Isolation and characterization of lactic acid bacteria ... - Rombio.eu

Romanian Biotechnological Letters
Copyright © 2011 University of Bucharest
Vol. 16, No.6, 2011, Supplement
Printed in Romania. All rights reserved
SHORT COMMUNICATION
Isolation and characterization of lactic acid bacteria from romanian
fermented vegetables
Abstract
Received for publication, October 7, 2011
Accepted, November 21, 2011
1 SILVIA SIMONA GROSU-TUDOR, 1* MEDANA ZAMFIR
1 Institute of Biology Bucharest of Romanian Academy,
296 Splaiul Independentei, 060031 Bucharest, P.O. Box 56-53, ROMANIA
* Corresponding author, phone: (021)2239072; fax: (021)2239071;
e-mail: medana.zamfir@ibiol.ro
Fermentation was used since ancient times as an easy method of vegetables’ preservation,
which also maintains and/or improves the nutritional and sensory properties of vegetables. The
spontaneous fermentation process is mainly carried out by lactic acid bacteria (LAB). The aim of our
study was to isolate and characterize the LAB involved in such spontaneous fermentations. Brine and
vegetable samples were collected from 20 fermentations carried out at household level. They were
analysed for acidification and plated on VRBG agar medium (for enumeration of enterobacteriaceae)
and MRS agar medium (for enumeration and isolation of LAB). The cell morphology, growth at
different temperatures, capsular polysaccharide production, mucoidness and ropyness of the colonies
for the Gram-positive, catalase-negative isolates (139) were investigated. LAB were prevalent in all
end-samples, as represented by their MRS counts, whereas enterobacterial counts were low in most
cases, indicating good fermentation quality. All isolates grew well at 37ºC and 45ºC, in only 24h, while
the growth at 10ºC was slower for some of them (72h). The cells are rod-shaped for most of the isolates,
while the others are cocci and coccobacilli. All except five isolates were shown to produce capsular
polysaccharide and 31 developed mucoid colonies on sucrose-based media.
Key words: lactic acid bacteria, fermented vegetables
Introduction
Fermented vegetables represent a frequently used food in Romania, especially during
the winter. The preservation is based on the property of lactic acid bacteria (LAB) to ferment
sugars to lactic acid, causing an acidification and hence, a stabilization of the end product.
The most common vegetables used are cucumbers, cabbage, and bell peppers (paprika). Green
tomatoes, carrots, and cauliflower are used too, sometimes mixed with fruits (water melon,
apples, or pears). During the spontaneous fermentation, LAB mostly found belong to
Lactobacillus sp. (L. plantarum, L. brevis), Leuconostoc sp. (L. mesenteroides) and
Pediococcus sp. (P. pentosaceus, P. cerevisiae) (Miambi & al. [1], Chao & al. [2], Nakayama
& al. [3], Kim & al. [4]).
Traditional preservation of vegetables and fruits by the process of lactic acid
fermentation has numerous advantages beyond those of preservation. The proliferation of
LAB in fermented vegetables enhances their digestibility and increases the vitamin levels and
vitamin bioavailability. These beneficial organisms contribute to the organoleptic properties
of the fermented product and to its preservation by in situ production of antimicrobial
substances such as lactic and acetic acid, hydrogen peroxide, bacteriocins etc. (De Vuyst &
Vandamme [5]). Their main end product, lactic acid, not only keeps vegetables and fruits in a
state of perfect preservation but can also promote the growth of healthy microbiota
throughout the intestine. Public awareness of effects of diet on gut health and a healthy gut
microbiota is resulting in the consumption of products that mantain health in addition to
148

SILVIA SIMONA GROSU-TUDOR, MEDANA ZAMFIR
preventing disease. In Romania some fermented vegetables (especially sauerkraut) are
sometimes used as “therapeutic agents”, especially for stomach disorders.
Many LAB strains have the ability to produce exopolysaccharides (EPS), with an
important role in the rheology and texture properties of fermented food products, and thus of
interest for food applications as in situ produced, natural bio-thickeners (De Vuyst &
Vaningelgem [6]). It has been suggested that some EPS produced by LAB have prebiotic
activity (Ruijssenaars [7], Salazar [8]), contributing to the promotion of human
gastrointestinal health.
