Sarah BazzoccoIsmo MattilaSylvain GuyotCatherine M.G.C. RenardAnna-Marja Aura

Factors affecting the conversion of applepolyphenols to phenolic acids and fruitmatrix to short-chain fatty acids by humanfaecal microbiota in vitro

Received: 26 June 2008Accepted: 23 September 2008Published online: 15 October 2008

S. Bazzocco Æ I. Mattila Æ A.-M. Aura (&)VTT, Technical Research Center ofFinlandP.O. Box 1000, Tietotie 202044 VTT, FinlandE-Mail: anna-marja.aura@vtt.fi

S. GuyotINRA, UR117Rennes, France

C.M.G.C. RenardINRA, UMR408Avignon, France

j Abstract Proanthocyanidins(PAs) in apples are condensedtannins comprised mostly of())-epicatechin units with someterminal (+)-catechins. PAs, espe-cially those having a long chain-length, are absorbed in the upperintestine only to a small extent andare passed to the colon. In thecolon they are subjected tomicrobial metabolism by colonicmicrobiota. In the present article,the ability of human microbiota toferment apple PAs is studied.Freeze-dried fruit preparations(apple, enzymatically digestedapple, isolated cell-walls, isolatedPAs or ciders) from two varieties,Marie Menard and Avrolles, con-taining PAs of different chainlengths, were compared. Fermen-tation studies were performed inan in vitro colon model usinghuman faecal microbiota as aninoculum. The maximal extent ofconversion to known microbialmetabolites, was observed at late

time point for Marie Menardcider, having short PAs. In thiscase, the initial dose also contrib-uted to the extent of conversion.Long-chain PAs were able toinhibit the in vitro microbialmetabolism of PAs shown as lowmaxima at early time points.Presence of isolated PAs alsosuppressed SCFA formation fromcarbohydrates as compared withthat from apple cell wall or faecalsuspension without substrates.The low maximal extents at earlytime points suggest that there is acompetition between the inhibi-tory effect of the PAs on microbialactivity, and the ability to convertPAs by the microbiota.

j Key words procyanidin –cell-wall – Malus domesticaBorkh – in vitro fermentation –gut microbiota

Introduction

Dietary polyphenols are ubiquitous plant derivedsecondary metabolites. Proanthocyanidins (PAs) arecondensed tannins comprised mainly of ())-epicate-chin units, although some of the terminal units mayalso be of (+)-catechin [41]. In apples flavan-3-ols(including condensed tannins and monomeric cate-

chins) and hydroxycinnamic acids correspond to themajor polyphenol classes in the fruit [18]. The esti-mated daily intake of flavan-3-ols from human dietvaries between 100 and 550 mg [3, 20].

Low amounts of different flavan-3-ol monomers,PA dimers and trimers have been detected in rat urineas sulphate metabolites [44], or in human plasmaafter consumption of red wine or cocoa as glucu-ronidated, sulphated and methylated conjugates [21,

ORIGINAL CONTRIBUTIONEur J Nutr (2008) 47:442–452DOI 10.1007/s00394-008-0747-2

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40]. Thus most of the PAs are likely to reach colonand subjected there to microbial conversion. Poly-phenols, proanthocyanins among them, can be con-verted to phenolic acids by colonic microbiota, whichincreases the occurrence and significance of phenolicacids as one of the major group of circulatingmetabolites [24]. When polymeric 14C-labeled PAswere incubated with human faecal microbiota in vitro,9–22% of the label was found in the metabolite pool,and ethyl acetate soluble metabolites represented2.7% of the initial radioactivity [12]. When 14C-la-beled PAs were given by gavage to rats, only a smallproportion of the label was found in liver (1–1.5%),urine (1–2%) and carbon dioxide (1–2%) [1]. Poly-meric PAs were catabolised by human colonic mic-robiota in vitro producing several phenolic acids: 3-hydroxyphenylpropionic acid, 3-phenylpropionicacid, 4-hydroxyphenylpropionic acid and 4-hydroxy-phenylacetic acid as major microbial metabolites [12].Chocolate consumption increased the human urinaryexcretion of 3-hydroxyphenylpropionic acid, 3,4-dihydroxyphenylacetic acid, 3-hydroxyphenylaceticacid, 3-methoxy-4-hydroxybenzoic acid (vanillic acid)and 3-hydroxybenzoic acid [39]. However, the effect ofdegree of polymerization on microbial conversion ofPAs to phenolic acids has not been studies adequately.

