Food and Nutrition Sciences, 2013, 4, 721-726
http://dx.doi.org/10.4236/fns.2013.47092 Published Online July 2013 (http://www.scirp.org/journal/fns)
Use of Starter Cultures in Olives: A Not-Correct Use Could
Cause a Delay of Performances
Marianne Perricone, Maria Rosaria Corbo, Milena Sinigaglia, Antonio Bevilacqua*
Department of Agriculture, Food and Environmental Science, University of Foggia, Foggia, Italy.
Received December 10th, 2012; revised January 13th, 2013; accepted January 21st, 2013
Copyright © 2013 Marianne Perricone et al. This is an open access article distributed under the Creative Commons Attribution Li-
cense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
The role of lactic acid bacteria for a correct course of fermentation of table olives has been extensively reviewed and it
is a common idea that the inoculum of selected strains could exert a strong benefit for product quality; however, the
basic assumption of this research is that a not-correct preparation of starter could affect its performances in brines and
delay the benefit of a starter inoculum. Thus, some selected strains of LAB (lactic acid bacteria) and yeasts were inocu-
lated into brines of Bella di Cerignola olives (Spanish style), as a single starter (LAB or yeasts) or a combined prepara-
tion (LAB + yeasts) and compared to a natural fermentation, focusing on the performances of the process (acidification,
production of lactic acid) and trying to verify the basic assumption. LAB and yeasts were grown in the opportune lab
media and then directly inoculated in brine after the lye treatment. LAB exerts a kind of benefit (increased content of
lactic acid) only for 2 - 3 weeks; then, the inoculated samples behave in the same way as not-inoculated ones. Thus, this
research underlined that a starter culture, not prepared in the correct way, could not be useful to guide the fermenta-
tion.
Keywords: Olives; Lactic Acid Bacteria; Yeasts; Delay in Fermentation; Starter
1. Introduction
Fermentation of table olives starts spontaneously and is
strongly influenced by olive cultivar, indigenous micro-
biota and methodological factors, such as temperature
and salt concentration in the brine [1].
Moreover, olive fermentation relies on a complex mi-
crobiota, including Enterobacteriaceae, lactic acid bacte-
ria and other genera of Gram positive microorganisms
and yeasts; the traditional fermentations are usually based
on the maintenance of this association [2].
In a natural olive fermentation after an initial stage,
characterised by the growth of Gram negative bacteria,
lactic acid bacteria and yeasts coexist until the end of
fermentation; in particular, in Bella di Cerignola olives,
yeasts were found at concentrations of approximately 5 -
6 log·cfu/g on olives and 6 log·cfu/ml in the brines [3,4].
On the other hand, Hernandèz, Martin, Aranda, Pèrez-
Nevado and Còrdoba [5] recovered a yeast number of
about 3.0 log·cfu·g/l and 4.9 log·cfu/ml in Spanish green
olives and their brines, respectively. The role of lactic
acid bacteria for a correct course of fermentation of table
olives has been extensively reviewed and it is a common
idea that the inoculum of selected strains could exert a
strong benefit for product quality [2].
For a better course of the process and to control natu-
rally occurring microflora, many authors recommended
the inoculation of brine with commercial strains; the use
of suitable Lactobacillus plantarum starter cultures has
the potential to improve the microbiological control of
the process, increase the lactic acid yield and, accord-
ingly, provide the production of Spanish-style fermented
green olives of consistently high quality [6]. Recently,
some authors proposed a mixed starter, containing a com-
bination of LAB and yeasts [4].
During the fermentation, weak acidification rates are
often registered, because most of the sugars and nutrients
are lost by the effect of lye treatment and strong washing,
thus pH is often unsuitable for a safe storage of product
[7].
However, many times a delay or a lack of fermentation
of olives, especially those of the Manzanilla variety, oc-
curs and there is no explanation for this phenomenon;
many variables can affect the progress of lactic acid fer-
mentation such as salt concentration, temperature, nutria-
*Corresponding author.

Use of Starter Cultures in Olives: A Not-Correct Use Could Cause a Delay of Performances 722
nutrient content, inhibitors and others. Earlier investiga-
tions related the difficulties of LAB growth in olive
brines with the presence of polyphenols [8].
However, many times the incomplete acidification can-
not be related to phenols (Bevilacqua, unpublished re-
sults); the basic assumption of this research is that a not-
correct preparation of starter could affect its perform-
ances in brines and delay the benefit of a starter inocu-
lum.
