Optimization of sourdough process for improved sensory profile and texture of wheat bread

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LWT 39 (2006) 1189–1202 www.elsevier.com/locate/lwt

Optimization of sourdough process for improved sensory profile and texture of wheat bread K. Katina, R.-L. Heinio¨, K. Autio, K. Poutanen VTT Biotechnology, P.O. Box 1500, FIN-02044 VTT, Finland Received 27 December 2004; received in revised form 5 August 2005; accepted 16 August 2005

Abstract The aim of the study was to determine optimum sourdough process conditions for improved flavour and texture of wheat bread. The influence of process conditions and the starter culture on the characteristics of wheat sourdough bread was established by using response surface methodology. Influence of fermentation temperature (16–32 1C), ash content of flour (0.6–1.8 g/100 g), and fermentation time (6–20 h) were considered as independent factors and their effects were studied in sourdough bread fermented with Lactobacillus plantarum, Lactobacillus brevis, Saccharomyces cerevisiae or with a combination of yeast and lactic acid bacteria. Intensity of sensory attributes, specific volume and bread hardness were considered as the main responses. Ash content of flour and fermentation time were the main factors determining the intensity of sensory attributes. The possibility to enhance intensity of overall flavour, aftertaste and roasted flavour without excessive pungent flavour and without reduced fresh flavour in wheat bread containing 20 g sourdough/100 g of wheat dough was demonstrated by choosing e.g. Lb. brevis for a starter and by utilization of high ash content of flour, long fermentation time and reduced temperature. Bread specific volume was improved 0.2–0.5 ml/g and hardness was reduced (after 4 days of storage) up to 260 g by using low ash content of flour and by optimizing fermentation time according to the microbial strain. Lactic acid fermentation had more profound influence on both desired and undesired flavour attributes, as well as textural features of bread in comparison with yeast fermentation. r 2005 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved. Keywords: Sourdough; Flavour; Texture; Optimization; Sensory profile

1. Introduction Use of sourdough has been reported to improve bread flavour, volume and shelf-life, especially in rye baking (Lorenz & Brummer, 2003). In wheat baking, use of sourdough is optional and more demanding way to improve bread quality because most of consumers accept only mild acidity in wheat products. Thus, controlled acidity level of wheat sourdough and subsequent bread is premise for improved flavour. Utilization of sourdough as quality improver is complex, which is well described in earlier papers reporting contradictory results on the influence of sourdough on bread quality. For example, usage of sourdough has been reported either to decrease (Armero & Collar, 1996; Rouzaud & Martinez-Anaya, Corresponding author. Tel.: +358 9 456 5184; fax: +358 9 455 2103.

E-mail address: Kati.Katina@vtt.fi (K. Katina).

1997; Salovaara & Valjakka, 1987) or to increase (Corsetti et al., 1988, 2000; Crowley, Schober, Clarke, & Arendt, 2002; Hansen & Hansen, 1996) bread volume and shelf-life (Corsetti et al., 1998, 2000). Accordingly, sourdough fermentation has been reported enhance both desired and undesired flavour attributes (Collar, de Barber, & Martinez-Anaya, 1994; Hansen & Hansen, 1996; Meignen et al., 2001; Salovaara & Valjakka, 1987; Thiele, Ga¨nzle, & Vogel, 2002). Furthermore, industrial utilization of wheat sourdough as bread improver has not gained wide acceptance in many countries, which might also reflect difficulties to utilize wheat sourdough successfully. For example, in France breadmaking with sourdough has been estimated to be only 3% of all the bread manufactured (Poitrenaud, 2003). In Finland, yeasted preferments are used widely but use of wheat sourdough is very rare, despite of long tradition for eating acidic rye bread (Valjakka, Kerojoki, & Katina, 2003).

0023-6438/$30.00 r 2005 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.lwt.2005.08.001

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However, sourdough is a potential option to improve bread quality also in wheat baking due to the natural, additive-free image of products containing sourdough (Salovaara, 1998). Furthermore, recent results demonstrate the effectiveness of sourdough fermentation in improving the nutritional value of cereal products (Liukkonen et al., 2003). Requirement for effective use of wheat sourdough is that influence and interactions of sourdough process conditions on biochemical activity of sourdough and subsequent bread quality are understood and optimized accordingly. Bread flavour remains still a challenge due to its complicated nature. Especially when sourdough is utilized, improvement of wheat bread flavour requires a carefully controlled process to avoid e.g. excessive acidity (perceived as a sour or pungent flavour), and still enhance the positively charged flavour characteristics, such as roasted flavour of bread crust. Starter culture, ash content of flour, fermentation temperature and dough yield have been reported to influence bread flavour (Gobbetti et al., 1995; Hansen & Hansen, 1996; Martinez-Anaya, Collar, & de Barber, 1995; Meignen et al., 2001). However, the influence of the concurrent interactions of fermentation time, temperature and ash content of flour on the specific sensory attributes of sourdough breads determined by using quantitative descriptive analysis has not been reported before. Traditionally, extensive pungent (or sour) flavour formation has been controlled by limiting amount of sourdough (5–10 g/100 g of dough) to be used in subsequent bread. This approach, however, limits also amount of sourdough originated flavour precursors as well as important flavour compounds in the final bread dough. Recently, controlled sourdough process with moderate acidity and high amount of flavour precursors has been developed by utilizing yeast and lactic acid bacteria (LAB) with special technological properties (Mori, Okada, Onishi, & Takaki, 2001). Other possibility would be to adjust processing conditions to produce maximum amount of flavour precursors such as amino acids and flavour active volatile compounds with minimum amount of acidity development (Katina, Poutanen, & Autio, 2004). Improved volume and shelf-life of sourdough breads has been suggested to be dependent on the nature and intensity of the acidification process (Clarke, Schober, & Arendt, 2002). Systematic studies to improve bread volume and shelf-life by using optimized sourdough process are also rare (Clarke, Schober, Angst, & Arendt, 2003), because the interactions of the process parameters have not yet been thoroughly studied. The present study was designed: (1) to determine the influence and interactions of sourdough process parameters (ash content of flour, fermentation time and temperature) on the flavour attributes, the volume and the shelf-life of subsequent wheat sourdough breads fermented with single strains of lactobacillus or yeast or with a combination

