lable at ScienceDirect

LWT - Food Science and Technology 57 (2014) 663e670

Contents lists avai

LWT - Food Science and Technology

journal homepage: www.elsevier .com/locate/ lwt

Chemical composition changes in four green olive cultivars duringspontaneous fermentation

Hajar Kiai, Abdellatif Hafidi*

Food Sciences Laboratory, Department of Biology, Faculty of Sciences-Semlalia, P.O. Box: 2390, 40090 Marrakech, Morocco

a r t i c l e i n f o

Article history:Received 29 April 2013Received in revised form10 February 2014Accepted 10 February 2014

Keywords:Olive fermentationMoroccan PicholineLanguedoc PicholineSevillanaAscolana

* Correspondingauthor. Tel.:þ212524434649(OfficeE-mail address: a.hafidi@uca.ma (A. Hafidi).

http://dx.doi.org/10.1016/j.lwt.2014.02.0110023-6438/� 2014 Elsevier Ltd. All rights reserved.

a b s t r a c t

Changes in some physico-chemical characteristics (pH and free acidity) and chemical composition(sugars, total phenolic and flavonoid contents) of four olive cultivars during spontaneous fermentation inbrine were investigated. The cultivars were typical of the Moroccan market: “Moroccan Picholine”,“Languedoc Picholine”, “Ascolana” and “Sevillana”. The physico-chemical changes of olives and brinesduring fermentation process were monitored. A similar pattern of pH was noticed for the “MoroccanPicholine”, “Languedoc Picholine” and “Ascolana” cultivars with a final pH ranging between 4.4 and 4.6.The profile of free acidity measured throughout fermentation period in brines was in agreement with thepH trend. The concentration of sugars, total phenolic and flavonoids contents in olives flesh and brinesduring fermentation is reported. The loss of flavonoids, sugars and total phenolic contents in the oliveflesh by the end of fermentation process was up to 60%, 63% and 79% in “Languedoc Picholine”, “Sev-illana” and “Moroccan Picholine”, respectively. The main phenols identified and quantified in thedifferent brines at the end of brining process were hydroxytyrosol, tyrosol, (þ)-catechin and quercetine.The highest total phenolic content and antioxidant activity were obtained in Moroccan Picholine brineafter 71 days of fermentation.

� 2014 Elsevier Ltd. All rights reserved.

1. Introduction

The olive tree (Olea europaea L.) is one of the most cultivatedfruit trees in the Mediterranean area. Its products, olive oil andtable olives constitute an important part of the Mediterraneandiet. Either in Mediterranean-style meals such as pizzas andsalads or as an appetizer accompanying beverages, table olives aregreatly appreciated for their nutritive values and sensory char-acteristics. The worldwide production of table olives has beenestimated to more than 2,400,000 tons for the 2010e2011 season(IOOC, 2012) and this is shared between Mediterranean countries(more than 80% of world production), USA, Australia and SouthAmerica.

Table olive differs from other fermented crops (beans, cabbage,carrots, and pumpkins) by its chemical composition. It has a bittertaste mainly due to the presence of a glucoside compound calledoleuropein. It contains low proteins (10e20 g/kg), sugars (26e60 g/kg) and high fat levels (120e300 g/kg), mainly oleic acid, althoughthese values can change depending on the olive cultivar (�Skevin

:515); fax:þ212524436769.

et al., 2003), the ripening stage (Sakouhi et al., 2008), the agricul-tural techniques (Marsilio et al., 2006) and the preparationmethods (Montaño, Casado, De Castro, Sánchez, & Rejano, 2005).

The beneficial effects of table olives consumption have beenattributed to monosaturated fatty acids, tocopherols, phenoliccompounds and phytosterols (Bianchi, 2003) known by theirimportant biological properties. Several works describe a protectiveaction of the a-tocopherol on human health against different pa-thologies, contributing to reduce the negatif effects of inflamma-tory diseases by defending the body against free radicals (Bogani,Galli, Villa, & Visioli, 2007). In addition, phenolic compounds havebeen reported to show chemoprotective effects against certaincancers, e.g. breast and colorectal cancer (Bouallagui, Han, Isoda, &Sayadi, 2011; Sirianni et al., 2010) and also contribute to loweringrisks of coronary heart diseases (Covas et al., 2006).

There are three main types of preparations widely usedworldwide to produce edible olives: green or Spanish-style olives,black ripe or Californian-style olives and Greek-style or naturalblack olives in brine (IOOC, 2004). More than fifty percent of thetable olives are prepared according to the Spanish-style green ol-ives (Ruiz Barba & Jiménez Díaz, 2012). The procedure consists oftreating olive fruits with lye (20e50 g/l) to hydrolyze oleuropeinand consequently eliminate partially the bitterness and increase

H. Kiai, A. Hafidi / LWT - Food Science and Technology 57 (2014) 663e670664

skin permeability (Garrido Fernandez, Garcia-Garcia, & BrenesBalbuena, 1992). The olives are left in sodium hydroxide solutionuntil the lye has penetrated two-thirds to three-fourths of thedistance from the olive surface to pit. Then, olives are washed once,twice or three times with tap water in order to eliminate the excessof alkali. Finally, washed fruits are stored in brine (100e120 g/lNaCl), where lactic fermentation occurs (Hurtado, Reguant,Bordons, & Rozès, 2012; Sánchez-Gómez, García, & Rejano, 2006;Vergara, Blana, Mallouchos, Stamatiou, & Panagou, 2013). Theinitial treatments (lye treatment and washing) are olive variety-dependent, due to the different physical properties (texture, size)of each cultivar (Montaño, Sánchez, Casado, de Castro, & Rejano,2003).

