Laboratoire de Valorisation de la Biomasse et Production des Protéines chez les Eucaryotes, Centre de Biotechnologie de Sfax, Route Sidi Mansour,P «1177» 3018 Sfax, Université de Sfax, TunisiaUnité de technologie et qualité, Institut de l’olivier de Sfax, Route de l’aéroport km 2, B.P. 1087 3018 Sfax, Université de Sfax, Tunisia
r t i c l e i n f o
rticle history:eceived 8 October 2010eceived in revised form 1 March 2011ccepted 2 April 2011vailable online 20 April 2011
eywords:live oil
a b s t r a c t
The aim of this work was to investigate and evaluate culture filtrates of different enzymatic formula-tions in terms of their individual and synergistic effects with regard to the quality and extraction yieldsof Tunisian olive oil from the Chemlali Sfax variety. The formulations, which contained a number ofhydrolytic enzymes, particularly pectinases, xylanases and cellulases were tested both separately and incombination. The results demonstrated that when compared to those of the control, the oil extractionyields of both types of olives (green and black) treated by the enzymatic formulations were enhanced by1.5%. Interestingly, the synergistic effect of different activities (pectinase, cellulase and xylanase) was able
ectinaseellulase and xylanaseolyphenolsigments
to improve the extraction of polyphenols to 10% and of pigment compounds, namely chlorophylls andcarotenoids, by 25 and 30%, respectively. The olive oil stability, particularly the one from green olives,which was treated by the enzymatic mixture, was observed to increase up to 3.5 h. Overall, the find-ings presented in the current study demonstrate that the enzymatic formulations under investigationexhibited promising properties and attributes which make them potential strong candidates for futureapplication in the oil industry.
. Introduction
Olive oil production is an important agricultural activity in manyarts of the world. It is one of the primary driving engines of theconomy of a wide range of Mediterranean countries, prominentmong which is Tunisia. Tunisian production of olive oil has reachedn average of 150,000 tonnes per year, with the dominance of thehemlali variety that has a very harmonious sensorial quality and
high economically importance (about 60% of the total produc-ion). Moreover, olive oil exports constitute about 40% of Tunisia’sotal agricultural exports. However, and due to the high costs asso-iated with its production, processing and extraction processes,live oil is considered relatively expensive when compared to mostther vegetable oils. The cost is higher for the following operationnits: harvesting and processing (about 60euros/ton of olive forach operation).
Nevertheless, virgin olive oil with excellent nutritional, sen-orial and functional qualities is regarded by consumers as moreesirable.
Several factors are known to affect the composition of olivefruits, among them, the ripening cycle of the fruit and the natureof the cultivar [1]. The effects of agronomic and climatic conditionshave also been demonstrated [2]. The average composition of anolive fruit is 50% water, 20% oil, 20% carbohydrates (pectic, cellu-losic and hemicellulosic substances), with the rest being organicacids, pigments phenolic compounds and minerals [3].
In the oil-bearing cells, the major part of the oil is located inthe vacuoles, where it is free, and the remaining part is bound ordispersed in the cytoplasm and is, therefore, not directly accessi-ble in the extraction process and lost in the waste. Several studieshave been conducted to find effective methods for the recovery ofthe oil enclosed in the cell and a the need to destroy the cell wallsthrough the use of specific enzyme to the breakdown of the indi-vidual types of polysaccharides in the cell wall structure has oftenbeen emphasized as a workable solution. In this respect, Vierhuiset al. [4] indicated that the major polysaccharides present in thecell wall of olive fruit are pectic and hemicellulosic ones.
