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Effect of bioprocessing on the in vitro colonic microbial metabolism of phenolic acids from rye bran fortified breads Ville Mikael Koistinen, Emilia Nordlund, Kati Katina, Ismo Mattila, Kaisa Poutanen, Kati Hanhineva, and Anna-Marja Aura J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b05110 • Publication Date (Web): 16 Feb 2017 Downloaded from http://pubs.acs.org on February 18, 2017

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Journal of Agricultural and Food Chemistry

Effect of bioprocessing on the in vitro colonic microbial metabolism of phenolic acids from rye bran fortified breads Ville M Koistinena, Emilia Nordlundb, Kati Katinab,c, Ismo Mattilab,d, Kaisa Poutanenb, Kati Hanhinevaa, Anna-Marja Aurab* a

University of Eastern Finland, Institute of Public Health and Clinical Nutrition, P.O. Box 1627,

FI-70211 Kuopio, Finland b

VTT Technical Research Centre of Finland, P.O. Box 1000, Tietotie 2, Espoo, FI-02044 VTT,

Finland c

University of Helsinki, Department of Food and Environmental Sciences, P.O. Box 66 (Agnes

Sjöbergin katu 2), FI-00014 University of Helsinki, Finland. d

Steno Diabetes Center, Niels Steensens Vej 2, DK-2820 Gentofte, Denmark

*Corresponding author (Tel: +358 40 820 8731; Fax: +358 207227071; Email: [email protected])

1 ACS Paragon Plus Environment


ABSTRACT 2

Cereal bran is an important source of dietary fiber and bioactive compounds, such as phenolic

3

acids. We aimed to study the phenolic acid metabolism of native and bioprocessed rye bran fortified

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refined wheat bread and to elucidate the microbial metabolic route of phenolic acids. After

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incubation in an in vitro colon model, the metabolites were analyzed using two different methods

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applying mass spectrometry. While phenolic acids were released more extensively from the

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bioprocessed bran bread and ferulic acid had consistently higher concentrations in the bread type

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during fermentation, there were only minor differences in the appearance of microbial metabolites,

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including the diminished levels of certain phenylacetic acids in the bioprocessed bran. This may be

10

due to rye matrix properties, saturation of ferulic acid metabolism or a rapid formation of

11

intermediary metabolites left undetected. In addition, we provide expansion to the known metabolic

12

pathways of phenolic acids.

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Keywords: rye, bran, bioprocessing, phenolic acid, fecal microbiota, in vitro colon model


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INTRODUCTION 14

Cereal bran is an important source of dietary fiber (DF). The DF complex within the bran also

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contains several classes of phytochemicals. The importance of the intake of cereal dietary fiber for

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health is well established: epidemiological studies have indicated that consumption of whole grain

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foods shows consistently decreased risk of type 2 diabetes and cardiovascular diseases1, and the DF

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complex has been suggested to be an underlying factor in the risk reduction.2-5 Among the

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phytochemicals of the bran DF complex, phenolic acids are mostly covalently bound to the bran

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matrix.6,7 Hydroxycinnamic acids are the most abundant phenolic acids, comprising mostly of

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ferulic acid but also of sinapic and 4-coumaric acids. Benzoic acid derivatives (benzoic and vanillic

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acids) are present in small quantities.7,8 It is likely that the microbial metabolites circulating in the

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bloodstream, originating from the vast pool of phytochemicals contained in cereal fiber and other

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food sources, has an impact on our health.9 Therefore, the identification of those metabolites is

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necessary to enable studies of the mechanisms underlying the correlations observed thus far.

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Bioprocessing of bran with enzymes and/or microbes enhances its technological functionality and

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nutritional properties, thus assisting in increasing DF consumption. Bioprocessing of wheat bran

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has been used for improvement of the properties of wheat dough and bread containing bran, i.e.

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bread volume, crumb texture and shelf life.10,11 In addition, sourdough fermentation of bran

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increases the amount of folates and free phenolic acids in breads.12,13 Bioprocessing of wheat bran

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with enzymes and yeast fermentation has also shown to alter the kinetics of phenolic acids and their

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microbial metabolites from wheat bran fortified breads via changes in their in vitro

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bioaccessibility.14 Subsequently, bioprocessing alters the pharmacokinetic profiles of both phenolic

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acids and their microbial metabolites in healthy volunteers.15 Bioprocessing of rye bran also has an

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impact on the release of phenolic acids in humans, as has been shown by Lappi et al.16



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The main microbial colonic metabolites from native and bioprocessed wheat bran fortified breads in

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vitro are reported to include 3-(3-hydroxyphenyl)propionic acid, dihydrocaffeic acid (3-(3,4-

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dihydroxyphenyl)propionic acid) and 3-phenylpropionic acid.14 The anaerobic conversion route of

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isolated 8-O-4-diferulates and 5-5-diferulates in the human in vitro colon model has been proposed

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earlier by Braune et al,17 who showed that the abovementioned hydroxylated phenylpropionic acids

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were formed from one ferulic acid unit of 8-O-4-diferulates, whereas the other unit of the diferulate

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(vanilpyruvic acid) was converted to 2-(3,4-hydroxyphenyl)- and 2-(3-hydroxyphenyl)acetic acids

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and the corresponding lactic acids. Pekkinen et al18 showed sulfate derivatives of bioprocessed rye

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bran phytochemicals (dihydroferulic acid, hydroxylated phenylpropionic and -acetic acids) and

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hippuric acid, a glycinated benzoic acid, in urinary excretion of mice by using LC–MS

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metabolomics. However, the microbial metabolites of phenolic acids have not been studied from

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rye bran, which naturally contains the diferulates. Hanhineva et al19 investigated the microbial

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metabolite content (focusing on lignans and benzoxazinoids) of rye bran soluble extract and its non-

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extractable residue using a liquid chromatography–mass spectrometry (UPLC–QTOF–MS)

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metabolomics platform and showed that the extract and bran residue differed in their metabolic

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profiles.