During fermentation, important physico-chemical changesoccur. Water-soluble compounds such as carbohydrates, phenoliccompounds mainly oleuropein and hydroxytyrosol glucoside andother nutriment, diffuse from olives to the brine, while salt goesfrom the brine into the flesh until it reaches a steady state by theend of brining process. The fermentable substrates (glucose,fructose, mannitol, sucrose, etc) are the main energy source offermentative microorganisms, which will provide organic acids(mainly lactic acid) essential for the stability and preservation oftable olives during fermentation and storage. However, thephenolic compounds undergo quantitative and qualitativetransformations during olive processing, mainly, the alkalin hy-drolysis and/or the microbial degradation of oleuropein intohydroxytyrosol and elenolic acid glucoside during debitteringand brining processes (Brenes & de Castro, 1998; Servili et al.,2006).

Several studies reported chemical changes in table olives duringspontaneous or controlled fermentation of different olive cultivars(cv.) such as Chétoui variety (Ben Othman, Roblain, Chammen,Thonart, & Hamdi, 2009), Kalamata and Semidana cv. (Piga, DelCaro, Pinna, & Agabbio, 2005), Itrana, Peranzana, Cellina di Nardo,Nocellara del Belice and Bella di Cerignola cv. (Tofalo et al., 2012),Brandofino, Castriciana, Nocellara del Belice, and Passa-lunara cv.(Aponte et al., 2010), Manzanilla, Hojiblanca and Gordal cv.(Montaño et al., 2003), andmany other olive varieties. Inspite of thelarge volumes of the Moroccan table olive production and alsoexportations, the nutritional and the chemical characterizations ofthe Moroccan fermented olives are very scarce. To our knowledge,the chemical changes occurring during fermentation of Moroccanolives fromMoroccan Picholine, Languedoc Picholine, Sevillana andAscolana, which are the predominant cultivars in Morocco, werenot yet investigated.

The aim of the present work is to investigate the physico-chemical characteristics (pH and free acidity) and chemicalcomposition (sugars, total phenolic and flavonoids contents)changes occurring in brine and olive flesh during spontaneousfermentation of the four most used green olive cultivars inMorocco (i.e. Moroccan and Languedoc Picholine, Sevillana andAscolana).

2. Material and methods

2.1. Chemicals and standards

Ethyl acetate, methanol, acetonitrile and phosphoric acid,FolineCiocalteu reagent, sodium carbonate, sodium hydroxide,sodium nitrite, aluminum chloride, sulfuric acid phenol, hydrox-ytyrosol, tyrosol, (þ)-catechin, quercetine, and glucose, werepurchased from Sigma Aldrich (Germany). All standards usedwere of 95% minimum purity, the other reagents were of analyt-ical grade except those used in the HPLC analysis, which were ofHPLC grade.

2.2. Processing and sampling

2.2.1. Table olive processingAs is customary in the industrial procedure, olives were stored

for 24 h at room temperature (25 �C) before processing to avoidsloughing of fruits when treated with lye. Olives were immersed in20 (g/l) NaOH solution. The alkaline treatment lasted for 4e6 h forAscolana and Sevillana cultivars and for 10e12 h for Moroccan andLanguedoc Picholine varieties at room temperature until the lyepenetrated two-thirds of the distance to the pit. Awashing stepwasfollowed with two water changes at 4 and 12 h. After the washingstep, olives were covered with a 70e80 (g/l) NaCl solution forAscolana and Sevillana cultivars and 100e120 (g/l) NaCl solution forMoroccan and Languedoc Picholine varieties and left to undergothe lactic fermentation process.

2.2.2. Brine and olive samplesOlives and brines samples from Spanish-style green olives of

Moroccan and Languedoc Picholine, Sevillana and Ascolana culti-vars were kindly provided by an olive manufacturer located inMarrakech, Morocco, during the 2009e2010 season. Samples werecollected at different time of fermentation (2, 5, 10, 17, 24, 31, 38, 45,56 and 71 days after brining), transported to the laboratory andstored in the freezer until analysis.

2.3. Extraction

The olive fruit pulp was crushedwith hammer crusher in bath ofice (4 �C). A quantity (l0 g) of the obtained paste was extracted for15 min with 20 ml of methanol/water 80:20 (v/v) at room tem-perature. The mixture was centrifuged at 4000 g for 10 min and thesupernatant was recovered. The residues were re-extracted underidentical conditions for further two times. The supernatants werecombined, filtered and washed with hexane to remove oil. Thisextract was used for sugars; total phenolic and flavonoids contentdetermination in olive flesh.

Sugars, total phenolic and flavonoids contents were determineddirectly from brine samples. A volume of 50 ml of brine sampleswas extracted three times with ethyl acetate (v/v) at room tem-perature. The extracts was concentrated to dryness in a rotaryevaporator and dissolved in 5 ml methanol for antioxidant activitydetermination and HPLC analysis.

2.4. Physicochemical analysis

The pH of samples was measured using a Multilab P5 (WTW,Germany). Free acidity was determined by titration of 10 ml ofbrinewith NaOH (0.2mol/l) in the presence of phenolphthalein andexpressed as percent (w/v) of lactic acid Garrido-Fernandez,Adams, and Fernandez-Diez (1997).

2.5. Sugars content

Sugars content was determined according to the method of Raoand Pattabiraman (1989) using glucose as a standard, with somemodifications. Briefly, 200 ml aliquot of appropriately dilute samplewas assayed with 1 ml of sulfuric acid (18 mol/l) and 200 ml ofphenol (50 g/l). Themixturewas vortexed and kept 5min in awaterbath at 95 �C. The flask content was cooled at room temperatureand diluted with the addition of 1 ml of distillate water. After15 min the absorbance of the mixture was measured at 480 nm.Sugars content was expressed as g of glucose equivalent (GLE) perliter of brine and as g of GLE per 100 g of flesh dry weight (dw). Thesugars content of olive flesh and brines were analyzed in triplicate.

