Process Biochemistry 40 (2005) 691697

Sourdough bread production with lactobacilli and S. cerevisiae isolated from sourdoughs
Hlya Gl a , Sami zelik b , Osman Sa d c, , Muharrem Certel d g
b a Agricultural Faculty, Department of Food Engineering, University of ukurova, 01330 Adana, Turkey Agricultural Faculty, Department of Food Engineering, University of Sleyman Demirel, 32260 Isparta, Turkey c Engineering Faculty, Department of Food Engineering, University of Erciyes, 38039 Kayseri, Turkey d Agricultural Faculty, Department of Food Engineering, University of Akdeniz, 07070 Antalya, Turkey

Received 12 November 2003; received in revised form 29 December 2003; accepted 30 January 2004

Abstract The interactive effects of lactic acid bacteria (LAB) and yeasts were identied through isolation of sourdough sponges. Fourteen sourdough samples were collected from different bakeries in Isparta. Carnobacterium divergens (Lactobacillus divergens) (6.1%), Lactobacillus brevis (15.1%), Lactobacillus amylophilus (6.1%), Lactobacillus sake (6.1%), Lactobacillus acetotolerans (6.1%), Lactobacillus plantarum (3.0%), Pediococcus pentosaceus (6.1%) and P. acidilactici (6.1%), Tetragenococcus halophilus (Pediococcus halophilus) (3.0%) were isolated from sourdoughs as LAB while Saccharomyces cerevisiae (27.0%), S. delbrueckii (2.7%), Torulopsis holmii (10.8%) and T. unisporus (2.7%) were also isolated from sourdough sponges as yeasts. Seven kinds of bread were made with the inoculation 1.5% S. cerevisiae and/or 1.5% lactobacilli (Lb. amylophilus, Lb. brevis, Lb. plantarum, Lb. sake and Lb. acetotolerans). Six kinds of bread were produced with the mixture of the above ve different LAB and S. cerevisiae, and the seventh bread made with S. cerevisiae (commercial culture) alone as control bread. The sample prepared with 1.5% Lb. amylophilus and 1.5% S. cerevisiae showed the best results on rheological properties of bread while that with 1.5% mixed LAB and 1.5% S. cerevisiae was the poorest. LAB used in sourdough breads increased shelf life and delayed staling. In sensory analyses, 1.5% Lb. sake and 1.5% S. cerevisiae application were preferred well by the panel. 2004 Elsevier Ltd. All rights reserved.
Keywords: Sourdough; Lactic acid bacteria; Yeasts; Bread making

1. Introduction Sourdough is an acidic sharp tasting mixture of our and water for making bread from cereal ours [1]. Sourdough bread is a traditional product with a great potential which can only be achieved if the interactions between the lactic acid bacteria (LAB) and yeasts that populate the sourdough are understood [2]. The major focus of recent studies on cereal fermentations was the characterisation of sourdough fermentations based on wheat or rye our as raw materials [3]. Wheat and rye sourdoughs are ecosystems where fundamental interactions between LAB and yeasts take place. As a general rule, LAB are the predominant organisms although
Corresponding author. Tel.: +90-352-4374937; fax: +90-352-4375264. E-mail address: o sagdic@yahoo.com (O. Sa d). g 0032-9592/$ see front matter 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2004.01.044

in many cases yeast cells are also present in signicant numbers [3]. Most LAB isolated from sourdough belong to the genus Lactobacillus, but species of Pediococcus, Leuconostoc and Enterococcus are occasionally found or used in sourdough processes [2,4]. Lactobacillus sanfranciscencis (synonym Lb. brevis ssp. lindneri), Lactobacillus plantarum and Lactobacillus brevis are frequently isolated from sourdough [2]. Several species of yeasts are also found in sourdoughs; Saccharomyces cerevisiae is frequently present or is added. In particular, S. exiguus, Torulopsis holmii, Candida krusei, Pichia norvegensis and Hansenula anomala are generally found in yeast genera from the sourdoughs [5]. Sourdough fermentation is generally evaluated by the measurement of parameters such as pH, acidity and microora [6]. Bread produced with spontaneous sourdoughs with low pH and a high ratio of lactic and acetic acids have the highest volumes and the lowest rates of staling during storage [7,8]. Sourdough LAB and yeasts have been shown

