Quality Properties of Wheat Breads Incorporated with Dried Sourdoughs Produced with Different Fermentation and Drying Methods

M. Hendek Ertop; Ş.M. İlter; F. Yilmaz; C. Baltaci; A. Gündoğdu

doi:10.3136/fstr.24.971

Abstract

The aim of this study was to evaluate the effect of the dried sourdoughs produced with two different fermentation methods [Spontaneous fermentation(SPF) and starter of lactic acid bacteria-added fermentation(STF)], and three different drying methods (Drying in oven;D_O, Freeze drier;D_FD, and Spray drier;D_SD) on various quality properties of the wheat bread. The bread samples prepared by addition of the dried sourdoughs at the rates of 3%, 6% and 12%, and control bread prepared without sourdough were compared in terms of bioactivity, shelf life, physicochemical and sensory properties. The addition of the dried sourdough affected the specific volume, pH and acidity of the bread samples significantly (p < 0.05), and a slow down moisture loss occurred during shelf life. The a* value of crust, antioxidant activity, and sensory properties were improved compared to the control bread. The use of dried sourdough at the rate of 12% gave the most effective results. When the fermentation and drying methods were evaluated together, the combinations of SPF/D_O, SPF/D_FD and STE/D_SD gave the best results.

Introduction

Sourdough is one of the oldest biotechnological processes which is used to enhance quality of cereal based food products. It has been used for 5000 years, and more than three million tons have been manufactured every year (Vogel et al., 2011). Due to the increase in consumer demand for additive-free food products, the sourdough fermentation method has gained importance. Sourdough is a mixture of flour and water that is fermented with lactic acid bacteria (LAB) and yeast. They are very complex biological ecosystems due to their microbial compositions and the interactions between bread-making processes and ingredients. The typical characteristic of sourdough is mainly due to its microbiota, basically represented by LAB/yeasts, and their interaction (De Vuyst et al., 2014). Several metabolites produced by the microbiota, such as organic acids, exopolysaccharides and/or enzymes, affect the characteristics of sourdough and provide enhanced aroma profile, good internal structure, high volume, and long shelf life to the end product. Sourdough fermentation also has beneficial effects such as extending microbiological shelf life, retarding staling and improving nutritional features (Martinez Anaya, 1996; Katina et al., 2006; Gocmen et al., 2007; Chavan and Chavan, 2011).

The main method used to sourdough production is spontaneous fermentation also known as multi-stage fermentation and back-slopping method which is one of the oldest known techniques. It is a labor intensive and time-consuming process. The use of specific cultures especially LAB such as Lactobacillus brevis, Lactobacillus plantarum, Lactobacillus delbruecki, Lactobacillus sanfranciscensis, and Lactobacillus fermentum and the control of the fermentation process have also seen in recent years. However, the method requires microbiological background, proper starter culture and laboratory for the users. Therefore, the production of sourdough in powder form will be necessary for the industrial and domestic usage. It will also meet the expectations of today's customers regarding natural and healthy products. The various techniques, such as freeze drying, spray granulation, fluidized drying, spray drying and tumble drying can be used for the purpose. It is important to provide microbial stability and vitality throughout the process. Spray drying and tumble drying have been widely tested in several studies (Golshan Taftı et al., 2013). In spray drying, the liquid sourdough is pulverized in a hot air stream. The water content is evaporated. During this process, because of the fact that the particles are not exposed to hot air stream for a long time, the browning reaction does not occur (Chavan and Chavan, 2011). Lyophilization, known as freeze drying, is one of the methods used for preserving the vitality of microorganisms such as Lb. delbrueckii, Lb. fructivorans, Lb. plantarum, and Lb. brevis (Hammes and Gänzle, 1998). However, lyophilization may also have disadvantages regarding loss of aromatic compounds (Kirchhoff and Schieberle, 2001).

The aim of this study was to evaluate the effect of dried sourdough on the physicochemical and sensorial features of bread. Two types of fermentation, spontaneous fermentation (SPF) and starter (LAB) added fermentation (STF) were performed in sourdough production. Drying was carried out in the freeze dryer, spray dryer and in the oven. The protection of microbial vitality of dried sourdoughs was ensured during the drying processes. The powder sourdoughs were used at rates of 3%, 6%, and 12% in bread formula, and they were compared with the control bread.

Materials and Methods

The wheat flour (14.3% moisture, 0.63% ash, 11.3% protein, 59.2% water absorption) supplied from a milling company (Cesur Milling Co., Trabzon). The salt and instant active dry yeast (Dr. Oetker) were from a local supermarket. The chemicals were supplied by Merck (Germany) and DPPH (2,2,-diphenyl-1-picrylhydrazyl) radical by Sigma (Germany).