Thus, some selected strains of LAB and yeasts were
inoculated into brines of Bella di Cerignola olives, as a
single starter (LAB or yeasts) or a combined preparation
(LAB + yeasts) and compared to a natural fermentation,
focusing on the performances of the process (acidifica-
tion, production of lactic acid) and trying to verify the
basic assumption.
2. Materials and Methods
2.1. Strains
4 strains of Lactobacillus plantarum were used in this
study: the strains c1, c16 and c19 isolated from a natural
fermentation of Bella di Cerignola olives [9], whereas
the isolate DSMZ 2601 was purchased from a Public
Collection (Deutsche Sammlung von Mikroorganismen
und Zellkulturen, Braunschweig, Germany).
Moreover, 3 strains of Candida spp. (labelled as 22-
51-66 and isolated from a natural fermentation of Bella
di Cerignola olives) were used as selected yeasts; in par-
ticular the strains 22 and 51 were identified as C. famata,
while the strain 66 as C. guilliermondii [2].
The strains of Lb. plantarum, stored at −20˚C in MRS
broth (Oxoid, Milan, Italy) added with 33% of sterile
glycerol (J.T. Baker, Milan, Italy), were grown over 2
passages in MRS broth, incubated at 30˚C for 48 h. The
strains of Candida spp. were maintained on Sabouraud
Dextrose Agar slants (Oxoid), stored at 4˚C and trans-
ferred monthly; before each assay, the strains were
grown in Sabouraud broth (Oxoid), incubated at 25˚C for
48 h.
2.2. Inocula Preparation
LAB cocktail was prepared as follows: 50 ml of the cul-
ture of each strain were centrifuged at 4000 rpm for 10
min. then, the supernatant was discarded and 50 ml of
saline solution (9% of NaCl) was added to the pellet (cell
suspension).
The cocktail was prepared by mixing 50 ml of cell
suspension for each LAB strain to obtain a final volume
of 200 ml; this volume was used for the inoculation of 10
l of brine (the level of each strain was about 8 log·cfu/
ml).
Concerning yeast cocktail, 70 ml of the culture of each
strain were centrifuged at 4000 rpm for 10 min; then, the
pellet was suspended in 70 ml of brine. Yeast cocktail
was prepared by mixing cell suspension of each strain (ca.
6 - 7 log·cfu/ml), thus attaining a final volume of 210 ml.
This suspension was used for the inoculation of 10 l of
brine (high inoculum); for the batches inoculated with a
low level of yeasts (low inoculum), the suspension was
diluted at 5 log·cfu/ml and then used for brine inocula-
tion.
2.3. Olive Preparation and Fermentation
The fermentation was performed on Bella di Cerignola
olives, size GGG (i.e. 70 to 80 olives/kg). After har-
vesting, olives were subjected to grading, to remove
fruits with visible damages, washed with tap water and
treated with lye (1.3%) for 12 to 15 h. After 2 washings
(2 to 3 h and 12 to 13 h), olives were brined (9% of NaCl)
and inoculated with LAB and/or yeasts.
The fermentation took place in vessels of 25 l, con-
taining 10 kg of olives and 10 l of brine; olives were
maintained dipped in the brines through a grid. The ves-
sels were stored at room temperature for the entire fer-
mentation and mixed periodically to avoid the formation
of a mould layer on the top of the brine. NaCl was
checked periodically and maintained for the entire fer-
mentation at 8% - 9%.
Table 1 shows the different batches prepared for the
analysis.
Although performed at laboratory level, the conditions
used for the fermentation (ratio olive/brine 1:1, use of a
grid, mixing periodically the brines, fermentation at room
temperature) mimic the conditions encountered through-
out olive production in Apulian region.
2.4. Microbiological Analyses
Microbiological analyses were performed both on olives
and brines throughout the entire fermentation; 20 g of
olives were diluted with sterile saline solution (0.9%
NaCl) and homogenized through a Sterilmixer (Pbi In-
ternational, Milan, Italy) at 16,500 rpm for 2 min. Serial
dilutions of olive homogenates and brines were per-
formed and plated onto selective media. All the media
and the supplements were purchased from Oxoid.
Table 1. Batches prepared for the fermentation assays (T,
olives; S, brines).
