Chiang Mai J. Sci. 2015; 42(1) 1

Chiang Mai J. Sci. 2015; 42(1) : 1-16http://epg.science.cmu.ac.th/ejournal/Contributed Paper

Phenolics Production from a Novel Substrate PalmOil Mill Effluent by Aspergillus niger IBS-103ZA:Evaluation of Fermentation Conditions andAntioxidant ActivityZulkarnain Mohamed Idris [a, b], Parveen Jamal*[a, c], Md Zahangir Alam [a],Abdul Haseeb Ansari [c] and Anumsima Ahmad Barkat [a][a] Bioenvironmental Engineering Research Centre (BERC), Department of Biotechnology Engineering,

Faculty of Engineering, International Islamic University Malaysia (IIUM), P.O. BOX 10,50728 Kuala Lumpur, Malaysia.

[b] School of Bioprocess Engineering, Kompleks Pusat Pengajian Jejawi 3, Universiti Malaysia Perlis,02600 Arau, Perlis, Malaysia.

[c] International Institute for Halal Research and Training (INHART), International Islamic University Malaysia,P.O. BOX 10, 50728 Kuala Lumpur, Malaysia.

*Author for correspondence; e-mail: jparveen@iium.edu.my, jparveen3@yahoo.com

Received: 28 July 2013Accepted: 7 January 2014

ABSTRACTThis paper introduces palm oil mill effluent (POME) as a novel substrate for

production of phenolics under liquid state fermentation by Aspergillus niger IBS-103ZAwith selected fermentation conditions. The statistical analysis showed a significantimprovement at optimum fermentation conditions of 5.39% (w/v) sucrose, 2.22% (w/v)MnSO4 and a temperature of 35°C. The antioxidant activity of the fermented extract(FE) of POME was evaluated through several in-vitro assays: inhibition of β-carotenebleaching, ferric reducing antioxidant power (FRAP), and inhibition of oxidativehemolysis in human erythrocytes. The results were compared with the syntheticcompound, butylated hydroxytoluene (BHT) and the unfermented POME extract (UFE).It was found that the FE has the highest antioxidant activity, based on FRAP assay(FRAP value, 1088.27±34.25 μmol FeSO4.7H2O/g dry extract) and inhibition of oxidativehemolysis (IC50, 0.097±0.020 mg/ml). In case of inhibition of β-carotene bleaching, theFE showed 79.10±5.55% inhibition at a concentration of 0.1 mg/ml.

Keywords: phenolics, palm oil mill effluent, fermentation, oxidative hemolysis,antioxidant activity

1. INTRODUCTIONThe global trend towards effective

utilization of agro-industrial wastes for theproduction of a number of metabolites of

commercial value has increased significantlyin recent years. In Malaysia, the by-productsof palm oil industry have attracted

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considerable interests for the bioconversionof these wastes into biosurfactant, fertilizer,fuel, water reclamation, citric acid production,etc. [1, 2, 3].The production of phenoliccompounds by utilizing POME can, thus, bea viable alternative for recovery of valuablesources of natural antioxidants as well as aprofitable means of waste management byrecycling to value-added by-products.

Phenolics compounds are plant-basedsecondary metabolites, diverse in structurewith a wide phylogenetic distribution.Phenolic compounds can be classified intovarious groups as a function of the numberof phenol-rings that they contain, based onthe structural elements which bind thesering to one another. These compounds,as one of the most widely and plentifullyavailable phytochemicals, play a key rolein the management of various diseasesassociated with oxidative stress [4, 5].Phenolic compounds are considered aseffective substances in explaining theirprotective effects against cancer andcardiovascular diseases. Their anti-agingbenefits (controlling inflammation, boostingthe immune system, and improving bloodcirculation) demonstrate a safe and naturalway to enhance longevity and alleviate signsof aging. Due to their ability to promotebenefits for human health, they are the objectsof great interest of pharmaceutical, cosmeticsand food industry.

Phenolic compounds are usuallyrecovered from natural sources throughsolid-liquid extraction using organic solventsin heat-reflux systems [6, 7]. Other techniquesto obtain these compounds such as the useof supercritical fluids, high pressuresprocesses, microwave-assisted and ultrasound-assisted extraction have also been proposedrecently [8]. Production of phenoliccompounds through fermentation hasgained interest as an alternative to the

conventional extraction and hydrolysismethods because it is able to providehigh quality and high activity extractswhile precluding any toxicity associatedwith the organic solvents. Response surfacemethodology (RSM) has been used as asuccessful statistical tool for optimizationof media compositions and processconditions in many fermentation areas ofbiotechnology with the purpose to highproduction yield with relatively less cost.The RSM is a collection of mathematicaland statistical techniques for designingexperiments, building models, evaluatingthe effects and determining the optimumconditions of factors of differentbiotechnological bioprocess [9]. However,no study has so far been carried out on theuse of RSM for optimization of the mediacomponents and process parameters forphenolics production from POME underliquid-state fermentation by Aspergillus niger.

