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Preparation of sourdough bread using a blend of bacterial culture and baker’s yeast
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Faqir M. Anjum, Imran Pasha, Kashif Ghafoor, M. Issa Khan and M. Ali Raza Institute of Food Science and Technology, University of Agriculture, Faisalabad, Pakistan Abstract Purpose – Wheat is the staple food in many parts of the world and bread is one of the most important products of wheat flour. There is a need for innovations in bread making to increase its shelf life and consumer’s attraction. Fermentation is mostly done by yeast but it does not produce appreciable amounts of organic acids, which are required to enhance the shelf life of bread. The present study aims to determine the effect of bacterial and yeast culture blends on the quality and shelf life of sourdough bread and to observe the sugar utilization during fermentation. Design/methodology/approach – Three treatments were made using different blends of bacterial cultures (homo-fermentative and hetero-fermentative) and baker’s yeast compared with a control having only baker’s yeast. Chemical analysis, sugar utilization (Sucrose, glucose and fructose) through high performance liquid chromatography, sensory characteristics (both internal and external) and microbial count (Bacterial and fungal count) for each treatment were conducted at different storage intervals. Findings – The hetero-fermentative bacteria i.e. Lactobacillus plantarum along with baker’s yeast exhibited the best results regarding the utilization of sugars during fermentation (after 3 h of fermentation 0.0158 mg/ml sugar remained), objective evaluation of bread and its sensory characteristics. The bread prepared using the blend of hetero-fermentative bacteria (0.5 per cent) and yeast (0.5 per cent) also showed greater resistance against bacteria (9101 cfu/g after 60 h of storage) and mold (1.1 102 cfu/g after 60 h of storage) growth. Research limitations/implications – Hetero-fermentative bacteria along with baker’s yeast can be utilized in sour dough to improve major bread characteristics. This study is a step further in improving the shelf life of sourdough. Originality/value – Presently only baker’s yeast is being used by bread industry for fermentation purpose but a blend of bacterial culture along with baker’s yeast can give better performance for better quality and shelf life of the bread. Keywords Bakery products, Bacteria, Food products Paper type Research paper
Nutrition & Food Science Vol. 38 No. 2, 2008 pp. 146-153 # Emerald Group Publishing Limited 0034-6659 DOI 10.1108/00346650810863028
Introduction Wheat (Triticum aestivum L.) is a main food crop in most parts of the world including Pakistan. Among the cereal grains wheat is preferred due to the presence of gluten, which possesses unique properties of forming cohesive and elastic dough. Bread is one of the most important products of the wheat flour. One of the main problems encountered by bread producers is its short shelf life. There is a need of innovation in bread making for attracting more people who are more reluctant for traditional products. All consumers today have a considerable portion of their nutritional needs meet through fermented foods (Steinkraus, 1994). Fermented foods include alcoholic food/ beverage food, vinegar, pickled vegetables, sausages, cheese, yogurts, leavened and sour dough breads. Sourdough is an acidic and sharp taste mixture of flour and water for making bread from cereal flours. 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. Among various steps involved in bread making, fermentation is an important one. Dough fermentation mostly carried out by yeasts that metabolizes sugars in dough and produces CO2, ethanol and other chemical compounds. Several yeast species are found in sourdoughs, the most prevalent being Saccharomyces cerevisiae, Saccharomyces exiguus, Candida milleri and Candida krusei (Hammes et al., 2005). Lactic acid production depends on both starter culture and yeast (Esteve et al., 1994). Yeasts alone do not produce appreciable amounts of either lactic acid or acetic acid. LAB are added for acidification (Gobbetti et al., 1994). Sourdough LAB usually belongs to the genus Lactobacillus, but occasionally, Leuconostoc spp. and Enterococcus spp. are found (Gobbetti, 1998; Hammes and Ganzle, 1998). Some species of LAB have been regarded as dominant in different types of sourdoughs, depending on the technology used for their production (Hammes and Ganzle, 1998). A number of species of LAB and yeast are responsible for dough transformation. These microorganisms are usually contaminants originating from flour or the environment. The dough acidified with homo-fermentative LAB (Lactobacillus bulgaricus, Lactobacillus lactis and Lactobacillus ceremoris) contains small quantity of acetic acid. Homo-fermentative bacteria do not produce CO2, so yeast must be added. Heterofermentative LAB especially Lactobacillus brevis and Lactobacillus plantarum produce lactate, ethanol and CO2, and give increased volume up to a level i.e. 20 per cent (Schleining, 1995). Sourdough fermentation is generally evaluated by the measurement of parameters such as pH, acidity and microflora (Wick et al., 2003). 