Food Chemistry 131 (2012) 1430–1436

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Food Chemistry

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Fungal enzymes as a powerful tool to release antioxidants from olivemill wastewater

Manel Hamza, Sonia Khoufi ⇑, Sami SayadiLaboratoire des Bioprocédés Environnementaux, pôle d’excellence régional (PER, AUF), Centre de Biotechnologie de Sfax, University of Sfax, B.P. ‘‘1177’’, 3018 Sfax, Tunisia

a r t i c l e i n f o

Article history:Received 27 April 2011Received in revised form 30 September2011Accepted 7 October 2011Available online 13 October 2011

Keywords:Fungal enzymeHydrolysisOlive mill wastewaterPhenolic compoundsHydroxytyrosolAntioxidant activity

0308-8146/$ - see front matter � 2011 Elsevier Ltd. Adoi:10.1016/j.foodchem.2011.10.019

Abbreviations: HT, hydroxytyrosol; OMW, olive mdextrose agar slants; BHT, butylated hydroxytolupicrylhydrazyl.⇑ Corresponding author. Tel./fax: +216 74 874 452.

E-mail address: sonia.khoufi@cbs.rnrt.tn (S. Khoufi

a b s t r a c t

Enzymatic hydrolysis of olive mill wastewater compounds (OMW) using culture broth of Aspergillus niger,Trichoderma atroviride and Trametes trogii was tested in order to release free simple phenolic compoundshaving high antioxidant activity. HPLC analysis of treated and untreated OMW indicated the release oflarge amounts of free hydroxytyrosol (HT). Enzymatic pre-treatment of OMW by A. niger and T. atroviridebroth cultures which are rich in b-glucosidase increased the amount of HT respectively from 0.05 up to1.1 and 0.50 g/L. However, the use of T. trogii broth culture showed an oxidation of phenolic compoundsdue to its high laccase activity. The ethyl acetate extract of OMW treated by A. niger broth culture withIC50 of 45 lg/mL showed the strongest antioxidant potency compared to untreated OMW with IC50 of501 lg/mL.

� 2011 Elsevier Ltd. All rights reserved.

1. Introduction

The three-phase and the continuous olive-oil extraction pro-cesses generate a dark-coloured effluent usually termed olive millwastewater (OMW). This effluent is characterised by a high pollut-ing load and the presence of recalcitrant phenolic compounds(Casa et al., 2003). Due to the presence of these compounds,OMW disposal constitutes a large-scale environmental problemparticularly in the Mediterranean area where about 3 � 107 m3 ofthis effluent are produced annually. OMW has broad spectrum tox-icity against microorganisms (Mekki, Dhouib, Feki, & Sayadi, 2008).However, olive biophenols are most widely studied as antioxidantsand free radical scavengers (Ziogas, Tanou, Molassiotis, Diamanti-dis, & Vasilakakis, 2010) and OMW is rich in these compounds.Several researchers identify OMW as a potential source for therecovery of antioxidants (Feki, Allouche, Bouaziz, Gargoubi, & Say-adi, 2006; Fki, Allouche, & Sayadi, 2005; Ziogas et al., 2010).Hydroxytyrosol [4-(2-hydroxyethyl)-1,2-benzenediol,2] (HT) isthe main natural phenolic compound occurring in OMW and it isalso present in virgin olive oil, albeit in very small amounts andmainly in the form of natural derivatives (Bianco & Uccella,

ll rights reserved.

ill wastewater; PDA, potatoene; DPPH, 1,1-diphenyl-2-

).

2000). It originates in all likelihood from the hydrolysis of oleurop-ein by means of enzyme during the mill process (Bianco & Uccella,2000).

HT is characterised by a highly antioxidant activity, which issimilar to that of the main synthetic antioxidants such as 2,6-di-tert-butyl-p-hydroxytoluene (BHT) and 3-tert-butyl-6-hydroxyani-sole (BHA) (Fki et al., 2005). In vitro, it also inhibits the oxidation oflow-density lipoproteins (Visioli & Galli, 2002) and confers bothcell protection (Galli, Zsebo, & Geissler, 1994) and dietetic proper-ties to virgin olive oil (Visioli & Galli, 2002).