Traditionally fermented foods, including fermented vegetables, can be a rich source of
new LAB strains, with interesting functional properties and with potential applications in food
industry and health. In this context, the aim of our study was to isolate and characterize new
LAB strains from various fermented vegetables, in a search for new EPS-producing strains.
Materials and methods
Bacterial strains and media. Brine and vegetable samples were collected from 20
spontaneous vegetable fermentations (Table 1) carried out at a household level in the region
of Valenii de Munte (Chiojdu) and Bucharest. For fermentations 19 and 20, nine additional
samples were also collected during the 5 weeks of fermentation, at diferent time intervals.
Samples were analysed for acidification (pH measurements) and plated on VRBG agar
medium (for enumeration of enterobacteriaceae, Mossel & al. [9]) and MRS agar medium (for
enumeration and isolation of LAB, de Man & al. [10]). Colonies were randomly picked up,
purified and tested for catalase production and Gram-staining. Gram-positive, catalasenegative
isolates were stored at -75°C in liquid MRS medium supplemented with 25%
(vol/vol) of glycerol as cryoprotectant and used in our further experiments.
Characterization of LAB isolated from Romanian fermented vegetables. To determine
the mesophilic/thermophilic character, the strains isolated from Romanian fermented
vegetables were incubated in MRS at different temperatures: 10°C, 37°C and 45°C. After 24–
72 h of incubation at these temperatures, the pH and the optical density at 600 nm was
measured. If the growth of the strains occurs between 10°C and 37°C and there is no growth
observed at temperatures higher than 45°C, they are mesophilic, while the strains showing a
good growth at 45°C, but not able to grow at 10°C are thermophilic (G. Zarnea [11]).
The cell morphology of the isolates was observed by microscopical examination of the Gramstained
slides.
In order to evaluate the capacity to produce exopolysaccharides, the isolates were cultivated
on four different media: MRS with 20 g liter -1 of glucose, modified MRS (mMRS) with 50g
liter -1 of sucrose instead of glucose, ST (Dave & Shah [12]) with 10 g liter -1 of lactose and
modified ST (mST) with 50 g liter -1 of sucrose instead of lactose. After 24 h of incubation at
37°C, the mucoidness and ropyness of the colonies was observed. The mucoid colonies have a
glistening and slimy appearance on agar plates, but are not able to produce strands when
extended with a stik, whereas the ropy colonies form a long filament by this method
(Dierksen & al. [13]).
Capsular polysaccharide (CPS) formation was evaluated by the Chinese ink negative staining
technique (Mozzi & al. [14]) after growing the strains on MRS with different carbon sources
and at different temperatures (10°C, 37°C and 45°C). Ten microliters of a fresh culture was
mixed with a drop of Chinese ink and spread on a slide in a thin film, coverred with a cover
glass and examined.
Romanian Biotechnological Letters, Vol. 16, No. 6, Supplement (2011) 149

Isolation and characterization of lactic acid bacteria from romanian fermented vegetables
Results and discussions
The final pH of the brines of all end-samples of the different spontaneous vegetable
fermentations carried out was on average pH 3.6. LAB were prevalent in all end-samples, as
represented by their MRS counts [ca. 10 9 (cfu/ml)], whereas enterobacterial counts were low
in most cases, indicating good fermentation quality (data not shown).
A total of 150 colonies were picked up from the MRS plates, purified and checked for
catalase production and Gram staining. A number of 139 isolates were shown to be Grampositive
and catalase-negative. The latter isolates were used for further characterization.
All the isolates grew very well at 37°C, reaching an OD 600nm of over 1.2 after 24 h of
incubation. Generally, 37°C is considered the optimal temperature for most species of lactic
acid bacteria (Zarnea, [11]). The isolates were able to grow well also at 10°C, 35 isolates
reaching an OD 600nm of over 0.5 after only 24 h of incubation. The other isolates needed 48 h
(95 isolates) or even 72 h (9 isolates) of incubation to reach the same OD value. At 45°C, all
isolates showed a good growth after 24 hours of incubation (OD 600nm over 0.5) . The pH
values of the bacterial cultures ranged between 3.0 and 5.0 for the isolates incubated at 37°C
and 45°C, and around 5.0 for the isolates incubated at 10°C. Since all isolates could grow at
all tested temperatures, we could not make, at this point, a distinction between their
thermophilic/mesophilic character.