It is for these reasons that the objectiveof this paper is to optimize the mediacomponents and process parameters(sucrose, MnSO4 and incubation temperature)for the phenolics production from POMEunder liquid-state fermentation by Aspergillusniger IBS-103ZA using central compositedesign (CCD) under response surfacemethodology (RSM). Sucrose, MnSO4 andincubation temperature are selected forfurther optimization study based onscreening results from Plackett-Burmananalysis and one-factor-at-a-time (OFAT)method [10]. The antioxidant activity of thefermentation extracts will also be evaluatedby several in-vitro antioxidant assays. Thus,the production of phenolic acids fromPOME by liquid-state fermentation usinglocal isolated strain would be a new suitableand sustainable technology of utilizingsuch organic residues and solving theenvironmental problems as well.

Chiang Mai J. Sci. 2015; 42(1) 3

2. MATERIALS AND METHODS2.1 Chemicals and Reagents

2,2’ -azobis - (2 -amidinopropane)dihydrochloride (AAPH), β-carotene,butylated hydroxytoluene (BHT), Folin-Ciocalteu reagent, phosphate buffer saline(PBS), 2, 4, 6-tripyridyl-S-triazine (TPTZ),and other solvents and reagent of analyticalgrade were purchased from Merck Chemicals(Darmstadt, Germany).

2.2 Preparation of Raw MaterialPOME was collected from East Oil Mill,

Sime Darby Plantation Sdn. Bhd, Cary Island,Selangor, Malaysia and was stored at 4.0 °Cin the laboratory cold room for further use.The POME sample having 4.0% (w/v) oftotal suspended solid (TSS) was preparedby removing or adding distilled water fromthe original sample on the basis of materialbalances [11].

2.3 Fungal Strain and Preparation ofInoculum

The culture of Aspergillus niger IBS-103ZA(IMI 385267) strain was obtained from thelab stock [11]. The culture was maintainedat 4.0°C on potato-dextrose agar (PDA),with regular subculturing at interval of30 days. Inoculum preparation (sporesuspension) was done according to thesuggested method [2]. The strain of theAspergillus niger was first cultured on fourPDA plates for 7 days in an incubator at32.0°C. About 100 ml of sterilized waterwas used for suspension inoculum. The sporesin the culture plates were harvested withsterilized water followed by filtration usingfilter paper to remove the mycelia from sporesuspension. The spore count of the suspensionwas determined by hemocytometer, whichshowed 1 × 106 spores/ml.

2.4 The Central Composite DesignThe central composite design (CCD)

under the RSM was employed in order toillustrate the nature of the response surfacein the experimental region and elucidate theoptimal conditions of the most significantindependent variables. Three variablesnamely sucrose concentration, MnSO4

concentration and incubation temperaturewere selected for RSM of CCD based onthe Plackett-Burman design and ‘one-factor-at-a-time’ (OFAT) analysis [10]. In this study,the independent variables were examined atthree different levels; low (-1), basal (0) andhigh (+1). According to the CCD (Table 1),the experimental design consisted 19 runs(5 runs at the center point) were performedand their observations were fitted to thefollowing second order polynomial ordermodel:

Y = β0 + β1A + β2B + β3C + β11A +β22B + β33C + β12AB + β13AC + β23BC (1)

where, Y is the dependent variable (totalphenolic content); A, B and C are independentvariable (sucrose concentration, MnSO4

concentration and incubation temperature);βo is the regression coefficient at centerpoint; β1, β2 and β3 are linear coefficients;β11, β22 and β33 are the quadratic coefficients;and β12, β13 and β23 are the second orderinteraction coefficients. The developedregression model was evaluated byanalyzing the values of regression coefficients,analysis of variance (ANOVA), p- and F-values.The quality of fit of the polynomial modelequation was expressed by the coefficientof determination, R2. The statisticalsoftware package Design-Expert® 6.0.8(Stat Ease Inc., Minneapolis, USA) was usedto generate a regression model to predictthe optimum combinations considering

4 Chiang Mai J. Sci. 2015; 42(1)

the effects of linear, quadratic andinteraction on the total phenolic content.

A final experiment was conducted tovalidate the CCD model developed.

