Impact of Sorghum and Nabag (Ziziphusspina-Christi) Pulp Fruit Lactic Acid Bacteria Sourdoughs on Fermentation Properties of Dough, Quality and Shelf Life of Wheat Bread

Sourdoughs were produced from sorghum and nabag flour using Lactobacillus plantarum and L. brevis and added to a basic bread formulation (10% and 20% addition levels). Dough fermentation, quality and shelf life of wheat bread were examined. Acidification characteristics (pH and total titratable acidity), total bacteria count, fermentation end-products (malic acid, lactic acid, acetic acid, citric acid, succinic acid, fumaric acid and ethanol) and soluble carbohydrates (arabinose, galactose and glucose) contents were measured during both sorghum sourdough and nabag sourdough. Some differences between L. plantarum and L. brevis in acidification properties, fermentation end-products and soluble carbohydrates availability were observed both in sorghum sourdough and nabag sourdough. Addition of sorghum and nabag sourdough starters progressively decrease pH and increased TTA values compared to the control dough and bread. Addition of sorghum and nabag sourdough significantly decreased dough water absorption and increased maximum gas fermentation height, total gas volume, gas retention volume, thereby sorghum and nabag sourdough has positive effect to improve of the fermentation properties of dough. The results showed that dough prepared with 10% and 20% sorghum and nabag sourdough starters had a positive impact on bread quality properties, whereas nabag sourdough starters showed higher volume and moisture content and better textural properties during storage than samples of sorghum sourdough and control.


Introduction
Sourdough fermentation is one of the oldest biotechnological processes for the production of bread. Sourdough, a mixture of flour and water fermented with lactic acid bacteria and/or yeast, are use to improve the quality of wheat bread [1]. The addition of sourdough previously served to improve flavour, texture, shelf life and nutritional properties of bread [2]. Sorghum [Sorghum bicolor (L.) Moench] is a traditional crop in Africa that is safe for consumption by celiac patients [3,4]. In recent years sorghum has received increasing attention due to its functional properties, prevention of chronic diseases, polyphenolic compounds, and as a potential raw material in gluten-free diet [4,5]. Fermentation of gluten-free flours has previously been shown to improve overall bread quality [2,6]. The positive effects are linked to metabolites produced by lactic acid bacteria (LAB) during sourdough fermentation, including organic acids, exopolysaccharides (EPS) and enzymes. There is consensus regarding the positive effects of sourdough addition for bread production, including improvement in bread volume, crumb structure, flavor and shelf-life [7,8].
Lactic acid fermentation is used in fermentation of milk, vegetables (cucumber, cabbage, and cassava), cereal (wheat,

Materials
High-gluten wheat flour, white sorghum flour, dry yeast, salt, sugar, and butter we purchased in a local market. Dry nabag fruit was purchased from a local farm in Wad Medani City, Gezira State, Sudan. Moisture, ash, and protein contents were determined by AACCI 2000 Approved Methods 44-15.02A, 08-01.01, and 46-13.01, respectively. Dry nabag fruit were pitted manually, grounded into powder by a hammer mill and stored at -20°C until needed.

Microorganisms and Growth Conditions
The strains L. plantarum and L. brevis were previously isolated from wheat sourdoughs at the Laboratory of Baking Science and Ingredient Functionality Research, School of Food Science and Technology, Jiangnan University, P. R. of China were used in this study. The strains were used singly as culture starters in sourdough fermentation. Lactobacillus strains were grown in MRS broth at 37°C for 24 h and stored at 4°C until use; pure stocks were stored at -20°C in glycerol (1:1 v/v).