Owing to its application in food as well as in cosmetic and phar-macological industries, substantial interest has been given to theproduction of HT both from natural sources or by synthetic orchemical procedures. Natural HT was recovered by bench-scalepurification from OMW (Capasso, Evidente, Avolio, & Solla, 1999).Espin, Soler-Rivas, Cantos, Tomàs-Barberàn, and Wichers, (2001)discussed the synthesis of HT from tyrosol using mushroomtyrosinase as a biocatalyst and ascorbic acid as a reducing agent.Another enzymatic method was also described, based on oleurop-ein hydrolysis by b-glucosidase and destabilisation of the aglyconestructure under defined temperatures and pH conditions (Briante,La Cara, Febbraio, Patumi, & Nucci, 2002). Biological strategies haveopened the channel for the bioconversion, using whole cells for HTproduction (Bouallagui & Sayadi, 2006). All these studies describedthe production of HT from a synthetic precursor molecule such astyrosol or oleuropein. The problem of HT recovery from raw OMWis that HT concentration is very low (from 0.2 to 0.4 g L�1) and does

M. Hamza et al. / Food Chemistry 131 (2012) 1430–1436 1431

not support recovering processes. This concentration represent thefree form of HT. HT conjugated forms are also present in OMWas oleuropein, HT-glycosides and verbascoside. To increase HTconcentration in this by-product, clean biotechnological processescould be used for the enhancement of HT release from itsconjugated forms.

The importance of microbial enzymes in biotechnology applica-tions has grown significantly in the last two decades, particularlyin the bioconversion of industrial by-products (Bhanja, Kumari, &Banerjee, 2009). Fungi are microorganisms well known for thewide range of enzymes they produce and which are used in manyindustrial processes.

To our knowledge, there are few reports on the effect of fungalenzyme addition on the polyphenolic composition of OMW. There-fore, the objective of this work was to evaluate the possibility ofapplying raw growth culture media (without any purification step)from three strains of fungi – Aspergillus niger CTM 10099, Tricho-derma atroviride CTM 10476 and Trametes trogii CTM 10156 – onOMW, with the aim of increasing the concentration of antioxidantcompounds such as HT.

2. Materials and methods

2.1. Chemical and raw materials

Fresh OMW was sampled from a three-phase discontinuousextraction factory located in Sfax, Tunisia. Samples were collectedand stored at �20 �C. Wheat bran was obtained locally and storedat 4 �C.

Ethyl acetate, acetonitrile and orthophosphoric acid were HPLC-grade solvents and purchased from Prolabo (France). HT, used as astandard, was purified from OMW in our laboratory (Allouche, Da-mak, Ellouz, & Sayadi, 2004). 1,1-Diphenyl-2-picrylhydrazyl(DPPH) and HPLC standards (tyrosol, caffeic acid, p-coumaric acidand oleuropein) were purchased from Fluka (Buchs, Switzerland).Finally, p-nitrophenol, p-nitrophenyl-b-D-glucopyranoside, p-nitro-phenyl acetate, 2,6-dimethoxyphenol, butylated hydroxytoluene(BHT) and b-glucosidase pure enzyme from A. niger (EC 2328898)were purchased from Sigma–Aldrich (St. Louis, MO). Viscozyme,a commercial enzyme preparation, was provided by Novo-NordiskA/S (Bagsvaerd, Denmark).

2.2. Biological material

A. niger CTM 10099, T. trogii CTM 10156 and T. atroviride CTM10476 were taken from the «Collection Tunisienne de Microorgan-ismes», Centre de Biotechnologie de Sfax (Sfax, Tunisie). Strainswere maintained on potato dextrose agar slants (PDA).

2.3. A. niger cultivation

A spore stock suspension was obtained by growing the funguson PDA at 30 �C for 5 days. The conidia were harvested from thesurface by adding sterile water containing 2% (v/v) Tween 80 andscraping the surface with a sterile spatula. The obtained spore con-centrated solution was stored in 25% (v/v) glycerol at �20 �C.

