J Sci Food Agric 1997, 73, 296È300

Hydrolysis and Fermentation by Rat GutMicroorganisms of 2-O-b-D-Xylopyranosyl(5-O-Feruloyl)-L-Arabinose Derived fromGrass Cell Wall ArabinoxylanGundolf Wende,* Callum J Buchanan and Stephen C Fry”

ICMB, Division of Biological Sciences, The University of Edinburgh, Daniel Rutherford Building,The KingÏs Buildings, MayÐeld Road, Edinburgh, EH9 3JH, UK

(Received 18 April 1996 ; revised version received 24 July 1996 ; accepted 17 September 1996)

Abstract : It has been shown that a common side-chain of grass cell wall ara-binoxylans is 2-O-b-D-xylopyranosyl-(5-O-feruloyl)-L-arabinose [X(F)A]. The sta-bility of X(F)A was determined by incubation of ( feruloyl-U-14C)-labelled X(F)Aand (pentosyl-1-3H)-labelled X(F)A anaerobically with rat caecal contents andchromatographic analysis of the radioactive products. The ester linkage washydrolysed very rapidly to form ferulic acid (which was stable) and the disaccha-ride (XA). The 3H-XA was further metabolised, but 3H-monosaccharides did notaccumulate. In the end-products of fermentation of (pentosyl-3H)-labelled X(F)A,67% of the 3H was present in bacterial polymers. In contrast, when free [1-3H]arabinose was incubated with rat caecal contents, 74% of the 3H quickly becamevolatile, probably as It is concluded that X(F)A is highly susceptible to3H2O.(feruloyl)esterase activity produced by bacteria present in the rat caecum, andthat the disaccharide produced is further fermented, but not via the productionof extracellular arabinose and xylose.

Key words : arabinose, arabinoxylan, cell walls (plant), fermentation, feruloylgroups, feruloylesterase, oligosaccharides, tritium labelling.

INTRODUCTION

Plant cell walls are an important source of nutrients forruminants (Akin 1993). In addition, dietary carbon isrecovered from the polysaccharides of plant cell walls inthe lower gut of omnivores (Buchanan et al 1994a,b).Before utilisation, the cell walls need to be digested,through the intervention of microbes. However, micro-bial degradation is subject to restrictions ; cell wallsobtained from various botanical sources, and subjectedto diverse pre-treatments, di†er in their susceptibility todigestion.

One factor that has often been suggested to limit thedigestibility of the polysaccharides in plant cell walls is

* Present address : Department of Plant Pathology, Universityof Wisconsin, 1630 Linden Drive, Madison, WI 53706-1598,USA.” To whom correspondence should be addressed.

the presence of phenolic material. Even in young,growing plant tissues that are not ligniÐed, the cell wallsfrequently contain phenolic groups such as feruloyl andp-coumaroyl esters (Harris and Hartley 1976 ; Hartleyand Jones 1977 ; Fry 1982 ; Tanner and Morrison 1983 ;Wallace and Fry 1994). In grasses, much of the ferulateis esteriÐed to O-5 of the a-L-arabinofuranose side-chains of arabinoxylans, forming 5-O-feruloyl-L-ara-binosyl (FA) units (Kato and Nevins 1985 ;Mueller-Harvey et al 1986 ; Ishii and Hiroi 1990). FAitself probably contributes relatively little to resistingmicrobial digestion (Buchanan et al 1996). However, ithas recently been shown that, in grass arabinoxylans,the feruloylated side-chains are usually more elaboratethan FA. In particular, the feruloylated arabinoseresidue is often xylosylated, forming 2-O-b-D-xylopyranosyl-(5-O-feruloyl)-L-arabinose [X(F)A](Himmelsbach et al 1994 ; Saulnier et al 1995 ; Wendeand Fry 1997a). In addition, X(F)A is itself the core of

296J Sci Food Agric 0022-5142/97/$09.00 1997 SCI. Printed in Great Britain(

Microbial digestion of a feruloyl oligosaccharide 297

numerous more complex feruloylated oligosaccharideside-chains that are attached to the xylan backbone(Wende and Fry 1997b).

We have therefore now tested whether X(F)A is ableto resist microbial degradation. X(F)A, that was either14C-labelled in its feruloyl residue or 3H-labelled in itstwo pentose groups, was incubated with fresh rat caecalcontents. The microorganisms present in the rat caecumnormally digest a high proportion of the non-starchpolysaccharides of plant cell walls, which escapedigestion by the enzymes of the stomach, pancreas andsmall intestine (Gray et al 1993 ; Buchanan et al1994a,b). The experiments described in the presentpaper tested the resistance of both the feruloyl esterlinkage and the Xyl-(1 ] 2)-Ara bond of X(F)A todigestion by the microorganisms of the rat gut.

