Postprint of LWT – Food Science and Technology, Volume 78, May 2017, Pages

165-171. DOI: 10.1016/j.lwt.2016.12.040

Oleuropein Hydrolysis by Lactic Acid Bacteria in Natural Green Olives

Authors: Eva Ramírez, Manuel Brenes, Antonio de Castro, Concepción Romero and

Eduardo Medina*

Food Biotechnology Department. Instituto de la Grasa (IG-CSIC). Campus

Universitario Pablo de Olavide, Building 46. Ctra. Utrera, km 1. 41013 Seville (Spain).

*Correspondence to: Tel: +34 954611550. Fax: +34 954616790. E-mail address:

emedina@ig.csic.es (E. Medina).

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Abstract

The presence of phenolic compounds in raw olives, particularly the bitter glucoside

oleuropein, requires transforming this substance into other non-bitter to make the fruit

palatable. An alkali treatment is currently carried out to hydrolyze the oleuropein but a

high volume of wastewater is generated. The aim of this study was to develop a natural

product without an alkali treatment and with appropriate organoleptic characteristics

similar to those of Spanish-style green olives. Mild heat treatments (60 °C for 10

minutes) were sufficient to inactivate the β-glucosidase activity which prevented the

formation of antimicrobial compounds and thereby the growth of lactic acid bacteria

was promoted. By contrast, heating released a high concentration of oleuropein in fruits

that remained at a very high level even after 6 months of fermentation. The inoculation

of the brines of Manzanilla olives with selected lactic acid bacteria with

oleuropeinolytic activity was insufficient for reducing the high concentration of the

bitter glucoside. However, favorable results were obtained with varieties such as Gordal

and Aloreña, which have lower oleuropein contents. The product obtained showed a

very attractive color, similar to that of Spanish-style green olives and lighter than

natural green olives, which is a positive aspect for consumer acceptability.

Keywords: Table olives; phenolic; oleuropein; hydrolysis; lactic acid bacteria.

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1. Introduction

Table olives have been a component of the Mediterranean diet for centuries and their

consumption is increasing worldwide because of their nutritional and palatable

characteristics. Among the different types of commercial table olives, Spanish-style

green olives are the most popular. The presence of phenolic compounds in the raw fruit,

particularly the bitter glucoside oleuropein, requires a step to transform this compound

into other non-bitter. In alkaline conditions, the ester bond of the oleuropein is broken

with the consequent formation of hydroxytyrosol and elenolic acid glucoside, both non-

bitter compounds (Brenes, & de Castro, 1998). Sodium hydroxide treatment allows for

the sweetening of Spanish-style green olives in a short period of time and favors a

product with a characteristic color, flavor and aroma due to spontaneous fermentation in

brines. Precisely, lactic acid fermentation is a critical step in the process of Spanish-

style green olives because it is responsible for the organoleptic properties of fruits and

guarantees their preservation.

On the other hand, natural green olives, which are not treated with alkali, lose their

bitterness slowly for months or even a year. The main microbiota in the brine of these

olives is formed by yeasts and sometimes lactic acid bacteria when conditions are

favorable (Garrido-Fernández, Fernandez-Díaz, & Adams, 1997). Some of these

microorganisms possess β-glucosidase and esterase activity in their metabolism and

many researchers have studied the possibility of using them to hydrolyze the oleuropein

molecule and thus to accelerate the sweetening of the fruits. Ciafardini, Marsilio, Lanza

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and Pozzi (1994) inoculated model solutions rich in oleuropein with 3 different strains

of Lactobacillus plantarum isolated from natural black olives. These strains possessed

β-glucosidase activity and hydrolyzed the phenolic glucoside in vitro. Similar tests were

carried out later and confirmed that some Lactobacillus plantarum strains were able to

hydrolyze oleuropein through the action of β-glucosidase forming its aglycone, and

subsequently esterase activity released the final products of hydrolysis, elenolic acid and

hydroxytyrosol (Marsilio, Lanza, & Pozzi, 1996; Ghabbour et al., 2011; Zago et al.,

2013; Tofalo et al., 2014.). Some of these strains were able to hydrolyze 90% of the

oleuropein in the model solution, obtaining hydroxytyrosol as the final hydrolysis

product, while other researchers only detected the formation of an intermediate

metabolite, oleuropein aglycone (Santos, Piccirillo, Castro, Kalogerakis, & Pintado,

2012). Enzymatic transformations of oleuropein have also been studied in vitro by

different yeast strains isolated from natural green olives (Bautista-Gallego et al., 2011;

Restuccia et al., 2011; Tofalo, Perpetuini, Schirone, Suzzi, & Corsetti, 2013) and black

olives (Bonatsou, Benítez, Rodríguez-Gómez, Panagou, & Arroyo-López, 2015), with

Wicheramomyces anomalus being the microorganism with the highest activity.

