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111







THE EVOLUTION OF LACTIC ACID BACTERIA COMMUNITY DURING
THE DEVELOPMENT OF MATURE SOURDOUGH

Tanja D. ugi-Petrovi, Nataa M. Jokovi and Dragia S. Savi



In order to follow the composition and changes in lactic acid bacteria (LAB) po-
pulation of rye flour sourdough that was continuously propagated by a repeated inocu-
lation, sixty-two strains of LAB were isolated and characterized. The LAB were the only
bacteria detected, both at the end of the second propagation step and in the stage of
mature sourdough (after two weeks of continuous daily refreshment). The stable ecolo-
gical system in rye sourdough could be established from the second propagation step
onward. The predominant genera of LAB during the development of sourdough were lac-
tobacilli, which were grouped in eight clusters. Heterofermentative lactobacilli were in
majority in both propagation step two and a mature sourdough participating 56% and
70% of total bacterial count, respectively. The identification based on a phenotypic cha-
racterization that was carried out by using a set of 36 tests, showed that the lactobacilli
contained in the two sourdough steps did not clearly belong to any known species of the
genus Lactobacillus. In addition, the structure of the bacterial population were monito-
red by two statistical techniques (Hierachical Cluster Analysis and Principal Component
Analysis), being applied to phenotypical characteristics of the isolates.

KEY WORDS: Lactic acid bacteria, sourdough, Hierachical Cluster Analysis, Principal
Component Analysis

INTRODUCTION

Sourdough fermentation is a process in which a mixture of flour and water is fermen-
ted with lactic acid bacteria (LAB) and yeast. It is an ancient way to improve flavor, tex-
ture and microbiological shelflife of bread and has a natural, additive-free image. Both
nutritional and technological quality could be considerably enhanced in cereal foods rich
in dietary fibers by utilizing sourdough.
The LAB included in sourdough fermentation may originate from flour (spontaneous
fermentation), preceding sourdough or a commercial starter culture. Spontaneous sour-
dough fermentation begins with aerobic fermentation immediately upon mixing flour and

Tanja D. ugi-Petrovi, B.Sc., Nataa M. Jokovi, M.Sc., Dr. Dragia S. Savi, Prof., savic@junis.ni.ac.rs,
Laboratory for Food Science and Biotechnology, Faculty of Technology, Bulevar oslobodjenja 124, 16000
Leskovac, Serbia
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water. Once oxygen is depleted, anaerobic fermentation begins with the growth of LAB
and yeasts. The production of acids by LAB enables their rapid growth when the pH
value has dropped to low for other microorganisms to grow. So, the LAB become the
most abound microflora in the sourdough, and they are therefore responsible for the final
stages of sourdough (1, 2). This type of processing is still the basic practice for preparing
so-named predoughs. When the fermented dough is used as an inoculum for a succeeding
fermentation run, the adaptation of the microbial assotiation to the process becomes
greater (2).
The microflora of spontaneously fermented dough depends on the microflora of raw
materials, and is variable in terms of kind, origin and storage conditions of the flour, as
well as the technological parameters of the fermentation process applied. The impact of
these parameters during a continuous propagation of sourdough causes the selection of a
characteristic microbiota (3). The wholemeal rye flour may contain 10
4
-10
6
cfu of unspe-
cified bacteria per gram (1, 4, 5), in which 10
2
- 10
3
cfu g
-1
belong to LAB (5). According
to our previous research (1), LAB participated in one third of rye flour bacterial
populations were detected on MRS agar. In mature sourdough, LAB ranged between 10
7