2.1. Preparation of sourdoughs The dough yield value (DY), which represents the proportion between flour and water, was 200 for the sourdoughs (Chavan and Chavan, 2011).

Sourdough prepared by spontaneous fermentation: The traditional “back-sloping method” was used. 200 g wheat flour and 200 g water were mixed and fermented spontaneously at 26°C until the dough reached a pH value below 4.5. At the end of the stage 20 g of sourdough was taken and added to fresh dough. This process was repeated four times until the counts of LAB microbiota reached 10⁹–10¹⁰.

Sourdough prepared by starter: Lb. delbrueckii (B-763), Lb. brevis (B-3065) and Lb. plantarum (B-4496) (ARS Culture Collection, Illinois, USA) were activated on MRS broth to obtain a cellular suspension of 10⁹ CFU/mL. Bacterial suspensions were washed two times. The bacterial suspensions were centrifuged at 4000×g and the supernatants discarded. LAB fermentation starters were prepared using a wheat flour:water ratio of 1:1 (w/w) (Wu et al., 2012). It was added at the rate of 1%. The sourdough was fermented at 26°C until the dough reached to pH 4.5 and to the counts of LAB microbiota min 10⁷.

2.2. Drying methods Drying infreeze drier (D_FD); Sourdough was spread as a thin layer and placed in a freeze dryer (Xianou-12N, China). The drying was performed at the temperature of −68°C under vacuum. The moisture content was periodically controlled. When the moisture content of about 4–5% was reached, the process was ended, and then it was grinded (IKA, Germany).

Oven drying (D_O); The low temperature (37±1°C) and short drying time were used in this study. The sourdough was spreaded as a thin layer on the baking paper and placed into the oven (Nüve FN400, Turkey) shelves. When the moisture content of about 4–5% was reached, the process was ended, and then it was grinded (IKA, Germany).

Drying in spray drier (D_SD); The sourdough was homogenized and pulverized with the nozzle (1 mm diameter). It was dried up to the moisture content of about 4–5% at 135±5°C spray drier (LabPlant SD 06, UK) temperature (Ghandi et al, 2012).

2.3. Enumeration of LAB LAB numbers were determined in elective media MRS agar (Merck, Germany). Appropriate dilutions were plated on MRS agar and incubated anaerobically at 35°C for 48 h. The colonies were then counted (CFU/g).

2.4. Preparation of bread samples The bread production formula and the straight dough method of Keswet et al. (2003) were used as slightly modified. The water (59% based on flour), salt (1.5% based on dry matter), instant active dry yeast (2%), flour (300 g) and dried sourdough (3%, 6%, and 12% based on flour) were mixed (KitchenAid KSM150PSER, Belgium) for 15 min. The powder sourdough was mixed with water and kept in the room's temperature for 30 min before adding it to the dough. The dough devided to two pieces after preparing. The baking experiment was performed as two repetitions and two paralel. Bread production followed the main steps as premixing, kneading, fermentation for 40 min, shaping, proofing for 50 min, and finally baking for 30 min at 180°C.

2.5. Properties of bread samples pH meter was used for the measurement of pH. Total titratable acidity (TTA) was determined after homogenization of 10 g of the sample with 90 mL of distilled water, and calculated by titration with 0.1 N NaOH (Rizzello et al., 2016). The volumes were determined by applying the rapeseed displacement method. The bread was weighed. The specific volumes of bread were calculated using the equation conducted by Artan et al., 2010). The color properties of samples were measured by a chlorimeter (Konica-Minolta, CR400, Japan) as L*, a* and b* on five different points from the crust and crumb. The average values were calculated (Torrieri et al., 2014).

2.5.1. Antioxidant activity For the extraction, 50 mL of 80% methanol solution and 5 g sample were mixed at 37°C for 2 h (Banu et al, 2010). Antioxidant activity was determined by using the DPPH radical scavenging assay according to (Karamach et al., 2002) as inhibition%.

2.5.2. Sensory evaluation Crust and crumb properties such as color, shape, elasticity, and taste, smell and overall consumption attributes of bread were evaluated together. Bread evaluation was performed by a panel of 20 male and female untrained volunteers (students and staff) randomly selected. The bread samples were labeled randomly with three-digit numerical codes and given to the panelists in the same room condition under the fluorescent light, and they were served as same portion (Keswet et al., 2003). The hedonic scale used by Olapade and Adetuyi (2007) was slightly modified. Each attribute was scored using a 10-point hedonic scale (1 and 10 representing extreme dislike and extreme like, respectively).

2.6. Bread shelf life

2.6.1. Moisture and moisture loss For measurement of moisture, a slice was taken from the bread after 3 h of baking (Poinot et al., 2008). Moisture was determined by moisture content analyzer (Ohaus MB45, Sweden) at 110°C. Moisture loss (ML) during shelf life was also determined between 2nd and 8th days.

