hammer mill equipped with a 500 µm

sieve (Ultra Centrifugal Mill ZM200). Three different fine milling options were then applied for

sample preparation. As Restch hammer mill equipped with a 200 µm sieve did not allow final

particle size below 300 µm without heating damage, it was compared with cryo-milling and jet-

milling, two alternative ultrafine milling approaches. Cryo-milling was achieved with a

Freezer/Mill 6800 (Spex SamplePrep, Stanmore, UK). Jet milling of samples was performed

5
using a lab-scale Jet mill (FrymaKoruma, Rheinfelden, Switzerland). After milling, powdered

samples were packed in sealed aluminum pouches and kept at -20 °C before analysis.

2.4 Raw material analyses

Moisture on native raw materials was measured by oven drying at 102 °C during 3 hours.

Granulometry analysis of powdered samples was performed by laser diffraction using a HELOS

device (Sympatec GmbH, Clausthal-Zellerfeld, Deutschland).

2.5 Measurement of antioxidant capacity

The QUENCHER procedure was adapted from initial protocol set-up by Serpen et al. (2007).

Stock solution of ABTS reagent was prepared by mixing 192 mg of ABTS with 33 mg of

potassium persulfate in 25 mL of water. The solution was then left to stir overnight, protected

from light. ABTS reactant was prepared daily by dilution of the appropriate amount of stock

solution in ethanol:water 50:50 to reach the target absorbance (i.e. 0.8 at 734 nm if not stated

otherwise).

Volumes and weightings recommended in the native QUENCHER protocol were doubled for

convenience reasons: a 20 mg (unless stated otherwise) portion of powdered sample (instead of

10 mg) was transferred to a Teflon tube and the reaction was started by adding 12 mL of ABTS

reagent (instead of 6 mL). Doubling weight and volumes were previously checked to give similar

final TEAC values. All samples were thoroughly vortexed, then settled in a HulaMixerTM device

(Thermo Fisher Scientific, Waltham, USA) for constant mixing during 20 minutes. Solutions were

finally centrifuged 2 min at 9200 g. Final absorbance was read on the clear supernatant at 734

nm using a Beckman DU800 spectrophotometer (Beckman Coulter International, Brea, Canada),

exactly 35 minutes after adding the ABTS radical solution. All measurements were performed on

triplicate preparations. When necessary, powdered samples were previously diluted with

6
cellulose. In such case, proper mixing was ensured using the HulaMixerTM device during 60

minutes.

Within each day of measurement and/or each new dilution of ABTS stock solution, a reference

sample (whole wheat flour cryo-milled) was run in duplicate and average TEAC value obtained

was compared with validation data obtained on the same reference. If the reference was out of

limits defined as +/- 3*SD (with SD the standard deviation obtained on the reference sample

independently analyzed 6 times under varying conditions), all measurements were repeated.

When deviation on triplicate values reached more than 5 %, measurements on the related

sample were repeated as well. Test series did not contain more than 4 test tubes to ensure

proper handling and effective measurement before the 35 minutes end point. Finally, calibration

curves were performed using liquid solutions of Trolox in ethanol: water 50:50.

2.6 Statistical analyses

Statistical analyses were all performed using R Software (Version 3.2.3). Parameter impact was

addressed through multiple way analysis of variance (ANOVA), with interactions, and tested for

significance by the Tukey HSD test. Differences were considered significant at the 95 % level, i.e.

if p < 0.05. Data modelling was performed using the lm function in R. Models used are detailed

in the Results and Discussion section.

3. Results and Discussion

3.1 Impact of sample weight and ABTS stock solution

The standard QUENCHER procedure is performed on only 10 mg of material per sample, which

might prove difficult in industrial applications, especially with electrostatic freeze-dried powders

or very dense powders. To avoid this constraint, weights and volumes were doubled. This led to

working with 12 mL of ABTS which was already a maximum limitation for regular centrifuge

tubes and for pipetting in a single step. The impact of three different weight targets (10, 20 and

7
40 mg) on the final TEAC calculated was thus studied on a refined wheat flour (chosen for its

particle size naturally comprised between 100 and 300 µm, avoiding any grinding issues). To

avoid dilution in cellulose of high TEAC-exhibiting samples, which is time-consuming and may

induce reproducibility issues (Del Pino-García et al., 2015), use of a high absorbance ABTS

solution was evaluated, so as to increase the ratio of ABTS radical concentration to antioxidant

concentration. Two working solutions of ABTS were thus prepared, one with an absorbance at