starter (yeast+LAB); and (2) to improve flavour, volume and shelf-life of wheat bread by using an optimized sourdough process. 2. Materials and methods 2.1. Microbial strains The LAB used in the studies were Lactobacillus plantarum VTT E -78076 and Lactobacillus brevis VTT E-95612. Yeast was Saccharomyces cerevisiae VTT B81047. The selected strains originated from Finnish sour rye baking. Combination starter contained both studied LAB and S. cerevisiae. Growth ability of LAB and yeast, and preparation of culture filtrate for sourdoughs was done as described earlier (Katina et al., 2004). 2.2. Preparation of sourdoughs Wheat flours (Oululaisen Mylly, Finland) with different ash content (0.6 g/100 g, 1.2 g/100 g, 1.8 g/100 g, d.w) were used in the experiments. Sourdough was prepared by mixing 600 g of tap water, 400 g of wheat flour and the inoculum of LAB or yeasts (106–107 cfu/g) in a large beaker (2000 ml) and covered with aluminium foil. The size of inoculum for LAB was 107 cfu/g of sourdough and for yeasts 106 cfu/g of sourdough. Ash content of flour, fermentation time and temperature were as indicated in the experimental design (Table 1). Ready sourdoughs were immediately transferred to a cooling cabinet (4 1C) and used in subsequent baking without delay (maximum storage time 1 h at 4 1C).

Table 1 Composition of various runs of central composite design Run

Temperature (Te, 1C)

Time (Ti, h)

Ash content of flour (A, g/100 g, d.w)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

16 32 16 32 16 32 16 32 16 32 24 24 24 24 24 24 24

6 6 20 20 6 6 20 20 12 12 6 20 12 12 12 12 12

0.6 0.6 0.6 0.6 1.8 1.8 1.8 1.8 1.2 1.2 1.2 1.2 0.6 1.8 1.2 1.2 1.2

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2.3. Baking Sourdoughs were prepared according to the experimental design (Table 1) and used in the subsequent baking at the level of 20 g/100 g of bread dough. The recipe for the control bread without sourdough (g) used in this study was wheat flour with an ash content of 0.6 g/100 g (1250), wholemeal rye flour (200), wholemeal wheat flour (100) and wheat flour with an ash content of 1.2 g/100 g (100), fresh yeast (55), salt (30), fat (25) and water (1073). The recipe for sourdough breads (g) was sourdough (578), wheat flour with an ash content of 0.6 g/100 g (1019), wholemeal rye flour (200), wholemeal wheat flour (100) and wheat flour with an ash content of 1.2 g/100 g (100), fresh yeast (55), salt (30), fat (25) and water (726). Thus, the amount of flour and water was same in the control bread and in the sourdough breads. Breads were prepared by mixing (sourdough), flour, water, sugar, salt, yeast (fresh yeast, Suomen Hiiva Oy, Finland), fat (baking margarine, Raision Yhtyma¨, Finland) and emulsifier (Panodan, Danisco Ingredients, Denmark) for 3+5 min with a Diosna spiral mixer (DIOSNA Dierks & So¨hne GmbH, Germany). After a floor time of 30 min at +28 1C and relative humidity of 76% the dough was divided into 400 g loaves and moulded mechanically. The loaves were proofed in pans (60 min at +35 1C, RH 75%) and baked at 230 1C for 20 min. After 2–6 h of cooling, sensory evaluation and determination of specific volume were performed. For shelf-life evaluation, loaves were stored in plastic bags at 20 1C for 4 days. TPA-test was performed after 24 h and 96 h storage. 2.4. Analytical methods 2.4.1. Sensory evaluation Descriptive analysis was used to determine the sensory profiles of the bread samples. The control for the sourdough bread samples was wheat bread without sourdough. For creating the vocabularies of the sensory attributes, several sourdough breads deviating as much as possible from each other were baked for model breads. A

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four-member expert panel, specialized in cereal products, described the differences between the breads in a roundtable session. The selected attributes of the sensory profile described the texture and flavour (simultaneous perception of odour, taste and trigeminal nerve response) characteristics of the different sourdough breads as extensively as possible. For the bread samples eight descriptors were relevant: softness, springiness and moistness of the bread crumb, degree of the roasted flavour of the bread crust, and intensity of pungent flavour and degree of fresh flavour, intensity of overall flavour and aftertaste of the bread crumb. The assessors were instructed to evaluate first the intensity of the roasted flavour of the crust by breaking it up from the pan bread and thereafter the intensities of the crumb attributes from the rest of the bread. Detailed definitions of the attributes are presented in Table 2. The attribute intensities were rated on continuous unstructured, graphical intensity scales by the panel. The scales were 10 cm in length and verbally anchored at each end, the left side of the scale corresponding to the lowest intensity (value 0) and the right side to the highest intensity (value 10) of the attribute. The descriptive panel consisted of ten trained assessors with proven skills. All sensory work was carried out at the sensory laboratory of VTT Biotechnology, which fulfils the requirements of the ISO standards (ISO 1985, 1988). All assessors of the internal sensory panel have passed the basic taste test, the odour test and the colour vision test. They have been trained in sensory methods at numerous sessions over several years, and their evaluation ability is routinely checked using individual control cards for each assessor. The panel was particularly familiarized with the sensory descriptors and the attribute intensities by using verbal definitions describing the ends of the intensity scales of the attributes. The same panel has also frequently been used in our previous studies on cereals. The 17 bread samples prepared according to the experimental design consisted of 14 samples and three replicates of the center point sample and the control bread without sourdough. Owing to the high number of samples, the samples were assessed in three sessions during one day.