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to compete for carbon sources which inuence acid production by bacteria. Acidication of the dough, proteolysis of gluten and moderate hydrolysis of starch are LAB activities which vary among sourdough strains and which may affect the physicochemical changes throughout shelf life of bread [8,9]. Some researchers have studied the physicochemical and microbiological characteristics of sourdoughs and sourdough bread [1,6,815]. In Turkey, sourdough is generally prepared using wheat our [16]. In this study, LAB and yeasts were isolated from sourdough samples obtained from Isparta province in Turkey. The appropriate isolates of LAB and yeasts were then used for bread production. The objective was to study various combinations of LAB and yeast starters and to observe their inuence on certain characteristics of sourdough bread. 2. Materials and methods 2.1. Collection of samples Fourteen sourdoughs were obtained from different bakeries in Isparta, Turkey. Samples were aseptically collected into sterilised jars. The collected sourdough samples had been fermented for bread production by bakeries. The wheat our samples used for bread production were obtained from the local markets in Isparta. Combinations of microorganisms isolated from the sourdoughs and commercial yeast (Compressed bakers yeast, from Pakmaya, Turkey) were used as starter cultures for the experimental breads (Table 1). For bread production, the dough were prepared with type V wheat our. According to Turkish Standard (TS 4500) [17], type V our contains 1.20% of ash and 8.5% of dry gluten with 0.08% acidity.

2.2. Production of experimental breads A conventional straight-dough baking procedure was conducted [15]. An amount of 1.2% NaCl and 1.5% (w/w) yeast and 1.5% (w/w) lactic acid bacteria were added to each 100 g our and mixed at 60 rpm for 2025 min. The amount of water was adjusted according to the water absorption determined by a farinograph. The dough was left for bulk fermentation for 1 h at 30 1 C and 75% relative humidity. At the end of the fermentation time, the dough was divided in to 100 g pieces and each piece was rounded before moulded by hand. The moulded dough pieces were proofed for 1 h at 301 C and 85% relative humidity before baking at 240250 C for 3540 min in a rewood-heated oven. The seven bread groups were produced and coded as T1, T2, T3, T4, T5, T6 and T7 (control). The experimental breads were produced at Degirmenci Bakery Plant in Isparta, Turkey. 2.3. Physicochemical analyses The pH values of the sourdough samples were determined electrometrically with a WTW 526 pH meter (WTW Instruments, Germany). Titratable acidity (% lactic acid) was measured as suggested by Uluz [18]. Fermentation time (FT) was experimentally applied by bakeries and is shown in Table 2. Moisture and total solid contents were determined by heating the sample in an oven at 105 C until a constant weight was obtained. The yields of dough, yields of bread and volume of bread and specic volume of bread were determined according to Elgn et al. [16] and Anon. [19]. The penetrometer (FT-327, USA) values of breads were measured after 24, 48 and 72 h of bread production. All analyses were performed in duplicate.

Table 1 Combination, acid production ability and ratios of starters used in bread making Codes of breads T1 T2 T3 T4 T5 T6 Combined starters Lb. amylophilus EL 1 S. cerevisiae EM 2 Lb. brevis EL 2 S. cerevisiae EM 2 Lb. plantarum BL 2 S. cerevisiae EM 2 Lb. sake YL 2 S. cerevisiae EM 2 Lb. acetotolerans TL 1 S. cerevisiae EM 2 Lb. amylophilus EL 1 Lb. brevis EL 2 Lb. plantarum BL 2 Lb. sake EM 2 Lb. acetotolerans TL 1 S. cerevisiae EM 2 S. cerevisiae Acid production ability (pH) 3.23 3.74 3.25 3.63 3.38 3.23 3.74 3.25 3.63 3.38 Ratios (%) (LAB + yeast) 1.5 + 1.5 1.5 + 1.5 1.5 + 1.5 1.5 + 1.5 1.5 + 1.5 1.5 (LAB) + 1.5 (yeast)

T7 (control)

1.5 (commercial yeast culture)