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Applications of mass spectrometry are widely used in metabolomics. Aura et al.20,21 have used the

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GC×GC–TOF–MS method coupled with a compound library NIST08 in the preliminary

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identification of small (below 1000 Da) microbial phenolic metabolites. This is possible in the

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analysis by GC×GC–TOF–MS, since the retention times of the compounds do not change in the

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constant operation conditions of the analysis. In addition, the GOLM Group tool in Guineu

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program22 can be used in further identification of functional groups in a molecule, when complete

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structure cannot be revealed.20 Owing to its high sensitivity, the use of liquid chromatography

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coupled with mass spectrometry is one of the most widely used methods in plant metabolomics.23


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UPLC–QTOF–MS has been previously used by Hanhineva et al. in the identification of novel

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phytochemicals.24,25

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The aim of the present study was to elucidate how bioprocessing with enzymes and yeast influences

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the microbial metabolites of phenolic acids originating from rye bran. This was done by introducing

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bioprocessed and native rye bran fortified white wheat breads in an in vitro colon model and

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profiling the fermented samples with GC×GC–TOF–MS coupled with identification tools and

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UPLC–QTOF–MS, which enables the further examination of novel metabolites and offers

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additional verification to compound identification. Having refined wheat bread as the vehicle of

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introducing rye bran into the in vitro model may cause some interactions, such as introducing

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wheat-based compounds into the samples, thus potentially affecting the results. However, the same

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wheat bread was used as a basis for both bread types and the total phenolic acid content in rye bran

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is nearly 40 times higher than in white wheat bread26, similar differences existing for other

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phytochemical classes as well;27 therefore, rye bran can be considered the main contributor to the

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phytochemical pool investigated in this study. Since ferulic acid is the most abundant phenolic acid

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in rye bran, the emphasis was in identification of the potential metabolites of its derivatives. In

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particular, the metabolic route of ferulic acid into its microbial products was investigated. Targeted

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analysis with external standard compounds was used for quantitation of the most common phenolic

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microbial metabolites with GC×GC–TOF–MS. MATERIALS AND METHODS

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Bioprocessing of rye bran and bread baking. Bioprocessing of rye bran was performed and the

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breads were baked as described in Nordlund et al.28 Shortly, rye bran was first treated with a

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hydrolytic enzyme mixture of Depol 740L (Biocatalysts; dosing 200 nkat/g bran, based on xylanase

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activity) and Grindamyl A 1000 (dosing 75 nkat/g bran, based on amylase activity) at 65% water

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content in 40 °C for four hours, mixing at 0.5, 1, 2, 3, and 4 hour intervals. Baker’s yeast (1.25%) 5 ACS Paragon Plus Environment


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(Sunnuntai, Raisio, Finland) was added into the bran slurry and the mixture was fermented in 20 °C

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for 20 hours without mixing. For preparing the wheat bread with native or bioprocessed bran, 35%

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of the wheat flour was replaced with the bran based on dry matter. Bioprocessed bran was added

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into the dough in a wet form directly after bioprocessing and baked to breads, as described earlier.28

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In vitro digestion and colon model. Fresh wheat breads enriched with native or bioprocessed rye

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bran were first ground using a mincer (MG450, Kenwood Ltd, Hampshire, UK) and digested

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enzymatically using porcine digestive enzymes (salivary α-amylase, pepsin and pancreatin) in an in

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vitro digestion model mimicking the upper intestine. Starch and protein contents of the samples

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were reduced by salivary α-amylase, pepsin and pancreatin digestion, and the digestion products

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were removed by dialysis.28,29 After the dialysis, the non-digestible residues were freeze-dried and

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characterized for starch, protein, fat and DF components as described earlier.28 The characterization

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was repeated for the samples after the colonic fermentation using the same method. The content of

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free and total phenolic acids were acquired by treating the samples with HCl and NaOH,

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respectively, as described previously by Mateo Anson et al.14 and analyzing them with HPLC

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coupled with diode array detector as described by Mattila et al.26 The release and conversion of

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phenolic acids were then studied in an in vitro colon model in strictly anaerobic conditions. In vitro

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fecal fermentation experiments were performed as described in Nordlund et al.28 using pooled

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human fecal suspension (10% w/v) from five healthy volunteers, who had given a written consent.

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The collection and handling of the samples were performed according to the guidelines of the VTT

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Ethical committee.

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Analyses of phenolic acids. The phenolic acid contents of bran and bread samples were determined

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with HPLC as described by Mateo Anson et al.14 Phenolic acids formed by microbial conversion in

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the in vitro colon model were analyzed as described in Aura et al.20 using two-dimensional gas

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chromatography coupled with a time-of-flight detector (GC×GC–TOF–MS). In the targeted 6 ACS Paragon Plus Environment

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analysis, the phenolic acids were quantitated with 18 authentic standards as described in Nordlund

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et al30 and using 2-hydroxycinnamic acid (mainly trans; Aldrich Inc. H2,280-9; 97%; St. Louis,

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USA) as an internal standard as described earlier.20 N-Methyl-N-(trimethylsilyl)trifluoroacetamide

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(MSTFA) from Pierce (Rockford, USA) and methoxyamine 2% hydrochloride in pyridine (MOX;

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Pierce, Rockford, USA) were used as the silylation reagents.