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 (Barber et al., 1992; Corsetti et al., 1998). Sourdough LAB and yeasts have been shown to compete for carbon sources which influence acid production by bacteria. Acidification 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 (Gelinas et al., 1999). Mixed culture of LAB and yeast (Saccharomyces cerevisae) offer more protection against bread spoilage. The demand for sourdough bread is increasing in Europe for its natural taste and good health effects (Brummer ad Lorenz, 1991). In wheat bread, sourdoughs are mainly used to improve flavor (Hansen and Hansen, 1996). Compounds formed by blending microorganisms often complement each other and work to the exclusion of unwanted microorganisms. Mixed culture permit better utilization to subtract because they possess wide range of enzymes and are able to attack great variety of compounds (Zeikus and Johnson, 1991). Texture and crumb gains characteristic are two major quality attributes of bread product (Crowley et al., 2002). Generally, bread spoils in 70 h due to the development of rope and molds. The Shelf life of bread can be increased by maintaining hygienic conditions during processing and storage (Wassermann, 1969). In addition to it, the use of LAB may contribute to the production of safer foods by inhibiting microbial pathogens or by removing chemicals or toxic substances. Present study was conducted to determine the effect of bacterial starter cultures blend on the quality and shelf life of bread and to observe the changes in the sugar, acidity and pH during dough fermentation by different bacteria as compare to that of yeast alone.
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Materials and methods Procurement of raw material Bacterial culture L. lactis was obtained from cheese industry; L. plantarum (NRRL4496) and L. bulgaricus (NRRL-548) were donated by the microbial genomics and bioprocessing research unit, USDA, USA. Wheat flour, sugar, yeast, shortening and salt were procured from local market.
148 Chemical analysis Chemical analysis (Moisture, Ash, Crude protein, Crude fat and Crude fiber) was done according to the methods of AACC (2000). Nitrogen Free Extract (NFE) was calculated subtracting the percentage of moisture, ash, crude protein, crude fat and crude fiber from 100. Bread preparation Bread was made according to the method described in AACC (2000) by using bacterial starters having four treatments given below: Treatments T0 ¼ Control 1 per cent yeast. T1 ¼ 0.5 per cent L. bulgaricus þ 0.5 per cent yeast; T2 ¼ 0.5 per cent L. lactis þ 0.5 per cent yeast; T3 ¼ 0.5 per cent L. plantarum þ 0.5 per cent yeast. Total titratable acidity and pH Total titratable acidity (TTA) and pH were measured after dough fermentation according to the method described in AACC (2000). Determination of sugars Different sugars like Glucose, Fructose and Sucrose were estimated by using high performance liquid chromatography having Aminex HP x 0.87H column following method described by Torbjorn and Hageral (1989). The mobile phase was 0.005 N H2SO4 and the sample injection volume was 20 mL at a flow rate of 0.6 ml/min using refractive index detector. Objective evaluation of bread Bread samples were evaluated for their weight, volume (rapeseed displacement method) and weight to the volume ratio as described in AACC (2000). Sensory evaluation of bread Sensory evaluation of bread carried out by a panel of eight judges at various storage intervals. The Shelf life of all breads assessed and identification of fungal colonies on spoiled bread was carried out according to the method described by Cappuccino and Sherman (1996).
Microbiological count Microbiological count with colony counter at 0, 24, 48, 72 h of storage was carried out by counting and adding up the moulds and bacteria colonies. The media used for fungal colonies and bacterial colonies were saborand agar and nutrient agar, respectively. Statistical analysis Data obtained were subjected to statistical analysis as described by Steel et al. (1997).