MATERIALS AND METHODS

Radiochemicals

L-[1-3H]Arabinose (97 MBq kmol~1) was custom-produced by Amersham International (Bucks, UK).Cinnamic acid (15 MBq kmol~1) was prepared bytreatment of L-[U-14C]phenylalanine (AmershamInternational) with phenylalanine ammonia-lyase (EC4.3.1.5). Cell suspension cultures of tall fescue grass(Festuca arundinacea Schreber) were incubated asdescribed (Wende and Fry 1997a). A 4-day-old culture(100 ml) was supplied either with 47 MBq of L-[1-3H]arabinose for 5 h or (aseptically) with 2 MBq of (E)-[U-14C]cinnamate for 7 days. The cells were then packedinto a column and washed for at least 24 h with slowly-Ñowing 80% ethanol, then dried. The alcohol-insolubleresidue (500 mg) was incubated with 25 ml of 0É1 M

TFA at 100¡C for 1 h. TFA was removed by drying invacuo in a SpeedVac, and the water-soluble productswere then subjected to preparative paper chromatog-raphy in BAW. X(F)A was located by its Ñuorescence,eluted in water (Eshdat and Mirelman 1972) and furtherpuriÐed by PC in BEW and low-pressure reversed-phase chromatography on silicaC18-substitutedcolumns (BondElut, Varian, Analytichem, Harbor City,CA, USA; 100 mg packing) (Wende and Fry 1997a). Inthe 3H-labelled X(F)A, the xylose residue had a speciÐcactivity of 15É0 MBq kmol~1 while the arabinosemoiety had a speciÐc activity of 37É8 MBq kmol~1(Wende and Fry 1997b).

Paper chromatography

PC was performed on Whatman 3MM paper by thedescending method in BAW (butan-1-ol/acetic acid/water (12 : 3 : 5, v/v/v)) or BEW (butan-1-ol/ethanol/water (20 : 5 : 11, v/v/v)).

Assay of radioactivity

3H and 14C were assayed as described by Wende andFry (1997a,b).

Fermentation studies

All manipulations of medium and gut contents wereperformed under strictly anaerobic conditions by use of

Caecal contents were taken from a freshly killed ratN2 .(Wistar male D300 g body weight). Routinely, caecalcontents were used at 50% concentration : D5 ml of ratcaecal content was made up to 10 ml with modiÐed (nocarbohydrate) medium of Scott and Dehority (1965). Toslow down the hydrolysis and fermentation in someexperiments, 10% caecal contents were used (1 mlcaecal contents made up to 10 ml with medium). ThepH of the medium is 7É8 and the rate constant for alka-line hydrolysis of X(F)A is 8 mM~1 s~1 (Wende and Fry1997a) ; therefore, the non-enzymic hydrolysis of X(F)Ain the medium would be negligible (initial rate>1% h~1).

Rat caecal contents (10 ml ; 10 or 50% originalconcentration) were mixed with 1 ml of medium con-taining 40 kBq of 14C- or 3H-labelled X(F)A, or 6 MBqof [3H]arabinose, and incubated at 37¡C with constantslow bubbling of at D5 ml min~1. At each samplingN2time, 1 ml of the suspension was added to 4 ml offreshly-prepared methanol/formic acid (4 : 1, v/v). Thesample for time zero was mixed with 4 ml methanol/formic acid Ðrst and then the radiochemical (4 kBq or0É6 MBq) was added. After centrifugation at 1000 ] gfor 10 min, the pellet was washed twice with mediumand the supernatants were combined.

For investigation of the volatility of the radioactivefermentation products, three portions (iÈiii, each 100 kl)of the methanol-soluble fraction were assayed for radio-activity : (i) was assayed directly for total radioactivity ;(ii) was mixed with 1É25 ml of 1 M NaOH, then driedand assayed ; (iii) was mixed with 1É25 ml of TFA, thendried and assayed. From the data, the following weredetermined : (A) non-volatile products ((i)È(iii)) ; (B)

and/or radioactive non-acidic volatiles, eg meth-3H2Oanol or ethanol ((i)È(ii)) and (C) volatile acids, eg acetateor butyrate ((ii)È(iii)).

RESULTS

( feruloyl-14C)-Labelled X(F)A was very rapidlydegraded by rat caecal contents. Its half-life was about0É2 min and 3 min in the presence of 50% and 10%caecal contents, respectively (Fig 1). The radioactiveproducts were mainly methanol-soluble and non-volatile : very largely free [14C]ferulic acid but probablywith a trace of 14C-FA at short incubation times (Fig 2).

298 G W ende, C J Buchanan, S C Fry

Fig 1. Degradation of ( feruloyl-14C)-labelled X(F)A by ratcaecal contents. The caecal contents were diluted to 50% (…)

or 10% of their natural concentration.(L)

The rapid degradation was conÐrmed for (pentosyl-3H)-labelled X(F)A. At early sampling times, themethanol-soluble, radioactive products included a sub-stance that approximately co-migrated with xylobiose(XX) and was presumably the disaccharide b-D-Xylp-(1 ] 2)-L-Ara, and a trace of free monosaccharide(arabinose and/or xylose ; not resolved) (Fig 3a,b). Inaddition, an unidentiÐed product with a very lowpolarity higher than that of ferulic acid) accumulat-(RFed. With more prolonged incubation times, thedisaccharide disappeared and was partially replaced by

Fig 2. PC in BAW of methanol-soluble products after treat-ment of 14C-labelled X(F)A with 50% caecal contents.Samples were analysed after (a) 0 min or (b) 5 min incubation.