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The LC–MS analysis was performed on a 1290 Infinity Binary UPLC system (Agilent

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Technologies, Santa Clara, CA, USA) coupled with an Agilent 6540 Q-TOF (quadrupole time-of-

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flight) mass spectrometer, as described previously by Hanhineva et al.31 After the reversed-phase

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separation (Zorbax Eclipse XDB-C18 column), the electrospray ionization was carried out in the

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positive and negative mode. The collision energies for MS/MS analysis were chosen as 10, 20 and

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40 V, for compatibility with databases.

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Statistical analyses. For the GC×GC–TOF–MS data, all the responses from the colon model were

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measured in triplicates. Two-Way ANOVA was used with repeated measures using a Bonferroni

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adjustment to test for differences between samples (p < 0.05). The non-targeted metabolite profiling

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and statistics were performed using an in-house developed Guineu program.22 Statistically

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significant phenolic acid metabolites (FDR q value < 0.05) with maximal similarity above 700 were

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included in the heat maps, which display the profiles of the individual metabolites with color

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intensities and the time-point specific significances (t-test p-values) against the corresponding

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control. The Fold test function of Guineu calculates and shows the fold change (FC) as the maximal

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ratio of the average peak area from the GC×GC–TOF–MS chromatogram at each time point against

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the corresponding fecal control. The clustering was performed by Guineu according to the

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similarities of the metabolite profiles in the course of time.

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For the UPLC–QTOF–MS data, the initial search for compounds was carried out using the Find by

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Molecular Feature algorithm in MassHunter Qualitative Analysis version 7.0 (Agilent 7 ACS Paragon Plus Environment


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Technologies). The extraction mode was set to small molecules with a mass peak threshold at 300

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counts and absolute compound peak height at 3000 counts. The allowed negative ion species were

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singly charged ions and chloride adducts and for the positive ions, singly charged ions and sodium

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adducts were allowed. The peak spacing tolerance for isotope grouping was selected as m/z 0.0025

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(absolute) and 7 ppm (relative), with an isotope model for common organic molecules. The

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extracted molecular entities were then exported to Mass Profiler Professional version 2.2 (Agilent

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Technologies), where peak alignment, data clean-up and biostatistical analysis was performed on

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the data. The following parameters were used: minimum absolute abundance of a signal at 5000

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counts, minimum number of detected ions at 2, retention time tolerance at 0.1% (relative) and 0.15

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min (absolute), and mass tolerance at 5.0 ppm (relative) and 2.0 mDa (absolute). Entities appearing

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in at least 50% of replicates in at least one sample group were included for further analysis. To

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account for the high variability of signal intensities, Z-transform was used as the baselining method.

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The resulting compounds were reanalyzed in MassHunter Qualitative Analysis; for this recursive

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analysis, compound mass tolerance was set at ± 15 ppm and retention time tolerance at ± 0.10 min.

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The dataset was then transferred back to Mass Profiler Professional, where the same parameters as

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previously were applied for the second analytical procedure. For the remaining entities, a statistical

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analysis was carried out in the software using one-way ANOVA (for relevant time / sample type

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pairs) with an asymptotic p-value computation (p < 0.05) and Benjamini–Hochberg false discovery

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rate (FDR) as the multiple testing correction. The entity list was then exported to Excel (Microsoft

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Corporation) for further filtering and sorting.

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Multiple Experiment Viewer software (version 4.9) was used to visualize the results from UPLC–

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QTOF–MS and to provide a k-means clustering analysis in order to categorize compounds based on

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their relative intensities between different bread types. 4346 molecular features obtained from the

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RP negative mode were included in the analysis. Before the analysis, each row (feature intensity)

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was normalized based on the following formula: x = (x – x̄row) / SDrow. For the chart, the color scale 8 ACS Paragon Plus Environment

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limits were set to –2 and 2 in normalized values. The amount of clusters was adjusted with visual

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inspection by choosing the maximum number of distinct clusters (in this case, k = 7) with the

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compounds within one cluster being relatively more abundant in particular bread samples. The

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principal component analysis (PCA) was performed in SIMCA version 14 based on the same

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dataset. To create the PCA-x model, mean-centered scaling and model auto-fitting was used, thus

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determining three significant components for the analysis. A three-dimensional score scatter plot

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was then created to visualize the differences between the samples.

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Identification of compounds. For the GC×GC–TOF–MS data, the tentative identification of the

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metabolites that were not verified by external standards was performed using NIST08 and GOLM

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metabolome database32 as described in Aura et al.20 For the LC–MS data, the accurate mass,

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MS/MS fragmentation data, and predicted chemical formulae were used to screen for putative

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identifications in freely available databases, primarily METLIN33 and PubChem34. The MS/MS

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fragmentation pattern of a compound was compared with that of a standard, or when no standard

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was available, with databases and previously published literature. RESULTS

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Characterization of the breads after enzymatic digestion. Wheat breads fortified with native rye

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bran (NB) and bioprocessed rye bran (BB) were digested in an enzymatic digestion model to obtain

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residues that mimicked the digesta entering the human caecum. The proportion of soluble and

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insoluble DF components increased in the dry matter as the starch was digested, with the exception

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of fructan content, which slightly decreased in the in vitro enzymatic digestion and dialysis (Table

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1). The content of all free phenolic acids and a substantial amount of the bound phenolic acids

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decreased in the enzymatic digestion model and dialysis, and the decrease was enhanced by

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bioprocessing (Table 2). The following changes in bound phenolic acids occurred in NB fortified

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bread: the content of ferulic acid decreased by 50%, sinapic acid by 59%, and 4-coumaric acid by 9 ACS Paragon Plus Environment


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83%. The bound phenolic acid contents in the digestion of BB fortified bread decreased even more,

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as the content of ferulic acid decreased by 87%, sinapic acid by 90%, and 4-coumaric acid by 94%

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in the residue of BB fortified bread samples. Thus, the total phenolic acid contents entering the

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colon model were 0.635 mg/g d.w. and 0.160 mg/g d.w. in the digestion residues of the NB and BB

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fortified wheat breads, respectively, and only bound phenolic acids were present.