Olive oil has been used since ancient times particularly for
culinary and therapeutic purposes and has often been consideredas one of the healthier oils for consumption. It is considered tohave a high nutritional value and beneficial potential against awide array of diseases such as atherosclerosis, coronary heart and
ertain cancers [5]. It contains a wide range of bioactive compoundsuch as phenolics, namely hydroxytyrosol and oleuropein, and largemounts of monounsaturated to polyunsaturated fatty acids. Dueo its promising properties and attributes, olive oil has attracted aot of attention in modern academic and scholarly research. Of spe-ial interest to the aims of the present study, several approachessing enzymatic pre-treatment have been proposed to improveil extraction in terms of both the quantity and quality of prod-ct [3,6,7]. We shall insist on the fact that the use of enzymes isurrently forbidden in the processing of olive oil according to reg-lations of IOC and EC. Most of the currently available commercialnzymatic formulations used in such pre-treatment processes areich in pectinases, cellulases and xylanases and are mainly pro-uced by Gist Brocades (Cytolase 0, Rapidase adex D, Bioliva) andovozymes (Olivex, Glucanex, Novoferm) [8]. It is worth noting in
his context that several factors have significant influences on thefficiency of extraction. These factors include malaxation tempera-ure and time of olive paste exposure to air contact [9], the ripenessf the fruit, the climatic conditions, and the cultivar [10] as well ashe enzymes involved in the process [3,7].
Furthermore, recent research has also addressed a number ofenetic and biochemical factors which have often been describedo provide valuable information to breeders and agronomists aboutritical issues such as species and their biochemical properties asell as oil performances. The authors of the present study have, for
nstance, previously reported on the use of the Amplified Fragmentength Polymorphism (AFLP) technique as a linkage tool for thetudy of variation and diversity in olive tree species at Tunisia [11].
The present study is a contribution to the ongoing researchn the improvement of olive oil production and extraction yieldsn terms of both quantity and quality. It was undertaken with
particular focus on the technological aspects involved in oliveil extraction processes. It aimed to investigate and evaluate theffects of using a different of fungal enzymatic preparations on theuantity and quality of the oil extraction yield of the Chemlali-fax olive oil. Two different pectinase formulations (E1, E2) fromur CT1 mutant of Penicillium occitanis, a combination of cellu-ase and xylanase (E3) from Trichoderma sp., and the binary andernary mixture combinations E4 and E5 from the previous formu-ations were applied at constant proportions. The effects associated
ith the type of enzymatic formulation on olive oil quality andield were determined and evaluated using well-established crite-ia relating to acidity, extinction index at 232 and 270 nm, refractionndex, oxidative stability, acid composition and the polyphenols,arotenoids, chlorophylls, and unsaponifiable matter contents.
. Materials and methods
.1. Olive variety and experimental process
The experimental assays conducted in the present study wereerformed on Tunisian olive tree fruits from the Chemleli-Sfax vari-ty during the 2006/2007 season the ripening degree of which wasstimated by color (green and black olives). The following stagesere carried out to process the olive fruit samples: (1) cleaning
nd leaf removal; (2) milling by a hammer mill at 3000 rpm tobtain fine paste; (3) paste malaxation at 20 rpm for 30 min afterhich, and in the beginning of the kneading step, the corresponding
nzymes (200 ml/ton olives) were added; (4) paste centrifugationt 3500 tr/min for 2 min; (5) separation by natural decanting in twohases, oil and waste water.
.2. Enzyme preparation
Three enzymatic formulations (E1: pectinase 1, E2: pectinase, E3: xylanase and cellulase) and two binary (E1 + E3 = E4) andernary (E1 + E2 + E3 = E5) combinations were added at constant
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proportions to paste samples at the beginning of the malaxationstep. All these enzymes were water soluble and obtained frommicroorganisms that were not pathogenic. Hydrolytic activitieswere produced in the liquid medium through using either citruspectin or gruel (by-product from wheat manufactory) as carbonsources for the production of pectinase enzymes, called E1 and E2,respectively, by the CT1 mutant of P. occitanis [12] and throughusing wheat bran to produce cellulase and xylanase enzymes simul-taneously, called E3, by RutC30 mutant of Trichoderma reesei [13].
2.3. Micro organisms
The CT1 mutant was selected from a CL100 wild type strain aftera single round of mutagenesis by Nitrous acid HNO2 [12] of theCL100 strain of P. occitanis, which was certified as safe by G. Thirabyfrom Toulouse-France. The RutC30 mutant was isolated in RutgersUniversity [13] from T. reesei, which is also a GRAS strain [14], isobtained from international collection. Both strains were propa-gated on potato dextrose agar (PDA) and spores were maintainedin 20% glycerol at −80 ◦C.
