Skip to main content
NCBI home page
Journal of Food Science and Technology logoLink to Journal of Food Science and Technology
. 2017 Feb 9;54(3):778–785. doi: 10.1007/s13197-017-2521-z

Enrichment of maize and triticale bran with recombinant Aspergillus tubingensis ferulic acid esterase

Eunice N Zwane 1, Petrus J van Zyl 2, Kwaku G Duodu 3, Shaunita H Rose 1, Karl Rumbold 4, Willem H van Zyl 1, Marinda Viljoen-Bloom 1,✉
PMCID: PMC5334237  PMID: 28298692

Abstract

Ferulic acid is a natural antioxidant found in various plants and serves as a precursor for various fine chemicals, including the flavouring agent vanillin. However, expensive extraction methods have limited the commercial application of ferulic acid, in particular for the enrichment of food substrates. A recombinant Aspergillus tubingensis ferulic acid esterase Type A (FAEA) was expressed in Aspergillus niger D15#26 and purified with anion-exchange chromatography (3487 U/mg, K m = 0.43 mM, K cat = 0.48/min on methyl ferulate). The 36-kDa AtFAEA protein showed maximum ferulic acid esterase activity at 50 °C and pH 6, suggesting potential application in industrial processes. A crude AtFAEA preparation extracted 26.56 and 8.86 mg/g ferulic acid from maize bran and triticale bran, respectively, and also significantly increased the levels of p-coumaric and caffeic acid from triticale bran. The cost-effective production of AtFAEA could therefore allow for the enrichment of brans generally used as food and fodder, or for the production of fine chemicals (such as ferulic and p-coumaric acid) from plant substrates. The potential for larger-scale production of AtFAEA was demonstrated with the A. niger D15[AtfaeA] strain yielding a higher enzyme activity (185.14 vs. 83.48 U/ml) and volumetric productivity (3.86 vs. 1.74 U/ml/h) in fed-batch than batch fermentation.

Keywords: Ferulic acid esterase, Aspergillus niger, Aspergillus tubingensis, Triticale, Maize

Introduction

The growing global food industry requires the development of new technologies to add value and create new market opportunities for the agricultural sector. Ferulic acid is the most abundant hydroxycinnamic acid in plant cell walls (Balasundram et al. 2006; Mathew and Abraham 2004) and exhibits better inhibition of lipid and protein oxidation compared to other naturally occurring phenolic antioxidants such as gallic acid, caffeic acid, malvidin and epicatechin, among others (Graf 1992; Heinonen et al. 1998). Due to its low toxicity, ferulic acid has been approved for use as an antioxidant additive in cosmetics, foods and beverage industries (Graf 1992). In the food industry, ferulic acid is, among others, used as a precursor for the production of vanillin, a flavouring agent (Priefert et al. 2001). Ferulic acid represents up to 1.4% of dry weight of cereal grains and is covalently linked with various carbohydrates as a glycosidic conjugate, ester or amide (Kumar and Pruthi 2014). However, ferulic acid can only be released from these substrates through alkaline hydrolysis or enzymatic hydrolysis. Alkaline treatment is quite effective, with 83% of the bound ferulic acid released from corn bran, but it requires downstream purification of the ferulic acid from the brown hydrolysate (Zhao et al. 2014). The use of microbial enzymes for the fractionation of lignocellulose represents a natural process to release ferulic acid from plant substrates, but poses challenges as most of the ferulic acid in plants is covalently linked with lignin and other biopolymers (Kumar and Pruthi 2014).

Ferulic acid esterases (FAEs; EC 3.1.1.73), also known as feruloyl, cinnamic acid or cinnamoyl esterases, are able to release ferulic acid and other cinnamic acids from plant cells (Benoit et al. 2006; Fazary and Ju 2008). Ferulic acid esterases are classified based on their substrate specificity for methyl esters, i.e. methyl ferulate [MFA], methyl p-coumarate [MpCA], methyl sinapate [MSA] or methyl caffeate [MCA], and oligosaccharides, e.g. (O-{5-O-((E)-feruloyl)-α-L-arabinofuranosyl}-(1,3)-O-β-D-xylopyranosyl-(1,4)-D-xylopyranose (FAXX). Type A FAE typically has a preference for substrates with a methoxy substitution in the meta position (C3 and/or C5) of the aromatic ring (e.g. MSA and MFA) as well as substrates with a hydroxyl group at the C4 position of the aromatic ring (e.g. MpCA), as reported for Aspergillus awamori (Koseki et al. 2006) and Aspergillus niger (Faulds and Williamson 1993; Zwane et al. 2014). The phenolic acids in maize bran, representing about 4–5% of the cell wall on a mass basis, are mostly ferulic acid and lower concentrations of p-coumaric acid and other phenolic acids esterified to the cell wall polymers (Lapierre et al. 2001). Triticale, a hybrid of wheat and rye, also contains a variety of cinnamic acids, including ferulic acid and p-coumaric acid. The release of these phenolic acids—a natural source of antioxidants—will improve the health benefits of high-fibre brans and therefore represent a significant value-addition to these substrates (Hosseinian and Mazza 2009). In the farming industry, the dimeric and trimeric forms of ferulic acid that are either esterified or etherified to plant residues, have been reported to inhibit digestion of these residues in ruminants (Yu et al. 2005). However, the addition of a multi-enzyme cocktail that included a purified Aspergillus FAE increased the cell wall digestibility of oat hulls by 86%.