Total Bacterial Count, pH, and TTA and Metabolite Formation in Sourdough
Total bacterial count (colony forming units CFU/mL) on the sourdough starters were analyzed using standard microbiological dilutions, plating and enumeration techniques. Briefly, 1 mL of sourdough starter was homogenized with 9 mL sterile physiological saline solution containing 0.85% NaCl (w/v), the homogenate was decimally diluted in the same solution, 1 mL of each dilution inoculated in MRS agar and incubated at 37°C for 28 h. pH and titratable acidity (TTA) were determined on sourdough starters, doughs and breads following the previously reported method [8]. Briefly, sample aliquots (10g) were homogenized with 90 mL of sterile distilled water. The pH value was recorded, and the acidity was titrated with 0.1mol/L NaOH to a final pH of 8.6. The TTA was expressed in milliliters of 0.1mol/L NaOH. To determine the concentration of organic acids and sugars, for sample preparations, 7% perchloric acid were added to sourdough and incubated at 4°C overnight. Precipitated protein was removed by centrifugation.
Organic acids were determined by HPLC using the REZEX 8µ 8% H, organic acid column 300×7.8 mM (Phenomenex, USA) coupled to a refrective index detector and UV detector (210 nm) were used for detection. As the elution fluid with 0.01 N H 2 SO 4 was used, at a flow rate of 0.6 mL/min. The temperature of the column was maintained at 65°C. Malic acid, lactic acid, acetic acid, citric acid, succinic acid, fumaric acid and ethanol were determined using external standards. Sugars were analyzed with an Aminex 87H column (300 mm* 7.8 mm, Bio-Rad, Mississauga, Canada) at a temperature of 70°C and a flow rate of 0.4 ml min -1 with 5 mM H 2 SO 4 as the eluent. The injection volume was 5µl. Rhamnose, galactose and glucose were used external standards.

Fermentation Properties of Composite Dough
Fermentation properties were analyzed using a Rheofermentometer F3 (Chopin, Villeneuve -La -Garenne Cedex, France) recording the following parameters: maximum height of dough (Hm [mm]), height of maximum gaseous release (H'm [mm]), time to maximum dough development (T1) and coefficient of gas retention using the method described [16]. A dough piece (250 g) was placed in a movable basket of the gas meter with a 1500 g cylindrical weight, and the cover of the vat was fitted with an optical sensor. The test was conducted at 38°C for 3 h.

Bread Making
Bread ingredients were added on a percent wheat flour basis (Table 2). Briefly, the bread basic recipe included 500 g of wheat flour, 1% salt, 1% baker's yeast (Saccharomyces cereviseae), sugar 3%, and 5% shortening. Sorghum sourdough starter was added at 10% SSD LP , 20% SSD LP , 10% SSD LBr and 20% SSD LBr and nabag sourdough starter at 10% NSD LP , 20% NSD LP , 10% NSD LBr and 20% NSD LBr . The control consisted of 100% wheat flour bread without sorghum sourdough starter and nabag sourdough starter. Shortening was incorporated 2 min after the end of first mixing. Mixing was done in a spiral mixer (SM-25, Sinmag Bakery Equipment, Wuxi Co., Ltd., Wuxi City, Jiangsu, China) and comprising two steps. The first step consisted of 3 min at low speed and 3.5 min at high speed. The second step consisted of 0.5 min at low speed and 3 min at high speed. The dough was divided into 60 g round pieces. Dough pieces were proofed for 90 min, at 35°C and 85% relative humidity (SM-40SP, Sinmag Bakery Equipment, Wuxi Co., Ltd., Wuxi City, Jiangsu, China) and baked at 180°C for about 25 min in an oven (SM-603T,Sinmag Bakery Equipment, Wuxi Co., Ltd., Wuxi City, Jiangsu, China). After baking, the breads were packed in plastic bags. The breads were stored at room temperature up to 7 days.

Physical Characteristics of Breads
Breads were evaluated for loaf weight, loaf volume (rapeseed displacement), and specific volume (mL/g) one hour after removal from the oven. The bake off expressed as percentage was calculated by recording the weight of dough and baked loaf according to the method [17].