2.4. Fermentation conditions
The basal medium used was a slightly modified Mandelsmedium [in 12] buffered at pH 5.5. It was composed of (g l−1):(NH4)2SO4, 1.4; MgSO4, 0.3; KH2PO4, 2; CaCl2, 0.3; NaNO3, 5; andTween 80, 1 ml; and 1 ml l−1 of trace elements solution (g l−1) CoCl2:2; MnSO4 H2O: 1.6; and ZnSO4H2O: 1.4; FeSO4 7H2O: 0.5).
For enzyme production, 100 ml of basal medium was inoc-ulated in 500 ml erlenmeyer flasks with spore suspensions(106 spores/ml). In order to produce pectinase and xylanase-cellulase activities, the carbon source concentrations were settledto 1% (w/v) for citrus pectin, gruel and to 2% for wheat bran,respectively. The cultures were incubated on a rotary shaker setat 150 rpm and 30 ◦C for 5 days. The culture broths were filtered ona 0.45 �m Millipore membrane and then used for various furtheranalyses: pectinolytic, xylanolytic, and cellulolytic activities, andreducing sugar and protein contents.
2.5. Determination of reducing sugars and proteins
Reducing sugars were determined using the 3.5 dinitrosalicylicacid (DNS) method of Miller [15] and expressed as galacturonicacid equivalents. Protein contents were estimated by the method ofBradford [16] using crystalline bovine serum albumin as standard.
2.6. Pectinolytic activity assays
Exo-pectinolytic activities were determined by measuring theamount of reducing sugars released after 1 h of incubation at 50 ◦Cwith 0.9% citrus pectin in citrate buffer pH 4,8 as described byAguilar and Huitron [17]. One unit (UI) of exo-pectinolytic activitywas defined as the amount of enzyme that liberated one micromoleof galacturonic acid per minute.
Endo-pectinolytic activities were determined by estimating therelative change in viscosity measured by the Ostwald viscosime-ter [18]. One unit of endo-pectinolytic activity was defined as theamount of enzyme necessary to reduce the viscosity of the citruspectin solution by 50% in 5 min.
Pectin lyase activity was determined according to the methoddescribed by Pitt [19]. One unit of pectate lyase is the amount ofenzyme causing a change in absorbance of 0.01 under the condi-
tions of the assay.
PME activity was determined by the continuous titration of car-boxyl groups formed during pectin hydrolysis using 0.01 M NaOH[20]. An automatic pH-stat (718 STAT titrino, ‘�’ Metrohm, Herisau,
Fig. 1. Effect of different enzymatic formulations (E1, E2, E3, E4 and E5) on theextraction yield of olive oil from two types of olive (green and black) from the Chem-
N. Hadj-Taieb et al. / Biochemic
witzerland) was used. Routine assays were performed using 30 mlf 0.5% (w/v) pectin solution (DE 67%) in acetate buffer pH 6.5 at0 ◦C. PME activity in units (U) is defined as the amount of enzymeequired to release 1 �mol of carboxyl groups per min under theonditions described above.
.7. Cellulolytic and xylanolytic activities assays
Xylanase activity was determined by measuring the releasef reducing sugars from birchwood xylan (1%, w/v) by theinitrosalycylic-acid method [15]. One unit of xylanase was defineds the amount of enzyme required to release 1 �mol of xylose fromirchwood xylan per min under standard assay conditions (50 ◦C,0 mM phosphate buffer, pH 7).
Total cellulase (usually named “filter paper” or FP) activityas determined as recommended by the IUPAC biotechnology
ommission following the protocol of Mandels et al. modified byontenecourt and Eveleigh [21]. One unit of activity was defined
s the amount of enzyme required to release 1 �mol glucose (DNSrocedure) per min.
.8. Statistical analysis
A one way analysis of variance (ANOVA) was performed to com-are means of acidic composition and oil yield between the controlnd different enzymatic treatments. ANOVA was done separatelyor each maturity stage: statistical analysis was performed usingPSS version 13.0 (SPSS, Version 13.0, 2001, LEAD Technologies,nc., USA).
