Journal of the Science of Food and Agriculture J Sci Food Agric 86:1005–1013 (2006)

DOI: 10.1002/jsfa.2449

Effect of irrigation and lactic acid bacteria

inoculants on the phenolic fraction, fermenta-
tion and sensory characteristics of olive (Olea
europaea L. cv. Ascolana tenera) fruits
Vincenzo Marsilio,1∗ Riccardo d’Andria,2 Barbara Lanza,1 Francesca Russi,1
Emilia Iannucci,1 Antonella Lavini2 and Giovanni Morelli2
1 CRA – Istituto Sperimentale per la Elaiotecnica, Contrada Fonte Umano 37, 65013 Città S. Angelo (PE), Italy
2 CNR – Istituto per i Sistemi Agricoli e Forestali del Mediterraneo, CP 101, 80040 San Sebastiano al Vesuvio (NA), Italy

Abstract: Plant productivity, phenolic profile and natural fermentation, with and without lactic acid
bacteria inoculants, were investigated in olive fruit of Ascolana tenera growing under different irrigation
regimes. A rainfed control and two treatments receiving a water depth equivalent to 33 and 66% of the
estimated crop evapotranspiration (ETc) from the pit hardening stage and a treatment with 66% of ETc
during the entire season (25 May-4 October), were tested. Olive vigour increased with irrigation, which
also induced a fresh yield enhancement, mainly due to the increase in fruit size. The differences between
treatments were less evident for fruit number per tree and pulp/stone ratio. The phenolic content increased
in water deficit-stressed olives and differences were detected in the concentrations of individual phenolic
compounds. Olive oil content and fatty acids composition appeared scarcely affected by irrigation. Values
of pH, acidity, sugars, phenolic composition and microbial population were monitored during olive
fermentation. Extracts of freshly harvested olive fruits exhibited significantly higher phenolic content
than extracts of processed olives. The interaction of irrigation and fermentation appeared to have a
significant effect on the chemical and sensory characteristics of the end product. The bitter taste of olive
fruit was positively correlated with the level of total phenolics, but negatively correlated with the overall
acceptability for direct consumption. The fermentation process was accelerated in the presence of lactic
acid bacteria inoculants, indicating the potential use of these microorganisms as quality control markers
in table olives during processing.
 2006 Society of Chemical Industry

Keywords: Olea europaea L.; Ascolana tenera; water use; phenolics; fermentation; sensory evaluation

INTRODUCTION applying a deficit water supply from May to September

The low rainfall and the scarce water availability in
the traditional olive-growing Mediterranean regions
often expose the olive tree to a severe water stress.
The problem of water as a limiting factor for crop
production is most relevant for table olives since
irrigation also improves uniformity and fruit size.
Therefore, the development of irrigation strategies
for optimising olive management under conditions
of limited water resources is of crucial importance
for improvement of the long-term profitability within
the concept of sustainable olive growing. The use of
irrigation techniques able to reduce water volumes
has been studied and encouraging results have
been obtained by suspending/reducing the water
supply during fixed phenological stages of the olive
vegetative–productive cycle. Goldhamer1 obtained a
10% yield reduction and a 45% water saving by
on cv. Manzanilla. Alegre2 reported that a restitution
of 25% of crop evapotranspiration (ETc) from the
pit hardening stage until the beginning of ripening
resulted in a 16% yield loss and a 47% water saving
compared with the fully irrigated control. Many papers
have reported the influence of water deficit on olive
fruit development and fruit composition.3 – 7 Some
show a clear effect of water deficit on phenolic
compounds in olives. It seems that a low water
availability during olive ripening increases the phenolic
content in olive fruit, affecting bitterness, flavour,
colour and its aromatic potential characteristics.8 – 10
More recently, phenolic compounds have attracted
interest also for their strong antioxidant scavenging
abilities.11 – 15 A lower total phenolic content was found
in fresh olive fruit of cv. Nocellara del Belice irrigated
with 66% of ETc during the dry season, compared with

