The straight dough bread making and experimental design
The ingredients were added based on a % wheat flour basis. Bread dough recipe included500 g of wheat flour,100 g of sweet potato flour, 280 g of water, 5 g of salt, 5 g of instant dry yeast (Saccharomyces cereviseae), and finally butter (incorporated 2 minute after the end of first mixing). Mixing was done in a spiral mixer (Sinmag Bakery Equipment Wuxi CO., Ltd) comprising of two steps: first step, the mixer was adjusted to 3 min low speed and 3.5 high speed. The second step was 0.5 min low speed and 3 min high speed. The overall mixing time was between 3.5 and 6.5 min and the dough was divided into small round pieces weighing60 g each by Sinmag bread divider (Sinmag Bakery Equipment Wuxi CO., Ltd). Dough pieces were optimally proofed for 90 min at 35 ° C with 85% relative humidity in an electric proofer (Sinmag Bakery Equipment Wuxi CO., Ltd) and baked for 16 minutes in an electric oven (Sinmag Bakery Equipment Wuxi CO., Ltd). Top and bottom temperature were 180°C and 200°C respectively. Enzyme and additive were added immediately during mixing. The addition of TGM was calculated on the basis of the amount of protein content of GMP present in each sample. The recommended level of use for food is generally 1 U of TGM/g of protein. Levels of AA addition were selected based on multiple preliminary experimental run. Experiment was designed as follows: control (100% WF), described as target; T1 (80% WF + 20 % SPF), described as baseline; T2 (80% WF + 20 % SPF + 0.5% TGM); T3 (80% WF + 20 % SPF + 1% TGM); T4 (80% WF + 20 % SPF + 1.5 % TGM); T5 (80% WF + 20 % SPF + 0.1% AA) ;T6 (80% WF + 20 % SPF + 0.3% AA) and T7 (80% WF + 20 % SPF + 0.5% AA).
Sulfhydryl and disulfide content of composite sweet potato – wheat dough
Sulfhydryl (SH) (free and buried SH) and SH- total (SH and reduced SS) groups of proteins in composite doughswere determined by method of (L.Ellman, 1959) in the manner described by Y. Huang, Hua, and Qiu (2006)with some modifications. Chemicals were prepared as follows: Tris-glycine buffer (10.4g Tris, 6.9g glycine, 1.2g EDTA and 10 g sodium dodecylsulfate (SDS)per liter, pH 8.0, denoted as Tris-Gly), Ellman’s reagent (5,5’-dithiobis-2-nitrobenzoic acid) (DNTB) (Sigma Aldrich) in Tris-Gly (4 mg/ml), 12% trichloroacetic acid (TCA), and 8 M urea containing 5 M GuHCl in Tris-Gly (Urea-GuHCl). Freeze dried composite doughs (100 mg) were solubilized in 10 ml of Tris-Gly buffer by magnetic stirring at room temperature. For SH determinations, 4 ml of sample solution was added to 3 ml of Tris-Gly buffer and 0.1 ml Ellman’s reagent, vortexed, and incubated at 25 oC for 1 h (Gujral & Rosell, 2004). For SH-Total determination, 2 ml of sample solution was added to 0.05 ml of 2-mercaptoethanol and 4 ml of Urea-GuHCl and the mixture was incubated for 1 h at 25 oC. Then, 10 ml of 12 % TCA was added, vortexed and incubated again for another 1 h under same conditions. The mixtures were centrifuged at 4000 rpm for 15 min. The precipitate was resuspended twice in 5 ml of 12% TCA and centrifuged to remove 2-mercaptoethanol. The final precipitate was dissolved in 10 ml of Tris-Gly buffer and 0.08 ml of Ellman’s reagent was added and then samples were let to stand for 1 h to develop color. Absorbance was measured at 412 nm against reagent blanks in which sample was replaced by the buffer in same amounts. Calculations were based on a more accurate TNB2- extinction coefficient of 14,150 /M-cm (Y. Huang et al., 2006)according to the following equation (1):
µM SH/g = A412 x 70.67 x D/ C; (1)
Where:
A412 = the absorbance at 412 nm; C = the sample concentration in mg solids/ml; D = the dilution factor, 2.03 and 10.08 for SH and total SH –Total, respectively; and 70.67 is derived from 106/ (1.415 x 104); 1.415 x 104 is the molar absorptivity and 106 is for conversions from the molar basis to the nmol/ml basis and from mg solids to g solids.