Fig. 1. Changes in pH values in brines of four green olives cultivars during 71 days offermentation. (A) Moroccan Picholine, (C) Languedoc Picholine, (:) Sevillana and(-) Ascolana. Each value is the mean of three determinations � SD.

H. Kiai, A. Hafidi / LWT - Food Science and Technology 57 (2014) 663e670 665

2.6. Total phenolic content

Total phenolic content were determined colorimetrically usingFolineCiocalteu reagents according to the method of Catalano,Franco, De Nobili, and Leita (1999) using tyrosol as a standard,with slight modifications. The extract (100 ml) was mixed with3.9 ml of distilled water and 100 ml of Folin-Ciocalteau reagent andallowed to stand at ambient temperature, for 3 min. 1 ml sodiumcarbonate solution (200 g/l) was added to the mixture. The tubeswere left for 60 min in the dark at room temperature; absorbancewas measured at 725 nm using a visible spectrophotometer. Theconcentrations were expressed as g of tyrosol equivalent (TYE) perliter of brine and as g of TYE per 100 g of flesh dry weight (dw). Thetotal phenolic content of olive flesh and brines were measured intriplicate.

2.7. Total flavonoids content

Total flavonoids were measured by a colorimetric assay devel-oped by Zhishen, Mengcheng, and Jianming (1999). 200 ml ofdiluted sample was added to 0.8 ml of distillate water. First 0.06 mlof sodium nitrite (50 g/l) was added to the flask. After 5 min,0.04 ml of aluminum chloride (100 g/l) was added. At 6 min, 0.4 mlof 1 mol/l sodium carbonate was added to the mixture. Immedi-ately, the flask content was diluted with the addition of 0.5 ml ofdistillate water and thoroughly mixed. Absorbance of the mixturewas determined at 510 nm against prepared water blank. Theconcentrations were expressed as g of (þ)-catechin equivalent(CAE) per liter of brine and as g of CAE per 100 g of flesh dry weight(dw). The total flavonoids content of olive flesh was analyzed intriplicate.

2.8. Antioxidant capacity determinations

Two methods were used to evaluate the antioxidant capacitiesof the studied brines by the end of fermentation using DPPH� andreducing power (RP) assays.

2.8.1. Free radical scavenging assayThe antioxidant activity of the extracts was evaluated based on

hydrogen-donating or radical-scavenging ability using the stablefree radical 2,2-diphenyl-1-picrylhydrazyl (DPPH$). A 0.1 ml ofphenolic extracts was added to 3 ml of 0.4 g/l methanolic solutionof DPPH$ The mixture was shaken vigorously and left to stand for60 min at room temperature in the dark. The decrease in absor-bancewasmeasured at 517 nm, against methanol as a blank. DPPH$scavenging effect was calculated as a percentage of DPPH$ discol-oration using the following equation:

% Inhibition ¼h1�

�Asample=Acontrol

�i*100

where Acontrol was measured as the absorbance of DPPH$ withoutsample. The sample concentration providing 50% inhibition (IC50)was calculated from the graph plotting inhibition percentageagainst phenolic extracts concentration.

2.8.2. Reducing power assayThe capacity of the extracts to reduce Fe3þ was evaluated ac-

cording to the method of Oyaizu (1986). The phenolic extract (1 ml)was mixed with 2.5 ml of 0.2 mol/l sodium phosphate buffer (pH6.6) and 2.5 ml of 10 g/l potassium ferricyanide. The mixture wasincubated at 50 �C for 20 min, added with 2.5 ml of 100 g/l tri-chloroacetic acid (w/v) were added, the mixture was centrifuged at3000 g for 10 min. The upper layer (2.5 ml) was mixed with 2.5 ml

of deionized water and 1 ml of 1 g/l of ferric chloride, and theabsorbance was measured at 700 nm (higher absorbance indicateshigher reducing power). Extract concentration giving 0.5 ofabsorbance (EC50) was calculated from the graph of absorbance at700 nm against extract concentration.

2.9. HPLC analysis

Identification and quantification of phenolic monomers wascarried out using a high-performance liquid chromatograph(Knauer) equipped with a (K-1001) pump and a PDA detector(200e700 UV-Vis) operating at 280 nm. The column was(4.6 � 250 mm) (Eurospher II 100-5), and the temperature wasmaintained at 25 �C. The flow rate was 1 ml/min and the samplevolume injected was 10 ml. A mixture of acidified water (A) andacetonitrile (B) was used as mobile phase for a total running timeof 60 min. The identification of phenolic compounds was fulfilledby comparison of retention times and UV-vis spectra with thestandards.

3. Results and discussion

3.1. pH and free acidity changes during olives fermentation

The pH and free acidity of the brine are crucial parameters fromtechnological and sanitary point of view when green olives areprocessed according to the Spanish-style and must be controlledthroughout the brining process.

