Pakistan Journal of Nutrition 6 (6): 562-565, 2007 ISSN 1680-5194 Asian Network for Scientific Information, 2007

Effect of Sourdough Bacteria on the Quality and Shelf Life of Bread

Salim-ur-Rehman1*, Haq Nawaz2, Sarfraz Hussain3, Muhammad Mushtaq Ahmad4, Mian Anjum Murtaza3 and Mian Saeed Ahmad1 1 Institute of Food Science and Technology, University of Agriculture, Faisalabad, Pakistan 2 Institute of Animal Nutrition and Feed Technology, University of Agriculture, Faisalabad, Pakistan 3 Department of Food Science and Technology, University of Sargodha, Sargodha, Pakistan 4 Ayub Agriculture Research Institute, Faisalabad, Pakistan
Abstract: Bread dough is usually fermented with yeast but in the present study sourdough lactic acid bacteria (Lactobacillus bulgaricus) alone and in combination with yeast (Saccharomyces cerevisiae) were used to determine their effect on the shelf life and sensory characteristics of bread at different intervals of storage. Lactic acid bacteria improved the sensory characteristics of bread such as volume, evenness of bake, character of crust, grain of bread, colour of bread crumb, aroma, taste and texture of bread and extended shelf life of bread by inhibiting the growth of microbes. Key words: Lactic acid, microbes, bread, shelf life, sensory characteristics

Introduction
Dough is usually leavened by bread yeast, which ferment dough sugar and produces mainly carbon dioxide and alcohol. However, other gas producing microorganisms e.g., wild yeasts, coliform bacteria, saccharolytic Clostridium species, heterofermentative lactic acid bacteria and various naturally occurring mixtures of these organisms have been used for leavening of dough instead of bread yeast alone (Vollmer and Meuser, 1992; Bratovanova, 1996). Sourdough is an important modern fermentation of cereal flours and water based upon an earlier spontaneous process (Vogel et al., 1999). A slack flour dough is inoculated with microbial starter, mother culture, which is constantly renewed in a cyclical way, using specified recipes and ripening conditions (Hammes and Ganzle, 1995; Ottogalli et al., 1996). In addition to yeast, certain strains of the Lactic Acid Bacteria (LAB) play a key role in fermentation of bread dough. Although, yeast shows a high demand for amino acids but total amino acid concentrations are not affected by lactic acid bacteria (Thiele et al., 2001). The sourdough fermentation is central to acceptability in flavour, as chemically acidified breads prepared with pure commercial starter cultures are not well scored in sensory preference assessments (Lund et al., 1989; Rehman et al., 2006). The synergistic metabolic activities of microorganisms produce acidification or souring influencing the final characters of bread, notably the texture (Martinez et al., 1990; Rcken and Voysey, 1995; Corsetti et al., 2000) and generate typical flavour compounds yielding typical sourdough sensory attributes (Gobbetti, 1998; Katina et al., 2006). Sourdough bread may vary in flavouring compounds as a result of ingredients selection and lactic acid bacterial fermentation (Schieberle and Grosch, 1985; Schieberle 562

and Grosch, 1992; Schieberle and Grosch, 1994). Moreover, lactic acid bacteria contribute to the production of safer foods by inhibiting the growth of pathogenic microbes or by removing chemicals or toxic contaminants. Certain lactobacillus bacteria, in the process of souring of dough, produce an enzyme that breaks down a protein to be toxic to people with celiac disease (Cagno et al., 2002; Gobbetti et al., 1995). Therefore, the present study was conducted to determine the effect of Lactobacillus bulgaricus alone and in combination with yeast (Saccharomyces cerevisiae) on the sensory characteristics and shelf life of bread.

Materials and Methods

Collection of raw materials: Pure culture of lactic acid bacteria (Lactobacillus bulgaricus) was isolated from yoghurt starter cultures purchased from the local market of Faisalabad. Wheat flour, salt, sugar and yeast were also procured from the local market. Purification of cultures: Streak plate method was used to get pure culture of lactic acid bacteria. Then a single colony was transferred to the fresh medium and a pure culture (stock culture) was obtained (Cappuccino and Sherman, 1996). Proximate analysis of flour: The commercial flour samples were analyzed for moisture, total ash, crude protein, crude fiber, crude fat and nitrogen free extract (AACC, 2000). Rheological characteristics of dough: The rheological properties of flour samples were determined by Brabender Farinograph and Amylograph (AACC, 2000).