2.6.2. Enumeration of mold growth during shelf life For the determination of mould growth during 8 days, the method used by Dal Bello et al. (2007) and Hager et al. (2012) was slightly modified. The loaf of bread was sliced as 20 mm thickness, then exposed to the air for 5 min on each side and then packed in a plastic fridge bag and sealed with heat. During which procedure a small and uniform slot was left open to ensure comparable aerobic conditions in each bag. The samples were stored at the room temperatures and examined for sponteneously mould growth during the storage period. The dilutions prepared from samples were plated on Yeast Extract Glucose Chloramphenicol (Merck) agar and incubated at 27°C for 48 h and the colonies were counted.

2.7. Statistical analysis All experiments were carried out in both parallel and repetition samples. The results are presented as mean and standard deviation values. The analyses of variance (ANOVA) (SPSS 17.0.1, Chicago, IL, USA) were used for the comparison (p < 0.05) of results. “Tukey's studentized range test” was used for multiple comparisons.

Results and Discussion

3.1. Some physicochemical properties and LAB counts of dried sourdoughs The results shown that, while the fermentation methods did not affect (p < 0.05) the color change, the drying methods affected it (Table 1). Moreover, the process conditions of drying methods directly acted on appearance of dried sourdoughs. Visually noticeable differences were also determined in a* and b* values of samples. L* values of D_O samples for both fermentation methods was lower than D_FD or D_SD samples. D_O samples for both fermentation methods had the highest a* and b* values. During the drying process, which lasted for 5–6 h at the temperature of 36–38°C in the oven, the melanoidin pigments occurred. The dried sourdough because of the Maillard reaction appeared as cream color and its a* and b* values measured as higher. Since the D_SD samples were suddenly exposed to hot air condition, the water was dehydrated in 2–3 s, all chemical events associated with color were prevented and the structure color remained white. It was determined that D_SD showed the lowest moisture value.

Table 1. Some physicochemical properties and LAB counts of dried sourdoughs

Fermentation method	Drying method	L*	a*	b*	pH		Moisture (%) LAB counts (log CFU/g)
Fermentation method	Drying method	L*	a*	b*	Wet	Dried	Dried	Wet	Dried
SPF	D_FD	88.04±0.10^a	0.19±0.01^c	13.57±0.52^b	4.42±0.06^ab	4.49±0.04^a	5.14±0.08^b	9.30 ^a	8.60 ^b
	D_O	86.18±0.17^b	0.84±0.03^a	16.27±0.61^a	4.55±0.02^ab	4.46±0.10^a	5.95±0.04^a		7.70 ^c
	D_SD	88.95±0.29^a	0.21±0.00^b	6.05±0.26^c	4.58±0.00^a	4.51±0.06^a	4.80±0.04^b		9.70 ^a
STF	D_FD	88.30±0.25^a	0.17±0.02^c	14.83±0.11^b	4.18±0.09^b	4.14±0.11^a	5.02±0.07^b	8.05 ^b	8.30 ^b
	D_O	86.75±0.30^b	0.86±0.00^a	18.50±0.25^a	4.30±0.05^ab	4.20±0.01^a	6.02±0.00^a		7.48 ^c
	D_SD	89.03±0.12^a	0.23±0.01^b	6.37±0.13^c	4.16±0.12^b	4.25±0.02^a	4.85±0.10^b		9.60 ^a

SPF: spontaneous fermentation; STF:starter (lactic acid bacteria) added fermentation; Dfd: Freeze drier; Do: Oven; Dsd:Sprey drier LAB:Lactic acid bacteria

a–c Means with different superscripts in the same column are significantly different (p < 0.05)

The pH of dried samples was determined before and after drying and found that their acidity level was protected. Since the sourdough that can be added to bread dough should have approximately 4.0–4.5 pH acidity level (Torrieri et al., 2014).

LAB in both sourdough and dried sourdough were counted for the purpose of observing the effects of drying methods on the number of LAB (Table1). While the number of LAB in the wet sourdough produced with SPF method was 9.30 log CFU/g, it was in produced with STF method was 8.05 log CFU/g. The LAB number of dried samples was counted between 7.48–9.70 log CFU/g. According to results, drying in the spray drier which was obtained the highest number of the microbiota was more effective than other drying methods in terms of the vitality of the LAB. On the other hand, the long time drying in the oven reduced the LAB numbers.

3.2. Bread characterization

3.2.1. Physicochemical quality properties of breads The bread samples produced dried sourdoughs and the control bread was compared in terms of various attributes (Table 2). The statistically significant differences (p < 0.05) in pH and TTA values of the bread samples were found (Table 2). Furthermore, the decrease in pH and increase in TTA were determined which was depended on the usage rate for all dried samples. Generally, lower pH values were obtained with the usage of dried sourdoughs produced with STF.

