UDC 635.1/.6:579.64:579.

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APTEFF, 37, 1-192 (2006) BIBLID: 14507188 (2006) 37, 3-11
Original scientifc paper
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Dr. Gordana R. Dimic, Assoc. ProI., University oI Novi Sad, Faculty oI Technology, 21000 Novi Sad, Bulevar
Cara Lazara 1, Serbia
CHARACTERISTICS OF THE Leuconostoc mesenteroides
subsp. mesenteroides STRAINS FROM FRESH VEGETABLES
Goraana R. Dimic
Strains synthesizing extracellular polysaccharide dextran on a medium with 10%
sucrose were isolatea from aifferent kina of vegetables (cabbage, cucumber, cauliower,
kohlrabi, carrot, green beans, red beet, pepper, eggplant, radish). Carbohydratefermentation bohydratefermentation fermentation
was examined using a bioMerieux API 50 CHL test system. Among micropopulations with
characteristic spherical cell morphology, 94.9% belonged to Leuconostoc mesenteroides
subsp. mesenteroides and 5.1% were identifed as Leuconostoc mesenteroides subsp.
dextranicum. According to fermentation of pentoses L. mesenteroides strains were divi-
ded into three groups with a certain number of biotypes; 10 strains were tested on acid
production.
KEYWORDS: Leuconostoc, vegetable, lactic acid bacteria,
carbohydrate metabolism, Iermentation
INTRODUCTION
The Iermentation oI vegetables is in our country still perIormed in the traditional way
(spontaneous Iermentation), however, the quality oI the fnished product is not uniIorm.
Very oIten, due to low initial number oI lactic acid bacteria, or their low activity and im-
possible control, the result oI the process is uncertain.
Several kinds oI Iacultatively anaerobic lactic acid bacteria participate in the Iermen-
tation oI vegetables. Among these bacteria Leuconostoc mesenteroides subsp. mesenteroi-
des, Lactobacillus plantarum and L. brevis are the most important. They are naturally
present on vegetables.
The genus Leuconostoc is mostly associated with the sliminess oI sugar syrup which
can occur during sugar production (1, 2). The slime is polysaccharide dextran Iormed by
4
polymerization oI glucose units Irom sucrose. Much more important has this group oI
microorganisms in lactic acid Iermentation oI Iood, together with other lactic acid bacteria
(3 - 8).
HeteroIermentative Leuconostoc mesenteroides subsp. mesenteroides (Iurther L. me-
senteroides) disintegrates glucose to D(-) lactic acid and is the most responsible Ior the
creation oI anaerobic conditions, especially in the initial phase oI Iermentation and its
proper development.
The aim oI the work was to determine the strains oI L. mesenteroides and to learn
more about their phenotype characteristics concerning the role oI these bacteria in produc-
tion oI Iermented vegetables.
EXPERIMENTAL
Isolation procedure
The samples oI Iresh vegetables (green beans, caulifower, kohlrabi, cabbage, cucum-
ber, carrot, eggplant, pepper, radish and red beet) were mashed and suspended into liquid
medium with 10 sucrose (50 ml) and incubated Ior 24 to 48 hours at the temperature oI
25C. In the presence oI dextranogenous bacteria the medium became thick and lactic. The
selective medium contained (g/l): consume sucrose 100 g; yeast extract 2.5 g; K
2
HPO
4
5 g;
(NH
4
)
2
SO
4
0.2 g; MgSO
4
7H
2
O 0.2 g; NaCl 0.6 g. Pure cultures were obtained on agar
medium oI the same composition.
Classihcation ana characteri:ation of isolates
The strains were determinated by the procedure described by Garvie (9). Several phe- The strains were determinated by the procedure described by Garvie (9). Several phe- determinated by the procedure described by Garvie (9). Several phe-
notype characteristics including cell morphology, catalase activity, CO
2
production Irom
glucose, and dextran production were studied. The growth in the presence oI 6.5 NaCl
were tested in deep agar.
Pure culture were cultivated on MRS agar Irom lactic acid bacteria in order to inves-
tigate carbohydrates Iermentation. For this purpose the API 50 CHL test was used, giving
the possibility to test 49 diIIerent substrates. Inoculum was prepared with a culture diluted
to match a 2 McFarland turbidity standard (5041 API System, S.A., La Balme les Grottes,
Montalieu-Vercieu, France). All tests were read aIter 24 and 48 hours oI incubation at
30C.