However, there are few studies on a pilot plant scale. Servili et al. (2006) selected 5

strains isolated from Italian natural black olive brines and found that a strain of

Lactobacillus pentosus (1MO) was able to sweeten olives in only 8 days. Kaltsa,

Papaliaga, Papaioannou and Kotzekidou (2015) isolated five strains of Lactobacillus

plantarum from natural green and black olives and found that Lactobacillus LP15 had a

high β-glucosidase activity, whereas Lactobacillus LP20 had esterase activity in low

salt fermentations. Nevertheless, no industrial application of these starters is known.

The production and consumption of organic and/or natural foods has increased in recent

years, but not those of ecological and/or natural olives. There are several reasons for this

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phenomenon and among them is the need to remove the bitterness of the olives without

the use of a NaOH solution and to provide a desirable product for consumers. The aim

of this work was to develop a new natural green olive process to obtain a final product

with appropriate organoleptic characteristics (color, firmness and flavor), and if

possible, similar to those of the type of table olives universally accepted, such as

Spanish-style green olives.

2. Material and Methods

2.1. Raw material

Fruits of the Manzanilla, Hojiblanca, Gordal and Aloreña cultivars (Olea europaea L.)

in the ripening stage corresponding to the green–yellow color on the surface were

supplied by local farmers.

2.2. Effect of temperature on the β-glucosidase activity and phenolic compound

concentration in olives

The olive fruits (Manzanilla cv.) were dipped in a water bath at different temperatures

(50, 60 and 70 ºC) for 3, 5, 10, 15 and 40 minutes. Then, the fruits were rapidly chilled

in cool water. Ten grams of olive pulp were put in 10 mL of distilled water and

homogenized in an Ultra-turrax (IKA-T25, S25N-18G). The mixture was centrifuged at

12000 × g for 5 min at 4 ºC. The aqueous phase was filtered through a 0.22 µm pore

size nylon filter and phenolic compounds were analyzed as described below.

Simultaneously, raw olives (Manzanilla and Hojiblanca cv.) were treated at the

following heating conditions before brining: i) No heat treatment (control); ii) 60 ºC for

10 min; iii) 70 ºC for 5 min; and iv) 80 ºC for 5 min. After treatments, the olive fruits

were placed in containers of 3 L capacity with 1.8 kg of fruit and 1.2 L of brine (50 g/L

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NaCl and 5 g/L of acetic acid). After 24 hour of brining, the vessels were inoculated

with a cocktail of 4 selected strains of lactic acid bacteria (Lactobacillus brevis Z17BP,

Lactobacillus pentosus ATCC 8041, Lactobacillus plantarum ATCC 14917 and

Leuconostoc mesenteroides LM51) to reach an initial population of 5 x 107 CFU/mL.

These strains were chosen and tested as described in sections 2.3 and 2.4. The vessels

were left at ambient temperature for six months and were periodically analyzed to know

their microbiological and chemical evolution.

In another experiment, Manzanilla and Hojiblanca olives from two different seasons

(2010/2011 and 2011/2012) were heat-treated at 60 ºC for 15 minutes. Subsequently,

the oleuropein concentration in the pulp of the fruit was analyzed as described below.

2.3. Strain sources and maintenance

A total of 105 lactic acid bacteria (LAB) strains from different origins were used in this

work. Some strains were purchased from the American Type Culture Collection (ATCC

strains) and the Spanish Type Culture Collection (CECT strains), although most of them

were isolated by the authors from olive brines belonging to diverse cultivars, locations

and manufacturing methods. All the strains were routinely cultured in MRS broth

(Oxoid, Basingstoke, UK) under anaerobic conditions (AnaeroGen, Oxoid), and kept

frozen at -80 ºC in broth with glycerol (200 g/L). Catalase-negative bacteria that were

able to grow on MRS agar with sodium azide (0.2 g/L) were considered LAB.

2.4. Depletion of oleuropein by the different strains in synthetic broth and brine model

solution

A minimum medium mimicking the conditions prevailing in the brines of natural olives

was formulated in order to favor the utilization of oleuropein by the isolates. The

composition was: glucose (Panreac, Barcelona, Spain) 1 g/L; neutralized bacteriological

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peptone (Oxoid) 10 g/L; yeast extract (Oxoid) 4 g/L; sodium chloride (Panreac) 50 g/L;

and oleuropein (Sigma, Aldrich, St. Louis, MO) 5 g/L in an acetic-acetate buffer pH 4.

