Influence of fermentation time on characteristics of sourdough bread

Sourdough is used in the manufacture of numerous baked products. The microorganisms used in this preparation of sourdoughs included two strains from the Lactobacillus paracasei (1 and 2) and two strains from the Saccharomyces cerevisiae group (1 and 2). Samples of raw dough were analyzed for pH, titratable acidity and plate counts and samples of resulting bread were analyzed for pH, titratable acidity and specific volume. The samples were analyzed every 2 h, between 4 and 10 h of fermentation. After 10 hours of fermentation, the lowest values of pH were for dough with LC2 and bread with SC1. Titratable acidity values increased over time, with the highest levels of acidity were found in the dough and bread with yeasts. Lactic acid bacteria showed the highest microbial counts over time. With the exception of SC2, the greatest microbial increases occurred at 10 hours of fermentation. LC1 showed the lowest volume across all time points (p < 0.05). The largest volumes were found in breads after 6 hours of fermentation. SC1 showed the best specific volume values across all times tested.


INTRODUCTION
Due to increasing consumer demand for more natural, tastier and healthier food, the traditional process of sourdough bread production has enjoyed renewed success in recent years (Brümmer, Lorenz, 1991;Thiele et al., 2002;Lopez et al., 2003).Sourdough is employed in the manufacture of a number of baked products, such as breads, cakes and crackers (De Vuyst, Gänzle, 2005).Gocmen et al. (2007) and Thiele et al. (2002) reported that the application of sourdough to wheat breads produced several effects, including leavening, acidification, improvement of the dough properties, flavor of the bread, texture, delayed firmness and staling, increased resistance to microbial spoilage and improved nutrient availability of cereals.It was suggested that all these benefits were attributable to the acid lactic bacteria (LAB) and yeasts naturally present in sourdough.
Moreover, it has been noted that when sourdough is added, there are changes in the fundamental rheological properties of wheat dough, making it soft, less elastic and therefore easily extendable (Clarke et al., 2002).The impact of changes in the rheology of dough must be considered in order to choose an appropriate fermentation time and obtain good quality bread.The dough must contain a large volume of gas and also reserve gas retention for oven rise (Bloksma, 1990).
However, there are other factors to be taken into account, such as the type of flour, the sourdough fermentation conditions (pH and temperature) and the selection of starter cultures with specific and desirable metabolic properties (De Vuyst, Vancanneyt, 2007).In this study, the influence of fermentation time on sourdough in terms of pH, titratable acidity, microbiological counts and volume of dough and bread, was analyzed.

Microorganisms
The microorganisms used included two strains from the Lactobacillus paracasei (1 and 2) and two strains from the Saccharomyces cerevisiae (1 and 2).The lactic acid bacteria from the Lactobacillus paracasei (LP1 -9.4 log CFU/g and LP2 -11.11 log CFU/g) and yeasts from Saccharomyces cerevisiae 1 (SC1 -8.18 log CFU/g and SC2 -7.16 log CFU/g) were isolated from grape sourdough, phenotypically and genotypically characterized, freeze dried and applied in this study.

Sourdough preparation
Separate sourdoughs were prepared with each strain.The amount of microorganisms used was 1% (w/w on flour basis, Plessas et al., 2008) and fermented for 48 hours.Dough 1 (D1) was prepared by mixing 60 mL of tap water, 50 g of wheat flour and 0.25 g of NaCl incubated at 30 °C.After 24 hours' incubation, sourdough 1 (SD1) was obtained.Subsequently, 100 g of SD1 was mixed with 200 g of wheat flour and 100 mL of tap water to form dough 2 (D2).After 24 hours incubation, sourdough 2 (SD2) was produced, according to the procedure of Paramithiotis et al. (2005), with modifications.

Dough and bread analyses
Samples of raw dough were analyzed for pH, titratable acidity (TTA) and plate counts while samples of bread were analyzed for pH, titratable acidity and specific volume.These analyses were carried out every 2 hours, between 4 and 10 hours of fermentation.
The pH value was recorded and acidity titrated using 0.1N NaOH (Brasil, 2004).The TTA was expressed in mL 0.1 N NaOH/100 g.The evaluation of growth was determined by counting the number of viable cells in logarithms (log UFC/g) using the pour plate method (Silva et al., 2010).Aseptically, 25 g of each dough was mixed with 225 mL of 0.1 g 100 g -1 of sterile peptone water in a bag mixer (model P, Interscience, St. Nom, France).The necessary dilutions of LAB and yeast were performed.LAB were grown on MRS Agar at 30 ºC/48 hours and yeasts were grown on PDA Agar with tartaric acid (10%) at 25 ºC/72 hours.All experiments were carried out in triplicate.