Given the potential commercial importance of plant-derived ferulic acid and antioxidants in various industries (Benoit et al. 2008; Fazary and Ju 2007; Koseki et al. 2009), it is imperative to develop enzyme production and treatment processes that are both efficient and cost-effective, particularly given the high cost of enzyme production. A genomic copy of the Aspergillus tubingensis ferulic acid esterase Type A (faeA) gene was previously cloned and expressed in Aspergillus niger D15#26 under control of the A. niger gpdA promoter (Zwane et al. 2014). The current study describes the evaluation of the crude A. niger D15[AtfaeA] extract for the release of phytochemicals from triticale and maize bran, as well as the upscaled production of the recombinant A. tubingensis AtFAEA in bioreactors.

Materials and methods

Strain and culture conditions

All chemicals were of analytical grade and unless stated otherwise, sourced from Sigma-Aldrich (UK). Recombinant A. niger D15 strains were selected and maintained on minimal agar medium (2 g/l neopeptone, 18 g/l agar, 1 g/l yeast extract, 0.4 g/l MgSO4.7H2O, 10 g/l glucose, 2 g/l casamino acids, 20 ml 50 × AspA [300 g/l NaNO3, 26 g/l KCl, 76 g/l KH2PO4, pH 6] and 1 ml/l of 1000 x trace elements (Rose and Van Zyl 2002) at 30 °C for 120 h. Spores were harvested with physiological saline solution (NaCl, 0.9% w/v). The A. niger D15#26 and recombinant A. niger D15[AtfaeA] strains were cultivated in liquid minimal medium (5 g/l yeast extract, 0.4 g/l MgSO4·7H2O, 2 g/l casamino acids, 20 ml 50 x AspA, 1 ml/l 1000 x trace elements and 10 g/l maize bran as sole carbohydrate source). The growth media were inoculated with 1 × 106 spores/ml and incubated on a rotary shaker at 125 rpm at 30 °C for 5 days. Samples were centrifuged at 7600×g for 15 min at 4 °C and the crude supernatant used for enzyme treatments.

Enzyme purification

Shake flasks containing 250 ml minimal medium was inoculated with 1 × 106 spores/ml and incubated on a rotary shaker at 125 rpm at 30 °C for 5 days. Cultures were centrifuged at 7300×g at 4 °C for 15 min. The enzyme was purified from the supernatant with anion-exchange chromatography using an ÄKTAPrime system (GE Healthcare Life Sciences, Uppsala, Sweden) and DEAE-Sepharose column (GE Healthcare Life Sciences) pre-equilibrated with 50 mM sodium phosphate buffer (pH 6.5). The bound AtFAEA was eluted with 10 column volumes of a 0–500 mM NaCl gradient in 50 mM sodium phosphate buffer (pH 6.5). The eluted fractions were heated for 3 min at 100 °C in the presence of protein loading buffer and separated by SDS-PAGE (12% w/v) (Sambrook et al. 1989), using 5 µl peqGOLD prestained protein marker. Electrophoresis was carried out at 100 V at ambient temperature and the proteins visualised with Fast Blue (Sigma-Aldrich, South Africa). Protein sequencing and identification of protein species, provisionally identified as AtFAEA, were performed by the Centre for Proteomic and Genomic Research (Cape Town, South Africa).

Characterisation of AtFAEA

For enzyme activity assays, fresh 4-nitrophenyl ferulate (4NPF) substrate solution was prepared by vortexing 0.1 M potassium phosphate buffer (pH 6.5) containing 2.5% v/v Triton X-100 and 11 mM 4NPF (dissolved in DMSO) in a 9:1 ratio. Standard FAE assays (De Vries et al. 2002) were performed with 0.1 ml enzyme and 2 ml 4NPF substrate solution incubated at 37 °C for 1 h, with the release of 4NP measured spectrophotomerically at 410 nm (xMark Microplate Spectrophotometer, Bio-Rad, San Francisco, USA). One unit (1 U) of FAE activity was defined as the amount of enzyme that released 1 µmol 4NP from 4NPF in 1 min (Hedge et al. 2009). Total protein concentrations were quantified with the Qubit assay and Qubit 2.0 fluorimeter (Invitrogen, Carlsbad, USA) as per the supplier’s specification.

