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Starter Cultures
Starter Cultures
Fermentation
Introduction
Table olive fermentation is carried out by homo- and hetero-fermentative lactic acid bacteria and
yeasts found on olives. The micro flora and therefore the fermentation process depend on the
cultivar itself as well as on industrial and agricultural practices.
An approach to improve and control the fermentation process, for a more predictable process, is
the use of starter cultures.
A starter culture is a concentrated preparation of viable microorganisms which are added to a raw
material and whose metabolic activity has desired effects for fermentation as for example
improved aroma, texture and flavor (Borcakli et al., 1995).
The application of starter cultures can further be used to initiate the fermentation process as well
as to control undesired microbial activity, accelerate fermentation, removal of fermentable sugars
for prevention of secondary fermentation and prolonged shelf-life by inhibiting spoilage
microorganisms (Alperden and zay, 1993). Inhibit of spoilage bacteria works by competition for
nutrients, production of inhibitors (anti-microbial compounds such as organic acids, carbon
dioxide, hydrogen peroxide, diacetyl, ethanol or bacteriocins) [1].
Further advantages of starter cultures are that they can have probiotic properties, degradation of
anti-nutritional factors, the improvement of protein digestibility, and bio-availability of
micronutrients, and the nutritional enrichment of food through the biosynthesis of vitamins,
essential amino acids, and other nitrogen compounds.
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Strains
Lactic acid bacteria (LAB)
Lactic acid bacteria are the main fermenting bacteria found in lye treated table olives. They
produce lactic acid from the sugars present in the olives which inhibits growth of acid sensitive
bacteria such as gram-negative and coliform bacteria. Currently they are the first choice for the
use of starter cultures.
The main starter cultures used in experimental studies application in table olive fermentation are
listed in table 1.
It was observed that the use of LAB starter cultures in Spanish-style green olive fermentation can
positively affect this technology by accelerating the fermentation and, thus, decreasing the time
available for spoilage microorganisms growth [2]. Moreover, the flavor of inoculated olives was
proved to be unambiguously enhanced. Trials to confirm these results at industrial scale are
presently underway.
Lactobacillus plantarum and L. pentosus
The most widely used strains are Lactobacillus plantarum (Etchells et al., 1966; Leal-Snchez et al.,
2003; Chorianopoulos et al., 2005; Lamzira et al., 2005; Marsilio et al., 2005; Sabatini et al., 2008),
L. pentosus (de Castro et al., 2002; Panagou et al., 2003, 2008; Servili et al., 2006) or both (Snchez
et al., 2001; Panagou and Tassou, 2006) [3]. They were shown to increase lactic acid formation and
improve microbial control and result in the production of high quality fermented olives.
Gas pocket causing bacteria like Leuconostoc and heterofermentative Lactobacillus bacteria could
be reduced by the use of 5*1010 - 2*1010 starter cultures of Lactobacillus plantarum [4].
Application of Lb. plantarum starter cultures also lead to a faster pH decrease in green table olive
processing, reducing spoilage risk during the first days of fermentation [5].
Borcakli et al. also showed in 1995 that fermentation time with L. plantarum strains could be
reduced to 5.5 months by pre-treating olives with water (pH 4.5) for three days and further
covering in 6% NaCl brine pH 4.5 at 15 C (aeration provided).
Sanchez et al. used L. pentosus CECT 5138 to initiate fermentation at alkaline pH (>9), showing a
rapid growth and acidification that reduced the Enterobacteriaceae population and thereby the
risk of spoilage [6].
Panagou et al. showed that use of L. plantarum and freeze-dried L. pentosus from the commercial
Vege-Start 10 (Chr. Hansens Biosystems, Horsholm, Denmark) accelerated fermentation process
and reduced the survival period of Gram-negative bacteria by 5 days compared with the
spontaneous process, thus minimizing the likelihood of spoilage for the anaerobe fermentation of
natural black olives (cv. Conservolea). Fermentation conditions were 6% (w/v) NaCl (maintained
constantly), 20C for 30 days. L. pentosus showed a better brine acidification than the L. plantarum
strain [7].
Hurtado et al. (2010) found that L. pentosus showed better fermentation performances than L.
plantarum for controlling Arbequina table olive fermentation at 20C for 52 days in a brine
containing 8% NaCl (w/v) [3].
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Genus
Lactobacillus
Leuconostoc
Pediococcus
Enterococcus
coryniformis
cremoris
paramesenteroides
pentosaceus
faecium
casseliflavus
Manzanillo
In general L. plantarum strains can be used alone, or in combination with other strains like
Debaromyces hansenii or S. cerevisiae.