Salim-ur-Rehman et al.: Effect of Sourdough Bacteria on the Quality and Shelf Life of Bread
Table 1: Treatments Treatments T0 Control bread (1% yeast) T1 3 ml lactic acid starter culture T2 0.25% yeast + 2 ml lactic acid starter culture T3 0.50% yeast + 1.5 ml lactic acid starter culture T4 0.75% yeast + 1 ml lactic acid starter culture. 1 ml = 1.5106 (L. bulgaricus) lactic acid producing bacteria Table 2: Proximate composition of commercial flour Constituent Quantity (%) Moisture 12.20 Ash (db) 0.65 Crude protein (db) 12.87 Crude fibre (db) 0.54 Crude fat (db) 0.94 NFE (db) 85.0 db: dry basis Table 3: Farinographic and amylographic characteristics of flour Characteristics Quantity Water absorption (%) 64.0 Arrival time (min) 2.0 Departure time (min) 9.0 Peak time (min) 3.0 Dough stability (min) 7.0 Softening of the dough (BU) 50.0 Tolerance index (BU) 30.0 Amylographic (BU) 1500.0 Table 4: Effect of different treatments on volume, weight and weight to volume ratio of bread Weight to Treatments Volume (CC) Weight (g) volume ratio T0 585b 153 3.83ab T1 587ab 152 3.86a T2 588a 153 3.84b a T3 590 154 3.80c T4 583bc 153 3.81cd Table 5: Effect of lactic acid characteristics of bread Characteristics (score) T0 Volume (10) 8.2b Crust Colour (8) 7.0b Symmetry of form (5) 4.0c Evenness of bake (3) 2.2 Character of crust (4) 3.5b Grain of bread (15) 13.2c Colour of crumb (10) 8.5c Aroma (10) 8.0d Taste (20) 16.0c Texture (15) 11.8d starter T1 8.5a 7.5a 4.5a 2.2 3.8a 13.7b 8.9a 8.7a 18.0a 12.5b culture T2 8.0c 6.8c 4.2b 2.3 3.7a 13.4c 8.8b 8.4b 17.0b 12.0c on T3 8.0c 6.9cd 4.3b 2.2 3.3c 13.4c 8.8b 8.2c 16.5c 11.3e sensory T4 8.0c 7.0b 4.2b 2.3 3.3c 14.0a 8.5c 8.2c 16.2d 12.8a

till clean up stage and allowed to ferment for two and a half hours. Then the dough was divided into 100 g dough ball, moulded, placed in a pan and kept in a proofer at 30C and 85% relative humidity for 60 minutes. The bread was baked in a gas heated oven at 220C for 15 minutes. The bread was depanned and allowed to cool for 2 hours. The bread was sliced and packed in a polythene bag. Objective evaluation of bread: Bread was evaluated for its weight, volume and weight to volume ratio. The volume of a loaf was measured by rape seed displacement method using loaf volume meter (AACC, 2000) and weight of fresh loaf was measured with a digital balance. Sensory evaluation of bread: The bread was evaluated for sensory characteristics at 0, 24, 48, 72, 96 h storage intervals by panel of judges (Land and Shepher, 1988). Microbial identification: Total bacterial and mould colony counts and identification of moulds were carried out at 0, 24, 48, 72, 96 h storage intervals (Cappuccino and Sherman, 1996). Statistical analysis: Data obtained on different parameters were subjected to statistical analysis using analysis of variance technique. When F-test was statistically significant a least significant difference (LSD0.05) was used to test the treatments effect (Steel et al., 1997).

