ISOLATION AND FAST PRE-SELECTION OF YEASTS FROM
SPONTANEOUS FERMENTATION OF CALABRIAN TABLE OLIVES
Andrea CARIDI
Department of AGRARIA , Mediterranea University of Reggio Calabria, Loc. Feo di Vito, 89124 Reggio Calabria, Italy
The aim of this research was to pre-select yeast
strains from Calabrian table olives. The main
traits
of
interest
are:
(a)
the
quality
improvement
by
interaction
with
natural
phenolic
compounds
possessing
positive
sensorial characteristics; (b) the shelf-life
extension by production or preservation of
natural
antioxidant
compounds.
Eighteen
samples of Calabrian table olives produced by
spontaneous
fermentation
were
collected.
Using Yeast Extract-Peptone-Dextrose (YPD)
Agar a total of 54 yeast strains were isolated
from the table olive samples and their brines.
The new strains were collected in Microbank at
-80°C. To perform a fast pre-selection, a
simple phenotypic-based methodology was
designed to allow all the 54 strains to be
simultaneously
tested.
So,
their
main
phenotypic characteristics were detected using
standard methods [1-3] and two new media,
designed to study the parietal adsorption
activity and used similarly to a chromogenic
grape-skin-based medium [4]. To prepare the
two media, 200 g of homogenised olive pulps
or olive seeds were suspended in 1 litre of
distilled water, treated at 110°C for 5 min to
extract olive pigments, and filtered through
gauze. The filtered extract was measured using
a graduate cylinder and the corresponding
amount of the following ingredients was
added:
citric
acid
monohydrate
100
g/l,
disodium hydrogen phosphate 50 g/l, dextrose
40 g/l, casein peptone 15 g/l, yeast extract 9
g/l. The solution was divided into test tubes (5
mL per tube) and heated at 110°C for 5 min.
Agar 40 g/l was dissolved in distilled water,
divided into test tubes (5 mL per tube) and
sterilized by autoclaving at 121 °C for 15 min.
Then, one test tube containing the medium and
one
containing
the
agar
solution,
both
maintained at 50°C in a water bath, were
poured together in Petri dishes (60×15 mm).
After careful mixing with a sterile L-shaped
plastic spreader, the medium was allowed to
solidify. The yeast strains were inoculated in
the Petri dishes containing the two media by
spreading over the surface using a sterile
L-shaped plastic spreader. After 10 days of
incubation at 25 °C, the biomass was carefully
mixed and spread on a sterile loop to prepare a
flat surface to be photographed. The colour
assessment was performed on the photographs
of the yeast biomass spread on the loop,
measuring
their
red,
green,
and
blue
components with Adobe Photoshop CS for
Windows XP (Adobe Systems, Inc., San Jose,
CA, USA).
All the 54 strains resulted positive towards the
catalase activity and the growth in YPD broth
at pH 4.30. The other tests showed an
interesting biodiversity, reported in the table
beside. This way, only
the best
strains
remaining at the end of this pre-selection (18)
will be soon selected using Oxitester CDR with
a great saving of money.
[1] Garrido Fernández, A. et al. - Tables
Olives: Production and Processing. Chapman
& Hall, London, UK, 1997.
[2] Arroyo-López F.N. et al. – Review. Yeasts
in table olive processing: desirable or spoilage
microorganisms? International Journal of Food
Microbiology, 160, 42-49, 2012.
[3] Bautista-Gallego J. et al. - Fermentation
profiles of Manzanilla-Aloreña cracked green
table olives in different chloride salt mixtures.
Food Microbiology, 27, 403-412, 2010.
[4] Caridi A. - Improved screening method for
the selection of wine yeasts based on their
pigment adsorption activity. Food Technology
and Biotechnology, 51(1), 137-144, 2013.