The cell morphology of all 139 isolates was evaluated by microscopical observations
using the immersion objective. The cells from most of the isolates (95) were rod-shaped,
from other 32 were coccoid, from eleven were cocobacillar and from one isolate had a
diplococcal shape (Table 1).
Table 1. Overview of the spontaneous vegetables fermentations and characterization of the isolates
Fermented product
Time of
sampling
No. of
isolates
Mucoid
isolates
(MRS
suc)
CPS
positive
(MRS
gluc)
Cell morphology
1. Cucumber and plums 8 weeks 4 0 4 rods (2)
cocobacilli (2)
2.Green tomato 4 weeks 4 0 4 rods (3)
cocci (1)
3.Cucumber and beans 8 weeks 1 1 0 rods (1)
4.Cucumber and carrots 6 weeks 0 0 0 -
5.Green tomato 8 weeks 6 0 4 rods (6)
6.Cucumber 12 weeks 0 0 0 -
7.Green tomato, beets and 8 weeks 1 0 0 cocobacilli (1)
cabbage
8.Cucumber and
cauliflower
6 weeks 6 1 2 rods (4)
cocobacilli (2)
9.Green tomato 4 weeks 4 2 3 rods (3)
cocobacilli (1)
10.Green tomato, apple,
pear, and cucumber 2 weeks 9 2 9
rods (2)
cocci (4)
cocobacilli (3)
11.Green tomato, apple,
pear and cucumber 2 weeks 12 3 5
rods (7)
cocobacilli (2)
cocci (3)
12.Green tomato, carrots 8 weeks 5 1 4 rods (5)
and celery
13.Cabbage and beet 4 weeks 5 2 3 rods (5)
14.Cabbage 8 weeks 3 0 3 rods (3)
15.Green tomato, carrots
and cauliflower 8 weeks 8 3 7 rods (8)
150 Romanian Biotechnological Letters, Vol. 16, No. 6, Supplement (2011)

SILVIA SIMONA GROSU-TUDOR, MEDANA ZAMFIR
16.Cabbage 4 weeks 5 0 5 rods (5)
17.Pepper filled with 8 weeks 9 0 6 rods (9)
cabbage
18.Cucumber 4 weeks 6 2 6 rods (6)
19.Green tomato,
cauliflower, carrots,
pepper and celery
5 weeks 32 11 23
rods (16)
cocci (15)
diplococci (1)
20.Cauliflower 5 weeks 19 3 10 rods (10)
cocci (9)
Most of the isolates (87) grew well in all four media used (MRS, mMRS, ST, and
mST), but it was observed a preference for sucrose as a carbon source. Thirty-one strains
developed mucoid colonies on MRS with sucrose (Table 1, Fig 1a) and less or no mucoid on
media with glucose or lactose as a carbon source (data not shown). In some cases, due to the
small size of the colonies, the mucoidness and ropyness phenotype could not be detected.
a. b.
Fig 1. Mucoidness (a) and ropynes (b) of the colonies developed on agar plates by the isolate 52 and isolate 371,
respectively.
Results are in accordance with those obtained by Van Geel-Schutten & al. [15] who
screened several Lactobacillus strains of different origins (fermented food, human dental
plaque etc.) for mucoid phenotype in MRS medium supplemented with high concentrations
(100 g liter -1 ) of different sugars: glucose, fructose, maltose, raffinose, sucrose, galactose or
lactose. The MRS supplemeted with sucrose was the best medium for detecting the mucoide
phenotype. The autors attributed the high percentage of positive isolates to the high content of
sugar used in the medium. Also, Smitinont & al. [16] observed slimy colonies on agar media
containing sucrose as a sole carbon source.