∗ Correspondence to: Vincenzo Marsilio, CRA – Istituto Sperimentale per la Elaiotecnica, Contrada Fonte Umano 37, I-65013 Città S.
Angelo (PE), Italy
E-mail: vincenzo.marsilio@tdcolive.it
Contract/grant sponsor: Ministero delle Politiche Agricole e Forestali (MiPAF)
(Received 10 June 2005; revised version received 8 August 2005; accepted 24 November 2005)
Published online 6 March 2006
 2006 Society of Chemical Industry. J Sci Food Agric 0022–5142/2006/$30.00 1005
V Marsilio et al.
a not irrigated control.6,7 Better fermentation rates
and higher bacterial growth were also found during
olive processing, probably as a consequence of a lower
phenolic content. However, the effects of the irrigation
regime on the physicochemical and microbiological
characteristics of olives for direct human consumption
have not been fully ascertained. Fermented table
olives show very good safety, generally related to the
growth of lactic acid bacteria resistant to invasion by
spoilage, toxic or food-poisoning microorganisms.16
However, little is still known about the influence of
agronomic practices on the fermentation and evolution
of phenolic compounds. The objective of this work
was to study the phenolic content and the evolution
of the individual phenolic compounds in olive fruits of
Ascolana tenera under different irrigation strategies.
In addition, the effect of using a selected lactic
acid bacteria starter culture on the fermentation
and organoleptic characteristics of fruits was also
investigated.
MATERIALS AND METHODS
Experimental plot
The trial was carried out in 2003 in an 11-year-
old olive (Olea europaea L. cv. Ascolana tenera)
orchard, planted at the CNR Institute for Agricultural
Mediterranean Systems experimental station (14◦ 43
E, 41◦ 06 N; elevation 250 m). Trees were trained
using the monocone system17 with a planting density
of 6 × 3 m.
Irrigation strategies consisted in a rainfed control
(T0 ), two treatments receiving a water depth equiva-
lent to 33 and 66% (T33p and T66p , respectively) of the
estimated ETc starting from the pit hardening stage
(4 August) and a treatment watered with a volume of
66% (T66w ) of the ETc during the entire season, from
25 May to 4 October. Irrigation water was delivered
daily at 4 L h−1 per tree using a system with four drip
nozzles (two per side), set in a line along the rows at
a distance of 0.50 and 1.00 m from the trunk. The
seasonal irrigation volume was 48, 95 and 198 mm for
T33p , T66p and T66w , respectively, and the total useful
volume of rain (>5 mm in a 24 h period) was 68 mm.
Plant growth was assessed by measuring plant
height, canopy volume and pruned wood. Fresh yield
was analysed by determining the fruit weight and the
fruit number per tree. On a sample of 50 fruits, the
mean fruit weight and volume, pulp/stone ratio and
polar and equatorial diameters of the whole fruit were
also determined.
Soil
The soil texture of the site was a sandy loam with a
water content of 36% (by volume) at the field capacity
(−0.03 MPa) and of 21% at wilting point (−1.5 MPa).
The pH was 7.2 and the organic matter and CaCO3
contents were 1.76 and 1%, respectively.
The ETc was estimated according to the class ‘A’
pan evaporation18 with readings adjusted with a pan
1006
coefficient (kp ) of 0.8, a crop coefficient (kc ) of 0.6
and a green ground covered coefficient (kr ) of 0.85,
as suggested by Fereres et al.19 Soil water content
was estimated at 15 day intervals during the irrigation
season, using TDR equipment and self-constructed
probes20 installed near three representative plants.
Three profiles down to a depth of 1.4 m were
monitored at distances of 0.75, 1.25 and 1.75 m from
the tree trunk.
Climatic conditions
The environment is characterised by good precipita-
tion in spring (40–50 mm per month), scarce or no
rainfall from mid-June to mid-August and frequent
rains thereafter. The daily mean temperature gener-
ally increases from 10 to 12 ◦ C in April to 24–25 ◦ C at
the end of July, decreasing to 18–20 ◦ C in September.
The yearly mean pan evaporation is about 1250 mm
and values increase from about 3 mm day−1 in April
to about 8 mm day−1 in July, starting to decrease from
the beginning of August. In 2003, the evapotranspi-
ration was higher than the polyannual mean in June
and temperatures showed peaks in June and August
(Fig. 1).
Processing
Olive fruits from T0 , T66p and T66w were hand-
harvested at their mature-green stage of ripening
(in mid-October) and immediately transported to
the CRA – Istituto Sperimentale per la Elaiotecnica
(CRA-ISEL) in Pescara, Italy, to be processed as
table olives according to the Greek method, a simple
natural process that does not use chemicals.21 The
olives from treatment T66w were divided into two
lots and then directly brined in a 5% (w/v) NaCl
solution, the same concentration as used for T0 and
T66p . The NaCl content was kept constant during the
fermentation by periodic addition of dry salt. After
1 week of brining, one lot (T66w−i ) was inoculated
with a starter culture of Lactobacillus plantarum B1-
2001 (our collection), whereas the other (T66w ) was
40 18
2003
'83-'03
Air temperature (°C)
30 14
E0 A (mm d-1)
20 10
10 6
0 2
May June July Aug. Sep. Oct.
Figure 1. Maximum and minimum (bars) air temperature (10-day
mean) and evaporation (10-day mean) in 2003 compared with the
period 1983–2003.
J Sci Food Agric 86:1005–1013 (2006)

