Bioprocessing of Wheat Bran in Whole Wheat Bread Increases the Bioavailability of Phenolic Acids in Men and Exerts Antiinflammatory Effects ex Vivo1-3

The Journal of Nutrition January 1, 2011 | Anson, Nuria Mateo; Aura, Anna-Marja; Selinheimo, Emilia; Mattila, Ismo; Poutanen, Kaisa; van den Berg, Robin; Havenaar, Robert; Bast, Aalt; Haenen, Guido R M M 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 healthy men 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 were measured 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 associated with the risk for developing some diet-related disorders such as type 2 diabetes, cardiovascular events, and obesity, disorders that are commonly referred to as metabolic syndrome Hyperglycemic and pro-oxidative conditions observed in those metabolic disorders may promote the excessive production of reactive oxygen species and advanced glycation end leading to tissue damage and malfunction, the main endogenous inducers of inflammation (2). The main mediators of the inflammatory process are the cytokines, which act in local and intercellular communications required in the integrated immune response. Numerous cytokines have been identified, but it is the “balance” between pro-inflammatory cytokines (IL-1/3, TNFa, IL.6) 1L-2, IL-8, and IFNy), and antiinflammatory cytokines IL.10) IL-4 anf] TGF/3) that is thought to be a determinant in the outcome of disease (3).

Whole grain foods tend to have a iow gjycernic index, resulting in lower postprandial glucose responses and insulin demand compared with refined cereal products. Whereas the low glyc?©mie index of whole grain is a generally recognized health benefit, the role of phytochemicals present in whole grain is still under debate (1). A high consumption of phytochemicals has been recently associated with iow adiposity and oxidative stress ,n obese VounS adults (4). Several of the phytochemicals m whole grain have been reported to exert antioxidant and antiinflammatory effects, such as some phenolic compounds reviewed elsewhere (5-8). Among the phenolic compounds found in wheat gram, ferulic acid is the most abundant and is strongly correlated with the antioxidant capacity of different wheat fractions (9). The outermost part of the grain, the bran, is rich in ferulic acid; however, its bioaccessibility or intestinal release from that matrix is very low. The low bioaccessibility is explained by the structural position of most of the ferulic acid, which is covalently bound to the indigestible polysaccharides of the cell walls constituting the fiber (10). Innovative processing techniques, such as bioprocessing, have been developed to increase the bioaccessibility of phenolic compounds from wheat bran (11).

In this study, we investigated the effect of bioprocessing of bran in whole wheat bread on the bioavailability of phenolic compounds, plasma antioxidant capacity, and antiinflammatory potential.

Materials and Methods Chemicals. Benzoic acid, 3-hydroxybenzoic acid, 3,4-dimethoxybenzoic acid, 3-(4-hydroxyphenyl)propionic acid, 3-(3,4-dihydroxyphenyl)propionic acid, 3,4-dihydroxytoluene, and 3-coumaric acid were products from 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-phenylpropionic acid, and 3,4-dihydroxybenzoic acid were from Fluka. Ferulic acid and 4-coumaric acid were provided from Extrasynthese. 3-(3-Hydroxy phenyl) propionic acid was purchased from Alfa Aesar. 2,2,2-Trifluoro-N-merhylN-trimethylsilyl-acetamide was from Pierce. All chemicals were of analytical grade.

Participants. Eight healthy men were recruited and enrolled in the study. They were between 21 and 55 y old and had a BMl between 20 and 30. They did not smoke or use any medication. Blood donation 3 mo before the start of the study, consumption of S3 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 of the study, and written informed consent was obtained. The study was approved by the Medical Ethical Commission of the Maastricht 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 ingrethents were supplied by B??hler. The 2 breads had similar concentrations of bran, total dietary fiber, and general macron utrients. 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 with enzyme treatment, consisting of cell-wall-degrading enzymes, mainly xylanase, cellulose, ??-glucanase, and feruloyl-esterase, for 20 h at 200C. Further details on the bioprocessing technique, bread making, and macronutrient analysis can be found elsewhere (11). The concentrations of phenolic acids in the breads (Table ??) were measured by HPLC and diode array as previously described (11).

Study design. The design was a blind cross-over study, with randomization 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 before the 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 the evening 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. Upon the 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 during the 24 h as a 0- to 24-h sample. Urinary collectors of 2 L capacity containing 1 g sodium ascorbate were provided for die 0- to 12-h period and for the 1 2- to 24-h period. The urine was pooled and aliquots were stored at -800C until analysis.

