INTERNATIONAL JOURNAL OF AGRICULTURE & BIOLOGY

ISSN Print: 15608530; ISSN Online: 18149596

09056/AWB/2009/113329332
http://www.fspublishers.org

Full Length Article

Isolation and Characterization of Starter Culture from

Spontaneous Fermentation of Sourdough
M. SAEED1, F.M. ANJUM, TAHIR ZAHOOR, HAQ NAWAZ AND SAJJAD-UR-REHMAN
National Institute of Food Science and Technology, University of Agriculture, Faisalabad-38040, Pakistan
Institute of Animal Nutrition and Feed Technology, University of Agriculture, Faisalabad-38040, Pakistan
Department of Microbiology, Faculty of Veterinary Sciences, University of Agriculture, Faisalabad-38040, Pakistan
1
Corresponding authors e-mail: saeedifst@yahoo.com

ABSTRACT
Wheat flour dough samples were collected from different bakeries located in Faisalabad city, Pakistan. Spontaneous
fermentation of dough samples was carried out for 24 h at 30C in the laboratory. The isolates were Gram stained and the
Gram positive were identified to genus level using morphological, physiological tests. Identification based on classical
identification methods and API 50 CH assimilation profiles showed that the lactobacilli contained in sourdoughs belonged to
three groups (Lactobacillus brevis, L. plantarum & L. fermentum). Yeasts were also characterized on the basis of
morphological and biochemical criteria. Conventional identification methods and API 20 C AUX assimilation profiles
revealed that Saccharomyces cerevisiae was the only yeast species present on the sourdoughs.
Key Words: Sourdough; API 50 CH; API 20 C AUX; Lactobacilli; Yeast

INTRODUCTION
Bacterial starters have been produced for a variety of
fermented products to improve their sensory and other
quality characteristics. Spontaneous fermentation has been
used for the production of fermented foods based on the
microflora present in the raw material (Vogel et al., 2002).
The quality of end-product was dependent on the types and
number of microorganism in the raw material. Spontaneous
fermentation was optimized through back slopping i.e.,
inoculation of the raw material with a small quantity of a
previously performed successful fermentation, which means
dominance of the best adapted strains (Harris, 1998). The
direct addition of selected starter cultures to raw materials is
a milestone in the production of fermented foods, which
may help control the overall standardization of the
fermentation process and quality of the end product. Strains
with the specific physiological and metabolic properties
were isolated from natural habitats or from successfully
fermented products for use in the industrial productions
(Oberman & Libudzisz, 1998).
Lactic acid bacteria (LAB) are a group of Grampositive bacteria, catalase-negative, non-motile, nonsporeforming rods or cocci and produce lactic acid as the
major end product during the fermentation. They are strictly
fermentative, microaerofile, acidophilic, salt-tolerant with
complex nutritional requirements for carbohydrates, amino
acids, peptides, fatty acids, salts, nucleic acids derivatives
and vitamins. The natural habitat of these organisms

includes humans, animals and plants. Their long history of

safe use (Holzapfel et al., 2001), commonly referred to as
the GRAS (Generally Recognized As Safe) status, has led to
a wide range of industrial applications i.e., flavor, texture
and preservative qualities of many fermented foods such as
cheese, yoghurt, sausages, sourdough breads. Several
species belonging to the genera Leuconostoc, Weissella,
Pediococcus, Lactococcus, Enterococcus and Streptococcus
have been isolated from sourdough, but Lactobacillus
strains are the most abundant. The overall flavor of the
bread is affected by the content of lactic and acetic acid.
Homofermentative LAB are able to convert hexoses almost
completely into lactic acid (>85%), whereas
heterofermentative LAB degrade hexoses into lactic acid,
acetic acid/ethanol and CO2. The temperature also affects
the ratio of lactic acid to acetic acid in addition to type of
starter culture. Lactic and acetic acids are also produced by
heterofermentative LAB from pentoses (Hammes & Vogel,
1995). Yeasts are also present in sourdoughs such as S.
exiguous, Torulopsis holmii, Candida krusei, Pichia
norvegensis and Hansenula anomala but S. cerevisiae is
frequently present or added (Gobbetti et al., 1994).
The starter cultures for the production of fermented
products are not presently produced in Pakistan and are
imported for industrial use. Mostly yeasted preferments are
being used for the production of white bread. The use of
LAB as starter culture may help to improve the quality and
shelf life of the products. The LAB of the naturally
fermented sourdoughs may be used in the production of