Specific volume
Loaf volume was measured using the rapeseed replacement method described by Hallén et al. (2004).Each loaf was placed in a metal container with a known volume (V C ).The container was then topped up with rapeseed, the loaf removed and the volume of the rapeseed measured (V R ).Loaf volume (V L ) was then calculated and recorded according to Equation 1. Eq.1 After cooling for 1 h, the same loaves used for measuring volume were weighed on digital scales, W (g). Specific volume (V S ) of bread was calculated according to Equation 2.

Statistical analysis
Statistical analyses were performed using the Statistica® software version 8.0 (Statsoft Inc., Tulsa, OK, USA).The behavior of the raw dough and bread, prepared using sourdough with LC1, LC2, SC1 and SC2, over the period between 4 and 10 hours of fermentation was studied by a simple linear regression model, given by Y= β 0 + β 1 X + Ɛ , where X is the fermentation time, expressed in hours.Differences between the means were established using one-way analysis of variance (ANOVA) followed by Tukey's test.Differences at the 5% level of significance (p < 0.05) were considered statistically significant.

Dough and bread evaluation
The estimated equation of analysis of linear regression for pH, titratable acidity, and plate counts for raw dough, between 4 and 10 h of fermentation, are shown in Figure 1 (A, B and C) whereas pH, titratable acidity and specific volume for breads, between 4 and 10 h of fermentation, are shown in Figure 2 (A, B and C).
The pH values of dough and bread declined with longer fermentation time.The initial pH of dough and breads at 4 h of fermentation ranged from 4.51 to 4.73 ± 0.01 and 4.45 to 4.74 ± 0.01, respectively.At the end of the fermentation period of 10 h, the lowest pH values were for dough with LP2 (3.44 ± 0.04) and for bread with SC1 (2.99 ± 0.05).The analysis of regression indicated that the rates of decrease in pH of dough (Figure 1A) and of bread (Figure 2A), were statistically equal (p > 0.05).
Therefore, the null hypothesis (H 0 ) was not rejected by the F test at the significance level of 5%, where H 0 indicates that angular coefficients were equal.In the analysis of pH of dough, a higher coefficient of determination was observed for yeasts (SC2: 0.8832; SC1: 0.8723; LP2: 0.8416: LP1: 0.8094), while for the pH of breads LAB had the highest r 2 (LP2: 0.9512; LP1: 0.9162; SC1: 0.9061; SC2: 0.8831).Among many important effects, the drop in pH value caused by organic acids exerted an influence on the viscoelastic behavior of dough (Wehrle, Arendt, 1998).
The acidification process, affected by the application of sourdough, is mainly used to improve quality, taste and flavor of wheat breads (Brümmer, Lorenz, 1991;Katina et al., 2006a;Arendt et al., 2007) and to delay staling (Katina et al., 2006b;Plessas et al., 2007).The use of salt has a marked effect on both LAB growth and acid production and can be used as a method of controlling overall bread acidity (Simonsom et al., 2003).
LAB showed the highest counts over time.With the exception of SC2, the highest microbial growth occurred after 10 hours of fermentation, the highest value was for LP2 with 8.91 log CFU/g, followed by LP1 with 8.66 log CFU/g, SC1 with 8.03 log CFU/g.At the same time, SC2 showed a drop of 0.21 log CFU/g.At 6 hours of fermentation, LP2 obtained 8.66 log CFU/g and LP1 8.33 log CFU/g.Yeasts showed 7.18 log CFU/g for SC1 and 7.08 log CFU/g for SC2 (Figure 1C).
Plate counts of LAB and yeasts during spontaneous fermentation of maize flour were previously studied by Edema and Sanni (2008).LAB counts increased steadily from 4.62 log CFU/g at mixing (0 h) to 6.45 log CFU/g after 48 hours fermentation while yeast counts increased from 4.18 to 6.64 log CFU/g over the same period of fermentation.
For plate counts, the analysis of regression indicated that the angular and linear coefficients of LP1/LP2 were statistically the same (p > 0.05), as were the linear coefficients of LP2/SC2 and LP2/SC2.All other contrasts revealed a statistical difference between treatments (p < 0.05).The highest coefficient of determination (r 2 ) for plate count was with SC1 at 0.9674, indicating that the adjustment of the regression explains 96.74% of total variation in Y, but only 3.26% for the residual variation (error), considered a good fit.