Acid production.
The chosen strains oI L. mesenteroides were plated in liquid medium Ior the Iollowing
oI acid production, which contained: (g/l): tripton 5 g; yeast extract 2.5 g; glucose 15 g;
salt solution A and B 5 ml (salt A- K
2
HPO
4
10 ; KH
2
PO
4
10 ; salt B- MgSO
4
7H
2
O
0.4; NaCl 0.2; FeSO
4
0.2 ; MnSO
4
4H
2
O 0.2 ). During seven days oI incuba-
tion at 25qC, the total acidity and pH oI the medium were determined. The total acidity is
expressed as the titratable acidity and calculated as the lactic acid.
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RESULTS AND DISCUSSION AND DISCUSSION
From diIIerent kinds oI vegetables, 59 Leuconostoc strains synthesizing mucous dex-
tran on sucrose medium were isolated. The production oI dextrans is signifcant only Ior
two species oI this genus, L. mesenteroides and L. dextranicum (10 - 12). On 10 sucrose
agar, the isolates Iormed several types oI mucous colonies, but in general two types could
be defned: a) colonies with clearly expressed macromorphological characteristics - con-
vex, with fat edges, smooth, shiny, semitransparent (Fig. 1a) and b) spread colonies (Fig.
1b). It is known that dextrans obtained with diIIerent strains oI the same species are not
identical in chemical structure.
Fig. 1. L. mesenteroides on 10 sucrose agar:
a) compact colonies; b) spread colonies
The diIIerence in size oI colonies was particularly expressed Ior individual strains.
Diameter oI colonies ranged Ior three days cultures Irom 3.6 to 7.5 mm in average. Spheri-
cally shaped cells are grouped mostly in pairs, shorter and longer chains (Fig. 2a), whereas
at certain strains the aggregation oI cells (Fig. 2b) is noticeable.
All strains showed negative catalase test and produced gases Irom glucose, and 94.9
grow in the presence oI 6.5 NaCl. Considering the ability to disintegrate pentose, these
bacteria possessed the characteristics oI L. mesenteroides, as a key Ior classifcation and
diIIerentiation oI mucus producing Leuconostocs arabinose was used (9, 13). Only 5.1
oI strains isolated Irom vegetables were not able to use arabinose, and did not grow in the
presence oI higher salt concentrations. They belonged to the less tolerant L. dextranicum.
In order to improve phenotypic species characterization oI the genus Leuconostoc
Milliere et al. (14) reported that the tolerance to 6.5 NaCl varied, except Ior L. cremoris,
which could not grow at this concentration. Lactic streptococci which are taxonomically
and ecologically connected with leuoconostocs are excluded, because they do not disin-
tegrate pentose and are salt sensitive. Besides, they Ierment glucose almost completely to
lactic acid (homoIermentative bacteria).
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Fig. 2. L. mesenteroides - micromorphological characteristic (1000):
a) chains; b) aggregation
The investigations oI carbohydrate metabolisms using bioMerieux API system based
on Iermentation oI pentoses: arabinose, xylose and ribose, resulted in dividing the strains
oI L. mesenteroides into 3 groups with certain number oI biotypes. The results are pre-
sented in Tables 1, 2 and 3.
Group A consists oI 5 biotypes with totally 28 strains (Table 1). The characteristic
oI this group is that all strains showed positive reaction to Iermentation oI L-arabinose.
Considering the number oI substrates they disintegrate, Iollowing biotypes A1 are distin-
guished: galactose, mannitol, amygdalin and A5: mannitol, amygdalin, lactose. All these
reactions were negative Ior isolates A3.
Table 1. DiIIerentiating characteristics oI L. mesenteroides strains (group A)
Characteristic A1 A2 A3 A4 A5
Galactose + +
Mannitol + + +
Amygdalin + + + +
Lactose +
D-Rafnose +
Group B is characterized by a smaller number oI strains (23), but by a higher variabil-
ity concerning Iermentation characteristics (Table 2). All oI them, beside L-arabinose, use
D-xylose and ribose. Regarding the Iermentative activity, the biotype B3 was dominant,
and the lowest activity showed B6, with the ability to use only salicine. In comparison the
group A, the participation oI representatives Iermenting lactose was somewhat higher.