Wheat bran, an industrial by-product, was used as sole carbonsource for the growth of fungal mycelia under liquid state condi-tions. A. niger fermentation on wheat bran was conducted in500-mL shake flask experiments containing 100 mL of liquid med-ium for the fungal growth. The optimised quantity of wheat branwas 6.7 g per 100 mL of culture medium. The composition of themedium was optimised with the aim of improving b-glucosidaseproduction by A. niger. This culture medium consists of 1 g/LKH2PO4, 2.5 g/L K2HPO4, 0.3 g/L MgSO4 7H2O, 0.3 g/L CaCl2,3.75 g/L urea, 3.75 g/L wheat peptone, 1 g/L yeast extract, 1 mL/L

Tween 80 and 1 mL/L trace elements. The pH was adjusted to 4by adding HCl (2 M) solution. Shake flask experiments containinga mixture of wheat bran and liquid medium are closed by plugsErlenmeyer gas, and then sterilised by autoclaving (20 min,121 �C). The sterilised Erlenmeyer flasks were inoculated with A.niger spores at a concentration of 6 � 108 spores/mL of mediumand then inoculated at 30 �C with agitation at 160 rpm for 10 days.

2.4. T. atroviride and T. trogii cultivation

Fungal conidia were used to inoculate a PDA solid medium andcultivated at 30 �C for 7 days. The inoculation culture was preparedby transferring two loops of the grown mycelia with conidia toshake flasks (250 mL) each filled with 50 mL malt extract (20 g/L)medium. Flasks were shaken at 160 rpm and incubated at 30 �Cfor 72 h. A 2% (v/v) inoculum was transferred to the productionculture, which contained 100 mL of medium and wheat bran in a500-mL Erlenmeyer flask. The medium was the same as for A. nigercultivation for b-glucosidase production. Fungal cultures weregrown at 30 �C with agitation (160 rpm) for 10 days.

2.5. Fungal enzyme preparation

Fungal culture broths were withdrawn every day from theexperimental fermentations. The volume of the obtained enzymepreparation decreased with cultivation time (from 100 to 90 mL).These samples were filtered (Whatman GF/D) and the clarifiedcrude enzyme preparations were kept at �4 �C until their use forbiotransformation and/or enzyme assays. Enzyme solution of b-glucosidase purified from A. niger (EC 2328898) was prepared bydissolving 1.5 mg of enzyme powder in 0.8 mL of citrate buffer(20 mM, pH 4.5).

The enzymatic profile of the commercial enzyme preparation,Viscozyme, determined in our laboratory was as follows: pectinase(105 IU/mL), xylanase (47 IU/mL), CMCase (19 IU/mL), b-glucosi-dase (27000 IU/mL) and esterase (170 IU/mL).

2.6. Enzyme assays

The b-glucosidase activity was determined according to Nork-rans (1950) but with the use of 5.0 mM substrate instead of1.0 mM. A sample volume of 0.1 mL was mixed with 0.9 mL of5.0 mM p-nitrophenyl-b-D-glucopyranoside in 50 mM sodium cit-rate buffer (pH 4.8) and kept at 50 �C for 10 min. The reactionwas stopped by additing 2.0 mL of 1.0 M Na2CO3 followed by10 mL of water and the absorbance was measured at 405 nm. Inthe blank, 0.1 mL of water were used instead of the enzyme sam-ple. The activity was defined as lmoL of p-nitrophenol producedper minute under the assay conditions (IU). Activities were mea-sured in triplicate and were expressed in International Unit permillilitre (IU/mL) with 1 IU being defined as the amount of enzymethat catalyses the release of 1.0 lmol of p-nitrophenol per minute.

Esterase activity was carried out according to the method ofMackness, Walker, Rowlands, and Price (1983). The reaction mix-ture contained 50 lL of crude enzyme, 50 lL of 150 mM p-nitro-phenyl acetate in ethanol and 2.9 mL of Tris–HCl buffer (9.2 mM,pH 7.5), to give a final concentration of 2.5 mM p-nitrophenyl ace-tate in a total volume of 3.0 mL. The reaction was initiated by add-ing the enzyme extract and incubated at 25 �C for 4 min.Absorbance was determined at 405 nm. A standard curve was pre-pared in the range of 0–220 nmol of p-nitrophenol.

Laccase activity was determined by using 10 mM DMP in100 mM sodium tartrate buffer, pH 5.0 (e469 = 27,500/M cm, basedon DMP concentration) (Yaropolov, Skorobogatko, Vartanov, & Var-folomeyev, 1994). The enzymatic reactions were carried out atroom temperature (22–25 �C) and one unit of enzyme activity

1432 M. Hamza et al. / Food Chemistry 131 (2012) 1430–1436

was defined as the amount of enzyme oxidising 1 lmol of sub-strate per minute.