left to ferment spontaneously in addition to T0 and
T66p .
Chemical analyses
Determination of pH, total titratable acidity and sugars
The initial pH and its changes throughout the
fermentation time were followed by the use of a pH
electrode (NeoMet) connected to a digital readout
unit (NeoMet, Model 79P) (Istek, Seoul, Korea) The
electrode was dipped well inside the fermenting brine,
leaving some space above the base of the beaker.
Total titratable acidity (TTA) was determined by
titrating up to pH 8.3 with 0.1 mol L−1 NaOH and
expressed as percentage of lactic acid.
Sugars were quantified using Fehling’s reagent
according to the standard method.22
Phenol analysis
The total phenolics content and the phenolic
composition were determined according to the
procedures described previously for olives.23 Briefly,
an aliquot (0.5 mL) of olive pulp methanolic extract
introduced into a test-tube was mixed with 1 mL
of 0.5 mol L−1 Folin–Ciocalteu reagent and 3 mL
of 200 g L−1 Na2 CO3 solution. The volume was
made up to 10 mL with distilled water. The solution
was stirred and allowed to stand for 30 min, then
centrifuged for 10 min at 3500 × g. The absorbance
of the supernatant was measured at 725 nm and data
were expressed as mg caffeic acid equivalent kg−1
fresh weight. Phenolic composition was determined by
gas chromatography (GC)–flame ionisation detection
(FID) and GC–mass spectrometric (MS) analyses.
Fruits, destoned and immediately frozen in liquid
nitrogen, were triturated in a blender. Approximately
5 g of the powder were extracted four times with
aqueous ethanol (800 mL L−1 ) containing 5 g L−1
sodium metabisulfite. An ethanolic solution of
resorcinol (0.5 g L−1 ) was added as internal standard.
The combined supernatants were concentrated under
reduced pressure and washed with hexane. The
phenols were then extracted with ethyl acetate–water,
filtered over anhydrous sodium sulfate and evaporated
to dryness at 30 ◦ C. The dry residue was converted into
trimethylsilyl derivatives and analysed by GC–FID
and GC–MS. A Carlo Erba (Milan, Italy) Model 5160
gas chromatograph equipped with a flame ionisation
detector and an HP1 capillary column (Hewlett-
Packard, Palo Alto, CA, USA) (30 m × 0.32 mm i.d.,
0.1 µm film thickness) was used with hydrogen as
carrier gas. The column temperature was programmed
from 70 to 90 ◦ C at 20 ◦ C min−1 and from 90 to 300 ◦ C
at 4 ◦ C min−1 and held at 300 ◦ C for 40 min. The
sample (0.3 µl) was injected in the on-column mode.
A Hewlett-Packard Model 5890 gas chromatograph
interfaced to a Model 5970 mass-selective detector
was used for GC–MS analysis. The chromatographic
conditions were the same as described above. Electron
ionisation mass spectrometry (EI-MS) was performed
at 70 eV with helium as carrier gas. Compounds were
J Sci Food Agric 86:1005–1013 (2006)
Irrigation and biochemical changes in olive processing
identified by comparing both their retention times and
mass spectra with those of authentic compounds or
reference standards.
Oil extraction and fatty acid analysis
Oil was extracted using a Soxhlet apparatus after
drying 50 g of olive flesh at 100 ◦ C for 24 h. The fatty
acids of each oil sample were methylated according
to the official methods.24 The fatty acid methyl esters
(FAMEs) were analysed with a Carlo Erba 8560 Mega
2 series gas chromatograph, equipped with a flame
ionisation detector and a Supelco (Bellefonte, PA,
USA) fused-silica capillary column (60 m × 0.32 mm
i.d., 0.2 µm film thickness). The oven temperature was
programmed as follows: increased from 120 to 165 ◦ C
at 30 ◦ C min−1 , held at 165 ◦ C for 10 min−1 , increased
from 165 to 200 ◦ C at 5 ◦ C min−1 , held at 200 ◦ C for
10 min, increased from 200 to 220 ◦ C at 5 ◦ C min−1
and finally held at 220 ◦ C for 10 min. The detector
temperature was 260 ◦ C. Hydrogen was used as carrier
gas at a pressure of 50 kPa. FAMEs were identified
by comparison of their retention time with those of
commercial standards (Sigma, St Louis, MO, USA)
and quantified according to their percentage area.
Microbiological analysis
Inoculum preparation
Lactobacillus plantarum was grown on autoclaved
MRS broth (Oxoid, CM 0359 Basingstoke, UK)
and counted on MRS agar (Oxoid, CM 0361) by
a standard plate counting method. Cells from an
overnight culture were pelleted by centrifugation,
washed twice with sterile water, resuspended at a
concentration of ca 1010 colony-forming units (CFU)
mL−1 and used to inoculate the fermenting brine
at a ratio of 40 mL L−1 . A preliminary NaCl test
tolerance was carried out by using MRS broth with
NaCl concentrations up to 100 g L−1 .
Fermentation
Brine samples were taken at time intervals during the
fermentation and analysed for microbiological counts.
Lactic acid bacteria and yeasts were enumerated in
1.0 mL aliquots of the appropriate serial dilution
added to pour plates of MRS agar and malt extract agar
(Oxoid, CM 0059), respectively, incubating at 30 ◦ C
for 72 h. Coliforms were determined with the most
probable number technique using 2% Brilliant Green
Bile broth (Oxoid, CM 0031) and incubating at 37 ◦ C
for 24 h. Each dilution was performed in duplicate and
culture responses were expressed as CFU mL−1 . All
fermentations were carried out in duplicate at room
temperature.
Sensory evaluation
According to the methodology described previously,25
the organoleptic characteristics of the processed olives
were evaluated by a panel of eight members from
staff of CRA-ISEL, selected and specifically trained
in descriptive analysis of olive fruits and olive oil.
1007
V Marsilio et al.