Shelf Life
Standard baking tests were performed on three loaves for each bread type at each baking trial 2 h after baking (day 0) as previously described [18]. The remaining loaves were packed and further evaluation was carried out after 1, 3, 5 and 7 days of storage. Moisture content of the samples were determined according to AAC method 44-15A. Crumb texture analyses, three slices of 25 mm thickness were sliced from the center of each three loaves of each bread type. The textural characteristics of the bread were measured with a Texture Analyzer TA.XT2i (Stable Microsystems, Godalming, U.K.) equipped with an aluminum 25 mm diameter cylindrical probe. Breads were sliced using a bread slicer SM-302N (Sinmag Bakery Equipment, Wuxi Co., Ltd., Wuxi City, Jiangsu, China) set at 12.5 mm thick. Bread slices taken from the center of each loaf were used to evaluate the crumb texture. A stack of two slices was prepared and the top slice was compressed to 50% of its original thickness. The test conditions were: pre-test speed, 1 mm/sec; test speed, 3 mm/sec; posttest speed, 3 mm/sec; and trigger force (auto mode), 5 g. The measurements obtained for three loaves of one batch over storage time were averaged into one value (one replicate). TPA was repeated after 1, 3, 5 and 7 days of storage at room temperature. The following parameters were recorded: hardness, gumminess, chewiness, resilience and cohesiveness.
The microbial shelf life of breads was determined using the method described [19]. Each loaf was sliced transversely in a sterile manner to obtain uniform slices of 25 mm thickness. Each side of the slice was exposed to air for 5 min, packed in a plastic bag and heat sealed. A tip of a pipette was inserted to ensure comparable aerobic conditions in each bag. Bags were incubated at room temperature and examined over a 7-day storage period. Mould growth was quantified as the number of slice surfaces, i.e. both front and back of the slice, showing aerial mycelia as a percentage of total bread slices.

Statistical Analysis
Analysis of variance was conducted using SPSS Statistical program v. 16.0 (SPSS Inc., Chicago, USA) and mean separation by Duncan's multiple range with a significance level of 0.05%. The results were reported as mean (at least in triplicates, n=3) and standard deviation for each treatment.

Fermentation Properties of Dough
Sorghum and nabag sourdoughs affected significantly (P≤0.05) dough fermentation properties are summarized in (Table 3). The addition of 10% SD LBr increase (P<0.05) of a maximum dough development (Hm) with two treatments T2 (83.7mm) and T4 (84.1mm), (T2 90% wheat flour and 10% SSD LBr ; T4 90% wheat flour and 10% NSD LBr ) compared with a control T1 (79.2mm), but was a decreased upon the addition of 20% SD LBr , T3 (64.7mm) and T5 (67.7mm). The decrease of Hm with increasing SD was attributed mainly to the dilution of gluten. The addition of SD LP increase (P<0.05) to a maximum dough development (Hm) with three treatments T6, T7 and T8 (T6 90% wheat flour and 10% SSD LP ; T7 (80% wheat flour and 20% SSD LP ) and T8 (90% wheat flour and 10% NSD LP ) 83.8, 83.8 and 84.4mm, respectively, decreased T9 (80% wheat flour and 20% NSD LP ) 76.8±0.3mm, compared with the control. Hm compared to sorghum sourdough starters were lower with the nabag sourdough starters. Overall, sorghum sourdough and nabag sourdough also reduced the time of development of maximum dough height (T1). The levels of gas produced increased; indicating the presence of gas (increased H'm) but the dough did not retain it (decrease in Hm). There was a significant increase (P<0.05) in the overall effect of the treatments on total gas volume and gas retention volume compared to the control (Table 3). Total gas volume and gas retention volume were improved by the addition of more sorghum sourdough and nabag sourdoughs indicating that there was no lack of gas produced. The results could be partially explained by increased gas production by active LAB in the sorghum and nabag sourdoughs. Gas retention ratio (R3) decreased with the treatments compared to the control (Table 3) suggesting that the dough prevented gas release efficiently. In conclusion, the addition of sorghum sourdough and nabag sourdough in the dough ehnaced maximum gas fermentation height, total gas volume and gas retention volume whiles improving the fermentation properties of the dough when combined with the LAB fermentation.