Table 2 The attributes, definitions and anchors used in the descriptive analysis Attribute

Definition

Anchors

Texture Softness of bread crumb Springiness of bread crumb Moistness of bread crumb

Degree of softness in crumb by pressing between fingers Degree of springiness in crumb by pressing between fingers Degree of moistness in crumb by feeling with fingers and in mouth

Soft–hard Not springy–springy Dry–moist

Flavour Degree of roasted flavour of bread crust Intensity of pungent flavour of bread crumb Degree of fresh flavour of bread crumb Intensity of overall flavour of bread crumb Intensity of aftertaste of bread crumb

Degree of roasted/burnt odour and taste of bread crust Degree of pungent (sour, vinegar-like) odour and taste of bread crumb Degree of fresh odour and taste of bread crumb Intensity of overall odour and taste of bread crumb Taste of crumb staying in mouth after tasting

Not roasted–roasted Not pungent–pungent Not fresh, musty–fresh Low–high Low–high

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The freshly baked bread samples were presented to the assessors as 2.5 cm slices from three-digit coded white cardboards in random order. The control bread without sourdough was thus introduced in evaluations as a hidden, randomized sample among other samples. Each assessor was provided with six bread samples to be evaluated in one session. Water was provided for cleansing the palate between the samples. The panel was instructed to sniff each sample prior to tasting it, and they were requested to swallow the samples. Scores were recorded and collected using a computerized data system (Compusense Five, Ver 4.2, CSA, Computerized Sensory Analysis System, Compusense Inc., Canada). Means of the raw data obtained were calculated. The significance of each descriptive attribute in discriminating between the samples was analysed using analysis of variance (ANOVA), and Tukey’s honestly significant difference (HSD) test (significance of differences at Po0:05). A two-way ANOVA was applied as the general linear model (GLM) procedure for the bread samples by using the SPSS software (SPSS Ver. 10.0, SPSS Inc.). ANOVA was used to test statistical differences in sensory attributes between the samples, and the statistical difference between the sessions. When the difference in ANOVA among the samples was statistically significant, pairwise comparisons of these samples were analysed using Tukey’s test. 2.5. Texture analysis Bread volume was determined by rape seed displacement (4 loaves). Crumb firmness was measured at days 1 and 4 to assess the potential shelf-life of the breads. Bread crumb firmness during storage was determined as maximum compression force (40% compression, AACC 1998, modified method 74-09) using the texture profile analysis (TPA) test (Texture Analyser, Stable Micro Systems, Godalming, England). Eight bread slices (originating from 3 loaves) were measured and results were expressed as mean values. The height of each bread slice was 2.5 cm and edges of the slice were cut off before measurement. Mean values of volume and hardness were calculated and used in modelling.

sourdoughs and sponge fermentation processes applied in Finnish bakeries. The results were analysed by a multiple regression method (MLR or PLS), which describes the effects of variables in second-order polynomial models. For each response (flavour attributes, specific volume and hardness) a quadratic model was used: Y ¼ b0 þ b1 Te þ b2 Ti þ b3 A þ b11 Te2 þ b22 T12 þ b33 A2 þ b12 TenTi þ b13 TenA þ b23 TinA þ
. This model took into account the effects of the variable alone (e.g.: Ti), the effects of the interactions between two variables (e.g.: TinTe) and quadratic effects of the variables alone (Ti2). Regression analysis was calculated and the response surfaces were plotted with the Modde 4.0 and 6.0 (Umetrics AB, Umea˚, Sweden). The fit of model to the experimental data was given by the coefficient of determination, R2, which explains the extent of the variance in a modelled variable that can be explained with the model. Each model was validated by calculating predictive power of model, Q2, which is a measure of how well the model will predict the responses for a new experimental condition. Q2 is based on prediction of residual sum of squares (PRESS). For determining Q2, the computations are repeated several times, each time omitting different objects from the calculation of the model. PRESS is then computed as the squared difference between observed Y and predicted values (cross validation of R2). Large Q2 indicates that the model has good predictive ability and will have small prediction errors. Q2 should be 40.5 if conclusion are to be drawn from the model (Lingren, 1995). The replicates at the center point made it possible to estimate pure error of the analyses, which was used to predict whether the models gave significant lack of fit (Carlsson, 1992). Validity of model is considered to be good if lack of fit value is 40.25. Reproducibility of models was evaluated by comparing variation of the response under the same conditions (pure error), at the centre points to the total variation of the response with following equation: 1—(meansquare (pure error)/mean square (total SS corrected). Only models with high reproducibility and with no significant lack of fit were included in this study. 3. Results

