The Journal of Nutrition

Nutritional Immunology

Bioprocessing of Wheat Bran in Whole WheatBread Increases the Bioavailability of PhenolicAcids in Men and Exerts AntiinflammatoryEffects ex Vivo1–3

Nuria Mateo Anson,4,5* Anna-Marja Aura,6 Emilia Selinheimo,6 Ismo Mattila,6 Kaisa Poutanen,6

Robin van den Berg,7 Robert Havenaar,5 Aalt Bast,4 and Guido R. M. M. Haenen4

4Maastricht University, 6200 MD Maastricht, The Netherlands; 5TNO Quality of Life, 3700 AJ Zeist, The Netherlands; 6VTT

Biotechnology, Technical Research Centre of Finland, Espoo, FI-02044 VTT, Finland; and 7Unilever Research Vlaardingen, NL-3130 AC

Vlaardingen, The Netherlands

Abstract

Whole grain consumption has been linked to a lower risk of metabolic syndrome, which is normally associated with a low-

grade chronic inflammation. The benefits of whole grain are in part related to the inclusion of the bran, rich in phenolic acids

and fiber. However, the phenols are poorly bioaccessible from the cereal matrix. The aim of the present study was to

investigate the effect of bioprocessing of the bran in whole wheat bread on the bioavailability of phenolic acids, the

postprandial plasma antioxidant capacity, and ex vivo antiinflammatory properties. After consumption of a low phenolic

acid diet for 3 d and overnight fasting, 8 healthymen consumed 300 g of whole wheat bread containing native bran (control

bread) or bioprocessed bran (bioprocessed bread) in a cross-over design. Urine and blood samples were collected for 24 h

to analyze the phenolic acids and metabolites. Trolox equivalent antioxidant capacity was measured in plasma. Cytokines

weremeasured in blood after ex vivo stimulation with LPS. The bioavailabilities of ferulic acid, vanillic acid, sinapic acid, and

3,4-dimethoxybenzoic acid from the bioprocessed bread were 2- to 3-fold those from the control bread. Phenylpropionic

acid and 3-hydroxyphenylpropionic acid were the main colonic metabolites of the nonbioaccessible phenols. The ratios of

pro-:antiinflammatory cytokines were significantly lower in LPS-stimulated blood after the consumption of the bioprocessed

bread. In conclusion, bioprocessing can remarkably increase the bioavailability of phenolic acids and their circulating

metabolites, compounds which have immunomodulatory effects ex vivo. J. Nutr. 141: 137–143, 2011.

Introduction

High whole grain consumption has been inversely associatedwith the risk for developing some diet-related disorders such astype 2 diabetes, cardiovascular events, and obesity, disordersthat are commonly referred to as metabolic syndrome (1).

Hyperglycemic and pro-oxidative conditions observed inthose metabolic disorders may promote the excessive productionof reactive oxygen species and advanced glycation end products,leading to tissue damage and malfunction, the main endogenousinducers of inflammation (2). The main mediators of the

inflammatory process are the cytokines, which act in local andintercellular communications required in the integrated immuneresponse. Numerous cytokines have been identified, but it is the“balance” between pro-inflammatory cytokines (IL-1b, TNFa,IL-6, IL-2, IL-8, and IFNg), and antiinflammatory cytokines(IL-10, IL-4, and TGFb) that is thought to be a determinant inthe outcome of disease (3).

Whole grain foods tend to have a low glycemic index,resulting in lower postprandial glucose responses and insulindemand compared with refined cereal products. Whereas thelow glycemic index of whole grain is a generally recognizedhealth benefit, the role of phytochemicals present in whole grainis still under debate (1). A high consumption of phytochemicalshas been recently associated with low adiposity and oxidativestress in obese young adults (4). Several of the phytochemicals inwhole grain have been reported to exert antioxidant andantiinflammatory effects, such as some phenolic compoundsreviewed elsewhere (5–8). Among the phenolic compoundsfound in wheat grain, ferulic acid is the most abundant and isstrongly correlated with the antioxidant capacity of differentwheat fractions (9). The outermost part of the grain, the bran, is

1 Supported by the European Commission in the Communities 6th Framework

Programme, Project HEALTHGRAIN (FOOD-CT-2005-514008). It reflects the

author’s views and the Community is not liable for any use that may be made of

the information contained in this publication.2 Author disclosures: N. Mateo Anson, A-M. Aura, E. Selinheimo, I. Mattila, K.