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In vitro release and conversion of phenolic acids. The non-targeted metabolite profiling by LC–

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MS revealed a considerable change in the overall metabolite content after the in vitro fermentation

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of the rye bran but with only a minor overall difference between the native and bioprocessed rye

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bran, as shown in the principal component analysis (PCA) with three components (Figure 1).

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According to the PCA, there was a clear difference in the metabolite content between the NB and

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BB fortified breads in the initial time point of the fermentation, but these differences became less

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profound as the fermentation progressed. In the clustering analysis, cluster 1 (n = 1183) showed

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features highly abundant only in the initial time point of the fermentation in both types of rye bran,

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indicating their origin from the breads and a consequent rapid metabolism by the microbiota (Figure

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2). In cluster 2 (n = 860), a similar pattern could be seen with the exception of survival of some of

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the compounds until 2 hours of fermentation. Clusters 3 (n = 390), 4 (n = 799), and 5 (n = 533)

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showed features likely to be metabolites formed from the compounds in clusters 1 and 2 with

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different time profiles: the features in cluster 3 were products of more rapid metabolism while the

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features in cluster 5 had been formed in a longer-lasting metabolic process. Cluster 6 (n = 417)

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showed a distinct group of features with their levels increasing in the fecal control towards the end

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of the incubation.

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The metabolite profiling by GC×GC–TOF–MS showed a number of different significant

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metabolites, some of which were fully identified with chemical standards (IDs 1–19). However,

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some metabolites could only be identified on the basis of their mass spectra and functional groups 10 ACS Paragon Plus Environment

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and were given putative names, or in the case of no matching spectra, classified e.g. as phenolic or

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carboxylic acids. The results below concentrate on the unambiguously and tentatively identified

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compounds and their structural relevance in the metabolism of phenolic acids, mainly ferulic acid

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derivatives.

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The changes in the metabolite profiles, as analyzed by the non-targeted GC×GC–TOF–MS

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approach, are visualized in heat maps as metabolite-specific differences between the BB and NB

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breads (Figure 3A) or between the NB or BB bread and the fecal control (Figure 3B and 3C,

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respectively). Figure 4 shows the released phenolic acids and the quantitated or semi-quantitated

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significant microbial phenolic metabolites from NB and BB fortified breads. According to Figure

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3B and 4, the concentration of sinapic acid dropped markedly after the initial time point, but ferulic

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and vanillic acids were released steadily during the incubation and the levels of caffeic and 4-

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coumaric acids increased towards the end of the incubation. Vanillic acid showed high folds at two

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separate

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methoxyphenyl]propionic acid) had a significant difference from the fecal background at 2 hours

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(fold change 40.4; Figure 3B).

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The

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hydroxyphenyl)propionic, and 3-(4-hydroxyphenyl)propionic acids at the later time points in both

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bread types indicate demethylation and subsequent dehydroxylation either at 3’ or 4’ positions.

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Further,

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hydroxyphenyl)lactic acid at the later time points were also among the significant metabolites

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formed from the NB but not from the BB fortified bread, signifying the subtle differences in the

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microbial conversion of phenolic acids from different rye matrices. In addition to the phenolic acid

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metabolites, 4-methylcatechol (Figure 3B and 3C) was released or converted from both NB and BB

time

significant

points.

Structurally

formation

3-hydroxybenzoic

of

acid

similar

dihydroferulic

dihydrocaffeic,

(m-salicylic

acid),

acid

(3-[4-hydroxy-3-

3,4-dihydroxyphenylacetic,

3-phenyllactic


acid,

and

3-(3-

3-(4-


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fortified breads. The proposed structural transformations of phenolic acids of rye bran to their

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microbial metabolites are shown in Figure 5.

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Effect of bioprocessing on release and conversion of phenolic acids. Bioprocessed rye bran

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bread exhibited a smaller number of microbial metabolites in the GC×GC–TOF–MS than the native

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bran bread, as visualized on the heat map (Figure 3B). The direct comparison of the fortified breads

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did not reveal significant differences in the phenolic acid metabolites apart from ferulic acid (0, 2, 4

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and 8 h) and benzoic acid (4 h). Bioprocessing enhanced the release of ferulic acid significantly, as

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its concentrations in the fecal suspensions were from the beginning to the end of the incubation

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constantly higher in samples containing BB fortification than NB fortification. However, for other

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phenolic acid precursors, such as 4-coumaric, sinapic and vanillic acid, there were few significant

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differences in the levels between BB and NB breads for different time points.

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The concentration of dihydrocaffeic acid reached the maximum at 4 h and 8 h for the NB and BB

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fortified breads, respectively. The formation of 3-(3-hydroxyphenyl)propionic acid and 3-(4-

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hydroxyphenyl)propionic acid increased during the incubation, but the difference between the NB

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and BB fortified breads was not significant at the end of the incubation (Figure 4). Two metabolites,

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3,4-dihydroxyphenylacetic acid and 4-hydroxyphenylacetic acid, were formed transiently but

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significantly less from the BB fortified bread than from the NB fortified bread. Benzoic acid

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formation was quite similar from both breads, except at the 4 h time point, indicating no major

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effect of bioprocessing on the release of benzoic acid or conversion rate from its precursors.