None of the mucoid colonies showed a ropy phenotype. There were, however, two
isolates (338 and 371) that developed ropy colonies on sucrose containing media (Fig 1b).
Some LAB (e.g. Lactococcus lactis spp. cremoris Ropy352, Lactobacillus casei CG11) were
described in the literature to express both ropy and mucoid phenotypes, depeding on the
growth conditions (Dierksen & al. [13], Cerning & al. [17]). In Lactococcus lactis spp.
cremoris Ropy352, it has been shown that this ability is due to the production of two
exopolysaccharides with different chemical composition (Knoshaug & al. [18]).
It was shown that ropy strains of LAB yield fermented milk products having smoother body,
higher viscosity, and less syneresis than products made with nonropy strains (Bouzar & al.
Romanian Biotechnological Letters, Vol. 16, No. 6, Supplement (2011) 151

Isolation and characterization of lactic acid bacteria from romanian fermented vegetables
[19]) and are of particular interest in Scandinavian milk products such as viili and langmjolk.
Ropy starter cultures also provide benefits for yogurt production (Broadbent & al. [20]). In
Mexico, where the milk supply routinely faces shortages, yogurt can be made with less total
milk solids if a ropy starter culture is used (Wacher-Rodante & al. [21]).
In fermented vegetables, however, ropiness might be a disadvantage for the
appearance of the final product. It was previously shown that depending on the culture
medium and conditions, some strains can produce excessive ropiness and undesirable
characteristics (Toba & al. [22]).
The polysaccharide caspule formation was investigated for the 139 isolates, by
microscopical observation after staining with Chinese ink. Ten out of the 139 isolates,
randomly selected, were tested for capsule production on different media (MRS, mMRS, ST,
and mST) and at different incubation temperatures (10°C, 37°C, and 45°C). Because no
signifiant differences were observed in the presence/absence of the polysaccharide capsule in
these different conditions, MRS medium was further used for the growth of tested isolates and
incubation was performed at 37°C.
Twenty six isolates showed a clear, large capsule (Fig 2), 72 showed a medium
capsule, 36 showed a small capsule and for 5 of them no capsule was observed. CPS of
different sizes was detected in all mucoid and ropy isolates. It is, however, difficult to make a
precise correlation between the ropy/mucoid phenotype, CPS production and production of
exopolysaccharaides (Van der Meulen & al. [23]).
Fig 2. Capsular polysaccharide sorrounding the cells of the isolate 326
Capsule formation can be found among both nonropy and ropy strains. Streptococcus
thermophilus OR901, a strain isolated from commercial yogurt and able to form ropy strands
from the cell mass, appeared encapsulated using light microscopy with Indian ink staining
(Ariha & al. [24]). This was due to the production of two EPS with the same monosaccharide
composition and branching linkage, but with different molecular mass. The EPS with the
higher molar mass was assumed to affect physical (texture and viscosity) properties of yogurt
and the EPS with lower molecular mass was thought to be located in the capsule, given that it
was only released from the cell surface after sonication (Ariha & al. [24]). Thick-layered CPS
have been shown to protect bacteria against phage infection (Kang & Cottrell, [25]).
However, the involvement of CPS in the phage infection process of LAB is still unclear. The
industrial interest in bacterial strains producing CPS has increased in recent years because the
use of these strains for milk fermentation improves cheese moisture retention and cheesemelting
ability (Broadbent & al. [26]). Thus, substantial efforts have been devoted to
developing methods to optimize CPS biosynthesis.
152 Romanian Biotechnological Letters, Vol. 16, No. 6, Supplement (2011)

SILVIA SIMONA GROSU-TUDOR, MEDANA ZAMFIR
Conclusions
This study was undertaken to isolate and characterize new lactic acid bacteria involved in the
spontaneous Romanian vegetables fermentations. It was proven that fermented vegetables are
a rich source of LAB, 139 isolates being obtained from 20 different fermented products. All
isolates except five were shown to produce capsular polysaccharide and 31 developed mucoid
clonies on sucrose-based media and some of them might be further selected as EPS-producing
strains and tested for their potential applications in food industry (for the production of food
with improved rheological properties) or health (prebiotics).
