BiochemicalSociety Transaidions (1995)23 253s
Release of the antioxidant, ferulic acid, from plant material
by specific &erases.
CRAIG B. FAULDS and GARY WILLIAMSON
.
.
Figure 1. The release of f e w from m e t h m
human- f
1
Assays were carried out at 37°C and
pH 6.5 with mixing.
0 = 96 h grown samples; W = 120 h grown samples.
Dept. Food Molecular Biochemistry, Institute of Food
Research, Norwich Research Park, Colney, Nonvich NR4
7UA, U.K.
Hydroxycinnamic acids, such as ferulic, p-coumaric and
caffeic acid, are common constituents of plant cell walls,
certain of which are used as forage or processed as foods [l].
Ferulic acid represents up to 2.5% (w/w) of cell walls in
grasses [2]. These hydroxycinnamic acids can be toxic to
many soil and ruminal micro-organisms [3]. A few microbes
on the other hand can survive and utilize these compounds as
a carbon source [4].
Due to the phenolic nucleus and extended side-chain
conjugations, ferulic acid has been found to form a resonance
stabilised phenoxy radical which accounts for its potent
antioxidant potential [5], and has been suggested as a ’natural’
antioxidant food additive [6]. The nitrite scavenging effect of
wheat bran has also been shown to be due to the ferulic acid
content in the dietary fibre [q.
Ferulic acid is ester-linked to sugars in various plant cell
wall polysaccharides, but only a few microbial species have
been reported to produce esterases which cleave the linkage.
These cinnamoyl esterases have recently been isolated and
characterised from bacteria and fungi [8] and show differences
in both physical properties and substrate specificity. Enzymes
have been isolated from the ruminal fungus Neocullimastix [9],
which, although releasing free ferulic acid from low molecular
weight feruloylated oligosaccharides, released very little from
plant cell wall material. All the ferulic acid esterases have
been shown to act together with other plant cell wall
hydrolases to release ferulic acid from plant cell wall
preparations in a synergistic interaction [8-91. The release of
ferulic acid must therefore involve a range of enzymes
including ferulic acid esterase. Ferulic acid esterases are
present in the ruminal gut, but are they present in the human
gut?
Human faecal samples were obtained after incubation in
anaerobic medium [lo]. The soluble faecal slurry sample
(0.75 ml) was incubated under rotation at 37°C in the presence
of methyl ferulate (0.75 mM) and 100 mM MOPS @H 6.5) in
a total of 2.5 ml. Boiled slurry was used as a control.
Samples were removed over a 4 hour time perid and the
release of ferulic acid determined by HPLC 1111.
The
amount of ferulic acid released increased over time, both
samples increasing to 4 nmoYml slurry after 4 hours incubation
This corresponds to 0.16% hydrolysis of the
(fig. 1).
substrate over the time period. This value is low compared
to hydrolytic ram measured in other microbial systems,
especially Aspergillus [8], but does show the existence of a
’ferulic acid esterase-type enzyme’ in the human gut
microflora.
Studies on the substrate specificities of ferulic acid esterases
have shown the existence of multiple enzymes from one
source, each showing different specificities towards both plant
cell wall derived feruloylated oligosaccharides and model
substrates. Rumen micro-organisms are known to secrete
multiple forms of hydrolytic enzymes, and three cinnamoyl
esterases have been identified in Neocullinwfix, giving this
organism a unique advantage in the biodegradation of
0
1
2
3
4
Incubation time (hours)
phenolic-containing and phenolic cross-linked polysakharides
[Ill.
The presence of multiple esterases in human gut
microflora has yet to be determined.
So why is ferulic acid removed, and what happens in the gut
once the acid is free? Removal of phenolic acid esters
increased the degradation of wheat bran by human faecal
bacteria three-fold, rendering the polymers more susceptible to
microbial attack [12].
A vital question to answer is whether ferulic acid and other
hydroxycinnamic acids can act as physiologically significant
antioxidants. The understanding of such mechanisms would
facilitate the transfer of in vitro observations on ferulic acid in
antioxidant action [6] into in vivo benefits of having sufficient
free or bound ferulic acid in the diet.
We wish to thank the BBSRC for funding and Peter Rya’en
and Jim Robenson of IFRN for providing samples.
[I] H e m , K. (1989) Crit. Revs. Food Sci. Nutr. 28:315-347
(21 Hartley, R.D. &Jones, E.C. (1977) Phytochem. 16:1531-1534
[3] Chesson, A., Stewart, C.S. & Wallnce, R.J. (1982)
AppLEnviron. Microbiol. 44597-603
[4] McCnrthy, A.J. (1987) FEMS Microb. Revs.46:145-163
[5] G n f , E. (1992) Free Rad. Biol. Medicine 13:435-448
[a] Scott, B.C., Butler, J., Halliwell, B. & Aruomn, 0.1.(1993) Free Rad.
Res. C o r n . 19241-253
[71 Meller, M.E., Dahl, R. & B8ckmpn, O.C. (1988) Food Chem.
Toxic. 26:841-845
[8] Faulds, C.B. &Williamson, G. (1994) Microbiol. 140:779-787
[9] Bornemm, W.S., Ljungdahl, L.G.,Hartley, R.D. & Akin, D.E.
(1992) A w l . Environ. Microbiol. 583762-3766
[ 101 Livese.y, G., Johasoo, I.T., Gee,J.M., Smith, T., Lee, W.E., H i h a ,
K.A. Meyer, J. &Turner, S.C. (1993) Eur. J. Clin. Nutr. 47:419430
[ 111 Johnson,K.G., Harrison, B.A., Schneider, H., MacKeazie, C.R. &
Fontann, J.D. (1988) Enzyme Microh.Techno1. 10:403-409
1121 Steveas, B.J.H & S e l v d m , R.R. (1988) cubobydr. Res. 183:311319