The attributes assessed were colour, odour, acid/sour,
bitter and two descriptors encompassing the texture
(firmness and crispness). Each attribute was evaluated
on a non-structured line scale running from 0 (none)
to 10 (strong presence of the attribute). The panellists
had also to indicate their comments and their global
assessment. Each sample (olives and brine) was
served in a cupping-glass with a three-digit code.
The evaluations were performed in separated booths
under normal daylight at ambient temperature. The
panellists used water to clean the palate between
samples. Results were averaged prior to data analysis.
Data analysis
Data were subjected to analysis of variance and mean
separation was obtained using the least significant
difference (LSD) test. The relationships between
chemical and sensory properties were also explored.
The significant level was set to 5% (P < 0.05).
RESULTS AND DISCUSSION
Soil water reserve (Fig. 2) decreased in deficit
irrigation treatments (T0 , T33p and T66p ) reaching,
during the season, values near the wilting point at
the beginning of August. Soil humidity was partially
recovered after irrigation, reaching values of about
50% of available water in T66p at the end of the season,
whereas the treatment T33p was constantly lower. The
T66w readings never were below 50% of soil available
reserve, indicating that this treatment was not exposed
to high water stress. Irrigation had a positive effect on
the production per tree. Fresh and dry pulp of the fruit
and fruit volume increased with the irrigation, whereas
the pulp/stone ratio varied slightly (Table 1). Fruit
shape was not influenced by the irrigation regime. The
lower weight of olive fruits that received irrigation only
during the phase of cell expansion (T66p ) indicated
that a water stress during the cell division phase had a
negative effect on the final fruit size. This implies
that to obtain the highest yield of olive for table
consumption, an adequate amount of water has to be
supplied during the initial stages of fruit development.
Fresh yield enhanced linearly (R2 = 0.93) according
to the crop water availability (Fig. 3). The yield
behaviour was primarily due to the increase in fruit size
(R2 = 0.98), while the trend was less evident for the
mean fruit number per tree (R2 = 0.71). Evidently,
the degree of water stress during the flowering and
the fruit set phenological stages were not sufficient to
affect the latter parameter significantly. Similar results
were also observed in other olive cultivars.26
As far as the plant vegetative aspect is concerned,
T33p treatment had little or no effect on canopy volume
compared with the rainfed control, while differences
between T66p and T66w were not significant. The
weight of pruned wood increased with irrigation in a
similar pattern of canopy volume. Data indicated that
a seasonal water depth up to 66% of ETc should be

40 T0 T33p T66p T66w 10 Yield (kg pt -1) Mean fruit weight (g) Fruit per plant (No. *10 -2)
soil water content (m3 m−3)

35 F.C. y = 0.0134x + 4.7118

8 R2 = 0.98
30 Irrig.
6
25
4 y = 0.0044x + 1.673
W.P.
20 R2 = 0.71
BF BFs BPH H
15 2
y = 0.0061x + 0.6806
125 150 175 200 225 250 275 300 R2 = 0.93
DOY 0
50 100 150 200 250 300
Figure 2. Volumetric soil water content The main phenological stages seasonal water volume
are also reported. BF = beginning of flowering; BFs = beginning of
fruit-set; BPH = beginning of pit hardening; H = harvest; F.C. = field Figure 3. Yield per plant, mean fruit weight and mean number of
capacity; W.P. = wilting point; DOY = day of the year. fruits per plant.

Table 1. Main plant and fruit characteristics of cv. Ascolana tenera depending by irrigation treatments

Canopy Pruned wood Fresh pulp Dry pulp Moisture Vol.c

volume (m3 ) (kg per plant) weight (g) weight (g) (%) P/Sa φ pol/eqb (cm3 )

T0 8.79 1.57 4.44 1.17 73.66 4.24 1.21 5.58

T33p 9.57 2.57 5.32 1.31 75.46 4.63 1.19 6.30
T66p 10.65 3.26 5.65 1.32 76.52 4.58 1.23 6.70
T66w 11.21 3.63 6.85 1.57 77.09 4.87 1.19 8.75
LDS(0.05) 1.85 1.04 1.11 0.14 – nsd ns 1.12
a
P/S = pulp/stone ratio.
b
φ pol/eq = fruit polar to equatorial diameter ratio.
c
Vol. = fruit volume
d ns = not significant (P < 0.05).