and 10
8
cfu g
-1
(6).
Microbiological studies have revealed that more than 50 species of LAB occur in
sourdough (2). Sourdough LAB originate generally from the genera Lactobacillus,
Leuconostoc, Pediococcus or Weissella and the majority of the strains belong to the ge-
nus Lactobacillus (3). Since sourdough is a suitable substrate for most lactobacilli, more
than half of the species in this genus occur in sourdoughs or in related cereal fermen-
tations (7). Homofermentative (Lb. acidophylus, Lb. delbrueckii subsp. delbrueckii, Lb.
amilovorus), as well as heterofermentative (Lb. sanfranciscensis, Lb. panis, Lb. pontis)
lactobacilli, were found in rye spontaneously fermented sourdoughs (8, 9).
The isolation of the strains from microbial communities and their identification is still
a necessary approach when the characterization of physiological and technological pro-
perties of microbial community members is of interest, as in the case of sourdoughs.
Sourdoughs are microbial systems that have not been thoroughly investigated and new
species of LAB may occur in this ecosystem due to the continuous propagation in the
sourdough system (9). In this paper, the composition and changes in LAB microflora of a
rye flour sourdough that was continuously propagated by a repeated inoculation, were
examined. The results represent the resumption of those previously obtained (1). Statis-
tical procedures applied to physiological characteristics of the isolates were used to re-
present the composition of LAB and detect physiological factors affecting their differen-
tiation.

MATERIALS AND METHODS

Sourdough production and propagation

Doughs were made by mixing 100 g rye flour ("itopromet" Zajear, Serbia with
11.7 % and 0.99 % water and ash content, respectively) and 60 cm
3
of sterile tap water in
aseptic conditions (prior to work a mixer dish and blades were moistened with ethanol
and flamed). Sourdough formation started by a spontanous fermentation followed by se-
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veral propagation steps using 10% of previously fermented dough (calculated on a total
dough weight) as inoculum for the next fermentation. Each of the sourdoughs were
fermenting for 24 h at 30
o
C.

Enumeration and isolation of lactic acid bacteria

For isolation end enumeration of the bacteria, 10 g of dough were homogenized with
90 cm
3
of sterile 0.85% saline. Serial dilution was spread plated on MRS (Torlak, Bel-
grade, Serbia) and incubated under the anaerobic incubation (GasPak, BBL, Cockeys-
ville) at 30
o
C for 48 h.
The isolation of bacteria was done both at the end of the second propagation step and
after two weeks of a daily refreshment (the mature sourdough stage). After enumeration,
the colonies were randomly isolated from MRS plates, transferred to MRS broth and,
after the incubation (48 h, 30
o
C), purified three times by streaking on the MRS agar and
then checked for morphology, Gram stain and catalase test (determined by transferring
fresh colonies from agar medium to a glass slide and adding 5% H
2
O
2
). Each of Gram
positive and catalase negative cultures were separated for further examination.

Physiological characterisation of lactic acid bacteria

A set of 36 tests (including morphology, Gram staining characteristic and a catalase
test) was used to clasify the isolates. The tests used to determine catalase activity, gas
production, arginine and esculine hydrolisis, the ability to acidify, cloth and reduce lac-
mus milk (1%), growth at different temperatures (15
o
C and 45
o
C) and the concentration
of NaCl (4, 6.5 and 8%) were performed by using previously described methods (6, 10).
Other tests were observing the growth on entero and citrate agar (HIMEDIA, Mombai,
India) and the ability to form diacetyl (11) and exopolysaccharides (formation of slimy
colonies on MRS agar with sucrose as carbon source, 20 g/dm
3
).
Acid production from carbohydrates (L-arabinose, D-xylose, galactose, mannitol, tre-
halose, mannose, raffinose, lactose, maltose, sucrose, glucose, fructose, rhamnose, sorbo-
se, ribose, salicin, cellobiose, melibiose, inuline, sorbitol, Sigma) was evaluated by the
procedure as follows: filter sterilized solution of sugar was added to basal MRS medium
(without glucose) and with 0.16 g/dm
3
bromcresol-purple to a final concentration of 10
g/dm
3
. The filter sterillized (0.22 m, Millipore, Saint-Quentin, France) sugar solution
was dispensed (0.9 cm
3
) in the microtube (1.5 cm
3
). The cells suspension (0.1 cm
3
) obtai-
ned by centrifuging 5 cm
3
of 16-h old MRS broth culture and resuspension of the sedi-
ment in 5 cm
3
sterile saline was used to inoculate microtube. The apperance of yellow
color in medium after the incubation (48 h at 30
o
C) was considered as a positive result.