Acknowledgements
The authors acknowledge their financial support of the Postdoctoral Research Project
PD_33/2010 of the Romanian National Research Plan (PNII-RU). Part of this work was
supported by the project no. RO1567-IBB05/2011 from the Institute of Biology Bucharest of
Romanian Academy.
References
1. E. MIAMBI, J.P. GUYOT, F. AMPE, Identification, isolation and quantification of representative
bacteria from fermented cassava dough using an integrated approach of culture-dependent and cultureindependent
methods. Int. J. Food Microbiol., 82, 111, 120 (2003).
2. S.H. CHAO, Y TOMII, K. WANTANABE, Y.C. TSAI, Diversity of lactic acid bacteria in fermented
brines used to make stinky tofu, Int. J. Food Microbiol. 123, 134, 141(2008).
3. J. NAKAYAMA, H. HOSHIKO, M. FUKUDA, H. TANAKA, N. SAKAMOTO, S. TANAKA, K.
OHUE, K. SAKAI, K. SONOMOTO, Molecular monitoring of bacterial community structure in longaged
nukadoko: pickling bed of fermented rice bran dominated by slow-growing lactobacilli, J. Biosci.
Bioeng., 104, 481, 489 (2007).
4. H.J. KIM, M.I. VAZQUEZ ROQUE, M. CAMILLERI, D. STEPHENS, D.D. BURTON, K. BAXTER,
G. THOMFORDE, A.R. ZINMEISTER, A randomized controlled trial of a probiotic combination and
placebo in irritable bowel syndrome with bloating. Neurogastroenterol & Motility, 17, 687, 696 (2005).
5. L. DE VUYST and E.J. VANDAMME, E.J. (Eds.) Bacteriocins of Lactic Acid Bacteria. London.
Blackie Academic and Professional (1994).
6. L. DE VUYST and F. VANINGELGEM, Developing new polysaccharides. In Mc Kenna, B.M. (Ed.),
Texture in Food –Volume 2: Semi-solid Foods. Woodhead Publishing Ltd. Cambridge, UK, 275, 320
(2003).
7. H.J. RUIJSSENAARS, F. STINGELE, S. HARTMANS, Biodegradability of food-associated
extracellular polysaccharide. Curr. Microbiol., 40, 194, 199 (2000).
8. N. SALAZAR, M. GUEIMONDE, A.M. HERNANDEZ-BARRACO, P. RUAS-MADIEDO, C.G. DE
LOS REYES-GAVILAN, Exopolysaccharides produced by intestinal Bifidobacterium strains act as
fermentable substrates for human intestinal bacteria. Appl. Environ. Microbiol., 74, 4737, 4745 (2008).
9. D.A.A. MOSSEL, I. EELDERINK, M. KOOPMANS, F. VAN ROSSUM, Influence of carbon source,
bile salts and incubation temperature on recovery of Enterobacteriaceae from foods using MacConkeytype
agars. J. Food Protection, 42, 470, 475 (1979).
10. J.C. DE MAN, M. ROGOSA, M.E. SHARPE, A medium for the cultivation of lactobacilli, J. Appl.
Bacteriol., 23, 130, 135 (1960).
11. G. ZARNEA, Tratat de Microbiologie Generală, vol. I şi II; Ed. Academiei Române, Bucureşti.
(1984).
12. R.I. DAVE, N.P. SHAH, Evaluation of media for selective enumeration of Streptococcus thermophilus,
Lactobacillus delbrueckii ssp. bulgaricus, Lactobacillus acidophilus and Bifidobacteria. J. Dairy Sci.,
79, 1529, 1536 (1996).
13. K.P. DIERKSEN, W.E. SANDINE, J.E. TREMPY, Expression of ropy and mucoid phenopypes in
Lactococcus lactis, J. Dairy Sci., 80, 1528, 1536 (1997).