H. Gl et al. / Process Biochemistry 40 (2005) 691697 Table 2 Some physicochemical and microbiological characteristics of sourdough and dough samples after fermentation Samples FT (h) pH Acidity (%) Total solid (%) Counts of microorganisms (log10 cfu g1 ) Total bacteria 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Mean S.D. 6.0 6.0 6.5 6.5 12.0 18.0 7.0 3.5 11.0 8.0 4.5 4.5 12.0 12.0 8.39 4.05 4.24 4.16 3.66 3.68 3.70 3.95 3.82 3.96 3.65 3.94 5.55 5.30 3.99 3.96 4.11 0.59 5.0 5.6 6.4 6.4 12.7 9.0 14.0 7.6 7.1 5.4 5.6 4.2 13.6 14.0 8.33 3.65 38.3 53.0 54.0 58.0 59.0 53.2 56.0 58.0 58.0 61.3 44.0 49.5 50.0 50.0 53.02 6.31 5.97 8.36 7.41 7.57 6.66 8.12 7.15 7.01 7.86 8.05 8.83 9.09 9.54 9.57 7.94 1.07 Yeasts 7.31 8.46 7.24 7.62 6.33 8.40 7.15 9.96 7.85 7.95 8.77 9.11 8.88 8.96 8.14 0.97 LAB 5.72 7.26 5.28 5.79 7.94 8.36 7.44 6.81 5.81 8.59 7.26 7.21 9.66 9.57

693

7.34 1.39

S.D.: standard deviation.

2.4. Microbiological analysis A 10 g of sourdough sample was homogenised with 90 ml of 0.85% (w/v) sterilised NaCl (Merck, Darmstadt, Germany) solution by use of a Waring (Waring Commercial Blender, USA) blender. Further decimal dilutions were prepared with the same diluents. Total aerobic mesophilic bacteria (TAMB) were enumerated on plate count agar (Merck, Darmstadt, Germany) following the pour-plate method and incubated at 30 C for 72 h [4]. Yeast and moulds were determined on Malt Extract Agar (Oxoid, UK) acidied with 10% lactic acid (Merck, Darmstadt, Germany) to pH 3.5 following the surface plate method, with incubation at 25 C for 3 days. LAB were counted on MRS agar (Merck, Darmstadt, Germany) using the pour-plate method after incubating at 30 C for 3 days [20], 10 ppm of cycloheximide (Merck, Darmstadt, Germany) was added to the media for prevention of the growth yeasts and moulds [10]. 2.5. Isolation and identication of LAB from sourdoughs Isolation was made from serial dilutions of 0.1 ml of each of the sourdough samples by plating on MRS agar [20]. LAB isolates were randomly selected from MRS agar. The isolated strains were identied according to selection criteria: morphology, Gram reaction, catalase, homo- or heterofermentation, growth capacity at 15 and 45 C or/and growth capacity at 4, 6.5, 7 and 10% of salts, hydrolysis of arginine and sugar fermentations. Gram-positive, catalase-negative rods were characterised according to Kandler and Weiss [21], Schillinger and Lcke [22], Baumgart [23] and Wood and Holzaphel [24]. Gram-positive, catalase-negative cocci were identied using procedures of Wood and Holzapfel [24].

Carbohydrate fermentation proles for isolates were determined in modied MRS broth without glucose and beef extract, containing chlorophenol red (0.04 g l1 ) as a pH indicator and supplemented with 1% of each carbohydrate [20,22]. The isolates were preserved in litmus milk and stored at 18 C [24]. The lactobacilli isolates used as starters were cultivated in MRS broth at 30 C for 24 h. 2.6. Isolation and identication of yeasts from sourdoughs Yeasts were isolated by plating on Malt Extract Agar acidied with tartaric acid to pH 3.5. Plates were incubated for 3 days at 25 C, and then yeast colonies were counted. Pure cultures were obtained using the conventional streaking method. The puried cultures were routinely maintained on Malt Extract Agar slants and kept at 4 C. The yeast isolates were subjected to various morphological, physiological and biochemical tests, which included sugar fermentation (glucose, fructose, galactose, lactose, maltose, d-mannitol, sucrose, trehalose, d-xylose, l-(+)-arabinose and sorbitol), urease tests, growth at 37 C, presence of pseudohyphae using Yeast Malt Extract medium, growth at liquid medium and size of vegetative cells by direct microscobic examination. Identication was made by reference to standard descriptions in Harrigan and Mc Cance [25], Smith and Yarrow [26], Beuchat [27] and Deak [28]. The isolates were preserved in slant Malt Extract Agar and stored at 4 C. The S. cerevisiae used for bread making as starters were grown in Malt Extract broth at 30 C for 24 h. 2.7. Determination of acidity produced by the LAB Acid production was determined in a sterilised solution containing 10% our and 2% sucrose. A 1% inoculum from