Determination of TTA and pH

Total titrable acidity (TTA) and pH of sourdough were determined on an aliquot of 10
g sourdough, blended with 90 cm
3
destilled water. The pH of this suspension was deter-
mined by using HANNA HI 9025 meter. For TTA determination, the same aliquot was
titrated against 0.1M NaOH to final pH 8.5. TTA was expressed as the amount (cm
3
) of
NaOH used.
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Statystical analysis

The relationship among the isolated strains was determined by Hierachical Cluster
Analysis (HCA) and Principal Component Analysis (PCA) using Statistica 7.0 (StatSoft
Inc. USA) for Windows. The results of phenotypical tests were coded as + positive, - ne-
gative or +- weak positive or delayed (positive after 7 days of incubation) reaction. For
morphology, three codes were used cocci, + long bacillus cells and +- common bacillus
cells. HCA was carried out using the algorithm Unweighed Pair-Group Average Link-
age Analysis. Distances between the clusters were assessed using Percent of dissagre-
ement and its translation in similarity level assuming that 0% disagreement=100% si-
milarity. PCA was implemented using the state-of-the-art algorithm known as NIPALS
(None Linear Iterative Partial Least Squares).

RESULTS AND DISCUSSION

The total cell count of the continuous rye flour fermentations was determined over 3
propagation steps (PS) and in the stage of mature sourdough (MD) after two weeks of
daily propagations. The additional characterization was achieved by the pH and TTA
measurement at the end of the first three propagation steps, as well as in mature sour-
dough (Figure 1). The significant increase of the number of bacteria was observed only at
the end of the first 24 h fermentation after flour and water mixing, reaching the level of
ca 8 log cfu g
-1
which varied slightly during the propagation period and the formation of
the MD (Figure 1). The pH values that reached 3.7 at the end of PS2 were maintained till
the end of the study, and TTA were ca 13 from the second refreshment onward (Figure
1), thus demonstrating the acid production in the sourdough.


Fig. 1. The kinetics of pH (), TTA () and the number of lactic acid bacteria () in start
dough (SD), sourdough et the end of propagation steps (PS1, PS2 and PS3) and mature
sourdough (MD) after two weeks of continuous daily propagations

The acidity, the viable count, and the composition of LAB microflora during repeated
succeeding spontaneus fermentations were related to the first 2-propagation step. The lac-
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tic acid bacteria did not dominate microflora in the initial dough (as a consequence of not
dominating in rye flour, as well), making one-third of bacterial population that can be
detected on MRS agar (Figure 2A), but their acid resistance enabled them to continue
growing during a natural sourdough fermentation. The LAB dominated the entire sour-
dough microflora (ca 80%, Figure 2A) at the end of the first propagation step, but maxi-
mum values of acidification were not achieved yet. Maximum acidification was achieved
and varied slightly after the second propagation steps (Figure 1) when LAB were the only
bacteria found in sourdough (Figure 4A). The number of bacteria found in sourdough
(8.0-8.7 log cfu g
-1
) during all propagation steps was in accordance with previously pu-
blished results where counts of soudoughs LAB determined by enumeration on MRS ran-
ged between 10
7
-10
8
(6, 9). It can be assumed that the stable ecosystem of mature sour-
dough could be established from the second propagation step onward, when the pH and
total titrable acidity of sourdoughs were ca 3.5 and ca 13, respectively as the consequence
of the action of LAB that were at the level of 8.0-8.7 log cfu g
-1
.



Fig. 2. Participation of LAB in microflora determined on MRS (A), and particule genera
in lactic acid bacteria population (B) in rye flour - RF, propagation steps 1 - PS1 (1) and
2 - PS2 and mature sourdough - MD