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Based on the UPLC–QTOF–MS results, 4-hydroxyphenylacetic acid was formed less from the BB

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than from the NB fortified bread from the 4 h time point onwards (Figure 4); it showed a significant

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difference from the fecal background beginning from the 6 h time point. 3-hydroxyphenylacetic

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acid, on the other hand, was present in the samples at steady levels from the beginning of the

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fermentation, having minor but statistically significant differences between each of the three sample 12 ACS Paragon Plus Environment

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groups (data not shown). Caffeic acid was released consistently during the fermentation; however,

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the difference between the BB and NB breads was significant only at the 2 h time point (higher

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level in the NB fortified bread). Dihydroferulic acid had higher levels in the BB fortified bread

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from the initial time point until 6 h, but the levels became lower than NB at the 24 h time point. For

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those phenolic acids that were detected with both methods used in this study, the levels detected in

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the UPLC–QTOF–MS analysis were mainly consistent with the results from GC×GC–TOF–MS

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(data not shown); due to the semi-quantitative nature of the UPLC–QTOF–MS data, the GC×GC–

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TOF–MS results quantitated with a standard (IDs 1–19) were prioritized. DISCUSSION

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Metabolic routes of phenolic acids from rye bran. The metabolic routes of phenolic acids, such

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as O-diferulates, ferulic acid, 4-coumaric acid and vanillic acid are connected and therefore the

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conversions cannot be fully distinguished from each other. The formation of dihydroferulic and

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caffeic acid from ferulic acid is in accordance with the results presented by Braune et al,17 who

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identified dihydroferulic acid as a precursor of dihydrocaffeic acid. Based on the delayed increase

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in the levels of 4-coumaric acid, resembling that of caffeic acid (Figure 4), we suggest that it may

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also be metabolized from ferulic acid via caffeic acid by demethylation and subsequent

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dehydroxylation (Figure 5), as suggested by Grbić-Galić and Young.35

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The non-targeted GC×GC–TOF–MS metabolomics profile visualized in the heat maps (Figure 3B

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and 3C) together with the UPLC–QTOF–MS results (Figure 4) revealed the formation of

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dihydroferulic acid, indicating an early reduction of a double bond in the side chain of ferulic acid.

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Ferulic

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methoxyphenyl]propionic acid) and vanillic acid (4-hydroxy-3-methoxybenzoic acid) have the

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same substitution pattern with differences only in the side chain. The subsequent formation of 3-(3-

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hydroxyphenyl)propionic acid and 3-(4-hydroxyphenyl)propionic acid is most likely a result of

acid

(4-hydroxy-3-methoxycinnamic

acid),

dihydroferulic


acid

(3-[4-hydroxy-3-


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metabolism of dihydroferulic acid by 3-demethylation and 3- or 4-dehydroxylation. However, the

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formation of vanillic acid (Figure 4) suggests that vanillic acid may appear via several routes: a

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direct release from the rye matrix and alpha- or beta-oxidation of dihydroferulic acid, which could

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explain the two peaks in the time course of vanillic acid concentration. Beta-oxidation of ferulic

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acid into vanillic acid has been previously suggested to occur in the liver36 and in the gut, as

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proposed recently by Feliciano et al.37

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The formation of dihydroxylated phenylacetic and phenyllactic acids are in line with results of

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Braune et al.,17 who suggest that they originate from one of the two units of 4-O-8-diferulate,

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whereas free ferulic acid and the ferulic acid unit of 4-O-8-diferulate are precursors of hydroxylated

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phenylpropionic acids, also present in the visualized metabolite profiles. Even though the in vitro

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metabolic route of isolated diferulates was presented by Braune et al.17 earlier, this has not been

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elucidated from a natural cereal source before. The metabolite profile of the urinary excretion of

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bioprocessed rye bran from humans and mice showed the same compounds as sulfated derivatives

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verifying the bioavailability of microbial metabolites from bioprocessed rye bran. In addition,

287

hippuric acid, a hepatic metabolite of benzoic acid, indicated the presence of post-colonic hepatic

288

metabolites.18,38

289

As part of the identification process of significant metabolites, we characterized some potential

290

ferulic acid and diferulate microbial metabolites previously not reported from rye. Two additional

291

isomers of 3-hydroxyphenylpropionic acid in addition to the previously known 3-(3-

292

hydroxyphenyl)propionic acid, namely 3-(2-hydroxyphenyl)propionic acid (melilotic acid) and 3-

293

(4-hydroxyphenyl)propionic acid (phloretic acid), were detected. However, the levels of 3-(2-

294

hydroxyphenyl)propionic acid, identified only from the UPLC–QTOF–MS data, were not higher in

295

the NB and BB samples compared with the fecal control (data not shown), thus indicating that the

296

compound is not a metabolite originating solely from rye bran. As discussed previously, 3-(314 ACS Paragon Plus Environment

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hydroxyphenyl)propionic acid and 3-(4-hydroxyphenyl)propionic acid are likely to be resulting

298

mainly from ferulic acid metabolism. These metabolites are also reduction products of caffeic, 3-

299

coumaric and 4-coumaric acids,17,39 but 3-coumaric acid has not been detected in rye40 and the

300

amount of caffeic and 4-coumaric acid in rye is significantly less than that of ferulic acid.26