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H. Gl et al. / Process Biochemistry 40 (2005) 691697 Table 3 Species of LAB isolated from the sourdough samples Species Carnobacterium ssp. C. divergens Lactobacillus ssp. Lb. brevis Lb. amylophilus Lb. sake Lb. acetotolerans Lb. plantarum Lactobacillus sp. Pediococcus ssp. P. pentosaceus P. acidilactici Pediococcus sp. Tetragenococcus ssp. Tt. halophilus Total
a b

an overnight lactobacilli culture was used and incubation proceeded for 24 h at 30 C. Acidity was measured with a pH meter. Acid production was used as a characteristic for selection of starter cultures. 2.8. Preparation of LAB and yeast cultures Ten milliliter of MRS and Malt Extract Broths (Merck, Darmstadt, Germany) were inoculated with LAB (1%) and yeast (1%) cultures, respectively. LAB and yeast were incubated at 30 C for 24 h. Second, 100 ml of MRS and Malt Extract Broths were then inoculated with 10% of LAB and yeast cultures and were incubated at 30 C for 24 h. The nal cell concentrations of LAB and yeast were 106 to 107 cfu ml1 on MRS and Malt Extract agars. After incubation, microbial cells were obtained by centrifuging (6000g for 15 min) the culture. Supernatants were removed and microbial cell pellets were used in preparation of dough. 2.9. Organoleptic evaluation Organoleptic characteristics of the bread samples were assessed by a panel of researchers at the University of Suleyman Demirel, Isparta, Turkey. This was done within 24 h of baking. The panellists were asked to rate the bread samples for external properties (volume, crust colour, shape, crust properties, border properties) and internal properties (crumb colour, taste, avour, texture, pore size, chewing properties). The external and internal characteristics were evaluated over 30 and 70 total points, respectively [16]. 2.10. Statistical analyses Analyses of variance were performed on the data obtained at different stages of manufacture in accordance with SPSS for Windows, version 7.5. Properties of sourdough bread produced by the isolated cultures were subjected to analyses of variance using one-way ANOVA. Differences between means were separated using Duncans multiple range test [29].

Number of isolates 2 2 23 5 2 2 2 1 11 7 2 2 3 1 1 33

%a 6.1 6.1 69.7 15.1 6.1 6.1 6.1 3.0 33.3 21.2 6.1 6.1 9.0 3.0 3.0 100

%b 100 100 100 21.7 8.7 8.7 8.7 4.4 47.8 100 28.6 28.6 42.8 100 100

Percentage of strains of the genus. Percentage of strains over the total of isolated strains (33).

The log cfu g1 of total mesophilic aerobic bacteria, yeasts and LAB counts are listed in Table 2. The total bacterial counts of the samples ranged from 5.97 to 9.57 log cfu g1 . The total bacterial count was the highest in 14th sourdough. The yeast counts of the samples ranged from 6.33 to 9.96 log cfu g1 . In addition, the LAB counts of the samples were 5.28 to 9.66 log cfu g1 . The highest LAB count was in 13th sourdough, 9.66 log cfu g1 . 3.2. LAB and yeasts isolated from sourdoughs Thirty-three LAB isolates were obtained from the sourdough samples (Table 3). The LAB were identied as Carnobacterium (two isolates), Lactobacillus (23 isolates), Pediococcus (seven isolates) and Tetragenococcus (one isolate). Lb. brevis was the predominant bacteria among the LAB in the samples; this organism represented 15% of the isolates. Okada et al. [10] and D rak [14] reported g that Lb. brevis is the most frequent isolates in sourdoughs. Carnobacterium divergens, Lactobacillus amylophilus, Lactobacillus sake, Lactobacillus acetotolerans, Lb. plantarum, Lactobacillus sp., Pediococcus pentosaceus, P. acidilactici, Pediococcus sp. and Tetragenococcus halophilus were also isolated among LAB. Results on the composition of species of lactic microora in the sourdoughs were different from those reported for other sourdoughs [10,30]. D rak [14] g and Kurucu [15] reported that Lb. brevis, Lb. divergens, Lb. plantarum and P. pentosaceus are isolated from sourdoughs. In the previous studies conducted on sourdoughs, detection of Lb. amylophilus, Lb. sake and Lb. acetotolerans were not reported. In general, some researchers reported the isolation of Lb. sanfranciscensis from sourdoughs [31,32]. This bacterium was not detected in the present study. A total of thirty-seven yeasts were isolated from the sourdough samples (Table 4). The yeasts were identied as