Poutanen, R. v. d. Berg, R. Havenaar, A. Bast, and G. R. M. M. Haenen, no

conflicts of interest.3 Supplemental Table 1 is available with the online posting of this paper at jn.

nutrition.org.

* To whom correspondence should be addressed. E-mail: Nuria.mateoanson@

farmaco.unimaas.nl.

ã 2011 American Society for Nutrition.

Manuscript received June 17, 2010. Initial review completed July 19, 2010. Revision accepted October 23, 2010. 137First published online November 24, 2010; doi:10.3945/jn.110.127720.

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0.DC1.html http://jn.nutrition.org/content/suppl/2010/12/20/jn.110.12772Supplemental Material can be found at:

rich in ferulic acid; however, its bioaccessibility or intestinalrelease from that matrix is very low. The low bioaccessibility isexplained by the structural position of most of the ferulic acid,which is covalently bound to the indigestible polysaccharides ofthe cell walls constituting the fiber (10). Innovative processingtechniques, such as bioprocessing, have been developed to increasethe bioaccessibility of phenolic compounds from wheat bran (11).

In this study, we investigated the effect of bioprocessing ofbran in whole wheat bread on the bioavailability of phenoliccompounds, plasma antioxidant capacity, and antiinflammatorypotential.

Materials and Methods

Chemicals. Benzoic acid, 3-hydroxybenzoic acid, 3,4-dimethoxybenzoic

acid, 3-(4-hydroxyphenyl)propionic acid, 3-(3,4-dihydroxyphenyl)pro-

pionic acid, 3,4-dihydroxytoluene, and 3-coumaric acid were productsfrom Aldrich. Hippuric acid, 4-hydroxybenzoic acid, 2-(3-hydroxyphenyl)

acetic acid, and 2-(3,4-dihydroxyphenyl)acetic acid were purchased from

Sigma. Heptadecanoic acid, vanillic acid, sinapic acid, 3-phenylpropionicacid, and 3,4-dihydroxybenzoic acid were from Fluka. Ferulic acid and

4-coumaric acid were provided from Extrasynthese. 3-(3-Hydroxyphenyl)

propionic acid was purchased from Alfa Aesar. 2,2,2-Trifluoro-N-methyl-

N-trimethylsilyl-acetamide was from Pierce. All chemicals were of ana-lytical grade.

Participants. Eight healthy men were recruited and enrolled in the study.

They were between 21 and 55 y old and had a BMI between 20 and 30.They did not smoke or use any medication. Blood donation 3 mo before

the start of the study, consumption of $3 glasses/d of alcohol, vegetarian

lifestyles, or allergies to food components were exclusion criteria in the

recruitment. The participants were informed of the purposes and risks ofthe study, and written informed consent was obtained. The study was

approved by theMedical Ethical Commission of theMaastricht Academic

Hospital and Maastricht University (reference MEC 08–3-079).

Bread supplementation. The 2 types of bread given in the intervention

were control bread or bioprocessed bread. They consisted of 82%whole

wheat flour, 16% bran, 1% yeast, and 1% salt. The ingredients weresupplied by Buhler. The 2 breads had similar concentrations of bran,

total dietary fiber, and general macronutrients. In the control bread, the

bran was native. In the bioprocessed bread, the bran was bioprocessed by

yeast fermentation (Baker’s Yeast, Finnish Yeast Ltd.) combined withenzyme treatment, consisting of cell-wall-degrading enzymes, mainly

xylanase, cellulose, b-glucanase, and feruloyl-esterase, for 20 h at 208C.Further details on the bioprocessing technique, bread making, andmacronutrient analysis can be found elsewhere (11). The concentrations

of phenolic acids in the breads (Table 1) were measured by HPLC and

diode array as previously described (11).