Similar FAE assay conditions were used to quantify enzyme activity under different pH conditions, using either 100 mM citrate–phosphate buffer (pH 3–7) or 100 mM Tris–HCl (pH 7–9). For pH stability, the recombinant AtFAEA was incubated in the respective buffers at 4 °C and the residual FAE activity measured after 24 h. The FAE activity was evaluated at 30–70 °C in 0.1 M potassium phosphate buffer (pH 6), whilst temperature stability was determined at 30–70 °C after incubation for 1 h in 0.1 M potassium phosphate buffer (pH 6, without substrate).

Substrate specificity was determined by incubating 1.7 µg purified AtFAEA in a total volume of 2 ml with 2 mg/ml of either hydroxycinnamic acid (MFA, MpCA, MSA or MCA) or FAXX prepared in 0.1 M potassium phosphate buffer, pH 6. The reaction was incubated at 37 °C for 1 h and terminated by the addition of 500 µl methanol and 400 µl distilled water. Kinetic parameters (K m and K cat) for MFA were calculated from initial rate data determined for 50 mU AtFAEA at substrate concentrations ranging from 0.2 to 0.8 mM at 50 °C, pH 6 for 15 min. The corresponding hydroxycinnamic acids, i.e. ferulic acid (FA), sinapic acid (SA), coumaric acid (pCA) and caffeic acid (CA), were quantified by HPLC with a Nucleosil C18 column with a 5 µM particle size (Supelco, Bellefonte, USA) at a flow rate of 1 ml/min at room temperature with 70% v/v acetonitrile as mobile phase. Detection was done by a Surveyor Plus UV/VIS detector (Thermo Electron Corporation, Elandsfontein, South Africa) at 320 nm.

Enzyme treatment of triticale and maize bran

Triticale bran (X Triticosecale Wittmack ex A. Camus cultivar US2007) was provided by the Department of Process Engineering (Stellenbosch University, South Africa), while commercial maize bran was donated by Sasko (Pioneer Foods, Paarl, South Africa). Duplicate batches of 20 mg triticale or maize bran were treated with 0.3 mg/ml (0.2 U FAE) of the crude extract from A. niger D15#26 or A. niger D15[AtfaeA] in 1.0 ml 50 mM potassium phosphate buffer (pH 6) for 24 h at 40 °C, while incubated on a rotary platform at 250 rpm (Shin and Chen 2006). The control experiments were treated with growth medium alone (i.e. no crude extract). The supernatant was collected by centrifugation (13,000×g, 10 min) and analysed for ferulic acid, p-coumaric acid and caffeic acid with reverse-phase HPLC using a modification of the method described by Kim et al. (2007). The crude extracts were filtered through a 0.45 μm polytetrafluoroethylene (PTFE) syringe filter and the filtrate subjected to HPLC using a Waters 1525 binary HPLC pump, YMC-Pack ODS AM-303 (5 μm particle size) column and 2487 dual wavelength absorbance detector (Waters Associates, Milford, MA, USA). The injection volume of 10 μl was separated at a flow rate of 0.8 ml/min at 25 °C and using a mobile phase of 0.1% acetic acid in water (solvent A) and 0.1% acetic acid in acetonitrile (solvent B). The linear gradient of solvent B was increased from 8 to 10% in 2 min, then to 30% in 25 min, followed by an increase to 100% in 1 min, kept at 100% for 4 min, and returned to the initial conditions. Breeze software (Waters Associates) was used for data collection and analysis.

HPLC-grade standards of caffeic acid, p-coumaric acid and ferulic acid were prepared in 0.1% acetic acid in acetonitrile at concentrations of 200, 150, 100, 50, 25 and 0 mg/l. Standards of 10 μl aliquots were chromatographed singly and as mixtures by injection into the HPLC system. Standard calibration curves were obtained for each phenolic acid by plotting peak areas versus concentrations. Regression equations that showed high degree of linearity (r2 > 0.995) were obtained for each phenolic acid from the calibration curves. Phenolic acids in the samples were identified by comparing the retention time of the unknown with those of the standard phenolic acids. The concentrations of the identified phenolic acids were calculated using the regression equations.