3. Results and discussion 3.1. Physicochemical and microbiological characteristics of the doughs and sourdoughs Table 2 shows the fermentation time (FT), pH, titratable acidity (%) and total solid (%). The 11th sourdough sample had the highest pH value (5.55), and the mean pH values in the sourdoughs were similar to those reported in earlier studies on sourdoughs [4,14]. The 12th sample had the lowest acidity (4.2%) while the 75th and 14th sourdough samples had the highest (14.0%). Mean solid content of the samples was 53.02 6.31.

H. Gl et al. / Process Biochemistry 40 (2005) 691697 Table 4 Species of yeasts isolated from the sourdough samples Species Saccharomyces ssp. S. cerevisiae S. delbrueckii Saccharomyces sp. Torulopsis ssp. T. holmii T. unisporus Total
a b

695

Number of isolates 32 10 1 21 5 4 1 37

%a 86.5 27.0 2.7 56.8 13.5 10.8 2.7 100

%b 100 31.3 3.1 65.6 100 80.0 20.0

Percentage of strains of the genus. Percentage of strains over the total of isolated strains (37).

Saccharomyces (32 isolates) and Torulopsis (ve isolates). Saccharomyces cerevisiae was predominant among the yeasts in the samples; this organism represented 27% of the isolates. Collar et al. [13] reported that S. cerevisiae was the most frequently isolated yeast genera in sourdoughs. S. delbrueckii, Saccharomyces sp., Torulopsis holmii and T. unisporus were other frequently isolated yeast genera among yeasts. It was not reported that S. delbrueckii was isolated from sourdough. Again, S. cerevisiae, Torulopsis holmii and T. unisporus were also identied by some researchers [33]. The acid production ability (as pH) of all isolates was determined for the selection of the starter cultures for experimental sourdough breads production (Table 1). Results of the screening procedure of pH values showed that a few lactobacilli, especially Lb. amylophilus EL 1, Lb. brevis EL 2, Lb. plantarum BL 2, Lb. sake YL 2 and Lb. acetotolerans TL 1, grew well according to the selection criteria (Table 1). All of the ve starter organisms of Lb. amylophilus EL 1, Lb. brevis EL 2, Lb. plantarum BL 2, Lb. sake YL 2 and Lb. acetotolerans TL 1 gave the highest pH value compared to other strains. 3.3. Characteristics of breads produced by isolates Bread samples T1, T2, T3, T4, T5, T6 and T7 (control) were produced from the sourdoughs soured with combined cultures and commercial cultures as indicated in Table 1. During the phase of sourdough production using a constant