Biomass spread on a loop
of the strains 882 (up) and
871 (down) grown on Olive
Seed Agar (left) and on
Olive Pulp Agar (right)
Petri plates of strains
882
(up)
and
871
(down) grown on Olive
Seed Agar (left) and on
Olive Pulp Agar (right)
Session 2A: yeasts in food biotechnology:
biodiversity and ecology in foods and beverages
Board number S2A - 49
Legend - For each test, red highlight means negative result, no highlight means positive result, yellow highlight means negative but not excluding result
Legend - For each test, red highlight means negative result, no highlight means positive result, yellow highlight means negative but not excluding result
Re d
Gre e n
Blue
Re d
Gre e n
Blue
832, brine
+
+
+
+
-
+
-
-844, olive
+
+
+
+
-
+
-
-833, olive
-
-
+
+
-
+
-
-834, olive
+
+
+
-
-
-
-
-845, brine
+
+
-
-
-
-
-
-835, olive
-
+
+
+
+
+
+
+
836, olive
+
+
+
+
+
+
-
-854, brine
+
+
+
+
+
-
+
+
861, brine
-
+
+
+
+
-
-
-877, olive
-
+
+
+
+
+
-
-878, olive
-
+
+
+
+
+
-
-880, olive
-
-
+
+
+
-
-
-859, brine
-
-
+
+
+
+
+
-881, olive
-
+
+
+
+
+
+
+
882, olive
-
+
+
+
+
+
+
+
885, olive
-
-
+
+
+
+
+
-864, brine
-
-
+
+
+
+
+
+
886, olive
-
-
+
+
+
+
+
+
887, olive
-
-
+
+
+
+
-
+
865, brine
-
-
+
+
+
+
-
-888, olive
-
-
+
+
+
+
+
+
891, olive
-
+
+
-
-
+
+
+
857, brine
+
+
+
+
-
-
+
-892, olive
+
+
+
+
+
+
+
-893, olive
+
+
-
-
-
+
+
-867, brine
-
-
-
-
-
-
+
-894, olive
-
-
-
-
-
-
-
+
895, olive
-
+
-
-
-
-
+
+
870, brine
-
-
-
-
-
-
-
-898, olive
-
-
-
-
+
-
-
-914, olive
-
-
+
-
+
-
-
+
915, brine
-
-
-
-
-
-
-
+
916, brine
-
-
-
-
-
+
+
+
871, brine
-
-
-
-
-
+
-
+
900, olive
-
+
-
-
+
-
+
+
902, olive
-
-
-
-
-
-
+
+
873, brine
-
-
-
-
-
-
-
+
874, brine
-
-
-
-
+
-
-
+
904, olive
-
+
-
-
-
-
+
+
905, olive
-
+
-
-
-
+
+
+
875, brine
-
+
-
-
-
-
-
+
908, olive
-
+
+
+
+
+
+
+
909, olive
-
+
-
-
+
-
-
+
XV, Ottobratica , San Giorgio Morgeto
913, brine
-
+
-
-
+
-
-
-837, olive
+
-
-
-
-
-
+
+
839, brine
+
-
-
-
+
+
+
+
847, olive
+
-
-
-
+
+
+
+
840, olive
+
+
+
+
+
-
-
+
841, olive
-
+
-
-
+
+
+
+
848, olive
-
+
+
+
+
+
+
+
856, brine
+
+
-
-
-
+
-
+
842, olive
+
-
-
-
-
+
+
+
849, olive
-
-
+
+
+
+
+
-850, brine
-
+
+
+
+
+
+
-Selected Yeasts
54
39
29
25
28
23
23
25
22
18 (yellow)
Final
Se le ction