Cell-wall polysaccharides in apples are mainly pecticpolysaccharides and cellulose, which enter the colonand are fermented by the microbiota to short-chain fattyacids (SCFA) [8, 31]. There has been studies concerningthe fermentability of cell-wall polysaccharides and iso-lated PAs separately, however, these components areconsumed together in the actual fruit and are known tobind spontaneously to each other [25]. This associationmay cause differences in the microbial metabolism, asdescribed by Aprikian et al. [2], when the preparationsare introduced together than if they are administeredseparately. Furthermore, butyrate, one of the SCFA,has been associated with local beneficial effects oncolon health: improved cell proliferation and inductionof apoptosis enhancing healthy tissue turnover [23].

Therefore this work aims to compare microbialmetabolism of PAs and cell-wall polysaccharidesalone and in combination. PA conversion to phenolicacids is also studied from natural apple or cidermatrices. The products were selected to elucidatethe effect of PA chain length on the fermentation byhuman gut microbiota.

Materials and Methods

j Materials

Apples (Malus domestica Borkh) of the Marie Menardand Avrolles varieties were obtained from the exper-

imental orchard of Institute Francais de ProductionsCidricoles (Sees, Orne, France) in 2005. Several applematerials (crude apple powder, enzymatically pre-di-gested apple powder, purified cell-walls, purified PAextracts and freeze-dried cider) were prepared fromfruits of these two distinct apple varieties.

Reagents for analysis of the microbial metabolitesof flavanols were as follows: Heptadecanoic acid andsuccinic acid-2,2,3,3-d4 used as the internal standard,was purchased from Sigma-Aldrich Inc., (St. Louis,USA) and the following compounds were used asstandards: benzoic acid (BA), 3-hydroxybenzoicacid (3-OHBA), 3-(4-hydroxyphenyl)propionic acid(4-OHPPr) and 3-(3,4-dihydroxyphenyl)propionicacid (3,4-diOHPPr) were products from Aldrich,(Steinheim, Germany). 4-Hydroxybenzoic acid (4-OHBA), 2-(3-hydroxyphenyl)acetic acid (3-OHPAc)and 2-(3,4-dihydroxyphenyl)acetic acid (3,4-diOHPAc)were purchased from Sigma (St. Louis, USA). 3-Phen-ylpropionic acid (3-PPr) and 3,4-dihydroxybenzoic acid(3,4-diOHBA), were from Fluka (Buchs, Switzerland)and 3-(3-hydroxyphenyl)propionic acid (3-OHPPr) waspurchased from Alfa Aesar (Karlsruhe, Germany). N-Methyl-N-trimethylsilyl-trifluoracetamide (MSTFA)from Pierce (Rockford, USA) was used as the silylationreagent.

j Methods

Preparation of apple products

The preparation steps of apple samples are describedin Fig. 1. Apple powders were prepared from a batch

Marie Ménard/ Avrolles apples (Malus domestica Borkh)

Ciders

Freeze-drying

Enzymatic digestion byalimentary enzymes at

37°C in anaerobic conditions

PA acetone extract Cell-walls

Time course of metabolites formed by faecal microbiotain vitro at 37°C for 0-24 h in anaerobic conditions

Fig. 1 Schematic diagram of preparation of apple samples prior to in vitrofermentation with human faecal microbiota

S. Bazzocco et al. 443Factors affecting the conversion of apple polyphenols to phenolic acids and fruit matrix

of 10 fruits as described by Renard et al. [38]. PAacetone extracts were prepared by successive metha-nol and aqueous acetone extractions from the freeze-dried apple powders (150 g) according to Sanoneret al. [41] and purified as described in Guyot et al.[17]. Cell-walls for the fermentation experiments wereisolated from peeled fresh apples of the both varietiesby the phenol-buffer method described in Renard[36]. Ciders were prepared from apple juices from15 kg of Avrolles or Marie Menard apples by crushingand pressing the fruits. The juices were clarifiedenzymatically by using polygalacturonase and pec-tinmethylesterase enzyme mixture (Rapidase C80L;DSM Food, Seclin, France) and microfiltrated (poly-vinylidine difluoride membrane, 0.45 mm, MilliporePellicon, Millipore Corp.) as described by Hubertet al. [22]. The juices were fermented with driedSaccharomyces uvarum SRC-73 strain with a dose of5Æ105 cfu/ml until the density of the juice was below1,000 g/l. The two cider batches (2 l) were concen-trated under vacuum to remove ethanol and freeze-dried.

In vitro enzymatic digestion of apple samples

Enzymatic in vitro digestion [4] was performed underanaerobic conditions at 37�C with magnetic stirring(250 rpm) for the freeze-dried apple samples. Mouth(neutral), stomach (acidic) and duodenum (neutral)were mimicked by successive additions of porcineenzymes from Sigma (St. Louis, USA): salivarya-amylase (mouth), pepsin (stomach stage) andpancreatin, bile and mucin (duodenum), respectively.Samples after digestion were washed with distilledwater under anaerobic conditions, rapidly frozenusing liquid nitrogen and freeze-dried prior to fer-mentation in the in vitro colon model. Apple cell-wallpreparations, freeze-dried ciders or PA extracts werenot digested enzymatically (Fig. 1).