Batch and fed-batch fermentations

Batch and fed-batch fermentations were conducted in a 2-l bench-top bioreactor (Inforsag, Switzerland) with a working volume of 1.5 l. Pre-cultures of A. niger D15[AtfaeA] were prepared with approximately 108 spores/ml inoculated in 500 ml Erlenmeyer flasks containing 100 ml inoculum medium (10 g/l NaNO3, 0.8 g/l MgSO4.7H2O, 2 g/l KH2PO4, 0.1 g/l CaCl2.2H2O, 5 g/l yeast extract, 5 g/l tryptone, 10% v/v Struktol J673 antifoam and 1 ml/l trace elements) and incubated overnight at 30 °C at 125 rpm on a rotary shaker. Duplicate batches of 1 l minimal medium were inoculated with 100 ml of the pre-culture and the protein concentration, enzyme activity, dissolved oxygen and glucose consumption were monitored. The fermentation temperature was controlled at 30 °C (±0.1 °C), aeration with oxygen at 0.8 volume per volume per minute (v/v/m) and pH at 6 with 4 M KOH and 2 M H2SO4. The dissolved oxygen was maintained above 30% saturation by adjusting the agitation between 400 and 800 rpm. An initial glucose concentration of 30 and 10 g/l glucose was used for batch and fed-batch fermentations, respectively. The glucose feed for fed-batch fermentation was 50 g glucose in 250 ml water (200 g/l), initiated after depletion of the initial glucose charge and fed over 7 h.

Samples were taken every 12 h, centrifuged for 5 min at 37,600×g (Sorval RC-6 Plus, Thermo Scientific) and the supernatant stored at −80 °C for ferulic acid esterase assays as described above. Total biomass was based on 10 ml samples taken at the end of the fermentation, vacuum-filtered using pre-weighed filters (Whatman no. 1) and dried to constant weight at 105 °C for 24 h. Total protein content was measured with the BioRad Protein Assay (BioRad, Hemel, Hempstead, UK) and residual glucose determined with HPLC (CarboPac™ PA1 column, Dionex, MA, USA).

Results and discussion

Purification and characterisation of recombinant AtFAEA

The A. tubingensis AtfaeA gene was previously cloned and successfully expressed in A. niger D15#26 under the constitutive A. niger gpd promoter (Zwane et al. 2014). In this study, the recombinant A. niger D15[AtfaeA] strain was cultured in shake-flasks with maize bran as sole carbohydrate source, followed by purification and characterisation of the enzyme. Anion exchange chromatography enabled 7-fold purification and a 48.2% recovery of FAE activity (Table 1). The specific activity of the purified AtFAEA on 4NFP as substrate was 77.6 U/mg protein, which was higher than recombinantly expressed feruloyl esterases from Aspergillus usamii (44.9 U/mg) (Gong et al. 2013), A. nidulans (21.7 U/mg) (Debeire et al. 2012), A. awamori (9.01 U/mg) (Koseki et al. 2005) and A. oryzae (0.57 U/mg) (Koseki et al. 2009). The purified AtFAE displayed a 36 kDa protein species on SDS-PAGE (Fig. 1), similar to the A. niger FAEA (Faulds et al. 1998), and peptide mass fingerprinting (PMF) confirmed the presence of AtFAEA with a protein score of 146 and a 100% confidence interval (data not shown).

Table 1.

Purification of FAEA from A. niger D15[AtfaeA]

Purification step Total FAE activity (U)a Specific activity (U/mg protein) Purification Recovery (%)
Culture filtrate 675.0 ± 0.5 11.1 ± 0.7 Onefold 100.0
Anion exchange 325.0 ± 0.8 77.6 ± 0.3 Sevenfold 48.2
Open in a new tab

aDetermined with 4NPF as substrate

Fig. 1.

Fig. 1

Open in a new tab

SDS-PAGE analysis of fractions from anion-exchange chromatography: Lane M, peqGOLD protein marker; AtFAE, pooled fractions 18–22 that displayed the highest FAE activity; F1 to F41, selected fractions; Load, culture filtrate

Optimum FAE activity was displayed at pH 5–6 (Fig. 2a) and 50 °C (Fig. 2b), with the enzyme remaining stable for at least 24 h at pH 5–7 and at least 1 h at 30 °C to 60 °C (data not shown). Optimum FAE activity at pH 6 was also reported for other Aspergillus FAEAs (Benoit et al. 2008), whereas the higher enzyme stability in acidic conditions (retaining more than 80% activity at pH 3–7) corresponded to an A. awamori FAE (Kanauchi et al. 2008). The optimum FAE activity at 50 °C is within the temperature range of 35–55 °C reported for other fungal FAEAs, including that of A. niger (Benoit et al. 2008). However, the native A. niger FAE was able to tolerate 80 °C (Sundberg et al. 1990), whereas the AtFAEA was stable only up to 60 °C.

Fig. 2.

Fig. 2

Open in a new tab

Effect of pH (a) and temperature (b) on ferulic acid esterase (FAE) activity in A. niger D15[AtfaeA]. Error bars represent standard deviations from triplicate experiments

With MFA as substrate, the purified AtFAEA displayed a K m of 0.43 mM (Table 2), compared to 0.76 mM reported for the A. niger FAEA (De Vries et al. 1997). The latter also reported low levels of activity (0.97 U/ml) for the A. tubingensis faeA expressed in the A. niger NW154 strain. Our results indicate that the protease-deficient A. niger D15#26 strain was a better host for over-expression of AtfaeA, yielding a specific activity of 3487 U/mg total protein on MFA as substrate.

