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

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LWT - Food Science and Technology

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Chemical composition changes in four green olive cultivars during

spontaneous 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 a b s t r a c t

Article history: Changes in some physico-chemical characteristics (pH and free acidity) and chemical composition
Received 29 April 2013 (sugars, total phenolic and flavonoid contents) of four olive cultivars during spontaneous fermentation in
Received in revised form brine were investigated. The cultivars were typical of the Moroccan market: “Moroccan Picholine”,
10 February 2014
“Languedoc Picholine”, “Ascolana” and “Sevillana”. The physico-chemical changes of olives and brines
Accepted 10 February 2014
during fermentation process were monitored. A similar pattern of pH was noticed for the “Moroccan
Picholine”, “Languedoc Picholine” and “Ascolana” cultivars with a final pH ranging between 4.4 and 4.6.
Keywords:
The profile of free acidity measured throughout fermentation period in brines was in agreement with the
Olive fermentation
Moroccan Picholine
pH trend. The concentration of sugars, total phenolic and flavonoids contents in olives flesh and brines
Languedoc Picholine during fermentation is reported. The loss of flavonoids, sugars and total phenolic contents in the olive
Sevillana flesh by the end of fermentation process was up to 60%, 63% and 79% in “Languedoc Picholine”, “Sev-
Ascolana illana” and “Moroccan Picholine”, respectively. The main phenols identified and quantified in the
different 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 brine
after 71 days of fermentation.
Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction
The olive tree (Olea europaea L.) is one of the most cultivated
fruit trees in the Mediterranean area. Its products, olive oil and
table olives constitute an important part of the Mediterranean
diet. Either in Mediterranean-style meals such as pizzas and
salads or as an appetizer accompanying beverages, table olives are
greatly appreciated for their nutritive values and sensory char-
acteristics. The worldwide production of table olives has been
estimated 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 South
America.
Table olive differs from other fermented crops (beans, cabbage,
carrots, and pumpkins) by its chemical composition. It has a bitter
taste mainly due to the presence of a glucoside compound called
oleuropein. It contains low proteins (10e20 g/kg), sugars (26e60 g/
kg) and high fat levels (120e300 g/kg), mainly oleic acid, although

these values can change depending on the olive cultivar (Skevin
* Corresponding author. Tel.: þ212 5 24 43 46 49 (Office: 515); fax: þ212 5 24 43 67 69.
E-mail address: a.hafidi@uca.ma (A. Hafidi).
et al., 2003), the ripening stage (Sakouhi et al., 2008), the agricul-
tural techniques (Marsilio et al., 2006) and the preparation
methods (Montaño, Casado, De Castro, Sánchez, & Rejano, 2005).
The beneficial effects of table olives consumption have been
attributed to monosaturated fatty acids, tocopherols, phenolic
compounds and phytosterols (Bianchi, 2003) known by their
important biological properties. Several works describe a protective
action 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 have
been reported to show chemoprotective effects against certain
cancers, e.g. breast and colorectal cancer (Bouallagui, Han, Isoda, &
Sayadi, 2011; Sirianni et al., 2010) and also contribute to lowering
risks of coronary heart diseases (Covas et al., 2006).
There are three main types of preparations widely used
worldwide to produce edible olives: green or Spanish-style olives,
black ripe or Californian-style olives and Greek-style or natural
black olives in brine (IOOC, 2004). More than fifty percent of the
table olives are prepared according to the Spanish-style green ol-
ives (Ruiz Barba & Jiménez Díaz, 2012). The procedure consists of
treating olive fruits with lye (20e50 g/l) to hydrolyze oleuropein
and consequently eliminate partially the bitterness and increase

http://dx.doi.org/10.1016/j.lwt.2014.02.011
0023-6438/Ó 2014 Elsevier Ltd. All rights reserved.