2.6. Experimental design and statistical methods To study the effects of the main factors on properties of sourdough bread the following parameters were selected as independent variables: temperature (Te, 16–32 1C), ash content of flour in g/100 g (A, 0.6–1.8 g/100 g) and fermentation time (Ti, 6–20 h). A central composite design was used to arrange experiments and three replicates were made at the centre point of design to allow estimation of the pure error at sum of the square. The experimental design is presented in Table 1. Levels of variables were selected on the basis of commonly used ones in real

Seventeen experiments were performed according to the experimental design and intensity of sensory attributes, specific volume and bread firmness for single starters and for the combination starter (yeast and LAB) were determined at each experimental point. For each response group a quadratic equation was formed with relevant terms (Po0:05) to obtain as high R2 and Q2 values as possible. Based on these equations, behaviour of response can be predicted within the experimental area and presented as a response surface. The coefficients of a particular model, and R2 and Q2 values, and lack of fit values obtained are

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Table 3 Effects of factors, expressed as their corresponding coefficients obtained in the models for sensory attributes, in sourdoughs fermented with Lb. brevis, Lb. plantarum, S, cerevisiae or with combination startera Sensory attributes Lb. plantarum Dr* IP* DFr Ifl IA Lb. brevis Dr IP DFr Ifl IA S. cerevisiae Dr IP DFr Ifl IA Combination starter IP DFr IFl IA

Regression equationsb

Goodness of fit and predictive power

0.59+0.044Ab+0.004Te+0.006Ti 0.047+0.163A+0.016Ti 5:48 þ 1:28A þ 0:12Ti
0:007TenTi
0:67TinA

R2 R2 R2 R2 R2

¼ 0:88, ¼ 0:78, ¼ 0:96, ¼ 0:95, ¼ 0:91,

Q2 Q2 Q2 Q2 Q2

¼ 0:81 ¼ 0:58 ¼ 0:63 ¼ 0:87 ¼ 0:75

0.44 0.24 0.27 0.25 0.25

R2 R2 R2 R2 R2

¼ 0:91, ¼ 0:96, ¼ 0:77, ¼ 0:85, ¼ 0:85,

Q2 Q2 Q2 Q2 Q2

¼ 0:75 ¼ 0:84 ¼ 0:58 ¼ 0:75 ¼ 0:70

0.35 0.33 0.77 0.65 0.27

1:28
1:14A
0:045Te þ 0:008TenTi
0:15Ti 5:82
1:89A þ 0:074AnTi 5.2+0.95A
0.28Ti+0.015Ti2 3.72+0.88A
0.32Ti+0.016Ti2

R2 R2 R2 R2 R2

¼ 0:75, ¼ 0:88, ¼ 0:91, ¼ 0:85, ¼ 0:75,

Q2 Q2 Q2 Q2 Q2

¼ 0:61 ¼ 0:67 ¼ 0:66 ¼ 0:68 ¼ 0:61

0.77 0.25 0.38 0.25

4.47
0.069A+0.33Ti 8.44
0.17A+0.015Ti2 4:73 þ 0:013A þ 0:076Ti þ 0:04AnTe 2.92+0.79A+0.043Ti

R2 R2 R2 R2

¼ 0:80, ¼ 0:76, ¼ 0:89, ¼ 0:85,

Q2 Q2 Q2 Q2

¼ 0:56 ¼ 0:63 ¼ 0:77 ¼ 0:76

0.39 0.52 0.64 0.26

8:92 þ 0:84A þ 0:007TenTi þ 0:02Ti2 1:77 þ 0:94A þ 0:04Ti þ 0:01TenTi 2.67+1.08A+0.035Ti
0.005Ti2 2:01
1:11A
0:072Te þ 0:356Ti þ 0:013TenTi þ 0:84A2
0:023Ti2 9.45
0.84A+0.013Ti2 3.70+0.99A+0.065Ti 2.16+1.01A+0.11Ti 3.72+0.88A
0.32Ti 2

Lack of fit

a

Only values of significant coefficients are presented (95% confidence level), *, logarithm of the response is used; DR, degree of roasted flavour in bread crust; IP, intensity of pungent flavour in bread crumb; DFr, degree of fresh flavour of bread crumb; IFl, intensity of overall flavour; IA, intensity of aftertaste. b A, ash content of flour; Te, temperature; Ti, time; TenT1, time–temperature interaction; A2, quadratic effect of ash content of flour; Ti2, quadratic effect of time; R2, measure of fit of the model; Q2, predictive power of the model.

presented in Tables 3 and 4. Using these tables, values of measured variables such as the degree of roasted flavour (e.g. bread with Lb. brevis fermented sourdough) can be predicted by the following equation for the experimental area under consideration: Degree of roasted flavourprd ¼ 2:67 þ 1:08nA þ 0:035nTi þ
0:005nTi2 , where prd ¼ predicted, A ¼ ash content of flour and Ti ¼ time. If logarithm transformation of responses is used (to normalize standard distribution of data), logarithm of the particular response value is predicted from the equation. Selected response surface figures were also presented to illustrate these equations and figures were made by choosing two main variables (e.g. time and temperature) exhibiting the highest influence on the particular response (e.g. specific volume) for x and z-axis. The coefficients of correlation of the measured responses are also presented by using Lb. brevis and combination starter fermented sourdough breads as an example (Table 5).