Determination of phenolic acids. Urine and plasma sample preparation was performed based on a previous method with modifications (12). The main modifications were in the hydrolysis conditions. The hydrolysis solution contained ^-glucuronidase (>3000 U) and sulfatase (>100 U) from Helix pomatia (Sigma-Aldrich) in 0.15 moI/L acetate buffer, pH = 4.1, and the incubation was for 16 h at 370C. The internal standard 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 (45?°C, 1 h) and 25 ??L of 2,2,2trifluoro-N-methy!-N-trimethylsilyl-acetamide (45?°C, 1 h) before the injection in the GC X 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 the trapezoidal method was used to calculate the area under the curve (AUCo.,)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 concentration of uric acid was determined by HPLC and UV detector (15).

Ex vivo induced inflammatory response. Blood samples drawn before the bread ingestion (0 h or baseline) and at 1.25, 6, and 12 h after bread ingestion were incubated in triplicate with LPS from Escherichia colt (purchased from Sigma Aldrich) in a final concentration of 1 /xg/L for 24 h at 370C and 5% CO2 as in a previous method (16). Cytokines (IL-10, IL-6, TNFor, IL- I??, IFNy, and IL-8) were determined in the supernatant with kits from Millipore BV following the instructions of the manufacturer and with Luminex XMAP Technology. IL-8 was above the highest limit of quantification (>20 /ig/L). For each participant, the cytokine determination (mean of triplicate determinations) was relative to the baseline value, i.e. LPS-stimulared blood that was drawn at time Oh or before the bread ingestion. Thus, differences from baseline values are observed when the values were < 1 or > 1 . The ratios of pro-:antiinflammatory cytokines were calculated by dividing each TNFa, IL-6, and IL- 1/3 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), ?? = 0.20, and an anticipated difference between treatments (?) equal to the expected SD of that difference (sa). 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, 3,4dimethoxybenzoic acid, and 4-hydroxyphenylpropionic acid AUC0., (if applicable) and urinary excretions, and 1L-6:IL-10 and IL-1/3:IL-10 cytokine ratios. To detect significant differences in me other measured outcomes, a larger sample size is needed. The study of secondary outcomes is merely exploratory and no adjustments for multiple hypothesis testing have been made. Assumptions of normal distribution (Kolmogorov-Sinirnov test) and homogeneity of the variance (Levene test) could not be made for all the data, partly due to the limited sample size (?« = 8). Accordingly, a distribution-free method, the WUcoxon’s signed-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 in medians and the variation of the data are given as the IQR (difference between the 25th and 75th quartile). The data of the ex vivo inflammatory response were analyzed using a linear mixed-effects model for longitudinal 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 x time interaction. The random factor was the subject. The estimated marginal means 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 significant digits. The software used for the analyses was SPSS 17.0 for Windows. go to web site whole wheat bread

Results Pharmacokinetics. The following phenolic compounds were detected and quantified in blood plasma: ferulic acid, vanillic acid, 3-hydroxyphenylpropionic acid (3-OHPP), phenylpropionic acid (PP), 3, 4-dihydroxy benzoic acid, 3,4-dimethoxybenzoic acid, 3-hydroxybenzoic acid, 4-hydroxybenzoic acid, benzoic acid, hippuric acid, and 3,4-dihydroxytoluene.

The relative bioavailability, i.e. AUCo-t of the compound from the bioprocessed bread relative to that from the control bread, was 2.7 for ferulic acid, 1.8 for vanillic acid, and 1.8 for 3,4-dimethoxybenzoic acid (Table 2). There were no significant differences in the time to maximum plasma concentrations (tmax) ?°f these compounds between the breads. The maximum plasma concentrations (Cmax) of ferulic acid, Vanillic acid, and 3,4-dimethoxybenzoic acid were significantly higher after the ingestion of bioprocessed bread than after the ingestion of control bread (Table 2). Ferulic acid was the phenolic acid with the highest C1113x and the largest difference from the baseline and the control. The Cmax and AUCu-t of ferulic acid after the ingestion of bioprocessed bread was 3-fold that of the control bread (Fig. 1). Plasma concentrations of 3-OHPP and PP rose from baseline values later than 6 h after ingestion (Fig. 2). Cm3x and tmax could not be accurately determined due to the relatively late increase in plasma concentration and lack of data during night time when blood was not collected.