XIV, Nocellara , San Giorgio Morgeto
XVI, Roggianella , San Giorgio Morgeto
XVII, Sinopolese , Seminara
XVIII, Carolea , Seminara
VII, Nocellara Messinese , Moschetta
VIII, Geracese , Gioiosa Ionica
IX, Carolea , Villa San Giovanni
X, Carolea , San Giorgio Morgeto
XI, Carolea , San Giorgio Morgeto
XII, Carolea , Gioia T auro
XIII, Carolea , Martone
I, Geracese , Marina di Gioiosa Ionica
II, Geracese , Mammola
III, Carolea , Siderno
IV, Geracese , Gerace
V, Carolea , Marina di Gioiosa Ionica
VI, Geracese , Locri
C olour Adsorption on O live Se e d Agar
C olour Adsorption on O live Pulp Agar
Growth in YPD Broth with
the Addition of NaC l 11%
Sample , C ultivar, Place
Strain and
O rigin
C O
2
Production
Biomass Colour on Olive Seed Agar, Photographs of the Loops,
Red Component, Method: LSD at 95.0%
The values less than or equal to the mean are highlighted in yellow
Strains Count Mean Homogeneous Groups
835-3 4 101.25 X 836-3 4 114.00 X 880-4 4 115.00 X 861-4 4 115.25 XX 882-5 4 116.00 XXX 877-4 4 116.75 XXXX 887-6 4 119.75 XXXXX 878-4 4 120.50 XXXXXX 840-17 4 121.00 XXXXXX 881-5 4 121.25 XXXXXX 859-5 4 122.50 XXXXXXX 886-6 4 123.75 XXXXXXX 892-8 4 124.25 XXXXXX 865-7 4 124.25 XXXXXX 857-8 4 124.75 XXXXX 885-5 4 125.00 XXXXXX 864-6 4 125.50 XXXXXX 833-2 4 125.75 XXXXXXX 888-7 4 127.00 XXXXXXXX 832-1 4 127.50 XXXXXXXX 854-3 4 128.00 XXXXXXXX 848-17 4 128.75 XXXXXXXX 850-18 4 130.25 XXXXXXXX 844-1 4 131.00 XXXXXXXXX 834-2 4 131.25 XXXXXXXX 908-14 4 132.25 XXXXXXXX 891-7 4 133.50 XXXXXXXX 914-11 4 133.75 XXXXXXXX 849-18 4 134.25 XXXXXXXX 842-18 4 135.50 XXXXXXXX 867-9 4 136.75 XXXXXXXX 839-16 4 137.00 XXXXXXXXX 845-2 4 137.50 XXXXXXXX 856-17 4 138.50 XXXXXXXXX 837-16 4 138.50 XXXXXXXXX 900-12 4 139.25 XXXXXXXXX 916-11 4 141.50 XXXXXXXXX 893-8 4 142.25 XXXXXXXXX 874-13 4 142.50 XXXXXXXX 898-10 4 143.75 XXXXXXX 847-16 4 144.25 XXXXXX 913-15 4 144.75 XXXXXXX 905-13 4 145.25 XXXXXXX 841-17 4 145.50 XXXXXXX 909-14 4 147.00 XXXXXXX 915-11 4 147.75 XXXXXX 870-10 4 148.00 XXXXX 871-12 4 149.25 XXXXX 873-13 4 150.75 XXXX 894-9 4 153.25 XXX 904-13 4 154.00 XX 895-9 4 155.00 X 902-12 4 167.75 X 875-14 4 169.50 X
Biomass Colour on Olive Seed Agar, Photographs of the Loops,
Green Component, Method: LSD at 95.0%