Study design. The design was a blind cross-over study, with random-

ization of the participants in the 2 periods and treatments. Between the 2

periods there was a washout period of at least 1 wk. The participants

were asked to avoid the consumption of phenol-rich foods for 3 d beforethe intervention day. Whole grain products, fruit and fruit-containing

products, vegetables, nuts and seeds, chocolate, wine, tea, and coffee

were excluded from the diet. The participants received a standardized

meal, low in phenolic compounds, consisting of wheat noodles theevening prior to the intervention day. After overnight fasting, the first

blood sample was taken in the morning for the baseline measurements

(time 0 h). Directly after that, between 0830 and 0900 h, the participants

consumed 300 g of control bread or 300 g of bioprocessed bread. Uponthe consumption of the bread, blood was taken after 0.25, 0.5, 0.75, 1,

1.25, 1.5, 2, 3, 4, 5, 6, 9, 12, and 24 h. During the intervention day, food

and drinks were restricted to water and a standardized meal in the

evening, which was again the noodle meal. Urine was collected duringthe 24 h as a 0- to 24-h sample. Urinary collectors of 2 L capacity

containing 1 g sodium ascorbate were provided for the 0- to 12-h period

and for the 12- to 24-h period. The urine was pooled and aliquots werestored at 2808C until analysis.

Determination of phenolic acids. Urine and plasma sample prepara-

tion was performed based on a previous method with modifications (12).The main modifications were in the hydrolysis conditions. The hydrol-

ysis solution contained b-glucuronidase (.3000 U) and sulfatase

(.100 U) from Helix pomatia (Sigma-Aldrich) in 0.15 mol/L acetate

buffer, pH = 4.1, and the incubation was for 16 h at 378C. The internalstandard added to the plasma samples was 2-coumaric acid and to the

urine samples was heptadecanoic acid. After extraction, the samples

were derivatized with 25 mL of MOX (458C, 1 h) and 25 mL of 2,2,2-trifluoro-N-methyl-N-trimethylsilyl-acetamide (458C, 1 h) before the

injection in the GC 3 GC-time-of-flight/MS, which is a 2-dimensional

GC coupled to a time-of-flight mass spectrometer as described previously

(13). For the pharmacokinetic analysis, the integral approximation of thetrapezoidal method was used to calculate the area under the curve

(AUC0-t)8 of the compound in plasma from its concentration over time

(0–24 h).

Determination of antioxidant capacity in plasma. Plasma was

deproteinated with the addition of 10% trichloroacetic acid to the plasma

in a 1:1 ratio. Trolox equivalent antioxidant capacity was determined in

the deproteinated plasma, as previously described (14). The concentrationof uric acid was determined by HPLC and UV detector (15).

Ex vivo induced inflammatory response. Blood samples drawnbefore the bread ingestion (0 h or baseline) and at 1.25, 6, and 12 h after

bread ingestionwere incubated in triplicate with LPS fromEscherichia coli(purchased from Sigma Aldrich) in a final concentration of 1 mg/L for 24 h

at 378C and 5%CO2 as in a previous method (16). Cytokines (IL-10, IL-6,TNFa, IL-1b, IFNg, and IL-8) were determined in the supernatant with

kits fromMillipore BV following the instructions of the manufacturer and

with Luminex XMAP Technology. IL-8 was above the highest limit of

quantification (.20 mg/L). For each participant, the cytokine determina-tion (mean of triplicate determinations) was relative to the baseline value,

i.e. LPS-stimulated blood that was drawn at time 0 h or before the bread

ingestion. Thus, differences from baseline values are observed when thevalues were,1 or.1. The ratios of pro-:antiinflammatory cytokines were

calculated by dividing each TNFa, IL-6, and IL-1b value by the IL-10

values at each time.

Statistical analysis. The sample size (n) or number of participants

needed for the study to detect an effect on the primary outcome, ferulic

acid bioavailability, was calculated as indicated elsewhere for crossover

designs (17). The parameters in the formula were a = 0.05 (2-tailed), b =0.20, and an anticipated difference between treatments (D) equal to the

expected SD of that difference (sd). A post hoc power analysis indicated

that for the primary outcome, the sample size of the study was adequate,and also for the secondary outcomes: vanillic acid, sinapic acid, 3,4-

dimethoxybenzoic acid, and 4-hydroxyphenylpropionic acid AUC0-t (if

applicable) and urinary excretions, and IL-6:IL-10 and IL-1b:IL-10

TABLE 1 Phenolic acid contents in a 300-g serving of bread1

Control bread Bioprocessed bread

Total Free Total Free

mg/300 g serving

Ferulic acid 230 6.5 220 28

Sinapic acid 17 0.89 17 2.6

4-Coumaric acid 5.4 0.25 4.4 0.35

Vanillic acid 4.9 1.6 5.3 2.3

1 Values are means, n = 2, CV , 5%.

8 Abbreviations used: AUC, area under the curve; Cmax, maximum plasma

concentration; 3-OHPP, 3-hydroxyphenylpropionic acid; PP, phenylpropionic

acid; tmax, time to maximum plasma concentration.