2.5 Extraction of Phenolic CompoundsThe extraction was carried out according

to the suggested method [12] with a slightmodification. The samples were acidified topH 2 with 6 N of HCl and washed threetimes with hexane to remove the lipidfraction at a hexane to water phase ratio of2:3. The mixture was vigorously shakenand centrifuged for 5 min at 3000 rpm.The phenolic compounds were extractedfour times with ethyl-acetate at a solvent towater phase ratio of 1:1, and the mixture wasvigorously shaken and centrifuged for 5 minat 3000 rpm. The phases were separatedand the ethyl-acetate was evaporated to dry

mass under vacuum at 40°C. The dry residuewas dissolved in methanol and kept at -80.0°Cuntil further analysis.

2.6 Determination of Total PhenolicContent

The total phenolic content (TPC) wasdetermined based on the suggested method[13, 14] with modification using the Folin-Ciocalteu reagent with gallic acid as astandard. In 15 ml test tube, 2.37 ml ofdistilled water, 0.03 ml of sample extract orblank and 0.15 ml of Folin-Ciocalteu reagentwere added and vortexed. After 1 min,0.45 ml of 20% saturated sodium carbonate

Table 1. Experimental design using CCD showing coded and actual values along with theexperimental and predicted values of phenolics production for media components and processparameters optimization.

Run

12345678910111213141516171819

SucroseA % (w/v)

+1(8) -1(4) -1(4) 0(6) -1(4) -1(4) 0(6)+1(8) -1(4) 0(6) 0(6) 0(6) 0(6) 0(6)+1(8)+1(8)+1(8) 0(6) 0(6)

MnSO4

B % (w/v)

-1(1.5) -1(1.5)+1(3.5) 0(2.5)+1(3.5) -1(1.5) 0(2.5)+1(3.5) 0(2.5) 0(2.5) 0(2.5) 0(2.5) 0(2.5) -1(1.5) 0(2.5) -1(1.5)+1(3.5)+1(3.5) 0(2.5)

Incubation temperatureC (°C)

-1(30) -1(30)+1(40) 0(35) -1(30)+1(40) 0(35) -1(30) 0(35) 0(35) -1(30) 0(35) 0(35) 0(35) 0(35)+1(40)+1(40) 0(35)+1(40)

Total phenolic content(GAE mg/l)

Experimental819.74848.91772.86942.12831.20892.05943.96809.74912.47943.60909.34942.68940.35924.97870.79765.56771.69825.99898.92

Predicted808.63866.62781.10937.96806.75883.65937.96815.28919.36937.96921.65937.96937.96905.19875.37787.14751.11857.24898.08

Chiang Mai J. Sci. 2015; 42(1) 5

(Na2CO3) was added, and then the mixturewas vortexed and allowed to stand at40.0 °C for 30 min. The absorbance wastaken at 750 nm. The total phenolic contentwas expressed as mg of gallic acid equivalentper liter (GAE mg/l). All measurementswere measured in triplicate.

2.7 Ferric Reducing Antioxidant Power(FRAP) Assay

FRAP assay was based on the reductionof Fe3+-TPTZ to a blue complex, Fe2+-TPTZ(Benzie & Strain, 1996). The assay was adaptedfrom the suggested methods [15, 16] withslight modification. From original stock andsample concentrations, a 1:10 dilution wasmade with ethanol followed by two foldserial dilutions. The FRAP reagent wasfreshly prepared by mixing acetate buffer(300 mM, pH 3.6), a solution of 10 mMTPTZ in 40 mM HCl, and 20 mM FeCl3 at10:1:1 (v/v/v). The reagent (300 μl) and 10 μlof extracts or BHT or standards solutionswere added to each well and mixedthoroughly. The absorbance was taken at593 nm after 10 min. The antioxidantpotential of the extracts and the BHT weredetermined against a standard curve ofFeSO4.7H2O (concentrations ranging from0.5 to 3.0 mM) and expressed as mMFeSO4.7H2O per litre of sample. All analyseswere run in triplicate.

2.8 Inhibition of βββββ-Carotene BleachingThe antioxidant activity of the extracts

was evaluated by using the β-carotene-linoleatemodel system [17] with a slight modification[18, 19] for the 96-well microplate reader.From original stock and sample (or standard)concentrations, a 1:10 dilution was madewith ethanol followed by two fold serialdilution. Briefly, β-carotene (0.5 mg) wasdissolved in 2 ml of chloroform. After that,25 μl of linoleic acid and 200 mg of Tween

40 were added and the mixture was leftstanding at room temperature for 15 min.Chloroform was removed using a rotaryevaporator. Oxygenated distilled water(100 ml) was added and then the mixturewas shaken to form an emulsion. Theoxygenated distilled water was obtained bybubbling water with compressed oxygen gasfor at least 2 hours at room temperature.The test mixture was prepared fresh andused immediately. Aliquots of 250 μl of theemulsion were transferred into wells and35 μl of different sample concentrations ofextracts were added. The plate was incubatedat 45 °C. As soon as the emulsion wasadded to each well, the zero time absorbance(A0) was measured at 470 nm. A secondabsorbance (A1) was measured after 120 min.A blank, without β-carotene was preparedfor background subtraction. All analyseswere run in triplicates. BHT was used as astandard. Lipid peroxidation (LPO) inhibitionwas calculated using the following equation:

LPO inhibition (%) = (∆A1/∆A0) × 100 (2)

2.9 Inhibition of Erythrocyte HemolysisMediated by Peroxyl Free Radicals

Erythrocyte resistance to oxidative stress(hemolysis) was carried out according to thesuggested method [20, 18] with slightmodification. From original stock andsample (or standard) concentrations, a 1:10dilution was made with ethanol followed bytwo fold serial dilution. Briefly, blood wasobtained from a healthy adult male of75 kg and was centrifuged at 3000g and 4 °Cfor 5 min to separate the erythrocytes fromthe plasma. The erythrocytes were washedtwice with 10 mM phosphate buffer saline(PBS) at pH 7.4 and centrifuged at 3000gfor 2 min. Five percent (5%) of the suspensionerythrocytes in PBS were used for the test.In each well of 96-well plate, 100 μl of

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erythrocyte suspension, 160 μl of 200 mM2 , 2 ′ - a z o b i s ( 2 - a m i d i n o p r o p a n e )dihydrochloride (AAPH), and 30 μl ofdifferent sample concentrations of extractswere incubated at 37°C. The samplemixture was shaken gently while incubating.After 3 h of incubation, the sample mixturewas centrifuged at 3000g for 10 min andthe supernatant was taken to read theabsorbance at 540 nm. A sample mixturewithout sample extract was used as control.All analyses were run in triplicates. The BHTwas used as a standard. The percentage ofhemolysis inhibition was calculated by usingthe following formula:

Hemolysis inhibition (%) = [(∆AC - ∆AS)/∆AS)] × 100 (3)

where, AC- absorbance of the control; AS-absorbance of the sample or standard.

2.10 Statistical AnalysisAll measurements were carried out in

triplicate. Statistical analysis was performedusing one-way analysis of the variance(ANOVA), and the significance of thedifference between means was determinedby Duncan’s multiple range tests. Differencesat p<0.05 were considered statisticallysignificant. The results were presented asmean values ± SD (standard deviations).The analysis of tendency for IC50 value wasperformed using logarithmic function withMicrosoft Excel using data dose-responsecurve.

3. RESULTS AND DISCUSSION3.1 Optimization of Media Componentsand Process Conditions by CCD UnderRSM

The RSM is a very useful tool todetermine the optimal level of parametersand their interaction. Three independent

variables; Sucrose, MnSO4 and incubationtemperature were optimized for themaximum production of phenolics fromPOME. Experiments were carried out asper the design matrix of the CCD (Table 1),and the mean total phenolic contentobtained from the fermentation culture wasused as response. For predicting the optimalvalues of phenolics produced within theexperimental constraints, a second orderpolynomial model was fitted to theexperimental results by the Design Expertsoftware. The model developed is as follows:

Y = -1214.11 + 123.69A + 284.52B +87.40C - 10.15A2 - 56.74B2 - 1.12C2 + 8.31AB-0.96AC - 2.13BC (4)

where, Y is the total phenolic content asyield, which is a function of sucroseconcentration (A), MnSO4 concentration(B), and incubation temperature (C).

The adequacy of the model was checkedusing ANOVA, which was tested usingFisher’s statistical and the results are shownin Table 2. The regression equation obtainedafter ANOVA showed a determinationcoefficient of 0.9487 for phenolicsproduction (a value of R2> 0.75 indicatedthe aptness of the model), which indicatesthat the statistical model can explain 94.87%of variability in the response. The value ofthe adjusted determination of coefficientwas very high (89.74%), which indicates ahigh significance of the model [2, 21].The goodness of a model (statisticallysignificant model) can be checked by thedetermination of coefficient (R2) andcorrelation coefficient (R). The R2 value isalways between 0 and 1. The closer the R2 isto 1, the stronger is the model and it predictsthe better response [22].The value of R2 isalso a measure of fit of the model and itcan be mentioned that only about 5% of the

Chiang Mai J. Sci. 2015; 42(1) 7

total variations were not explained by thephenolics yield. In this experiment, the valueof R was 0.9740 which indicated a closeagreement between the experimentalresults and the theoretical values predictedby the model equation. The significantmodel fit shows that the predicted responsesurface fits the model points well.Moreover, the observed lack of fit (F-value= 372.55) predicts that the five centerpoints in this design are so close that theyoverlap and some are hidden below theresponse plane. The high lack of fitvalue shows that the differences betweenthe actual data points and the response

plane are greater than the differencesbetween the center points and the centerpoints are fitting better than the modelpoints. Usually, this happens when therepeating data are very similar to each other.The response measurements from thereplicates show that this amount of variationwas expected from the process. However,the decisions about using the model willhave to be made based on whether themodel works for the use to which theresearchers intend toput it. This is morea measure of the R2 (predicted) and thestandard deviation of the model thananything else.