Analysis of apple samples

Polyphenols in crude apple powders (before and afterin vitro digestion, BD and AD, respectively) and in theciders were analyzed after thiolysis by reversed phaseHPLC according to Guyot et al. [16]. The purified PAextracts were similarly analyzed according to Guyotet al. [17]. Simple sugars (glucose, fructose andsucrose) were measured colorimetrically using theBoehringer analysis kit (R-Biopharm, St Didier au

Mont d’or, France). Alcohol insoluble solids (AIS)from freeze-dried apples and digested apples wereprepared according to Renard [36] using 70% ethanolextraction and submitted to pre-hydrolysis in 13 Msulphuric acid (1 h, room temperature) [43]. Cidersand polyphenol extracts were directly submitted topolysaccharide analysis. Myo-inositol was used asinternal standard and the individual neutral sugarswere analyzed by gas chromatography as alditol ace-tates [13]. Uronic acids were determined after acidhydrolysis (Seaman procedure) of cell walls by spec-trophotometric m-hydroxydiphenyl assay describedwith galacturonic acid as external standard byBlumenkrantz and Asboe-Hansen [9]. Complex carbo-hydrates were quantified as sugars detected after acidhydrolysis in the AIS, and monomeric and polymericcarbohydrates were expressed as total amounts ofmonomeric sugar units (lmol/g apple sample).

j In vitro fermentation of apple products

Fermentation experiments were performed understrictly anaerobic conditions according to Aura et al.[5, 6] using 10% (w/v) faecal suspension based on amethod described by Barry et al. [8]. Faecal sus-pension was prepared using phosphate buffer (pH5.5) by pooling the faeces of four (Experiment 1) orfive (Experiment 2) healthy donors. Suspension wasthen diluted to the concentrations above and appliedto the samples (100 or 25 mg). Three replicatesamples were incubated in a water bath at 37�C for0, 1, 2, 4, 6, 8 and 24 h and stirred magnetically(250 rpm). Four 2 ml aliquots were drawn from thebottles and microbial metabolites and SCFA wereanalyzed.

Analysis of the microbial metabolites

Microbial metabolites of phenolic compounds wereanalyzed using heptadecanoic acid and succinic acid-2,2,3,3-d4 as internal standards by GC-MS usingselective-ion-mode (SIM) after extraction twice with3 ml ethyl acetate, evaporation of the solvent andsubsequent silylation as follows: Dichloromethane(100 ll) and MSTFA (30 ll) was added to the sam-ples, and incubated (5 min, 50�C) [7]. The metaboliteformation was calculated as lmol of formed meta-bolite at each time point per 10 ml. The extent ofmetabolite formation (%) was calculated from Eq. 1,

ExtentSubstrateðtÞ ¼P

Metabolitef SubstrateðtÞ �MetaboliteFeacal controlðtÞg½lmol�Total PA content ½lmol� � 100% ð1Þ

444 European Journal of Nutrition (2008) Vol. 47, Number 8� Steinkopff Verlag 2008

in which t = time point; R = sum of the followingmetabolites: 3,4-diOHPPr, 3-OHPPr, 3-PPr, BA, 3,4-diOHPAc, 3-OHPAc, metabolites, which differed fromthe faecal background in the presence of substrate;PA: Proanthocyanidin. SCFA were analyzed by GCafter diethylether extraction according to Aura et al.[6]. Total SCFA formation was a sum of acetic, pro-pionic and butyric acids. The relative proportions ofthe individual SCFA was calculated in respect to thetotal SCFA formation at time point 24 h. All resultswere expressed as averages and standard deviationsfrom three replicate measurements.

Results

j Effect of enzymatic digestion on PAs andcarbohydrates

The characterisation of the phenolic compounds fromMarie Menard and Avrolles apples powders before(BD) and after (AD) enzymatic digestion, cell-wallpolysaccharides, PA extracts and ciders is describedin Table 1. The total flavan-3-ol contents and

caffeoylquinic acid contents were expressed, becausethey were known precursors of phenylpropionic acidderivatives. The apple PAs consisted mainly of ())-epicatechin units regardless of the variety or theproduct. The gap between total polyphenol contentand the sum of flavan-3-ols and caffeoylquinic acidwas very small in almost all the apple samples, exceptfor ciders, which also contained p-coumaroylquinicacid 1.08 and 2.04 mg/g d.w. in Marie Menard andAvrolles, respectively. The other significant compo-nents were phloridzin (0.60 and 0.98 mg/g d.w.;Table 1) and phloretin xyloglucoside (0.70 and2.00 mg/g d.w.) in Marie Menard and Avrolles ciders,respectively. However, their microbial metabolites arenot known and thus they were not included in theprecursors of phenolic acid metabolites. The cell-wallpreparations did not contain precursors of microbialphenolic acid metabolites.