664 H. Kiai, A. Hafidi / LWT - Food Science and Technology 57 (2014) 663e670
skin permeability (Garrido Fernandez, Garcia-Garcia, & Brenes
Balbuena, 1992). The olives are left in sodium hydroxide solution
until the lye has penetrated two-thirds to three-fourths of the
distance from the olive surface to pit. Then, olives are washed once,
twice or three times with tap water in order to eliminate the excess
of alkali. Finally, washed fruits are stored in brine (100e120 g/l
NaCl), where lactic fermentation occurs (Hurtado, Reguant,
Bordons, & Rozès, 2012; Sánchez-Gómez, García, & Rejano, 2006;
Vergara, Blana, Mallouchos, Stamatiou, & Panagou, 2013). The
initial 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 changes
occur. Water-soluble compounds such as carbohydrates, phenolic
compounds mainly oleuropein and hydroxytyrosol glucoside and
other nutriment, diffuse from olives to the brine, while salt goes
from the brine into the flesh until it reaches a steady state by the
end of brining process. The fermentable substrates (glucose,
fructose, mannitol, sucrose, etc) are the main energy source of
fermentative microorganisms, which will provide organic acids
(mainly lactic acid) essential for the stability and preservation of
table olives during fermentation and storage. However, the
phenolic compounds undergo quantitative and qualitative
transformations during olive processing, mainly, the alkalin hy-
drolysis and/or the microbial degradation of oleuropein into
hydroxytyrosol and elenolic acid glucoside during debittering
and brining processes (Brenes & de Castro, 1998; Servili et al.,
2006).
Several studies reported chemical changes in table olives during
spontaneous or controlled fermentation of different olive cultivars
(cv.) such as Chétoui variety (Ben Othman, Roblain, Chammen,
Thonart, & Hamdi, 2009), Kalamata and Semidana cv. (Piga, Del
Caro, 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), and many other olive varieties. Inspite of the
large volumes of the Moroccan table olive production and also
exportations, the nutritional and the chemical characterizations of
the Moroccan fermented olives are very scarce. To our knowledge,
the chemical changes occurring during fermentation of Moroccan
olives from Moroccan Picholine, Languedoc Picholine, Sevillana and
Ascolana, which are the predominant cultivars in Morocco, were
not yet investigated.
The aim of the present work is to investigate the physico-
chemical characteristics (pH and free acidity) and chemical
composition (sugars, total phenolic and flavonoids contents)
changes occurring in brine and olive flesh during spontaneous
fermentation of the four most used green olive cultivars in
Morocco (i.e. Moroccan and Languedoc Picholine, Sevillana and
Ascolana).
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, were
purchased from Sigma Aldrich (Germany). All standards used
were of 95% minimum purity, the other reagents were of analyt-
ical grade except those used in the HPLC analysis, which were of
HPLC grade.
2.2. Processing and sampling
2.2.1. Table olive processing
As is customary in the industrial procedure, olives were stored
for 24 h at room temperature (25  C) before processing to avoid
sloughing of fruits when treated with lye. Olives were immersed in
20 (g/l) NaOH solution. The alkaline treatment lasted for 4e6 h for
Ascolana and Sevillana cultivars and for 10e12 h for Moroccan and
Languedoc Picholine varieties at room temperature until the lye
penetrated two-thirds of the distance to the pit. A washing step was
followed with two water changes at 4 and 12 h. After the washing
step, olives were covered with a 70e80 (g/l) NaCl solution for
Ascolana and Sevillana cultivars and 100e120 (g/l) NaCl solution for
Moroccan and Languedoc Picholine varieties and left to undergo
the lactic fermentation process.
2.2.2. Brine and olive samples
Olives 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 in
Marrakech, Morocco, during the 2009e2010 season. Samples were
collected at different time of fermentation (2, 5, 10, 17, 24, 31, 38, 45,
56 and 71 days after brining), transported to the laboratory and
stored in the freezer until analysis.
2.3. Extraction
The olive fruit pulp was crushed with hammer crusher in bath of
ice (4  C). A quantity (l0 g) of the obtained paste was extracted for
15 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 the
supernatant was recovered. The residues were re-extracted under
identical conditions for further two times. The supernatants were
combined, filtered and washed with hexane to remove oil. This
extract was used for sugars; total phenolic and flavonoids content
determination in olive flesh.