The medium was sterilized by filtration and 250 μL were dispensed into sterilized vials.

Cultures of the strains were carried out in MRS broth, firstly without NaCl, and a

second culture in MRS with 50 g/L of NaCl to adapt the isolates. After overnight

incubation at 30 ºC, 1 mL of each culture was centrifuged. The pelletized cells were

washed with 1 ml of saline solution (9 g NaCl/L) and centrifuged again. Finally the

pellets were re-suspended with 0.5 mL of saline solution and 10 μL of suspension were

used to inoculate the vials. With this method, the expected initial inocula were between

107-108 cfu/mL. The duplicate vials for each strain were incubated at 30 ºC in anaerobic

conditions for 10 days and then analyzed to know the remaining concentration of

oleuropein.

In another experiment, brine from heat-treated olives aseptically stored for two months

containing a concentration of 11.36 mM oleuropein was used as a brine model. The

brine was sterilized by filtration and distributed in 250 µl volumes into sterile vials.

Inoculation with the selected strains, incubation and analysis were performed as

described above for the minimum medium. Uninoculated brines were used as control.

2.5. Depletion of oleuropein by selected strains during olive fermentations

Twelve fermentation vessels were filled with raw olives (Manzanilla cv.) and covered

with acidulated brine (100 g NaCl /L and acetic acid 3 g/L). Except for control A, the

rest of the fruits suffered a heat treatment at 60 ºC for 15 minutes with the aim of

inactivating the endogenous enzymatic activity. The specific treatments were: no heat

treatment and no inoculation (Control A); heat-treated but no inoculation (Control B);

inoculated with Lactobacillus plantarum CECT 748 (treatment C); inoculated with

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Lactobacillus pentosus LAB 80 (treatment D); inoculated with L. pentosus LAB 64

(treatment E); and inoculated with L. plantarum LAB 46 (treatment F). LAB 80, LAB

64, and LAB 46 were classified according to Torriani, Felis & Dellaglio (2001).

Inoculation was carried out at day 7 and the experiment was performed in duplicate.

Cultures of the different strains in MRS broth plus 50 g NaCl/L were incubated at 30 ºC

for 24 h, then centrifuged and the pellets resuspended in saline for washing the cells,

next they were centrifuged again, and finally the pellets were re-suspended with brine

from the corresponding vessels to be inoculated, and injected into the approximate

center of each one. The vessels were left at ambient temperature and periodically

analyzed to know their microbiological and chemical evolution. After 6 months of

fermentation, color, firmness and chemical analyses were performed. The olives were

pitted and washed twice with water for 24 hours prior to packaging with new brine (85

g NaCl/L and 10 g/L of acetic acid). After 1 month of equilibration, the olives were

analyzed by six table olives judges, which are members of the Table Olive Sensory

Panel of the Instituto de la Grasa with over 25 years of experience on this field. The

tests were carried out in a standardized testing room according to the “Method for

sensory analysis of table olives”, COI/OT/MO 1/Rev.2 No 1 (IOC, 2010), using the

profile sheet also included in this methodology. This method employs the descriptors

related to the perception of negative sensations (abnormal flavor and other defects),

gustatory attributes (salty, acidic, bitter) and kinesthetic sensations (hardness,

fibrousnesses, crunchiness), in order to commercially classify the olives.

In another experiment, raw olives of Manzanilla, Hojiblanca, Gordal and Aloreña

varieties were brined (NaCl 100 g/L and acetic acid 3 g/L) in fermentation vessels for 6

month as control. In parallel, a heat treatment (65 ºC for 15 minutes) was applied to all

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olive varieties and the vessels were inoculated with LAB 80 at day 7. The rest of the

analyses were similar to those described above.

2.6. Analysis of phenolic compounds in olive brine and pulp

The analysis of phenolic compounds in the brine and aqueous phase was carried out by

mixing 0.25 mL of brine, 0.25 mL of internal standard (2 mM syringic acid in water),

and 0.5 mL of deionized water. Phenolic compounds were extracted from the olive pulp

with dimethyl sulfoxide (DMSO) according to Kumral et al. (2013). 10 g of olive pulp

were put in 30 mL DMSO and homogenize in an Ultra-turrax (IKA-T25, S25N-18G).

After 30 min, the mixture was centrifuged at 10000 × g for 5 min, and 0.25 mL of the

supernatant was diluted with 0.5 mL of DMSO and 0.25 mL of 0.2 mM syringic acid in

DMSO (internal standard). Finally, all samples were filtered through a 0.22 µm pore

size nylon filter and an aliquot (20 µL) was injected into the chromatograph.