Table 2. Some physicochemical quality properties and antioxidant activities of bread samples

Fermentation method	Drying method	pH			TTA*			Volume (cm³)			Weight (g)			Specific volume (cm³/g)			Inhibition%
Fermentation method	Drying method	3%	6%	12%	3%	6%	12%	3%	6%	12%	3%	6%	12%	3%	6%	12%	3%	6%	12%
SPF	D_FD	5.83^b	5.67^bc	5.53^b	3.72^c	4.61^b	5.32^ab	1280^b	1375^b	1430^b	414^bc	430^a	432^a	3.092^b	3.197^b	3.310^b	8.74^b	14.35^b	25.13^a
	D_O	5.78^c	5.70^b	5.52^b	3.82^c	4.42^b	5.30^ab	1330^a	1410^a	1450^a	422^ab	425^a	427^a	3.152^a	3.317^a	3.396^a	9.02^ab	16.97^ab	22.16^b
	D_SD	5.69^d	5.65^cd	5.54^b	3.81^c	4.30^b	5.22^b	1180^cd	1210^e	1292^e	414^bc	416^bc	411^c	2.850^d	2.909^e	3.143^d	9.15^ab	18.11^a	25.85^a
STF	D_FD	5.71^d	5.62^d	5.42^c	4.63^a	5.12^a	5.51^a	1150^d	1300^c	1340^c	419^ab	413^c	427^a	2.745^f	3.044^d	3.138^d	9.33^ab	15.62^ab	25.17^a
	D_O	5.80^c	5.66^c	5.36^d	4.32^b	4.53^b	5.37^ab	1200^c	1270^d	1325^d	426^a	420^b	420^b	2.817^de	3.024^d	3.155^d	10.19^a	17.88^a	23.96^ab
	D_SD	5.70^d	5.53^e	5.42^c	4.24^b	4.50^b	5.53^a	1205^c	1280^cd	1345^c	409^c	407^d	410^c	2.946^c	3.145^c	3.280^c	10.17^a	17.93^a	26.74^a
Control bread		6.01^a	6.01^a	6.01^a	2.9^d	2.9^c	2.9^c	1130^e	1130^f	1130^f	403^c	403^d	403^d	2.804^e	2.804^f	2.804^e	7.82^c	7.82^c	7.82^c

* TTA:Total titratable acidity

SPF: spontaneous fermentation; STF:starter (lactic acid bacteria) added fermentation

D_FD: Freeze drier; DO: Oven; DSD:Sprey drier

a–f Means with different superscripts in the same column are significantly different (p < 0.05)

In the present study, the volumes of bread samples which were contained dried sourdough were higher than the control bread statistically significant (p < 0.05) (Table 2). There was an increase in volume in the bread samples according to usage rates. The previous studies indicated that the addition of sourdough increases the volume of the bread (Dal Bello et al., 2007; Wu et al., 2012; Hendek Ertop and Şeker, 2018). The improving effects of the dried sourdough in bread making performance is closely dependent on the fermentation process and interaction of microbiota. While the gas production depended on only yeast fermentation in the control bread, the CO₂ production and volume formation depend on the yeast and heterofermentative LAB in the bread samples produced with dried sourdough. When the fermentation methods and drying methods were evaluated together, the highest bread volume was obtained when dried sourdough was produced by combining the SPF/D_O, SPF/D_FD and STF/D_SD.

The weight of bread samples that contained dried sourdough were higher than the control bread. Moreover, the increase in weights was proportional to the usage rate. Evaluation of the moisture contents together revealed that the bread containing dried sourdough had higher moisture contents than the control bread. It means that the usage of dried sourdough increased the moisture retention capacity and the weight of bread.

The use of dried sourdough also increased the specific volume of the bread dependent on the usage rate. The highest specific volume was achieved by SPF/D_O combination. The previous studies indicated that the addition of sourdough increased the volume and specific volume of the bread (Dal Bello et al., 2007; Wu et al., 2012). This effect may have depend on various factors. The gluten structure in acidic doughs that contain sourdough has a better gas retention capacity (Gobbetti et al., 1995). It is caused by the solubility of pentosans during the sourdough process (Corsetti et al., 2000) and by the increase in endogenous enzyme activities through lower pH related to the use of sourdough (Clarke et al., 2003). Moreover, the effect was associated with its improvement of the water retention capacity of the dough (Gobbetti et al., 1995).

Color is one of the most remarkable features indicating bread's quality and affecting consumer preferences. Chemical reactions such as Maillard reaction and caramelization cause color changes at different levels in the bread's structure, especially in crust during baking (Artan et al, 2010). L*, a*, and b* values were measured in order to observe the effects of dried sourdough use on bread's color features (Table 3). Although the L* values of sourdough breads were found higher than control bread, no explicit relation was determined between L* and b* values of breads and usage of dried sourdough. The a* value of bread samples increased depending on the usage of dried sourdough. The highest a* value was obtained in the bread contained dried sourdough produced with SPF/D_FD combination. It was followed by STF/D_SD and SPF/D_O combinations, respectively.”