As shown in Fig. 1, the pH of Moroccan Picholine, LanguedocPicholine and Ascolana brines, increased rapidly during the firstfive days of fermentation. This rise may be due to the diffusion ofresidual lye to the brine; during this stage lactic acid bacteria(LAB) would have not yet proliferated. In fact, during naturalSpanish-style green table olive fermentation, a succession of themicroorganism species is reported, depending on their respectivephysiology and nutrition requirements (Arroyo-López, Bautista-Gallego, Rodríguez-Gómez, & Garrido-Fernández, 2010). At thebeginning of fermentation (2e3 days), Gram-negative bacteria,mainly from the Enterobacteriaceae family, were reported toappear and multiply, because of the high pH subsequent to thealkali treatment. However, as fermentation progresses, Gram-negative bacteria are rapidly inhibited due to the acidificationprocess originated by LAB (Abriouel, Benomar, Lucas, & Gálvez,2011). Our results demonstrate an important pH decreasewithin the next five days. Obtained values fall from 5.2 to 4.7 and

Fig. 3. Changes in sugar content in olive flesh of four green olives cultivars during 71days of fermentation. (A) Moroccan Picholine, (C) Languedoc Picholine, (:) Sevillanaand (-) Ascolana. Each value is the mean of three determinations � SD.

H. Kiai, A. Hafidi / LWT - Food Science and Technology 57 (2014) 663e670666

4.8 units for Moroccan Picholine and Ascolana brines, respec-tively. Comparatively, the pH of Sevillana brine dropped from 5 atthe beginning of fermentation to 4.4 units after ten days offermentation. Over the time, the pH of the brines of the fourtested cultivars decreased progressively until reaching a steadystate after 45 days of brining, with values ranging from 4.2 to 4.6.The decrease in pH is attributed to the transformation of sugarsduring the fermentation process (mainly glucose, fructose andsucrose) into organic acids. LAB, particularly Lactobacillus pen-tosus and Lactobacillus plantarum grow massively during briningprocess and produce lactic acid until the brine reaches a pHbelow 4.5 by the end of fermentation. Yeast species (mainlyPichia anomala, Pichia membranifaciens, Saccharomyces cerevisiae,Debaryomyces hansenii and Candida boidinii) coexist with LABduring brining (Arroyo-López et al., 2010). Furthermore thediffusion of some acid phenols and degradation of otherphenolic compounds into acids such as elenolic acid obtainedfrom the alkaline hydrolysis of oleuropein may contribute tothe pH lowering during the initial phase of olive tablefermentation.

The pH changes reflect the free acidity rates expressed as g oflactic acid per 100 ml of brine, the lower the pH, the higher the freeacidity. During the first fifteen days of fermentation, the rate of freeacidity increases progressively in Ascolana, Moroccan and Lan-guedoc Picholine brines and sharply in the Sevillana brine (Fig. 2),followed by a moderate rise in the next ten days. After that, the freeacidity rate becomes more or less stable until the 55th day offermentation from which the free acidity rate increase again in alltested brines. Sevillana brine presents the highest rate of acidityfollowed by Ascolana, Moroccan Picholine and Picholine Langue-doc. Differences in the average values of final free acidity werenoticeable among cultivars. Particularly, Sevillana and Ascolanapresented high acidity values (0.8 and 1 g lactic acid/100 ml ofbrine) compared to Moroccan and Languedoc Picholine in whichthe acidity remain around 0.5 g lactic acid/100 ml of brine. Theacidities obtained for Ascolana and Sevillana brines are similar tothose obtained for Manzanilla and Hojiblanca cultivars, respec-tively (Montaño et al., 2003).

3.2. Changes in fermentative substrates

Changes of fermentative substrates in olive flesh and brine arereported in Figs. 3 and 4. Sugars diffused into the brine and theirconcentrations increased quickly and reached maximum values

Fig. 2. Evolution in free acidity in brines of four green olives cultivars during 71 days offermentation. (A) Moroccan Picholine, (C) Languedoc Picholine, (:) Sevillana and(-) Ascolana. Each value is the mean of three determinations � SD.

of 6.8, 4.6, 3.4 and 3.3 g GLE/l in Sevillana, Ascolana, LanguedocPicholine and Moroccan Picholine brines, respectively after 25days of fermentation. Actually, the sugars contents values inbrines are the result from the diffusion from the olives and alsothe consumption by fermentative microorganisms. The diffusionof fermentative substrates depends on various parameters suchas skin permeability, salt concentration, fruit to brine ratio andtemperature (Garrido-Fernandez et al., 1997). After that a veryfast decrease of sugars was recorded for all brines. By the 45thday of processing, sugars content reached a steady state for thefour tested cultivars. Residual sugars concentrations in thebrines at the end of fermentation period were in the range 1.3e4 g GLE/l.

In the opposite side, total sugars in the olive flesh underwenta sharp decrease throughout fermentation. It dropped from12.6 g GLE/100 g dw to 2.6 g GEL/100 g dw, declined from10.7 g GLE/100 g dw to 2.6 g GLE/100 g dw and failed from6.7 g GLE/100 g dw to 1.4 g GLE/100 g dw for Sevillana, Ascolana,Moroccan and Languedoc Picholine olive cultivars, respectivelyafter 71 days of processing. The reduction rates of sugars con-tents by the end of fermentation were almost similar for all thestudied cultivars and ranged from 71% to 79%. The numerousglycosides in olive flesh (including oleuropein) release solublesugars when hydrolyzed. It is worthy to note that there is arelative correlation between the consumption of sugars and theaccumulation of lactic acid.

Fig. 4. Changes in sugar content in brines of four green olives cultivars during 71 daysof fermentation. (A) Moroccan Picholine, (C) Languedoc Picholine, (:) Sevillana and(-) Ascolana. Each value is the mean of three determinations � SD.

Fig. 5. Evolution of olive flesh total phenolic content during spontaneous fermentationof four green olive cultivars. (A) Moroccan Picholine, (C) Languedoc Picholine, (:)Sevillana and (-) Ascolana. Each value is the mean of three determinations � SD.

Fig. 7. Evolution of olive flesh flavonoids content during spontaneous fermentation offour green olive cultivars. (A) Moroccan Picholine, (C) Languedoc Picholine, (:)Sevillana and (-) Ascolana. Each value is the mean of three determinations � SD.