301

4-hydroxyphenylacetic acid (Fig. 5; identified with UPLC–QTOF–MS) has previously been

302

reported as a metabolite by human colonic bacteria of two flavonoids, namely daidzein and

303

naringenin,41 but it has not been reported as a metabolite from rye before. The origin and pathway

304

of this compound remains to be elucidated with targeted experiments. However, it may be a

305

dehydroxylation product of 3,4-dihydroxyphenylacetic acid (homoprotocatechuic acid), which has

306

been identified as a rye metabolite by Nordlund et al.30 as well as in this study. Braune et al.17

307

identified 3,4-dihydroxyphenylacetic acid as a metabolite of dehydrodiferulic acid. Another novel

308

metabolite from rye bran observed in this study, 4-methylcatechol (3,4-dihydroxytoluene) (Fig. 3B;

309

identified with GC×GC–TOF–MS), has also been described as a metabolite of 3,4-

310

dihydroxyphenylacetic acid,42 thus linking also this compound to diferulic acid metabolism.

311

Effect of bioprocessing on phenolic acids. We have shown earlier that bioprocessing of rye bran

312

with enzymes and yeast causes degradation of aleurone and endosperm cell walls.28 Thus, ferulic

313

acid is released more easily from the BB enriched bread than from the NB enriched bread.

314

Solubilization of rye arabinoxylan has also been shown to increase the release of ferulic acid by

315

human microbial esterases in vitro,43 which could explain the lack of detected diferulates from the

316

colon model samples. In parallel to these studies, the hydrolysis of arabinoxylan bioprocessing

317

increased the proportion of free ferulic acid in bran in this study. This was expected to show as

318

different profiles of phenolic acid metabolites between the bread types during the colonic

319

fermentation.



320

The in vitro colonic microbial metabolomes of wheat breads containing native rye bran (NB) and

321

bioprocessed rye bran (BB) had only subtle differences: some of the discriminative compounds (for

322

at least some time points) included ferulic acid, dihydroferulic acid, dihydrocaffeic acid, 3,4-

323

dihydroxyphenylacetic acid and 4-hydroxyphenylacetic acid (Figure 4). The clustering of the

324

GC×GC–TOF–MS data showed only ferulic and 4-hydroxyphenylacetic acids as significant

325

phenolic acid metabolites along with other compounds (Figure 3A). Furthermore, the differences

326

between the NB and BB samples could be seen in the initial time point for the whole metabolite

327

pool, but were diminished in the later time points in the UPLC–QTOF–MS results, possibly due to

328

the diversity of the detected molecular entities in the data (Figure 1). This result is in agreement

329

with the human study by Lappi et al.,16 where the same breads were used and no difference in the

330

benzoic, phenylpropionic, and phenylacetic acid metabolites was observed between the breads.

331

The bioprocess-induced changes can also enhance the production of colonic metabolites as has been

332

shown previously by Mateo Anson et al,14,15 who compared native and bioprocessed wheat bran

333

fortified wheat breads in the release and conversion of phenolic acids in vitro. The bioprocessed

334

wheat bran bread showed a higher concentration of 3-phenylpropionic acid, considered as the end

335

product of the colonic metabolism of ferulic acid, than native wheat bran bread at the end of the

336

incubation.14 In a human study, the bread fortified with native wheat bran had a larger variety of

337

microbial metabolites in plasma compared with native bran,15 indicating the importance of the

338

cereal matrix in delivering substrates for the conversion by colonic microbiota, as also suggested by

339

Vitaglione et al.3 Similar observations were also seen in mice fed with wheat bran aleurone fraction

340

by Pekkinen et al.44

341

The lack of significant differentiating metabolites between the NB and BB fortified breads in this

342

study may be due to the saturation of ferulic acid metabolism due to its slow release from the

343

remaining fiber matrix, which could result in the increased ferulic acid levels in both bread types 16 ACS Paragon Plus Environment

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344

compared with fecal background at the end of incubation. It might also explain why markedly

345

increased levels in the BB fortified bread were only seen for ferulic acid but not for its metabolites.

346

The metabolism of phenolic acids may have a different pattern in the rye bran compared to wheat

347

bran. In rye bran, the higher content of soluble DF and a softer matrix may cause native rye bran to

348

behave in a more similar way to bioprocessed bran, thus resulting in less difference in the

349

appearance of metabolites in the colonic digestion. It is possible that some of the transformations

350

were so rapid that they occurred before the 2-hour time point of the fermentation.

351

The GC×GC–TOF–MS data did show more intermediary metabolites for native rye bran bread than

352

for bioprocessed rye bran bread during the time course of metabolism (Figures 3B and 3C).

353

Bioprocessing increased the proportion of free ferulic acid, which was lost in the dialysis, and

354

possibly reduced the proportion of diferulates. This change in the proportion of diferulates may

355

reduce the amount of 3-hydroxylated and 3,4-dihydroxylated phenylacetic acids and corresponding

356

lactic acids. This may be the reason why several lactic acid derivatives were absent in the heat map

357

of BB-bread-derived metabolites and why such a small number of significant metabolites was

358

present in the heat map of BB bread. Low concentrations of hydroxylated phenylacetic acids in the

359

further analysis of the BB fortified breads confirm this observation. These observations suggest that

360

bioprocessing may not only affect the time course of the ferulic acid metabolism but also emphasize

361

the metabolic route of ferulic acid via the elimination of the diferulate precursors, seen as the

362

diminished appearance of specific metabolites of diferulates, such as phenyllactic and phenylacetic

363

acids, compared to the ferulic acid metabolites (phenylpropionic acids).