The apple varieties had been chosen to present dif-ferent average degree of polymerization (aDPn). MarieMenard apple products contained short PA chains(aDPn 2.2–9.5), while Avrolles apples contained longpolymers (aDPn 7.4–71.2). The PA extract showed ahigher aDPn than the apple material as the monomers

Table 1 Composition of apple powders (BD before and AD after digestion), cell-wall preparations, PA extracts and ciders

Marie Menard Avrolles

Apple powder Apple powder

BD AD Cell-walls PA extract Cider BD AD Cell-walls PA extract Cider

Polyphenol content mg/g dry weight apple sampleTotal flavan-3-ols 31 25 ND 777 35 24 56 ND 883 1Caffeoylquinic acid 6 1 ND 35 31 1 0 ND 6 5Coumaroylquinic acid 0 0 ND ND 1 1 0 ND 0 2Phloridzin 0 0 ND ND 1 1 0 ND ND 1Phloretin xyloglucoside 0 0 ND ND 1 1 0 ND ND 2Total polyphenols 38 26 ND 817 68 28 57 ND 903 11

Characteristics of PAsAverage degree of polymetization (aDPn) 4.3 8.2 NA 9.5 2.2 35.2 71.2 NA 35 7.4Catechin units (%) 3 2 NA 2 2 0 0 NA 0 Traces())-Epicatechin units (%) 97 98 NA 99 98 100 100 NA 100 100

Soluble carbohydrates in apple sampleFructose (mg/g) 307 29 NA NA NA 226 27 NA NA NAGlucose (mg/g) 92 74 NA NA NA 94 54 NA NA NASucrose (mg/g) 208 58 NA NA NA 153 27 NA NA NASoluble carbohydrates (lmol/g) 3,433 911 NA NA NA 2,673 608 NA NA NA

Polymeric carbohydrates (mg/g apple sample) 215 545 1,000 1,000 1,000 116 627 1,000 1,000 1,000Carbohydrate composition (mg/g polymers)Rhamnose 9 6 9 1 3 11 6 11 0 3Fucose 9 8 9 0 0 12 6 12 0 0Arabinose 130 123 130 3 2 106 99 106 2 0Xylose 52 67 52 1 13 52 58 52 2 8Mannose 27 6 27 1 7 26 5 26 0 8Galactose 46 52 46 2 5 60 70 60 1 4Glucose 304 286 304 16 159 281 347 281 18 111Galacturonic acid 194 105 194 1 4 229 128 229 0 4Polymeric carbohydrates (lmol/g) 1,120 2,425 5,209 172 1,265 611 3,006 5,269 154 919

aDPn average degree of polymerization of total flavan-3-ols, including monomeric flavan-3-ols

S. Bazzocco et al. 445Factors affecting the conversion of apple polyphenols to phenolic acids and fruit matrix

and small oligomers had been extracted in the dis-carded methanol fraction. The lowest aDPn were foundin the ciders. The total polyphenol content decreased inMarie Menard apples after enzymatic digestion andseparation of solids, whereas it increased in corre-sponding Avrolles apples after enzymatic digestion andremoval of soluble components (Table 1).

Freeze-dried apple samples contained solublesugars, the amount of which decreased after digestionand washing the residue. The alcohol insolublefraction (AIS) increased in the enzymatic digestion,which was expected. Cell-walls were purified frompolyphenols and soluble carbohydrates, and containedonly alcohol insoluble polymeric carbohydrates. Cidersamples were applied as such into the colon model, be-cause most of the digestible carbohydrates were alreadyfermented in the cider making process.

j Profiles of microbial metabolites

The apple samples (Fig. 1) were fermented with freshhuman faecal inoculum from different donors in twoexperiments, one for each variety using doses indi-cated in Table 2. The microbial metabolite profiles areshown for Marie Menard apple samples (Experiment1) in Fig. 2 and for Avrolles series (Experiment 2) inFig. 3. Metabolite dynamics can be observed fromMarie Menard experiment (Fig. 2) better than fromAvrolles apple experiment (Fig. 3), because MarieMenard samples showed distinctively higher amountsof microbial metabolites than corresponding Avrollessamples. Primary metabolite (3,4-diOHPPr, Fig. 2a) is