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Results and discussion The chemical composition of commercial flour was 12.20 per cent moisture, 0.62 per cent ash, 11.59 per cent crude protein, 0.50 per cent crude fiber, 0.91 per cent crude fat and 74.18 per cent NFE. TTA and pH were determined for each treatment. The lowest pH (5.29) and the highest TTA (4.9 per cent) were obtained by T3 while the highest pH (6.13) and lowest TTA (2.3 per cent) were in T0. These results were in close agreement with those obtained by Javanainen and Linko (1993). Halm et al. (1996) carried out controlled fermentation experiments using six strains of Lactobacillus fermentum and one strain of S. cerevisiae. They found that for most of the inoculated samples the required pH of 3-7 was attained within 24 h of dough fermentation instead of 48 h as observed with spontaneous dough fermentations. Sucrose in samples was lower after 2 h as compared to 1 h fermentation Figure 1. It decreased further after 3 h of fermentation. The best utilization of sucrose was observed in case of T3 (0.1004 mg/ml remained) after 1 h of fermentation. After 2 h of fermentation maximum sucrose concentration was in T2 (1.5535 mg/ml remained) and minimum in T3 (0.0554 mg/ml remained) while after 3 h of fermentation the best utilization of sucrose was found to be again in T3 (0.0158 mg/ml remained), whereas lowest in T2 (1.3405 mg/ml
Figure 1. Sugar utilization in dough by blends of bacteria and yeast
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remained). These results indicate that the hetero-fermentative bacteria L. plantarum along with baker’s yeast permit the best utilization of sucrose in dough fermentation. Loaf volume of the bread does not show large variation with respect to the treatments. T0 and T3 attained maximum volume (569CC and 573CC respectively) followed by T2 (568CC) and T1 (557CC) attained the minimum volume. Regarding the weight, T2 attained the maximum weight (149 g) followed by T4 and T0 (148 g) but there was not much difference. Weight to volume ratio exhibited no wide range of difference among different breads. The mean scores for the effect of treatments and storage intervals on different bread characteristics are given in Tables I and II, respectively. The maximum mean score for volume of bread was obtained by treatment T3 (8.4) at 0 and 12 h of storage time and minimum by treatments T0 and T2 (7.53) at 60 h. The volume of bread was affected significantly by treatments as well as storage time. Analysis of variance showed that the effect of treatments and the storage time on colour of crust was highly significant. Maximum score was obtained by T3 (7.33) at 0 h and minimum by T0 (6.56) at storage interval of 60 h. The best storage time was 0 h followed by 12 h, whereas storage time of 36 and 60 h was found to be non significant to each other. Zubair (2000) found similar results for colour of crust of bread as affected by fermentation microorganism. The best results for the effect of treatments and storage intervals of bread characteristics like symmetry of form, evenness of bake, character of crust, formation of grain, aroma of bread and colour of crumb were shown by treatment T3 as indicated in Tables I and II. These findings regarding the sensory evaluation of the bread are in close proximity with the findings of Zleteva and Bratovanova (1994) who compared the bread prepared with LAB to that of control (No LAB). Taste of the product is an important quality parameter concerning the consumer’s acceptability of bread. The results for taste of bread were highly significant for both treatments and storage time. Taste of T3 (mean value 16.28) was the best while T2 (15.75) got the minimum score. The taste was best at 0 h (16.28) of storage and decreased with the passage of time. This might be due to growth of microorganisms and bread stalling. Taste of bread from all treatments was found to be very slightly Bread Crust Symmetry Evenness Character Crumb Treatments volume colour of bread of bake of crust Grain colour Aroma Taste Texture
Table I. Mean values for the effect of treatments on different bread characteristics
T0 T1 T2 T3
7.77c 7.95b 7.85bc 8.26a
6.82c 6.82c 7.01b 7.12a
3.72c 3.82b 3.82b 3.99a
2.12b 2.12c 2.08d 2.14a
3.24a 3.05b 3.19a 3.26a
12.87b 12.51d 12.71c 13.01a
8.17bc 8.14c 8.25ab 8.27a
7.77c 7.91b 7.83bc 8.14a
16.05b 16.08b 15.75c 16.28a
11.93b 12.13a 11.82c 12.20a