2.7. Bioconversion of OMW compounds by fungal enzyme preparations

Bioconversion reactions were conducted in 150-mL Erlenmeyerflasks with 20 mL of medium reaction: 5 mL of filtered fungal brothculture were added to 15 mL of OMW (pH 4.8). The concentrationof b-glucosidase in the medium reactions varied as a function ofcultivation time and the fungal strain. For the comparative study,Viscozyme and enzyme solution of A. niger-purified b-glucosidasewere added to OMW at a concentration of 1500 IU/mL in termsof b-glucosidase activity. The control was incubated with 5.0 mLof water instead of enzyme. The enzymatic reaction was performedat 50 �C for 2 h under static conditions. During the course of hydro-lysis, samples were collected every 30 min, and then extractedwith solvent for subsequent analyses by HPLC. In order to assessthe repeatability of the hydrolysis, two parallel sets were carriedout under identical conditions.

2.8. Extraction of phenolic compounds from OMW

Liquid/liquid extraction with ethyl acetate was used for therecovery of OMW phenolic compounds (Khoufi, Aloui, & Sayadi,2008). The extraction of OMW samples (5 mL) was carried out ina separating funnel with added ethyl acetate (v/v). The mixture(solvent/OMW) was vigorously shaken for 10 min at room temper-ature and then centrifuged for 3 min at 3000 rpm. The organicphase was separated and filtered with a syringe filter (0.45 lM)for HPLC analysis.

2.9. HPLC analysis

HPLC was performed on a Shimadzu (Kyoto, Japan) C-R6A liquidchromatograph. The separation was carried out using a SupelcoC18 column (250 mm � 4.6 mm; Supelco, Bellefonte, PA). Com-pounds were eluted with a gradient, acetonitrile (70%)–H3PO4

(0.1%), where the acetonitrile concentration was varied as follows:0 min, 10%; 0–20 min, increased to 50%; 20–25 min, 50%; and 25–30 min, decreased to 10%. The column temperature was main-tained at 40 �C, and the flow rate was 0.5 mL/min. Sample detec-tion was achieved at 280 nm with a Shimadzu SPD 6AUVdetector connected to a Shimadzu C-R6A integrator. The injectionvolume was 20 lL. Compounds were identified and quantified bycomparison of retention times (RT) and peak areas of standardsanalysed under the same conditions: hydroxytyrosol (RT = 10 min),tyrosol (RT = 15 min), p-coumaric acid (RT = 25 min), caffeic acid(RT = 30 min) and oleuropein (RT = 38 min).

Table 1Optimal conditions and culture medium composition forb-glucosidase production by A. niger.

Parameter Results

Carbon source Wheat branOrganic nitrogen source Wheat peptoneInorganic nitrogen source UreaRatio C/N 10pH 4Temperature incubation 30 �CInoculum size 6 � 108 spores/mLAgitation 160 rpm.

2.10. DPPH radical method

The 1,1-diphenyl-2-picryl-hydrazyl radical (DPPH radical) scav-enging effects were evaluated according to the method used by Fkiet al. (2005). Four millilitres of each sample extract at differentconcentrations were added to 10 mL DPPH methanol solution(1.5 � 10�4 M). After mixing the two solutions softly and afterleaving for 30 min at room temperature, the optical density wasmeasured at 520 nm using a Shimadzu UV-160A spectrophotome-ter. The positive control BHT was tested over a range of concentra-tions. The antioxidant activity of each test sample and BHT wereexpressed in terms of concentration required to inhibit 50% DPPHradical formation (IC50 lg/mL) and calculated from the log-doseinhibition curve.