3.1. Sensory attributes Relatively high R2 values and Q2 were obtained in the flavour models (Table 3); typical R2 varied between 0.74 and 0.99 and typical Q2 values between 0.58 and 0.93. In general, the intensities of pungent and fresh flavour, the overall flavour and the aftertaste could be modified by using sourdough, because these attributes were significantly different (Po0:05) from each other among the sourdough breads and from the control bread without sourdough. Degree of roasted flavour of bread crust could be increased only with sourdoughs fermented by a single LAB starter. None of the texture attributes of sourdough breads assessed in the sensory evaluations was significantly different from each other, and thus only the above mentioned flavour attributes were included in the modelling. Sourdough breads fermented with Lb. brevis or Lb. plantarum had the most profound effect on sensory profile, since both of these strains intensified bread flavour as presented in Figs. 1 and 2. Influence of processing conditions on flavour attributes was different for Lb. plantarum and for Lb. brevis. With homofermentative

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Table 4 Effects of factors, expressed as their coefficients obtained in the models for specific volumes, and hardness after 4 days of storage in sourdough breads fermented with Lb. brevis, Lb. plantarum, S. cerevisiae or with combination startera Texture attributes

Regression equationsb

Goodness of fit and predictive power

Lack of fit

Lb. plantarum Specific volume Hardness after 4d

3.65+0.70A+0.004Te+0.40A2 552:47 þ 7:43Ti
0:57TenTi

R2 ¼ 0:84, Q2 ¼ 0:72 R2 ¼ 0:99, Q2 ¼ 0:96

0.61

R2 ¼ 0:99, Q2 ¼ 0:94

0.42

Hardness after 4 d

0:38
0:21A þ 0:02Te þ 0:02Ti þ 0:001AnTe
0:002AnTi
0:0005TenTi þ 0:078A2
0:003Te2
0:0005Ti2 975.88+25.38A
52.95Ti+1.52Ti2

R2 ¼ 0:92, Q2 ¼ 0:65

0.34

S. cerevisiae Specific volume Hardness after 4 d

5:78
0:48A
0:08Ti þ 0:002TenTi þ 0:002Te2 209:20 þ 49:86Te
10:21AnTi
1:29Te2 þ 1:50Ti2

R2 ¼ 0:88, Q2 ¼ 0:59 R2 ¼ 0:90, Q2 ¼ 0:56

0.75 0.28

Combination starter Specific volume* Hardness after 4 d

0:53
0:021A
0:00012TenTi 622.90
94.49A+9.03Te
11.86Ti

R2 ¼ 0:81, Q2 ¼ 0:61 R2 ¼ 0:88, Q2 ¼ 0:76

0.65 0.81

Lb. brevis Specific volume*

a

Only values of significant coefficients are presented (95% confidence level), * ¼ logarithm of the response is used. A, ash content of flour; Te, temperature; Ti, time; AnTe, ash content–temperature interaction; A2 ¼ quadratic effect of ash content; Ti2, quadratic effect of time; Te2, quadratic effect of temperature, R2, measure of fit of the model; Q2, predictive power of the model. b

Table 5 Correlation matrix between sensory attributes and texture properties in sourdough bread fermented with Lb. brevis or with combination starter IFl

IP

DFr

IA

DR

Volume

Hardness

Lb. brevis IFla IPb DFrc IAd DRe Volume Hardness

1 0.89
0.81 0.90 0.67 nsf ns

0.81 1
0.89 0.84 0.72 ns ns


0.81
0.89 1
0.65
0.67 ns ns

0.90 0.84
0.65 1 0.74 ns ns

0.67 0.72
0.67 0.74 1 ns ns

ns ns ns ns 1
0.68

ns ns ns ns ns 1

Combination starter IFla IPb DFrc IAd Volume Hardness

1 0.86
0.68 0.94 nsf Ns

0.86 1
0.86 0.86 ns ns


0.68
0.86 1
0.70 ns ns

0.94 0.89
0.70 1 ns ns

ndg nd nd nd nd nd

ns ns ns ns 1
0.55

ns ns ns ns
0.55 1

a

IFl, intensity of overall flavour. IP, Intensity of pungent flavour of bread crumb. c DFr, degree of fresh flavour of bread crumb. d IA, intensity of aftertaste. e DR, degree of roasted flavour of bread crust. f ns, no significant correlation at the level of 5%. g nd, not determined. b

LAB, enhanced intensity of overall flavour could be reached after short fermentation time, which was not the case with heterofermentative strain. With Lb. brevis, highest intensity of pungent flavour and decreased fresh flavour as well as highest degree of roasted flavour were obtained after 14 h of fermentation and with wholemeal wheat flour. With Lb. plantarum, highest intensities of

above-mentioned attributes were reached after 20 h fermentation. Effect of sourdoughs fermented with S. cerevisiae on both desired and undesired sensory attributes was considerably smaller in comparison with LAB-fermented sourdoughs (Fig. 3). Sourdough bread fermented with S. cerevisiae diminished roasted flavour of bread crust in the

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Fig. 1. Influence of fermentation time and ash content of flour on sensory attributes of sourdough bread fermented with Lb. plantarum (fermentation temperature 24 1C). C ¼ intensity of the attribute in control bread.

experimental region. Use of sourdough fermented with combination starter enhanced intensity of overall flavour, pungent flavour, aftertaste and diminished degree of fresh flavour. Response surface for these attributes (plots not shown) resembled ones obtained with Lb. brevis fermented sourdoughs in Fig. 2. However, sourdough fermented with combination starter did not alter roasted flavour in

comparison with control bread and this attribute was therefore not modelled. In general, ash content of flour was clearly the most important parameter influencing bread flavour in all sourdough types (Table 3, Figs. 1–3), as increasing ash content resulted in enhanced intensity of overall flavour, pungent flavour, roasted flavour and