Sugars, total phenolic and flavonoids contents were determined
directly from brine samples. A volume of 50 ml of brine samples
was extracted three times with ethyl acetate (v/v) at room tem-
perature. The extracts was concentrated to dryness in a rotary
evaporator and dissolved in 5 ml methanol for antioxidant activity
determination 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 of
brine with NaOH (0.2 mol/l) in the presence of phenolphthalein and
expressed 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 Rao
and Pattabiraman (1989) using glucose as a standard, with some
modifications. Briefly, 200 ml aliquot of appropriately dilute sample
was assayed with 1 ml of sulfuric acid (18 mol/l) and 200 ml of
phenol (50 g/l). The mixture was vortexed and kept 5 min in a water
bath at 95  C. The flask content was cooled at room temperature
and diluted with the addition of 1 ml of distillate water. After
15 min the absorbance of the mixture was measured at 480 nm.
Sugars content was expressed as g of glucose equivalent (GLE) per
liter of brine and as g of GLE per 100 g of flesh dry weight (dw). The
sugars content of olive flesh and brines were analyzed in triplicate.
 H. Kiai, A. Hafidi / LWT - Food Science and Technology 57 (2014) 663e670
2.6. Total phenolic content
Total phenolic content were determined colorimetrically using
FolineCiocalteu 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 with
3.9 ml of distilled water and 100 ml of Folin-Ciocalteau reagent and
allowed to stand at ambient temperature, for 3 min. 1 ml sodium
carbonate solution (200 g/l) was added to the mixture. The tubes
were left for 60 min in the dark at room temperature; absorbance
was measured at 725 nm using a visible spectrophotometer. The
concentrations were expressed as g of tyrosol equivalent (TYE) per
liter of brine and as g of TYE per 100 g of flesh dry weight (dw). The
total phenolic content of olive flesh and brines were measured in
triplicate.
2.7. Total flavonoids content
Total flavonoids were measured by a colorimetric assay devel-
oped by Zhishen, Mengcheng, and Jianming (1999). 200 ml of
diluted sample was added to 0.8 ml of distillate water. First 0.06 ml
of 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 ml
of 1 mol/l sodium carbonate was added to the mixture. Immedi-
ately, the flask content was diluted with the addition of 0.5 ml of
distillate water and thoroughly mixed. Absorbance of the mixture
was determined at 510 nm against prepared water blank. The
concentrations 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 in
triplicate.
2.8. Antioxidant capacity determinations
Two methods were used to evaluate the antioxidant capacities
of the studied brines by the end of fermentation using DPPH and
reducing power (RP) assays.
2.8.1. Free radical scavenging assay
The antioxidant activity of the extracts was evaluated based on
hydrogen-donating or radical-scavenging ability using the stable
free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH$). A 0.1 ml of
phenolic extracts was added to 3 ml of 0.4 g/l methanolic solution
of DPPH$ The mixture was shaken vigorously and left to stand for
60 min at room temperature in the dark. The decrease in absor-
bance was measured at 517 nm, against methanol as a blank. DPPH$
scavenging effect was calculated as a percentage of DPPH$ discol-
oration using the following equation:
h  i
% Inhibition ¼ 1  Asample =Acontrol *100
where Acontrol was measured as the absorbance of DPPH$ without
sample. The sample concentration providing 50% inhibition (IC50)
was calculated from the graph plotting inhibition percentage
against phenolic extracts concentration.
2.8.2. Reducing power assay
The 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 (pH
6.6) and 2.5 ml of 10 g/l potassium ferricyanide. The mixture was
incubated 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 at
3000 g for 10 min. The upper layer (2.5 ml) was mixed with 2.5 ml
665
of deionized water and 1 ml of 1 g/l of ferric chloride, and the
absorbance was measured at 700 nm (higher absorbance indicates
higher reducing power). Extract concentration giving 0.5 of
absorbance (EC50) was calculated from the graph of absorbance at
700 nm against extract concentration.
2.9. HPLC analysis
Identification and quantification of phenolic monomers was
carried 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 was
maintained at 25  C. The flow rate was 1 ml/min and the sample
volume injected was 10 ml. A mixture of acidified water (A) and
acetonitrile (B) was used as mobile phase for a total running time
of 60 min. The identification of phenolic compounds was fulfilled
by comparison of retention times and UV-vis spectra with the
standards.