At baseline, plasma concentrations of hippuric [2.8 ?µmol/L (range = 0.29-5.9 /??mol/L)] and benzoic [1.5 ?µmol/L (0.59-2.6 (Umol/L)] acids were high and they had irregular concentrationtime curves after bread intake. Irregular profiles, such as fluctuations from baseline with no clear increase following the bread ingestion, also occurred for 3-hydroxybenzoic acid, 4-hydroxybenzoic acid, 3,4-dihydroxybenzoie acid, and 3,4dihydroxytoluene (Supplemental Table 1).

Excretion of phenolic compounds. Among the compounds identified in urine, hippuric acid was present in the highest amount. Urine hippuric acid excretion did not differ after consumption of the 2 breads. The second highest phenolic compound found in urine was ferulic acid. The amounts of ferulic acid, sinapic acid, vanillic acid, and 3,4-dimethoxybenzoic acid found in urine after the ingestion of bioprocessed bread were approximately double of those found after the ingestion of control bread (Table 3). Excretions of other compounds were lower after the consumption of bioprocessed bread, including 3-coumaric acid (30%), 4-coumaric acid (20%), 4-hydroxyphenylpropionic acid (40%), and 4-hydroxybenzoic acid (50%) (Table 3). These compounds were low in urine and not detectable in plasma or fluctuating around baseline (Supplemental Table 1). 4-Hydroxyphenylpropionic acid and 4hydroxybenzoic acid were minor metabolites. Their rate of formation may have been lowered by the lower availability of their precursors, because less reached the colon or because these precursors might have been absorbed earlier and directed to hepatic metabolism.

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

Plasma total antioxidant capacity. There was a correlation between the changes from baseline in the ferulic acid plasma concentration over time and the changes in antioxidant capacity (rs = 0.25,- P < 0.01). Also, changes from baseline in plasma antioxidant capacity correlated with changes from baseline in uric acid concentration (rs = 0.47; P < 0.01).

Antiinflammatory effects. Blood drawn from the participant was subjected to an ex vivo LPS-chailenge to simulate an inflammatory response. Expression of IFNy by LPS stimulation was very low [110 ng/L (range = 7.7-660 ng/L)] compared with the other cytokines; IL-6 [11 ??ig/L (0.63-23 /Agri)], IL-l?? [Id /ig/L (0.39-6.9 /xg/L)], IL-10 [1.2 ??tg/L (0.30-3.1 ?µg/L)], and TNFa [1.2 ??tg/L (0.32-3.3 /xg/L)]. A linear mixed model was used to determine the effects of treatment (control bread, bioprocessed bread), time (1.25, 6, and 12 h after bread ingestion), and treatment over time (treatment X time interaction) on each of the outcome variables; IL-10, TNFa:IL-10, IL-6:IL-10, and IL-1/3:IL-10. There was an effect of the bread type on the IL-6:IL-10 (P = 0.015) and IL-10:IL-1O cytokine ratios (P = 0.010). The effect ??f time after bread ingestion was also significant for JL-6:IL-10 (P = 0.021) and IL- 1/3-.IL-IO (P < 0.001). There were no significant effects for the factors of period or treatment X time interaction (Fig. 3).

Discussion The technique of bioprocessing has been applied to bran for the enrichment of whole wheat bread. The aim of the present study was to investigate the effect of bioprocessing on the bioavailability of phenolic compounds, the postprandial plasma antioxidant capacity, and the ex vivo inflammatory response after the consumption of whole wheat bread.

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

The C^sub max^ 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 to some extent metabolites of ferulic acid from ??-oxidation and methylation reactions. This is supported by their structure resemblance (Fig, 4) and by the results in Table 3 that show an excretion of vanillic acid (30-49 /Amol) superior to its intake, which was ~5 mg (30 /xmol) (Table ?? ). The metabolism of ferulic acid to vanillic acid and some other metabolic conversions proposed in Figure 4 are in line with the findings of animal and human studies reviewed elsewhere (18).

The t^sub max^ of ferulic acid (90-1 OS min) after the ingestion of control or bioprocessed bread was within the range of 1-3 h reported after consumption of a high- bran breakfast cereal (12). The early tmax indicates that the absorption of ferulic acid mainly takes place in small intestine, despite the large proportion of ferulic acid that reaches colon bound to fiber (90%). Instead of a second phase in the absorption of ferulic acid from the colonic release, other compounds were detected to increase in plasma at late time points after ingestion (6-24 h). The main metabolites that increased after 6 h posterior to the bread ingestion were 3-0 HPP and PP. The time course of their plasma appearance and chemical structure (Fig. 4) confirm their colonic origin as reductive metabolites of ferulic acid, as also supported by other studies (1 1,18-20).