On the basis of the tests applied, sourdough anaerobe and/or facultative anaerobe iso-
lates fall within well-recognised LAB genera. The strains of genera Enterococcus found
in rye flour remained in the first two propagation steps, and the participation of entero-
cocci in LAB count were significantly reduced from 70% in rye flour to 3% in PS2, and
were not detected in MD (Figure 2B). Some enterococi were reported as common inhabi-
tants of vegetables (12) and were isolated from rye flour (E. faecium, E. avium, E. casse-
liflavus and E. durans - 4) and sourdoughs (4, 6).
Greater LAB diversity was observed at the end of the first 24 h fermentation and 6
genera were detected: Enterococcus, Streptococcus, Leuconostoc, Weisella, Pediococcus
and Lactobacillus (1). It was shown (16) that in the sourdough obtained by continuous
daily refreshments at 30
o
C, subdominant LAB such as E. faecium and P. pentosaceus are
stronger acidifiers than Lb. sanfranciscensis at the beginning of the sourdough produc-
tion. Those species inhibiting indigenous microorganisms other than LAB by lowering
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the pH, might prepare the environment for the establishment of typical species (e.g.
Lactobacillus spp.) of mature sourdoughs. Their most crucial role was played at the be-
ginning of the sourdough production (13). Nevertheless, once lactobacilli colonized
dough this determined a rapid decrement of LAB cocci which remained at subdominant
concentrations or at the concentration that enabled their detection (13). There is no work
reporting on the prevalence of enterococci strains in the sourdough environments at the
stage of mature sourdough.
After the first propagation step, lactobacilli were detected totaling up to one-third of
the entire LAB community and all of these were homofermentative. After the second
refreshment, lactobacilli comprised the greatest group of LAB (97%), while MD micro-
flora consisted only of the strains belonging to the genus Lactobacillus (Figure 2B). This
confirmed the already published reports (9, 6, 14) that rye and wheat sourdoughs micro-
bial populations were dominated mainly by bacteria of the genus Lactobacillus. The he-
terofermentative lactobacilli were first detected at the end of PS2 (56%) and in MD par-
ticipate with 70 % of bacterial count. With the increasing fermentation time, a shift
towards the predominance of hetrofermentative lactobacilli was reported in rye sour-
dough (14). A rather stable association of lactobacilli was a result of the selective pres-
sure exerted by the environmental conditions through a continuous propagation, adding
flour and water at regular intervals (14).
The identification of lactobacilli strains was not possible because they did not con-
form to any species description. A high percentage of disagreement was accounted bet-
ween our isolates and the obligately homofermentative (Lb. amilovorus and Lb. acido-
phylus), facultatively heterofermentative (Lb. plantarum) and obligately heterofermen-
tative (Lb. sanfransciscensis, Lb. brevis, Lb. fermentum, Lb. fructivorans, Lb. pontis, Lb.
panis) lactobacilli commonly isolated from sourdoughs. The main goal of this paper was
not to fully identify the isolates, since it is well known that it is very difficult to distin-
guish lactobacilli by their physiological properties (9, 16), especially in fermentations of
non-sterilized substrates. Environmental parameters lead to a well-adapted, stable flora
within this dough, and physiological properties could not be assorted to reference orga-
nisms from other habitats as a result of adaptation to different environments.
Statistical procedures based on phenotypic properties have been commonly used for
the analysis of sourdough microbial communities (6, 16). The techniques appeared to be
very effective in representation of the composition and a relative position of LAB com-
munities. In fact, in most papers statistical analysis was based on clustering of isolates
and on the presentation of distribution of isolates in graphical formats. This kind of re-
presentation had significant advantages, allowing a direct comparison between the groups
and detecting physiological factors affecting the differentiation.
Thirty-two and thirty strains were isolated at the end of PS2 and MD, respectively.
All of these were Gram-positive and catalase-negative, and all, but one, were rod-shaped.
The one remaining LAB isolate was coccus-shaped.
The similarity among strains of LAB isolated from PS2 and MD, as well as from
propagation step 1 (our previous work, 1), is summarized in the dendrogram shown in Fi-
gure 3, while the phenotypic characteristics of the clusters are summarized in Table 1.
Nine diferent LAB clusters were identified at the 80% similarity level. Within the
clusters, the profiles were not identical but very similar.
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Fig. 3. Dendrogram of similarities based on phenotypic tests of 80 LAB strains isolated at
the end of propagation steps 1 (1) and 2, and from mature sourdough. Note: Grouping
was performed by Hierachical Cluster Analysis using Unweighed Pair-Group Average
Analysis algorithm