Table 2. Statistical analysis (ANOVA) for evaluating the significant of the variables for responsesurface quadratic model on phenolics production.

*p < 0.05 indicate the model terms are significant and **p < 0.01 indicate the model terms are highly significant. R2=0.9487, Adjusted R2= 0.8974, Adequate Precision = 12.543, Coefficient of variation (CV) = 2.35.

SourceModel

ABCA2

B2

C2

ABACBC

Sum of squares70202.704838.285748.291389.074501.988796.612155.842212.02741.57910.68

df9111111111

Mean square7800.304838.285748.291389.074501.988796.612155.842212.02741.57910.68

F-value18.5011.4813.633.2910.6820.865.115.251.762.16

p-value (Prob > F)< 0.0001**0.0080**0.0050**0.1029

0.0097**0.0014**0.05010.0477*0.21740.1758

The purpose of statistical analysis is todetermine the experimental factors, whichgenerate signals that are large in comparisonto the noise. Adequate precision measuresthe signal to noise ratio. A ratio greater than4 is desirable. The value of adequate precisionof 12.543 is very high which indicates thatthis model can be used to navigate thedesign space. The coefficient of variation(CV) indicates the degree of precision withwhich the experiments are compared.The lower reliability of the experiment isusually indicated by high value of CV. In the

present study, lower value of CV (2.35)indicated a greater reliability of theexperiments performed. The Fisher varianceratio, the F-value (=Sr

2/Se2), is a statistically

valid measure of how well the factorsdescribe the variation in the mean of data.The greater the F-value indicates that thefactors explain adequately the variation inthe data about its mean, and the estimatedfactor effects are real. The quadratic regressionmodel was highly significant, as an evidentfrom the Fisher’s F-test with a very lowprobability value (Pmodel > F = 0.0001).

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The P-value denotes the significance of thecoefficients and is also important inunderstanding the pattern of the mutualinteractions between the variables. Value ofP-value > F less than 0.05 indicate that themodel terms are significant.

It was observed that the highly significant(P < 0.01) variables were the linear terms ofsucrose (A) and MnSO4 (B), and the squareterms of sucrose (A2) and MnSO4 (B2)respectively. The interactive term betweensucrose and MnSO4 (AB) was significant atthe level of P < 0.05. The linear and squareterms of incubation temperature, C and C2,and the interactive terms between sucroseand incubation temperature (AC) and MnSO4

and incubation temperature (BC) shown inthe ANOVA analysis were not significant(P > 0.05). Linear and quadratic effects ofsucrose and MnSO4 concentrations weresignificant, meaning that they can act aslimiting nutrient and little variation in theirconcentration would alter either the growthrate or the product formation rate or both toa considerable extent [23].

The response surface and the contourplots are the graphical representations of theregression equation. The three dimensionalresponse surface and their correspondingcontour plots for the phenolics productionagainst any two independent variables atzero levels are presented in Figure 1, Figure 2and Figure 3. Therefore, three responsesurfaces were obtained by consideringall possible combinations. The graphicalrepresentation provides a method tovisualize the relation between the responseand the experimental levels of each variable,and the type of interactions between testvariables in order to figure out optimumconditions. The optimum value of eachvariable was identified based on the humpin three-dimensional (3D) plot, or from thecentral point of the corresponding contour

plot. Each contour curve represents aninfinitive number of combinations of twotest variables with the other one maintainedat their respective zero level. The maximumpredicted value is indicated by the surfaceconfined in the smallest ellipse in thecontour diagram. Elliptical contours areobtained when there is a perfectinteraction between the independent variables[24].

Figure 1A shows the three dimensionalplot and its respective contour plot illustratingthe response surface from the interactionbetween sucrose concentration (A) andMnSO4 concentration (B). The resultsshowed the phenolics production, whichwas considerably affected by varying theconcentration of sucrose and MnSO4.It could be observed that the total phenoliccontent increased gradually with theincreasing sucrose concentration and MnSO4

concentration until the maximum phenolicsproduction of 944.57 GAE mg/lwas obtained at 5.34% (w/v) sucrose and2.21% (w/v) MnSO4, while keeping theincubation temperature at 35°C. Furtherincrease in the levels of both parametersresulted in a gradual decrease in yield oftotal phenolic content. Figure 2B is theresponse surface plot for variation in thephenolics production, as a function ofsucrose concentration and incubationtemperature while MnSO4 concentrationwas fixed at its zero level. The predictedtotal phenolic content decreased at higherand lower levels for both sucroseconcentration and incubation temperature.Maximum production was obtained nearthe center points of the response surface.The maximum phenolics production ofabout 941.70 GAE mg/l was predictedat 5.5% (w/v) sucrose and 34.17°C, whileMnSO4 concentration was fixed as 2.5%(w/v). Figure 3B depicts the effect of MnSO4