T1/S1Control
T2/S2 Batch inoculated with LAB
(6 - 7 log·cfu/ml)
T3/S3 Batch inoculated with yeasts
(high inoculum, 5 log·cfu/ml)
T4/S4Batch inoculated with LAB and yeasts (high inoculum)
T5/S5 Batch inoculated with LAB and yeasts
(low inoculum, ca. 3 log·cfu/ml)

Use of Starter Cultures in Olives: A Not-Correct Use Could Cause a Delay of Performances 723
The media and the conditions were the following:
1) Mesophilic bacteria: Plate Count Agar (PCA), in-
cubated at 30˚C for 24 - 48 h.
2) Spore-former bacteria: PCA, incubated at 30˚C for
24 - 48 h. The evaluation of spore-former bacteria was
carried out after heat-shocking olive homogenates and
brines at 80˚C for 10 min.
3) LAB: MRS Agar, added with 0.17 g/l of cyclo-
heximide (Sigma-Aldrich, Milan, Italy), incubated at 30˚C
under anaerobic conditions for 48 - 72 h.
4) Yeasts: Sabouraud Dextrose Agar, added of 0.1 g/l
of chloramphenicol (C. Erba, Milan, Italy), incubated at
25˚C for 2 - 4 days.
5) Pseudomonadaceae: Pseudomonas Agar Base, added
with CFC selective supplement (containing cetrimide, a
selective antibiotic for pseudomonas), incubated at 25˚C
for 48 - 72 h.
6) Staphylococci: Baird Parker Agar Base, added with
egg yolk tellurite emulsion and incubated at 37˚C for 24 -
48 h.
7) Enterobacteriaceae: Violet Red Bile Glucose Agar
(VRBGA), incubated at 37˚C for 18 - 24 h.
2.5. pH Evaluation
pH measurements were performed on olive homogenates
and brines through a pH-meter Crison 2001 (Crison In-
struments, Barcelona, Spain).
2.6. Determination of Organic Acids
The content of D- and L-lactic acids, as well as the con-
centration of acetic, citric, D- and L-malic acids, D-glu-
cose and D-fructose in the brines was determined
through enzymatic kits (Megazyme, Bray, Ireland) just
after the brining (beginning of the fermentation) and after
7, 21, 28, 35 and 42 days of fermentation. The analyses
were performed as suggested by the producer.
2.7. Statistical Analyses
All the analyses were performed in duplicate over 2 in-
dependent batches, labelled A and B. Data were submit-
ted to one-way analysis of variance (one-way ANOVA)
and Tukey’s test (P < 0.05) through the software Statis-
tica for Windows version 10.0 (Statsoft, Tulsa, Okla.,
USA).
3. Results
3.1. Microbiology
Figure 1(a) reports the growth of lactic acid bacteria
(LAB) in olives. In particular, in the batches inoculated
with the starter (T2-T4 and T5), cell counts were 4
log·cfu/g higher than in the control after 7 days, whereas
the difference was not significant after 21 days, as the
population attained 5 to 5.75 log·cfu/g in all the samples,
except for the batch inoculated with yeasts (T3) showing
a LAB count 1 log·cfu/ml lower.
Concerning brines (Figure 1(b)), LAB were ca. 6
log·cfu/ml just after inoculum in the batches T2, T4 and
T5. Moreover, after 7 days inoculated samples showed a
LAB count higher than un-inoculated ones, while the
differences were not significant after 14 days.
Figure 2 shows yeast evolution in brine. As expected,
after 7 days the highest cell number was observed in the
inoculated batches (T3, T4 and T5), with a cell load
ranging from 5.6 to 6.6 log·cfu/ml; otherwise, in the
un-inoculated samples (T1 and T2) yeast number was
approximately 4.3 to 5.3 log·cfu/ml. As reported for LAB,
the difference between inoculated and inoculated batches
was not significant after 14 days. A similar trend was
recovered in olives (data not shown).
Enterobacteriaceae were at 3 log·cfu/g in olives and 4
log·cfu/ml in brines, whereas pseudomonads, staphylo-
cocci and spore-former bacteria were at low levels (1 - 2
log·cfu/ml) or below the detection limit for the entire
running time (data not shown).
3.2. pH
Figure 3 shows pH values of olives. After 7 days, olives
showed a pH of about 7 in the batches T2, T5 as well as
in the control, whereas the pH was ca. 8 in the samples
T3 and T4. After 2 week pH was ca 6 - 6.5 and decreased
until 5 at the end of fermentation, without significant
differences amongst the different samples. A similar trend
was recovered in brines (data not shown).
