Full Length Research Paper
Biotransformation of ferulic acid to 4-vinyl guaiacol by
Hadiza Altine Adamu1,
Shahid Iqbal1,2, Kim Wei Chan1 and
1Laboratory of Molecular
Biomedicine, Institute of Bioscience, Universiti Putra
Malaysia, 43400 UPM Serdang, Selangor, Malaysia.
of Chemistry, University of Sargodha,
of Nutrition and Dietetics, Faculty of Medicine and Health
Sciences, Universiti Putra Malaysia, 43400 UPM, Serdang,
*Corresponding author. E-mail:
Tel: +603-89472115. Fax: +603-89472116.
Ferulic acid; 4VG, 4-vinyl guaiacol.
Accepted 5 August, 2011
Continuously growing demand for natural flavors has led to a
tremendous increase in biotransformation
process employing microorganisms
of different genera using ferulic acid (FA) as the
precursor. In this study, potential of Lactobacillus
farciminis (ATCC 29644) for biotransformation of FA to
4-vinyl guaiacol (4VG)
investigated. 4-vinyl guaiacol is a volatile phenol,
reported to have 40 fold higher economic value than FA and
is biotransformable to acetovanillone, ethylguaiacol and
vanillin. Biotransformation process started after 5 h
incubation of L. farciminis with FA in Man Regosa and
Sharpe (MRS) broth at 37°C under 5% CO2.
was observed at its maximum after 48 h. Formed 4VG was
identified by GC-MS (QQQ) and quantification was done by
HPLC UV-Vis. The impact of initial concentrations of FA and
bacteria on the production of 4VG was studied. The results
indicate that the production of 4VG is significantly
affected by initial concentration of FA, and empirically 1,
15 and 50 mg/l of FA yielded 0, 3.34 and 10.26 mg/l of 4VG,
respectively. The findings are a milestone towards safe high
yielding means of biotransforming some common
agro-industrial wastes to a value added product.
ferulic acid, 4-vinyl guaiacol, biotransformation.
impact of ferulic
acid steryl esters,
extracted from rice
bran oil, brought
ferulic acid (FA)
into focus during
the 1970s, which was
later on found to be
activities of FA
(Min et al., 2006; Max et
Ferulic acid is a
(Ghosh et al.,
2006), which may
be present either in
free or bound form
in plants (Zhao
It is present in
wheat, maize and
rice brans (Walton
et al., 2000; Mariod
et al., 2010).
Ferulic acid can be
made free by
(Walton et al., 2000; Min
et al., 2006).
of FA as the primary
source of carbon, by
bacteria of assorted
genera, has led to
the production of
as 4VG (Couto et
vanillic acid and
(Torres et al.,
There has been a
continuous rise in
demand for natural
because of the
synthetic ones (Okeke
and Venturi, 1999).
has gained momentum
during recent years
as a vital means of
Many fragrances and
flavors have been
technology so far
et al., 2002) using
microbial means (Brunati
et al., 2004).
4-vinylguaiacol is a
volatile phenol (Couto
et al., 2006) and is
reported to have 40
fold higher economic
value than FA and
can be biotransformed
vanillin (Landete et
al., 2010). It is
used in food and
for flavoring and in
ophthalmic field too
al., 2010). It is
present in pods of
juice, wine, raw
roasted peanuts and
white sesame seeds (IHBT,
2005). According to
Bohlin (1993), 4VG
glory) has been
reported to inhibit
species play major
role in industrial
processes due to
their ability to
with their generally
regarded as safe
(GRAS) status hence
their application as
et al., 2007).
No report describing the
for 4VG production
has been presented
so far. There have
been reports on the
production of 4VG
from FA but with
rates and low yields
of metabolites (Karmarkar
et al., 2000). Thus,
this study for the
first time seeks to
to biotransform FA
to 4VG. Coupled to
this condition that
is, initial FA and
been optimized to
improve the yield of
product. The work is
of high worth from
an industrial point
of view with wide
Materials and Methods
and vanillic acids were purchased from Sigma-Aldrich
(Germany), vanillin from MP Biomedicals (USA) and
vanillyl alcohol from Merck (Germany)
and liquid nitrogen from Malaysian Oxygen Berhad,
Petaling Jaya, Selangor, Malaysia.