Results and Discussion

Wheat flour contained moisture 12.20%, crude protein 12.87%, crude fibre 0.54%, crude fat 0.94%, ash 0.65% and NFE 85% (Table 2). The results were in close agreement with the findings of Ali (1980) who used amylolytic enzymes in wheat flour. The data on rheological studies are presented in Table 3. The perusal of the farinogram showed that flour had 64% water absorption, 2.0 min arrival time, 9.0 min departure time, 0.7 min dough stability, 3.0 min peak time, 30 B.U tolerance index and 50 B.U. softening of the dough. The results pertaining to objective assessment of bread are given in Table 4. Loaf volume showed significant variation as result of treatments. T1 and T3 showed maximum volume followed by T2 and T4 which produced minimum volume. Weight to volume ratio showed significant differences among treatments. Analysis showed that individual LAB used as starter culture was able to produce a characteristic sourdough. As the application of sourdough had a positive impact on bread volume (Clarke et al., 2002), it could be used in bread making process in combination with yeast in order to provide bread with desirable qualities (Robert et al., 2006). 563

Bread making process: Bread was prepared with some modification in the method as described in AACC (2000), by using lactic acid bacteria (Lactobacillus bulgaricus). Flour (100 g), sugar (4 g), oil (4 g), salt (1 g), water (64 ml; farinographic water absorption) and lactic acid starter culture/yeast were mixed together with Hobart mixer. Lactic acid starter culture was used in different proportions (Table 1). The dough was kneaded

Salim-ur-Rehman et al.: Effect of Sourdough Bacteria on the Quality and Shelf Life of Bread
Table 6: Effect of storage on sensory characteristics of bread Characteristics 0h 24 h 48 h 72 h 96 h Volume 8.20a 8.03a 7.72b 7.48bc 7.36c Crust Colour 7.04a 6.84ab 6.84ab 6.66b 6.36c Symmetry of form 4.14a 3.98b 3.76c 3.58d 3.22c Character of crust 3.64a 3.44b 3.30c 3.12d 2.94c Grain of bread 13.32a 13.14ab 13.00ab 12.88b 12.18c Colour of crumb 8.54a 8.46a 8.42a 7.78b 7.400c Aroma 7.46a 8.40a 7.58b 7.08c 6.30d Taste 15.64a 16.46ab 16.10b 14.82c 12.88d Texture 10.56a 11.40a 11.06b 10.66c 10.02d Table 7: Total bacterial count of bread at different intervals Treatments 0h 24 h 48 h 72 h T0 5101 7101 1.9102 2.5102 T1 5.3101 9.1101 T2 5.5101 9.5101 T3 8.9101 2.1102 T4 4101 1.2102 2102 Table 8: Total mould count of bread at different storage Treatment 0h 24 h 48 h 72 h T0 1.5102 1.8102 2.5102 2.8102 T1 5101 9101 1102 2.4102 T2 9101 1102 1.6101 2.1102 1 2 2 T3 710 1.210 2.510 2.5102 T4 1102 1.4102 2.2102 2.6102 storage 96 h 3102 2102 2.3102 2.4102 2.7102 intervals 96 h 3.9102 3.3102 2.3102 2.5102 3.3102

The breads prepared from different treatments were sensory evaluated for internal and external characteristics (Table 5, 6). The results revealed that the sensory characteristics of bread including volume of bread, crust colour, symmetry of form, character of crust, grain of bread, crumb colour, aroma, taste and texture were significantly affected by the different treatments (Corsetti et al., 2000). Maximum score for volume was noted in treatments T1 and minimum in treatments T2, T3 and T4. Maximum score for colour of crust was noted in T1 and minimum inT2. The effect of T4 was found nonsignificant with respect to T0. In ranking order, the mean value for T1 was at the top followed by T3 and T4, T2 and T0 for the symmetry of form of bread loaf. There was no effect of treatments and storage intervals on evenness of bake. For the character of crust, the T1 was placed at the top followed by T2 and T4. Although, T4 was most effective for grain formation but grain was adversely affected during storage. This might be due to staling effect. T0 was found to be least effective for grain formation. It has been generally acknowledged that holes of relatively small size were preferred in bakery products, whereas, large voids crumb distributions were undesirable (Cauvain, 1998).T1 was found best for the colour of bread crumb as well as for pleasant bread aroma. It was found that the taste of T1 was observed best followed by T2. Minimum score was obtained by T0. T4 was found best for texture of bread followed by T1 and T2. The score for various characteristics of bread decreased as storage period increased except 564