3. Results and discussion

3.1. Enzyme production

b-Glucosidase production by the mesophilic fungus A. niger wasenhanced by the optimisation of medium composition and fermen-tation conditions at flask level. Different substrates were tested assole carbon source for A. niger fermentation. The nature of nitrogensource was also optimised. Indeed, different organic and inorganicnitrogen supplements were incorporated in the medium to inves-tigate their effects on b-glucosidase production. The fermentationparameters, such as initial pH, incubation temperature and inocu-lum size, were also optimised. At each step of optimisation, theprocess conditions resulting in the highest enzyme yield were cho-sen as the control for the next experiment. The various solid andcereal-based substrates screened were wheat bran, barley bran,guar flour, saccharose, cellulose, and lactose. The result shows thatwheat bran, an available agricultural by product, is the best carbonsource and the urea and wheat peptone are the best sources ofnitrogen. Optimised cultivation conditions for A. niger on wheatbran were pH 4, incubation temperature of 30 �C and inoculum sizeof 6 � 108 spores/mL (Table 1). The same culture medium and cul-tivation conditions obtained for A. niger were used for T. atrovirideand T. trogii fermentations. A kinetic study of wheat bran fermen-tation by A. niger, T. atroviride, and T. trogii was investigated for10 days, in order to evaluate their enzyme production. For thecharacterisation of the enzyme profile of the three multienzymepreparations, only b-glucosidase, esterase and laccase activitieswere analysed.

Fig. 1 shows the typical b-glucosidase, esterase and laccase pro-duction profiles during the fermentation of wheat bran by A. niger,T. atroviride and T. trogii. These results show that the three strainsproduced different enzyme activities (Fig. 1). Indeed, wheat bran isa complex substrate rich in proteins (14%), carbohydrates (27%),minerals (5%), fat (6%) and B-vitamins (Haque, Shams-Ud-Din, &Haque, 2002). Kang, Park, Lee, Hong, and Kim (2004) have alsodemonstrated the production of cellulases and hemicellulases byusing derived wheat (bran and straw) as substrate. In this study,an increase of b-glucosidase was observed during the fermentationof wheat bran by the three strains. A. niger produced the highestactivity, followed by T. atroviride and T. trogii. A. niger producedapproximately 10 times more b-glucosidase than T. atroviride.Interestingly, esterase activity was detected in the three broth cul-ture preparations at the same order but laccase activity was pro-duced only by T. trogii and T. atroviride (Fig. 1). After 10 days ofcultivation, the highest b-glucosidase activity (6380 IU/mL) andlaccase activity (2246 IU/L) were obtained in the culture brothsof A. niger and T. trogii, respectively (Fig. 1a and 1c). However, T.atroviride produced the three enzymes at moderate levels (Fig. 1b).

(a)

(b)

(c)

Fig. 1. Evolution of b-glucosidase (BG), esterase and laccase activities in the culturebroths of A. niger (a), T. atroviride (b) and T. trogii (c) during 10 days of cultivation onwheat bran.

M. Hamza et al. / Food Chemistry 131 (2012) 1430–1436 1433

3.2. Enzymatic hydrolysis of OMW compounds

b-Glucosidases have also been studied due to their potential torelease flavour compounds, such as terpenes from odourless non-volatile glycosidic precursors in fruit juices and wines (Maicas &Mateo, 2005) and to release phenolic compounds with antioxidant,nutraceutical and flavourant properties from their glycosylatedforms in fruit and vegetable residues (Bhanja et al., 2009; Duenas,Hernandez, & Estrella, 2007). In the light of these studies, the effectof A. niger, T. atroviride and T. trogii broth filtrates on the hydrolysisof OMW macromolecules was studied.

3.2.1. Effect of A. niger, T. atroviride and T. trogii culture brothsFig. 2a gives the HPLC chromatogram of ethyl acetate extract of

raw OMW (control). This profile shows that fresh OMW is rich inoleuropein (peak 3) and another verbascoside compound not iden-tified (peak 4). Simple phenolic bioactive compounds such as HT(peak 1) and tyrosol (peak 2) are also present at low concentra-

tions. The concentration of HT was 0.05 g/L. This content corre-sponds to the free fraction of HT in raw OMW. Enzymaticanalysis shows that raw OMW contains a b-glucosidase activityin the range of 80 IU/mL (Table 2). This enzyme can be derivedfrom olive fruit (Jemai, Bouaziz, & Sayadi, 2009) and also fromthe activity of endogenous microorganisms (Ciafardini & Zullo,2000). This enzyme concentration (as well as other hydrolytic en-zymes) is not sufficient for the hydrolysis of conjugates present infresh OMW. HT has been shown to increase with time of OMWstorage, as a result of spontaneous fermentation and the cleavageof HT conjugates (Feki et al., 2006).

