**2. Experimental**

82 Recent Trends for Enhancing the Diversity and Quality of Soybean Products

be used: defatted soy flour (Mashayekh et al., 2008), physically modified soy flour (Maforimbo et al., 2008), soy flour and durum wheat flour mixture (Sabanis & Tzia, 2009), commercial soy protein isolate (Roccia et al., 2009), diffrerent kinds of soy protein powder (Qian et al., 2006) and part of soy seeds such as a hulls (Anjum et a., 2006). Based on these investigations results, different bread formulations are defined, and soy is used in portion up to 20%. When soy flour in wheat flour was to a level of 10% and in durum wheat flour up to 20%, the produced bread was without any negative effects in quality attributes such as colour, hardness and flavour, promising nutritious and healthy alternative to consumers (Sabanis & Tzia, 2009). By investigating the effect of defatted soy flour on sensory and rheological properties of wheat bread, Mashayekh et al. 2008, concluded that adding 3 and 7% defatted soy flour gives as good a loaf of bread as the 100% wheat bread and acceptable consumer attribute with rheological and sensory characteristics. Adding small quantity of soy protein powder of 3% to wheat flour did not change the sensory properties of bread and a large quantity of soy flour adding, exceeding 7%, can lead to stickiness and leguminous flavour (Roccia et al., 2009). The results of investigation (Anjum et al., 2006) of soy hulls usage showed the content of 4.5% soy hulls combined with wheat flour is acceptable and

The manufacture of bread from flour without gluten represents considerable technological difficulties (Jong et al., 1968; Schober et al., 2003) because gluten is the most important structure forming protein for making bread (Gujural et al., 2003; Moore et al,*.* 2004) and by using appropriate soy-wheat flour mixture these difficulties can be avoided. When soy was added to wheat, the soy globulins interact with wheat gluten proteins forming aggregates of high molecular weight. As reduction-reoxidation treatment facilitated the interaction of glutenin subunits and soy proteins (11S subunits), interaction probably occurs through the oxidation of SH groups (Maforimbo, 2008). Investigations of the changes in glutenin macro polymer content, protein composition and free sulfhydryl content showed that active soy flour decreased glutenin macro polymer content due to gluten depolymerization and glutenin macro polymer content increased by inactive soy flour because soy proteins became insoluble and precipitated together. Soy proteins were associated to wheat protein through physical interaction and covalent and non-covalent bonds during mixing and resting and these interactions produced large and medium-size polymers. This increased solubility of insoluble gluten proteins, producing a weakening of the gluten network (Perez et al., 2008) and decreasing availability of water to build up in gluten network (Roccia et all. 2009). Physicochemical status of soy protein in the product had a great influence on how wheat-soy proteins will interact (Perez et al., 2008). Incorporation of soy proteins changes the rheolocical and bread properties. The investigations showed that adding soy protein powder depresses loaf volume, gives poor crumb characteristics and decreases acceptability by consumers (Qian et al., 2006.) Ribotta et al. 2010. tested different additive combinations for improved bread quality obtained from soy-wheat flour in ratio of 90:10 w/w and found that the combination with transglutaminase showed a major improving effect on dough

Dough rheological properties have great relevance in predicting the mixing behaviour, sheeting and baking performance (Dobraszczyk & Morgenstain, 2003) and supplementation

suitable level by the consumers.

**1.2 Wheat-soybean bread manufacturing** 

rheological properties and crumb uniformity.

**1.3 Aims of investigations** 

### **2.1 Soybean seed**

The whole soybean seeds (*Glycine max* L.) cultivars ZP Lana, grown in Serbia in summer of 2006 were used. The seeds were purchased in local store "Green Apple" (Leskovac, Serbia) and milled. The particle size was determined by method of insemination via riddles with gaps size from 0.315 to 3.15 mm. The overall particle size was determined using equations:

$$100/d\_{sr} = \sum \Delta d\_i / d\_i \tag{1}$$

where *<sup>i</sup> d* is weight of fraction with appropriate particle size in %, and

$$d\_{\iota} = (d\_{\iota+1} + d\_{\iota+1})/2 \tag{2}$$

where *i*<sup>1</sup> *d* is bottom riddle gap size and *i*<sup>1</sup> *d* is upper riddle gap size.