1008 J Sci Food Agric 86:1005–1013 (2006)


8000
7000
6000
5000
mg kg-1
4000
3000
2000
1000
0 is <7%.
T0 T66p T66w
Figure 4. Evolution of total phenolics in olive pulp during the
fermentation; the coefficient of variation is <7%.
applied to obtain a significant vegetative response of
the olive tree in the area of the trial.
Chemical composition
The levels of total extractable phenolics from fresh
olive samples (Fig. 4) ranged from 7000 (T0 ) to 4350
(T66w ) mg kg−1 fresh pulp. Considering the relatively
large fluctuations in phenolic contents among differ-
ent olive cultivars and the seasonal variability within
the same cultivar, due to soil and climatic conditions,
such results are in reasonable agreement with other
values described in the literature.27 – 31 The accumula-
tion of phenolic compounds in unirrigated olive trees
could be related to complex metabolic mechanisms
which involve several factors including stomatal and
enzyme activities. Stresses to fruit tissue, in the form of
both water deficit and insect attack or cellular damage,
are known to result in activation of enzymes such as
phenylalanine ammonia lyase (PAL), which catalyses
the synthesis of phenolic compounds.32,33 Modulating
these biosynthetic pathways is likely to influence the
osmotic pressure in the fruit, facilitating the migration
of water from the peel to the pulp, so that wither-
ing phenomena are avoided. In addition, cross-links
between phenolic compounds and other plant cell
wall components increase, resulting in aggregation and
toughening of cell walls, which reduce the cell perme-
ability and water diffusion. Such mechanisms allow the
plant to utilise the best the available water resources.
The analysis of phenolic extracts by capillary GC
showed mainly significant amounts of secoiridoid phe-
nols, such as oleuropein, their hydrolytic derivatives
(oleuropein aglycones, oleoside 11-methyl ester and
hydroxytyrosol) and tyrosol. Vanillic acid and 3,4-
dihydroxyphenylglycol were also present at lower
concentrations. Quantitative data are presented in
Table 2. Differences observed between the total levels
of phenols as determined by GC and the total phenol
levels obtained by the Folin–Ciocalteu method are
presumably due to the principal differences between
the two methodologies since the latter is princi-
pally a measurement of reducing capacity of phenolic
J Sci Food Agric 86:1005–1013 (2006)
Irrigation and biochemical changes in olive processing
Table 2. Levels of biophenols and related compounds (mg kg−1 ) in
fresh fresh olive pulp in relation to different irrigation levelsa
15 days
30 days T0 T66p T66w
90 days
150 days
Tyrosol 460 70 52
210 days
Vanillic acid 9 10 9
Hydroxytyrosol 2210 350 385
3,4-Dihydroxyphenylglycol 10 20 tr
Oleuropein aglycones 80 30 28
Oleoside 11-methyl ester 20 tr tr
Oleuropein 1600 450 475
a Values are the means of duplicate analyses; the coefficient of variation
T66w-i
hydroxyl groups rather than an absolute quantita-
tive measurement of phenolic substances. Oleuropein
and its hydrolytic derivatives, aglycones and hydrox-
ytyrosol, were present at the highest concentrations,
accounting for 56% of total phenols in T0 and only for
19 and 20% in T66p and T66w , respectively. Such com-
pounds are known to be potent in vitro antioxidants
and capable of breaking peroxidative chain reactions,
so their accumulation contrasts with plant oxidative
phenomena due to water stress. Therefore, the pres-
ence of high levels of oleuropein and derivatives could
be a stress index for olive tree.34
The sugar content in fresh olive fruits increased
with irrigation and ranged from 2.74 (T0 ) to 3.22%
(T66w ), on fresh weight. Sugars form a major part of
the soluble solids of the olive tissue, contributing, with
the polyols, 20% or more to the dry weight.35 Such
compounds provide energy for metabolic changes
and act as a carbon source for microorganisms
for producing secondary metabolites responsible for
sensory characteristics of table olives. In addition,
they are an important component of the cell wall,
related to the textural properties of the fruit, and act
as precursors for olive oil biosynthesis.36
The irrigation did not cause significant variations in
oil accumulation in the fruit (from 11.2 to 10.8% in T0
and T66w , respectively) or fatty acid composition. Fatty
acids were myristic (14:0), palmitic (16:0), palmitoleic
(16:1), heptadecanoic (17:0), heptadecenoic (17:1),
stearic (18:0), oleic (18:1), linoleic (18:2), linolenic
(18:3), eicosanoic (20:0), eicosenoic (20:1), behenic
(22:0) and lignoceric (24:0) acids. The percentages
of saturated (myristic + palmitic + heptadecanoic +
stearic + eicosanoic + lignoceric), monounsaturated
(palmitoleic + heptadecenoic + oleic + eicosenoic)
and polyunsaturated (linoleic + linolenic) fatty acids
of lipid classes and the unsaturated/saturated,
monounsaturated/polyunsaturated and oleic/linoleic
acid ratios did not appear to be significantly influenced
by irrigation.
The squalene content decreased slightly with
irrigation, passing from 0.85% in T0 to 0.66% in T66w .
This compound has been shown to inhibit strongly the
activity of β-hydroxy-β-methylglutaryl-CoA reductase
(HMG-CoA reductase), a key enzyme in cholesterol
synthesis37 and chemically induced skin tumours.38
1009
V Marsilio et al.

Table 3. Levels of biophenols and related compounds (mg kg−1 ) in T66w and T66w−i olives during fermentationa

T66w T66w−i

Fresh 15b 30b 90b 150b 210b Fresh 15b 30b 90b 150b 210b

Tyrosol 52 63 79 91 111 130 52b 78 79 85 84 80

Vanillic acid 9 7 6 6 6 7 9 5 5 6 5 9
Hydroxytyrosol 385 513 617 746 825 848 385 489 592 695 844 842
3,4-Dihydroxyphenylglycol trc 24 21 21 10 25 tr 7 26 10 14 10
Oleuropein aglycones 28 157 152 97 35 13 28 156 118 65 22 8
Oleoside 11-methyl ester tr 18 23 10 tr tr tr 18 20 8 tr tr
Oleuropein 475 415 246 12 tr tr 475 7 6 tr tr tr
a Values are the means of duplicate analyses; the coefficient of variation is <7%.
b Days of fermentation.
c tr = trace.

Processing of olives resulted in a 35–40% loss of pH T0 pH T66p pH T66w pH T66w-i

total phenolics. The results indicate that they are TTA T0 TTA T66p TTA T66w TTA T66w-i
8.0 1.0
lost more during the first 3 months of fermentation 7.0
(Fig. 4). Of course, because there are complex 0.8
6.0
relationships between total phenolics and olive
5.0

TTA (g/L)
fermentation, the impact of processing conditions pH
0.6
on individual phenolics has been studied. The 4.0
evolution of individual phenolic compounds upon 3.0 0.4

processing of fully irrigated olive fruits (T66w ), 2.0

0.2
with and without lactic acid bacteria inoculants, is 1.0
reported in Table 3. The results showed a significant
0.0 0.0
decrease in oleuropein content during processing, 15 30 45 60 75 90 105 120 135 150 165 180 195 210
up to trace levels after 3 months of fermentation. days
The degradation rate of oleuropein was faster in the
presence of LAB starter culture, thereby explaining Figure 5. Evolution of pH and TTA during olive fermentation.