formed as a result of ring-fission of catechins and itshows a high profile only in samples containingpurified PAs or Marie Menard cider. Secondarymetabolites, 3-OHPPr and 3-PPr, (Figs. 2b and 3-PPr;Fig. 2c) are dehydroxylation products of the firstmetabolite. Some differences in benzoic acid (BA) andphenylacetic acid metabolites can also be observedbetween non-digested and digested Marie Menardapple samples (Fig. 2d–f). In contrast to phenylaceticacids, BA was released or formed from pre-digestedMarie Menard apple more than from non-digestedapples. Cider, having the shortest chain length PAs(Table 1), showed highest profiles of phenylpropionicand phenylacetic acid, whereas BA profile from ciderwas below that of the digested apple. Avrolles ciderand samples containing PA extract showed higherphenylpropionic acid profiles than correspondingapple samples (Fig. 3b, c), whereas formation of BAwas more pronounced in the samples containingAvrolles PA extract (Fig. 3d). When cell wall poly-saccharides and PA extract were fermented togetherin Avrolles series, the microbial metabolite profileswere in accordance with the purified PA extract(Fig. 3a–f). The postulated pathway of microbialmetabolism of apple PA is presented in Fig. 4,including the possible formation of benzoic acid fromphenylpropionic acids.

j Extent of microbial metabolism of PAs

The doses of samples applied to the in vitro colonmodel, their total amount of microbial metabolite

Table 2 Total amount of precursors of phenolic metabolites and carbohydrates per dose substrate in the in vitro colon model and maximal extents and time pointof maxima of microbial metabolism

Substrates Dose (mg) Phenolic precursorsa

(lmol per dose)Carbohydrates(lmol per dose)

Maximal extentb

(% of precursors)Time point ofmaxima (h)

Experiment 1Marie MenardApple powder 100 13.1 455 14 8Digested apple powder 100 9.0 334 10 6Cell-walls 100 0.0 521 ND NDPA extract 25 72.5 4 3 2Cider 100 21.1 127 44 24

Experiment 2AvrollesApple powder 100 9.0 328 5 8Digested apple powder 100 19.9 361 2 0Cell-walls 100 0.0 527 ND NDPA extract 25 79.3 4 2 4PA extract + cell-walls 25 + 100 79.3 531 2 8Cider 100 1.7 92 62 4

aSum of total flavan-3-ols (MW 290 g/mol) and chlorogenic acid (MW 354 g/mol), the known precursors of phenylpropionic acid derivatives and benzoic acidbCalculations according to Eq. 1. Sum of 3-(3,4-dihydroxyphenyl)propionic acid, 3-(3-hydroxyphenyl)propionic acid, 3-phenylpropionic acid, benzoic acid, 2-(3,4-dihydroxyphenyl)acetic acid and 2-(3-hydroxyphenyl)acetic acid. Experiments were performed using a pool of faeces from 4 or 5 donors in experiment 1 and 2,respectively

446 European Journal of Nutrition (2008) Vol. 47, Number 8� Steinkopff Verlag 2008

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Fig. 3 Microbial metabolite profiles of precursors (total flavan-3-ols andchlorogenic acid) from Avrolles apple samples. a 3-(3,4-Dihydroxyphenyl)propionicacid (3,4-diOHPPr); b 3-(3-Hydroxyphenyl)propionic acid (3-OHPPr); c 3-Phenyl-propionic acid (3-PPr); d Benzoic acid (BA); e 2-(3,4-Dihydroxyphenyl)acetic acid

(3,4-diOHPAc) and f 2-(3-Hydroxyphenyl)acetic acid (3-OHPAc). Symbols: Apple(dashed with filled diamond); Digested apple (dashed with diamond); Cell-walls(dashed with triangle); PA (dotted with filled square); PA and cell walls (dashed withopen square); Cider (dashed with filled circle); No substrate (dotted with open triangle)

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Fig. 2 Microbial metabolite profiles of precursors (total flavan-3-ols andchlorogenic acid) from Marie Menard apple samples. a 3-(3,4-Dihydroxy-phenyl)propionic acid (3,4-diOHPPr); b 3-(3-Hydroxyphenyl)propionic acid (3-OHPPr); c 3-Phenylpropionic acid (3-PPr); d Benzoic acid (BA); e 2-(3,4-

Dihydroxyphenyl)acetic acid (3,4-diOHPAc) and f 2-(3-Hydroxyphenyl)acetic acid(3-OHPAc). Symbols: Apple (dashed with filled diamond); Digested apple (dashedwith open diamond); Cell-walls (dashed with open triangle); PA (dotted with filledsquare); Cider (dashed with filled circle); No substrate (dotted with open triangle)

S. Bazzocco et al. 447Factors affecting the conversion of apple polyphenols to phenolic acids and fruit matrix

precursors (a sum of total amount of flavan-3-ols andcaffeoylquinic acid in lmol per dose) and the totalamount of sugar units (lmol per dose) are describedin Table 2. Extents of conversion were calculatedtaking into account the six microbial metabolites,which showed distinctive difference from faecalbackground in the presence of substrates (3,4-di-OHPPr, 3-OHPPr, 3-PPr, BA, 3,4-diOHPAc, 3-OH-PAc), and the total flavan-3-ol and caffeoylquinic acidcontents of the samples subjected to colonic micro-biota in vitro. Extents were calculated for all the timepoints as described in the Eq. 1 (results not shown)and maximal extents and their time points are de-scribed in Table 2. Metabolite profiles showed thehighest concentration of most of the metabolites forciders, resulting in high maximal extent of conversion44 and 62%, for Marie Menard and Avrolles, respec-tively (Table 2). Avrolles cider sample showed ahigher extent of conversion than Marie Menard cider,but also lower amounts of precursors.