364

Methodological considerations. Combining different analytical methods and a targeted and

365

untargeted approach, metabolite profiling is capable of showing the diversity of the metabolite pool

366

during the incubation with fecal microbiota. Whilst this multitude of compounds is beyond

367

quantitation and the use of authentic standards,19,20 a significant portion of it is potentially 17 ACS Paragon Plus Environment


368

bioavailable from the colon. The in vitro colon model used in this study applies fecal material from

369

several donors, which are pooled to have a consistent microbiota in the incubations. The microbial

370

composition varies only within the frame of intra-individual variation detected in the same subject

371

and does not change markedly in the course of incubation.45 The same microbial composition of the

372

fecal control for each time point is a prerequisite for microbial metabolomics of foods, beverages

373

and pure components to ensure that the responses at each time point are caused by substrates and

374

not by microbial variability. The choice of having a fecal background as the control in statistical

375

analyses is also required to indicate whether the observed compounds are indeed metabolites of the

376

studied food item or from another source, such as the diets of the donors.46,47

377

A large portion of phenolic acids was liberated from the bran matrix during bioprocessing and

378

dialysis and thus was unavailable for colonic fermentation. However, some of the liberated

379

compounds may be accessible for colonic metabolism in vivo despite of considerable absorption

380

occurring in the small intestine and because of enterohepatic circulation.48,49 Additional time points

381

in the initial stage of the fermentation, such as 0.5 h and 1 h, may have revealed additional

382

intermediary metabolites. Quantitative confirmation of some of the observed metabolites, such as

383

hydroxylated phenyllactic acids, demands targeted analysis and an authentic standard, which was

384

not available for the presented study design. Further, the formation of vanillic acid from

385

dihydroferulic acid remains to be confirmed in a model system using pure compounds introduced

386

into the colonic model.

387

The lack of difference in the levels of ferulic acid metabolites between the NB and BB fortified

388

breads may suggest that the rye bran matrix is behaving differently compared to wheat bran during

389

bioprocessing, resulting in less impact on the colonic microbial metabolism. Semi-quantitative

390

metabolite profiling using GC×GC–TOF–MS and UPLC–QTOF–MS was useful in the

391

identification of rye-related intestinal metabolites potentially available for absorption, including 18 ACS Paragon Plus Environment

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392

metabolites previously not reported from rye. Rye bran bioprocessing may have an impact on the

393

composition of metabolites with potential effects on human health, but more investigation is

394

required to determine the significance of these changes in vivo. ABBREVIATIONS

395

DF dietary fiber; AX arabinoxylan; BB bioprocessed bran; NB native bran; SCFA short chain fatty

396

acids; FA ferulic acid; SinA sinapic acid; 4-CA 4-coumaric acid; FC fold change; ID metabolite

397

identification number. CONFLICT OF INTEREST

398

The authors declare no conflict of interest. ACKNOWLEDGEMENTS

399

The authors thank Arja Viljamaa, Eeva Manninen, Siv Matomaa, Annika Majanen, Niina Torttila,

400

Airi Hyrkäs and Miia Reponen for skillful technical assistance. SUPPORTING INFORMATION DESCRIPTION

401

Supporting information. Table S1: Identification of the most significant microbial metabolites

402

from the GC×GC–TOF–MS data of native rye bran bread formed in the in vitro colon model. Table

403

S2: Identification of the most significant microbial metabolites from the GC×GC–TOF–MS data of

404

bioprocessed rye bran bread formed in the in vitro colon model. Table S3: Identification of the

405

phenolic acids and their metabolites from the UPLC–QTOF–MS data of in vitro fermented native

406

and bioprocessed rye bran bread.

407



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A. Metabolic transformations of dietary polyphenols: comparison between in vitro colonic and

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hepatic models and in vivo urinary metabolites. J. Nutr. Biochem. 2016, 33, 111-118.

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39. Scheline, R.R. Metabolism of phenolic acids by the rat intestinal microflora. Acta Pharmacol.

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Toxicol. 1968, 26, 189-205.

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40. Dykes, L.; Rooney, L. Phenolic compounds in cereal grains and their health benefits. Cereal

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42. Scheline, R. 4-Methylcatechol, a metabolite of homoprotocatechuic acid. Cell. Mol. Life Sci.

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44. Pekkinen, J.; Rosa, N.N.; Savolainen, O.; Keski-Rahkonen, P.; Mykkänen, H.; Poutanen, K.;

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Micard, V.; Hanhineva, K. Disintegration of wheat aleurone structure has an impact on the

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metabolite profile of diet-induced obese mice. Nutr Metab (Lond) 2014, 11, 1.

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45. Aura, A.; Maukonen, J. One Compartment Fermentation Model, In The Impact of Food

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46. Aura, A.; Martin-Lopez, P.; O’Leary, K.A.; Williamson, G.; Oksman-Caldentey, K.; Poutanen,

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K.; Santos-Buelga, C. In vitro metabolism of anthocyanins by human gut microflora. Eur. J. Nutr.

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2005, 44, 133-142.

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47. Bazzocco, S.; Mattila, I.; Guyot, S.; Renard, C.M.; Aura, A. Factors affecting the conversion of

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apple polyphenols to phenolic acids and fruit matrix to short-chain fatty acids by human faecal

537

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48. Rondini, L.; Peyrat-Maillard, M.; Marsset-Baglieri, A.; Berset, C. Sulfated ferulic acid is the

539

main in vivo metabolite found after short-term ingestion of free ferulic acid in rats. J. Agric. Food

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Chem. 2002, 50, 3037-3041.