14. F. MOZZI, M.I. TORINO, G.F. DE VALDEZ, Identification of exopolysaccharide-production lactic
acid bacteria. A method for the isolation of the polysaccharide in milk cultures. In: Spencer, F. Ragout,
Romanian Biotechnological Letters, Vol. 16, No. 6, Supplement (2011) 153

Isolation and characterization of lactic acid bacteria from romanian fermented vegetables
A. (Eds.) Food Microbiology Protocols. Methods in Biotechnology. Humana Press Inc., Totowa, NJ
(2001).
15. G.H. VAN GEEL-SCHUTTEN, F. FLESH, B. TEN BRINK, M.R. SMITH, L. DIJKHUIZEN,
Screening and characterization of Lactobacillus strains producing large amounts of exopolysaccharides,
Appl. Microbiol. Biotech., 50, 697, 703 (1998).
16. T. SMITINONT, C. TANSAKUL, S. TANASUPAWAT, S. KEERATIPIBUL, L. NAVARINI, M.
BOSCO, P. CRESCUTTI, Exopolysaccharide-producing lactic acid bacteria strains from traditional
Thai fermented foods: Isolation, identification and exopolysaccharide characterization, Int. J. Food
Microbiol., 51, 105, 111 (1999).
17. J. CERNING, Polysaccharides exocellulaires produits par les bacteries lactiques. In H. de Roissart, &
F.M. Luquet, Bacteries Lactiques. Aspects Fondamentaux et Techologiques., Vol I, 309-329, France
(1994).
18. E.P. KNOSHAUG, J.A. AHLGREN, J.E. TREMPY, Growth associated exopolysaccharide expression
in Lactococcus lactis subspecies cremoris Ropy352. J. Dairy Sci., 83, 633, 640 (2000).
19. F. BOUZAR, J. CERNING, M. DESMAZEAUD, Exopolysaccharide production in milk by
Lactobacillus delbrueckii spp. bulgaricus CNRZ 1187 and by two colonial variants. J. Dairy Sci., 79,
205, 211 (1996).
20. J.R. BROADBENT, D.J. MCMAHON, D.L. WELKER, C.J. OBERG, S. MOINEAU, Biochemistry,
genetics, and applications of exopolysaccharide production in Streptococcus thermophillus. A review, J.
Dairy Sci., 86, 407, 423 (2003).
21. C. WACHER-RODARTE, M.V. GALVAN, A. FARRES, F. GALLARDO, V.M.E. MARSHALL, M.
GARCIA-GARIBAY, Yogurt production from reconstitued skim milk powders using different polymer
and non-polymer forming starter cultures. J. Dairy Res., 60, 247, 254 (1993).
22. T. TOBA, H. UEMURA, T. MUKAI, T. FUJI, T. ITOH, S. ADACHI, A new fermented milk using
capsular polysaccharide-producing Lactobacillus kefiranofaciens isolated from kefir grains, J. Dairy
Res., 58, 497, 502 (1991).
23. R. VAN DER MEULEN, S.S. GROSU-TUDOR, F. MOZZI, F. VANINGELGEM, M. ZAMFIR, G.F
DE VALDEZ, L. DE VUYST, Screening of lactic acid bacteria isolates from dairy and cereal products
for exopolysaccharide production and genes involved. Int. J. Food Microbiol., 118, 250, 258 (2007).
24. H. ARIGA.,T.URASHIMA, E. MICHIHATA, M. ITO, N. MORIZONO T. KIMURA., S.
TAKAHASKI, Extracellular polysaccharide from encapsulated Strecptococcus salivarius spp.
thermophilus OR 901 isolated from commercial yogurt. J. Food Sci., 57, 625, 628 (1992).
25. K.S. KANG, I.W. COTTRELL, Polysaccharides, In H.J. Peppler and D. Perlman (ed), Microbial
technology: Microbial processes, 2nd ed., Vol. 1, Academic Press, Inc., New York, 417, 481 (1979).
26. J.R. BROADBENT, D.J. MCMAHON, C.J. OBERG, D.L. WELKER, Use of exopolysaccharideproducing
cultures to improve the functionality of low fat cheese. Int. Dairy J., 11, 443, 439 (2006).
154 Romanian Biotechnological Letters, Vol. 16, No. 6, Supplement (2011)