H. Kiai, A. Hafidi / LWT - Food Science and Technology 57 (2014) 663e670 667

3.3. Changes in phenolic compounds

Total phenolic content time course in olive flesh and brine aregiven in Figs. 5 and 6. As it can be observed from Fig. 5, the overallpatterns of total phenolic content for the studied olive cultivarswere quite similar. Olives as expected, showed an important loss intotal phenolic content during fermentation due to the diffusion ofthese compounds to the brine. Similar behavior has been reportedby other authors (Álvarez, López, & Lamarque, 2012; Ben Othmanet al., 2009; Romero, Brenes, Garcia, Garcia, & Garrido, 2004).

The phenolic content is variety dependant (Malheiro, Sousa,Casal, Bento, & Pereira, 2011; Tura et al., 2007). In general, culti-vars with small-size fruit have higher concentration of phenoliccompounds compared to large-sized fruit cultivars (Bianchi, 2003).Vossen (2007) reports that the phenolic content of olive cultivarsranges from the very high levels found in Koroneiki and Coratina tothe very low levels found in varieties such as Picual. The Moroccanand Languedoc Picholine cultivars were ranked in the high level oftotal polyphenols, followed by Ascolana variety (medium-highlevel of phenols content) and Sevillana cultivar (low phenolscontent).

During the first 20 days of fermentation, the daily decrease ratesof phenolic contents were estimated to 2%, 3%, 5% and 6%, forAscolana, Sevillana, Moroccan Picholine and Languedoc Picholine,respectively. Beyond the 24th day of processing, a slowing declineof phenolic content in the olive flesh of all tested cultivars isobserved. After 55 days of brining, phenolic content of Ascolana andSevillana cultivars attained a steady state. However, a sharp

Fig. 6. Evolution of brine total phenolic content during spontaneous fermentation offour green olive cultivars. (A) Moroccan Picholine, (C) Languedoc Picholine, (:)Sevillana and (-) Ascolana. Each value is the mean of three determinations � SD.

decrease in total phenolic was observed for Moroccan and Lan-guedoc Picholine. The total reduction of phenolic contents after 71days of processing were 40, 45, 59 and 63% for Ascolana, Sevillana,Languedoc and Moroccan Picholine, respectively. In fact, thediffusion of phenolic compounds from olive flesh to the brine de-pends on several parameters such as cultivar characteristics, fruitskin permeability, type of polyphenols present in olive flesh andtheir ability to diffuse outside the fruit. Oppositely, the totalphenolic contents in brines increased gradually in all fermenters torichmaximum concentrations of 4.7, 3.1, 4.2 and 3.2 g TYE/l after 17,24, 31 and 28 days of fermentation for Moroccan Picholine,Sevillana, Languedoc Picholine and Ascolana brines, respectively.After the 40th day of brining, the phenolic content in most brinesstart to decrease. This decline is may be due to the degradation ofphenolic acids by L. plantarum. It has been demonstrated thatL. plantarum contained phenolic acid decarboxylases, whichdecarboxylates p-coumaric, m-coumaric, ferullic and caffeic acidsto their corresponding vinyl derivatives (Barthelmebs, Divies, &Cavin, 2000; Cavin et al., 1997; Rodríguez, Landete, de las Rivas, &Muñoz, 2008). Furthermore, L. plantarum also displayed an induc-ible acid phenol reductase activity, able to reduce the vinylderivatives into ethyl derivatives, and to metabolize p-coumaricacid into phloretic acid (Rodríguez et al., 2008). Among thehydroxycinnamic acids, only gallic and protocatechuic acids weremetabolized to pirogallol and catechol, respectively (Rodríguezet al., 2008).

Fig. 8. Evolution of brine flavonoids content during spontaneous fermentation of fourgreen olive cultivars. (A) Moroccan Picholine, (C) Languedoc Picholine, (:) Sevillanaand (-) Ascolana. Each value is the mean of three determinations � SD.

Fig. 9. HPLC chromatograms recorded at 280 nm for the main phenolic compoundsidentified in the brines of Moroccan Picholine (a), Languedoc Picholine (b), Ascolana (c)and Sevillana (d) olives cultivars after 71 days of fermentation. 1: hydroxytyrosol; 2:tyrosol; 3: (þ)-catechin; 4: quercetine.

H. Kiai, A. Hafidi / LWT - Food Science and Technology 57 (2014) 663e670668

Changes in flavonoids content in the olive flesh and their cor-responding brines during the fermentation process are reported inFigs. 7 and 8. Languedoc Picholine olive flesh has showed thehighest flavonoids contents, compared to the other three varieties.It is noteworthy that the depletion followed a very fast kinetic inthe first 20 days. The daily decrease rates of flavonoids contentduring this first period of processing were 2.5%, 1.8%, 1.7% and 1.0%for Languedoc Picholine, Moroccan Picholine, Ascolana andSevillana, respectively. After the 25th day of fermentation, flavo-noids content in olives flesh decreased more slowly and reached asteady state around the 56th day of fermentation. The total loss offlavonoids content in all the studied cultivars is estimated to 60%after 71 days of brining. In the other side, the flavonoids contents inolive brines increase quickly during the first 20 days followed bymoderate increase thereafter. By the 45th day of fermentation, theflavonoids content of Ascolana and Sevillana brines reach a steadystate. However, a slight increase in flavonoids content in Moroccanand Languedoc Picholine brines was noticed. The final flavonoids

Table 1Concentrations of the main phenolic compounds identified in the brines of Moroccan Picfermentation, expressed in mg TYE/l.