Segovia Bravo et al. observed an improvement in the growth of L. pentosus when it was inoculated
together with S. cerevisiae in ozonated alkaline solutions reused as fermentation brines [6].
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The use of Enterococcus casseliflavus cc45 and L. pentosus 5138A applied with one day delay also
produced quicker acidification [6]. And the use of L. plantarum 1159 inoculation was useful,
together with other conditions, to prevent bloater damage in the presence of high counts of P.
anomala S18 [6].
Nevertheless, in most cases inoculation is carried out with wild strains of LAB isolated from
previous fermentations [2].
Enterococci
The co-inoculation of different Enterococcis with L. plantarum or L. pentosus was also suggested to
improve olive fermentation.
Lavermicocca et al. (1998) and Deiana et al. (1992) suggested the use of E. faecium associated with
L. plantarum or S. cerevisiae [12]. The inoculation of Spanish-style fermented Manzanilla olives
with Enterococcus casseliflavus together with L. pentosus showed a decrease of fermentation time
and reduced growth of spoilage microorganisms [13].
But as enterococci can cause infections in humans, its use in table olive fermentation has not been
recommended by the European Food Safety Authority [14].
Yeasts
Yeast are more abundant in directly brined green and Natural black olives because in olives that
are not lye treated, lactic acid bacteria are partially inhibited due to the presence of phenolic
compounds [15].
Yeasts can be used as biocontrol agents in table olive fermentation due to their yeast species
inhibiting factors. Pichia membranifaciens for example produces a toxin which is active against
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Tab. 2: Main characteristics of the positive and negative roles of yeasts isolated from table olive
fermentation and packing [17]
Fermentation
Positive role
Production of desirable
volatile
compounds and
metabolites
Antioxidant activity
Improvement of the
lactic acid
bacteria growth
Killer activity
Yeast species
Not related clearly with
yeast
species
Negative role
Gas pocked
spoilage/excessive
production of CO2
Yeast species
S. cerevisiae (c)
P. anomala (c)
P. anomala (e)
Polysaccharolytic
activity
D. hansenii (f)
R. minuta (f)
D. hansenii (j)
S. cerevisiae (i)
Polygalactunorase
activity
D. hansenii (g)
K. marxianus (g)
P. membranifaciens (h)
C. tropicalis (d)
Product with a
milder taste a less
self-preservation
R. glutinis (k)
R. minuta (k)
R. rubra (k)
Not related
clearly with
yeast species
Packing
Negative role
Production of CO2
Yeast species
P. anomala (l)
S. cerevisiae (a) (l)
I. occidentalis (a)
S. cerevisiae (a)
I. occidentalis (b)
Undoubtedly, the full potential of the commercial value of yeasts in table olive fermentations has
not yet been fully determined and further research according to yeast ecology, physiology,
biochemistry and molecular biology has to be conducted.
Application
First thing that need to be done is the selection of an appropriate culture. Various examples for
suitable strains are available in the literature. After selection, validation on a lab-scale basis and
validation at the factory-scale are recommended (Bevilacqua et al., 2012). These steps are critical
for the safety and quality of the final product [14].
Starter cultures are usually applied at a concentration range between 106 to 107 cfu/ml of brine
after bitter substances have been eliminated [3]. For L. paracasei an inoculation number of 109
cfu/ml was reported [3].
Especially the combination of lye treatment and starter culture application was found to increase
table olive quality. Activity increase could be obtained by chancing brine with brine adjusted to pH
4.5 every 10 to 15 days.
Temperature
For L. plantarum and L. pentosus the most appropriate temperature to obtain a good fermentation
range from 20 to 25C [3].
Fermentation at low temperatures
Duran-Quintana et al. (1999) demonstrated that by using a selected strain of L. plantarum as
starter it was possible to carry out a normal Spanish-style green olive fermentation at 12C by
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using 3% NaCl and a pH of 5.0. Similar results were obtained by Snchez et al. (2001), by using a
selected strain of L. pentosus inoculated in lye-treated green olives at alkaline pH [3].
Acceleration of fermentation
Servili et al. (2006) used L. pentosus 1MO starting cultures (inoculated at an initial cell
concentration of about 108 cfu/mL) for the fermentation of black olives (Itrana and Leccino cv.) at
28C, pH 6.0, 6% NaCl, with addition of 0.3% glucose and 0.05% yeast extract. With this approach a
debittering could be obtained in 8 days [3, 5]. Olives resulted in obtaining ready-to-eat, high
quality table olives, with decreased oleuropein and increased hydroxytyrosol concentration [19].