inoculum content, the ratio of all dough samples decreased gradually to a range of 3.654.24. The physicochemical characteristics of the experimentally produced breads are given in Table 5. The dough yield of the samples was the highest for the T4 sample (169.49 2.82%). Table 5 shows that the bread yield of the T6 sample (127.1014.80%) produced with mixed cultures was the lowest while this value of T7 (control) sample (140.492.82%) produced with S. cerevisiae (commercial culture) was the highest. However, the bread yield of T1 sample (139.50 0.50%) baked with Lb. amylophilus and S. cerevisiae was similar to the control sample. In addition, the volume yield of the bread samples was the highest for the control group (303.90 49.4%) whereas this value of sample T6 was the lowest (231.0 38.40%). But, the differences among moisture, yields of dough, bread and volume and specic volume of the bread samples were insignicant (P > 0.05). In this study, these values for the sample T1 produced with Lb. amylophilus and S. cerevisiae were similar to the control (T7) sample. But, Kurucu [15] reported that 2.5% Lb. plantarum and 5% S. cerevisiae showed the best volume efciency in bread. Some researchers reported that LAB increased shelf life of bread and delayed staling [5,8,13]. Crumb hardness of the bread samples was determined with a penetrometer for 24, 48 and 72 h (Table 5). The differences between hardness of the bread samples were insignicant (P > 0.05). The penetrometer value of the sample T6, produced with LAB mix and S. cerevisiae was the highest for all times while these microorganisms showed the best results in staling of bread. This value for the control sample was also the lowest after 48 and 72 h. The penetrometer value shows crumb hardness of bread, and while crumb hardness of bread is decreasing, staling of bread is delayed that mean shelf life of bread increases [16]. In a study on sourdough bread, use of 2.5% Lb. plantarum and 5% S. cerevisiae resulted in delay of staling while it decreased the crumb hardness signicantly [15]. Organoleptic attributes of the sourdough breads are shown in Table 6. Objective assessments did not indicate which bread had the best taste due to the fact that taste preference is personal. In the results, T4 sample baked with Lb. sake

Table 5 Physicochemical characteristics of bread produced by combined cultures (mean S.D.) Properties Breads T1 Moisture (%) Yield of dough (%) Yield of bread (%) Yield of volume (%) Specic volume (%) Penetrometer value 24 h 48 h 72 h S.D.: standard deviation. 37.00 168.82 139.50 267.50 1.915 2.80 2.15 0.50 75.70 0.53 T2 38.30 168.83 135.75 254.80 1.86 4.10 2.17 7.75 59.40 0.33 T3 37.70 168.17 130.20 270.00 2.08 3.20 1.50 13.30 13.30 0.11 T4 39.10 169.49 137.85 237.70 1.71 3.80 2.82 3.75 38.90 0.23 T5 37.80 167.67 137.40 250.90 1.83 0.60 1.00 0.10 69.10 0.50 T6 39.90 167.24 127.10 231.60 1.81 1.20 0.57 14.80 38.40 0.09 T7 39.45 168.17 140.10 303.90 2.16 1.55 1.50 2.60 49.4 0.31

1.93 0.07 2.82 0.09 2.75 0.09

2.22 0.11 2.55 0.09 3.02 0.14

2.10 0.12 2.84 0.12 3.01 0.19

2.57 0.05 2.56 0.09 2.75 0.11

2.94 0.11 2.99 0.11 3.00 0.23

3.57 0.12 3.35 0.11 3.65 0.14

2.63 0.12 2.41 0.11 2.72 0.21

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Table 6 Organoleptic properties of the breads produced by combined cultures (mean S.D.) Characteristics Breads T1 Total external properties Total internal properties Total general properties S.D.: standard deviation. 23.9 0.9 52.5 1.7 76.4 2.3 T2 22.3 0.8 51.8 1.9 74.1 2.3 T3 23.1 0.9 53.1 1.9 76.2 2.6 T4 24.3 0.8 55.9 2.0 80.2 2.6 T5 21.7 0.9 54.1 1.6 75.9 2.2 T6 23.2 0.8 53.2 1.8 76.4 2.2 T7 23.4 1.1 52.9 1.7 76.3 2.5

and S. cerevisiae had the best in terms of organoleptic properties. The total value (general properties) for the organoleptic properties of the sample T2 was the lowest (74.1 2.3). Kurucu [15] reported that the bread contained Lb. farciminis were preferred more than that of the Lb. plantarum counterparts. In contrary, Martinez-Anaya et al. [11] reported that Lb. plantarum and Lb. brevis affected positively the sensory properties of sourdough breads.

4. Conclusion Among experimentally produced breads, the T4 sample produced with Lb. sake and S. cerevisiae was the most preferred sample in terms of total sensory properties. Compared to the control treatment (T7 sample), LAB used in sourdough breads increased shelf life and delayed staling of the breads. Results of the bread production with LAB showed that LAB have important effects on the physicochemical, organoleptic and rheological characteristics of the breads.

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