#### **2.2 Chemicals**

Chemicals used for oil extraction were high quality chemicals (Centrohem, Serbia) and for HPLC and GC analysis they were analytical grade (Riedel-de Haën, Honeywell Specialty Chemicals, Germany).

### **2.3 Wheat and soybean flour and flour mixtures**

The wheat flour, Kikinda Mill, Serbia (WF) was bought from the local market. The soybean flour (SF) as »full-fat soy flour« was obtained by soybean seeds milling (IKA Model M120), to an overall particle size ( *dsr* ) of 0.4 mm. Quantities of 291, 285, 270, 240 and 210 g of wheat flour and 9, 15, 30, 60 and 90 g of soybean flour, respectively, were used to make flour mixture with 3, 5, 10, 20 and 30 % (w/w) soybean flour portion, without adding additives.

#### **2.4 Flour analyses**

Flour protein content was determined by the Kjeldahl method (Nx5.95). The moisture content was determined by Scaltec SMO 01 (Scaltec instruments, Germany) instruments: flour (5 g) was put into the disk plate analyzer, dried at 110oC to a constant weight, and the

The Main Components Content,

**2.8 GC analysis** 

with those of standards.

**2.9.1 Statistical analysis** 

**3. Results and discussion** 

**3.1 The main components content** 

used.

mixtures.

**2.9 Energetic value** 

Rheology Properties and Lipid Profile of Wheat-Soybean Flour 85

For GC analysis, fatty acids methyl esters were prepared. The lipid were alkaline hydrolyzed and methylated by methanol and BF3 as catalysts. The final fatty acids methyl esters concentration was about 8 mg/ml in heptane. For obtaining a methyl esters GC spectra, the HP 5890 SERIES II GAS-CHROMATOGRAPH, HP with FID detector and 3396 A HP integrator was used. Column was ULTRA 2 (25m x 0.32mm x 0.52 m) (Agilent Technologies, Wilington, USA), injector temperature of 320oC, and injector volume of 0.4 l. The carrier gas was He at a constant flow rate of 1 ml/min. The flame ionization detector was at 350oC and split ratio was 1:20. Oven temperature was initially 120oC and was maintained at 120oC, for 1 min, then increased by 15oC/min until 200oC, increased by 3 oC/min until 240oC, increased by 8oC/min until 300oC and maintained at 300oC for 15 min. The fatty acids were identified by comparison of retention times of the lipid components

Based on total carbohydrates (CHC), protein (PC) and lipid content (LC), the energetic value

STATISTICA, version 5.0 software was used to perform the statistical analysis: the means and standard deviations, the correlation coefficients and cluster analysis. The means and standard deviations were obtained by Descriptive Statistics, marking the Median & Quartiles and Confirm Limits for Means. In order to classify wheat flour and wheat-soybean mixtures, the cluster analysis and the Euclidean method with the complete linkage was

The results of wheat and soybean flour moisture, starch, protein, ash, lipid, gluten and carbohydrates content are showed in Table 1. Data are presented as means of three determinations with standard deviation. Based on these data, considering the soybean flour portion in mixtures and compared to wheat flour, it is evident the moisture (from 12.8 to 11.6%), the starch (from 76.6 to 56.8%), gluten (23.9 to 16.7%) and carbohydrates content (from 78.8 to 62.8%) decreased, while the protein (from 8.6 to 20.0%), ash (from 0.48 to 2.08%) and lipid (1.2 to 7.2%) content increased with increasing soybean flour portion in

Taking into account the protein, carbohydrates and lipid content, based on formula (3) for energetic value determination, it was obtained that the soybean flour increased energetic value in flour mixtures (from 1530 obtained for wheat flour, to 1544, 1554, 1579, 1625, 1674 kJ/100g, when soy flour portion was 3, 5 10, 20 and 30%, respectively) and the maximal

increasing of 9.4% was in mixture with soybean flour portion of 30%.