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 from
technological and sanitary point of view when green olives are
processed according to the Spanish-style and must be controlled
throughout the brining process.
As shown in Fig. 1, the pH of Moroccan Picholine, Languedoc
Picholine and Ascolana brines, increased rapidly during the first
five days of fermentation. This rise may be due to the diffusion of
residual lye to the brine; during this stage lactic acid bacteria
(LAB) would have not yet proliferated. In fact, during natural
Spanish-style green table olive fermentation, a succession of the
microorganism species is reported, depending on their respective
physiology and nutrition requirements (Arroyo-López, Bautista-
Gallego, Rodríguez-Gómez, & Garrido-Fernández, 2010). At the
beginning of fermentation (2e3 days), Gram-negative bacteria,
mainly from the Enterobacteriaceae family, were reported to
appear and multiply, because of the high pH subsequent to the
alkali treatment. However, as fermentation progresses, Gram-
negative bacteria are rapidly inhibited due to the acidification
process originated by LAB (Abriouel, Benomar, Lucas, & Gálvez,
2011). Our results demonstrate an important pH decrease
within the next five days. Obtained values fall from 5.2 to 4.7 and
Fig. 1. Changes in pH values in brines of four green olives cultivars during 71 days of
fermentation. (A) Moroccan Picholine, (C) Languedoc Picholine, (:) Sevillana and
(-) Ascolana. Each value is the mean of three determinations  SD.
666 H. Kiai, A. Hafidi / LWT - Food Science and Technology 57 (2014) 663e670
4.8 units for Moroccan Picholine and Ascolana brines, respec-
tively. Comparatively, the pH of Sevillana brine dropped from 5 at
the beginning of fermentation to 4.4 units after ten days of
fermentation. Over the time, the pH of the brines of the four
tested cultivars decreased progressively until reaching a steady
state after 45 days of brining, with values ranging from 4.2 to 4.6.
The decrease in pH is attributed to the transformation of sugars
during the fermentation process (mainly glucose, fructose and
sucrose) into organic acids. LAB, particularly Lactobacillus pen-
tosus and Lactobacillus plantarum grow massively during brining
process and produce lactic acid until the brine reaches a pH
below 4.5 by the end of fermentation. Yeast species (mainly
Pichia anomala, Pichia membranifaciens, Saccharomyces cerevisiae,
Debaryomyces hansenii and Candida boidinii) coexist with LAB
during brining (Arroyo-López et al., 2010). Furthermore the
diffusion of some acid phenols and degradation of other
phenolic compounds into acids such as elenolic acid obtained
from the alkaline hydrolysis of oleuropein may contribute to
the pH lowering during the initial phase of olive table
fermentation.
The pH changes reflect the free acidity rates expressed as g of
lactic acid per 100 ml of brine, the lower the pH, the higher the free
acidity. During the first fifteen days of fermentation, the rate of free
acidity 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 free
acidity rate becomes more or less stable until the 55th day of
fermentation from which the free acidity rate increase again in all
tested brines. Sevillana brine presents the highest rate of acidity
followed by Ascolana, Moroccan Picholine and Picholine Langue-
doc. Differences in the average values of final free acidity were
noticeable among cultivars. Particularly, Sevillana and Ascolana
presented high acidity values (0.8 and 1 g lactic acid/100 ml of
brine) compared to Moroccan and Languedoc Picholine in which
the acidity remain around 0.5 g lactic acid/100 ml of brine. The
acidities obtained for Ascolana and Sevillana brines are similar to
those 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 are
reported in Figs. 3 and 4. Sugars diffused into the brine and their
concentrations increased quickly and reached maximum values
Fig. 2. Evolution in free acidity in brines of four green olives cultivars during 71 days of
fermentation. (A) Moroccan Picholine, (C) Languedoc Picholine, (:) Sevillana and
(-) Ascolana. Each value is the mean of three determinations  SD.