PP can be further converted to benzoic acid by /3-oxidation and finally to hippuric acid by conjugation to glycine in the liver (21,22) (Fig. 4). Similar metabolic conversions have also been reported for other dietary phenolic compounds (20,23).

Both hippuric acid and benzoic acid were found in high concentrations in plasma. They were also high at baseline and a clear increase from baseline following ingestion of the breads was not observed. Sodium benzoate is widely used as preservative in many foods and beverages in relatively high amounts (maximal allowance of 0.1%). Therefore, it is difficult to exclude this compound from the diet, which could explain the high baseline values. Furthermore, benzoic acid can also be formed from many aromatic compounds, including phenylalanine and tyrosine from dietary protein (24). Altogether, this indicates that benzoic acid and hippuric acid are less specific metabolites of ferulic acid than the PP with different grades of hydroxylation, which originate in the colon. website whole wheat bread

There was a corr?©lation between changes in the plasma ferulic acid concentration and changes in the plasma antioxidant capacity. However, the variation in the plasma ferulic acid concentration can explain merely -6% (rs2 = 0.25 X 0.25) of the variation in the plasma antioxidant capacity. This is lower than that of the endogenous antioxidant uric acid (22%), which is formed from the metabolism of purines and was found in high concentrations in plasma (200-480 ?µmol/L).

Compared with the control bread, the bioprocessed bread led to lower pro-ianriinflarnmatory cytokine ratios (IL-6:IL-10 and lL-l??:JL-lQ) in the ex vivo LPS-stumiiated blood. The TNFa:lL-10 ratio did not differ between the breads. This could be due to the long LPS stimulation (24 h), which may be suboptimal for this cytokine, because the TNFa production peak occurs at 4-6 h in LPS-stimulated human whole blood (25). There was a significant effect of the time after bread ingestion on the production of IL-6: IL-10 and IL-IjSiIL- 10. Some colonie metabolites of ferulic acid have 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 these metabolites are sometimes lower and subjected to a large interindividual variation (Supplemental Table 1). The several possible colonic conversions of phenolic compounds to different metabolites are dependent of specific bacteria (23,27). A large inter-individual variability in the colonic metabolism among individuals can be expected because of differences in their bacterial populations, composition, and activity. The conversion rates may also vary greatly between individuals due to differences in transit times and substrate availability (28).

Besides the effect on cytokine modulation, phenolic compounds derived from cereal fractions have been reported to improve several cellular functions (Chemotaxis, ?¬ymphoproliferation, microbicidal activity) and the redox state of leukocytes (29).

It can be concluded that an optimized processing has a remarkable effect on the uptake of bioactive compounds from the whole grain food matrix. Although the antiinflammatory mechanism of phenolic compounds in whole grain is not fully elucidated, the present study shows that bioprocessing of bran increases the bioavailability of ferulic acid and other phenolic acids from whole wheat bread, and it has immunomodulatory effects. Further research is encouraged to optimize a staple food, such as bread, for the prevention of diet-related disorders.

Acknowledgments We thank the technical and intellectual support of Airi Hyrk?¤s, Marie-Jos?© Dritti], Lisette Bok, Wouter Vaes, and Javier Casta?±eda. 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 A.-M.A. analyzed data; N.M.A. wrote the paper; and K.P. had responsibility for final content. All authors read and approved the final manuscript.

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 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 In. nutrition.org 8 Abbreviations used: AUC, area under the curve; Cm11x. maximum plasma concentration; 3-OHPP. 3-hydroxyphenylpropionic acid; PP. phenylpropionic acid; tmax, time to maximum plasma concentration.

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[Author Affiliation] Nuria Mateo Anson,4,5* Anna-Marja Aura,6 Emilia Selinheimo,* Ismo Mattila,6 Kaisa Poutanen,6 Robin van den Berg,7 Robert Havenaar,5 AaIt Bast,4 and Guido R. M. M. Haenen4 4 Maastricht University, 6200 MD Maastricht, The Netherlands; 5TNO Quality of Life, 3700 AJ Zeisr, The Netherlands; 6VTT Biotechnology, Technical Research Centre of Finland, Espoo, f 1-02044 VTT, Finland; and ‘Unilever Research Vlaardingen, NL-3130 AC Vlaardingen, The Netherlands [Author Affiliation] Anson, Nuria Mateo; Aura, Anna-Marja; Selinheimo, Emilia; Mattila, Ismo; Poutanen, Kaisa; van den Berg, Robin; Havenaar, Robert; Bast, Aalt; Haenen, Guido R M M