Results were calculated against a cysteine standard curve and the values recorded were the mean of triplicates.
Fig 1.Effect of transglutaminase (TGM) and ascorbic acid (AA) on sulfhydryl and disulfide content of composite sweet potato – wheat dough: (a) SH (µmoles/g) content of dough treated with TGM, (b) Total - SH (µmoles/g) of dough treated with TGM, (c) SH (µmoles/g) of dough treated with AA, (d) Total – SH (µmoles/g) of dough treated with AA. control (100% WF), described as target; T1 (80% WF + 20 % SPF), described as baseline; T2 (80% WF + 20 % SPF + 0.5% TGM); T3 (80% WF + 20 % SPF + 1% TGM); T4 (80% WF + 20 % SPF + 1.5 % TGM); T5 (80% WF + 20 % SPF + 0.1% AA) ;T6 (80% WF + 20 % SPF + 0.3% AA) and T7 (80% WF + 20 % SPF + 0.5% AA). Bars represent ± SD for triplicate.
Baking characteristics of composite sweet potato – wheat breads
The textural characteristics of the composite bread were measured 2 h after baking using a Texture Pro CT V 1.4 Build 17 (Brookfield Engineering Laboratory, Middleboro, MA, USA) equipped with an aluminum 36 mm diameter cylindrical probe. Breads were sliced transversely using an electric bread slicer (Sinmag Bakery Equipment, Wuxi Co., Ltd., Wuxi City, Jiangsu, China) to obtain uniform slices of 12.5 mm thickness each. Bread slices taken from the center of each loaf were used to evaluate the crumb texture. A stack of two slices (25 mm total) was prepared and compressed to 50% of its original thickness. The test conditions were pretest speed, 2 mm/sec; test speed, 0.5 mm/sec; return speed, 0.5 mm/sec; and trigger load, 7 g. The results were recorded for the following parameters: hardness, chewiness, and gumminess. The weights of the loaves were determined with the aid of a weighing balance (electric scale). Loaf volume was measured using seed displacement method according to AACC Method (AACC 10-05.01). Loaves were placed in a container of known volume into which rapeseeds were run until the container was full. The volume of seeds displaced by the loaf was considered as the loaf volume. Bread Specific Volume was calculated according to the following formula: B.S.V = Loaf volume / Loaf weight.
Table 1 Effect of transglutaminase (TGM) and ascorbic acid (AA) on mixolab characteristics of composite sweet patato – wheat dough.
Hardness(g) / Gumminess
(g) / Chewiness
(mJ) / Volume
(ml) / Weight
(g) / BSV
(ml/g)
Control / 175 ± 32g / 135 ± 24h / 4.80 ± 0.9f / 52.92 ± 0.01e / 254.50 ± 2.06a / 4.80 ± 0.03a
T1 / 888 ± 43a / 702 ± 36a / 24.2 ± 1.61a / 54.66 ± 0.23d / 156.60 ± 2.87e / 2.86 ± 0.06cd
T2 / 716 ± 18b / 670 ± 50b / 21.5 ± 0.53b / 55.40 ± 0.32cd / 155.72 ± 1.83e / 2.83 ± 0.07cd
T3 / 500 ± 23d / 385 ± 17d / 13.8 ± 0.11d / 55.31 ± 0.13c / 164.51 ± 2.32de / 2.97 ± 0.13c
T4 / 668 ± 21c / 527 ± 12c / 18.7 ± 0.52c / 54.97 ± 0.28cd / 164.54 ± 2.03de / 3.02 ± 0.10c
T5 / 324 ± 35e / 355 ± 15e / 5.6 ± 0.56f / 59.60 ± 0.6b / 215.33 ± 8.02c / 3.59 ± 0.13b
T6 / 292 ± 3e / 274 ± 11f / 7.2 ± 1.73e / 60.46 ± 0.5a / 178.33 ± 2.80d / 2.61 ± 0.40d
T7 / 238 ± 52f / 196 ± 12g / 6.8 ± 0.38e / 60.10 ± 0.30ab / 231.66 ± 5.77b / 3.84 ± 0.11b
control (100% WF), described as target; T1 (80% WF + 20 % SPF), described as baseline; T2 (80% WF + 20 % SPF + 0.5% TGM); T3 (80% WF + 20 % SPF + 1% TGM); T4 (80% WF + 20 % SPF + 1.5 % TGM); T5 (80% WF + 20 % SPF + 0.1% AA) ;T6 (80% WF + 20 % SPF + 0.3% AA) and T7 (80% WF + 20 % SPF + 0.5% AA). Mean values within a column followed by different letters are significantly different (P ≤ 0.05) Mean (n=2) ± standard deviation.