The values less than or equal to the mean are highlighted in yellow
Strains
Count
Mean
Homogeneous Groups
835-3 4 90.25 X 836-3 4 109.25 X 882-5 4 114.25 XX 880-4 4 114.5 XXX 877-4 4 114.75 XXX 861-4 4 115.25 XXX 881-5 4 119.25 XXX 887-6 4 119.75 XXXX 859-5 4 121.25 XXXXX 865-7 4 121.5 XXXXX 878-4 4 122.0 XXXXX 888-7 4 122.0 XXXXX 908-14 4 122.5 XXXXX 886-6 4 122.75 XXXXX 840-17 4 123.75 XXXXX 864-6 4 124.75 XXXX 885-5 4 125.25 XXXXX 849-18 4 126.25 XXXXXX 854-3 4 127.5 XXXXXXX 892-8 4 128.5 XXXXXXXX 832-1 4 128.5 XXXXXXXX 833-2 4 128.75 XXXXXXX 857-8 4 129.0 XXXXXXXX 850-18 4 129.5 XXXXXXX 844-1 4 129.75 XXXXXXX 848-17 4 130.0 XXXXXXX 834-2 4 133.0 XXXXXXX 914-11 4 134.25 XXXXXXX 891-7 4 134.75 XXXXXXX 842-18 4 135.0 XXXXXXX 839-16 4 135.25 XXXXXXX 898-10 4 136.5 XXXXXX 837-16 4 136.75 XXXXXX 900-12 4 136.75 XXXXXX 845-2 4 137.75 XXXXXX 867-9 4 138.25 XXXXX 874-13 4 138.25 XXXXX 856-17 4 139.5 XXXX 841-17 4 140.0 XXXX 871-12 4 140.25 XXXX 870-10 4 140.5 XXXX 847-16 4 140.5 XXXX 916-11 4 140.75 XXXX 893-8 4 142.5 XXXX 913-15 4 143.5 XXXXX 905-13 4 143.75 XXXX 909-14 4 144.0 XXXX 895-9 4 146.25 XXXX 915-11 4 146.75 XXXX 902-12 4 150.75 XXX 894-9 4 151.0 XXX 873-13 4 152.25 XX 904-13 4 154.0 X 875-14 4 163.75 X
Biomass Colour on Olive Seed Agar, Photographs of the Loops,
Blue Component, Method: LSD at 95.0%
The values less than or equal to the mean are highlighted in yellow
Strains
Count
Mean
Homogeneous Groups
835-3 4 86.25 X 908-14 4 111.75 X 836-3 4 112.75 X 840-17 4 113.5 X 880-4 4 116.25 XX 861-4 4 119.25 XXX 887-6 4 120.25 XXXX 850-18 4 120.5 XXXXX 877-4 4 121.0 XXXXX 914-11 4 121.25 XXXXX 882-5 4 121.5 XXXXX 881-5 4 123.5 XXXXX 865-7 4 123.5 XXXXX 900-12 4 123.5 XXXXX 888-7 4 123.75 XXXXX 898-10 4 124.0 XXXXXX 892-8 4 124.75 XXXXXXX 854-3 4 125.5 XXXXXXXX 878-4 4 125.75 XXXXXXXXX 847-16 4 126.25 XXXXXXXXX 886-6 4 126.75 XXXXXXXXX 864-6 4 127.25 XXXXXXXXX 849-18 4 127.5 XXXXXXXXXX 874-13 4 128.25 XXXXXXXXXX 885-5 4 128.5 XXXXXXXXXX 859-5 4 128.75 XXXXXXXXXX 839-16 4 129.5 XXXXXXXXXX 913-15 4 130.0 XXXXXXXXXXX 848-17 4 130.25 XXXXXXXXXXX 841-17 4 130.25 XXXXXXXXXXX 909-14 4 130.25 XXXXXXXXXXX 857-8 4 131.75 XXXXXXXXXXX 902-12 4 131.75 XXXXXXXXXXX 833-2 4 132.0 XXXXXXXXXXX 867-9 4 132.5 XXXXXXXXXXX 870-10 4 133.25 XXXXXXXXXXX 