Maximal extent of conversion was reached muchlater (at 6–8 hours) with Marie Menard apple samplesthan with corresponding PA extract (2 h) (Table 2).The PA extracts were metabolised to the lowest extentand their conversion to phenolic acids was stoppedearly (2 and 4 hours, for Marie Menard and Avrolles,respectively). However, when Avrolles PA extract wasfermented in the presence of cell-wall preparation, theconversion time was doubled to 8 h compared withthat of PA extract alone without large extension ofconversion efficiency (Table 2). Both cell-wall prepa-rations were devoid of phenolics and no phenolic acidmetabolites were formed, when they were fermentedalone by faecal microbiota.

j SCFA formation

Avrolles apple samples showed larger differences inthe SCFA formation than corresponding Marie Me-nard samples, but the trends were similar (Fig. 5a, c):Non-digested apple had higher fermentation rate thanthe digested sample. Cell-wall preparations showedthe same rate and extent of SCFA formation as di-gested apple samples. Furthermore, Avrolles cideralso differed more in SCFA formation than MarieMenard cider as compared with the correspondingSCFA formation profiles of the fruit matrices. Pres-ence of PA extract was also able to suppress SCFAformation as compared with faecal control or with cellwall preparation alone (Fig. 5a, c).

Relative proportions of SCFA showed highest rel-ative proportion of acetate, intermediary for butyrateand lowest for propionate in all apple samplesregardless of the variety. Non-digested Marie Menardapples and cider showed the highest relative propor-tions of butyrate as compared with other apple sam-ples. However, the same result was not that evidentfor Avrolles cider due to high standard deviationsbetween replicates (Fig. 5b, d).

Discussion

j Effect of enzymatic digestion on compositionof apple samples

The effect of the enzymatic digestion and removal ofsoluble components on total flavan-3-ol content wasdependent on the chain length of PAs. When the chainlength was short as in Marie Menard apples, the totalflavan-3-ol content decreased and the average degree ofpolymerization (aDPn) increased after rinsing, indicat-ing loss of short-chain flavan-3-ols. As aDPn of Avrollesapple flavan-3-ols was already high, after isolation ofnon-digestible solids the PAs were even longer. Removalof soluble components increased the relative content oflong-chain PAs. The PA chain length in ciders wasshorter than in apples as a contribution of the affinity oflong-chain PAs for apple cell-walls [25]. The same phe-nomenon affects extraction to water during the rinsingprocedure after the enzymatic digestion. The gap bet-ween the amounts of total polyphenols and those of totalflavan-3-ols and caffeoylquinic acid decreased in thedigestion, reflecting removal of soluble monomericphenolic compounds. Cell-wall preparations, PA ex-tracts or ciders were not enzymatically digested, becauseremoval of soluble components after the digestion pro-cedure would not have been possible without losses ofcomponents under investigation.

Apple cell-wall AIS fractions had roughly the samecarbohydrate composition before and after enzymatic

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Fig. 4 Postulated microbial metabolites from apple PAs

448 European Journal of Nutrition (2008) Vol. 47, Number 8� Steinkopff Verlag 2008

digestion. The polysaccharides present in apple cell-walls are pectins (galacturonic acid, arabinose, ga-lactose, rhamnose), cellulose (glucose) and xylogal-acturonans (xylose, non- cellulosic glucose, galactose,fucose) [36]. In this study, some of the pectic poly-saccharides were lost during digestion, shown asdecrease in galacturonic acid. The loss was evident inboth Avrolles and Marie Menard apples.

j Relevance of dose in the in vitro fermentation

Daily intake of PAs varies according to the proposedsource: Auger et al. [3] reported that with moderateconsumption of red wine (180 ml/day), containing100.4 mg (0.35 mmol) flavanol units. Hammerstoneet al. [20] estimated that the daily intake per serving offood could vary from 147.1 to 164.7 mg (0.51–0.57 mmol flavanol units). A moderate red wine con-sumer, who eats three servings of food daily, couldhave approximately 100–550 mg of PAs (0.3–1.9 mmol flavanol units). According to Guyton andHall [19] 1,500 ml of chyme is emptied daily to caecumfrom ileum. Thus the average calculated concentrationof PAs entering the caecum could vary between 0.2 and1.3 lmol/ml. In the present study the dose of phenolicacid precursors varied from 1.7 to 79.3 lmol per 10 mlof faecal suspension corresponding 0.17–7.9 lmol/mlin the in vitro colon model. Thus the doses of PAsfrom fruit samples in the experiments were of the samemagnitude as the possible physiological concentra-

tions could be as calculated from the possible dailyintake data.