The chromatographic system consisted of a Waters 717 plus autosampler, a Waters 600

E pump, a Waters column heater module, and a Waters 996 photodiode array detector

operated with Empower 2.0 software (Waters Inc.). A Spherisorb ODS-2 column

(Waters Inc.), a flow rate of 1 mL/min and a temperature of 35 ºC were used in all

experiments. Separation was achieved by gradient elution using (A) water (pH 2.5

adjusted with 0.15% phosphoric acid) and (B) methanol. The initial compositions were

90% A and 10% B. The concentration of B was increased to 30% over 10 min and was

maintained for 20 min. Subsequently, B was raised to 40% over 10 min, maintained for

5 min, and then increased to 50%. Finally, B was increased to 60%, 70%, and 100% in

5-min periods. Initial conditions were reached in 10 min (García, Romero, & Brenes,

2014). Chromatograms were recorded at 280 nm. The evaluation of each compound was

performed using a regression curve with the corresponding standard. Standards were

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purchased from Sigma and Extrasynthese (Fenay, France) companies or isolated by

semi-preparative HPLC (Medina, Brenes, Romero, García, & de Castro, 2007). The

analyses were performed in duplicate.

2.7. Chemical parameters

The concentration of NaCl was analyzed by titration with a 0.1 N silver nitrate solution,

using a potassium chromate solution as indicator. The pH of the storage solutions was

measured in a Beckman model 45 pH-meter. Free acidity was measured by titration

using a Metrohm 670 Titroprocessor (Herisau, Switzerland) up to pH 8.3 with 0.2 M

NaOH and expressed as % (w/v) of lactic acid. The analyses were performed in

duplicate. Colorimetric measurements on olives were performed using a BYK-Gardner

Model 9000 Color-view spectrophotometer, equipped with computer software to

calculate the CIE L* (lightness), a* (redness) and b* (yellowness) parameters by

scanning the surface from 400 to 700 nm. The interference by stray light was minimized

by covering the samples with a box which had a matt black interior. The data of each

measurement were the average of 20 olives. Firmness was measured using a Kramer

shear compression cell coupled to an Instron Universal Testing Machine Model 1001

(Canton, USA). The crosshead speed was 200 mm/min. The firmness of the olives was

expressed as the mean of 10 measurements, each of which was performed on 3 pitted

olives and expressed as kN/100 g pitted olives.

2.8. Statistical analysis

Data were expressed as mean values and standard deviations (SD) were calculated.

Statistica software version 7.0 was used for data processing (Statistica for Windows,

Tulsa, OK, USA). Comparison among mean variables was made by the Duncan’s

multiple range tests and the differences considered significant when p < 0.05.

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3. Results and discussion

Recently, Ramírez, Medina, Brenes and Romero (2014) observed an important

influence of temperature on the optimal activity of β-glucosidase in model solutions.

The activity decreased drastically with heat treatments at temperatures higher than 50

ºC. In this work, it has been confirmed that heating Manzanilla olives at 50 °C,

regardless of the duration of the treatment, allowed for the formation of the dialdehydic

form of decarboxymethyl elenolic acid linked to hydroxytyrosol (HyEDA) (Figure 1).

Therefore, treatments at 50 °C, even for 40 minutes, were ineffective to inactivate the β-

glucosidase. A minimum temperature of 60 °C for 10 minutes or 70 ºC for 5 min were

required to inactivate the enzyme responsible for the formation of HyEDA. Previous

studies confirmed that higher temperature resulted in a decrease in enzyme activity

(Romero-Segura, Sanz, & Pérez, 2009; Kara, Sinan, & Turan, 2011). Also, Ramírez et

al. (2014) indicated that β-glucosidase activity is at its maximum at 30 °C and decreases

over 80% at 70 °C. The inactivation of β-glucosidase in model systems has previously

been reported (Romero-Segura et al., 2009) but not in raw olives.

Antimicrobial compounds like HyEDA are formed during the storage of natural green

olives in brine without alkaline treatment which inhibit the growth of lactic bacteria

(Medina et al., 2007). Hence, one of the aims of this work was to study the inactivation

of β-glucosidase thereby the growth of LAB could occur.