Fig 3. PC in BAW of methanol-soluble products formed bytreatment of (pentosyl-1-3H)-labelled X(F)A with 10% caecalcontents. Samples were taken after (a) 0 min, (b) 0É5 min or (c)4 min. FA, X(F)A, Ara, XX, xylose 1-phosphate (Xyl-1-P),glucose 6-phosphate (Glc-6-P) and ferulic acid (Fer) served asexternal markers. Larger loadings were applied to chromato-

grams (b) and (c) than to (a).

unidentiÐed, material that co-migrated withlow-RFsugar phosphates (Fig 3c).

After D1 h incubation, D20% of the 3H remained inthe methanol-soluble, non-volatile fraction, and D67%was in the methanol-insoluble fraction, presumablyincorporated into bacterial polymers. The remainingD13% of the 3H was volatile from alkaline solution,indicating that it was and/or alcohols3H2O low-Mr(Fig 4a).

The data showed that X(F)A was very rapidly de-feruloylated, and that the disaccharide produced,Xyl]Ara, was itself degraded. However, only smallamounts of free 3H-monosaccharide accumulated (Fig3). To determine the fate of any transiently formed freearabinose, we incubated [3H]arabinose with 50%

Microbial digestion of a feruloyl oligosaccharide 299

Fig 4. Volatility of methanol-soluble, radioactive fermenta-tion products formed from (a) (pentosyl-1-3H)-labelled X(F)Aand (b) L-[1-3H]arabinose. The curves show the total(undried) methanol-soluble radioactive material and the(…),methanol-soluble non-volatile material remaining after dryingfrom alkaline or from acidic solutions. The di†erences(>) (+)between the curves show: (A) non-volatile, methanol-solublematerial ; (B) 3H-alcohols and (C) volatile3H2O] low-Mr3H-acids.

caecal contents. The microorganisms very rapidly fer-mented the [3H]arabinose (Fig 5). After 1 h incubation,only 6% of the 3H was methanol-soluble and non-volatile (Fig 4b). About 74% was volatile from alkalinesolution (probably mainly formed by3H2Ofermentation), and D20% was methanol-insoluble.

DISCUSSION

The data show that the feruloyl disaccharide X(F)A wasvery rapidly de-esteriÐed, and that the free ferulic acidproduced was stable. This indicates the production ofhigh (feruloyl)esterase activity by rat gut micro-organisms. The disaccharide thus released was itself fer-mented. Therefore, all the enzymes necessary for a rapidbreakdown of X(F)A are produced by rat gut micro-organisms.

The radioactive end-products formed from (pentosyl-1-3H)-labelled X(F)A di†ered quantitatively from thoseformed from free [1-3H]arabinose. The 3H from freearabinose was quickly released as (D 74% within3H2O1 h). In contrast, at most 13% of the 3H from thedisaccharide moiety of X(F)A was released as 3H2O

Fig 5. PC in BAW of methanol-soluble products after fermen-tation of [3H]arabinose in rat caecal contents. Samples weretaken after (a) 0 min, (b) 5 min or (c) 120 min. Ara and Xyl-1-

P served as external markers.

within 1 h. The non-volatile material derived fromX(F)A included 3H-labelled bacterial polymers and 3H-labelled substances that co-migrated with sugar phos-phates. This indicates that the fermentation of thedisaccharide did not proceed via free monosaccharidesformed by the action of extracellular b-xylosidase.Instead, it appears more likely that the disaccharide wasmetabolised via a pathway occurring on or in the bac-terial cells ; indeed, it is known that some oligosac-charides can be taken up intact by rumen microbes(Martin and Russell 1987 ; Maas and Glass 1990). Themetabolic basis for the di†erent fates of the monosac-charide and the disaccharide is unknown. However, oneexplanation for the channelling of 3H from X(F)A intobacterial polymers and sugar phosphates is cleavage ofthe disaccharide by a phosphorylase rather than ahydrolase.

300 G W ende, C J Buchanan, S C Fry

The results show that X(F)A does not resist digestionby the enzymes produced by the microbes present in therat gut. In view of its rapid degradation in the rat, it isunlikely that X(F)A would survive appreciably longer inother degradative environments such as the rumen oranaerobic muds. There is therefore no evidence tosupport the hypothesis that the complex feruloylatedoligosaccharides now known to be present as side-chains of many grass arabinoxylans provide more e†ec-tive resistance to digestion than do the simple feruloylmonosaccharide (FA) side-chains previously investi-gated.

ACKNOWLEDGEMENT

The authors thank the BBSRC for funding this work.

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