734 nm around 0.8, as recommended (Re, Pellegrini, Proteggente, Pannala, Yang & Rice-Evans,

1999) and the other one at higher absorbance (i.e. 1.5). These conditions enabled to stay in the

(0.1 – 1.0) range for final absorbance measurement at 734 nm. Although the impact of initial

ABTS concentration has been reported (Arts, Dallinga, Voss, Haenen & Bast, 2004), working

above the 1.0 absorbance value whilst staying in a linear dose-response zone is possible with

modern spectrophotometers.

Results summarized in Figure 1 showed a clear impact of sample weight and of starting ABTS

absorbance. Working below adapted recommendations (i.e. at 10 mg for 12 mL ABTS) led to

higher TEAC and working above recommendation (i.e. 40 mg for 12 mL ABTS) to lower TEAC.

Using a more concentrated ABTS solution (Starting absorbance at 1.5 at 734 nm) also led to

systematically higher TEAC values than recommended set-up. Conditions assessed here never

led to less than 30 % or more than 90 % inhibition and were thus still in a reliable working range.

Significance of the impact of target weight and initial ABTS absorbance were investigated using

a two-way ANOVA Test with interactions (n = 18) (Table 1 – supplementary material). Both

target weight and initial absorbance had a highly significant impact (p < 0.0001) on TEAC. No

interaction was however found between both effects at the 95 % confidence level (p = 0.0774),

i.e. impact of target weight followed the same trend at both initial absorbance values and vice

versa.

Increasing the sample weight from 20 to 40 mg reduced the measured TEAC of the sample by

almost 50 % (Table 2 – supplementary material). Impact of the quantity weighted was

8
previously observed on wine pomace extracts (Del Pino-García et al., 2015). Authors reported

as well a decrease in TEAC with increasing weight, yet effect depended on the reaction medium

(100 % ethanol versus a 50:50 ethanol:water) and on the type of sample (hydrophilic

antioxidants-rich matrix versus hydrophobic antioxidant-rich matrix). It should be moreover noted

that this study was performed using extremely low amounts of sample which could have affected

some of the trends observed. The impact of sample weight observed in this study is also in line

with findings from Di Benedetto et al. (2015), who observed an inflection point when plotting

the % decrease in absorbance at 734 nm with increased flour/ABTS ratio. Authors attributed this

effect to a lowering of the reaction rate constant over a given value of insoluble antioxidants per

available ABTS+• radical. This hypothesis may likely apply to the results obtained here.

Increasing the ABTS stock solution absorbance from 0.8 to 1.5 corresponded to an ABTS

concentration increase of 56 µM to 97 µM, using a molar extinction coefficient of 1.5 x 104 M-1

(Arts et al., 2004). This led to a 32 % increase in sample TEAC values (Table 2 –

supplementary material). Arts and coworkers (2004) have previously reported that an increase

in the initial concentration of ABTS+• (working in a 0 – 45 µM range) resulted in a higher ABTS+•

consumption. Authors however observed that this effect decreased at high initial concentration

of ABTS+•. ABTS concentration in this study was beyond maximum assessed by these authors

and increasing the initial amount of ABTS+• did result in lower inhibition percentages. However,

after correction using appropriate calibration curves, TEAC values were still found higher at

higher ABTS concentration.

As visible Figure 2, the calculated TEAC followed a linear trend with the log of sample weight at

both ABTS concentration tested. Furthermore, as addressed using a statistical linear modelling,

no significant difference (at 95 % threshold) existed between adjusted slopes at both starting

ABTS solutions (p = 0.087) (Table 3 – supplementary material). Thus, a general model could

be built on refined wheat data, allowing to predict TEAC from one stock solution to another,

using the amount of sample weighted (adjusted R2: 0.9986). This indicates that a TEAC value

9
obtained at high starting ABTS absorbance could be corrected back to values as obtained using

the recommended set-up. Similarly, values obtained using higher sample amounts could also be

converted back to values as obtained using the proposed 10 mg in 6 mL ABTS (or 20 mg in 12

mL). Incidentally, aside from the fact that working at higher concentration of ABTS radicals

allows direct measurement of high TEAC samples without dilution, measurement variability was

also much reduced when going from a 10 mg aliquot to a 20 mg one or a 40 mg one, as clearly

visible Figure 1 and Figure 2. Possibility to further adjust obtained values to the reference set-

up would eventually facilitate comparison between samples and building of databases. This

modelling approach, although only yet applicable within the experimental set-up of this study,

seems promising and should be investigated further.