842-18 4 133.75 XXXXXXXXXX 837-16 4 133.75 XXXXXXXXXX 871-12 4 134.0 XXXXXXXXX 832-1 4 134.5 XXXXXXXXX 856-17 4 135.25 XXXXXXXX 895-9 4 135.5 XXXXXXX 844-1 4 135.75 XXXXXX 891-7 4 137.25 XXXXXX 916-11 4 138.75 XXXXXX 905-13 4 139.75 XXXXX 834-2 4 140.0 XXXX 893-8 4 141.25 XXX 894-9 4 146.0 XX 845-2 4 146.75 XX 915-11 4 146.75 XX 873-13 4 147.5 X 904-13 4 148.0 X 875-14 4 159.0 X
Biomass Colour on Olive Pulp Agar, Photographs of the Loops, Red Component, Method: LSD at 95.0%
The values less than or equal to the mean are highlighted in yellow
Strains Count Mean Homogeneous Groups
841-17 4 105.5 X 888-7 4 110.25 X 849-18 4 122.75 X 848-17 4 123.25 X 850-18 4 124.75 XX 882-5 4 126.5 XXX 891-7 4 127.0 XXX 916-11 4 127.0 XXX 832-1 4 127.75 XXXX 842-18 4 127.75 XXXX 885-5 4 128.5 XXXXX 859-5 4 128.75 XXXXX 881-5 4 129.25 XXXXX 839-16 4 129.25 XXXXX 833-2 4 130.0 XXXXXX 836-3 4 130.25 XXXXXX 835-3 4 130.5 XXXXXX 887-6 4 130.75 XXXXXX 905-13 4 131.25 XXXXXXX 864-6 4 132.0 XXXXXXXX 877-4 4 132.25 XXXXXXXX 847-16 4 133.75 XXXXXXXX 886-6 4 134.0 XXXXXXXX 844-1 4 134.0 XXXXXXXX 908-14 4 134.5 XXXXXXXXX 893-8 4 134.75 XXXXXXXX 878-4 4 135.75 XXXXXXXX 892-8 4 137.0 XXXXXXXX 856-17 4 137.75 XXXXXXX 865-7 4 138.0 XXXXXXX 871-12 4 138.25 XXXXXXX 857-8 4 139.25 XXXXXXX 854-3 4 141.0 XXXXXXX 861-4 4 141.25 XXXXXX 894-9 4 142.5 XXXXXX 875-14 4 142.5 XXXXXX 837-16 4 143.5 XXXXXXX 867-9 4 144.0 XXXXXX 902-12 4 144.25 XXXXXX 880-4 4 145.75 XXXXXX 914-11 4 146.75 XXXXXX 898-10 4 148.25 XXXXX 904-13 4 148.25 XXXXX 895-9 4 149.0 XXXXXX 834-2 4 149.0 XXXXXX 874-13 4 149.5 XXXXX 909-14 4 150.25 XXXXXX 900-12 4 150.75 XXXXXX 870-10 4 152.0 XXXXX 913-15 4 153.25 XXXX 915-11 4 154.75 XXXX 840-17 4 158.25 XXX 845-2 4 160.0 XX 873-13 4 164.0 X
Biomass Colour on Olive Pulp Agar, Photographs of the Loops,
Green Component, Method: LSD at 95.0%
The values less than or equal to the mean are highlighted in red
Strains
Count
Mean
Homogeneous Groups