Olive brines Phenolic compounds (mg TYE/l)

Hydroxytyrosol Tyrosol

Moroccan Picholine 2208 � 34 157 � 5Languedoc Picholine 2185 � 30 177 � 15Ascolana 1440 � 22 168 � 8Sevillana 814 � 12 65 � 2

Values are given as mean of three repetitions � standard deviation.

concentrations in the brines by the end of the brining process werein the range 1.45e2.46 g CAE/l (Fig. 8).

3.4. Simple phenolic compounds and antioxidant activities of olivebrines

As a consequence of the diffusion from the olive flesh, animportant enrichment of phenols and flavonoids occurs in brinesduring fermentation. Furthermore, LAB led to the conversion ofsugars into organic acids, mainly lactic acid. Consequently, by theend of fermentation olive brine becomes a rich medium of highadded value products such as natural phenolic compounds andorganic acids (lactic acid). This latter can be used in industriesincluding in foods. It can be also used for the production of ethyllactate (green solvent for the food industry) and/or the productionof poly (lactic acid) polymers. Such uses necessitate adequatetechniques for its extraction and purification from fermentationmedia. Recently, inorganic nanofiltration membranes were pre-sented as an efficient tool to dewater lactic acid (Duke, Lim, Castroda Luz, & Nielsen, 2008). As far Agroindustrial by-products containimportant amounts of natural phenolic compounds particularlywhich are well known for their high antioxidant and antimicrobialactivities (Bouaziz et al., 2008), the extraction and purificationtechnologies of such nutraceuticals from these by-products arebecoming a very active field of scientific research (Galanakis, inpress).

A high performance liquid chromatographiy technique wasused to identify and quantify the major phenolic compounds ofolive brine. The studied brines contained hydroxytyrosol, tyrosol,(þ)-catechin and quercetine with some quantitative differencesamong cultivars (Fig. 9; Table 1). The phenols amounts found inbrines after 71 days of fermentation ranged from w1e3 g TYE/l,following the order Moroccan Picholine > LanguedocPicholine > Ascolana > Sevillana (Table 1). Hydroxytyrosol wasthe main simple phenolic compound identified in all brines, itsproportion was up to 84% of total simple phenolic compounds.Actually, this finding is in a good agreement with the literaturedata where hydroxytyrosol was found to be the most abundantphenol in green table olives wastewaters: lye, washing waste-water and brine (Parinos, Stalikas, Giannopoulos, & Pilidis, 2007).This compound results from the hydrolysis of oleuropein, whichis the major phenolic in fresh green olive fruit. Moreover, anincrease of hydroxytyrosol amounts in brines during the briningprocess is reported due to its diffusion from the olives into thebrine and also because of the acid hydrolysis of oleuropein andhydroxytyrosol-4-b-glucoside, phenols that decreased during thebrining process (Romero et al., 2004). Thus, at the end of pro-cessing, hydroxytyrosol become the main phenol in brine(Table 1). As far as this natural phenolic compounds accounts fora powerful natural antioxidant, the table olive brines are ex-pected to have interesting biological and antioxidant activities.These latter activities were investigated in the four studiedbrines using the DPPH� and the reducing power (RP) methods.

holine, Languedoc Picholine, Ascolana and Sevillana olives cultivars after 71 days of

Total

(þ)-Catechin Quercetine

180 � 5 142 � 9 2687 � 53102 � 11 131 � 12 2595 � 68138 � 6 43 � 2 1789 � 3856 � 3 57 � 8 992 � 25

Table 2Total phenolics, IC50 (DPPH� assay) and EC50 (RP assay) in the brines of MoroccanPicholine, Languedoc Picholine, Ascolana and Sevillana olives cultivars after 71 daysof fermentation.

Olive brines Total phenolics(g TYE/l)

DPPH� assay IC50

(mg/ml)RP assay EC50(mg/ml)

MoroccanPicholine

3.7 � 0.05 0.124 � 0.003 0.038 � 0.02

LanguedocPicholine

3 � 0.12 0.129 � 0.004 0.044 � 0.01

Ascolana 2.5 � 0.06 0.232 � 0.002 0.083 � 0.004Sevillana 1.8 � 0.05 0.559 � 0.035 0.187 � 0.008

Values are given as mean of three repetitions � standard deviation.

H. Kiai, A. Hafidi / LWT - Food Science and Technology 57 (2014) 663e670 669

Table 2 shows the TPC, IC50 (DPPH� assay) and EC50 (RP assay) ofthe four studied brines by the end of fermentation. The TPC ofvarious brines after 71 days of brining process were in the range of1.8 � 0.05 to 3.7 � 0.05 g TYE/l. The order of TPC among differentcultivar brines is as follows: Moroccan Picholine > LanguedocPicholine > Ascolana > Sevillana. The IC50 ranged from0.124 � 0.003 to 0.559 � 0.035 mg/ml, and the EC50 values rangedfrom 0.038 � 0.02 to 0.044 � 0.01 mg/ml. The higher TPC valuecorresponded with higher Scavenging of free DPPH� radical and theRP values and lower IC50 and EC50 values. The brines of Moroccanand Languedoc Picholine cultivars showed the higher total phenoliccontent and the greatest antioxidant activity followed by Ascolanaand Sevillana brines.

