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This paper must be cited as:

Verdú Amat, S.; Vasquez, F.; Ivorra Martínez, E.; Sánchez Salmerón, AJ.; Barat Baviera,
JM.; Grau Meló, R. (2015). Physicochemical effects of chia (Salvia Hispanica) seed flour on
each wheat bread-making process phase and product storage. Journal of Cereal Science.
65:67-73. doi:10.1016/j.jcs.2015.05.011.

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http://dx.doi.org/10.1016/j.jcs.2015.05.011

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 1 Physicochemical effects of chia (Salvia Hispanica) seed flour on each wheat bread-

2 making process phase and product storage.

3 Samuel Verdú1*, Francisco Vásquez 3, Eugenio Ivorra2, Antonio J. Sánchez2, Jose M.

4 Barat1, Raúl Grau1

6 1
Departamento de Tecnología de Alimentos. Universidad Politècnica de València,

7 Spain.

8 2
Departamento de Ingeniería de Sistemas y Automática, Universidad Politècnica de

9 València, Spain

10 3
Departamento de Tecnología de Alimentos de Origen Vegetal. Centro de Investigación

11 en Alimentación y Desarrollo A,C, Hermosillo, Sonora, Mexico.

12

13

14

15 *Author for correspondence: Samuel Verdú

16 Address: Edificio 8G - Acceso F - Planta0
17 Ciudad Politécnica de la Innovación
18 Universidad Politécnica de Valencia
19 Camino de Vera, s/n
20 46022 VALENCIA – SPAIN
21 E-mail: [email protected]
22 Phone: +34 646264839
23

1
24 Abstract

25 Some chia seed flour effects relating to different bread-making process phases and

26 variables were studied by distinct image analysis and physicochemical techniques.

27 Wheat flours with three different degrees of substitution (5%, 10% and 15%) were

28 tested. In technological terms, the aim was to study the influence and properties of chia

29 flour on several relevant parameters, such as pasting properties, growth kinetics and

30 internal crumb structure during dough fermentation; and baking process, mass loss,

31 water activity and texture profile of the end product during its storage. Some changes in

32 pasting properties were observed. The effects obtained by image analysis techniques

33 proved that addition of chia improved gas retention in dough and cut the time required

34 to reach maximum dough development. A delay in hardness and water loss during

35 storage of breads was also observed. Bread presented reduced water activity, and

36 contained the same amount of moisture compared with the control. The mucilage

37 provided by chia has properties that can explain these observed effects given the

38 influence on water-holding capacity and its interactions with gluten proteins throughout

39 the gluten matrix-forming process.

40

41 Keywords: Chia, bread-making process, image analysis, wheat flour

42

43

44

45

2
46 1. Introduction

47 Some relevant factors affect productivity in the cereal by-product industry due to

48 changes between the properties and the behavior of the flour sort and other ingredients

49 used in production phases. Flour features, such as flour origin and quality, cultivation

50 and milling method, and cereal variety are some of the most influential factors.

51 Variability conferred by raw materials directly affects the process and product variables

52 (Cocchi et al. 2005), which are necessarily controlled to obtain high yields and

53 homogeneous characteristics in both the production chain and product quality. Some of

54 the most affected properties derive from the rheology of dough and batters, such as gas

55 retention capacity, water interactions and flow behaviors. How different strategies act

56 with these factors has been studied to solve the problems that appear in some phases of

57 industrial processes (Ahlborn et al. 2005). Work has been done mainly in those cases in

58 which flours present defects or low technological quality to reach the properties of

59 reference products, such as wheat flour with a small amount of gluten, flours without

60 gluten for gluten-free products, modifying properties during storage, etc. (Lazaridou et

61 al. 2007). In relation to the above objectives, one of the most studied areas is the

62 development of new ingredients, among which, several of the most important ones are

63 compounds that are situated in any plant part, like grains, which improve gas retention

64 in the dough matrix and water activity modification, among others (Houben et al. 2012).

65 The nature of those compounds is diverse, but the most influential ones can form

66 hydrocolloid structures. Hydrocolloids can induce some structural changes in the main

67 flour components from the bread-making process to product storage. So they are usually

68 added to dough to improve its viscoelastic properties, as well as the structure,

69 mouthfeel, acceptability and shelf life of bakery products (Ahlborn, et al. 2005).

70 Hydrocolloids are used either alone or combined to achieve specific synergies between

3
71 their respective functional properties. Numerous types of hydrocolloids are obtained

72 from the flour of whole grains and have been analyzed in this approach to take

73 advantage of the other active compounds and nutrients in these products (Del Rio et al.