j Identification of the microbial metabolites

The anticipated microbial metabolites of apple sam-ples were phenylpropionic acid derivatives (3,4-di-OHPPr, 3-OHPPr and 3-PPr), benzoic acid (BA) fromflavan-3-ols (Fig. 4) and hydroxycinnamic acids andacetic acid derivatives (3,4-diOHPAc and 3-OHPAc)from flavonols [5]. Deprez et al. [12] showed that themain microbial metabolites of synthesized labeledPAs were 3-OHPPr, 4-OHPPr, 4-OHPAc and 3-PPr. Inour study 4-OHPPr concentrations in apple samplesdid not differ from those in the faecal background and4-OHPAc was not among the standards. Caffeoylqui-nic acid can be converted to 3-OHPPr, which can bemetabolized to benzoic acid via b-oxidation [15].Thus caffeoylquinic acid metabolism by gut micro-biota can contribute to the concentrations of thesemicrobial metabolites. These results are in accordancewith the data concerning urinary metabolites in vivoafter intake of cocoa PAs [39], but naturally hepaticmetabolites such as hippuric acid (glycinated BA)or methoxylated phenolic acids, are not present inin vitro faecal incubations. The microbial metabolitesof the minor phenolic compounds in cider, phloretinand phloridzin (phloretin-2-O-glucoside) are not toour knowledge known. However, phloretin is formedfrom two aromatic hydroxylated rings coupled with

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Fig. 5 (a) Total short-chain fattyacid (SCFA) formation (lmol) and(b) relative proportions of SCFA (%)in samples containing Marie Menardapple products; (c) Total SCFAformation (lmol) and (d) relativeproportions of SCFA (%) in samplescontaining Avrolles appleproducts.Total SCFA contents are thesums of acetic (less densed dots),propionic (medium densed dots) andbutyric (dots with black background)acid contents in the samples and therelative proportions of individualSCFA were calculated as % of totalSCFA. Symbols: Apple (dashed withfilled diamond); Digested apple(dashed with open diamond); Cell-walls (dashed with open triangle); PA(dotted with filled square); PA andcell walls (dashed with open square);Cider (dashed with filled circle); Nosubstrate (dotted with open triangle)

S. Bazzocco et al. 449Factors affecting the conversion of apple polyphenols to phenolic acids and fruit matrix

CO–CH2–CH2-bridge [26] and it could be speculatedthat phloretin could contribute to the formation ofhydroxyphenylacetic acid and benzoic acid, if onlyoxygen ether bond of the bridge is broken similarly asester bonds are by colonic esterases [34].

It is possible that the low extent of ring-fissionmetabolites observed in Avrolles series was due tometabolites not detected in the targeted analysisperformed by GC-MS. A later study has shown that(+)-catechin and ())-epicatechin can also be con-verted mainly to 5-(3,4-dihydroxyphenyl)- and 5-(3-hydroxyphenyl)valeric acid metabolites instead ofcorresponding hydroxyphenylpropionic acid deriva-tives, depending on the human microbiota used as aninoculum [7]. Furthermore, ())-epicatechin and itsgalloylated derivatives can be converted to hydroxy-lated valerolactone derivatives [30]. However, valericacids or valerolactones are not commercially availableand thus were missed by the method used in thepresent study.

j Effect of chain length of PAs

Short PAs in cider exhibited a strong metabolite for-mation during the entire fermentation experimentand higher concentration of PAs could be toleratedwithout inhibition of metabolite formation. Theamount of precursors in Marie Menard cider was 12times higher than that in Avrolles cider. The Avrollescider sample showed a higher apparent extent ofconversion than the Marie Menard cider, but since itcontained lower amount of precursors, the extent wasmore dependent on the low precursor content thanhigh amount of detected metabolites. PAs in ciderswere shorter in chain length due to selective extrac-tion of lower molecular weight PAs [11] or due topoorer extractability of long-chain precursors to thecider [25].

The chain length of PAs has a more crucial role intheir microbial metabolism than the dose. The cal-culated extents confirmed the result: the highestmaximal extents were achieved, when the conversionto phenolic acids was allowed to last longer, whereaslow maximal extents were reached at early time points(2–4 h). Long chain length of PAs in Avrolles sampleswas shown as low amounts of metabolites. It is worthof note that different donors of faecal samples possesa different intestinal microbiota, which causes highindividual variation in concentrations of metabolites[10] and even change in the site of ring-fission [7].Thus comparisons between different fermentationexperiments using different inocula can be made onlywith great caution.