Fig. 3. Changes in sugar content in olive flesh of four green olives cultivars during 71
days of fermentation. (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, Languedoc
Picholine and Moroccan Picholine brines, respectively after 25
days of fermentation. Actually, the sugars contents values in
brines are the result from the diffusion from the olives and also
the consumption by fermentative microorganisms. The diffusion
of fermentative substrates depends on various parameters such
as skin permeability, salt concentration, fruit to brine ratio and
temperature (Garrido-Fernandez et al., 1997). After that a very
fast decrease of sugars was recorded for all brines. By the 45th
day of processing, sugars content reached a steady state for the
four tested cultivars. Residual sugars concentrations in the
brines at the end of fermentation period were in the range 1.3e
4 g GLE/l.
In the opposite side, total sugars in the olive flesh underwent
a sharp decrease throughout fermentation. It dropped from
12.6 g GLE/100 g dw to 2.6 g GEL/100 g dw, declined from
10.7 g GLE/100 g dw to 2.6 g GLE/100 g dw and failed from
6.7 g GLE/100 g dw to 1.4 g GLE/100 g dw for Sevillana, Ascolana,
Moroccan and Languedoc Picholine olive cultivars, respectively
after 71 days of processing. The reduction rates of sugars con-
tents by the end of fermentation were almost similar for all the
studied cultivars and ranged from 71% to 79%. The numerous
glycosides in olive flesh (including oleuropein) release soluble
sugars when hydrolyzed. It is worthy to note that there is a
relative correlation between the consumption of sugars and the
accumulation of lactic acid.
Fig. 4. Changes in sugar content in brines of four green olives cultivars during 71 days
of fermentation. (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
Fig. 5. Evolution of olive flesh total phenolic content during spontaneous fermentation
of four green olive cultivars. (A) Moroccan Picholine, (C) Languedoc Picholine, (:)
Sevillana and (-) Ascolana. Each value is the mean of three determinations  SD.
3.3. Changes in phenolic compounds
Total phenolic content time course in olive flesh and brine are
given in Figs. 5 and 6. As it can be observed from Fig. 5, the overall
patterns of total phenolic content for the studied olive cultivars
were quite similar. Olives as expected, showed an important loss in
total phenolic content during fermentation due to the diffusion of
these compounds to the brine. Similar behavior has been reported
by other authors (Álvarez, López, & Lamarque, 2012; Ben Othman
et 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 phenolic
compounds compared to large-sized fruit cultivars (Bianchi, 2003).
Vossen (2007) reports that the phenolic content of olive cultivars
ranges from the very high levels found in Koroneiki and Coratina to
the very low levels found in varieties such as Picual. The Moroccan
and Languedoc Picholine cultivars were ranked in the high level of
total polyphenols, followed by Ascolana variety (medium-high
level of phenols content) and Sevillana cultivar (low phenols
content).
During the first 20 days of fermentation, the daily decrease rates
of phenolic contents were estimated to 2%, 3%, 5% and 6%, for
Ascolana, Sevillana, Moroccan Picholine and Languedoc Picholine,
respectively. Beyond the 24th day of processing, a slowing decline
of phenolic content in the olive flesh of all tested cultivars is
observed. After 55 days of brining, phenolic content of Ascolana and
Sevillana cultivars attained a steady state. However, a sharp
Fig. 6. Evolution of brine total phenolic content during spontaneous fermentation of
four green olive cultivars. (A) Moroccan Picholine, (C) Languedoc Picholine, (:)
Sevillana and (-) Ascolana. Each value is the mean of three determinations  SD.
667
Fig. 7. Evolution of olive flesh flavonoids content during spontaneous fermentation of
four 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 71
days of processing were 40, 45, 59 and 63% for Ascolana, Sevillana,
Languedoc and Moroccan Picholine, respectively. In fact, the
diffusion of phenolic compounds from olive flesh to the brine de-
pends on several parameters such as cultivar characteristics, fruit
skin permeability, type of polyphenols present in olive flesh and
their ability to diffuse outside the fruit. Oppositely, the total
phenolic contents in brines increased gradually in all fermenters to
rich maximum 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 brines
start to decrease. This decline is may be due to the degradation of
phenolic acids by L. plantarum. It has been demonstrated that
L. plantarum contained phenolic acid decarboxylases, which
decarboxylates p-coumaric, m-coumaric, ferullic and caffeic acids
to 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 vinyl
derivatives into ethyl derivatives, and to metabolize p-coumaric
acid into phloretic acid (Rodríguez et al., 2008). Among the
hydroxycinnamic acids, only gallic and protocatechuic acids were
metabolized to pirogallol and catechol, respectively (Rodríguez
et al., 2008).