evenness of bake which remained unchanged. The results are in close agreement with the findings of Maleki et al. (1980) who reported that larger loaf size produced softer bread. Moreover, the sourdough breads showed to have lower crumb firmness values (Corsetti et al., 2000; Clarke et al., 2002). The data on the bacterial colony counts in breads at different storage intervals are given in Table 7. Maximum numbers of bacterial colonies were observed in T0. T1 proved to be the most effective in inhibiting bacterial spoilage in bread. The colonies appeared after 48 h of storage in T1, T2 and T3. In T4, bacterial colonies appeared after 24 h (4101 cfu/g) and increased (2.7102 cfu/g) in bread at 96 h of storage. It indicated that lactic acid starter culture ([Link]) was more effective against the growth of other bacteria in bread (Mente et al., 2007). The mould colony counts in bread were observed at different storage intervals on saborauds agar media (Table 8). Maximum number of mould colonies were recorded in bread (T0) fermented only with yeast culture. T1 proved to be the most effective in inhibiting mould spoilage in bread followed by T2 (2 ml lactic acid starter culture+0.25% yeast) and T3 (1.5 ml lactic acid starter culture+0.5% yeast). Total colony count at 96 h of storage in treatment T2 was 2.3102 cfu/g of bread. The results showed that sourdoughs of different combinations of lactic acid starter culture and yeast were effective in enhancing the shelf life of bread by inhibiting the mould growth. The antifungal phenomenon might be due to the production of organic acids by lactic acid starter culture during sourdough fermentation (Rcken and Voysey, 1995). The results suggest that use of sourdough in bread production is beneficial in improving sensory properties, delaying firmness and preventing mould and bacterial spoilage (Martinez et al., 1990; Hammes and Gnzle, 1995; Gobbetti, 1998). Microscopic examination and staining of the samples with lactophenol cotton blue or lectophenol picric acid revealed that majority of the moulds; isolated during study belonged to Aspergillus flavous, Peniciellium and Rhizopus in all the spoiled breads. Conclusion: It was concluded that T1 (Lactobacillus bulgaricus) was most effective to inhibit microbial spoilage and extended the shelf life of bread. Sourdough affected the physical property of bread such as volume which was assessed objectively. However, the key differences between control and sourdough breads were observed in sensory characteristics i.e. Aroma, taste and texture.

References
AACC, 2000. Approved Methods of the American Association of Cereal Chemistry. Am. Assoc. Cereal Chem. Inc, St. Paul, Minnesota.