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Fig. 2. Influence of fermentation time and ash content of flour on sensory attributes of sourdough bread fermented with Lb. brevis (fermentation temperature 24 1C). C ¼ intensity of the attribute in control bread.

aftertaste. Increased ash content also diminished fresh flavour. In addition, increased fermentation time enhanced intensity of overall flavour, pungent flavour, roasted flavour and aftertaste, and diminished fresh flavour in

most sourdough types (Table 3). However, for some flavour attributes such as intensity of overall flavour (Lb. plantarum, S. cerevisiae), degree of roasted flavour (Lb. brevis), degree of fresh flavour (Lb. brevis and combination starter), intensity of pungent flavour (S. cerevisiae),

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Fig. 3. Influence of fermentation time and ash content of flour on sensory attributes in sourdough breads fermented with S. cerevisiae (fermentation temperature 24 1C). C ¼ intensity of the attribute in control bread.

quadratic effect of time was observed. This indicates that there is an optimum time window in which maximum/ minimum value of the attribute in question will be reached.

The influence of fermentation temperature was in general much smaller in comparison with time and flour quality. Increased fermentation temperature enhanced

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roasted flavour in sourdough breads fermented with Lb. brevis and increased intensity of pungent flavour in sourdough breads fermented with Lb. brevis or with S. cerevisiae. Also, interaction of fermentation time and temperature was recognized in the intensity of overall flavour, fresh flavour and aftertaste in sourdough breads fermented with Lb. plantarum indicating that influence of time on the attribute in question was dependent on the level of temperature (Table 3). Interaction of time and temperature was also significant in formation of pungent flavour in breads fermented either with Lb. brevis or with S. cerevisiae. 3.2. Specific volume of breads Relatively high R2 values and Q2 were obtained in the volume models (Table 4); typical R2 values varied between 0.81 and 0.99 and typical Q2 values between 0.59 and 0.94. In general, specific volume of bread could be improved by 0.2–0.5 ml/g, if optimized sourdough process was utilized. Increasing ash content of flour diminished bread volume in all sourdough types, but influence of fermentation time and temperature was strain-dependent. On the sourdough breads fermented with Lb. brevis, the strong quadratic effect of time was observed indicating that optimum volume was reached, if fermentation time was 10–14 h (Table 4, Fig. 4). For the sourdough bread fermented with Lb. brevis, strong quadratic effect was observed also for fermentation temperature, optimum volume required temperature window of 22–26 1C as exemplified in Fig. 4. For Lb. plantarum fermented sourdough breads, optimum volume was reached with medium ash content of flour and with elevated temperature levels (Table 4). For the sourdough bread fermented with S. cerevisiae, optimum volume was reached in 8 h and at 16 1C as

Fig. 4. Influence of fermentation time and temperature on specific volume of sourdough bread fermented with Lb. brevis (ash content of flour 0.6 g/ 100 g). C ¼ specific volume of control bread.

Fig. 5. Influence of fermentation time and temperature on specific volume of sourdough bread fermented with S. cerevisiae with 0.6 g/100 g ash content of flour. C ¼ specific volume of control bread.

increased time and temperature both diminished bread volume (Fig. 5). Significant quadratic effect of temperature, however, was observed for S. cerevisiae fermented bread (Table 4) indicating that utilization of room temperature (20–24 1C) diminished bread volume with all fermentation times as presented in Fig. 5. For sourdough bread fermented with combination starter, optimum volume was reached with short fermentation time and high temperature due to significant interaction of these parameters (Table 4). Among most of studied sourdough types significant interaction of time and temperature was observed (Table 4) indicating that influence of temperature on bread volume was dependent on the length of fermentation time. 3.3. Firmness of breads Relatively high R2 values and Q2 were obtained in the hardness models (Table 4); typical R2 values varied between 0.88 and 0.99 and typical Q2 values between 0.55 and 0.99. After 1 day of storage, firmness of sourdough breads did not differ significantly (Po0:05) from the control bread. However, after 4 days of storage, hardness of sourdough bread could be decreased by 100–190 g (depending on the sourdough type) in comparison with the control bread, if optimized sourdough conditions were utilized. The softest bread texture after 4 days of storage was obtained in sourdough bread fermented with S. cerevisiae for 12 h at 32 1C and with low ash content of flour (0.6 g/ 100 g), as presented in Fig. 6. For LAB-started sourdough breads the softest bread texture after storage was obtained in 20 h with low ash content of flour (0.6 g/100 g) (Fig. 7). In general, fermentation time had a strong linear effect on softness of bread crumb among all sourdough types, as increased fermentation time decreased firmness. Quadratic effect of time was observed with breads fermented by Lb. brevis or by S. cerevisiae indicating that there is an

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overall intensity of bread flavour, intensity of aftertaste and with degree of roasted flavour and negatively with degree of fresh flavour. Intensity of overall flavour correlated positively with intensity of aftertaste and negatively with degree of fresh flavour. Degree of roasted flavour correlated positively with intensity of aftertaste and negatively with degree of fresh flavour. Significant negative correlation was observed between volume and hardness of the sourdough bread. Sensory attributes were not significantly correlated with bread volume or with bread hardness in any of the studied sourdough breads. 4. Discussion

Fig. 6. Influence of fermentation time and temperature on hardness of sourdough bread fermented with S. cerevisiae after 4 days of storage (Ash content of flour, 1.2 g/100 g). C ¼ hardness of control bread after 4 days of storage.