Physical Characteristics of Breads
Addition of sorghum and nabag sourdoughs starters in breads ensured higher specific volumes and improved the overall texture when compared to the control (non-sourdough treatment) (Table 4a) indicating that LAB starters improved the form ratio of bread. These results were similar to those reported [19,22] who reported that breads with higher specific volume values had softer crumb texture. However, there are combined reports in the literature on sourdough fermentation and its effect on bread specific volume. The specific volume of bread appears to be highly dependent on the type and level of acidification [23]. The positive effect of sourdough in bread volume has been associated with improved gas holding capacity of the dough [24]. The highest bread SV (6.20±0.1g/mL) was obtained with 20% nabag sourdough with LP (T9) compared to control. Addition of nabag sourdough starters in breads ensures higher specific volume compared to sorghum sourdough starters (Table 4a). This means nabag sourdough starters improved the form ratio of the breads compared to the sorghum sourdough starters. The loaf volume of the bread was significantly improved (p<0.05) through the addition of sorghum and nabag sourdough starters, with the exception T6 (T6 90% wheat flour and 10% sorghum sourdough starters with LP) which is similar to the control. The nabag sourdough starters improved gluten gave the bread its structural framework; indicating that gluten is needed to improve the loaf volume of breads in practical applications. The results obtained by addition of nabag sourdough starters gave the breads the positive baking characteristics; meanwhile, the weight of the bread is reduced during this transformation. The results presented in (Table 4) indicate that (T4 10% nabag sourdough starters with LBr) had the lowest ratio loss value (12.7%), compared with the other samples, which ranged from (18.3 to 12.7%), resulting in the lowest loss ratio value by the addition of 10%, 20% nabag sourdough starters compared with the addition of 10%, 20% sorghum sourdough starters. These data suggest that nabag sourdough starters can be substituted to improve the loaf volume, and loaf height of composite flour bread.

pH and Titratable Acidity of Dough and Breads
Incorporation of sorghum and nabag sourdough starters into bread making process resulted in a decrease in pH and Sourdoughs on Fermentation Properties of Dough, Quality and Shelf Life of Wheat Bread an increase of TTA values compared to the control (Table  4b). In mixed doughs, proofed doughs and breads, increasing the amounts of sorghum and nabag starters led to a progressive decrease of pH and increased TTA values compared to the non-acidified control mixed doughs, proofed doughs and breads. The pH decrease in mixed doughs, proofed doughs and breads from 5.54, 5.36 and 5.44 (control) to 4.74, 4.24 and 4.14 (T9: 20% nabag sourdough starter with LP) with an increase in titratable acidity from 3.91, 3.98 and 3.85mL (control) to 8.35, 9.69 and 8.90mL (T9 20% nabag sourdough starter with LP). Breads produced with addition of nabag sourdough starters showed higher TTA values and lower pH values in comparison to the addition of sorghum sourdough starters. The results of the present study indicate that fermented nabag with LP is needed to obtain some of the pH and titratable acidity of the breads. The drop in pH associated with acid production could cause an increase as the activity of proteases and amylases in the dough, thus leading to a reduction in staling [25] with subsequent extension of shelf-life [19]. Among other benefits from sourdough fermentation is antimicrobial activity as a result of low pH and antibacterial compounds produced by LAB.  Treatments as described in Table 2. C TTA is reported as mL NaOH (0.1N)/10g bread dough or bread.