Here, the increase of bioactive compounds concentration wastested through the enzymatic hydrolysis of OMW during 2.0 h at50 �C by using culture broth of A. niger, T. atroviride and T. trogii.Batch fermentations on wheat bran with the three strains wereinvestigated under the same controlled conditions. Fig. 3 showsthe evolution of HT from hydrolysed OMW using the different fun-gal extracellular broths recovered at different times of cultivation.As shown from this figure, significant amounts of HT were releasedafter the different enzymatic pre-treatments of OMW. Fig. 2 showsthat HPLC chromatograms exhibited an increase of HT peak (1)with a concomitant decrease of the oleuropein peak (3) after enzy-matic pre-treatment of OMW. The highest concentrations of HTwere obtained in the presence of A. niger culture broths.

A progressive increase of HT concentration in OMW was ob-served with cultivation time of A. niger which could correlate withthe increase of b-glucosidase activity in the culture broth. Goodcorrelation between high b-glucosidase activity and HT release inOMW was observed (Figs. 1 and 3) for the three strains. These re-sults can confirm that b-glucosidase plays a significant biochemicalrole in catalysing the hydrolysis of the glycosidic bonds in oligoand polysaccharides present in OMW. The esterase activity mea-sured in the three broth cultures showed activities between 100and 200 IU/mL (Fig. 1). This enzyme is able to hydrolyse esterbonds between phenolic compounds and polysaccharides. Oleu-ropein is a heterosidic ester of b-glucosylated elenolic acid andHT. Thus, HT is thought to have originated from oleuropein, viaits aglycone, by the opening of the elenolic acid ring with a finalrearrangement into the secoiridoid compound and many forms ofelenolic acid (Walter, Fleming, & Etchells, 1973). This result is inagreement with previous studies of Capasso et al. (1999), and Bri-ante et al. (2002).

During the enzymatic experiments, maximum HT concentra-tions of about 1.1, 0.5 and 0.2 g/L were obtained in the presenceof culture broths of A. niger at Day 10 (6381 IU/mL), T. atrovirideat Day 4 (365 IU/mL) and T. trogii at Day 2 (29 IU/mL), respectively.These concentrations were 21.6-, 10.2- and 4.1-fold higher thanthe control (Table 2). An increase of tyrosol was also observed inthe case of T. atroviride (Fig. 2e). This result shows that T. artrovirideenzymes are also active on polymers containing tyrosol as a pheno-lic entity such as ligostroside. However, the use of T. artroviride cul-ture broth which exhibited moderate b-glucosidase and laccaseactivities resulted in the decrease of HT concentration in OMW(from 0.5 to 0.03) at the end of fermentation (Fig. 2e). This degra-dation is explained by the oxidation of HT by laccase which is verysensitive to oxidative agents than tyrosol (Chakroun, Mechichi,Martinez, Dhouib, & Sayadi, 2010). The use of T. trogii culture brothwhich showed high laccase activity resulted in the decrease of thetotal phenolic compounds concentration (Fig. 1c and 2f). This re-sult shows that laccases are effective for the transformation or oxi-dation of simple and complex phenolic compounds and can beregarded as an efficient tool for the detoxification of phenolicwastewaters as OMW. Oxidative transformation of natural andsynthetic phenolic mixtures by Trametes versicolor laccase was alsoinvestigated by Canfora, Iamarino, Antonietta Rao, and Gianfreda(2008).

Fig. 2. HPLC chromatogram of phenolic compounds (detection at k = 280 nm) extracted from raw (a) and hydrolysed OMW by purified b-glucosidase (b) and by enzymepreparations collected from the fermentation of wheat bran by A. niger at Day 10 (c), T. atroviride at Day 4 (d) and Day 10 (e) and T. trogii at Day 10 (f) of cultivation. 1,hydroxytyrosol; 2, tyrosol; 3, oleuropein; 4, unidentified compound.

1434 M. Hamza et al. / Food Chemistry 131 (2012) 1430–1436

From this part of the study, we can conclude that OMW is asource of hydrolysable phenols, such as oleuropein, ligstroside orverbascoside, which possess glucosidic and ester links betweenthe main phenol components and polysaccharides and/or lignin(Bianco & Uccella, 2000; Walter et al., 1973). Consequently, higherquantity of HT is freed when enzymatic hydrolysis is carried out onOMW by using specific enzyme preparations.