Fig. 7. Influence of fermentation time and ash content of flour on hardness of sourdough bread fermented with Lb. brevis after 4 days of storage (fermentation temperature 24 1C). C ¼ hardness of control bread after 4 days of storage.

optimum time window for reaching maximum softness. With breads fermented with S. cerevisiae or with combination starter, increasing temperature decreased bread firmness (Table 4). Furthermore, negative quadratic effect of temperature was observed in sourdough breads fermented with S. cerevisiae. Increased ash content of flour increased firmness in sourdough breads fermented with either Lb. brevis, S. cerevisiae or combination starter. 3.4. Correlation of textural measurement, sensory attributes and acidity values of bread In general, sensory attributes of sourdough breads correlated strongly (positively or negatively) with each other (r ¼ 0:6520:94) as exemplified in Table 5 for breads fermented with Lb. brevis or with combination starter. Intensity of pungent flavour correlated positively with

This work confirmed the effectiveness of sourdough in enhancing bread flavour attributes, especially if LABcontaining starters were utilized. The strong influence of increased ash content of flour and fermentation time in flavour modifications is most likely due to enhanced proteolysis, acidification and formation of volatile compounds, as reported earlier for fermentation conditions identical to those of this study (Katina et al., 2004). Furthermore, Czerny and Schieberle (2002) reported intensive formation of flavour active volatile compounds in wholemeal wheat flour during lactic acid fermentation in comparison to white wheat flour. Increased proteolysis is due to higher proteolytic activity of wholemeal flour (Loponen, Mikola, Katina, SontagStrohm, & Salovaara, 2004) and leads to the accumulation of amino acids into the sourdough and into the subsequent bread dough. Amino acids, such as leucine and proline, are well-known flavour precursors in yeast fermentation during dough proofing and in the Maillard reaction during baking (Thiele et al., 2002; Gassenmeier & Schieberle, 1995). However, desired flavour attributes (intensity of overall flavour, roasted flavour and intensity of aftertaste) correlated in general strongly with undesired flavour attributes such as pungent flavour and reduced fresh flavour. This is due to the fact that acidification, and especially formation of acetic acid, is the main factor enhancing pungent flavour (Salovaara & Valjakka, 1987; Spicher & Stephan, 1993) and diminishing fresh flavour (Heinio¨, Liukkonen, Katina, Myllyma¨ki, & Poutanen, 2003). On the other hand, acidification has been shown to be also a key factor in the induction of proteolysis, the main factor enhancing e.g. roasted flavour, during sourdough fermentation (Thiele et al., 2002). Furthermore, in utilization of wholemeal wheat flour with high ash content, outer layers of cereal kernel are introduced to wheat baking. Bran layer of cereal kernel has been reported to have more bitter flavour and intense aftertaste (Heinio¨, Katina, Wilhelmson, Myllyma¨ki, Rajama¨ki, Latva-Kala, 2003) in comparison to endosperm flour, most likely due to location of various phenolic compounds mainly in outer layers of cereal kernel. Phenolic compounds can have a major impact on flavour of cereals (Dimberg, Molteberg, Solheim, & Fro¨lich, 1996).

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Recently, it was demonstrated by Liukkonen et al. (2003), that sourdough fermentation of whomeal rye with mixed culture (yeast+LAB) considerably increased level of free phenolic compounds in sourdough, which might partly explain development of intense aftertaste and bitter flavour attributes. In addition, formation of peptides during intensive proteolysis, with undesired sensory characteristics, cannot be excluded (Martinez-Anaya, 1996). This dependence of proteolysis on the acidification (and possible liberation of phenolic compounds) in sourdough fermentation limits the possibility to enhance bread flavour, as maximum acceptable amounts of sourdough to be used in wheat baking (in terms of flavour) has been reported to be 5–10 g/100 g of dough (Lorenz & Brummer, 2003). Influence of processing conditions on bread flavour differed in some extent for Lb. plantarum and for Lb. brevis. Differences can be partly explained by correlating amino acids and organic acids of same sourdoughs (Katina et al., 2004) with sensory properties of subsequent sourdough breads (correlation data not shown). Formation of acetic acid in sourdough resulted slightly higher intensities of aftertaste (r ¼ 0:82), and different time pattern for formation of pungent flavour for Lb. brevis fermented sourdough bread. Acetic acid has been claimed to act as flavour enhancer in small concentrations by Molard, Nago, and Drapron (1979) and on the other hand, enhance pungent flavour in high concentrations (Molard & Cahagnier, 1980). Also, degree of roasted flavour was slightly more intense with Lb. brevis fermented sourdough and correlated highly with the level of proline (r ¼ 0:75) of this sourdough (Katina et al., 2004). With Lb. plantarum, roasted flavour correlated with formation of ornithine (r ¼ 0:80) and with reduce level of arginine formation in sourdoughs which may indicate ability of this strain to produce ornithine from arginine. The influence of sourdough fermented with yeast (yeasted preferment) or with combination starter on bread flavour attributes (both desired and undesired ones) was clearly smaller as compared with sourdoughs fermented with LAB. Inability of yeasted preferment or sourdough containing yeast to enhance roasted flavour is due to the fact that in yeast fermentation, accumulation of amino acids is modest, because of a high demand for amino acids by yeast metabolism. If white wheat flour is used, amount of amino acids actually decreases during fermentation (Katina et al., 2004). This finding confirms the very significant role of proteolysis during sourdough fermentation in enhancing subsequent bread flavour. An interesting finding is that the well-established effect of intensive formation of volatile compounds in yeasted preferments with low ash content of flour and with long fermentation time (Katina et al., 2004; Meignen et al., 2001) does not seem to correlate with enhanced intensities of bread flavour attributes. This can be partly explained the fact that all the analysed compounds are not flavour active compounds identified for example Schieberle and Grosch (1991).