Bread Shelf Life
Moisture content is a critical factor that affects bread quality, consumer acceptance and shelf life [25]. Addition of sorghum and nabag sourdough starters in breads ensures higher moisture content compared to control non-sourdough treated (Fig. 1A). In this respect several authors have reported beneficial effects of biological acidification on bread staling [26] due to the metabolite products of fermentation [27] and in particular due to proteolytic activity of LAB [26,28]. Moreover, the differences in moisture content between samples during storage may also substantiate these results. In fresh bread (day 0 of storage), the highest moisture content crumb in bread was obtained with 20% NSD LP (T9) (47.11±0.01%) compared to control (42.77±0.01%). Addition of nabag sourdough starters in breads ensured higher moisture content compared to the sorghum sourdough starters (Fig. 1A). This means nabag sourdough starters impacted the moisture content of the breads compared to the sorghum sourdough starters and control. In general, high bread water content has been reported to increase shelf life and delay starch retrogradation [26]. The moisture content of bread samples during storage time is reported in Fig. 1A. For sample T9 (80% wheat flour and 20% NSD LP ) higher moisture content in bread during storage compared to a control, and also higher compared other samples as shown in (Fig. 1A). Strain type has a significant effect on moisture content at all times during storage and sourdough starters with LP samples always showed a higher moisture compared sourdough starters with LBr. The production of organic acids [26], bacterial hydrolysis of starch and proteolysis of gluten subunits [26] are activities involved in bread staling which may explain the different effects of LAB starters. Indeed, sample T9 (80% wheat flour and 20% NSD LP ) showed a constant moisture content whereas other samples a decrease in moisture content was observed after the first days of storage. As reported [26], moisture redistribution throughout the loaf during storage has an effect on bread freshness. The results presented in (Fig. 1B) indicate that (T9 80% wheat flour and 20% NSD LP ) had the lowest ratio loss values to moisture, compared with the control during storage and higher ratio loss in T2 (T2 90% wheat flour and 10% SSD LBr ), T3 (T3 80% wheat flour and 20SSD LBr ), T6 (T6: 90% wheat flour and 10% SSD LBr ) and T7 (T7 80% wheat flour and 20% SSD LP ), resulted in the lowest ratio loss value; addition 10%, 20% nabag sourdough starters compared with the addition 10%, 20% sorghum sourdough starters. These data suggest that, addition of nabag sourdough starters can improve the shelf life of breads.  Hardness and gumminess of bread samples during storage time is reported in (Fig. 2). In fresh bread (0 day of storage), addition of sorghum and nabag sourdough starters decreased (p<0.05) crumb hardness and gumminess for all treatments compared to the control ( Fig. 2A, B). Fermentation plays an important role in sorghum and nabag as it provides an improvement of nutritional quality. However, T9 (80% wheat flour and 20% NSD LP ) showed decreased crumb hardness and gumminess compared to the control. During storage for1 days, T3 (80% wheat flour and 20% SSD LBr ) and T6 (80% wheat flour and 20% SSD LP ) increased (p<0.05) crumb hardness and gumminess, decreased for other samples compared to a control. At 3 day of storage, T6 (80% wheat flour and 20% sorghun sourdough starter with LP) increased hardness and T2 (90% wheat flour and 10% SSD LBr ), T3 Sourdoughs on Fermentation Properties of Dough, Quality and Shelf Life of Wheat Bread (80% wheat flour and 20% SSD LBr ) and T6 (80% wheat flour and 20% SSD LP ) increased (p<0.05) gumminess as shwon in (Fig. 2B). After 5 and 7 days, the hardness increased in T2, T3, T6 and gumminess at 5 day but increased in T2 and T3 on day 7. In fresh bread (0 day of storage) addition of sorghum and nabag sourdough starters decreased (p<0.05) crumb chewiness for all treatments compared to the control (Fig. 2C). T3 and T6 increased (p<0.05) crumb chewiness on 1day storage, on 3 days of storage, T2, T3 and T6 increased chewiness. After 5 and 7 days, chewiness increased in T2 and T3 compared to the control. During storage for 1, 3, 5 and 7 days, addition of nabag sourghun starters decreased (p<0.05) crumb hardness, gumminess and chewiness compared to a control. This means nabag sourdough starters increased the shelf-life of wheat bread. Addition of sorghum and nabag soudough starters increased (p<0.05) resilience for all treatments compared to the control (Fig. 2D) on 0 day storage. During storage for1, 3 and 5 days, T5 (80% wheat flour and 20% NSD LBr ) and T9 (80% wheat flour and 20% NSD LP ) increased (p<0.05) resilience compared to a control (Fig. 2D). Addition of sorghum and nabag soudough starters increased (p<0.05) cohesiveness for all treatments compared to the control (Fig. 2E). During storage for1, 3 and 5 days T9 (80% wheat flour and 20% NSD LP ) increased (p<0.05) cohesiveness compared to a control (Fig. 2E). Addition of 10%, 20% nabag sourdough starters with LBr and LP increased the resilience and cohesiveness during the 7days storage time and decresed resilience and cohesiveness when sorghum sourdough starters were added. The results of the present study indicate that the addition of nabag sourdough starters to improves of textural properties of bread during storage time. The additions of nabag sourdough starters significantly improve the quality and shelf life of wheat bread.
Microbial shelf life studies were conducted on breads (data not shown). The first mould growth was observed on day five for wheat bread (control), giving the breads four days shelf life T2 (90% wheat flour and 10% SSD LBr ) showed mould growth from day seven on, resulting in a shelf life of six days, while the mould growth was not observed in samples (T3,T4,T5,T6,T7,T8 and T9). The results suggest that use of sourdough in bread production is beneficial in improving sensory properties and preventing mould and bacterial spoilage compared to the control. In general, mould growth was not observed in sourdough breads with pH level < 4.9 and higher pH-levels promoted mold growth. Antimould activity of different LAB strains has been reported [29] and some strains appear to be active even with higher pH-levels [30]. However, role of acidity in the performance of different antimould strains requires further studies to evaluate potential of sourdough to promote microbiological shelf-life without deteriorating flavour. It was concluded that L. plantarum was most effective to inhibit microbial spoilage and extended the shelf life of bread compared with the L. brevis. Comparatively, sorghum sourdough breads has the lower shelf life than nabag sourdough breads, indicating that nabag sourdoughs improves the shelf life of breads.