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Table 2. DiIIerentiating characteristics oI L. mesenteroides strains (group B)
Characteristic B1 B2 B3 B4 B5 B6
Mannitol +
Amygdalin + +
Arbutin + + +
Salicin + + + + +
Cellobiose + + +
Lactose + + +
-Gentibiose + + + +
Gluconate + +
5 keto-
gluconate
+
The last group C included the strains Iermenting L- arabinose and D- xylose. Five
such strains were identifed (Table 3). According to characteristics they were grouped in
three biotypes, and the most active one was C2.
Table 3. DiIIerentiating characteristics oI L. mesenteroides strains (group C)
Characteristic C1 C2 C3
Amygdalin +
Arbutin + +
Salicin +
Cellobiose + +
Melobiose + +
D-RaIfnose + +
Gluconate +
Amygdalin showed to be mostly Iermented by all three groups L. mesenteroides (Ta-
ble 4). Reactions identical Ior all the strains are not presented. The examination showed
that the strains oI group A were the most Irequently present on green beans (8.93), and
the strains oI group B on caulifower and carrot (Table 5). Similar results Ior lactic acid
bacteria were reported by Valdez et al. (15).
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Table 4. Number oI positive strains with Iermentation ability oI isolated L. mesenteroides groups
Source of
fermentation
Group
A B C
Amygdalin 26 18 3
Arbutin 19 4
Cellobiose 19 4
D-RaIfnose 1 4
Galactose 12
Gluconate 8 1
Lactose 1 13
Mannitol 25 1
Melobiose 4
Salicin 22 3
E-Gentibiose 20
5 keto-gluconate 1
Table 5. Frequence oI L. mesenteroides groups in vegetables
Vegetable Group [%]
A B C
Pepper 5.36 1.78 1.78
Caulifower
5.36 8.93
Cabbage
7.14 1.78
Green Bean 8.93 3.57 3.57
Cucumber 7.14 5.36
Egg plant 7.14
Radish 3.57 1.78
Red Beet 1.78 3.57
Kohlrabi 3.57 5.36 3.57
Carrot 8.93
The acid production by 10 L. mesenteroides strains oI groups A and B is presented
in Table 6. The analysis oI results shows that the highest percent acidity, expressed as the
content oI produced lactic acid, was obtained aIter the frst day oI Iermentation, while the
production was aIterwards slower. The biggest decrease oI pH value was recorded aIter
the frst day, compared to the initial value oI 6.5.
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Table 6. Acid production and pH values oI L. mesenteroides
attained aIter growth Ior 1 and 7 days
Strain 1. day 7. day
Acid (%) pH Acid (%) pH
A1/43 0.50 3.90 0.56 3.70
A1/33 0.50 3.90 0.58 3.85
A1/39 0.36 3.95 0.49 3.90
A4/56 0.50 3.95 0.58 3.70
A4/74 0.52 3.95 0.58 3.70
A5/64 0.50 3.90 0.59 3.75
B1/61 0.52 3.85 0.59 3.70
B1/80 0.38 4.00 0.52 3.70
B1/52 0.40 3.90 0.54 3.80
B3/18 0.40 3.95 0.52 3.90
Initial pH 6.5
Regarding the Iermentative activity, fve strains are standing out, with highest produc-
tion oI acid, 0.59 (A5/64, B1/61) and 0.58 (A1/33, A4/56, A4/74). Strain A1/39 pro-
duced the lowest amount oI acid during the seven days oI Iermentation (0.36 0.49).
CONCLUSION
Research oI Iermentation Ieatures oI lactic acid bacteria is the frst step in the selection
oI strains capable to produce suIfcient amount oI acid. The aim oI Iurther investigation
was to choose the most suitable strains oI L. mesenteroides as starter cultures Ior the con-
trolled Iermentation oI vegetables.
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175-179.
Leuconostoc mesenteroides
subsp. mesenteroides
.
(, , , , , -
, , , , ) -
10 . -
biorieux PI 50 CHL
.
, 94,9 Leuconostoc mesenteroides subsp. mesenteroides,
5,1 Leuconostoc mesenteroides subsp. dextranicum. -
11
L. mesenteroides -
; 10 L. mesenteroides
.
Received 5 May 2006
Accepted 29 September 2006