Fig. 8. Evolution of brine flavonoids content during spontaneous fermentation of four
green olive cultivars. (A) Moroccan Picholine, (C) Languedoc Picholine, (:) Sevillana
and (-) Ascolana. Each value is the mean of three determinations  SD.
668 H. Kiai, A. Hafidi / LWT - Food Science and Technology 57 (2014) 663e670
Fig. 9. HPLC chromatograms recorded at 280 nm for the main phenolic compounds
identified 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.
Changes in flavonoids content in the olive flesh and their cor-
responding brines during the fermentation process are reported in
Figs. 7 and 8. Languedoc Picholine olive flesh has showed the
highest flavonoids contents, compared to the other three varieties.
It is noteworthy that the depletion followed a very fast kinetic in
the first 20 days. The daily decrease rates of flavonoids content
during this first period of processing were 2.5%, 1.8%, 1.7% and 1.0%
for Languedoc Picholine, Moroccan Picholine, Ascolana and
Sevillana, respectively. After the 25th day of fermentation, flavo-
noids content in olives flesh decreased more slowly and reached a
steady state around the 56th day of fermentation. The total loss of
flavonoids content in all the studied cultivars is estimated to 60%
after 71 days of brining. In the other side, the flavonoids contents in
olive brines increase quickly during the first 20 days followed by
moderate increase thereafter. By the 45th day of fermentation, the
flavonoids content of Ascolana and Sevillana brines reach a steady
state. However, a slight increase in flavonoids content in Moroccan
and Languedoc Picholine brines was noticed. The final flavonoids
Table 1
Concentrations of the main phenolic compounds identified in the brines of Moroccan Picholine, Languedoc Picholine, Ascolana and Sevillana olives cultivars after 71 days of
fermentation, expressed in mg TYE/l.
Olive brines Phenolic compounds (mg TYE/l)
Hydroxytyrosol
Moroccan Picholine 2208  34
Languedoc Picholine 2185  30
Ascolana 1440  22
Sevillana 814  12
Values are given as mean of three repetitions  standard deviation.
concentrations in the brines by the end of the brining process were
in the range 1.45e2.46 g CAE/l (Fig. 8).
3.4. Simple phenolic compounds and antioxidant activities of olive
brines
As a consequence of the diffusion from the olive flesh, an
important enrichment of phenols and flavonoids occurs in brines
during fermentation. Furthermore, LAB led to the conversion of
sugars into organic acids, mainly lactic acid. Consequently, by the
end of fermentation olive brine becomes a rich medium of high
added value products such as natural phenolic compounds and
organic acids (lactic acid). This latter can be used in industries
including in foods. It can be also used for the production of ethyl
lactate (green solvent for the food industry) and/or the production
of poly (lactic acid) polymers. Such uses necessitate adequate
techniques for its extraction and purification from fermentation
media. Recently, inorganic nanofiltration membranes were pre-
sented as an efficient tool to dewater lactic acid (Duke, Lim, Castro
da Luz, & Nielsen, 2008). As far Agroindustrial by-products contain
important amounts of natural phenolic compounds particularly
which are well known for their high antioxidant and antimicrobial
activities (Bouaziz et al., 2008), the extraction and purification
technologies of such nutraceuticals from these by-products are
becoming a very active field of scientific research (Galanakis, in
press).
A high performance liquid chromatographiy technique was
used to identify and quantify the major phenolic compounds of
olive brine. The studied brines contained hydroxytyrosol, tyrosol,
(þ)-catechin and quercetine with some quantitative differences
among cultivars (Fig. 9; Table 1). The phenols amounts found in
brines after 71 days of fermentation ranged from w1e3 g TYE/l,
following the order Moroccan Picholine > Languedoc
Picholine > Ascolana > Sevillana (Table 1). Hydroxytyrosol was
the main simple phenolic compound identified in all brines, its
proportion was up to 84% of total simple phenolic compounds.