.9. Olive oil analysis
.9.1. Quality indicesFree acidity, given as % of oleic acid, peroxide value (PV)
xpressed as milliequivalents of active oxygen per kilogram of oilmequiv. O2/kg), and K232 and K270 extinction coefficients calcu-ated from absorption at 232 and 270 nm, were measured, followinghe analytical methods described in IOOC Regulations [22]. Allarameters were determined in triplicate for each sample.
.9.2. Fatty acid determinationFor the determination of fatty acid composition, the methyl
sters were prepared by vigorous shaking of a solution of oiln hexane (0.2 g in 3 ml) with 0.4 ml of 2 N methanolic potashnd analyzed by gas chromatography (Shimadzu. GC-17A) andquipped with a FID detector. A fused silica capillary column (30 mength × 0.32 mm diameter), coated with Carbowax (Polyethylenelycol) phase was used. Nitrogen was employed as carrier gasith a flow through the column of 1 ml/min. The temperatures of
he injector and detector were set at 230 and 250 ◦C respectively,hereas the oven temperature was 180 ◦C. An injection volume of
�l was used [22].
.9.3. Total phenol contentPhenolic compounds were isolated by extraction of a solution
f oil in hexane with a water–methanol mixture (60/40), threeimes. The Folin Ciocalteau reagent (Merck) was added to a suitableliquot of the combined extracts and the absorption of the solutiont 725 nm was measured. Values are given in milligrams of cafeiccid per kilogram of oil [23].
.9.4. Pigment contentThe chlorophyll fraction at 670 nm and the carotenoid fraction
t 470 nm were evaluated from the absorption spectrum of eachirgin olive oil sample (7.5 g) dissolved in cyclohexane (25 ml) [24].
lali variety as compared to a reference oil (control). Error bars indicate standarddeviation. Ns: not significantly (p < 0.05); ***value with three asterisks is signifi-cantly different from the control (p < 0.001).
The chlorophyll and carotenoid contents are expressed as mg perkg of oil.
2.9.5. Oxidative stabilityOxidative stability was evaluated by the Rancimat method [25],
which is a fast and reliable analytical procedure. Stability wasexpressed as the oxidation induction time (h), measured with theRancimat 679 apparatus (Metrohm Co., Basel, Switzerland), usingan oil sample of 5 g warmed to 100 ◦C and an air flow of 20 l/h. Withthis well-established methodology, the volatile oxidation productswere stripped from the oil and dissolved in cold water, whose con-ductivity increased progressively. The time taken to reach a fixedlevel of conductivity was measured.
2.9.6. Ripening indexThis parameter wax evaluated following the method described
by Uceda and Frias [26]. It is a simple technique, based on the assess-ment of the color of the skins of 100 olives which are randomlydrawn from 1 kg of the sample. The first stage of ripening is knownas the “green stage” (RI = 0.5) corresponding to green mature fruitsthat have reached their final size. Afterwards, chlorophyll pigmentsin the olive skin are progressively replaced by anthocyanines dur-ing fruit ripening. These make it possible to identify purple stageand black stage (RI = 5.5).
2.9.7. Refractive indexThe refractive index or index of refraction (RI) is a ratio of the
speed of light in a vacuum relative to that speed through a givenmedium. This was evaluated using a refractometer apparatus.
RI = velocity of light in a vacuumvelocity of light in medium
3. Results and discussion
3.1. Efficiency of enzyme formulation
Before the addition of the enzymes under investigation, the oilyield (amount of oil released by 100 kg of olive treated) obtained forthe black olives (14%) was observed to be greater than that for thegreen olives (8.5%) (Fig. 1). After the enzymatic formulations wereapplied, however, both types of olives were observed to undergo anoticeable increase in terms of yield, which attained 15.7% and 10%,
respectively. Pectinase, xylanase and cellulases were, for instance,observed to enhance oil yields at different proportions. The find-ings indicated that the highest levels of increase in terms of yieldwere obtained with the addition of formulation 5, which contained
82 N. Hadj-Taieb et al. / Biochemical Engineering Journal 62 (2012) 79– 85
Table 1Enzymatic activities (pectinase, cellulase and xylanase) in the different formulations(E1, E2, E3, E4 and E5).