Analysis of regression of specific volume (Figure 2C) indicated that all the linear coefficients were statistically different (p < 0.05).The highest coefficients of determination (r 2 ) for yeasts were SC1 and SC2 (0.7975; 0.7909).LC1 and LC2 showed the lowest volume across all hours tested (p < 0.05), where this occurred because they constitute facultative heterofermentative lactic bacteria.
Lactobacillus can be divided into three groups, with the final product as a criterion of their fermentation: Lactobacillus thermophilic obligatory homofermentatives, that ferment only hexoses and lactic acid (Lactobacillus delbrueckii subsp.bulgaricus, Lactobacillus delbrueckii subsp.lactis and Lactobacillus helveticus); Lactobacillus    a-d Different superscript letters in the same column between samples denote significant differences (Tukey's test, p < 0.05).mesophilic facultative heterofermentatives, which are able to ferment other carbon sources beyond these hexoses, producing organic acids, CO 2 , alcohol and H 2 O 2 (Lactobacillus casei, Lactobacillus paracasei and Lactobacillus plantarum); and Lactobacillus mesophilic obligatory heterofermentatives that use principally hexoses and pentoses as carbon source, fermenting hexose and lactic acid, acetic acid, ethanol and CO 2 and pentoses to lactic acid and acetic acid (Lactobacillus brevis, Lactobacillus fermentum) (Fox et al., 2000).The species Lactobacillus casei, Lactobacillus paracasei and Lactobacillus rhamnosus belong to the "Lactobacillus casei Group" and are of great commercial value to the food industry (Ferrero et al., 1996).
The highest specific volumes in bread were found at 6 hours of fermentation (Table 1).SC1 showed better results across all times, followed by SC2.SC1 obtained at 6 hours of fermentation showed the best results, with 2.71 mL/ g (p < 0.05).Saccharomyces cerevisiae is used in the preparation of bread because it produces a dough with structure and has the ability to retain gas.After 6 hours of fermentation, an influence inversely proportional to TTA increase, with a decrease in volume over time can be observed.Sanz-Penella et al. (2012) used Bifidobacteria pseudocatenulatum as a starter in sourdough fermentation and reported that fermented dough with 20% sourdough yielded TTA values of 9.23 ± 0.32mL NaOH 0.1 N/100 g and pH of 4.57 ± 0.11; while bread with LAB and yeast yielded TTA of 10.60 ± 0.41 mL NaOH 0.1 N/100 g and pH of 4.96 ± 0.06.The sample volume increased with 20% sourdough content showed a significantly lower bread specific volume than the control, with 2.22 ± 0.12 mL/g.Crowley et al. (2002) reported that bread specific volume significantly in bread containing 20% sourdough (3.40 ± 0.08 mL/g).
However, Collar et al. (1994) developed lower volume breads when using a high percentage of sourdough with L. plantarum and L. brevis as starters.The acidification of the sourdough and partial acidification of the bread dough have an impact on structure-forming components such as gluten and starch.During incubation of sourdough and dough fermentation, biochemical changes occur in the carbohydrate and protein components of flour owing to the action of microbial and endogenous enzymes (Rollan et al., 2005).There is strong consensus with regard to the positive effects of the addition of sourdough on bread volume and crumb structure (Arendt et al., 2007).

CONCLUSION
The pH values of the dough and bread declined with longer fermentation time.Saccharomyces cerevisiae 2 showed the highest production of acidity of dough over all times studied.LAB showed the highest microbiological counts at between 4 and 10 hours.Lactobacillus paracasei 1 showed the lowest specific volume for all hours tested (p < 0.05).The best fermentation time of breads found in this study was 6 hours, evidenced by the largest volumes.The two strains exhibited different characteristics in the analyses performed, despite being of the same species.Food Sci. Technol., v.39, n.3, p.256-265, 2006.

FIGURE 1 -
FIGURE 1 -Evolution of pH (a), titratable acidity, (b) and plate counts (c) of raw bread dough between 4 and 10 hours of fermentation.

FIGURE 2 -
FIGURE 2 -Evolution of pH (a), titratable acidity (b) and specific volume (c) of bread between 4 and 10 hours of fermentation.