Results and discussion
Effect of TGM and AA on sulfhydryl content of composite sweet potato – wheat dough
Changes in SH and SH- total groups of proteins in composite dough produced by the action of the TGM and AA were measured before and after TGM and AA treatments Fig 1. Substitution of WF with SPF significantly (p≤0.05) affected SH and SH- total content in the composite dough. SH and SH-total dropped from 2.87 (control) to 2.10 and from 45.27(control) to 35.80 for T1 respectivelyFig a and b. However, progressive decrease in the amount of SH and increase in SH-Toatal was observed after TGM treatment. SH content from T1 dough significantly (p≤0.05)decreased when TGM was added from 0.5% to 1.5% Fig 1a whereas SH- total increased with enzyme concentration Fig 1b. Opposite trend was noted on addition of AA from 0.1% to 0.5% (% flour basis), although the rates of change were different from one additive concentration to another. SH drastically increased with AA concentration Fig 1c. It is clear that SH progressively increased with a slight increase of SH- total in dough treated with AA. Our results are surprising since they are not in agreement with the mechanism of AA on glutenins that AA inhibites SS/SH interchange by blocking the thiol groups. Therefore, we recommend further studies on this particular additive in composite dough. Joye, Lagrain, and Delcour (2009a)reported that intermolecular SS bonds can be reduced by addition of reducing agents, releasing the component glutenin subunits (GS) which have similar solubility to gliadins. From our knowledge, this is not the case since in our study the amount of SH- total increased with addition of AA, from 0.1 to 0.5 (% flour basis). However, our results are in agreement with Koehler (2004) and E. Maforimbo et al. (2007) who showed that increasing ascorbic acid concentrations caused an increase in the concentrations of free SH groups. Gujral and Rosell (2004)Suggested that the change in SH content of composite dough could be due to the disappearance of the lysine groups exposed to the enzyme reaction. However, the crosslinking reaction may also bring some amino acids close to each other as polymeric protein molecules become more compact. Thus, the sulphur containing amino acids may come close to each other resulting in the formation of SS bonds by oxidation (Gauche, Vieira, Ogliari, & Bordignon-Luiz, 2008; Gujral & Rosell, 2004; Rasiah et al., 2005). Steffolani et al. (2010)Reported that TGM catalyses the formation of isopeptidic bonds, which modifies the three-dimensional structure of the gluten protein. Therefore the possibility of forming disulfide bonds depends on protein structure as well as the S-S formation and TGM doses present in the protein network.
3.4.Effect of TGM and AA on baking characteristics of composite sweet potato – wheat breads
Breadcrumb texture properties were evaluated using a texture profile analysis in terms of crumb hardness; gumminess and chewiness. Results are illustrated in Table 5. Incorporating sweet potato flour dilutes wheat gluten, which consequently weakens the gluten network. Crumb hardness, gumminess and chewiness significantly increased with wheat flour substitution. All values were higher in T1 (20% SPF) than the control (WF). Bread specific volume (BSV) significantly reduced with the substitution of wheat flour with sweet potato flour. Our results are consistent with those reported by (Greene.J.L & Bovell-Benjamin.A.C, 2004; Kun-Lun Wu. et al., 2009; Trejo-González et al., 2014). In order to improve the baking qualities of composite sweet potato – wheat breads, dough was treated with TGM and ascorbic acid. It is obvious that both additives significantly improvedthe composite sweet potato – wheat breads and their effects differ from one additive to another according to their concentration. Compared with the T1 (20% SPF), crumb hardness and gumminess of breads from dough treated with TGM and AA significantly reduced as their concentrations increased, with breads from dough treated with AA 0.5% (T5) having the lowest crumb hardness and gumminess. A slight increase in crumb hardness and chewiness from dough treated with TGM 1.5% (T4) were observed. It can be assumed that the enzyme would positively contribute when its concentration is below 1.5%. Both additives were found to have antagonistic effect on crumb chewiness. Increasing TGM concentration significantly reduced crumb chewiness, while the opposite was observed for breads from dough treated with AA.