However, Gassenmeier and Schieberle (1995) reported intensive formation of flavour active compounds, 3methylbutanol and 2-phenylethanol in yeasted, liquid preferment (low ash content of flour) after fermentation of 16 h at 30 1C. In this study, similar conditions did not modify significantly sensory properties of crumb in comparison to the control bread without preferment, perhaps due to the use of different yeast strains (sourdough yeast versus commercial baker’s yeast) or due to different sensory evaluation procedure. Zehentbauer and Grosch (1998) reported lower degree of roasted flavour of bread crumb containing 10 g of yeasted preferment/100 g of dough and made with 18 h fermentation in comparison to one made without preferment and fermented for 3 h. Also in our study, degree of roasted flavour was smaller in bread containing preferment fermented 6–20 h in comparison to bread made with straight dough (fermented 90 min). Also, Lorenz and Brummer (2003) reported influence of pure yeast fermentation on enhancement of bread flavour being unlikely. In practice, enhancement of the desired flavour attributes without excessive pungent flavour and loss of fresh flavour could be obtained with Lb. brevis started sourdough fermented at 24 1C for 20 h and made with flour having an ash content of 1.6 g/100 g. According to the models obtained, predicted values would be 6.7 for intensity of overall flavour (5.3 for control), 4.4 for intensity of pungent flavour (3.5 for control), 5.5 for degree of fresh flavour (6.6 for control) 4.9 for intensity of aftertaste (3.6 for control) and 7.5 for degree of roasted flavour of crust (6.5 for control bread). However, such sourdough would be in an intensive phase of metabolism and would require an instant method (such as cooling down or salting) to slow down fermentation and to preserve the required levels of metabolites responsible of the desired levels of flavour attributes. Enhanced intensity of overall flavour without development of pungent flavour or reduced fresh flavour could be obtained with Lb. plantarum started sourdough breads fermented at 32 1C for 6 h and made with flour having an ash content of 1.6 g/100 g. According to the models obtained, predicted values would be 7.0 for intensity of overall flavour (5.3 for control), 3.6 for intensity of pungent flavour (3.5 for control), 7.2. for degree of fresh flavour (6.6 for control) 3.2 for intensity of aftertaste (3.6 for control) and 6.6 for degree of roasted flavour of crust (6.5 for control bread). The study confirmed the effectiveness of LAB-containing starters in improving bread volume as compared with pure yeasted preferments (Clarke et al., 2003; Corsetti et al., 2000; Gobbetti et al., 1995). However, the big influence of the level of process parameters as well as the mutual interactions of these parameters in determining bread volume have not been reported before. Achievement of optimum volume will require individual adjustment of these levels for each starter, as the influence of process parameters was strain-dependent.

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The positive effect of sourdough in bread volume has been linked to better gas holding capacity of gluten in acidic dough containing sourdough (Gobbetti, Corsetti, Rossi, 1995), solubilization of pentosans during the sourdough process (Corsetti et al., 2000), altered activities of endogenous enzymes due to utilization of sourdough and subsequent low pH (Clarke et al., 2003), and faster yeast fermentation in the presence of LAB (Gobbetti, Corsetti et al., 1995). Increasing acidity, however, leads finally also to a stepwise degradation of gluten proteins (Thiele, Ga¨nzle, & Vogel, 2003) and thus results in softer, less elastic dough with poorer gas holding properties, if fermentation is allowed to continue long enough to obtain intensive acidification (Clarke et al., 2003). Accordingly, the acidity level of sourdough and subsequent bread dough must be carefully controlled to attain increased bread volume as recognized by Clarke et al. (2003) and confirmed by the results of this study. Increased softness after storage was at least partly due to the improved specific volume (significant positive correlation) of these breads. Acidification, however, was related to the improved softness, as pH of sourdoughs correlated positively with firmness values in all types of sourdough bread. Biological acidification may aid in maintaining bread freshness because it influences moisture redistribution throughout the loaf during storage (Corsetti et al., 2000). Furthermore, formation of exopolysaccharides and dextrins during fermentation has also been proposed to enhance the shelf-life by decreasing starch recrystallization (Korakli, Rossman, Ga¨nzle, & Vogel, 2001; MartinezAnaya, 1996; Rouzaud & Martinez-Anaya, 1997). Improved softness of sourdough breads during storage also requires controlled acidity levels, as positive influence is obtained only in moderate acidity. According to our results, an overall improvement of bread volume, softness and flavour with the same sourdough is not likely. This is due to the fact that optimum extraction rate of flour differs for textural improvement and for flavour independent of sourdough type, as the former requires use of low ash content flour and the latter high ash content flour, respectively. In addition, the requirement for fermentation time is different for texture and flavour improvement. Efficient use of sourdough thus requires consideration of sourdough’s final utilization in wheat baking, and sourdough can be tailored either for flavour improvement or texture improvement. Improvement of any of the quality attributes of wheat bread, however, is based on tuned metabolic activity of sourdough, because positive effects in flavour, texture or shelf-life are obtained only in the optimized conditions of sourdough fermentation. 5. Conclusion Significant influence of sourdough process conditions on bread flavour and texture was clarified in this study by using experimental design and mathematical modelling.

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