3.2 Impact of granulometry on TEAC

The QUENCHER methodology is especially relevant on cereal-based matrices, for which the

major part of phenolic acids are chemically bound to the dietary fiber polysaccharides (Fardet,

2010). However, bran-rich materials contain hard particles that are difficult to mill below the 300

µm recommended particle size, especially without intensive sample heating and potential starch

damage. Traditional hammer milling was thus compared to alternative ultrafine milling

approaches: cryo-milling and jet-milling. Particle size impact was assessed on rice grains, whole

grain wheat flakes and whole grain wheat extruded pellets. Products were chosen as they were

easily milled with the hammer mill. Hammer milling, Cryo-milling and Jet milling allowed 99 % of

product particles to be below 500, 250 and 25 µm, respectively.

As shown in Figure 3 a clear impact of particle size was observed on all three samples. Shifting

from maximum 500 µm to maximum 250 µm (i.e. 2 fold reduction) using cryo-milling led to a

significant increase of measured TEAC from 20 to 90 %. Decreasing maximum particle size

again by a 10-fold using the jet mill led to further increase in TEAC from 14 to 40 %. These

effects are not surprising considering the increase in total surface area/volume ratio achieved

10
with a ten-fold reduction in particle size. When using jet milling instead of cryo-milling, mean

particle diameter (D[4,3]) shifted from 28.5 µm to 5.3 µm for wheat flakes and from 53.9 µm to

6.3 µm for rice samples. This reduction of more than 80 % in size however generated twice less

difference in TEAC estimation than the shift from large particles as obtained on the Restch (500

µm) to cryo-milling. It is thus likely that increase of method sensitivity when decreasing particle

size reaches a plateau beyond a certain size reduction. The wheat extruded pellets were not

further impacted by the use of jet milling as compared to cryo-milling. This may be explained by

the fact that fine particles remained stuck at the bottom of the jet mill and that the powdered

sample obtained was hence not homogeneous.

As illustrated Figure 4, jet milling enabled to obtain fine but also unimodal and monodisperse

particle size distributions, centered around 5-10 µm. Such extreme particles sizes may however

generate difficulties in handling, as they may either stick to the milling equipment (as observed

here) or be oversensitive to working environment (e.g. moisture uptake). Cryo-milling enabled

wider and bimodal distribution but the equipment was anyway much more appropriate for lab

scale than was the jet-milling device and was thus selected for the preparation of cereal

products.

3.3 Application of QUENCHER to industrial-scale testing

To illustrate relevance of the methodology for raw material testing in a factory environment, a

total of 31 different cereals from 8 different geographical sources (Table 1) were cryo-milled and

assessed for their TEAC. Raw materials were originally supplied either milled (i.e. as flours) or

as whole grains. Samples also included some cereal bran.

As shown in Figure 5, antioxidant capacity of these grains were overall in accordance with

literature (Serpen et al., 2008, Gökmen et al., 2009, Serpen & Gökmen, 2012). Some crops, as

barley, displayed lower TEAC value than reported by Serpen and coworkers (2008), i.e. 31

versus 55-60 µmol TE/g, however this range is likely to be enlarged as data is still scarce. Crop

11
to crop/geographical variability was only minor considering the data set here as TEAC values

from the 9 different whole wheat cultivars assessed only extended from 24 to 30 µmol TE/g.

Whole grain cereals displayed higher TEAC values than refined ones and the highest TEAC

were found for pure bran samples. This result also confirmed previous data (Serpen et al., 2008),

and are expected as cereal antioxidant compounds (as e.g. phenolic compounds) are known to

be concentrated in the bran section (Vitaglione, Napolitano & Fogliano, 2008).