Table 2.

Substrate specificity of purified AtFAEA

Substrate Specific activity (U/mg protein)
Synthetic
MFAa 3.487 ± 0.5
MpCA 446 ± 0.1
MSA 432 ± 1.5
MCA 0
Natural
FAXX 9.837 ± 5.3
Open in a new tab

aMFA, methyl ferulate; MpCA, methyl p-coumarate; MSA, methyl sinapate; MCA, methyl caffeate; FAXX, O-{5-O-((E)-feruloyl)-α-L-arabinofuranosyl}-(1,3)-O-β-D-xylopyranosyl-(1,4)-D-xylopyranose

Release of phenolic acids from maize and triticale bran

Corn bran represents one of the best possible source of ferulic acid among agricultural crops, with up to 26 mg/g substrate reported for refined maize bran (Zhao and Moghadasian 2008). In this study, treatment with a crude A. niger D15[AtfaeA] extract released 26.56 mg/g ferulic acid from maize bran to, whereas 8.86 mg/g ferulic acid was released from triticale bran (Table 3). Noteworthy is that the A. niger D15[AtfaeA] preparation also released significantly higher levels of p-coumaric and caffeic acid from triticale bran, confirming that these phenolic acids are susceptible to ferulic acid esterases (Poidevin et al. 2009). In contrast, less p-coumaric and caffeic acid were extracted from the maize bran, relative to the control treatments.

Table 3.

Relative concentrations of selected phenolic acids after treatment of maize and triticale bran with crude extracts

Treatment Phenolic acids (mg/g substrate)
Ferulic acid p-Coumaric acid Caffeic acid
Maize bran
Control 7.63 ± 0.00* 0.99 ± 0.00 2.65 ± 0.00
Aspergillus niger D15[pGTP] 2.71 ± 0.00 0.07 ± 0.00 1.16 ± 0.00
Aspergillus niger D15[AtfaeA] 26.56 ± 0.00 0.59 ± 0.00 1.21 ± 0.00
Triticale bran
Control 0.16 ± 0.00 0.15 ± 0.00 0.48 ± 0.00
Aspergillus niger D15[pGTP] 3.75 ± 0.00 0.27 ± 0.00 0.98 ± 0.00
Aspergillus niger D15[AtfaeA] 8.86 ± 0.00 3.83 ± 0.00 3.43 ± 0.00
Open in a new tab

* Values are the means of duplicate measurements, together with standard deviations

Batch and fed-batch fermentations

The production of FAEA by the recombinant A. niger D15[AtfaeA] strain was scaled up to 2-l batch fermentations to increase the FAEA production levels. The maximum biomass reached was 8.68 g/l with an overall growth rate of 0.20 g/l/h. The extracellular protein concentration increased from 106.38 (±5.96) mg/l at the point of sugar depletion (20 h) to 201.31 (±86.30) mg/l after 44 h (Fig. 3a). The extracellular FAE activity followed the same trend and increased from 62.65 (±5.03) U/ml after 20 h to 83.48 (±19.72) U/ml after 44 h. The volumetric productivity of the enzyme production peaked at 6.89 (±0.01) U/ml/h at 10 h before dropping to 1.90 (±0.45) U/ml/h at 44 h (Table 4).

Fig. 3.

Fig. 3

Open in a new tab

Time course for the production of FAEA by A. niger D15[AtfaeA] using a 2-l bioreactor under batch (a) and fed-batch (b) conditions: (filled triangle) total extracellular protein concentration (mg/l), (filled square) FAE activity (U/ml), and (filled circle) volumetric productivity (U/ml/h). Error bars represent standard deviations from duplicate experiments

Table 4.

Comparison of A. niger D15[AtfaeA] performance in batch and fed-batch fermentations at maximum volumetric enzyme activity

Batch fermentation Fed-batch fermentation
Run time (h) 44 48
Biomass (g/l) 8.68 ± 0.30* 10.57 ± 0.50
Volumetric enzyme productivity (U/ml/h) 1.90 ± 0.45 3.86 ± 0.03
Yield (g FAE/gglucose) 0.29 ± 0.13 0.18 ± 0.01
Volumetric activity (U/ml) 83.48 ± 19.72 185.1 ± 1.33
Open in a new tab

* Values are the means of duplicate measurements from duplicate treatments, together with standard deviations

The A. niger D15[AtfaeA] was subsequently evaluated in a fed-batch fermentation with a 10 g/l initial glucose charge and a 20% glucose feed (final glucose concentration of 50 g/l, 40% dissolved oxygen). The batch phase of this fermentation was comparable to the batch fermentation described above with 58.32 (±9.52) U/ml FAE activity after 12 h (Fig. 3b), whereas the volumetric productivity of enzyme production decreased from 4.86 (±0.79) U/ml/h at 12 h to 3.86 (±0.03) U/ml/h at the end of the fermentation (Table 4). The fed-batch process yielded 0.18 g FAEA/gglucose with 10.57 g/l biomass produced at an overall growth rate of 0.22 g/l/h.