A new experiment with Manzanilla and Hojiblanca olives was carried out. The olives

were subjected to different heat treatments in which the inactivation of the enzyme β-

glucosidase was ensured (no HyEDA formation). Subsequently, they were put directly

in brine and preserved at room temperature for 6 months. The characteristics of the final

product after six months storage are given in Table 1. Control brines from unheated

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Manzanilla olives had higher pH and lower free acidity than olives subjected to heating,

which suggests that lactic acid fermentation took place in the latter brines but not in the

former. It must be noted that the brines were inoculated and acid formation did not

occur in the control brines. By contrast, the three heat treatments applied prevented the

formation of HyEDA and allowed for the growth of LAB. These data are in agreement

with previous results that showed a lower concentration of HyEDA in scalded olives

than in unheated fruits (Montedoro et al., 2002).

Regarding Hojiblanca olives, pH and free acidity were relatively similar in both heat-

treated and control olive brines (Table 1), which means that lactic acid fermentation

occurred in all cases. These results are in agreement with the low concentration of

HyEDA found in the control brine of the Hojiblanca olives in comparison with

Manzanilla olives (Fig. 2). According to previous results, the Hojiblanca variety has a

lower concentration of oleuropein and lower β-glucosidase activity than Manzanilla

olives (Ramírez et al., 2014). Medina et al. (2007) indicated that a concentration above

0.25 mM of HyEDA is needed to inhibit the growth of Lactobacillus pentosus in green

natural olives, and this level was not reached in the control brine of Hojiblanca olives of

the tested batch (Fig. 2).

In relation to the color, it was observed that the heat-treated Manzanilla olives had a

yellow color on their surface after 6 months of brining (Table 1), with no significant

differences among the three heat treatments applied. Conversely, the color of unheated

olives was brown. The values of the parameters L* and b* were lower and the

parameter a* higher in untreated olives than those thermally treated. With regard to the

Hojiblanca variety, the color parameters L* and b* showed no significant differences

between treatments, while the parameter a* had a significantly higher value for the

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control olives, which were more reddish. Therefore, this color enhancement occurred in

the two olive varieties, and was more significant for Manzanilla.

The disadvantage of exposing olives to a heat treatment is the possibility of altering

certain physical parameters such as firmness (Brenes, García, & Garrido, 1994),

resulting in a final product with low consumer acceptance. No significant differences

were observed in relation to the firmness of the Manzanilla olives subjected to

treatments of 60 °C for 10 minutes and 70 °C for 5 minutes with respect to untreated

fruits (Table 1). By contrast, a temperature of 80 °C for 5 minutes originated a

significant decrease in firmness. Moreover, all heat treatments caused a reduction in the

firmness of Hojiblanca olives, although in this case it can be a positive effect because

this variety is characterized by a very tough and fibrous firmness.

Surprisingly, both olive varieties showed a higher concentration of oleuropein in the

brines of heat-treated olives than in untreated ones (Table 1). No explanation has been

found, although this phenomenon could be produced due to the breakdown of cell

structures during heating that could allow the release of oleuropein, in particular from

dimers, trimers or oligomers containing this substance (Cardoso et al., 2006). This

surprising result was confirmed with olives of several varieties from two seasons (Table

2). In all cases, a heat treatment of the raw material gave rise to an increase in the

concentration of free oleuropein in the pulp of the fruit, with this effect being more

marked in olives of the 2010/2011 than in those of the 2011/2012 season. Apart from

the effect on oleuropein concentration as a consequence of the heat treatment, it is also

remarkable the differences between seasons in oleuropein concentration of fresh olives

without heat treatment (Table 2). This variability among seasons in polyphenol

composition is not uncommon and has been previously reported (Ramírez et al., 2014).

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As mentioned above, a soft heat treatment (60 ºC for 15 min) on raw olives inhibits the

formation of anti-lactic acid bacteria compounds (HyEDA) and gives rise to fruit with a

good yellow color. However, this treatment also produces a higher concentration of

oleuropein in brines and olives thereby maintain a strong bitterness in the final product.

Some researchers have isolated LAB with β-glucosidase activity (Bleve et al., 2015)

and capable to hydrolyze oleuropein (Servili et al., 2006). In this study, the isolation and

selection of 105 strains from table olive brines according to their oleuropeinolytic

capacity were carried out. More than half of the isolates did not have the ability to

hydrolyze oleuropein (Fig. 3), around 40 % of isolates hydrolyzed between 10 and 60 %

of the initial concentration, and only 5 strains were capable of hydrolyzing a high

percentage of oleuropein, between 60 and 90%. A strain of Lactobacilllus plantarum

from the CECT (748), and microorganisms isolated from table olive brines such as

Lactobacillus pentosus (LAB 64, LAB 80) and Lactobacilllus plantarum (LAB 46,

LAB 94), were selected according to their high oleuropeinolytic capacity. It has to be

said that, among the 105 isolates, the different strains belonging to the species L.

plantarum and L. pentosus showed diverse oleuropeinolytic capacity. Therefore, this

trait is strain-dependent. These species have previously been described as typical leader

microorganisms of the lactic fermentation of Spanish-style green olives (De Castro,

García, Romero, Brenes & Garrido, 2002).