and confirming a significant role of such bacteria in

the olive debittering.39 Oleuropein aglycones were
in T0 , probably owing to the antibacterial activity
also consumed more rapidly in the presence of
of phenolic compounds largely accumulated in olive
LAB inoculants, whereas other phenolics showed a
fruit, and needed more than 5 months to reach values
similar trend during fermentation, with or without
around 4.0. In T66w , the pH decreased to 4.0 after
LAB inoculants. Tyrosol and hydroxytyrosol levels
2 months of fermentation and reached a final value of
increased constantly, whereas the oleoside 11-methyl
3.8 after 5 months. In the presence of LAB inoculants
ester and 3,4-dihydroxyphenylglycol contents changed
(T66w−i ), the pH decreased rapidly to 4.0 after 15 days
slightly during fermentation. Hydroxytyrosol was
of fermentation and then remained constantly below
quantitatively the main phenol compound detected
this value, reaching a final value of 3.6. In parallel with
in the end products and its concentration was around
the pH decrease, total acidity increased, reaching final
850 mg kg−1 . Such data are in accordance with results
values of 0.7% in T0 and T66p , 0.8% in T66w and 0.9%
in the literature.40,41
in T66w−i .
Sugars, as expected, declined significantly during
These findings reflect the differences in the
fermentation owing to the microbial metabolism. After
microbial composition of the fermenting brines,
30 days of fermentation, reductions of 69, 62 and
summarized in Fig. 6. The growth of coliforms was
72% of the initial sugar concentrations were found
absent or low in all the brines. The number of
in unirrigated (T0 ), partially irrigated (T66p ) and fully
yeasts was similar up to 90 days of fermentation in
irrigated olives (T66w ), respectively, whereas in T66w−i
T0 and T66p ; thereafter their number was higher in
a consumption of 92% was detected due to the effect
T0 where the microbiological profile was substantially
of LAB inoculants, which give rise to higher acidity,
characterised by an equilibrium between yeasts and
so inhibiting the potential spoilage organisms.
lactic acid bacteria. These latter reached the stationary
phase after 15 and 30 days of fermentation in the
Fermentation inoculated (T66w−i ) and uninoculated olives (T66w ),
The pH of all the fermented brines was measured respectively. In T66w−i , lactic acid bacteria dominated
at time intervals and the results are plotted versus the whole fermentation process, which was shifted
time in Fig. 5. The pH decreased to around 4.0 in towards the production of a higher acidity, so
T0 and T66p and to 3.8 and 3.6 in T66w and T66w−i , improving the stability of the end product. A tendency
respectively. The lowering of pH occurred very slowly for lower yeast counts was found in the inoculated

1010 J Sci Food Agric 86:1005–1013 (2006)

 Irrigation and biochemical changes in olive processing

T0 T66p
12 12

10 10
yeasts LAB
log CFU ml−1

log CFU ml−1

8 8

6 6

4 4

2 2

0 0
0 50 100 150 200 250 0 50 100 150 200 250
fermentation (days) fermentation (days)

T66w T66w-i
12 12

10 10

log CFU ml−1

log CFU ml−1

8 8

6 6

4 4

2 2

0 0
0 50 100 150 200 250 0 50 100 150 200 250
fermentation (days) fermentation (days)

Figure 6. Evolution of microbial population in the fermenting brines.

Table 4. Average marks scored by the tasters for the sensory evaluation of the olives. Values are means of eight independent values ± standard
deviation

Colour Odour Acid Bitter Firm Crisp Overall

T0 3.8 ± 1.1 3.5 ± 0.7 7.9 ± 1.8 8.6 ± 0.4 7.4 ± 1.3 7.6 ± 0.9 3.3 ± 1.0
T66p 4.1 ± 1.1 3.8 ± 1.4 6.4 ± 1.3 6.3 ± 0.6 6.5 ± 1.4 6.8 ± 1.1 5.0 ± 1.1
T66w 3.8 ± 1.1 3.9 ± 1.0 5.1 ± 1.2 5.4 ± 1.2 6.3 ± 1.2 7.4 ± 0.7 5.2 ± 0.7
T66w−i 3.7 ± 0.5 5.2 ± 1.0 6.8 ± 1.1 4.2 ± 1.1 5.4 ± 0.7 7.1 ± 0.7 6.5 ± 0.4
LSD0.05 nsa 0.75 0.68 0.85 0.64 0.57 0.83
a ns = not significant (P < 0.05).

brine, probably because of the predominance of olives were characterized by a high odour intensity, a
lactobacilli and a lower pH. less bitter taste and good textural attributes, whereas
Overall the data suggested that both irrigation for those uninoculated a significant decrease in the
treatments and inoculation with LAB starter culture odour intensity and a higher bitter taste were observed
affected the fermentation, enhancing the acidification (Fig. 7). Overall, the olives inoculated with LAB
rate and lowering the pH, so avoiding the risk of the inoculants were better appreciated by the tasters as
appearance of ‘gas pocket’ spoilage. their scores were significantly higher than those for
uninoculated olives.
Sensory analysis The bitterness of olive fruits decreased with the
The sensory evaluation of the olives was carried out irrigation regime (T0 > T66p > T66w ), according to
after 7 months of fermentation. Panellists assessed the bitter compound levels. A positive relationship
each attribute indicating also which was acceptable or was found between phenolic content and bitter taste
not, apart from the overall acceptability. Results of the (R2 = 0.94). The latter was negatively correlated with
organoleptic test are presented in Table 4. For all the the overall acceptability (R2 = 0.97).
samples, there were significant differences among the
indicators. T0 olives were perceived to be less odorous
and too bitter, sour and firm and therefore were CONCLUSIONS
considered inedible and received the lowest scores. This study confirmed the importance of irrigation for
Olives from T66p and T66w were judged scarcely table olive production. An irrigation level of 66% of
acceptable for direct consumption. The inoculated ETc during the whole season gave the best results