To cite this paper: Saeed, M., F.M. Anjum, T. Zahoor, H. Nawaz and S.U. Rehman, 2009. Isolation and characterization of starter culture from spontaneous
fermentation of sourdough. Int. J. Agric. Biol., 11: 329332

SAEED et al. / Int. J. Agric. Biol., Vol. 11, No. 3, 2009

aseptically transferred into sterile cryotubes containing acidwashed glass beads and stored at -80C (De Man et al.,
1960).
Isolation and identification of yeasts. Yeasts were isolated
by plating on PDA acidified to pH 3.5. Plates were
incubated for 3 days at 25C and then yeast colonies were
counted. Representative colonies were isolated and purified
by streak plating using the same medium. The yeast isolates
were subjected to various morphological and physiological
tests, which included urease test, growth at 37C, presence
of pseudohyphae using yeast extract malt extract media,
growth and size of vegetative cells in a liquid medium by
direct microscopic examination. Identification was made
with reference to standard descriptions (Harrigan &
McCance, 1990; Beuchat, 1993; Deak, 1993). Yeast isolates
were further characterized for their assimilation patterns
using API 20 C AUX gallery (Biomerieux). The purified
cultures were routinely maintained on PDA slants and kept
at 4C.

novel fermented foods such as sourdough bread, which is

likely to have superior quality and longer shelf life.
Therefore, the present study was designed to characterize
the suitable starter culture for the production of sourdough
bread.

MATERIALS AND METHODS

Sample collection. Fifteen sourdough samples were
collected from different bakeries in Faisalabad, Pakistan
using sterilized jars. The samples were kept at 48C and
transported in special cool boxes within hours to the
Institute of Food Science and Technology, University of
Agriculture, Faisalabad, Pakistan for analysis.
Viable count. A 10 g of sourdough sample was
homogenized with 90 mL of sterile diluent (5 g peptone, 8.5
g NaCl, 1000 mL distilled water, pH 7.00.2). Further
decimal dilutions were prepared with the same diluent. Total
aerobic mesophilic bacteria were enumerated on plate count
agar (PCA, Oxoid, Basingstoke, Hampshire, UK) (Lonner
et al., 1986). MRS (deMan Rogosa Sharpe) agar (Oxoid,
UK) was used for total LAB counts (Man et al., 1960). A 10
mg L-1 of cycloheximide (Merck, Darmstadt, Germany) was
added to the media for prevention of the growth of yeasts
and moulds. Acidified potato dextrose agar (PDA, Oxoid,
UK), pH 3.7, was used for enumeration of yeasts (Okada et
al., 1992).
Isolation and identification of lactobacilli. Presumptive
lactobacilli were selected from higher dilution nutrient agar
plates and tested for cell morphology, Gram reaction and
catalase formation by adding 3% H2O2 directly onto each
plate. The isolates were purified by successive streaking on
the Rogosa agar and MRS agar media before
characterization. The agar plates were incubated
anaerobically (BBL Gas Pak, H2 & CO2; Becton-dickinson,
Cockeysville, MD, USA) for 24 h at 30C for the isolation
of lactobacilli. The Gram positive and catalase negative
strains were subjected to the following physiological and
biochemical tests: gas (CO2) formation from glucose,
arginine hydrolysis, growth in 2%, 4% and 6.5% NaCl,
growth at 5, 15 and 45C (Harrigan & McCance, 1990). The
fermentation pattern among carbohydrates was determined
by using the API 50 CH gallery with the API 50 CHL
medium (Biomerieux, France). Anaerobiosis in the
inoculated tubes was obtained by overlaying with sterile
paraffine oil. The inoculated galleries were incubated at
30C and the observations were made after 24 and 48 h. The
identification of the isolates was facilitated with the use of a
computer program, APILAB PLUS, version 3.2.2.
(Biomerieux) and reference to Bergeys Manual of
Systematic Bacteriology (Wood & Holzapfel, 1995). The
pure bacterial isolates were inoculated into MRS broth,
incubated for 24 h at 30C, centrifuged (Sigma, 3K30,
Germany) at 3000 rpm for 15 min and the supernatant was
decanted. The cell pellets were resuspended in sterile MRS
broth containing 10% (v/v) glycerol. The suspension was