The selected strains were then inoculated in model brines with high oleuropein

concentration obtained from olives subjected to a heat treatment. These brines did not

contain the antimicrobial compound HyEDA so that no inhibitory activity against LAB

could be expected. Surprisingly, the percentage of oleuropein hydrolyzed by the

selected LAB after 10 days was much lower than that found in the minimum medium.

The hydrolysis of oleuropein in model brines was only around 20% for strain CECT

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748 and 30% for LAB 64, LAB 80 and LAB 46. LAB 94 presented the lowest

hydrolysis capacity, which was around 13%. The model brines are likely to have

contained more nutrients than the minimum medium, and LAB could prefer these

nutrients instead of glucoside oleuropein. Furthermore, it is unknown whether the high

concentration of oleuropein in brines (11.36 mM) decreased the hydrolytic ability of

bacteria, although Medina et al. (2007) reported that an oleuropein concentration of 5.8

mM did not inhibit the growth of L. pentosus or L. plantarum.

The two strains of L. pentosus (LAB 64 and LAB 80), and the two strains of L.

plantarum (CECT 748 and LAB 46) with higher hydrolytic capacity in model brines

were also inoculated in pilot scale fermenters containing Manzanilla olives heated at 60

°C for 15 minutes. Except for the olives of control A (unheated), all heated olives

reached pH values of around 3.4 and 1.5% of free acidity as a consequence of lactic

fermentation (Table 3). The microbial growth of LAB was detected in all fermenters

after inoculation but not in control A. In control B (olives heated and uninoculated),

lactobacilli were detected from day 43; after this point spontaneous fermentation by

environmental bacteria took place. All heat-treated olives had a similar green-yellow

color which was better than the unheated control, which was brown. Regardless of the

heat treatment, firmness was similar in all cases. However, it must be noted that after 6

months of fermentation, selected strains hydrolyzed only 15-20% of total oleuropein in

the pulp and in the brine compared to control B (Table 3). Despite water-washing in

packaging, the panel test argued that the olives had an intense bitter taste and the

product was very different compared to common Spanish style green olives.

The later experiment was conducted with Manzanilla olives, which is a very bitter

variety. Further experiments were carried out with other less bitter varieties such as

Hojiblanca, Aloreña and Gordal. The strain L. pentosus LAB 80 was used as inoculums

15

because of its high capacity of oleuropein hydrolysis in the synthetic broth with

oleuropein. Besides, it was originally isolated from natural olives. As seen in Table 4,

all heat-treated olives fermented with a concomitant decrease in pH and increase in free

acidity. The color of control olives (unheated) of Manzanilla, Hojiblanca and Aloreña

varieties was darker than the corresponding heat-treated ones.

Again, the heat treatment caused an increase in the oleuropein concentration, especially

for the Manzanilla variety, which was less pronounced for Hojiblanca and Gordal

varieties, and at a minimum for the Aloreña variety (data not shown). However, when

the olives were pitted and washed, the Manzanilla variety was still bitter while the low

residual bitterness left in the Hojiblanca variety olives was not unpleasant to the

panelists, who detected no bitterness in Gordal or Aloreña. In contrast, the firmness of

the latter sweet varieties was very affected by the heat treatment (Table 4), which made

them unacceptable for consumers. Hence, less ripe olives or the use of calcium during

fermentation should be employed to get an acceptable product from these sweet olive

varieties.

4. Conclusion

The study of heat treatment in raw olives reveals that temperatures which are not too

high (60 °C for 10 minutes) are sufficient to inactivate β-glucosidase activity; prevent

the formation of antimicrobial HyEDA and promote the growth of lactic acid bacteria.

In addition, heated olives have a similar color to common Spanish-style green olives

and there is no significant loss in firmness. According to the results observed, heating

releases a high concentration of oleuropein in fruits which remains very high even after

6 months of fermentation, so the bitterness is very pronounced. The results indicate that

we must continue to search for strains of lactic acid bacteria with greater ability to

16

hydrolyze oleuropein, capable of metabolizing the high concentrations found in the

Manzanilla variety. However, favorable results were obtained for other varieties such as

Gordal and Aloreña, which have lower contents. This residual bitterness in the fruit

after the conservation process can be eliminated in the later stages of processing

(washing, pitting, packaging, etc.). The new product obtained shows a more attractive

color, similar to Spanish-style green olives and better than natural green olives, a

positive aspect to consider with regard to greater consumer acceptability.