841-17 4 85.25 X 847-16 4 96.5 X 839-16 4 97.75 X 888-7 4 102.75 XX 916-11 4 105.25 XXX 895-9 4 110.0 XXX 848-17 4 111.5 XXXX 902-12 4 111.75 XXXXX 904-13 4 111.75 XXXXX 849-18 4 111.75 XXXXX 882-5 4 112.0 XXXXX 837-16 4 112.25 XXXX 905-13 4 112.5 XXXX 842-18 4 113.0 XXXXX 891-7 4 115.25 XXXXX 885-5 4 115.75 XXXXXX 867-9 4 116.0 XXXXXX 864-6 4 116.0 XXXXXX 893-8 4 116.5 XXXXXXX 881-5 4 117.25 XXXXXXXX 908-14 4 117.5 XXXXXXXX 854-3 4 118.75 XXXXXXXXX 850-18 4 119.25 XXXXXXXXX 900-12 4 119.25 XXXXXXXXX 892-8 4 119.5 XXXXXXXXX 857-8 4 119.5 XXXXXXXXX 835-3 4 119.75 XXXXXXXXX 886-6 4 120.0 XXXXXXXXX 859-5 4 121.0 XXXXXXXXX 894-9 4 122.25 XXXXXXXX 871-12 4 122.25 XXXXXXXX 887-6 4 122.75 XXXXXXXX 909-14 4 124.0 XXXXXXXXX 877-4 4 124.0 XXXXXXXXX 856-17 4 125.0 XXXXXXXX 832-1 4 125.5 XXXXXXX 833-2 4 125.75 XXXXXXX 865-7 4 125.75 XXXXXXX 874-13 4 125.75 XXXXXXX 836-3 4 126.0 XXXXXX 844-1 4 127.0 XXXXX 915-11 4 127.0 XXXXX 875-14 4 127.75 XXXXXX 914-11 4 128.75 XXXXXX 861-4 4 129.75 XXXXXX 878-4 4 130.0 XXXXXX 898-10 4 132.0 XXXXXX 870-10 4 133.25 XXXXX 840-17 4 136.5 XXXX 873-13 4 137.75 XXX 880-4 4 139.0 XX 913-15 4 140.0 XX 834-2 4 148.75 X 845-2 4 162.75 X
Biomass Colour on Olive Pulp Agar, Photographs of the Loops,
Blue Component, Method: LSD at 95.0%
The values less than or equal to the mean are highlighted in yellow
Strains
Count
Mean
Homogeneous Groups
841-17 4 69.75 X 847-16 4 75.75 XX 902-12 4 80.5 XX 839-16 4 82.75 XXX 904-13 4 86.0 XXX 900-12 4 86.75 XXX 837-16 4 88.75 XXXX 916-11 4 92.0 XXXX 915-11 4 92.5 XXXX 905-13 4 93.25 XXXXX 895-9 4 93.25 XXXXX 842-18 4 93.25 XXXXX 874-13 4 93.5 XXXXX 909-14 4 95.25 XXXXXX 888-7 4 97.5 XXXXXX 856-17 4 97.75 XXXXXXX 835-3 4 99.0 XXXXXXX 908-14 4 100.0 XXXXXXX 894-9 4 100.5 XXXXXXXX 886-6 4 101.5 XXXXXXXX 848-17 4 101.75 XXXXXXXX 871-12 4 101.75 XXXXXXXX 891-7 4 102.0 XXXXXXXX 881-5 4 102.75 XXXXXXXX 882-5 4 103.25 XXXXXXXX 864-6 4 103.25 XXXXXXXX 914-11 4 103.5 XXXXXXXX 887-6 4 103.75 XXXXXXXX 854-3 4 104.0 XXXXXXXX 840-17 4 104.25 XXXXXXXX 873-13 4 104.25 XXXXXXXX 875-14 4 104.75 XXXXXXXX 885-5 4 105.25 XXXXXXX 892-8 4 106.0 XXXXXXXX 857-8 4 107.25 XXXXXXXX 859-5 4 107.5 XXXXXXXX 836-3 4 107.75 XXXXXXXXX 865-7 4 108.5 XXXXXXXXXX 867-9 4 110.0 XXXXXXXXXX 844-1 4 111.25 XXXXXXXXXX 861-4 4 112.0 XXXXXXXXXX 913-15 4 112.5 XXXXXXXXX 832-1 4 115.0 XXXXXXXXX 898-10 4 116.75 XXXXXXXX 893-8 4 117.0 XXXXXXX 877-4 4 117.25 XXXXXX 833-2 4 117.75 XXXXX 870-10 4 118.25 XXXX 849-18 4 119.25 XXXX 850-18 4 120.25 XXX 880-4 4 121.0 XX 878-4 4 122.75 X 834-2 4 144.25 X 845-2 4 155.5 X