Salim-ur-Rehman et al.: Effect of Sourdough Bacteria on the Quality and Shelf Life of Bread Ali, M.A., 1980. Effect of supplementation of flour from Pakistani wheat with amylolytic enzymes on the Quality Bread and Ratio. [Link]. (Hons.) Thesis, Dept. of Food Tech. University of Agriculture, Faisalabad. Pakistan. Bratovanova, P., 1996. Relationship between yeasts and lactic acid bacteria during fermentation of dough. Khranitelna promishlenost, 45: 7-11. Cappuccino, J.G. and N. Sherman, 1996. Microbiology: A Laboratory Manual. The Benjamin/Cummings Pub. Co. Inc. New York. Cagno, R. Di., M. De. Angelis, P. Lavermicocca and M. De. Vincenzi, 2002. Proteolysis by sourdough lactic acid bacteria. Effects on wheat flour protein fractions and gliadin peptides involved in human cereal intolerance. Appl. Environ. Microbiol., 68: 623-633. Cauvain, S.P., 1998. Bread-the product. In Technology of Breadmaking. S.P. Cauvain and L.S. Young (Eds). Blakie Academic and Professional. London, pp: 330-336. Clarke, C.I., T.J. Schober and E.K. Arendt, 2002. Effect of single strain and traditional mixed strain starter culture on rheological properties of wheat dough and bread quality. Cereal Chem., 79: 640-647. Corsetti, A., M. Gobbetti, B. De Marco, F. Balestrieri, F. Paletti, L. Russi and J. Rossi, 2000. Combined effect of sourdough lactic acid bacteria and additives on bread firmness and staling. J. Agri. Food Chem., 48: 3044-3051. Gobbetti, M., 1998. The sourdough microflora: Interactions of lactic acid bacteria and yeasts. Trends in Food Sci. Tech., 9: 267-274. Gobbetti, M., A. Corsetti and J. Rossi, 1995. Interaction between lactic acid bacteria and yeasts in sourdoughs using a rheofermentometer. World J. Microbiol. Biotech., 11: 625-630. Hammes, W.P. and M. Gnzle, 1995. The genus Lactobacillus. In The genera of Lactic acid bacteria. B.J.B. Wood and W.H. Holzapfel (Eds). Vol II. Blackie Academic and Professional London, pp: 19-54. Katina, K., E. Arendt, K.H. Liukkon, K. Autio, L. Flander and K. Poutanen, 2006. Optimization of sourdough process for improved sensory profiles and texture of wheat bread. LWT-Food Sci. Tech., 39: 1189-1202. Land, D.G. and R. Shepher, 1988. Scaling and Ranking Methods. In Sensory Analysis of Foods J.R. Piggott (Ed). Elsevier Applied Science, New York, pp: 155185. Lund, B., A. Hansen and M.J. Lewis, 1989. The influence of dough yield on acidification and production of volatiles in sourdoughs. LWT- Food Sci. Tech., 22: 150-153. Maleki, M., R.C. Hoseney and P.J. Mattern, 1980. Effects of loaf volume, moisture content and protein quality on the softness and staling rate of bread. Cereal Chem., 57: 138-140. 565 Martinez, M.A., B. Pitarch, P. Bayarri and C. Benedito de Barber, 1990. Microflora of sourdoughs of wheat flour bread and interaction between yeasts and lactic acid bacteria in wheat dough and their effects on bread quality. Cereal Chem., 67: 85-91. Mente, O., R. Ercan and M. Akelik, 2007. Inhibitor activities of two Lactobacillus strains, isolated from sourdough, against rope-forming Bacillus strains. Food Cont., 18: 359-363. Ottogalli, G., A. Galli and R. Foschino, 1996. Italian bakery products obtained with sourdough: Characterization of the typical microflora. Advan. Food Sci., 18: 131-144. Rehman, S., A. Paterson and J.R. Piggott, 2006. Flavour in sourdough breads: a review. Trends in Food Sci. Tech., 17: 557-566. Robert, H., V. Gabriel, D. Lefebvre, P. Rabier, Y. Vayssier and C. Fontagn-Faucher, 2006. Study of the behaviour of Lactobacillus plantarum and Leuconostoc starters during a complete wheat sourdough breadmaking process. LWT-Food Sci. Tec., 39: 256-265. Rcken, W. and P.A. Voysey, 1995. Sourdough fermentation in breadmaking. J. Appl. Bacteriol. Symp. Suppl., 79: 38S-48S. Schieberle, P. and W. Grosch, 1992. Changes in the concentration of potent crust odorants during storage of white bread. Flav. Fragr. J., 7: 213-218. Schieberle, P. and W. Grosch, 1994. Potent odorants of rye bread crust-differences from the crumb and from wheat bread crust. Zeitschrift fur LebensmittelUntersuchung und-Forschung, 198: 292-296. Schieberle, P. and W. Grosch, 1985. Identification of volatile flavour compounds of wheat bread crustcomparison with rye bread crust. Zeitschrift fur Lebensmittel-Untersuchung und-Forschung, 180: 474-478. Steel, R.G.D., J.H. Torrie and D. Dickey, 1997. Principles and Procedures of Statistics: A Biometrical Approach. 3rd Ed. McGraw Hill Book Co. New York. Thiele, C., M.G. Ganzle and R.F. Vogel, 2001. Contribution of sourdough Lactobacilli; yeast and cereal enzymes to the generation of amino acids in dough relevant for bread flavour. Publication No. C2001/1203-06R. Vollmer, A. and F. Meuser, 1992. Influence of starter cultures consisting of lactic acid bacteria and yeast on the performance of continuous sourdough fermenter. Cereal Chem., 69: 20-27. Vogel, R.F., R. Knorr, M.R.A. Mller, U. Steudel, M.G. Gnzle and M.A. Ehrmann, 1999. Non-dairy lactic acid fermentations: the cereal world. Antonie van Leeuwenhoek, 76: 403-411.