Storage Bread Crust Symmetry Evenness Character Crumb interval volume colour of bread of bake of crust Grain colour Aroma Taste Texture Table II. Mean values for the effect of storage intervals on different bread characteristics
0 12 36 60
8.07b 8.18a 7.90c 7.67d
7.17a 7.06b 6.81c 6.72c
4.14a 4.07a 3.72b 3.44c
2.24a 2.12b 2.07bc 2.03c
3.34a 3.21b 3.13c 3.06d
13.04a 8.52a 12.86b 8.30b 12.72c 8.14c 12.84d 7.87d
8.43a 16.61a 12.52a 8.24b 16.43b 12.39b 7.79c 16.11c 12.11c 7.18d 15.02d 11.07d
sour except that of the control. This was due to the production of minute quantities of organic acids by the LAB as evident by decrease in dough pH. Javanainen et al. (1993) also found that sourdough with a pH of 5.4-6 produces an acceptable lactic acid to acetic acid ratio and hence slight sour bread. Texture of bread was affected highly significantly for different treatments and storage intervals. The mean value for T3 (12.20) was the maximum while that of T2 (11.82) was the minimum. Texture was found to be best at 0 h (12.52) storage time and the minimum value (11.07) was attained at storage intervals of 60 h. Colony count of bacteria in bread at different storage intervals on nutrient agar media were conducted and maximum number of bacterial colony was observed in T0 in which there was 5 101 cfu/g bacterial count observed in bread at 0 h of storage and increased to 2.5 102 cfu/g at 60 h of storage (Table III). T3 showed a considerable resistance towards increase of bacteria even up to 60 h of storage (9 101 cfu/g of bread). In T3 and T1, first colony appeared after 12 h of storage. It exhibited that L. plantarum was more effective against the growth of other bacteria on bread. Rosenquist and Hansen (1998) found that the addition of sourdough into bread prevents the growth of Bacillus subtilis very effectively. Mold is widely spread in nature and moldiness is the main problem associated with the shelf life of bread. Maximum number of fungal colonies was observed in T0 in which there were 1.4 102 cfu/g fungal colonies in bread at 0 hour and increased to 3.5 102 cfu/g at 60 h of storage while T3 was proven to be the most effective (Table III). After 60 h of storage T3 had only 1.1 102 cfu/g of the bread which was much lower than other treatment. T1 and T2 were also found to resist fungal growth. Lavermicocca et al. (2000) found that antifungal compounds produced by L. plantarum were more effective against bread spoilage as compared to calcium propionate. LAB used in sourdough breads increased shelf life and delayed staling of the breads. Results of the bread indicated that LAB has important effects on the physicochemical, organoleptic and rheological characteristics of the breads (Gul et al., 2005). This study proves that the spectrum of dough sugars utilization can be broadened if bacterial cultures are used along with yeast for dough fermentation especially the Treatments T0
T1
T2
T3
Storage intervals
Bacterial count cfu/g
Fungal count (cfu/g)
0 12 36 60 0 12 36 60 0 12 36 60 0 12 36 60
5 101 9 101 1.9 102 2.5 102 x 4.8 101 5.3 101 9.1 101 2.3 101 4 101 5.5 101 9.5 101 x 2.8 101 5 101 9 101
1.4 102 1.9 102 2.6 102 3.5 102 4.5 102 8 101 2.1 102 2.8 102 9 101 1.5 102 1.8 102 2.5 102 2.1 101 3.9 101 0.1 102 1.1 102
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Table III. Total bacterial and fungal count in different treatments at different storage intervals
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hetero-fermentative bacterial cultures which may also prove to be helpful in improving textural attributes and sensory characteristics of bread. This is also a step further in improving the shelf life of bread. References AACC (2000), Approved methods of the American Association of Cereal Chemists, The American Association of Cereal Chemists Inc., St. Paul, MN. Barber, B., Ortola, C., Barber, S. and Fernandez, F. (1992), ‘‘Storage of packaged white bread. III. Effects of sourdough and addition of acids on bread characteristics’’, Z. Lebensmittel Unter. Forschung., Vol. 194, pp. 442-9. Brummer, J.M. and Lorenz, K. (1991), ‘‘European development in wheat sourdoughs’’, Cereal Foods World, Vol. 36, pp. 310-3. Cappuccino, J.G. and Sherman, N. (1996), Microbiology Lab Manual, The Benjamin/Cumminigs Pub. Co. Inc., New York, NY. Corsetti, A., Gobbetti, M., Balestrieri, F., Paoletti, F., Russi, L. and Rossi, J. (1998), ‘‘Sourdough lactic acid bacteria effects on bread firmness and