3.2.2. Effect of commercial enzyme preparationsBioconversion experiments using A. niger purified b-glucosidase

(EC 2328898) and Viscozyme were conducted using the same oper-ational conditions. These enzyme preparations were added toOMW at proportions giving a b-glucosidase concentration of

1500 IU/mL of medium reaction. The enzymatic hydrolysis ofOMW by using A. niger-purified b-glucosidase gave 1.15 g/L ofHT. This value is broadly similar to that obtained by raw A. nigerbroth culture without any purification step (Fig. 4). However, theincrease of the ortho-diphenol content using A. niger-purified b-glucosidase during the biotransfomation of OMW compounds hasbeen observed in many studies which have used this enzyme forthe hydrolysis of pure oleuropein (Ciafardini & Zullo, 2000), or Oleaeuropaea leaf extracts (Briante et al., 2002) or in green olives (Wal-ter et al., 1973).

Fig. 4 shows also the effect of Viscozyme on the HT release fromOMW. It was observed that Viscozyme increased the HT concentra-tion from 0.05 (control) to 1.2 g/L. In comparison to purified b-glu-

Table 2Maximum hydroxytyrosol (HT) concentrations (g/L) obtained after enzymatic treat-ment of OMW by using fungal culture broths and the corresponding b-glucosidase(IU/mL), esterase (IU/mL) and laccase activities (IU/L). (Mean values ± standarddeviation (n = 3)).

b-Glucosidase Esterase Laccase HT

Aspergillus niger 6380 ± 150 130 ± 20 0 ± 0.0 1.1 ± 0.2Trichoderma atroviride 365 ± 50 218 ± 20 840 ± 50 0.5 ± 0.2Tramates trogii 29 ± 10 230 ± 20 2246 ± 50 0.2 ± 0.1Raw OMW 80 ± 10 nd nd 0.05 ± 0.02

Nd, not detected.

0

0,2

0,4

0,6

0,8

1

1,2

Control

Day2Day3

Day4Day 5

Day 6Day

7Day

8Day

9

Day 10

Trametes trogii Trichoderma atroviride Aspergillus niger

Fig. 3. Evolution of hydroxytyrosol concentration in the phenolic fraction recoveredfrom OMW after hydrolysis treatment by extracellular broths collected during thefermentation of wheat bran by the three strains.

0

0,2

0,4

0,6

0,8

1

1,2

1,4

Purified -glucosidase

Viscozyme Enzymepreparation

Control

Hydr

oxyt

yros

ol (g

/l)

Fig. 4. Concentration of hydroxytyrosol in OMW before and after enzymatictreatment by purified b-glucosidase, Viscozyme and A. niger enzyme preparation.

Table 3Radical-scavenging effects of phenolic compounds onDPPH radical. (Values are means ± standard deviation ofthree determinations).

Extract and pure compounds IC50 (lg/mL)

BHT 28 ± 1.15Raw OMW EA extract 501 ± 13Hydrolysed OMW EA extract 45 ± 3.12Pure hydroxytyrosol 22 ± 2.02Oleuopein 173 ± 4.93

M. Hamza et al. / Food Chemistry 131 (2012) 1430–1436 1435

cosidase and A. niger preparation, Viscozyme gives the highest HTconcentration. This can be explained by the multienzymatic com-position of this commercial enzyme preparation, which containscellulases, arabinases, hemicellulases, gluconases and xylanases,capable of acting on a great number of components, in particularthe soluble and insoluble fibre fraction (Duenas et al., 2007), andthis causes modifications of several components and can releasemore simple phenolic compounds. These results indicated that A.niger enzyme preparation has the same effect as Viscozyme andpurified b-glucosidase for HT release.

Therefore, A. niger, generally recognised as a safe (GRAS) micro-organism, is an excellent producer of b-glucosidase, which is thekey hydrolytic enzyme for the monomerisation of OMW polyphe-nols. Hence, A. niger enzyme preparation is the best alternativefor increasing HT content in OMW via clean bioconversion reac-tion. Added to this, the enzyme preparation from A. niger is freefrom laccase, which is an oxidative enzyme for phenolic com-pounds. A high concentration of HT (1.1 g/L) was obtained after2 h of enzymatic reaction of OMW in the presence of A. niger en-zyme preparation at 50 �C, pH 4.8 and under static conditions.The obtained concentration of HT through this bioconversionmethod can be improved by optimisation of other parameters: en-zyme concentration, agitation condition, reaction time, etc.