Table 3. Color values of bread samples

Fermentation method	Drying method	Usage rate (%)	L*	a*	b*
SPF	D_FD	3	64.16±0.15	8.14±0.12	31.15±0.03
		6	64.08±0.22	8.65±0.06	31.29±0.15
		12	64.45±0.02	9.13±0.18	31.94±0.07
	D_O	3	64.91±0.07	6.86±0.09	28.06±0.11
		6	64.66±0.13	7.53±0.10	29.49±0.08
		12	65.64±0.25	8.87±0.15	29.58±0.14
	D_SD	3	64.49±0.09	7.05±0.25	22.52±0.16
		6	64.21±0.00	7.27±0.22	22.58±0.30
		12	67.79±0.25	8.48±0.06	23.09±0.55
STF	D_FD	3	64.71±0.02	7.96±0.16	30.01±0.07
		6	64.60±0.17	8.31±0.27	30.72±0.12
		12	65.33±0.03	8.33±0.21	31.28±0.33
	D_O	3	64.42±0.14	8.02±0.15	30.11±0.15
		6	65.16±0.02	7.14±0.13	30.12±0.18
		12	65.40±0.00	8.23±0.12	31.01±0.06
	D_SD	3	64.51±0.50	7.96±0.08	25.84±0.05
		6	65.58±0.23	7.94±0.28	26.20±0.20
		12	65.95±0.14	8.93±0.19	26.72±0.13
	Control		63.01±0.21	7.47±0.25	30.48±0.07

SPF: spontaneous fermentation; STF:starter (lactic acid bacteria) added fermentation

D_FD: Freeze drier; D_O: Oven; D_SD:Sprey drier

Chemical reactions responsible for browning, such as the Maillard reaction and caramelization, lead to color changes at different levels in crumb and crust during baking (Artan et al, 2010). The microbiota produces simple sugars as metabolites during sourdough fermentation. The participation of simple sugars in chemical browning reactions may have been responsible for the increase in the a* value in crust in particular. This is due to the bread crust being directly subjected to heat treatment in the oven which results in an appropriate environment for Maillard and caramelization reactions. The decrease in pH during fermentation has a positive effect on the browning reactions, probably due to the Amadori rearrangement requires H⁺. The level of fermentation has been reported to influence formation of the color. More compounds and brown pigments are formed in bakery products manufactured using fermented dough than those using unfermented dough (Martinez Anaya, 1996).

3.2.2. Antioxidant activity The antioxidant activity of bread samples was determined as inhibition% effect on the DPPH radical (Table 2). The bread samples contained dried sourdough had higher inhibition% values than the control bread. Inhibition% of the samples increased in relies on the usage rate of dried sourdough. Similar statements have been reported in the literature (Hendek Ertop and Şeker, 2018). The antioxidant properties of baked goods produced with sourdough are affected by the various factors and phytochemicals are mainly altered in during processing such as fermentation. LAB metabolism in sourdough can lead to lipid oxidation or strong antioxidative effects during fermentation. Homofermentative lactobacilli are known to increase lipid oxidation (Vermeulen et al., 2007). Also, some studies have shown that, the bakery products particularly their crusts have antioxidant features (Mıchalska et al., 2007).

Another perspective to be considered is the formation of bioactive peptides, which occur through sourdough fermentation, as a result of interactions between acidification and proteolysis (Rizzello et al., 2008). Bioactive peptides are specific protein fragments which exhibit mineral binding, antioxidative and antihypertensive effects. It has been shown that some selected LAB produce antioxidant peptides during sourdough fermentation (Coda et al., 2012).

3.2.3. Sensory properties of breads The panelists reported that the use of dried sourdough improved the color compared to that of the control bread. Especially the top color of the samples improved depended on the usage rate (p < 0.05) (Table 4). The panelists preferred bread which had higher a* value and high level of browning. They perceived them higher quality and awarded them higher scores. Bread prepared with SPF/D_FD, SPF/D_O and STF/D_SD combinations scored higher in terms of bread shape and this preference exhibited a parallel with the high volume bread given in Table 2. The panelists also reported that the bread prepared with SPF/D_O and SPF/D_FD combinations had better taste and aromatic profile. However, the satisfaction of some panelist in terms of the taste and smell perception of sourdough bread was generally lower compared to the control bread. The organic acids such as acetic and lactic acids and carbonyl and volatile compounds resulting from the sourdough fermentation constitute a unique aromatic profile in sourdough bread (Hansen and Hansen, 1994). Sourdough LAB has proteolytic activity and can release amino acids and small peptides from proteins, thereby enhancing flavor development (Gobbetti et al., 2005). This flavor profile was perceived as different from the control bread profile produced with only bakery yeast by panelists. This preference can be attributed to rarely consumption habits of the sourdough bread of the panelists. The panelists reported that the breads contained dried sourdough were less elastic than the control bread. The polymeric protein structure which is responsible for the elasticity of the dough and the bread might be affected by the proteolytic activities of LAB during fermentation; in this case, the elasticity might decrease.