staling’’, Journal of Food and Science, Vol. 63, pp. 347-51. Crowley, P., Schober, T.J., Clarke, C.I. and Arendt, E.K. (2002), ‘‘The effect of storage time on textural and crumb grain characteristics of sourdough wheat bread’’, Europen Food Research and Technology, Vol. 214, pp. 489-96. Esteve, C.C., Debarber, C.B. and Martinezanaya, M.A. (1994), ‘‘Microbial sourdough influence acidification properties and bread making potential of weak dough’’, Journal of Food Science, Vol. 59, pp. 629-33. Gelinas, P., McKinnon, C.M. and Pelletier, M. (1999), ‘‘Sourdough-type bread from waste bread crumb’’, Food Microbiology, Vol. 16, pp. 37-43. Gobbetti, M. (1998), ‘‘The sourdough microflora: interaction of lactic acid bacteria and yeasts’’, Trends in Food Science and Technology, Vol. 9, pp. 267-74. Gobbetti, M., Simonetti, M.S., Rossi, J., Cossignani, L., Corsetti, A. and Damiani, P. (1994), ‘‘Free D and L amino acids evaluation during sourdough fermentation and baking’’, Journal of Food Science, Vol. 59, pp. 881-4. Gul, H., Ozcelik, S., Sadic, O. and Certel, M. (2005), ‘‘Sourdough bread production with lactobacilli and S. cerevisiae isolated from sourdoughs’’, Process Biochemistry, Vol. 40, pp. 691-7. Halm, M., Osei-Yaw, A., Hayford, A.E., Kpodo, K.A. and Amoa-Awua, W.K.A. (1996), ‘‘Experiences with the use of starter culture in the fermentation of maize for ‘kenkey’ production in Ghana’’, World Journal of Microbiology and Biotechnology, Vol. 12, pp. 531-6. Hammes, W.P. and Ganzle, M.G. (1998), ‘‘Sourdough breads and related products’’, in Wood B.J.B. (Ed.), Microbiology of Fermented Foods, Blakie Academic and Professional, London, pp. 199-216. Hammes, W.P., Brandt, M.J., Francis, K.L., Rosenheim, J., Seitter, M.F.H. and Vogelmann, S.A. (2005), ‘‘Microbial ecology of cereal fermentations’’, Trends in Food Science and Technology, Vol. 16, pp. 4-11. Hansen, A. and Hansen, B. (1996), ‘‘Flavour of sourdough wheat bread crumb’’, Zeitschrift fur Lebenmittel Untersuchunhung und Forschung., Vol. 202, pp. 244-9. Javanainen, P. and Linko, Y.Y. (1993), ‘‘Mixed culture preferments of lactic and propionic acid bacteria for improved wheat bread shelf life’’, Journal of Food Science, Vol. 18, pp. 75-88. Lavermicocca, P., Valerio, F., Evidente, A., Lazaronir, S., Corsetti, A. and Gobbetti, M. (2000), ‘‘Purification and characterization of novel antifungal compound from sourdough
Lactobacillus plantarum strain 21B’’, Applied and Environmental Microbiology, Vol. 66, pp. 4084-90. Rosenquist, H. and Hansen, A. (1998), ‘‘The antimicrobial effect of organic acid sourdough and nisin against Bacillu subtilis and B. licheniformis isolated from wheat bread’’, Journal of Applied Microbiology, Vol. 85, pp. 621-31. Schleining, G., Zenz, H. and Wolf, J. (1995), ‘‘Investigation about sourdough for wheat bread using bacterial starter cultures’’, Ernahrung, Vol. 19, pp. 464-8. Steel, R.G.D., Torrie, J.H. and Dickey, D. (1997), Principles and Procedures of Statistics: A Biometrical Approach. 3rd ed., McGraw Hill Book Co., New York, NY. Steinkraus, K.H. (1994), ‘‘Nutritional significance of fermented foods’’, Food Research International, Vol. 27, pp. 259-67. Torbjorn, L. and Hageral, B.H. (1989), ‘‘Fermentation of lingo cellulose hydrolysis with yeast and xylose isomerase’’, Enzyme and Microbial Technology, Vol. 11, pp. 583-9. Wassermann, L. (1969), ‘‘Critical investigation of various ways of protecting bread against molds’’, Tagung fur Backerei-Technologic, Vol. 12, pp. 171-81. Wick, M., Stolz, P., Bo¨cker, G. and Lebeault, J.M. (2003), ‘‘Influence of several process parameters on sourdough fermentation’’, Acta Biotechnology, Vol. 23, pp. 51-61. Zeikus, J.G. and Johnson, E.A. (1991), Mix cultures in biotechnology, McGraw Hill. Inc., New York, NY, p. 307. Zlateva, D. and Bratovanova, P. (1994), ‘‘Freeze dried lactic acid Streptococci starter for manufacture of wheat bread’’, Khranitelna-Promishlenost, Vol. 43, pp. 20-4. Zubair, M. (2000), ‘‘Effect of different yeasts on sugar utilization during fermentation and the quality of bread’’, MSc (Hons) thesis. Institute of Food Science and Technology, University of Agriculture. Further reading De Vuyst, L. and Neysens, P. (2005), ‘‘The sourdough microflora: biodiversity and metabolic interactions’’, Trends in Food Science and Technology, Vol. 16, pp. 43-56. Corresponding author Imran Pasha can be contacted at:
[email protected]
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