All isolated lactobacilli grown at 45
o
C produced acid on glucose, maltose and sucrose
and could not be grown on entero agar (Table 1). The heterofermentative lactobacilli of
PS2 and MD dominated LAB microflora with 56% and 70% of total bacterial count,
respectively. Only the homofermentative lactobacilli of cluster VII showed the imposi-
billity of growing both in milk and fermenting lactose (Table 1). Eight strains of cluster
VIII were the only lactobacilli that could grow in the presence of 6.5 % and 8 % NaCl. It
was not possible to identify lactobacilli isolates on the species level scoring the results of
the tests applied with the species literature description (6, 8, 17).
One of two lactobacilli types of isolates from PS1 clustered together with isolates
from PS2 and MD with ca 80% interspecies similarity level (cluster VII on Figure 3 and
Table 1). The lactobacilli of this cluster from PS2 and MD explicitly differed from those
from PS1 (1) in producing acid from mannitol, as well as in disability to grow in the pre-
sence of 8% NaCl (Table 1). Another lactobacilli cluster from PS1, containing only two
isolates, was separated in cluster I and showed to be most related to cluster II (ca 70 %
similarity level) consisting of five isolates from PS2.
The coccoid isolate from PS2 deemed to belong to genus Enterococcus according to
its ability to hydrolise esculine and to grow at 15
o
C and 45
o
C, as well as to grow in entero
agar and in the presence of 6.5% NaCl (12, 18, 19). The identification of enterococcus
was not possible because they could not be related to known species description. Ente-
rococci isolated from PS1 and PS2 were comprised in one cluster with ca 78% interspe-
cies similarity level (Figure 3). When compared to those from PS1 (1), the enterococci
isolated from PS2 showed to be different in fermentation mannose and raffinose.
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Table 1. Physiological and biochemical characteristics of the clusters of the lactic acid
bacteria isolates from sourdoughs after propagation step 1 (1) and 2 and from mature
sourdough

Cluster No I II III IV V VI VII VIII IX
Number of LAB
1
2+0+0 0+5+0 0+4+6 0+5+8 0+6+5 0+3+2 8+5+4 0+3+5 8+1+0
Morphology
2
B B B B B BL B BS C
CO
2
from glucose - - + w w w - - -
Growth at 45
o
C + + + + + + w* w +
15
o
C - - -* - - + + + +
Arginine hydrolysis - - - + + + - - +
Esculin hydrolysis - - -* - w* - - + +
Growth with
4%NaCl
+ + +* -* + + + + +
6.5%NaCl - - - - - - w* + +
8%NaCl - - - - - - -* + -
Growth in milk
3
+a +ac -a* +acr* +acr* - -* +acr +acr
Entero agar - - - - - - - - +
Citrate agar - - - - - + -* + -*
Diacetile - - - - - - -* -* +*
EPS production - - - - - r - r -
Acid from
L-arabinose + + + + +* + - - w
D-xilose - w - -* w + - - -
galactose + + + +* + w + + +
mannitol - w - - + - +* + +*
trehalose + - - - +* - -* - +*
mannose +* - - - - - + + +*
rafinose + + + + + + - - +*
lactose + + + + + + - w +*
fructose + + - - + - + + +
rhamnose - - - - - - - - -
sorbose - - - - - - - - -
ribose - - -* w* w + -* +* -
salicine -* - - - - - - - +
celobiose w +* - - + - - + +
melibiose w w + +* + w - - +
inuline w w* - - +* - -* + +
sorbitol - - - - - - - + -
+ positive, - negative, w - weakly positive, All strains produce acid from maltose, sucrose and glucose.
1
the first number represents number of isolates from propagation step 1, the second from propagation step 2, and the third
from mature sourdough
2
C: cocci, B: rods, BL: long rods
3
acidification (a), clothing (c) and reducing (r) of lacmus milk, 1%
* - properties differ among strains of the same type

In addition, PCA was performed in order to evaluate the similarity among lactobacilli
isolates and detect physiological characteristics affecting their differentiation. The phy-
siological characteristics for isolated strains showed that four principal components (PC)
were able to explain 50 % of the variance (data not shown). The plot of the first two
components (Figure 4) made it possible to separate the 71 lactobacilli strains (10 from
PS1, 31 from PS2 and 30 from MD) into distinct groups (Figure 4A). Enterococus cluster
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was explicitly separated from all lactobacilli (data not shown) and was not included in
further statistical analysis.