3.3. Organic Acid Content
Figures 4(a) and (b) show the concentration of D- and
L-lactic acids in the brine; both L- and D-lactic acids
increased significantly after 21 days of fermentation,
then they decreased by 2 - 3 g/l; namely, in the sample
T2 (sample inoculated with LAB) the concentration of L
and D-lactic acids was the highest one (9.15 and 4.70 g/l),
whereas the acids were at the lowest levels in the sample
inoculated with a high level of yeasts.
Regarding acetic acid, its concentration increased
throughout the entire fermentation, without any differ-
ence amongst the various batches (Figure 5). Finally,
D-glucose and D-fructose decreased during the fermenta-
tion until to about 0.5 g/l or below the detection limit
(data not shown).
4. Discussion
4.1. Why This Research?
The use of a starter culture could shorten the processing

Use of Starter Cultures in Olives: A Not-Correct Use Could Cause a Delay of Performances 724
(a)
(b)
Figure 1. LAB microflora in olives (a) and brines (b). Mean values ± standard deviation.
Figure 2. Yeast microflora in brines. Mean values ± standard deviation.
time for the preparation of naturally green olives and
affects positively fermentation, acidification rate and
flavour of the product [7].
Many food producers select promising strains jointly
with Universities and Research Centres; the main result
of these collaboration is the selection of “the best strains”.
In the past a joint venture of the section of Applied Mi-
crobiology (University of Foggia) and some food produc-
ers of table olives of Apulian region resulted in the selec-
tion of some promising strains of lactic acid bacteria with
interesting functional traits [2] and the evaluation of the
technological and spoiling impact of yeast strains of
Bella di Cerignola table olives [2]. Following these results,
Many authors recommended the inoculation of brine
with starter colture; in particular, the use of suitable
strains of Lb. plantarum could assure a correct course of
fermentation [6-9]. Some benefits could be obtained also
by a controlled inoculum of yeasts [10]. However, the
focus of this study is different, as this research presents a
new perspective on the use of starter cultures.

Use of Starter Cultures in Olives: A Not-Correct Use Could Cause a Delay of Performances 725
Figure 3. pH of olives. Mean values ± standard deviation.
(a)
(b)
Figure 4. Content of D- and L-lactic acids (g/l) in the brine.
(a) D-lactic acid; (b) L-lactic acid. Mean values ± standard
deviation.
Figure 5. Content of acetic acid (g/l) in the brine. Mean
values ± standard deviation.
lactic acid bacteria and yeasts were suggested to produc-
ers for a controlled fermentation; however, what hap-
pened was that producers reported a decrease of the
technological impact of these strains under in vivo condi-
tions. After a brief evaluation of the problem in our lab,
we supposed that the same strains, showing optimal per-
formances in a lab fermentation, could be affected in a
real fermentation by the way of the preparation of inocu-
lum; thus, we prepared the starters in the same way re-
ported by producers (strain cultivation in lab media, fol-
lowed by harvesting and cell suspension in brine).
The initial planning included also some batches in-
oculated with starters, grown in media containing in-
creasing concentration of salt; then, we decided to delete
these samples, as the main aim of the research was to
verify if also under lab conditions the strains behave in
the same way than in factories.
What we found was that, as suggested by many pro-
ducers, the differences between inoculated and inocu-
lated samples were significant only for 2 - 3 weeks of
fermentation (cell number, pH, content of lactic acid);
after this time, the samples inoculated with the starter
showed the same trend than the uninoculated ones. A
practical implication of this result is that a preliminary
adaptation could be useful to assure the success of the
starter for the entire process. A particular kind of adapta-
tion is the back-slopping, i.e. inoculation often carried
out with brines from previous fermentations [11]. How-
ever, such lactic cultures exhibit a diversity of metabolic
activities, even among strains of the same species, in-
cluding differences in growth rate, adaptation to a par-
ticular substrate, antimicrobial properties, flavour and
quality attributes as well as competitive growth behav-
iour in mixed cultures, resulting in non-consistent and
low quality final products. This kind of protocol was
used in the past by Italian producer of table olives; al-
though the process run faster, the quality was not con-
stant.
Another element confirming the strong implications of
a not-correct use of the starter was the pH; the pH in a
safe table olive must be about 4.5. In our fermentation,
pH decreased until to about 5 after 28 days, then it re-
mained constant during the entire fermentation.
5. Conclusion
In conclusion, why this research? A possible answer:
underline the risk associated with a not-correct use of the
starter and the importance of a good step of starter
preparation. Using the starter only after a growth phase
in a laboratory medium could result a delay in this per-
formances in brines.
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