Methanol, ethanol, acetic acid and acetonitrile were
of HPLC grade and were procured from Fisher
farciminis (ATCC 29644), was purchased from
American Type Culture Collection (ATCC) and stored
in Merck’s MRS medium containing 30% (v/v) glycerol
Inoculum preparation and biotransformation
Colony count technique was used to determine total
viable cell count. L.
farciminis was observed to have a
cell density of 1 × 108 cells/ml. L.
farciminis was cultured following a method
reported by Sabu et al. (2006). About 5 ml of 18 h
culture was inoculated into 45 ml of MRS broth,
contained in a 250 ml conical flask and incubated at
37°C under 5% CO2 for 20 h. The inoculum
was then inoculated into MRS broth supplemented with
filter sterilized FA, which had been dissolved in 1M
NaOH solution and made up to pH 8.5 using 6M HCl in
a 100 ml total culture volume. This was then
incubated under same conditions. About 3 ml of the
sample was then withdrawn at intervals to determine
the concentration of FA degraded and that of 4VG
Ferulic acid conversion was expressed as:
FA conversion (%) = FAi – FAf
FAi = ferulic acid initial concentration,
FAf = ferulic acid final concentration.
Analysis of spent media
Identification of 4VG by gaschromatography–mass
Samples were analyzed following the method described by Couto et
al. (2006) with slight modifications. 1 ml mixture
of ether and hexane (1:1 v/v) was used to extract
the volatile phenol by vortexing 3 ml sample with
the mixture of ether and hexane for 5 min and the
organic layer obtained was concentrated under
nitrogen to about one third of the initial volume.
It was then injected into a gas chromatograph-mass
spectrometer (Thermo scientific-TSQ Quantum, USA)
with a thermo TR-5MS column (30 m × 0.25 mm ID
× 0.25 µm) (USA) and analyzed using X-calibur
software. Helium was used as carrier gas. Injection
temperature was set at 250°C, temperature gradient
was adjusted at 80°C for 2 min, 120°C for 4 min,
155°C for 4 min and heated at 250°C for 3 min,
injection volume employed was 1 µl and flow rate was
kept at 1 ml/min.
Quantification of 4VG by HPLC
Analysis of filtered spent media for quantification
of FA and 4VG was done using HPLC system (Agilent
1200 series, Germany) using C18 reversed phase
column (Zorbax) maintained at 22°C, UV-Vis detector
set at 280 nm. A linear gradient of two solvents was
chosen for the run: solvent A (4% acetic acid in
distilled water, v/v) and solvent B (acetic acid:
acetonitrile: methanol 1:5:94 v/v) from 0 to 52% of
solvent B for 30 min at a flow rate of 1 ml/min.
Identification was then carried out with respective
standards, while peak area was used as the tool of
The impacts of initial concentrations of bacteria
and FA on the production of 4VG were investigated as
biotechnological processes are significantly
influenced by initial concentrations of substrate
and microbes (Bloem et al., 2006; Faveri et al.,
2007). Microorganisms of different concentrations (1
to 5 ml) were grown in MRS media with various
concentrations (1, 5, 15, 25, 35 and 50 mg/l) of FA.
All the results in this study
were expressed as mean ±
standard deviation (SD) of 3 replicate measurements.
The significant differences (p < 0.05) among the
determined by one way analysis of variance (ANOVA)
using Minitab statistical software (Version
22.214.171.124, Minitab Inc, USA).