138 Anson et al.

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cytokine ratios. To detect significant differences in the other measured

outcomes, a larger sample size is needed. The study of secondary

outcomes is merely exploratory and no adjustments for multiplehypothesis testing have been made. Assumptions of normal distribution

(Kolmogorov-Smirnov test) and homogeneity of the variance (Levene

test) could not be made for all the data, partly due to the limited sample

size (n = 8). Accordingly, a distribution-free method, the Wilcoxon’ssigned-rank test, was used to detect the significant difference between

treatments (bioprocessed bread vs. control bread) within the subject.

Spearman’s rho was selected to assess the significance of correlation and

correlation coefficient rs between variables. The data are expressed inmedians and the variation of the data are given as the IQR (difference

between the 25th and 75th quartile). The data of the ex vivo inflam-

matory response were analyzed using a linear mixed-effects model forlongitudinal data. Each outcome was analyzed as a separate dependent

variable. The fixed factors of the model were: period (1,2), treatment (bread

type), time after ingestion (1.25, 6, 12 h), and treatment 3 time inter-

action. The random factor was the subject. The estimated marginalmeans were compared with assess the main difference between the

treatments and the 95%CI. The residuals were normally distributed with

homogeneity of variance between treatments. The data are expressed as

means and SD of the mean. All the values were rounded to 2 significantdigits. The software used for the analyses was SPSS 17.0 for Windows.

Results

Pharmacokinetics. The following phenolic compounds weredetected and quantified in blood plasma: ferulic acid, vanillicacid, 3-hydroxyphenylpropionic acid (3-OHPP), phenylpro-pionic acid (PP), 3,4-dihydroxybenzoic acid, 3,4-dimethoxybenzoicacid, 3-hydroxybenzoic acid, 4-hydroxybenzoic acid, benzoicacid, hippuric acid, and 3,4-dihydroxytoluene.

The relative bioavailability, i.e. AUC0-t of the compoundfrom the bioprocessed bread relative to that from the controlbread, was 2.7 for ferulic acid, 1.8 for vanillic acid, and 1.8 for3,4-dimethoxybenzoic acid (Table 2). There were no significantdifferences in the time to maximum plasma concentrations(tmax) of these compounds between the breads. The maximumplasma concentrations (Cmax) of ferulic acid, vanillic acid, and3,4-dimethoxybenzoic acid were significantly higher after theingestion of bioprocessed bread than after the ingestion ofcontrol bread (Table 2). Ferulic acid was the phenolic acid withthe highest Cmax and the largest difference from the baseline andthe control. The Cmax and AUC0-t of ferulic acid after theingestion of bioprocessed bread was 3-fold that of the controlbread (Fig. 1). Plasma concentrations of 3-OHPP and PP rosefrom baseline values later than 6 h after ingestion (Fig. 2). Cmax

and tmax could not be accurately determined due to the relativelylate increase in plasma concentration and lack of data duringnight time when blood was not collected.

At baseline, plasma concentrations of hippuric [2.8 mmol/L(range = 0.29–5.9 mmol/L)] and benzoic [1.5 mmol/L (0.59–2.6mmol/L)] acids were high and they had irregular concentration-

time curves after bread intake. Irregular profiles, such asfluctuations from baseline with no clear increase followingthe bread ingestion, also occurred for 3-hydroxybenzoic acid,4-hydroxybenzoic acid, 3,4-dihydroxybenzoic acid, and 3,4-dihydroxytoluene (Supplemental Table 1).