*EV* (*CHC PC*)17 *LC* 37 (3)

(EV) of wheat flour and white-soybean flour mixtures was calculated as:

moisture content was read out on the display. The ash content was determined by staking at 800oC during 5 h. For gluten content determination, the dough was prepared by adding a sodium chloride solution first; wet gluten was isolated by dough washing and weighed. The starch content was determined by polarimetry according to Grossfeld's method and the total carbohydrates content according to Luff-Schoorl's method (Trajković et al., 1983). The values for samples are from triplicate analysis and followed by standard deviation.

#### **2.5 The wheat and soybean flour lipid content**

The wheat or soybean flour (50g) were put into Erlenmeyer flask, 500 ml of trichloroethylene was added and extracted for 30 minutes, under reflux and by mixing (200 min-1) at approximately 88oC. The extract was separated by using Buchner funnel under weak vacuum. The plant material was extracted three more times by the same method; the extracts were mixed together and eluted by water in the separation funnel (3 x 10 ml). The eluted extracts' volume was recorded and an aliquot (3 ml) was taken for the dry residue determination test. This test was performed by drying at 110oC to a constant weight and the dry residue content was read out on the analyzer display (Scaltec SMO 01, Scaltec instruments, Germany). The lipid content in wheat and soybean flour was calculated based on average value of three measurements. The remnant of lipid extracts, after dry residue determination test, is evaporated under vacuum and obtained residue was used for HPLC and GC analysis.

#### **2.6 Rheology measurements**

The Brabender farinograph (Brabender Model 8 10 101, Duisburg, Germany) according to ISO 5530-1 test procedure, was used for water absorption values (WA value in ml/100g), development time (DT in minutes), dough stability (DSt in minutes), degree of softening (DSf in BU) and farinograph quality number (QN) determination. For extensograph measurement, the Brabender extensograph (Brabender, Model 8600-01, Duisburg, Germany) and test procedure ISO 5530-2 were used. The samples were prepared from wheat flour and wheat-soybean flour mixtures, distilled water and salt, and data for energy (E in cm2), resistance (R in EU), extensibility (Ex in mm) and ration number (R/Ex) were recorded on extensograph curve. To obtain amylograph data, such as gelatinization temperature (Tmax in oC) and gelatization maximum (max in AU), the amylograph (Brabender Model PT 100, Duisburg, Germany) and ISO 7973 test procedure were used.

### **2.7 HPLC analysis**

For HPLC analysis, Holčapek et al., (1999) modified HPLC method and the Agilent 1100 High Performance Liquid Chromatograph, a Zorbax Eclipse XDB-C18 column: 4.4 m x 150 mm x 5 m, Agilent technologies, Wilmington, USA and an UV/ViS detector were used. The flow rate of binary solvent mixture (methanol, solvent A, and 2-propanol/*n*-hexane, 5:4 by volume, solvent B) was 1 ml/min, with a linear gradient (from 100% A to 40% A+ 60% B in 15 min). The column temperature was held constant at 40oC. The components were detected at 205 nm. The free fatty acids (FFA), methyl esters (ME), monoacylglycerols (MAG), diacylglycerols (DAG) and triacylglycerols (TAG) were identified by comparing the retention times of the lipid components with those of standards. The samples of the reaction mixture were dissolved into a mixture of 2-propanol:*n*-hexane, 5:4 v/v and filtered through 0.45 m Millipore filters.