Moreover, the addition of L. pentosus 1MO showed a strong control of the spontaneous bacterial
strains, due to thefast pH reduction below pH 4.5. This aspect improves the safety of fermented
olives, while avoiding the possible growth of spoilage and/or pathogenic strains.
Due to the successful fermentation by L. pentosus 1MO (8 days are needed from harvesting to
eating is now already being applied at the industrial level.
Commercial starter
Vegestart60 (Chr. Hansen A/S, Horsholm, Denmark)
http://www.hopeland-cn.com/products_list.asp?nid=16
Example 1
An example for a starter culture application can be found in table 3 [8].
Tab. 3: Starter culture ingredients
Ingredient
Yeast extract powder
Dextrose monohydrate
Di potassium hydrogen phosphate
monohydrate anhydrous
Di ammonium phosphate (DAP)
Trisodium citrate dihydrate
Sodium acetate anhydrous
Magnesium sulphate
Manganese sulphate
Salt, coarse, heat sterilised
Water, potable, sterile
Lactic acid bacteria freeze-dried culture
(L. plantarum/L. pentosus) produced by
Hopeland Bio-Tech Co Ltd.
Chemical formula
N/A
C6H6O6
K2HPO4
Supplier
Amyl Media
Consolidated Chemical or Redox
Consolidated Chemical or Redox
g/L
4
20
2
(NH4)2HPO4
C6H5Na3O7*2H2O
CH3OONa
MgSO4*7H2O
MnSO4*H2O
NaCl
2
2
5
0.2
0.04
40
to 1 L
Available in aluminium
triple foil bags thermo
closed
Example 2
Ruiz-Barbaa and Jimnez-Daz established a protocol for a successful industrial-scale Spanish-style
green olive fermentation with a novel Lactobacillus pentosus-paired starter culture.
The used strains were L. pentosus LP RJL2 (a PLS producer) and L. pentosus LP RJL3 (fast and
predominant growth in fermentation brines, which produces high amounts of exopolysaccharides
(EPSs)). At industrial level, production of EPS improves the viscosity of the brines during the
Spanish-style green olive fermentation producing high quality olives with a typical flavor and
aroma (Fernndez Dez, 1983; Garrido Fernndez et al., 1995).
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Preparation for inoculation: L. pentosus LP RJL2 and LP RJL3 were subcultured overnight at 30C in
50 ml MRS broth containing 500 g/ml streptomycin (MRS-Str) or 10 g/ml rifampin (MRS-Rif),
respectively. The next day, 2 l of fresh MRS were inoculated with the respective strain and then
incubated at 30C for 16-18 h. These cultures at the early stationary phase of growth (ca. 5*109
cfu/ml) were used as inocula. Both strains were inoculated at a time in each fermentor at final
concentrations of 106 cfu/ml and 105 cfu/ml of LP RJL2 and LP RJL3, respectively, resulting in a
faster pH decrease and lactic acid increase and higher final concentration [20].
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A different approach is the fortification of previous fermented olives in storage brine with
autochthonous putative probiotic bacteria like Lactobacillus pentosus TOMC-LAB2 [23].
The strain has shown to survive under packing conditions for long period of times as well as to
colonize the olive surface which is the food finally ingested by consumers. This opens the
possibility for the development of a new and simply probiotic fortified olive product.
Conclusion
The use of starter cultures finds wide application for the production of alcoholic beverages and
dairy products. They could as well be applied for the fermentation of table olives for process
improvement.
More and more research is being conducted as the interest in developing starter cultures for table
olives fermentation increases, because starter culture application provides a promising attempt to
reduce production costs (energy), fermentation times, risk of spoilage (increased shelf-life), to the
improvement of process control, sensory quality, and safety attributes.
According to the literature some L. pentosus, L. paracasei, and L. plantarum strains have already
been successfully applied as starter cultures indicating the strong potential for their usage for olive
fermentation during industrial olive production (Servili et al., 2006).
Nevertheless the application of starter cultures in the field of table olives is quite far from reaching
the diffusion as it has in other sectors of food industry, although commercial preparations are
already available on the market.
Literature
[1] http://www.ncbi.nlm.nih.gov/pubmed/12036143/
[2] http://www.sciencedirect.com/science/article/pii/S0740002011002425
[3] http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3400274/
[4] http://www.okyanusbilgiambari.com/Bilimsel.Makale/Okyanus-OliveFermentation.pdf
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