Excretion of phenolic compounds. Among the compoundsidentified in urine, hippuric acid was present in the highestamount. Urine hippuric acid excretion did not differ afterconsumption of the 2 breads. The second highest phenoliccompound found in urine was ferulic acid. The amounts of fe-rulic acid, sinapic acid, vanillic acid, and 3,4-dimethoxybenzoicacid found in urine after the ingestion of bioprocessed breadwere approximately double of those found after the ingestion ofcontrol bread (Table 3). Excretions of other compounds werelower after the consumption of bioprocessed bread, including3-coumaric acid (30%), 4-coumaric acid (20%), 4-hydroxyphe-nylpropionic acid (40%), and 4-hydroxybenzoic acid (50%)(Table 3). These compounds were low in urine and notdetectable in plasma or fluctuating around baseline (Supple-mental Table 1). 4-Hydroxyphenylpropionic acid and 4-hydroxybenzoic acid were minor metabolites. Their rate offormation may have been lowered by the lower availability oftheir precursors, because less reached the colon or because theseprecursors might have been absorbed earlier and directed tohepatic metabolism.

Relative to the intake (Table 1), 10% of the ferulic acidcontent of the bioprocessed bread was excreted in 24 h urine,whereas this was only 4% in the case of the control bread. Theamount of sinapic acid in 24 h urine was 15 and 7% of the intakein the bioprocessed bread and control bread, respectively. For4-coumaric acid, this was 2% of the intake for both thebioprocessed and the control bread. The amount of vanillic acidexcreted in 24-h urine was 160 and 104% of the intake in thebioprocessed bread and the control bread, respectively.

Plasma total antioxidant capacity. There was a correlationbetween the changes from baseline in the ferulic acid plasmaconcentration over time and the changes in antioxidant capacity(rs = 0.25; P , 0.01). Also, changes from baseline in plasmaantioxidant capacity correlated with changes from baseline inuric acid concentration (rs = 0.47; P , 0.01).

Antiinflammatory effects. Blood drawn from the participantwas subjected to an ex vivo LPS-challenge to simulate aninflammatory response. Expression of IFNg by LPS stimulationwas very low [110 ng/L (range = 7.7–660 ng/L)] compared withthe other cytokines; IL-6 [11 mg/L (0.63–23 mg/L)], IL-1b[1.6 mg/L (0.39–6.9 mg/L)], IL-10 [1.2 mg/L (0.30–3.1 mg/L)],and TNFa [1.2 mg/L (0.32–3.3 mg/L)]. A linear mixed modelwas used to determine the effects of treatment (control bread,

TABLE 2 Pharmacokinetics of the main phenolic acids identified in plasma after the ingestion of the control bread or the bioprocessedbread in men1

AUC0-t Cmax tmax

Control Bioprocessed Control Bioprocessed Control Bioprocessed

mmol�min/L mmol/L min

Ferulic acid 240 (110) 640 (230)* 0.88 (0.15) 2.7 (0.63)* 90 (38) 100 (56)

Vanillic acid 39 (18) 70 (35)* 0.11 (0.03) 0.25 (0.18)* 100 (45) 120 (41)

3,4-Dimethoxybenzoic acid 5.4 (5.3) 9.9 (5.9)* 0.014 (0.04) 0.026 (0.02)* 150 (90) 120 (30)

1 Values are medians and IQR, n = 8. Baseline data were subtracted. *Different from control, P , 0.005 (Wilcoxon’s signed-rank test).

Bioavailability of phenolic acids from bread 139

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bioprocessed bread), time (1.25, 6, and 12 h after breadingestion), and treatment over time (treatment 3 time interac-tion) on each of the outcome variables; IL-10, TNFa:IL-10,IL-6:IL-10, and IL-1b:IL-10. There was an effect of the breadtype on the IL-6:IL-10 (P = 0.015) and IL-1b:IL-10 cytokineratios (P = 0.010). The effect of time after bread ingestion wasalso significant for IL-6:IL-10 (P = 0.021) and IL-1b:IL-10 (P ,0.001). There were no significant effects for the factors of periodor treatment 3 time interaction (Fig. 3).

Discussion

The technique of bioprocessing has been applied to bran for theenrichment of whole wheat bread. The aim of the present studywas to investigate the effect of bioprocessing on the bioavail-ability of phenolic compounds, the postprandial plasma anti-oxidant capacity, and the ex vivo inflammatory response afterthe consumption of whole wheat bread.

The bioavailabilities of ferulic, vanillic, sinapic, and 3,4-dimethoxybenzoic acids were higher from the whole wheatbread with bioprocessed bran than from the control bread,which was also a whole wheat bread with the same content inbran but native. The AUC0-t and Cmax of ferulic acid after theingestion of bioprocessed bread were ~3 fold of those after thecontrol bread.