Table 4. Sensory evaluation results of bread samples [A, Spontaneous fermentation; B, Starter (LAB) added fermentation]

Usage rate (%)	Top color	Bottom color	Top shape	Bottom shape	Crumb color	Crumb texture	Taste	Smell	Elasticity	Overall eating
D_O 3	6.57±0.11^cde	6.43±0.15^cd	6.71±0.20^c	6.86±0.13^cd	6.86±0.13^c	7.00±0.21^de	6.57±0.24^cd	7.00±0.32^abc	8.00±0.26^ab	6.14±0.16^ef
D_O 6	6.86±0.10^bcde	7.29±0.10^bc	7.14±0.39^c	7.29±0.03^bcd	7.14±0.13^bc	7.29±0.13^cde	7.00±0.25^bcd	7.00±0.21^abc	7.57±0.24^ab	7.00±0.09^cd
D_O 12	7.71±0.20^ab	8.29±0.19^ab	8.83±0.11^a	8.57±0.28^ab	8.29±0.27^a	8.35±0.23^ab	8.00±0.31^b	7.66±0.15^a	7.0±0.20^ab	7.86±0.13^b
D_FD 3	7.29±0.08^bc	7.43±0.12^bc	6.57±0.18^c	6.43±0.19^cd	6.86±0.23^c	7.57±0.26^bcde	7.43±0.21^bcd	6.86±0.10^abc	8.00±0.31^ab	7.14±0.11^bc
D_FD 6	7.14±0.15^bcd	7.57±0.09^b	7.29±0.19^c	7.29±0.12^bcd	7.14±0.14^bc	6.71±0.25^e	7.14±0.11^bcd	6.86±0.22^abc	6.71±0.18^b	6.86±0.06^cde
D_FD 12	8.43±0.19^a	8.86±0.12^a	9.20±0.25^a	9.14±0.29^a	8.57±0.12^a	8.86±0.12^a	9.29±0.34^a	7.71±0.20^a	7.29±0.28^ab	9.29±0.10^a
D_SD 3	6.22±0.10^de	5.82±0.18^d	6.85±0.10^c	7.38±0.19^bc	8.03±0.23^ab	6.99±0.20^de	6.2±0.14^d	5.97±0.16^c	7.25±0.09^ab	6.00±0.22^f
D_SD 6	6.25±0.22^de	5.79±0.14^d	7.32±0.11^bc	7.21±0.22^cd	8.10±0.14^ab	7.85±0.06^abcd	6.47±0.16^cd	5.91±0.08^c	7.21±0.24^ab	6.12±0.04^f
D_SD 12	6.41±0.21^cde	6.2±0.22^d	8.48±0.21^ab	7.65±0.20^bc	8.15±0.23^ab	8.15±0.04^abc	6.85±0.14^bcd	6.51±0.12^bc	7.25±0.22^ab	7.12±0.12^c
Control bread	6.12±0.23^e	6.25±0.34^d	6.34±0.23^c	6.05±0.44^cd	6.15±0.31^c	6.55±0.20^e	7.72±0.23^bc	7.5±0.27^ab	8.20±0.25^a	6.35±0.17^def
(A)