5. Acknowledgement

This work was supported by Project AGL 2009-07512 from the Spanish government

and European Union (European Regional Development Funds). E.R. thanks MINECO

for her FPI fellowship.

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Figure caption

Figure 1. Concentration of the dialdehydic form of decarboxymethyl elenolic acid

linked to hydroxytyrosol (HyEDA) in the pulp of Manzanilla olives after several heat

treatments.

Figure 2. Concentration of the dialdehydic form of decarboxymethyl elenolic acid

linked to hydroxytyrosol (HyEDA) in the brine of Manzanilla and Hojiblanca olives

varieties without heat treatment (control) during storage in brine. Bars mean standard

deviation of duplicates.

21

Figure 3. Number of LAB isolates after 10 days of incubation according to the

percentage of oleuropein hydrolyzed in a minimum medium (with 0.5 % of oleuropein).

Value in the top of the bars indicates the number of LAB isolates with the same

behavior.

22

Table 1.

Chemical values of brines and physical characteristics in Manzanilla and Hojiblanca olives after 6 months of brining. The olive fruits were heat-treated before brining with different conditions.

Variety Heat Treatment

NaCl (%) pH

Free acidity

(%)

Oleuropein (mM)

HyEDA (mM)

Firmness (kN/100 g

pitted olives)

Color parameters

L* a* b*

Manzanilla

No heating 3.5 (0.2)

4.5 (0.0)

0.6 (0.1) 3.1 (0.9) 0.6 (0.2) 6.5 (0.5)a 46.7

(0.1)a6.5 (0.1)a 31.46 (2.0)a

60 °C/10min 3.4 (0.0)

3.7 (0.0)

1.4 (0.0) 11.8 (0.7) 0.0 (0.0) 6.1 (0.0)a 54.0

(0.9)b1.8 (0.1)b 37.7 (0.7)b

70 °C/5min 3.2 (0.0)

3.7 (0.0)

1.6 (0.0) 18.8 (1.4) 0.0 (0.0) 5.1 (0.0)a 54.2

(1.1)b1.6 (0.0)b 38.0 (0.8)b

80 °C/5min 3.4 (0.0)

3.7 (0.0)

1.7 (0.2) 9.4 (2.4) 0.0 (0.0) 3.9 (1.0)b 54.5

(1.0)b1.7 (0.1)b 37.8 (0.9)b

Hojiblanca

No heating 3.0 (0.0)

3.9 (0.0)

1.5 (0.0) 0.0 (0.0) 0.0 (0.0) 7.9 (0.5)a 51.8

(1.2)a5.0 (0.2)a 30.8 (0.3)a

60 °C/10min 3.0 (0.1)

3.6 (0.0)

1.9 (0.1) 1.9 (0.1) 0.0 (0.0) 4.5 (0.9)b 54.0

(0.3)a2.9 (0.2)b 32.3 (0.5)a

70 °C/5min 2.9 (0.0)

3.6 (0.1)

1.9 (0.0) 3.8 (1.4) 0.0 (0.0) 4.5 (0.9)b 51.2

(1.0)a2.3 (0.2)b,c 31.8 (1.4)a

80 °C/5min 3.1 (0.2)

3.7 (0.1)

1.7 (0.1) 3.0 (0.7) 0.0 (0.0) 4.3 (0.7)b 52.6

(1.0)a1.8 (0.3)c 32.9 (0.8)a

Standard deviation of duplicates in parentheses. Same letters within the same columns designate no statistically significant differences (p < 0.05) according to the Duncan's New Multiple Range Test. HyEDA means the dialdehydic form of decarboxymethyl elenolic acid linked to hydroxytyrosol.

23

Table 2.

Oleuropein concentration in the pulp of olives subjected to different heat conditions.

Season Variety

Heat conditionsOleuropein (mmol/kg

fruit)Temperature (°C) Time (min)

2010/11

Manzanilla 0 0 10.9 (0.5)60 15 55.3 (1.2)

Hojiblanca 0 0 8.3 (0.3)60 15 30.2 (0.2)

Gordal 0 0 1.7 (0.1)60 15 16.1 (0.9)

2011/12Manzanilla 0 0 68.2 (0.7)

90 30 77.4 (8.4)

Hojiblanca 0 0 24.9 (0.2)90 30 43.3 (2.3)

Standard deviation of duplicates in parentheses.