Chiang Mai J. Sci. 2015; 42(1) 9

concentration and incubation temperatureon total phenolic content while the sucroseconcentration was fixed at its zero level.The results showed that the total phenoliccontent decreased when the MnSO4

concentration in the media was at higherlevel. It was noticed that, the phenolicsproduction tended to increase as theincubation temperature increased. However,when the incubation temperature wasincreased above 35°C, the total phenoliccontent decreased gradually. The maximumtotal phenolics of 941.31 GAE mg/lwas predicted at 2.3% (w/v) MnSO4 and34.13°C, while sucrose concentration waskept at 6% (w/v). From examination of thecontour plots in Figure 1A, Figure 2A andFigure 3A, it was noticed that the phenolicsproduction was slightly more sensitive tochanges in the sucrose concentration andMnSO4 concentration than to change in theincubation temperature. The effect of theincubation temperature on the responsewas also insignificant (p > 0.05).

In order to verify the optimization resultsand to validate the statistical model and theregression equation, an experiment wasperformed in triplicate according to theoptimum values (5.39% (w/v) sucrose, 2.22%(w/v) MnSO4 at 35 °C) suggested by themodel for maximum total phenolic content.The predicted response for phenolicsproduction was 944.69 GAE mg/l, while theactual (experimental) response was 949.56GAE mg/l, which is slightly higher than thepredicted value thus proving the validity.From the optimization study, it is clearlyobserved that the production of phenolicsof 949.56 GAE mg/l with optimumcondition obtained by the CCD was slightlyincreased as compared to the production ofphenolics obtained by the Plackett-Burmandesign (914.69 GAE mg/l) and OFATmethod (940.80 GAE mg/l), respectively.

3.2 Ferric Reducing ActivityFerric reducing antioxidant power

(FRAP) assay measures the reductionpotential of an antioxidant reacting witha ferric tripyridyltriazine (Fe3+-TPTZ)complex and produce a colored ferroustripyridyltriazine (Fe2+-TPTZ). The reductionproperties are associated with the presenceof compounds which exert their action bybreaking the free radical chain by donatinga hydrogen atom or an electron [25, 26].Any electron-donating substances with a

Figure 1. (A) 2D contour plots and (B) 3Dresponse surface show the effect of sucroseconcentration (%, w/v) and MnSO4

concentration (%, w/v) on the phenolicsproduction (GAE mg/l) where theincubation temperature was at 35 °C.

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Figure 2. (A) 2D contour plots and (B) 3Dresponse surface show the effect of sucroseconcentration (%, w/v) and incubationtemperature (°C) on the phenolicsproduction (GAE mg/l) where the MnSO4

concentration was at 2.5% (w/v).

Figure 3. (A) 2D contour plots and (B) 3Dresponse surface show the effect of MnSO4

concentration (%, w/v) and incubationtemperature (°C) on the phenolicsproduction (GAE mg/l) where the sucroseconcentration was at 6.0% (w/v).

half reaction lower redox potential thenFe3+/Fe2+-TPTZ will drive the reaction andthe formation of the blue complex forward[27]. According to Benzie and Strain [25],the reduction of yellow Fe3+-TPTZ complexto blue-colored Fe2+-TPTZ occurs at lowpH.

FRAP values of the POME extractsand the standard BHT expressed in μmolFeSO4.7H2O/g dry extract are shown inFigure 4A. The highest FRAP value wasfound in fermented extract (FE) with1088.27±34.25 μmol FeSO4.7H2O/g dryextract. The lowest ferric reducing abilitywas observed to be in the unfermentedextract (UFE) with 263.88±14.57 μmolFeSO4.7H2O/g dry extract. The standardBHT had the FRAP value of 823.33±48.00μmol FeSO4.7H2O/g BHT. In this study,fermentation using Aspergillus niger was

proven to have significantly effect (p <0.05) on ferric reducing ability of thePOME extract. The FRAP value of thefermented extract (FE) was 4-fold higherthan the unfermented extract (UFE) andabout 1.3-fold higher than the standardBHT respectively. Biochemical changesthat occur during fermentation mightcontribute to the increase in antioxidantactivity of the fermented extract (FE) inPOME as reported by researchers [27, 28,and 29].