J Sci Food Agric 86:1005–1013 (2006) 1011

V Marsilio et al.
Colour
10,0
8,0
6,0
Crisp Odour
4,0
2,0 T66w
0,0
T66w-i
Firm Acid
Bitter
Figure 7. Sensory profiles of olives processed with and without LAB
inoculants. Values are the means of eight independent values.
in terms of total fresh yield and fruit size, whereas a
water stress during the phase of fruit cell expansion
negatively affected the final fruit size. The data clearly
indicated that a water deficit increased the phenolic
content in olive fruit, whereas the oil content and fatty
acids composition remained substantially unchanged.
During natural olive processing, a loss of total phe-
nolics occurred in the first 3 months of fermentation.
Hydroxytyrosol was the main phenolic compound
detected in the end product.
The use of selected lactic acid bacteria inoculants
accelerated the fermentation and the acidification
rate, positively influencing the flavour of the end
product. Sensorial acceptability was higher in olives
fermented with LAB culture, mainly owing to the
lower bitter taste of the commodity. Therefore, such
microorganisms play a positive role in table olive
processing and enhance the quality of the end product.
ACKNOWLEDGEMENT
This work was supported by funds from the
Italian MiPAF (Ministero delle Politiche Agricole e
Forestali), project ‘Collezione di microrganismi di
interesse agrario ed agroindustriale’.
REFERENCES
1 Goldhamer D, Regulated deficit irrigation for California canning
olives. Acta Hort 474:369–372 (1999).
2 Alegre S, Efecto de diferentes estrategies de riego deficitario
controlado durante la epoca estiva sobre la producion del
olivo (Olea europaea L.) cv Arbequina. PhD Thesis, University
of Leida (2001).
3 Rapoport HF, Costagli G and Gucci R, The effect of water
deficit during early fruit development on olive fruit
morphogenesis. J Am Soc Hort Sci 129:121–127 (2004).
4 Rallo L and Rapoport HF, Early growth and development of
the olive fruit mesocarp. J Hort Sci Biotechnol 76:408–412
(2001).
5 Patumi M, d’Andria R, Fontanazza G, Morelli G, Giorio P and
Sorrentino G, Yield and oil qualità of intensively trained trees
of three cultivars of olive (Olea europaea L) under different
irrigation regimes. J Hort Sci Biotechnol 74:729–737 (1999).
6 Patumi R, d’Andria R, Marsilio V, Fontanazza G, Morelli G
and Lanza B, Olive and olive oil quality after intensive
1012
monocone olive growing (Olea europaea L. cv Kalamata) in
different irrigation regimes. Food Chem 77:27–34 (2002).
7 d’Andria R, Marsilio V, Lavini A, Morelli G, Fragnito F, Ian-
nucci E, Russi F and Lanza B, Yield and quality of green table
olive of cv. Nocellara del Belice in relation to irrigation level.
Proc Giornate Sci SOI, Naples, 4–6 May 314–316 (2004).
8 Brenes M, Garcia P and Garrido A, Phenolic compounds
related to the black colour formed during the processing
of ripe olives. J Agric Food Chem 40:1192–1196 (1992).
9 Marsilio V, Campestre C and Lanza B, Phenolic compounds
change during california-style ripe olive processing. Food Chem
74:55–60 (2001).
10 Garrido AF, Fernandez Diez MJ and Adams MR, Table Olives.
Production and Processing. Chapman and Hall, London (1997).
11 Fogliano V, Ritieni A, Monti SM, Gallo M, Della Medaglia D,
Ambrosini ML and Sacchi R, Antioxidant activity of virgin
olive oil phenolic compounds in a micellar system. J Sci Food
Agric 79:1803–1808 (1999).
12 Visioli F and Galli C, Olive oil phenols and their potential effects
on human health. J Agric Food Chem 46:4292–4296 (1998).
13 Visioli F and Galli C, Biological properties of olive oil
phytochemicals. Crit Rev Food Sci Nutr 42:209–221 (2002).
14 Visioli F, Galli C, Galli G and Caruso D, Biological activities
and metabolic fate of olive oil phenols. Eur J Lipid Sci Technol
104:677–684 (2002).
15 Sajia A, Trombetta D, Tomaino A, Lo Cascio R, Trinci P,
Uccella N, Bonina F and Castelli F, In vitro evaluation of
the antioxidant activity and biomembrane interaction of the
plant phenols oleuropein and hydroxytyrosol. Int J Pharmacol
166:123–133 (1998).
16 Steinkraus KH, Classification of Fermented Foods. World Health
Organization, FAO, Rome (1995).
17 Fontanazza G, Olivicoltura Intensiva Meccanizzata. Edagricole,
Bologna (1993).
18 Doorenbos J and Pruitt WO, Crop Water Requirements. Irrigation
and Drainage. Paper No. 24. FAO, Rome (1977).
19 Fereres E, Pruitt WO, Beutel JA, Henderson DW, Holzapfel E,
Shulbach H and Uriu K, ET and drip irrigation scheduling,
in Drip Irrigation Management, ed. by Fereres E, University of
California, Div. of Agric. Sci. n. 21 259, pp. 8–13 (1981).
20 Clothier BE and Green SR, Root zone processes and the
efficiency use of irrigation water. Agric Water Manage. 25:1–12
(1994).
21 Marsilio V, Table olive production, processing and standard in
Italy. Olivae 49:6–16 (1993).
22 Ministero dell’Agricoltura e delle Foreste, Metodi Ufficiali di
Analisi per i Mosti, i Vini e gli Aceti. Istituto Poligrafico dello
Stato, Rome (1958).
23 Marsilio V, Seghetti L, Iannucci E, Russi F, Lanza B and
Felicioni M, Use of a lactic acid bacteria starter culture during
geen olive (Olea europaea L cv Ascolana tenera) processing. J
Sci Food Agric 85:1084–1090 (2005).
24 European Commission, Commission Regulation 2568/91/EEC on
the Characteristics of Olive Oil and Olive Residue Oil and on the
Relevant Methods of Analysis. G. U. CE L 248, 5 September
1991, and G.U. CE, L 295, 13 November (2003).
25 Marsilio V, Sensory analysis of table olives. Olivae 90:32–41
(2002).
26 d’Andria R, Lavini A, Morelli G, Patumi M, Terenziani F,
Calandrelli D and Fragnito F, Effects of water regime on five
pickling and double aptitude olive cultivars (Olea europaea L.)
J Hort Sci Biotechnol 79:18–24 (2004).
27 Esti M, Cinquanta L and La Notte E, Phenolic compounds in
different olive varieties. J Agric Food Chem 46:32–35 (1998).
28 Romani A, Mulinacci N, Pinelli P, Vincieri FF and Cimato A,
Polyphenolic content in five Tuscany cultivars of Olea europaea
L. J Agric Food Chem 47:964–967 (1999).
29 Servili M, Baldioli M, Selvaggini R, Miniati E, Macchioni A
and Montedoro GF, High-performance liquid chromatog-
raphy evolution of phenols in olive fruit, virgin olive oil,
vegetation water and pomace and 1D and 2D nuclear
magnetic resonance characterization. J Am Oil Chem Soc
76:873–882 (1999).
J Sci Food Agric 86:1005–1013 (2006)