24

Table 3.

Chemical values of brines and physical characteristics in Manzanilla olives after 6 months of brining. Oleuropein hydrolyzed percentage is calculated from mass balance of pulp and brines in comparison with control B.

Treatment NaCl (%) pH

Free acidity

(%)

Firmness (kN/100 g

pitted olives)

Color parameters Oleuropein hydrolyzed

(%)L* a* b*

Control A 6.2 (0.1)

4.4 (0.1)

0.4 (0.1) 6.6 (0.5)a 47.9

(0.1)a7.3 (0.1)a

29.8 (0.9)a –

Control B 7.9 (0.0)

3.3 (0.2)

1.6 (0.0) 6.3 (0.3)a 54.5

(0.9)b1.9 (0.1)b

39.6 (1.1)b –

C 6.5 (0.8)

3.4 (0.0)

1.5 (0.1) 6.5 (0.4)a 56.5

(0.5)c1.9 (0.1)b

39.5 (0.4)b 16.4 (0.5)

D 7.1 (0.0)

3.5 (0.1)

1.5 (0.1) 6.7 (0.5)a 56.7

(0.1)c1.7 (0.1)b

39.7 (0.6)b 14.8 (0.5)

E 7.0 (0.1)

3.4 (0.2)

1.5 (0.0) 6.2 (0.7)a 56.1

(0.2)c1.8 (0.0)b

39.3 (1.1)b 17.2 (5.4)

F 7.1 (0.1)

3.4 (0.1)

1.5 (0.1) 6.5 (0.5)a 55.8

(1.0)c1.8 (0.2)b

38.9 (0.8)b 20.9 (7.6)

Standard deviation of duplicates in parentheses. Same letters within the same columns designate no statistically significant differences (p < 0.05) according to the Duncan's New Multiple Range Test. Treatments: no heat treatment and no inoculation (A); heat-treated but no inoculation (B); C F heat-treated and inoculated with, L. plantarum CECT 748 (C); L. pentosus LAB 80 (D); L. pentosus LAB 64 (E); L. plantarum LAB 46 (F).

25

Table 4.

Chemical values of brines and physical characteristics in Manzanilla, Hojiblanca, Gordal and Aloreña olives after 6 months of brining. The olive fruits were heat-treated before brining (60 °C for 15 min) and inoculated with strain LAB 80.

Variety Heat treatment

NaCl (%) pH

Free acidity

(%)

Firmness (kN/100 g

pitted olives)

Color parameters Oleuropein concentration in pulp (mM)L* a* b*

ManzanillaNo 6.2

(0.1)4.2 (0.1)

0.6 (0.1) 6.4 (0.7)a 49.5

(0.5)a7.5 (0.7)a

35.0 (0.8)a 4.6 (0.7)

Yes 8.6 (0.1)

3.4 (0.2)

1.7 (0.2) 4.9 (0.8)b 58.1

(0.9)b2.1 (0.1)b

44.3 (0.3)b 15.3 (0.4)

HojiblancaNo 5.1

(0.0)4.3 (0.0)

0.5 (0.0) 7.6 (0.6)a 49.4

(0.5)a3.6 (0.7)a

31.7 (0.6)a 0.0 (0.0)

Yes 6.2 (0.6)

3.4 (0.1)

1.3 (0.1) 6.4 (0.4)b 50.7

(1.0)a2.1 (0.1)a

36.0 (0.7)b 6.2 (0.1)

GordalNo 5.7

(0.1)3.4 (0.2)

1.1 (0.1) 4.4 (0.7)a 54.8

(1.4)a6.8 (0.3)a

41.2 (1.3)a 0.1 (0.0)

Yes 5.6 (0.0)

3.4 (0.1)

1.2 (0.2) 2.1 (0.3)b 53.6

(1.2)a3.9 (0.2)b

43.3 (2.2)a 2.9 (0.0)

AloreñaNo 6.0

(0.0)4.1 (0.0)

0.8 (0.0) 4.6 (0.8)a 57.0

(0.8)a9.4 (0.0)a

36.0 (2.1)a 0.0 (0.0)

Yes 6.3 (0.0)

3.6 (0.2)

1.4 (0.1) 2.3 (0.2)b 62.3

(0.5)b2.0 (0.0)b

39.5 (0.7)a 2.4 (0.5)

Standard deviation of duplicates in parentheses. Same letters within the same columns designate no statistically significant differences (p < 0.05) according to the Duncan's New Multiple Range Test.

26

Figure 1

27

Figure 2

28

Figure 3

29