RESULTS AND DISCUSSION

Number of microorganisms. The microbiological
composition of the laboratory fermented sourdough samples
revealed that the total aerobic mesophilic bacteria (TAMB)
count ranged between 7.07105 to 6.89109, the lactic acid
bacteria (LAB) 6.24104 to 6.92107, the yeast 6.35103 to
7.95107 and coliform 3.47101 to 1.67104 (Table I). The
results obtained in the present study are in close agreement
with those reported for sourdoughs by Gobbetti (1998) and
Zinedine (2007).
The sourdoughs are ecosystems, where fundamental
interactions between LAB and yeasts take place. The
predominant organisms are LAB containing significant
numbers of yeast cells (Vogel et al., 1999). LAB are mainly
responsible for the acidification of the sourdough, whereas
the sourdough yeasts are very important for the production
of flavor compounds and for a well balanced flavor in
combination with the acids. The LAB may either originate
from natural flour contaminant, a fermented dairy product or
from a commercial starter culture containing characterized
strains of LAB produced through batch fermentation (Vuyst
& Neysens, 2005). These results suggested that the
sourdoughs tested in the study were contaminated with LAB
and yeasts. Moreover, most of the samples contained LAB
and yeast cells within the range reported by Vuyst and
Neysens (2005).
Composition of lactobacilli and yeast. The presumptive
lactobacilli were randomly isolated from the MRS agar
plates and divided into three groups based on their several
morphological and physiological characters. Lactobacilli
showed a characteristic cell morphology appearing as a
combination of thin, short and long rods in pairs or short
chains. The colonies on MRS agar were irregular, white and
rough sometime possessing a raised centre. Similar findings
have also been reported by Zahoor (2005) who isolated

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ISOLATION AND CHARACTERIZATION OF STARTER CULTURE / Int. J. Agric. Biol., Vol. 11, No. 3, 2009
Table I. Microbiological characteristics of laboratory
fermented sourdoughs (CFU g-1)

Table III. Fermentation of carbohydrates by the

isolates tested by API 50 CH gallery

Sourdough samples TAMB

LAB
7.07105
6.24104
S1
6.36106
5.67106
S2
5
8.1410
7.08105
S3
8.03107
7.15106
S4
9.25105
8.36105
S5
7.72106
6.20106
S6
5.88106
5.03105
S7
7.61108
7.00106
S8
6.77107
5.41106
S9
8
8.4410
5.67106
S10
8.25107
7.08105
S11
6.89109
6.92107
S12
7
7.6410
6.22106
S13
9.01107
6.05106
S14
7.55107
5.03107
S15
CFU = Colony forming units
TAMB = Total aerobic mesophillic bacteria
LAB = Lactic acid bacteria

Group
Sub group
L-arabinose
Cellobiose
Esculin
Galactose
Lactose
D-mannose
Melizitoze
Melibiose
D-raffinose
Saccharose
Trehalose
D-xylose
Rhamnose
Manitol
Sorbitol
Glycerol
Amygdaline
Salicin
Arbutin
Inulin

Yeast
6.35103
7.64104
7.83104
8.41103
8.01103
5.27107
7.95104
6.44105
6.35107
7.64106
7.83106
8.41106
8.01105
5.27107
7.95107

Coliform
1.22102
3.4710
2.90103
6.3510
1.06103
1.15102
2.44103
2.09101
4.53102
1.67104
3.87103
1.58102
2.39102
2.95102
3.62101