Figure 4. Scores plot (A) and loadings plot (B - the loadings less than 0.5 not presented)
from PCA of the physiological properties of lactobacilli isolated at the end of propagation
steps 1 (1) and 2, and from mature sourdough Note: The explained variance by PC1 and
PC2 were 21 % and 12 %, respectively. Numbers represent clusters from Figure 2

The clusters formed by HCA analysis (Figure 3) can be observed on plots from the
PCA (Figure 4A). Each cluster of strains was represented by the prediction interval
ellipse for its samples, as a measure of dispersion. The coordinates for the ellipse were
computed from the data and a number of observations given, and showed the prediction
interval for a single new observation (13). The prediction interval ellipse desribes the area
in which a single new observation can be expected to fall with the probability of 90 % (a
coefficient that controlled the ellipse was chosen to be 0.9). As shown in Figure 4A,
lactobacilli from clusters I and II formed by HCA could not be clearly separated by PCA
and made a single ellipse (I-II) representing related isolates.
The Principal component 1 (PC1) that explained 21 % of total variance in the data,
clearly separated clusters VII and VIII from other lactobacilli types, while PC2 (account
12 % of total variance) separated clusters I-II, III, VI and VII from clusters V and VIII
(Figure 4A). According to Figure 4B, PC1 distinguished the strains according to CO
2

production, growth at 15
o
C and 45
o
C, growth in medium containing 6.5 % NaCl, as well
as the acid production from mannose, raffinose, arabinose, fructose and mannose. PC2
differentiated the strains according to their growth in milk and fermentation of celobiose.
Only PC3 which explained 15% of the variation (data not shown) was defined by the
cell shape (long or common rods).
The growth in medium supplemented with 4% NaCl and acid formation from galac-
tose, trehalose, sucrose, galactose, salicin and ribose, as well as growth on citrate agar,
had low loadings on PC1 and PC2 (close to zero). Therefore, it could be concluded that
these properties had no significant influence on the attained classification of isolates. Ne-
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vertheless, the ribose and citrate fermentation had a significant influence on PC3 and
PC4, respectively (data not shown) and they could not be omitted from further studies of
the classification of isolates.
The lactobacilli of cluster VII were assembled in the upper left part of the graph (Fi-
gure 4A) that displayed homofermentetative bacilli with the ability to ferment fructose
and mannose but not lactose, raffinose, melibiose, xilose and arabinose (Figure 4B, Table
1). The strains grouped in the lower left part of the graph represented cluster VIII. The
isolates from this cluster showed growth in 8% NaCl, with the ability to hydrolyze escu-
lin, ferment inuline, and some strains had the potential to produce diacetile (Figure 4B,
Table 1).
Six strains isolated from PS2 and five strains from MD were located in the lower
right part of the graph (cluster V). They displayed a high activity in milk and arginine
hydrolysis. Two thermophilic isolates from PS1 and the majority of strains isolated after
PS2 (clusters I-II, III, IV and VI) which were located in the upper right part of the graph,
were characterized by disability to grow in 8% NaCl, to ferment sorbitol and to form
diacetyl.

CONCLUSION

In this study, the statistical analyses provided indices for the evaluation of the distan-
ces among the population members and their differentiation. Principal component 1 from
PCA succeeded in differentiating homo- from heterofermentative isolates with the excep-
tion of only cluster I-II. This cluster comprised homofermentative lactobacilli and was
located among other heterofermentative clusters (III, IV, V and VI). PCA differentiated
strains on the base of, mainly, pentose fermentation (Figure 4B) and the bias was intro-
duced because the strains from cluster I-II ferment arabinose (Table 1). Although the
strains may differ, pentoses (arabinose, xylose or ribose) were usually fermented by obli-
gately heterofermentative and seldom by homofermentative lactic acid bacteria. Further
investigation of the isolates (for example DNA-DNA hybridization, rRNK sequencing
and GC spectrum) is necessary to reveal whether the isolates belonged to some hitherto
unknown species of the genus Lactobacillus.

ACKNOWLEDGEMENT

This research was supported under the project PTR 2042 by the Ministry of Science
and Environmental Protection of the Republic of Serbia.

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APTEFF,40, 1-220 (2009) UDC: 664.654.1:664.64.016.3/.7
DOI: 10.2298/APT0940111Z BIBLID: 1450-7188 (2009) 40, 111-122
Original scientific paper
122



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Received 28 August 2009
Accepted 6 October 2009