30 Rivas G, Espin C and Wichers H, Oleuropein and related
compounds. J Sci Food Chem 80:1013–1023 (2000).
31 Bianco A, Muzzalupo I, Piperno A, Romeo G and Uccella N,
Bioactive derivatives of oleuropein from olive fruits. J Agric
Food Chem 47:3531–3534 (1999).
32 Kubota N, Phenolic content and L-phenylalanine ammo-
nia–lyase activity in peach fruit, in Modern Methods of Plant
Analysis. Vol 18. Fruit Analysis, ed. by HF Linkskens and
JF Jackson, Springer, Berlin, pp. 81–95 (1996).
33 Simmonds MSJ, Importance of flavonoids in insect-plant inter-
action: feeding and ovideposition. Phytochemistry 56:245–252
(2001).
34 Satta D, Nieddu G, Chessa I and Virdis F, Attività antiossidante
indotta dalla carenza idrica in olivo ed oleastro, Proc
Convegno Int Olivicoltura, Spoleto, Italy, 22–23 April (2002).
pp. 293–298.
35 Marsilio V, Campestre C, Lanza B and De Angelis M, Sugar
and polyol compositions of some European olive fruit varieties
(Olea europaea L.) suitable for table olive purposes. Food Chem
72:485–490 (2001).
J Sci Food Agric 86:1005–1013 (2006)
Irrigation and biochemical changes in olive processing
36 Donaire JP, Sanchez-Raja AJ, Lòpez-Gorge J and Recalde L,
Etudes physiologiques et biochimiques de l’olive. 1. Variation
de la concentration de divers metabolites pendant son cycle
evolutif. Agrochimica 21:311–321 (1977).
37 Bellosta S, Ferri N, Bernini F, Paoletti R and Corsini A, Non-
lipid-related effects of statins. Ann Med. 32:164–176 (2000).
38 van Dam RM, Huang Z, Giovannucci E, Rimm EB, Hunter DJ,
Colditz GA, Stampfer MJ and Willet WC, Diet and basal cell
carcinoma of the skin in a prospective cohort of men. Am J
Clin Nutr 71:135–141 (2000).
39 Panagou EZ, Tassou CC and Katsaboxakis CZ, Induced lactic
acid fermentation of untreated green olives of the Conservolea
cultivar by Lactobacillus pentosus. J Sci Food Agric 83:667–674
(2003).
40 Campestre C, Marsilio V, Lanza B, Iezzi C and Bianchi G,
Phenolic compounds and organic acid changes in black
oxidized table olives. Acta Hort 586:575–578 (2002).
41 Romero C, Brenes M, Garcia P, Garcia A and Garrido A,
Polyphenol changes during fermentation of naturally black
olives. J Agric Food Chem 52:1973–1979 (2004).
1013