I
II
III
A
B C
D
E
+
+
+
d
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
68
+
+
+
+
+
+
+
+
+
82
+
+
+
74
36
62
d
+
+
+
+
+
+
+
+
+
+
+
+
a-Methyl-D-glucoside +
+
+
+
N-Acetyl glucosamine +
+
+
+
-Gentibiose
+
+
D-Turanose
+
+
D-Arabitol
+
+
5-keto-gluconate
+
+
API = Analytical Profile Index
Symbols: +, positive reaction; -, negative reaction; d, weak reaction; 80,
80% strains positive

Table II. Characterization of lactobacilli isolates from

sourdoughs
Group
Subgroup
No. of isolates
Cell shape
Gram staining
Catalase reaction
CO2 from glucose
NH3 from arginine
Growth at 5C
Growth at 15C
Growth at 45C
Growth in 2% NaCl
Growth in 4% NaCl

I
II
A
B
C
17
13
7
Rods
Rods
Rods
+
+
+
+
+
+
80(10) 75(25)
+
+
+
+
+
35(60)
+
Growth in 6.5% NaCl Symbols: +, positive reaction; -, negative reaction; d,
80(10), 80% strains positive, 10% strains weakly positive

III
D
E
9
6
Rods Rods
+
+
+
+
+
+
+
+
+
+
45
weak reaction;

plantarum (belonging to group II) and 8% as L. fermentum

(belonging to group III). The present findings are in line
with those of Gobbetti et al. (1994) who isolated L. brevis
(49%), L. plantarum (21%) and smaller percentages of L.
fermentum with an overall percentage of heterofermentative
isolates of 58% as compared to 54% found in our study. The
results of the present study are also in concordance with the
several Italian (Gobbetti et al., 1994; Ottogalli et al., 1996),
German (Spicher, 1987) and Spanish (Barber et al., 1983)
sourdoughs showing the associations of hetero and
homofermentative strains. The results of the present study
are in contrary to the findings of Corsetti et al. (2001) who
reported that the sourdoughs consisted of either only one
species or a very complex association of several species
belonging to only one metabolic class. Similarly, Wood and
Holzapfel (1995) reported that the L. brevis and L.
fermentum were the dominating microorganisms in
fermenting plant material and sourdoughs. The differences
might be due to variation of the species present in the raw
material used in both studies.
After a preliminary morphological screening, the yeast
isolates were selected and examined through the API 20 C
AUX system. The results indicated that isolates
corresponded to S. cerevisiae, which were in agreement
with several taxonomic studies (Spicher, 1983; Gobbetti et
al., 1994; Gobbetti, 1998). A large number of S. cerevisiae
isolates is certainly related to the addition of bakers yeast to
the dough.

LAB from commercial curd samples. Within lactobacilli,

group I and II, which contained the largest number of
isolates showed growth at 15C and not at 45C but differed
for CO2 production from glucose and NH3 production from
arginine. The group III did not show growth at 15C but
grew at 45C and produced CO2 from glucose and NH3 from
arginine (Table II). The presumptive isolates belonging to
the above groups were further characterized using the API
50 CHL system. Sugar fermentation profiles in the API 50
CH (Table III) indicated that the isolates could be assigned
to three groups corresponding to individual species. In
general, all the strains fermented glucose, fructose, maltose,
ribose and gluconate. None of these fermented erythritol, Darabinose, L-xylose, adonitol, -methyl-xyloside, L-sorbose,
dulcitol, inositol, starch, glycogen, xylitol, D-xylose, Dtagatose, D-fucose, L-fucose, L-arabitol and 2-ketogluconate (Table III).
According to the species description of Spicher and
Schroder (1978), Kandler and Weiss (1986) and Hammes
and Vogel (1995), 46% of the isolates were identified as
Lactobacillus brevis (belonging to group I), 24% as L.

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CONCLUSION
The spontaneously fermented sourdough can be used
as a source for the isolation of starter cultures (L. brevis, L.
fermentum, L. plantarum) and for the production of several
fermented products. The sourdoughs contained both homo
and heterofermentative lactobacilli in coexistence, which
have great potential to impart the particular characteristics in
the final product through fermentation. The benefits of
sourdough incorporation in the bread production demand
that sourdough technology should be used on commercial
scale production of bakery products to improve their quality
and shelf life.

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(Received 16 February 2009; Accepted 19 February 2009)

332