The absence of multiple extraction steps was clearly the greatest asset of the QUENCHER

methodology. It indeed allowed screening in triplicate the 31 different cereal materials in less

than 2 days, as measuring one sample in triplicate only takes 40 minutes and as several

samples may be run in parallel. Easiness of preparation and limited equipment required easily

allows imagining regular batch monitoring in factory facilities. The use of a reference sample with

target value and upper and lower control limit also easily and quickly enabled to validate

analytical series or not. Finally, as highlighted by Del Pino-García et al. (2013), the method can

even be adapted to microplates, enabling even higher analytical throughput, although this would

add complexity to the equipment requirements.

If milling of the samples may be regarded as an issue, this could however be overcome when

considering that cereals are mostly supplied as flours, which easily adapted to the particle size

recommended originally (i.e. 100-300 µm). When it comes to supply of cereal grains, these are

generally milled in the factory, releasing either flour or cracked grains that can more easily be

milled down to recommended size with traditional laboratory milling equipment. In fact, in the

present study the milling and consequent particle size issue only arose when considering hard

raw grains as e.g. wheat grains or oat grains. Last but not least, the milling issue only concerned

bran-rich materials, i.e. whole grains.

4. Conclusions

12
The present work aimed at considering the relevance and application of the QUENCHER

methodology for industrial purposes, reviewing critical points as sample weight, necessity to

dilute high TEAC-exhibiting samples and particle size reduction. Changing the amount of sample

weighted significantly reduced TEAC values whilst working at higher ABTS radical concentration

than recommended significantly increased TEAC values. Interestingly, these effects could be

predicted using a general model built on refined wheat data (adjusted R2: 0.9986) which allowed

converting back TEAC values obtained using different sample weight and/or starting ABTS

absorbance to the recommended set-up. Although specific to refined wheat and to the

experimental set-up used in the present study, this modelling approach is promising, especially

when dealing with standard matrices, as it often happens in production.

Only cryo- and jetmilling enabled to reduce the granulometry of bran-rich samples to the particle

size range recommended and largely impacted TEAC values. Ultrafine milling is however neither

suitable for mainstream laboratory analyses nor for factory-implementable analytical

methodologies. For industrial application, the milling issue might however be less important

when considering i) that is was specific to bran-rich samples and ii) that TEAC values are usually

mostly expressed as compared to a reference, milled or treated in similar conditions,

disregarding the fact that they are optimal or not. Recent work from Di Benedetto et al. (2015)

furthermore highlights the possibility to use QUENCHER on samples of particle size between 0.2

and 0.5 mm through use of a proper calculation mode based on the slope value of the

regression line of ABTS+• response versus sample amount. This could be an interesting

alternative to evaluate hard samples resistant to milling.

The proposed model approach confirms that the QUENCHER method is very suitable for

industrial applications particularly in terms of speed and easiness of sample preparation and

thereby can replace other, more complex processes.

Acknowledgements

13
Special thanks are due to Enrico Chavez for guidance and relevant insight on statistical

treatment of the data, and to Denise Poete and Sabine Fontanella for their help on analytical

measurements. The authors would furthermore like to acknowledge Satya Jonnalagadda for

supply of the raw material and breakfast cereal samples.

14
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Caption to Tables

Table 1. Cereal grains from industrial origin analyzed for their antioxidant capacity

Caption to Figures

Figure 1. TEAC in µmol of Trolox Equivalent per g of fresh weight obtained on refined wheat

flour using a fixed volume of ABTS radical solution (12 mL) but different sample weight and

different starting absorbance for ABTS solution at 734 nm. Error bars were obtained using the

standard deviation calculated from the triplicate measurement performed under each condition.

Letters indicate significant difference at the 95 % level (Tuckey HSD)

Figure 2. TEAC of refined wheat flour as obtained using a fixed volume of ABTS radical solution

(12 mL) but different sample weight and different starting absorbance for ABTS solution at 734

nm. Data points represent the triplicate measurement performed under each condition

Figure 3. TEAC of two whole grain cereal products (wheat flakes and extruded wheat pellets)

and a raw material (rice grains) milled using either hammer milling (particles obatined below 500

µm), cryomilling(particles obatined below 250 µm) or jetmilling (particles obatined below 25 µm).