4. Conclusion

This work reports for the first time an investigation of physi-cochemical characteristics and chemical composition in industri-ally fermented green olives of Moroccan Picholine, LanguedocPicholine, Sevillana and Ascolana cultivars from Marrakech region.The chemical profile and the physicochemical characteristics pat-terns were similar between olive cultivars. Sevillana varietyshowed the lowest values of pH and the highest amount of lacticacid and sugars, while the physicochemical characteristics ofAscolana cultivar hardly differed from those of Moroccan andLanguedoc Picholine. The contents of sugars, phenolic compoundsand flavonoids contents have been determined in olive flesh andbrine. The results obtained in the present work denote that oliveprocessing induced an important loss in phenolic compounds andflavonoids from the olives. The HPLC analysis of phenols in thebrines report quantitative and qualitative differences in simplephenolic compounds among cultivars, with the prevalence ofhydroxytyrosol in the four studied brines by the end of fermenta-tion. Moreover, Moroccan and Languedoc Picholine brines exhibitthe highest antioxidant activity with the two methods used. Theobtained results suggest that table olive wastewaters (brine) can beconsidered as a potential source of cheap and available high addedvalue products if an appropriate method of treatment and valori-zation was applied.

Acknowledgment

The authors would like to acknowledge gratefully Mr. Said ElMallakh and all the employees of Agro-Hind society for theirvaluable cooperation.

References

Abriouel, H., Benomar, N., Lucas, R., & Gálvez, A. (2011). Culture-independent studyof the diversity of microbial populations in brines during fermentation ofnaturally-fermented Aloreña green table olives. International Journal of FoodMicrobiology, 144, 487e496.

Álvarez, D. M. E., López, A., & Lamarque, A. L. (2012). Industrial improvement fornaturally black olives production of Manzanilla and Arauco cultivars. Journalof Food Processing and Preservation. http://dx.doi.org/10.1111/j.1745-4549.2012.00751.x.

Aponte, M., Ventorino, V., Blaiotta, G., Volpe, G., Farina, V., Avellone, G., et al. (2010).Study of green Sicilian table olive fermentations through microbiological,chemical and sensory analyses. Food Microbiology, 27, 162e170.

Arroyo-López, F. N., Bautista-Gallego, J., Rodríguez-Gómez, F., & Garrido-Fernández, A. (2010). Predictive microbiology and table olives. In A. Mendez-Vilas (Ed.), Current research, technology and education topics in applied micro-biology and microbial biotechnology (pp. 1452e1461). Badajoz: FORMATEX.

Barthelmebs, L., Divies, C., & Cavin, J. F. (2000). Knockout of the p-coumaratedecarboxylase gene from Lactobacillus plantarum reveals the existence of twoother inducible enzymatic activities involved in phenolic acid metabolism.Applied and Environmental Microbiology, 66, 3368e3375.

Ben Othman, N., Roblain, D., Chammen, N., Thonart, P., & Hamdi, M. (2009). Anti-oxidant phenolic compounds loss during the fermentation of Chétoui olives.Food Chemistry, 116, 662e669.

Bianchi, G. (2003). Lipids and phenols in table olives. European Journal of LipidScience and Technology, 105, 229e242.

Bogani, P., Galli, C., Villa, M., & Visioli, F. (2007). Postprandial anti-inflammatory andantioxidant effects of extra virgin olive oil. Arteriosclerosis, 190, 181e186.

Bouallagui, Z., Han, J., Isoda, H., & Sayadi, S. (2011). Hydroxytyrosol rich extract fromolive leaves modulates cell cycle progression in MCF-7 human breast cancercells. Food and Chemical Toxicology, 49, 179e184.

Bouaziz, M., Lassoued, S., Bouallagui, Z., Smaoui, S., Gargoubi, A., Dhouib, A., et al.(2008). Synthesis and recovery of high bioactive phenolics from table-olivebrine process wastewater. Bioorganic & Medicinal Chemistry, 16, 9238e9246.

Brenes, M., & de Castro, A. (1998). Transformation of oleuropein and its hydrolysisproducts during Spanish-style green olive processing. Journal of the Science ofFood and Agriculture, 77, 353e358.

Catalano, L., Franco, I., De Nobili, M., & Leita, L. (1999). Polyphenols in olive millwastewaters and their depuration plant effluents: a comparison of the Folin-Ciocalteau and HPLC methods. Agrochimica, 43, 193e205.

Cavin, J. F., Barthelmebs, L., Guzzo, J., Van Beeumen, J., Samyn, B., Travers, J. F., et al.(1997). Purification and characterization of an inducible p-coumaric aciddecarboxylase from Lactobacillus plantarum. FEMS Microbiology Letters, 147,291e295.

Covas, M. I., Nyyssönen, K., Poulsen, H. E., Kaikkonen, J., Zunft, H. J., Kiesewetter, H.,et al. (2006). The effect of polyphenols in olive oil on heart disease risk factors.Annals of Internal Medicine, 145, 333e341.

Duke, M. C., Lim, A., Castro da Luz, S., & Nielsen, L. (2008). Lactic acid enrichmentwith inorganic nanofiltration and molecular sieving membranes by pervapo-ration. Food and Bioproducts Processing, 86, 290e295.

Galanakis, C. M. (2013). Emerging technologies for the production of nutraceuticalsfrom agricultural by-products: a viewpoint of opportunities and challenges.Food and Bioproducts Processing, 91, 575e579.

Garrido-Fernandez, A., Adams, M. R., & Fernandez-Diez, M. J. (1997). Naturally blackolives type. In A. Garrido-Fernandez, M. R. Adams, & M. J. Fernandez-Diez (Eds.),Table olives: Production and processing (pp. 289e363). London: Chapman andHall Publisher.

Garrido-Fernandez, A., Garcia-Garcia, P., & Brenes Balbuena, M. (1992). Therecycling of table olive brine using ultrafiltration and activated carbonadsorption. Journal of Food Engineering, 17, 291e305.

Hurtado, A., Reguant, C., Bordons, A., & Rozès, N. (2012). Lactic acid bacteria fromfermented table olives. Food Microbiology, 31, 1e8.