Although direct comparisons with other reports on FAE production are difficult due to variations in substrate and culturing conditions, the enzyme production by A. niger D15[AtfaeA] in the fed-batch reactor (185.14 U/ml) was significantly higher than the 0.97 U/ml reported for the A. tubingensis FAEA expressed in A. niger NW154 (Table 5). This confirms the suitability of the A. niger D15#16 strain for the expression of recombinant proteins, as previously reported (Rose and Van Zyl 2008).

Table 5.

Enzyme activity of a selection of recombinant fungal ferulic acid esterases

Enzyme Enzyme activity Recombinant host References
Aspergillus tubingensis FAEA 185.14 U/ml Aspergillus niger D15#26 This study
Aspergillus niger FAEA 3.16 U/ml Aspergillus tubingensis NW241 De Vries et al. 2002
Aspergillus tubingensisFAEA 0.97 U/ml Aspergillus niger NW154 De Vries et al. 2002
Aspergillus awamori FAEA 9 U/ml Pichia pastoris Koseki et al. 2006
Open in a new tab

Conclusions

Based on the hydrolysis profile of the methyl esters (MFA > MpCA > MSA), the recombinant AtFAEA has affinity for the C-5 feruloylated substrates and would thus be best at releasing ferulic acid from monocots such as wheat, oats, maize, barley, etc. where ferulic acid is esterified to the C-5 hydroxyl group of arabinopyranose residues. Furthermore, the relative stability of AtFAEA at pH 5–7 and 30–60 °C poses an advantage for applications in the food industry. Of particular importance is that the crude A. niger D15[AtfaeA] extract was able to release additional phenolic acids from maize and triticale bran without any pre-treatment of the substrate, which would support the cost-effective extraction of ferulic acid from plant substrates.

Enzyme production by the A. niger D15[AtfaeA] strain was significantly improved when cultured under either batch or fed-batch conditions, relative to shake flasks (Zwane et al. 2014), with the glucose-limited conditions in the fed-batch fermentation yielding higher extracellular FAE activity (185.14 vs. 83.48 U/ml) and volumetric productivity (3.86 vs. 1.90 U/ml/h) than the batch fermentation.

In conclusion, the crude A. niger D15[AtfaeA] extract was effective at extracting antioxidant-rich phytochemicals, represented in particular by ferulic acid, from triticale and maize bran. Treatment with FAEA can therefore increase the ferulic acid (and antioxidant) content and thus improve the nutritional value of these brans. Ferulic acid extracted from plant substrates using a natural (enzyme-based) process as opposed to alkaline treatment, can be used for the production of other ‘natural’ molecules such as vanillin, which is widely used in the food, pharmaceutical and cosmetic industries. An enzymatic process also has potential application in a multi-product biorefinery setup where the product(s) can serve directly as substrate(s) for other enzymes.

Acknowledgements

This work is based on the research supported in part by the National Research Foundation of South Africa (Grant 76597 to MVB and Grant 86423 to WHvZ). Prof Peter Punt, TNO Microbiology and Systems Biology, Netherlands, kindly provided the Aspergillus niger D15#26 host strain.

Author’s contribution

ENZ was the principal researcher responsible for experimental planning, execution, data collection and analyses, as well as drafting the manuscript. PJvZ assisted with the fermentation protocol; KGD with bran treatments and chemical analyses; SHR and KR with enzyme characterisation. WHvZ provided intellectual input together with MVB who prepared the final manuscript with all authors participating in the final editing.