In the present study the extents of PA conversionwere all in all very low for both the PA extracts. This

result is in agreement with the previous work withradiolabelled PAs performed by Deprez et al. [12], inwhich PAs were converted only to a small extent (2.7%of original dose). Furthermore, Gonthier et al. [14]showed in their rat study that the degree of polymeri-zation strongly decreased absorption and both thenumber of metabolites and extent of microbial metab-olism. Scalbert [42] has reported antimicrobial effectsand inhibitory action of long-chain PAs on cell-walldegrading enzyme activities. Furthermore, tannins ex-hibit binding to proteins causing haze in beverages [45]and inhibition of digestive enzymes [29]. Even hydro-lysable tannins (ellagitannins) have an ability to inhibitspecific bacteria [35] and proanthocyanidins exhibitedantimicrobial properties against ruminal bacteria [46].

j SCFA

SCFA formation from apple samples and cidersshowed similar profiles and extents regardless of thevariety. The differences between Marie Menard appleproducts were smaller in SCFA production than thosebetween the Avrolles apple products. SCFA profiles ofnon-digested apples were slightly above those of thedigested ones reflecting removal of starch and solublesugars after enzymatic digestion and rinsing proce-dures. Solid apple samples containing more polymericcarbohydrates showed expectedly higher amounts ofSCFA than ciders. Nevertheless, SCFA formation fromciders show that also beverages have a carbohydratematrix despite their soluble nature and the fermen-tation by yeast. SCFA formation from ciders origi-nated most likely from yeast glucans designated ashigh glucose content.

SCFA formation by colonic microbiota in vitro hasbeen shown extensively for various polysaccharidesincluding pectins [8, 31]. The presence of long-chainPAs were consistently able to inhibit the SCFA for-mation from cell-walls and from carbohydrates orig-inated from the faecal inocula as shown by resultsfrom the Avrolles series, a phenomenon which wasnot apparent, when the PAs were enclosed in thenatural apple matrix (Fig. 5a, c). This inhibition ismost likely due to inhibition of cell-wall degradingenzymes of the microbiota [42]. The enzymeshydrolyse the polysaccharides to monosaccharides,which is a step required prior to formation of SCFAby the microbiota. The inhibition of cell-wall poly-saccharide degradation by microbial enzymes in thepresence of PAs is in accordance with the inhibitionobserved for pectolytic enzymes, which was antici-pated to occur either by steric effects or through di-rect enzyme inhibition [37].

Butyric acid production was not high in digestedapple or apple cell-walls, which is in agreement with

450 European Journal of Nutrition (2008) Vol. 47, Number 8� Steinkopff Verlag 2008

literature. Pectic polysaccharides have not shownsignificantly higher relative proportions of butyrate ascompared with other polysaccharide preparations [8,31]. Dextrans (a-glucan) have been shown to increasethe relative proportion of butyrate in in vitro faecalfermentation model [32]. Thus higher relative pro-portion of butyrate may reflect the higher content ofglucans in non-digested apples (Table 1) or yeastglucans in ciders [28].

j Potential effects of non-fermented PAs

Since the degradation of PAs appears to occur at lowextent, it is likely that luminal tannins may causeenzyme inhibition, as described by McDougall andStewart [29]. Condensed tannin and pectin exhibitedan effect on lipid metabolism in the rat, by loweringplasma cholesterol level [27]. PA dimers and oligo-mers exhibit protective role in obesity and insulinresistance, focusing their role on the adipocytes,where PAs modify lipid synthesis, lipid degradation,glucose uptake, and adipose differentiation [33]. To-maru and co-workers [47] showed that dietary sup-plementation of cacao liquor PAs prevented elevation

of blood glucose levels in diabetic obese mice dose-dependently, suggesting benefits in preventing theonset of type 2 diabetes mellitus. Because PAs maychange the behavior of colonic fermentation, the im-pact of DF on fermentation or colonic transit may bealtered. Further studies are needed to determine thebiological significance of PAs and their microbialmetabolites.

Conclusions

Chain length is an important factor affecting themicrobial conversion of PAs to phenolic acids. Iso-lated, long-chain PAs were able to suppress bothphenolic acid and SCFA formation in vitro by faecalmicrobiota, suggesting inhibitory effects on luminalenzymes.

j Acknowledgments Hannele Virtanen is thanked for conductingSCFA analysis. Annika Majanen, Siv Matomaa, Marita Ikonen andAiri Hyrkas are gratefully acknowledged for the skilful technicalassistance. Dr Riitta Puupponen-Pimia is thanked for fruitfulcomments on the manuscript. European Commission (STREP-FLAVO Food-CT-2004-513960) is acknowledged for financial sup-port for the study.

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