IOOC, International Olive Oil Council. (2004). Trade standard applying to table olives.IOOC. (2012). IOOC statistic of table olive’s world production. Available at http://

www.internationaloliveoil.org/news/view/662-annee-2012actualites/225newsletter-marche-decembre-2011 (visited on February 4, 2013).

Malheiro, R., Sousa, A., Casal, S., Bento, A., & Pereira, J. A. (2011). Cultivar effect onthe phenolic composition and antioxidant potential of stoned table olives. FoodChemistry and Toxicology, 49, 450e457.

Marsilio, V., d’Andria, R., Lanza, B., Russi, F., Iannucci, E., Lavini, A., et al. (2006).Effect of irrigation and lactic acid bacteria inoculants on the phenolic fraction,fermentation and sensory characteristics of olive (Olea europaea L. cv. Ascolanatenera) fruits. Journal of the Science of Food and Agriculture, 86, 1005e1013.

Montaño, A., Casado, F. J., De Castro, A., Sánchez, A. H., & Rejano, L. (2005). Influenceof processing, storage time, and pasteurization upon the tocopherol and aminoacid contents of treated green table olives. European Food Research andTechnology, 220, 255e260.

Montaño, A., Sánchez, A. H., Casado, F. J., de Castro, A., & Rejano, L. (2003). Chemicalprofile of industrially fermented green olives of different varieties. FoodChemistry, 82, 297e302.

Oyaizu, M. (1986). Studies on products of the browning reaction. Antioxidativeactivities of browning reaction products prepared from glucosamine. JapaneseJournal of Nutrition, 44, 307e315.

Parinos, C. S., Stalikas, C. D., Giannopoulos, S., & Pilidis, G. A. (2007). Chemical andphysicochemical profile of wastewaters produced from the different stages ofSpanish-style green olives processing. Journal of Hazardous Materials, 145,339e343.

Piga, A., Del Caro, A., Pinna, I., & Agabbio, M. (2005). Anthocyanin and colourevolution in naturally black table olives during anaerobic processing. LWT e

Food Science and Technology, 38, 425e429.

H. Kiai, A. Hafidi / LWT - Food Science and Technology 57 (2014) 663e670670

Rao, P., & Pattabiraman, T. N. (1989). Reevaluation of the phenol sulfuric acid re-action for the estimation of hexoses and pentoses. Analytical Biochemistry, 181,18e22.

Rodríguez, H., Landete, J. M., de las Rivas, B., & Muñoz, R. (2008). Metabolism offood phenolic acids by Lactobacillus plantarum CECT 748T. Food Chemistry, 107,1393e1398.

Romero, C., Brenes, M., Garcia, P., Garcia, A., & Garrido, A. (2004). Polyphenolchanges during fermentation of naturally black olives. Journal of Agriculturaland Food Chemistry, 52, 1973e1979.

Ruiz Barba, J. L., & Jiménez Díaz, R. (2012). A novel Lactobacillus pentosus starterculture for Spanish-style green olive fermentation. Food Microbiology, 30,253e259.

Sakouhi, F., Harrabi, S., Absalon, C., Sbei, K., Boukhchina, S., & Kallel, H. (2008). a-Tocopherol and fatty acids contents of some Tunisian table olives (Olea europeaL.): changes in their composition during ripening and processing. Food Chem-istry, 108, 833e839.

Sánchez-Gómez, A. H., García, P., & Rejano, L. (2006). Trends in table olives pro-duction, elaboration of table olives. Grasas y Aceites, 57, 86e94.

Servili, M., Settanni, L., Veneziani, G., Esposto, S., Massitti, O., Taticchi, A., et al.(2006). The use of Lactobacillus pentosus 1MO to shorten the debittering processtime of black table olives (cv. Itrana and Leccino): a pilotscale application.Journal of Agriculture and Food Chemistry, 54, 3869e3875.

Sirianni, R., Chimento, A., De Luca, A., Casaburi, I., Rizza, P., Onofrio, A., et al. (2010).Oleuropein and hydroxytyrosol inhibit MCF-7 breast cancer cell proliferation

interfering with ERK1/2 activation. Molecular Nutrition & Food Research, 54,833e840.

�Skevin, D., Rade, D., �Strucelj, D., Mokrovãak, �Z., Nederal, S., & Ben�ci�c, D. (2003). Theinfluence of variety and harvest time on the bitterness and phenolic com-pounds of olive oil. European Journal of Lipid Science and Technology, 105, 536e541.

Tofalo, R., Schirone, M., Perpetuini, G., Angelozzi, G., Suzzi, G., & Corsetti, A.(2012). Microbiological and chemical profiles of naturally fermented tableolives and brines from different Italian cultivars. Antonie Leeuwenhoek, 102,121e131.

Tura, D., Gigliotti, C., Pedo, S., Failla, O., Bassi, D., & Serraiocco, A. (2007). Influence ofcultivar and site of cultivation on levels of lipophilic and hydrophilic antioxi-dants in virgin olive oils (Olea europaea L.) and correlations with oxidativestability. Scientia Horticulturae, 112, 108e119.

Vergara, J. V., Blana, V., Mallouchos, A., Stamatiou, A., & Panagou, E. Z. (2013).Evaluating the efficacy of brine acidification as implemented by the Greek tableolive industry on the fermentation profile of Conservolea green olives. LWT e

Food Science and Technology, 53, 113e119.Vossen, P. (2007). Olive oil: history, production and characteristics of the world’s

classic oils. HortScience, 42, 1093e1110.Zhishen, J., Mengcheng, T., & Jianming, W. (1999). The determination of flavonoid

contents in mulberry and their scavenging effects on superoxide radicals. FoodChemistry, 64, 555e559.