References

  1. Balasundram N, Sundram K, Samman S. Phenolic compounds in plants and agri-industrial by-products: antioxidant activity, occurrence, and potential uses. Food Chem. 2006;99:191–203. doi: 10.1016/j.foodchem.2005.07.042. [DOI] [Google Scholar]
  2. Benoit I, Navarro D, Marnet N, Rakotomanomana N, Lesage-Meessen L, Sigoillot JC, Asther M. Feruloyl esterase as a tool for the release of phenolic compounds from agro-industrial by-products. Carbohydr Res. 2006;341:1820–1827. doi: 10.1016/j.carres.2006.04.020. [DOI] [PubMed] [Google Scholar]
  3. Benoit I, Danchin EGJ, Bleichrodt RJ, de Vries RP. Biotechnological applications and potential of fungal feruloyl esterases based on prevalence, classification and biochemical diversity. Biotechnol Lett. 2008;30:387–396. doi: 10.1007/s10529-007-9564-6. [DOI] [PubMed] [Google Scholar]
  4. De Vries RP, Michelser B, Poulsen CH, Kroon PA, van den Heuvel RH, Faulds CB, Williamson G, van den Hombergh JP, Visser J. The faeA genes from Aspergillus niger and Aspergillus tubingensis encodes ferulic acid esterases involved in degradation of complex cell wall polysaccharides. Appl Environ Microbiol. 1997;63:4638–4644. doi: 10.1128/aem.63.12.4638-4644.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. De Vries RP, Kester HCM, van Kuyk PA. The Aspergillus niger faeB gene encodes a second feruloyl esterase involved in pectin and xylan degradation, and is specifically induced on aromatic compounds. Biochem J. 2002;363:377–386. doi: 10.1042/bj3630377. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Debeire P, Khoune P, Jeltsch J-M, Phalip V. Product patterns of a feruloyl esterase from Aspergillus nidulans on large feruloyl-arabino-xylo-oligosaccharides from wheat bran. Bioresour Technol. 2012;119:425–428. doi: 10.1016/j.biortech.2012.01.185. [DOI] [PubMed] [Google Scholar]
  7. Faulds CB, Williamson G. Ferulic acid esterase from Aspergillus niger: purification and partial characterisation of two forms from a commercial source of pectinase. Biotechnol Appl Biochem. 1993;17:349–359. [PubMed] [Google Scholar]
  8. Faulds CB, de Vries R, Visser J, Williamson G. Stability of feruloyl esterases from Aspergillus. Biochem Soc T. 1998;26:S165. doi: 10.1042/bst026s165. [DOI] [PubMed] [Google Scholar]
  9. Fazary AE, Ju YH. Feruloyl esterase as biotechnological tools: current and future perspective. Acta Biochim Biophys Sin. 2007;39:811–828. doi: 10.1111/j.1745-7270.2007.00348.x. [DOI] [PubMed] [Google Scholar]
  10. Fazary AE, Ju YH. The large-scale use of feruloyl esterases in industry. Biotechnol Mol Biol Rev. 2008;3:095–110. [Google Scholar]
  11. Gong YY, Yin X, Zhang HM, Wu MC, Tang CD, Wang JQ, Pang QF. Cloning, expression of a feruloyl esterase from Aspergillus usamii E001 and its applicability in generating ferulic acid from wheat bran. J Ind Microbiol Biotechnol. 2013;40:1433–1441. doi: 10.1007/s10295-013-1339-6. [DOI] [PubMed] [Google Scholar]
  12. Graf E. Antioxidant potential of ferulic acid. Free Radical Biol Med. 1992;13:435–448. doi: 10.1016/0891-5849(92)90184-I. [DOI] [PubMed] [Google Scholar]
  13. Hedge S, Srinivas P, Muralikrishna G. Single-step synthesis of 4-nitrophenyl ferulate for spectrophotometric assay of feruloylesterase. Anal Biochem. 2009;387:128–129. doi: 10.1016/j.ab.2009.01.001. [DOI] [PubMed] [Google Scholar]
  14. Heinonen M, Rein D, Satue-Gracia MT, Huang SW, German JB, Frankel EN. Effect of protein on the antioxidant activity of phenolic compounds in a lecithin-liposome oxidation system. J Agr Food Chem. 1998;46:917–922. doi: 10.1021/jf970826t. [DOI] [Google Scholar]
  15. Hosseinian FS, Mazza G. Triticale bran and straw: potential new sources of phenolic acids, proanthocyanidins, and lignans. J Funct Foods. 2009;1:57–64. doi: 10.1016/j.jff.2008.09.009. [DOI] [Google Scholar]
  16. Kanauchi M, Watanabe S, Tsukada T, Atta K, Kakuta T, Koizumi T. Purification and characterisation of ferulyol esterase from Aspergillus awamori G-2 strain. J Food Sci. 2008;73:458–463. doi: 10.1111/j.1750-3841.2008.00839.x. [DOI] [PubMed] [Google Scholar]
  17. Kim M, Hyun J, Kim J, Park J, Kim M, Kim J, Lee S, Chun S, Chung F. Relationship between phenolic compounds, anthocyanins content and antioxidant activity in colored barley germplasm. J Agr Food Chem. 2007;55:4802–4809. doi: 10.1021/jf0701943. [DOI] [PubMed] [Google Scholar]