The Cmax of ferulic acid was 10-fold and 100-fold of the Cmax

of vanillic acid and 3,4-dimethoxybenzoic acid, respectively(Fig. 2). Vanillic acid and 3,4-dimethoxybenzoic acid may be tosome extent metabolites of ferulic acid from b-oxidation andmethylation reactions. This is supported by their structureresemblance (Fig. 4) and by the results in Table 3 that show anexcretion of vanillic acid (30–49 mmol) superior to its intake,which was ~5 mg (30 mmol) (Table 1). The metabolism of ferulicacid to vanillic acid and some other metabolic conversionsproposed in Figure 4 are in line with the findings of animal andhuman studies reviewed elsewhere (18).

The tmax of ferulic acid (90–105 min) after the ingestion ofcontrol or bioprocessed bread was within the range of 1–3 hreported after consumption of a high-bran breakfast cereal (12).The early tmax indicates that the absorption of ferulic acidmainly takes place in small intestine, despite the large propor-tion of ferulic acid that reaches colon bound to fiber (90%).Instead of a second phase in the absorption of ferulic acid fromthe colonic release, other compounds were detected to increase

in plasma at late time points after ingestion (6–24 h). The mainmetabolites that increased after 6 h posterior to the breadingestion were 3-OHPP and PP. The time course of their plasmaappearance and chemical structure (Fig. 4) confirm their colonicorigin as reductive metabolites of ferulic acid, as also supportedby other studies (11,18–20).

PP can be further converted to benzoic acid by b-oxidationand finally to hippuric acid by conjugation to glycine in the liver(21,22) (Fig. 4). Similar metabolic conversions have also beenreported for other dietary phenolic compounds (20,23).

Both hippuric acid and benzoic acid were found in highconcentrations in plasma. They were also high at baseline and aclear increase from baseline following ingestion of the breadswas not observed. Sodium benzoate is widely used as preserva-tive in many foods and beverages in relatively high amounts(maximal allowance of 0.1%). Therefore, it is difficult toexclude this compound from the diet, which could explain thehigh baseline values. Furthermore, benzoic acid can also beformed from many aromatic compounds, including phenylala-nine and tyrosine from dietary protein (24). Altogether, thisindicates that benzoic acid and hippuric acid are less specificmetabolites of ferulic acid than the PP with different grades ofhydroxylation, which originate in the colon.

There was a correlation between changes in the plasmaferulic acid concentration and changes in the plasma antioxidantcapacity. However, the variation in the plasma ferulic acidconcentration can explain merely ~6% (rs

2 = 0.253 0.25) of the

FIGURE 1 Plasma concentration of ferulic acid over time after the

ingestion of the bioprocessed bread or the control bread in men.

Baseline data were subtracted. Values are medians and IQR, n = 8.

FIGURE 2 Plasma concentration of vanillic acid, 3,4-dimethoxybenzoic

acid, 3-OHPP, and PP over time after the ingestion of the control bread (A)

or bioprocessed bread (B) in men. Baseline data were subtracted. Values

are medians, n = 8.

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variation in the plasma antioxidant capacity. This is lower thanthat of the endogenous antioxidant uric acid (22%), which isformed from the metabolism of purines and was found in highconcentrations in plasma (200–480 mmol/L).

Compared with the control bread, the bioprocessed bread ledto lower pro-:antiinflammatory cytokine ratios (IL-6:IL-10 andIL-1b:IL-10) in the ex vivo LPS-stimulated blood. The TNFa:IL-10ratio did not differ between the breads. This could be due to thelong LPS stimulation (24 h), which may be suboptimal for thiscytokine, because the TNFa production peak occurs at 4–6 h inLPS-stimulated human whole blood (25). There was a significanteffect of the time after bread ingestion on the production of IL-6:IL-10 and IL-1b:IL-10. Some colonic metabolites of ferulic acidhave been reported to exert antiinflammatory effects in vitro,such as 3,4-dihydroxyphenylpropionic acid (26), 3-OHPP,and PP to a lesser extent (19). In vivo concentrations of thesemetabolites are sometimes lower and subjected to a large inter-individual variation (Supplemental Table 1). The several possi-ble colonic conversions of phenolic compounds to differentmetabolites are dependent of specific bacteria (23,27). A largeinter-individual variability in the colonic metabolism amongindividuals can be expected because of differences in theirbacterial populations, composition, and activity. The conversionrates may also vary greatly between individuals due to differ-ences in transit times and substrate availability (28).