#### **2.8 GC analysis**

84 Recent Trends for Enhancing the Diversity and Quality of Soybean Products

moisture content was read out on the display. The ash content was determined by staking at 800oC during 5 h. For gluten content determination, the dough was prepared by adding a sodium chloride solution first; wet gluten was isolated by dough washing and weighed. The starch content was determined by polarimetry according to Grossfeld's method and the total carbohydrates content according to Luff-Schoorl's method (Trajković et al., 1983). The values

The wheat or soybean flour (50g) were put into Erlenmeyer flask, 500 ml of trichloroethylene was added and extracted for 30 minutes, under reflux and by mixing (200 min-1) at approximately 88oC. The extract was separated by using Buchner funnel under weak vacuum. The plant material was extracted three more times by the same method; the extracts were mixed together and eluted by water in the separation funnel (3 x 10 ml). The eluted extracts' volume was recorded and an aliquot (3 ml) was taken for the dry residue determination test. This test was performed by drying at 110oC to a constant weight and the dry residue content was read out on the analyzer display (Scaltec SMO 01, Scaltec instruments, Germany). The lipid content in wheat and soybean flour was calculated based on average value of three measurements. The remnant of lipid extracts, after dry residue determination test, is evaporated under vacuum and obtained residue was used for HPLC

The Brabender farinograph (Brabender Model 8 10 101, Duisburg, Germany) according to ISO 5530-1 test procedure, was used for water absorption values (WA value in ml/100g), development time (DT in minutes), dough stability (DSt in minutes), degree of softening (DSf in BU) and farinograph quality number (QN) determination. For extensograph measurement, the Brabender extensograph (Brabender, Model 8600-01, Duisburg, Germany) and test procedure ISO 5530-2 were used. The samples were prepared from wheat flour and wheat-soybean flour mixtures, distilled water and salt, and data for energy (E in cm2), resistance (R in EU), extensibility (Ex in mm) and ration number (R/Ex) were recorded on extensograph curve. To obtain amylograph data, such as gelatinization temperature (Tmax in oC) and gelatization maximum (max in AU), the amylograph (Brabender Model PT 100,

For HPLC analysis, Holčapek et al., (1999) modified HPLC method and the Agilent 1100 High Performance Liquid Chromatograph, a Zorbax Eclipse XDB-C18 column: 4.4 m x 150 mm x 5 m, Agilent technologies, Wilmington, USA and an UV/ViS detector were used. The flow rate of binary solvent mixture (methanol, solvent A, and 2-propanol/*n*-hexane, 5:4 by volume, solvent B) was 1 ml/min, with a linear gradient (from 100% A to 40% A+ 60% B in 15 min). The column temperature was held constant at 40oC. The components were detected at 205 nm. The free fatty acids (FFA), methyl esters (ME), monoacylglycerols (MAG), diacylglycerols (DAG) and triacylglycerols (TAG) were identified by comparing the retention times of the lipid components with those of standards. The samples of the reaction mixture were dissolved into a mixture of 2-propanol:*n*-hexane, 5:4 v/v and filtered through

for samples are from triplicate analysis and followed by standard deviation.

**2.5 The wheat and soybean flour lipid content** 

Duisburg, Germany) and ISO 7973 test procedure were used.

and GC analysis.

**2.7 HPLC analysis** 

0.45 m Millipore filters.

**2.6 Rheology measurements** 

For GC analysis, fatty acids methyl esters were prepared. The lipid were alkaline hydrolyzed and methylated by methanol and BF3 as catalysts. The final fatty acids methyl esters concentration was about 8 mg/ml in heptane. For obtaining a methyl esters GC spectra, the HP 5890 SERIES II GAS-CHROMATOGRAPH, HP with FID detector and 3396 A HP integrator was used. Column was ULTRA 2 (25m x 0.32mm x 0.52 m) (Agilent Technologies, Wilington, USA), injector temperature of 320oC, and injector volume of 0.4 l. The carrier gas was He at a constant flow rate of 1 ml/min. The flame ionization detector was at 350oC and split ratio was 1:20. Oven temperature was initially 120oC and was maintained at 120oC, for 1 min, then increased by 15oC/min until 200oC, increased by 3 oC/min until 240oC, increased by 8oC/min until 300oC and maintained at 300oC for 15 min. The fatty acids were identified by comparison of retention times of the lipid components with those of standards.