  18. Koseki T, Takahashi K, Fushinobu S, Iefuji H, Iwano K, Hashizume K, Matsuzawa H. Mutational analysis of a feruloyl esterase from Aspergillus awamori involved in substrate discrimination and pH dependence. Biochim Biophys Acta. 2005;1722:200–208. doi: 10.1016/j.bbagen.2004.12.016. [DOI] [PubMed] [Google Scholar]
  19. Koseki T, Takahashi K, Handa T, Yamane Y, Fushinobu S, Hashizume K. N-Linked oligosaccharides of Aspergillus awamori feruloyl esterase are important for thermostability and catalysis. Biosci Biotechnol Biochem. 2006;70:2476–2480. doi: 10.1271/bbb.60207. [DOI] [PubMed] [Google Scholar]
  20. Koseki T, Fushinobu S, Ardiansyah SH, Komai M. Occurrence, properties, and applications of feruloyl esterases. Appl Microbiol Biotechnol. 2009;84:803–810. doi: 10.1007/s00253-009-2148-8. [DOI] [PubMed] [Google Scholar]
  21. Kumar N, Pruthi V. Potential applications of ferulic acid from natural sources. Biotechnol Rep. 2014;4:86–93. doi: 10.1016/j.btre.2014.09.002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Lapierre C, Pollet B, Ralet M, Saulnier L. The phenolic fraction of maize bran: evidence for lignin-heteroxylan association. Phytochemistry. 2001;57:765–772. doi: 10.1016/S0031-9422(01)00104-2. [DOI] [PubMed] [Google Scholar]
  23. Mathew S, Abraham TE. Ferulic acid: an antioxidant found naturally in plant cell walls and feruloyl esterases involved in its release and their applications. Crit Rev Biotechnol. 2004;24:59–83. doi: 10.1080/07388550490491467. [DOI] [PubMed] [Google Scholar]
  24. Poidevin L, Levasseur A, Pae G, Navarro D, Heiss-Blanquet S, Asther M, Record E. Heterologous production of the Piromyces equi cinnamoyl esterase in Trichoderma reesei for biotechnological applications. Lett Appl Microbiol. 2009;49:673–678. doi: 10.1111/j.1472-765X.2009.02734.x. [DOI] [PubMed] [Google Scholar]
  25. Priefert H, Rabenhorst J, Steinbuchel A. Biotechnological production of vanillin. Appl Microbiol Biotechnol. 2001;56:296–314. doi: 10.1007/s002530100687. [DOI] [PubMed] [Google Scholar]
  26. Rose SH, van Zyl WH. Constitutive expression of the Trichoderma reesei β-1,4-xylanase gene (xyn2) and the β-1,4-endoglucanase gene (egI) in Aspergillus niger in molasses and defined glucose media. Appl Microbiol Biotechnol. 2002;58:461–468. doi: 10.1007/s00253-001-0922-3. [DOI] [PubMed] [Google Scholar]
  27. Rose SH, van Zyl WH. Exploitation of Aspergillus niger for the heterologous production of cellulases and hemicelluloses. Open Biotechnol J. 2008;2:165–175. doi: 10.2174/1874070700802010167. [DOI] [Google Scholar]
  28. Sambrook J, Fritsch EF, Maniatis T. Molecular cloning: a laboratory manual. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press; 1989. [Google Scholar]
  29. Shin H, Chen RR. Production and characterisation of a type B feruloyl esterase from Fusarium proliferatum NRRL 26517. Enzyme Microb Technol. 2006;38:478–485. doi: 10.1016/j.enzmictec.2005.07.003. [DOI] [Google Scholar]
  30. Sundberg M, Poutanen K, Markkanen P, Linko M. An extracellular esterase of Aspergillus awamori. Biotechnol Appl Biochem. 1990;12:670–680. [Google Scholar]
  31. Yu P, McKinnon JJ, Christensen DA. Improving the nutritional value of oat hulls for ruminant animals with pretreatment of a multi-enzyme cocktail: in vitro studies. J Anim Sci. 2005;83:1133–1141. doi: 10.2527/2005.8351133x. [DOI] [PubMed] [Google Scholar]
  32. Zhao Z, Moghadasian MH. Chemistry, natural sources, dietary intake and pharmacokinetic properties of ferulic acid: a review. Food Chem. 2008;109:691–702. doi: 10.1016/j.foodchem.2008.02.039. [DOI] [PubMed] [Google Scholar]
  33. Zhao S, Yao S, Ou S, Line J, Wang Y, Peng X, Li A, Yu B. Preparation of ferulic acid from corn bran: its improved extraction and purification by membrane separation. Food Bioprod Process. 2014;92:309–313. doi: 10.1016/j.fbp.2013.09.004. [DOI] [Google Scholar]
  34. Zwane EN, Rose SH, van Zyl WH, Rumbold K, Viljoen-Bloom M. Over-expression of Aspergillus tubingensis faeA in protease-deficient Aspergillus niger enables ferulic acid production from plant material. J Ind Microbiol Biotechnol. 2014;41:1027–1034. doi: 10.1007/s10295-014-1430-7. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Food Science and Technology are provided here courtesy of Springer

RESOURCES