3.3 Inhibition of βββββ-Carotene BleachingIn the β-carotene bleaching assay,

linoleic acid produces hydroperoxides asfree radicals during incubation at 45°Cwhich attacks the highly unsaturatedβ-carotene. The oxidation of linoleic acidgenerates peroxyl hydroperoxides due to

Chiang Mai J. Sci. 2015; 42(1) 11

Figure 4. (A) Ferric reducing activity based on FRAP assay of unfermented extract(UFE), fermented extract (FE) and BHT. (B) Inhibition of β-carotene bleaching and (C)hemolysis inhibition as a function of unfermented extract (UFE), fermented extracts(FE) and BHT concentrations. BHT was used as positive control. Data are expressed asmeans ± SD (n = 3).

the abstracting of hydrogen atom fromdiallylic methylene group of linoleic acid[30]. Linoleic acid hydroperoxides attackthe β-carotene molecule and, as a result,it undergoes rapid decolorization. Thecorresponding decrease in absorbance canbe observed spectrophotometrically.

In this study, the antioxidant activitiesof the extracts against hydroperoxidesradicals (measured by the peroxidation ofβ-carotene) were increased with furtherincrease in theirs concentrations. At theirlowest concentrations studied, the extractsexhibited more than 50% of inhibitionagainst hydroperoxides radicals (UFE,61.79±2.43% at 0.00652 mg/ml; FE,61.06±1.88% at 0.00292 mg/ml). In case ofBHT, there were no significant differences(p > 0.05) on the percentages of inhibitionat lowest level; 0.0025 mg/ml (79.91±2.64%) and 0.025 mg/ml (82.25±2.32%).

At their highest concentration levels,the percentages of lipid peroxidation(LPO) inhibition were 88.69±1.70%(at 13.04 mg/ml) in the unfermented extract(UFE) while 90.48±1.45% (at 5.84 mg/ml)for the fermented extract with Aspergillus niger(FE). BHT showed about 97.48±1.31%inhibition against hydroperoxides radicalsat 5.0 mg/ml. In this study, it was found thatall extracts showed very high percentages ofinhibition at extremely low concentrations.To verify the observations, the percentagesobtained at 0.1 mg/ml were noted.The percentage was calculated based ondose-response curve of logarithmicfunction (BHT, y = 2.483 ln(x) + 93.79,R2= 0.952; UFE, y = 3.555 ln(x) + 83.78,R2= 0.845; FE, y = 3.702 ln(x) + 87.63,R2= 0.849) as shown in Figure 4B. Thefermented extract (FE) with 79.10±5.55%has higher antioxidant activity compared

12 Chiang Mai J. Sci. 2015; 42(1)

to unfermented extract (UFE) (75.59±2.22%). BHT has the highest antioxidantactivity with 88.07±3.08%. No significantdifference (p > 0.05) was observed onpercentage of inhibition between UFE andFE at 0.1 mg/ml. Higher inhibition againstlipid peroxidation by POME extracts canpossibly be explained by the presence ofpolar antioxidants, where the hydrophobicitycharacteristic of these antioxidants are able toexhibit greater inhibition activity shieldingthe emulsion by concentrating at the lipid:air surface [31].

3.4 Inhibition of Erythrocyte HemolysisAAPH is a peroxyl radical initiator that

generates free radicals by its thermaldecomposition and will attack theerythrocytes to induce the chain oxidationof lipid and protein that will disturb themembrane organization and eventuallyleading to hemolysis [32]. Erythrocytes areextremely subject to oxidative damage as aresult of high polyunsaturated fatty-acidcontent of their membranes and high cellularoxygen and hemoglobin (Hb) concentrations[33]. In vitro oxidative hemolysis of humanerythrocytes is used as a model to studyfree radical-induced damage of biologicalmembranes and the protective effect ofphenolic compounds and known antioxidants[18, 34]. Erythrocytes are excellent modelfor the study of biomembrane toxicityin vitro due to their ready accessibility, easepreparation, abundance of polyunsaturatedfatty acids and membrane proteins, andwealth of available information [35].

In this study, the protective effect ofPOME extracts and BHT at variousconcentrations on hemolysis by peroxylradical scavenging activity was investigated.It was found that the POME extracts,both the unfermented (UFE) and fermentedwith Aspergillus niger IBS-103ZA (FE)

displayed good inhibitory effects againsterythrocytes hemolysis as compared to thestandard BHT. At their highest concentrations,the inhibitions of erythrocytes oxidationfor the unfermented extract (UFE) andfermented extract (FE) were 82.75±2.49%(13.04 mg/ml) and 84.88±3.29% (5.84 mg/ml) respectively. For BHT, only 51.52±3.19%inhibition against erythrocytes hemolysiswas noticed at the concentration of5.0 mg/ml.