According to previous works (Bouallagui & Sayadi, 2006; Brianteet al., 2002; Capasso et al., 1999; Espin et al., 2001), synthesisprocedures for HT production are expensive and/or produce lowyields. For large-scale application of the proposed bio-process,1 m3 of OMW resulted in the production of 1.1 kg of HT. Preliminarycalculation cost of OMW hydrolysis by A. niger enzyme preparationgives a very interesting value of 695 US $/m3. Hence, this bio-process could open a new possible methodology to recover a highyield of HT at low cost.

3.3. DPPH radical-scavenging effect of OMW extract

The DPPH radical-scavenging effects of raw and hydrolysedOMW ethyl acetate extracts, BHT, pure HT and pure oleuropein

are shown in Table 3. Among the compounds present in OMW, itwas find that ortho-dihydroxylated aromatic components exhib-ited higher scavenging activity on DPPH, particularly HT and 3,4-dihydroxyphenylacetic acid (Fki et al., 2005). As shown in Table 3,HT is a powerful antioxidant and its activity (IC50 = 22 lg/mL) ishigher than that of a commercial antioxidant, BHT, (IC50 = 28 lg/mL). In particular, the antioxidant activities of raw and hydrolysedOMW extract were lower than that of BHT (Table 3). The radical-scavenging effect of raw OMW extract (IC50 = 501 lg/mL) is par-tially related to its content of caffeic acid, 3,4-dihydroxyphenylacetic acid and particularly to oleuropein. Ethyl acetate extract ofhydrolysed OMW exhibited a higher antiradical activity(IC50 = 45 lg/mL) in comparison to the ethyl acetate extract ofraw OMW and to oleuropein (Table 3). These results demonstratedthat the enzymatic hydrolysis of OMW compounds by A. niger en-zyme preparation leads to an increase of the antioxidant activity.This is due to the conversion of oleuropein and other verbascosidecompounds to HT, which exhibits exceptional scavenging capacityand strong radical absorption. Results in Table 3 proved that theantioxidant activity of HT and the ethyl acetate extract were higherthan that of oleuropein (IC50 = 173 lg/mL).

HT occurring after this type of bioconversion can be regarded ashigh value thanks to its high antioxidant properties (Table 3). Itwas demonstrated in this study and many others that the antioxi-dant activity of HT was higher than that of oleuropein (Soni, Bur-dog, Christian, Bitler, & Crea, 2006). Fki et al. (2005) proved thatOMW might be a natural source of useful substances, particularlyHT, which have potent scavenging action on superoxide radicals.

4. Conclusion

The present study has revealed that A. niger, T. trogii and T. atro-viride grow on an economic cultivation medium (wheat bran assole carbon source), producing extracellular enzyme preparationshaving different level of esterase, b-glucosidase and laccase activi-ties. These enzyme preparations were tested for the hydrolysis of

1436 M. Hamza et al. / Food Chemistry 131 (2012) 1430–1436

OMW phenolic compounds. The conversion of hydrolysable phe-nols, such as oleuropein, ligstroside or verbascoside into monomer-ics, such as HT, was demonstrated in this study. The best resultswere reached using A. niger culture broth, which exhibits high b-glucosidase activity. The concentration of free HT was increasedfrom 0.05 to 1.1 g/L. A comparative study using A. niger-purifiedb-glucosidase and Viscozyme has shown the same effect on HT re-lease as the raw enzyme preparation of A. niger.

Hence, the bioconversion reaction proposed in this study,allowing the recovery of natural HT, offers an alternative to con-ventional extraction methods and microbial fermentation. Thismethod is environmentally friendly and also improves the extrac-tion yield and the quality of the extracts. The high efficiency of HTas a potent antioxidant was confirmed by the DPPH radical-scav-enging method, encouraging the commercialisation of HT as apowerful natural antioxidant.

Acknowledgements

Thanks for the financial support provided by ‘‘ Contrats Pro-grammes Ministère de l’Enseignement Supérieur et de la Recher-che Scientifique, Tunisie ’’. We thank Mr. Adel Gargoubi for histechnical assistance in HPLC analysis and Mr. Anis Tounsi from ISETZaghouan, Tunisia for his help with English.

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