Fig. 2. Effect of different enzymatic formulations (E1, E2, E3, E4 and E5) on thepolyphenol composition (ppm) of Chemlali olive oil. Error bars indicate standarddeviation.
Xylanases (U/ml) 0 0 7Filter paper (U/ml) 0 0 1
combination of pectinase, xylanase and cellulase. The effect of for-ulation 5 is statistically significant (p < 0.001) on black olives but
n green olives the effect was slightly significant (p < 0.05). Thisncrease reached up to 1.5% for both types of olives, an increase
hich is comparable to the one obtained by Najafian et al. [3]hat ranged between 0.9% and 2.4%. The enzymes presented in theurrent study were also observed to allow for a better oil extrac-ion yield from ripe olives (black olives). In fact, the findings ofhe present work corroborate the assertion previously suggestedn earlier studies which emphasised the synergistic effect of dif-erent enzymatic activities with Rapidase adex D and Cytolase 05,27].
Furthermore, the applications of pectinases (E2) or xylanase andellulases (E3) were observed to exhibit similar performances inerms of oil yield enhancement with a slight advantage for the E2ormulation. In this context, the activities measured (exo pecti-ase and pectin esterase) were noted to be more abundant in E2Table 1).Endo pectinase activity, on the other hand, exhibited theame activity in the two formulations about 270 U/ml.
In fact, several previous studies demonstrated that the applica-ion of pectinase-rich commercial enzymes, such as pectinex ultraP-L and pectinase from the Novo Nordisk and Merk company, Bio-ivia [3,28], improves olive oil performance. In this context, Vierhuist al. [4] conducted a study on Italian olives and reported that thepplication of pectolytic enzymes might have reduced the com-lexation of phenolics with the pectic polysaccharides of the fruit,hus raising the concentration of free phenols in the paste and,hen, their release into the olive oil. In addition, the results of theresent work indicate that pectolytic activity was more efficienthen used in combination with both cellulase and hemicellulase
ctivities, which is also in accordance with previous reports in theiterature [7].
.2. Pigments (carotenoids, chlorophylls) and polyphenolsompounds
Polyphenols are biologically active components with antioxi-ant properties that affect the flavour and quality of the product10]. The effect of the enzymatic formulations under investigationn polyphenols content was evaluated. In fact, using pectinases forreen and black olives, the addition of the enzymes in the mixingrocess was observed to increase polyphenols content by about 10%nd 6%, respectively (Fig. 2). This increase was actually expectediven the wealth of green olives in phenolic compounds. Once more,e observed a slight advantage for E2, over E1, in all tested qualita-
ive parameters. It is worth noting, however, that compared to theddition of pectinase alone, the application of pectinases with cel-ulases and xylanases seemed to decrease the polyphenol contentncluding that of green olives. Other works have shown that com-
ercial enzyme preparations, such as those of olivex and cytolase [6], were successfully used to enhance the release of phenolics in
live oil. Such works confirmed the capacity of all different enzy-atic activities tested to degrade olive cell wall and, thus, their
bility to improve the phenolic compounds. These studies demon-trated that the application of pectinase-rich commercial enzymes,
Fig. 3. Effect of different enzymatic formulations (E1, E2, E3, E4 and E5) on thepigment composition (B-carotene ppm) of Chemlali olive oil. Error bars indicatestandard deviation.
such as pectinex ultra SP-L and pectinase from the Novo Nordiskand Merk company, Biolivia [3,28], improves olive oil performance.In this context, Vierhuis et al. [4] conducted a study on Italian olivesand reported that the application of pectolytic enzymes might havereduced the complexation of phenolics with the pectic polysaccha-rides of the fruit, thus raising the concentration of free phenols inthe paste and, then, their release into the olive oil.
The composition of photosynthesis pigments in the oil, such aschlorophylls and carotenoids, was also observed to improve whencompared to that of the control (without enzymes). In additionto having an antioxidant activity, the carotenoids in the oil wereprecursors of vitamin A. These substances were partly responsi-ble for the color of virgin olive oil. Fig. 3 shows that the additionof the enzymes resulted in an increase of about two times in thecontents of these substances in both types of olives. The formu-lation E3 which contained only xylanase and cellulose activitiescould improve carotenoids in the oils. In fact, previous studies havedemonstrated that the addition of commercial enzymes, such ascytolase 0, Rapidase adex D, and Biolivia, increased the pigmentcontent and chromatic parameters of oil. Notice that the apparentcolor (measured from the chromatic coordinates L, a, and b) hasbeen correlated with the pigment composition in virgin olive oil[29].