Usage rate (%)	Top color	Bottom color	Top shape	Bottom shape	Crumb color	Crumb texture	Taste	Smell	Elasticity	Overall eating
D_O 3	6.57±0.12^abc	6.29±0.26^a	6.71±0.12^cd	6.43±0.22^d	7.00±0.12^b	6.43±0.20^cd	5.86±0.09^f	6.14±0.13^b	6.43±0.18^cd	6.29±0.10^bcd
D_O 6	6.43±0.20^bc	6.71±0.19^a	6.57±0.16^cd	6.57±0.27^cd	7.00±0.21^b	7.14±0.13^bc	6.86±0.06^b	6.71±0.10^ab	6.71±0.10^bcd	7.00±0.20^abc
D_O 12	6.63±0.28^abc	6.00±0.12^a	7.00±0.17^c	6.57±0.14^cd	7.57±0.13^ab	6.71±0.09^c	6.57±0.10^bcde	6.71±0.08^ab	6.43±0.15^cd	6.71±0.13^abcd
D_FD 3	6.57±0.24^abc	6.14±0.15^a	7.00±0.21^c	6.71±0.20^bcd	7.00±0.19^b	6.71±0.12^c	6.00±0.09^ef	6.00±0.10^b	6.29±0.09^d	5.86±0.07^d
D_FD 6	6.14±0.19^c	6.86±0.13^a	6.14±0.12^d	6.43±0.18^d	6.00±0.11^c	5.71±0.08^d	6.00±0.12^ef	6.71±0.13^ab	6.29±0.18^d	6.29±0.18^bcd
D_FD 12	7.43±0.22^ab	7.00±0.13^a	6.86±0.13^cd	7.57±0.26^abc	7.86±0.08^a	7.14±0.13^bc	6.14±0.15^def	6.71±0.19^ab	6.25±0.14^d	7.14±0.18^ab
D_SD 3	5.94±0.17^c	6.02±0.19^a	7.12±0.29^c	7.85±0.14^a	7.93±0.16^a	7.84±0.14^ab	6.25±0.05^cdef	6.14±0.16^b	6.54±0.24^cd	6.25±0.17^cd
D_SD 6	5.86±0.18^c	6.22±0.19^a	8.25±0.25^b	7.72±0.19^ab	7.85±0.13^a	8.32±0.20^a	6.63±0.11^bcd	6.02±0.17^b	7.15±0.03^bc	6.82±0.09^abc
D_SD 12	7.56±0.18^a	6.33±0.21^a	9.16±0.15^a	7.65±0.15^abc	7.88±0.12^a	8.58±0.10^a	6.82±0.14^bc	6.78±0.13^ab	7.49±0.10^ab	7.20±0.12^a
Control bread	6.12±0.11^c	6.25±0.17^a	6.34±0.08^cd	6.05±0.20^d	6.15±0.13^c	6.55±0.20^c	7.72±0.11^a	7.50±0.19^a	8.20±0.11^a	6.35±0.23^abcd
(B)

D_FD : Freeze drier; D_O : Oven; D_SD :Sprey drier

3.3. Bread shelf life

3.3.1. Moisture content The weight of the bread samples contained dried sourdough was higher than the weight of the control bread. This can be interpreted as meaning that sourdough usage increased the water retention capacity of bread. In order to determine this, the initial moistures of the bread samples were measured, and moisture losses during shelf life were calculated by performing bread moisture analysis of days 0, 2, 4, 6, and 8. The moisture values of the bread samples contained dried sourdough were generally higher than those of control bread.

The highest moisture values were obtained from bread that contained dried sourdough D_SD for both types of fermentation. They were followed by bread that contained dried sourdough D_FD. For bakery products, not only initial moisture level but also the moisture's retention is important, during the shelf life. Retrogradation known as staling properties is indirectly related to moisture loss. In other words, slow moisture loss in the bread means more gradual staling. This is the main purpose behind the addition of the emulsifier, dried fat powder and water binder hydrocolloids used as texture-improving additives in bread making. The graphs indicating moisture changes during shelf life according to the different drying methods are given Fig.1 (A, B, and C). As shown in Fig.1, the highest moisture loss generally occurred on the second day in all breads. Subsequently, moisture loss in control bread continued rapidly. The study conducted previously by Hendek Ertop and Şeker (2018), it was found that the reduction of moisture was lower in the sourdough bread contrast to control bread prepared with only bakery yeast, during storage. In this study, similar result was obtained, while the control bread which not contained dried sourdough, it continued to lose moisture rapidly by the end of eight days of shelf life, all the other bread samples lost their moisture slowly, between days 2 and 4. The slope of moisture loss was shown in the graph. Generally, due to the bread consumption being on the first four days after baking, the moisture retention effect of sourdough is a considerable feature for baking industry and for customers. The lowest moisture loss was seen for the bread sample that contained 12% dried sourdough produced with SPF between days 2 and 4. The bread samples that contained dried sourdough had similar moisture levels at the end of their shelf life, and the ones were higher than the level of the control bread. Similar results were obtained for the bread samples containing sourdough dried in oven (D_O) (Fig.1A) and dried in spray drier (Fig.1C). Moisture loss profiles of all bread samples contained dried sourdough during their shelf life were similar in terms of higher moisture content and final moisture at the end of shelf life. When the bread samples were compared one with each other, the samples containing dried sourdough produced with SPF/D_O and STF/D_O combinations at the 3% level had higher moisture profiles. Moisture levels of all bread samples both initially and at the end of shelf life were higher than those of the control bread. Dried sourdoughs slowed the moisture loss in the bread samples, particularly on days 2 and 6. The use of dried sourdough produced with STF at rates of 6% and 12% in bread was found to be particularly effective.

Fig. 1.