Results and Discussion
L. farciminis (ATCC 29644) have earlier been reported in in vitro analysis, to have feruloyl esterase activity and has been identified as the enzyme responsible for microbial conversions of FA to vanillin (Bhathena et al., 2007) as it releases FA from plant cell walls making them available as substrates for phenolic acid decarboxylase, which transforms FA to 4VG (Landete et al., 2010). Numerous organisms such as Aspergillus, Bacillus, Candida, Corynespora, Fusarium, Pseudomonas are able to transform FA to a wide range of aromatic compounds. From our results, we do propose that the L. farciminis species used in this study utilizes the non-oxidative decarboxylation pathway for the production of 4VG from FA (Figure 1). To the best of our knowledge, the presence of phenolic acid decarboxylase in L. farciminis (ATCC 29644) has not been reported yet. Even though 4VG, as a breakdown product of FA was present in the culture medium, vanillin could not be detected. This may be due to the fact that vanillin is usually found at low concentration and is speedily metabolized, while the production of 4VG via decarboxylation of FA maybe a detoxification process in order to lower the concentration of inhibitory compounds (Baqueiro-Pena et al., 2010). The decarboxylation of FA due to one-carbon cleavage of FA has been chronicled for many lactic acid bacteria (Couto et al., 2006; Bloem et al., 2006). In this study, we report for the first time the production of high yields of 4VG from FA by non-oxidative decarboxylation using L. farciminis. The availability of agro-industrial wastes containing FA has been greatly highlighted in this work. Literature reports describe biotransformation of FA by different means including fungi, bacteria or genetically engineered microorganisms to other bioactives (Gosh et al., 2004; Li et al., 2008). L. farciminis in this study was able to biotransform FA to yield 4VG as the major degradation product; as detected by HPLC. The ability of lactic acid bacteria to degrade FA is in agreement with the findings of Bloem et al. (2006), whereby wine associated lactobacillus namely: Oenococcusoeni, L. hilgardi, L. brevis, L. plantarum and L. damnosus were observed to degrade FA with the production of vanillin and traces of 4VG. Also in a study conducted by Couto et al. (2006), 4VG from FA was produced from thirty two strains of LAB out of the thirty five strains tested. In this study, the influences of initial concentrations of FA on the production of 4VG were also studied.
Identification using GC-MS-QQQ (Figure 2) and quantification with HPLC from plotted standard curves was done. Experiments were performed in triplicates. HPLC analysis of the culture supernatant showed FA with a retention time of 14.1 min (Figure 3) and 4VG at 24.1 min (Figure 4). Results reveal a proportionate increase in production of 4VG with an increase in initial FA concentration. An initial FA concentration of 50 mg/l yielded about 10 mg/l of 4VG, while an initial FA of 1 mg/l yielded no 4VG. This result is in line with the findings of Couto et al. (2006), whereby the higher the hydroxyl-cinnamic acid content, the higher the concentration of volatile phenols produced. Substrate inhibition could not be determined as precipitation occurred, when FA concentration exceeded 50 mg/l. It has been reported that the growth of lactic acid bacteria is inhibited by hydroxycinnamic acids at 500 mg/l (Couto et al., 2006). The initial concentration of FA also influenced the time for production of 4VG, as 4VG production was observed at about 5 h (Figure 5) after incubation in cultures containing 5, 15, 25, 35 and 50 mg/l of initial FA, while none was observed for 1 mg/l initial FA. The production rate of 4VG was maximum after 48 h (Figure 6) incubation irrespective of the initial amount of FA used, after which the rate of 4VG formation started to decline but was still detectable at day 10 of incubation. The bioconversion rate of FA at 48 h of incubation ranged over 41 to 87% for initial FA concentrations of 5, 15, 25, 35 and 50 mg/l; with the lowest being 24% at an initial concentration of 5 mg/l, which is also in agreement with Couto et al. (2006). Initial FA concentration significantly influenced the production of 4VG; as compared to initial bacterial concentration (Figure 7). On the basis of findings, 50 mg/l of initial FA concentration was taken as the optimum amount.
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