The findings (Fig. 4) indicated that the amount of chlorophyllswas much more important for the green olive oil (1.15 ppm)than that of the black olives (0.3 ppm). Accordingly, the addi-tion of enzymes had a much more significant effect on greenolives than on black ones. Moreover, an additive or complementary
effect between the activities of the three enzymes was observedsince formulation E5 allowed for an increase of this compoundby about 0.35 ppm. The use of the enzymatic mixture has, there-fore, a positive effect on pigment transfers (of both chlorophylls
N. Hadj-Taieb et al. / Biochemical Eng
Fcd
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3i
tK
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ig. 4. Effect of different enzymatic formulations (E1, E2, E3, E4 and E5) on thehlorophylls composition (ppm) of Chemlali olive oil. Error bars indicate standardeviation.
nd carotenoids) from the fruit to the oil. The using of pecti-olytic activity seems to have an effect on the quantity of theseompounds. Indeed, the formulation E2 exopectinase-rich activitymproves the quantity of these compounds mainly the chlorophyllnd carotenoids
.3. Free acidity, peroxide value, and UV spectrophotometricndices and fatty acids composition
The K232 parameter is mainly indicative of the conjugation ofrienes and also of the presence of carbonylic compounds, while270 is related to the secondary oxidation rate.
able 2cidic composition (fatty acids) of olive oil treated by different enzymatic formulations (A
rom the control ***(p < 0.001); *(p < 0.05). A: Data are means of at least 3 replicates ± stan
Green olive C16:0 (ns) C16:1 (ns) C18:0 (ns) C18:1 (ns)
able 3ffect of different enzymatic formulations on the quantitative characteristics (acidity %;270) of Chemlali olive oil (A). A: Data are means of at least 3 replicates ± standard devia
Acidity % Refractive index (nD)
Green oliveControl 0.25 ± 0.03 1.4689 ± 0
E1 0.20 ± 0.02 1.4689 ± 0
E2 0.18 ± 0.02 1.4689 ± 0
E3 0.20 ± 0.02 1.4702 ± 0
E4 0.21 ± 0.01 1.4688 ± 0
E5 0.22 ± 0.01 1.4689 ± 0
Black oliveControl 0.25 ± 0.01 1.4688 ± 0
E1 0.18 ± 0.01 1.4688 ± 0
E2 0.16 ± 0.01 1.4684 ± 0
E3 0.20 ± 0.02 1.4684 ± 0
E4 0.23 ± 0.01 1.4689 ± 0
E5 0.21 ± 0.02 1.4684 ± 0
ineering Journal 62 (2012) 79– 85 83
These peroxide indices of oil quality were observed to decreasefollowing the application of the enzymes under study (Table 3).These results are in agreement with previously reported findingsin the literature [30]. Some other studies, however, seem to havereached different results [6].
Acidity is considered as an important criterion in the evaluationof oil quality. The oil acidity increase is a measure of oil degrada-tion, generally caused by fungal contamination during the storageof olives. Indeed, the acidity can change during the processing ofolives, which can bring some hydrolysis to the oils. Table 3 showsthat compared to those of the control, the addition of enzymesreduced the number of the free acid group (oleic acid) in the oil.The acid number decreased by 0.25–0.2% for the olive oil fromthe untreated olives (control) and the olives processed mainly byE1 and E2 formulations containing only the pectinolytic enzymes,respectively. Formulations 4 and 5, however, were observed tobring about slightly more acidity levels that did not exceed theindex.
With regard to oil composition in terms of fatty acid, no cleartrend was observed when the enzymes were added (Table 2). Thevalues were noted to be varied, which may be attributed to uncer-tainty with respect to the calculation of the composition. There isno statistically significant effect exerted by the enzyme aid on com-position of fatty acid in green olive. A strong increase of monoun-saturated fatty acid (C16-1) observed in black olive (p < 0.001).