Moisture losses of control bread and bread samples containing dried sourdough during shelf life; A: Dried in freeze dryer, B: Dried in oven, C:Dried in spray dryer [SPF; spontaneous fermentation, STF; starter culture (LAB) added fermentation]

3.3.2. Counts of mold growth Whereas any mold growth was not generally observed in first three days for bread samples, it was determined from the 3rd day (Table 5). Severely microbiologically improvement was not determined in bread included at 3% dried sourdough for the first three days. However, the best result was obtained in STF/D_SD combination, and any presence of mold was not seen even in the 5th day. The 6% usage rate has been generally effective on mold growth. While best result was obtained via D_O and D_SD for SPF, it was obtained via D_O for STF. The effects of dried sourdough in 12% usage rate were obviously seen. The best result in the usage rate was obtained with SPT/D_SD combination. However, it can be said that the use of dried sourdough is effective in this respect, because the bread is generally consumed in the first five days after producing. It was also revealed by previous studies that the microbial shelf life was extended by slowing the growth of molds in bread produced with LAB usage or sourdough fermented spontaneously (Dal Bello et al., 2007). The studies revealed that the usage level of the sourdough was the most effective factor for resistance to against to mold growth, and the rate of 50% was the most effective level. In this case, it was stated that the final pH of bread has values between 4.3–5.2 and the duration of bread resistance is 8–12 days (Plessas et al., 2007). In this study, sourdough was used at a max 12%. It can be said that, if used at a higher rate, the better results will be obtained in terms of mold growth.

Table 5. Mold growth in bread samples produced with dried sourdoughs during shelf life (log CFU/g)

Usage rate (%)	Fermentation method	Drying method	Day
3	SPF		0	1	3	5	7	9	11
		D_SD	—	—	—	>2	>2	>2	>2
		D_FD	—	—	—	>2	>2	>2	>2
		D_O	—	—	—	>2	>2	>2	>2
	STF	D_SD	—	—	—	—	>2	>2	>2
		D_FD	—	—	—	>2	>2	>2	>2
		D_O	—	—	—	>2	>2	>2	>2
6	SPF	D_SD	—	—	—	—	>2	>2	>2
		D_FD	—	—	—	>2	>2	>2	>2
		D_O	—	—	—	—	—	>2	>2
	STF	D_SD	—	—	—	>2	>2	>2	>2
		D_FD	—	—	—	>2	>2	>2	>2
		D_O	—	—	—	—	—	>2	>2
12	SPF	D_SD	—	—	—	—	—	—	>2
		D_FD	—	—	—	—	>2	>2	>2
		D_O	—	—	—	—	—	>2	>2
	STF	D_SD	—	—	—	—	>2	>2	>2
		D_FD	—	—	—	—	>2	>2	>2
		D_O	—	—	—	—	—	>2	>2
	Control		—	—	—	>2	>2	>2	>2

SPF: spontaneous fermentation; STF:starter (lactic acid bacteria) added fermentation

D_FD: Freeze drier; D_O: Oven; D_SD:Sprey drier

Conclusion

The main purposes of this study were to determine alterations in the physicochemical properties and in particularly microbiota of dried sourdough produced by two types of fermentation/three types of drying methods, and to evaluate the use of dried sourdough in bread. The results show that production of dried form is possible while still ensuring the survival of the microbiota. The drying processes led to a decrease in microbiota, however the counts of microbiota remained at an acceptable level (above 10⁵–10⁷ CFU/g) for microbiota reproduction in sourdough. Dried sourdoughs maintained their acidic profiles in the same way as wet sourdough and were able to be produced in a form suitable for use in dough. The dried sourdoughs were used in bread production at the rates of 3%, 6%, and 12%, and physicochemical quality properties of bread were determined. The bread volume of samples significantly increased relies on the usage rate of the dried sourdough. The use of the dried sourdough positively affected both the volume and the specific volume. It also led to an increase in the moisture content of bread and in moisture retention, especially on days 2 and 6 during the shelf life. This may be regarded as a “braking effect” in terms of moisture loss. This situation will represent a major improvement in terms of shelf life. It will therefore also contribute to a decrease in bread waste by delaying bread staling. One important quality parameter is also the browning level of bread crust. a* values increased with the amount of the dried sourdough used. This was confirmed by the panelists' preferences at the end of the sensory analysis. When the bread samples were compared, the total sensory analysis scores of all the bread containing dried sourdough were higher than the total scores of the control bread in general. Different combinations of fermentation and drying methods were tested in this study. Generally, sourdough produced by using SPF is better than the one produced by using STF. The powder products were superior in terms of various features. When all properties were taken into consideration, spray drying produced better results for STF, while oven drying produced better results for SPF. However, the aim of this study was to test the practicality of methods which can be used at an industrial scale rather than determining better combinations or better methods, and a positive result was obtained.

Acknowledgment The authors special thank to Republic of Turkey, Ministry of Food Agriculture and Livestock-TAGEM, and to Gumushane University for their support.

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