Error bars were obtained using the standard deviation calculated from the triplicate

measurement performed under each condition. Letters indicate significant difference at the 95 %

level (Tuckey HSD)

Figure 4. Particle size distribution, as measured by laser, of two whole grain cereal products

(wheat flakes and extruded wheat pellets) and a raw material (rice grains), and milled using

cryo-milling or jetmilling.

20
Figure 5. TEAC values of grain samples cryo-milled and analyzed by QUENCHER using double

weight and volumes as compared to reference procedure (20 mg sample weight in 12 mL ABTS).

Standard deviation displayed corresponds to triplicate measurements. WG : Whole Grain

21
Figures

25
ABTS initial absorbance at 0.8
ABTS initial absorbance at 1.5
d
20
a
a
TEAC (µmol TE/g)

15
b
b

10
c

0
10 20 40
Target weight (mg)

Figure 1.

22
 21

19

17
TEAC (µmol TE /g)

15

13

11

5
2 2.2 2.4 2.6 2.8 3 3.2 3.4 3.6 3.8
log(sample weight)

ABTS initial absorbance at 0.8 ABTS initial absorbance at 1.5

Figure 2.

23
 35
c < 500 um
< 250 um
30
b < 25um

25 b b
TEAC µmol TE / g product

a
a
20

15

c
10 b

5 a

0
Wheat flakes Wheat pellets Rice

Figure 3.

24
 1.4
Wheat Flakes_Cryomill
Wheat Flakes_Jet mill

1.2 Wheat pellets_Cryomill

Wheat pellet_Jet mill
Rice_Cryomill
1 Rice_Jet mill
Density distribution (log)

0.8

0.6

0.4

0.2

0
1 10 100 1000
Particle size (um)

Figure 4.

25
 Corn Bran US
Rice Bran US
WG Barley UK
WG Barley US
Brown Rice UK
Brown Rice AU
WG Rice US
WG Oat UK
WG Oat US
WG Oat AU
WG Corn US
WG Corn ME
WG Corn UK
WG Corn US
WG CornUS
WG Corn AU
WG Wheat UK
WG Wheat US
WG Wheat UK
WG Wheat US
WG Wheat US
WG Wheat UK
WG Wheat AU
WG Wheat FR
WG Wheat RU
Corn ME
Corn US
Rice US
Rice RU
Rice US
Rice FR
0 10 20 30 40 50 60 70
TEAC (µmol TE /g as is basis)

Figure 5.

26
Tables

Table 1.
Cereal Ingredient as labeled
Product application Country origin
type (WG: Whole Grain)
WG wheat Nesquik, Cheerios Australia (AU)
WG wheat Fitness France (FR)
WG wheat Nesquik France (FR)
WG wheat Fitness Russia (RU)
Wheat
WG wheat Shreddies, Shredded Wheat United Kingdom (UK)
(n=9)
WG wheat Cheerios United Kingdom (UK)
WG wheat Total USA (US)
WG wheat Multi Grain Cheerios USA (US)
WG white wheat Cinnamon Toast Crunch USA (US)
Corn Nesquik Mexico (ME)
Corn Honey Kix United States (US)
WG corn Cheerios, Nesquik Australia (AU)
WG corn Nesquik Mexico (ME)
Corn
WG corn Cheerios United Kingdom (UK)
(n=9
WG corn Honey Kix, Multi Grain Cheerios United States (US)
WG corn Honey Kix, United States (US)
WG corn Honey Kix, Multi Grain Cheerios United States (US)
Corn bran Honey Kix , Multi Grain Cheerios United States (US)
Rice Fitness France (FR)
Rice Fitness Russia (RU)
Rice Cinnamon Toast USA (US)
Rice Rice Rice Chex USA (US)
(n=8) WG Rice Multi Grain Cheerios USA (US)
Brown rice flour (WG) Cheerios Australia (AU)
Brown rice flour (WG) Cheerios United Kingdom (UK)
Rice bran Rice Chex USA (US)
WG oat Cheerios Australia (AU)
Oat
WG oat Cheerios United Kingdom (UK)
(n=3)
WG oat Multi Grain Cheerios, Cheerios USA (US)
Barley WG barley Cheerios United Kingdom (UK)
(n=2) WG barley Multi Grain Cheerios USA (US)

27