Figure 4C shows the dose-dependentinhibition of hemolysis, as a result ofprotection against the oxidative damage ofcell membranes of erythrocytes from human,induced by AAPH. Antioxidant activitywas also expressed in terms of IC50, theconcentration of extract needed to achieve50% inhibition against oxidative hemolysis oferythrocytes under experimental conditions.The IC50 value was calculated usingdose-response curve of logarithmic function(BHT, y = 6.51 ln(x) + 38.44, R2= 0.924;UFE, y = 10.86 ln(x) + 65.91, R2= 0.871;FE, y = 10.76 ln(x) + 75.13, R2= 0.906).In this study (Figure 4C), it was found thatthe fermented extract (FE) showed thelowest IC50 value (0.097±0.020 mg/ml)which has the strongest antioxidantactivity against oxidative hemolysis oferythrocytes compared to unfermentedextract (UFE) (0.23±0.035 mg/ml) andBHT (5.90±2.11 mg/ml). In this study,the IC50 values between FE and UFEwere found to be not significantly different(p > 0.05).

The erythrocytes morphology, beforeand after exposure to AAPH in the absenceand presence of POME extracts and BHTis shown in Figure 5. It was observed thatthe presence of AAPH greatly inducedhemolysis of erythrocytes as shown inFigure 5B. The number of echinocytes(cells lacking definite membrane spikes

Chiang Mai J. Sci. 2015; 42(1) 13

with irregular surface) formed increased ascompared due to normal hemolysis asshown in Figure 5A. In the presence ofextracts (at 5.84 mg/ml of FE and13.84 mg/ml of UFE respectively) asshown in Figure 5D and Figure 5E thenormal discoid shapes of most of theerythrocytes was maintained and lessechinocytes was produced. The presence ofBHT (at 5.0 mg/ml) as shown in Figure 5Cwas likely to induce more damage to thecell membrane with the increase in thenumber of echinocytes formed. From thisresult, it can be concluded that BHToffered less protection against hemolysisof the erythrocytes as compared toPOME extracts. In another word, BHTacts as pro-oxidant instead of antioxidant.

The protection capacity of POMEextracts and BHT against erythrocytesoxidation might be due to differentmechanisms. The ability to protect theerythrocytes from hemolysis is related notonly to a radical scavenging activity butalso to its ability to interact directly with cellmembranes (in the outer or innermembrane surface), including themodification of the protein profile [36].The lipid-soluble antioxidants can protectthe hydrophobic part of the cell membranewhile for the water-soluble or morehydrophilic antioxidants can quench theperoxyl radicals in the aqueous phasebefore the radicals attack the cell to causeperoxidation and hemolysis [37]. Phenolicshave been found to protect erythrocytesfrom oxidative stress or increase theirresistance to damage caused by the oxidants[38]. As shown in Figure 5D and Figure5E both POME extracts were veryefficient in protecting the erythrocytesfrom hemolysis induced by peroxylsradicals and this may be explained by thesynergistic antioxidant effect of phenolic

compounds that present in the POMEextracts. According to Cirico and Omaye[9], the antioxidant effect of phenolics isconcentration-dependent. Phenolics couldpresent good antioxidant effects among arange of concentration, but it is possiblethat pro-oxidant effects could appear atcertain concentrations.

In conclusion, the effect of selectedfermentation conditions on a novelsubstrate (POME) was evaluated for theproduction of phenolics. Optimization byCCD under RSM improved the contentfrom 639.90 ±4.19 to 944.69 GAE mg/l.

Figure 5. Evaluation of human erythrocytesmorphological changes after three hours ofincubation viewed under inverted lightmicroscope at magnification of 400x. (A)Erythrocytes without AAPH and extract.(B) Erythrocytes plus 200 mM AAPH. (C)Erythrocytes plus 5.0 mg/ml of BHT and200 mM AAPH. (D) Erythrocytes plus13.04 mg/ml of UFE and 200 mM ofAAPH. (E) Erythrocytes plus 5.84 mg/mlof FE and 200 mM of AAPH.

14 Chiang Mai J. Sci. 2015; 42(1)

The validation experiment improved itfurther to 949.56±3.82 GAE mg/l atoptimum fermentation conditions of5.39% (w/v) sucrose, 2.22% (w/v) MnSO4

and 35 °C with other fixed parameters. Itwas also found that the FE has the highestantioxidant activity, based on FRAP assay(FRAP value, 1088.27±34.25 μmolFeSO4.7H2O/g dry extract) and inhibitionof oxidative hemolysis (IC50, 0.097±0.020mg/ml), as compared to UF and BHT.

ACKNOWLEDGEMENTThe authors are grateful to the

Ministry of Higher Education (MOHE),Malaysia for financing this research undera research grant (IFRG 0701-14), ResearchManagement Center (RMC) and Departmentof Biotechnology Engineering, IIUM forsupporting and providing the necessaryfacilities.

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