Actually, this finding is in a good agreement with the literature
data where hydroxytyrosol was found to be the most abundant
phenol in green table olives wastewaters: lye, washing waste-
water and brine (Parinos, Stalikas, Giannopoulos, & Pilidis, 2007).
This compound results from the hydrolysis of oleuropein, which
is the major phenolic in fresh green olive fruit. Moreover, an
increase of hydroxytyrosol amounts in brines during the brining
process is reported due to its diffusion from the olives into the
brine and also because of the acid hydrolysis of oleuropein and
hydroxytyrosol-4-b-glucoside, phenols that decreased during the
brining 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 for
a 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 studied
brines using the DPPH and the reducing power (RP) methods.
Total
Tyrosol (þ)-Catechin Quercetine
157  5 180  5 142  9 2687  53
177  15 102  11 131  12 2595  68
168  8 138  6 43  2 1789  38
65  2 56  3 57  8 992  25
 H. Kiai, A. Hafidi / LWT - Food Science and Technology 57 (2014) 663e670
Table 2
Total phenolics, IC50 (DPPH assay) and EC50 (RP assay) in the brines of Moroccan
Picholine, Languedoc Picholine, Ascolana and Sevillana olives cultivars after 71 days
of fermentation.
Olive brines Total phenolics DPPH assay IC50 RP assay EC50
(g TYE/l) (mg/ml) (mg/ml)
Moroccan 3.7  0.05 0.124  0.003 0.038  0.02
Picholine
Languedoc 3  0.12 0.129  0.004 0.044  0.01
Picholine
Ascolana 2.5  0.06 0.232  0.002 0.083  0.004
Sevillana 1.8  0.05 0.559  0.035 0.187  0.008
Values are given as mean of three repetitions  standard deviation.
Table 2 shows the TPC, IC50 (DPPH assay) and EC50 (RP assay) of
the four studied brines by the end of fermentation. The TPC of
various brines after 71 days of brining process were in the range of
1.8  0.05 to 3.7  0.05 g TYE/l. The order of TPC among different
cultivar brines is as follows: Moroccan Picholine > Languedoc
Picholine > Ascolana > Sevillana. The IC50 ranged from
0.124  0.003 to 0.559  0.035 mg/ml, and the EC50 values ranged
from 0.038  0.02 to 0.044  0.01 mg/ml. The higher TPC value
corresponded with higher Scavenging of free DPPH radical and the
RP values and lower IC50 and EC50 values. The brines of Moroccan
and Languedoc Picholine cultivars showed the higher total phenolic
content and the greatest antioxidant activity followed by Ascolana
and 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, Languedoc
Picholine, Sevillana and Ascolana cultivars from Marrakech region.
The chemical profile and the physicochemical characteristics pat-
terns were similar between olive cultivars. Sevillana variety
showed the lowest values of pH and the highest amount of lactic
acid and sugars, while the physicochemical characteristics of
Ascolana cultivar hardly differed from those of Moroccan and
Languedoc Picholine. The contents of sugars, phenolic compounds
and flavonoids contents have been determined in olive flesh and
brine. The results obtained in the present work denote that olive
processing induced an important loss in phenolic compounds and
flavonoids from the olives. The HPLC analysis of phenols in the
brines report quantitative and qualitative differences in simple
phenolic compounds among cultivars, with the prevalence of
hydroxytyrosol in the four studied brines by the end of fermenta-
tion. Moreover, Moroccan and Languedoc Picholine brines exhibit
the highest antioxidant activity with the two methods used. The
obtained results suggest that table olive wastewaters (brine) can be
considered as a potential source of cheap and available high added
value products if an appropriate method of treatment and valori-
zation was applied.
Acknowledgment
The authors would like to acknowledge gratefully Mr. Said El
Mallakh and all the employees of Agro-Hind society for their
valuable cooperation.
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