Besides the effect on cytokine modulation, phenolic com-pounds derived from cereal fractions have been reported toimprove several cellular functions (chemotaxis, lymphoprolifera-tion, microbicidal activity) and the redox state of leukocytes (29).

It can be concluded that an optimized processing has aremarkable effect on the uptake of bioactive compounds fromthe whole grain food matrix. Although the antiinflammatorymechanism of phenolic compounds in whole grain is not fullyelucidated, the present study shows that bioprocessing of branincreases the bioavailability of ferulic acid and other phenolic

acids from whole wheat bread, and it has immunomodulatoryeffects. Further research is encouraged to optimize a staple food,such as bread, for the prevention of diet-related disorders.

AcknowledgmentsWe thank the technical and intellectual support of Airi Hyrkas,Marie-Jose Drittij, Lisette Bok, Wouter Vaes, and JavierCastaneda. G.R.M. M.H., R.v.d.B., A.B., R.H., and N.M.A.designed research; N.M.A. conducted research; I.M., E.S. and

TABLE 3 Urinary excretion in 24 h of all identified compoundsafter the ingestion of the control bread and thebioprocessed bread in men1

Control bread Bioprocessed bread

mmol/24 h

3,4-Dihydroxybenzoic acid 3.9 (0.90) 3.3 (0.63)

3,4-Dihydroxyphenylacetic acid 8.1 (4.7) 6.9 (3.5)

3,4-Dihydroxyphenylpropionic acid 1.9 (1.1) 2.4 (4.8)

3,4-Dihydroxytoluene 6.2 (8.4) 6.0 (7.9)

3,4-Dimethoxybenzoic acid 1.1 (0.85) 2.1 (1.9)*

3-Coumaric acid 1.5 (2.5) 1.1 (0.65)yy

3-Hydroxybenzoic acid 9.7 (5.6) 9.5 (6.4)

3-Hydroxyphenylacetic acid 20 (14) 14 (9.6)

3-OHPP 6.1 (4.1) 5.6 (3.5)

4-Coumaric acid 0.70 (0.40) 0.55 (0.30)y

4-Hydroxybenzoic acid 1.6 (3.2) 0.95 (0.48)yy

4-Hydroxyphenylpropionic acid 0.60 (0.53) 0.30 (0.30)yy

Benzoic acid 15 (9.3) 9.3 (7.5)

PP 0.90 (1.2) 0.5 (0.70)

Gallic acid 0.20 (0.08) 0.20 (0.10)

Hippuric acid 1500 (1500) 1100 (830)

Vanillic acid 30 (7.8) 49 (34)*

Sinapic acid 5.1 (3.7) 12 (3.2)**

Ferulic acid 51 (9.7) 110 (46)**

1 Values are medians and IQR, n = 8. Statistical differences from control: *P = 0.012,

**P = 0.004, yP = 0.023 (2-tailed), yyP = 0.008 (2-tailed) (Wilcoxon’s signed-rank test).

FIGURE 3 Ex vivo cytokine production in LPS-stimulated blood that

was drawn at 1.25, 6, and 12 h after the ingestion of bioprocessed

bread (B) and control bread (C) in men. Each data are relative to the

baseline value (LPS-stimulated blood drawn at 0 h) for each partici-

pant. Data are fitted linearly (R2 . 0.74) and are means and SD of the

mean, n = 8. Asterisks indicate the significance of the estimated

marginal mean difference from the mixed linear model: *C,B = 0.168

(95% CI: 0.035–0.301; P = 0.015), **C,B = 0.240 (95% CI: 0.060–

0.419; P = 0.010).

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A.-M.A. analyzed data; N.M.A. wrote the paper; and K.P. hadresponsibility for final content. All authors read and approvedthe final manuscript.

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FIGURE 4 Scheme of the possi-

ble metabolism of phenolic acids

from whole wheat bread in men.

Dotted lines represent b-oxidations

by either hepatocytes or entero-

cytes (11,18–20). *Amount recov-

ered in urine. 10 mmol. **Amount

recovered in urine . 100 mmol.

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