Conclusion
The effect of addition SSDLP, SSD LBr , NSD LP and NSD LBr on fermentation properties of dough, baking characteristics and shelf life of wheat bread was investigated. The results indicated that the addition of SSD LP , SSD LBr , NSD LP and NSD LBr in dough increased maximum gas fermentation height, total gas volume and gas retention volume whiles sorghum and nabag sourdough starters improved the fermentation properties of doughs. Addition of sorghum and nabag sourdough starters improved the specific volume compared to the control. The addition of 10%, 20% NSD compared with the addition of 10%, 20% SSD resulted in lower loss ratio value. These data suggest that NSD can be substituted to improve the loaf volume and loaf height of composite flour bread. The addition of sorghum and nabag sourdough starters with L. plantarum and L. brevis increases acidity and lowers pH of doughs and breads providing mildly acidic bread with improved shelf life. Breads produced with addition of nabag sourdough starters showed higher TTA values and lower pH values in comparison to the addition of sorghum sourdough. Addition of sorghum and nabag sourdough starter in breads higher moisture content when compared to control and sorghum sourdough starters. The results of the present study indicate that the addition of nabag sourdough starters can significantly improve the textural properties, quality and shelf life of breads during storage time. In conclusion, use of NSD in bread making ensures improvement in the bread quality, namely loaf volume, moisture content and crumb texture, and even the sensory quality of the fresh bread is enhanced compared to the control and sorghum starter. NSD has a positive effect on bread making and is key to making good bread without the use of chemical additive.