3.4. Autoxidation capacity, refractive index and unsaponifiable
content
In addition to conducting phenolic compounds analysis, thepresent study also investigated the resistance of the different
). Ns: not significantly; values with one or three asterisks are significantly differentdard deviation.
ig. 5. Rancimat stability of olive oil treated by different enzymatic formulationsE1, E2, E3, E4 and E5). Error bars indicate standard deviation.
amples under investigation to oxidation (or oxidative stability).his parameter determines the time required for the oil to starthowing signs of rancidity. The findings indicated that the oxidativetability determined for oils from the green olives was greater thanhose from the black ones. In fact, their oxidative stabilities wereetermined to be of about 5.5 h and 1.6 h, respectively (Fig. 5). Theddition of enzymes was observed to increase this time particularlyor green olives, which attained around 3.5 h. The mixing of cellu-ases and xylanases (E3) was noted to bring about a better stabilityf about 9 h. This finding is in agreement with previously reportedndings in the literature [6] although an increase in stability of only–2 h and over 23 h was described by De Faveri et al. [7] using othernzymes, such Olivex, which were based mainly on pectinases.
Concerning the index of refraction, it was observed to vary withhe oils’ degree of unsaturation, being lower with the oils havingigher levels of unsaturation. Olive oils generally have a refractive
ndex of about 1.47 in 1.466. The analysis performed to determinehis index showed that the oils that were treated and those thatere untreated by the enzyme had almost the same index of refrac-
ion, which was about 1.47. Similarly, the oils from black olives andhose from the green ones exhibited the same index (Table 3).
As far as the unsaponifiable fractions are concerned, theyere also observed to increase following the enzymatic treatment
Table 3). Formulations E3 and E5, in particular, were observed toe able to improve these unsaponifiable fractions in the green andlack olives by the order of 1.198 and 1.144, respectively.
. Conclusion
The findings presented in the current study demonstrate thathe use of enzymatic treatments during the malaxation of oliveaste could have important implications for olive oil in termsf yield and phenolic/antioxidant content. The main advantagesbserved are (1) an increased extraction efficiency of 1.5% for bothlive kinds from the Chemleli variety; (2) a synergistic effect of dif-erent activities (pectinase, cellulase and xylanase) that enhancehe ability to improve the extraction of polyphenols and pigmentchlorophylls, carotenoids) compounds; (3) a decrease in the acidumber by 0.25–0.2% for the olive oil from the untreated (control)lives and that from the olives treated by E1 and E2 formula-ions containing only the pectinolytic enzymes, respectively; (4) annhanced rancimat stability of oils to 3.5 h, particularly for greenlives, which may have important implications for olive oil stor-ge and shelf life. In nutshell, the use of the enzymes presented inhe current study, particularly pectinases secreted by a hyperpecti-
olytic mutant CT1 of P. occitanis, yielded satisfactory quantitativend qualitative results that are comparable to the findings of pre-iously studies that used different commercial enzymes, such ashose of Novo enzymes or Gist brocades. Moreover, the difference
[
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in the composition of pectinolytic activities of the formulations E1and E2 seems to have no effect on the quantity and quality of oliveoils. Considering the promising properties and attributes of theenzymatic formulations and combinations presented in the currentstudy, further studies are currently under way in our laboratoriesto further investigate the effect of the pectinolytic, cellulolytic andxylanolytic activities on the quantity and quality of olive oil yieldsat the initial and terminal times of oil extraction processes as well asover time. Since the enzyme treatment has contributed to increas-ing the quality and quantity of oil, the use of enzymes in the oliveoil process could be re examined by national and international leg-islations.
Acknowledgements
We deeply thank Professor Ahmed Rebai from the CBS for hiskind and expert help in statistical analysis. The authors wish toexpress their sincere gratitude to Prof. Anouar Smaoui from theSfax Faculty of Science for his careful proofreading and constructiveediting of the current paper. The technical staff of the “Technologyand Quality” research unit of the “l’Institut de l’Olivier” is thankedfor their skilled help.
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