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49. Borges, G.; Lean, M.E.; Roberts, S.A.; Crozier, A. Bioavailability of dietary (poly) phenols: a

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function 2013, 4, 754-762.

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Funding from Tekes (the Finnish Funding Agency for Innovation), Academy of Finland, and the

545

Faculty of Health Sciences (University of Eastern Finland) are gratefully acknowledged for the

546

financial support of this study.

547


Page 26 of 37

Page 27 of 37


FIGURE CAPTIONS 548

Figure 1. A three-dimensional principal component analysis (PCA) of the sample data obtained

549

from LC–MS in the RP negative mode. NB = bread with native rye bran, BB = bread with

550

bioprocessed rye bran, FC = fecal control. The number indicates the time point in hours.

551

Figure 2. A clustering analysis of the molecular features (n = 4346) detected in the RP negative

552

mode, including the tentatively identified phenolic acids.

553

Figure 3. A heat map of over-expressed (red) and under-expressed (blue) metabolites formed in the

554

in vitro colon model by human fecal microbiota. A. The metabolite profile from bioprocessed rye

555

bran bread against native rye bran bread. B. The metabolite profile from native rye bran bread

556

against fecal control. C. The metabolite profile from bioprocessed rye bran bread against fecal

557

control. The number indicates the identification number (ID) of the metabolite. FC is the fold

558

change between the response and the control (the ratio of average responses at the time point with

559

highest response); a negative value signifies downregulation. The significances are expressed with

560

asterisks (* p < 0.05; ** p < 0.01; *** p < 0.001). Metabolites identified with a standard are

561

signified with 1.

562

Figure 4. The levels of selected phenolic acids and their metabolites during the colonic

563

fermentation, analyzed with either the GC×GC–TOF–MS method (quantitation with standards) or

564

the LC–MS method (reversed-phase column, negative ionization mode; marked with an asterisk).

565

Figure 5. The proposed degradation of ferulic acid in the in vitro colon model by human fecal

566

microbiota, according to Braune et al.,17 Zhao and Moghadasian,36 Feliciano et al.,37 and the

567

presented study. The metabolic routes with a dashed arrow have not yet been verified. 8-O-4-

568

diferulic acid (marked with an asterisk) could not be identified in this study.



Page 28 of 37

TABLES Table 1. Dietary Constituents (g/100 g d.w.) of Native Rye Bran (NB) and Bioprocessed Rye Bran (BB) Fortified Wheat Breads Before and After Enzymatic Digestion in the Upper Intestinal Model.

bread with NB before digestiona

bread with BB before digestiona

bread with NB after digestion

bread with BB after digestion

starch g/100 g d.w.

48.7 (1)

47.4 (1)

9.9 (2)

9.6 (3)

protein g/100 g

14.2 (1)

15.1 (1)

17.3 (2)

16.7 (3)

fat g/100 g

9.8 (1)

10.1 (1)

18.5 (2)

16.9 (3)

soluble DF g/100 g

3.3 (1)

4.3 (2)

5.7 (3)

7.1 (4)

insoluble DF g/100 g

15.1 (1)

11.3 (1)

34.5 (2)

27.2 (3)

sum of soluble and insoluble DF

18.4 (1)

15.6 (1)

39.7 (2)

34.3 (3)

total AX g/100 g

9.7 (1)

10.9 (1)

14.30 (2)

14.25 (2)

soluble AX g/100 g

1.0 (1)

2.8 (2)

2.29 (3)

4.70 (4)

β-glucan g/100 g

1.5 (1)

0.5 (2)

3.0 (3)

1.2 (4)

fructan g/100 g

2.0 (1)

1.2 (2)

1.7 (3)

1.4 (4)

a

Published earlier in Nordlund et al 2013.28

For each constituent, values marked with the same number are not statistically different from each other (p > 0.05). The comparison was made between the breads containing brans NB and BB separately before and after digestion.


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Table 2. Free and Bound Individual Phenolic Acids and Total Phenolic Acids in Breads Before and

bread with BB

bread with NB

After the in Vitro Enzymatic Digestion. in bread (mg/g)

after in vitro digestion (mg/g)

average

SD

average

SD

free FA

0.016

0.000

0.000

-

bound FA

1.066

0.004

0.533**

0.007

free SinA

0.008

0.000

0.000

-

bound SinA

0.234

0.002

0.096*

0.006

free 4-CA

0.001

0.000

0.000

-

bound 4-CA

0.036

0.002

0.006*

0.000

total

1.361

0.635

average

SD

average

SD

free FA

0.162

0.001

0.000

-

bound FA

1.026

0.042

0.137**

0.004

free SinA

0.029

0.001

0.000

-

bound SinA

0.227

0.002

0.022*

0.001

free 4-CA

0.004

0.000

0.000

-

bound 4-CA

0.032

0.003

0.002*

0.000

total

1.480

0.160

Asterisks after the letter indicate the significance between the breads fortified with native and bioprocessed rye bran: * p < 0.05, ** p < 0.01. The total phenolic acid content corresponds to the sum of free and bound ferulic, sinapic and 4-coumaric acids. FA: ferulic acid; SinA: sinapic acid; 4CA: 4-coumaric acid. 569 29 ACS Paragon Plus Environment


FIGURES Figure 1.


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Figure 2.



Figure 3A.

570


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Figure 3B.



Figure 3C.


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Figure 4.



Figure 5.


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TOC GRAPHIC


Effect of Bioprocessing on the In Vitro Colonic ... - ACS Publications

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