74 2013). One grain with interesting properties for this area is chia seeds (Salvia

75 hispanica). Chia is an annual herb of the Labiatae family and was one of the basic

76 nourishments of Central American civilizations in pre-Columbian times (Ayerza and

77 Coates, 2005). Some studies have shown the potential uses of chia based on its

78 compositional profile (defatted chia seeds have 22% of fiber and 17% of protein). These

79 contents are similar to those of other oilseeds currently used in the food industry

80 (Cumbyet al. 2008; Vázquez-Ovando et al., 2010). The consumption of chia provides

81 numerous health benefits, such as a high content of oil, protein and bio-active peptides,

82 antioxidants, minerals and dietary fiber (Ixtaina et al. 2008). Some works have been

83 published with information about dough behavior and organoleptic acceptance, with

84 degrees of substitution between 4-5% with both chia by-products and whole seed flour

85 (Iglesias & Haros, 2013; Moreira et al., 2013). This percentage of substitution is based

86 on the daily bread intake recommended by the World Health Organization, which would

87 result in considerably improved nutrient contribution if all bread consumed contained

88 5% of chia. Those results report interesting technological properties apart from

89 nutritional improvement. The effect of high substitution degrees could be interesting

90 from a technological application point of view for the development of new formulas

91 based on gluten-free flours, and for possible improvements to different bread-making

92 process phases. From this technological viewpoint, the most important component of

93 these seeds is fiber content, which includes a polysaccharide gum with high-molecular-

94 weight mucilage. It has been proposed that the structure of the basic mucilage unit is a

95 tetrasaccharide with 4-O-methyl-a-D-glucoronopyranosyl residues which occurs in

4
 96 branches of β-D-xylopyranosyl in the main chain (Lin et al. 1994). This compound

97 presents a high water-holding capacity and forms an active hydrocolloid. The work of

98 Iglesias and Haros, 2013 observed how this compound could improve the dough volume

99 rate due to the formation of hydrophilic complexes between their ionic groups and

100 proteins as gluten, which favors gluten matrix formation. For all these reasons, chia

101 seeds present interesting features for testing some aspects of the bread-making process,

102 and to evaluate their plausible applications in order to improve some of these aspects.

103 Therefore, the purpose of the present study was to report further information about how

104 substituting wheat flour for chia seed flour at the 5%, 10% and 15% degrees affects

105 some important technological features; e.g., pasting properties of flour mixtures; dough

106 fermentation process; baking loss; physicochemical properties (texture profile, mass

107 loss and water activity) during storage of the end product for 0, 1, 3 and 7 days.

108

109 2. Material and Methods

110

111 2.1. Raw materials and dough preparation

112 The commercial wheat flour used was obtained from a local producer (Molí del Picó-

113 Harinas Segura S.L. Valencia, Spain) whose chemical composition was: 14.7±0.6% of

114 proteins, 1.1±0.03% of fat, 14.5±0.5% of water, and 0.32±0.1 of ash (w.b). The

115 alveographic parameters were also facilitated by the company, which were P = 94±2

116 (maximum pressure (mm)), L = 128±5 (extensibility (mm)), W = 392±11 (strength (J-4))

117 and 0.73 of P/L. Chia flour was obtained from a commercial black chia seed format

118 (BIOCESTA S.L., Valencia Spain) by milling in a stainless steel grinder (Retsch

119 GmbH, ZM 200, Haan, Germany) to obtain a particle size distribution as close to the

5
120 used wheat flour as possible. The particle size of flours was measured 6 times by laser

121 scattering in a Mastersizer 2000 (Malvern, Instruments, UK) equipped with a Scirocco

122 dry powder unit. The results are expressed as a maximum size in µm at 10%, 50% and

123 90% (d (0.1), d (0.5) and d (0.9), respectively) of the total volume of the analyzed

124 particles as their averages (D [4, 3]). The wheat flour results were d (0.1) = 25.5±1.1, d

125 (0.5) = 92.0±0.6, d (0.9) = 180.6±0.8 and D [4, 3] = 99.4±1.2. The chia flour results

126 were d (0.1) = 23.9±1.9, d (0.5) = 95.3±1.2, d (0.9) = 181.9±1.3 and D [4, 3] =

127 100.1±1.1. Chia flour contained 2±1% of proteins, 30.4±0.9% of fat, 8±0.3% of

128 moisture and 4.1±0.8% of ash (w.b). Four flour mixtures, which presented 0%, 5%,

129 10% and 15% wheat flour substitution degrees for chia flour, were prepared to carry out

130 the study.

131 The control formulation used to prepare dough was the following: 56% flour, 2%

132 refined sunflower oil (maximum acidity 0.2º Koipesol Semillas, S.L., Spain), 2%

133 commercial pressed yeast (Saccharomyces cerevisiae, Lesafre Ibérica, S. A., Spain), 4%

134 white sugar (≥ 99.8% of saccharose, Azucarera Ebro, S.L., Spain), 1.5% salt (refined

135 marine salt ≥ 97% NaCl, Salinera Española. S.A., Spain) and water 34.5%. This

136 formulation slightly increased the amount of water, following degree of substitution, to

137 1% in the case of 15% mixture, due to the low moisture of chia to maintain a constant

138 ratio between moisture (provided by wheat, chia and the added water) and dry matter

139 (provided by wheat and chia), observed in the control sample (0.89 g water/g dry

140 matter). Bread dough was made according to a closed process with no modifications

141 between mixes to observe the effect on a possible continuous industrial process. The

142 process was carried out by mixing all the ingredients in a food mixer (Thermomix®

143 TM31, Vorwerk, Germany) according to the following method: in the first phase, liquid

144 components (water and oil), sugar and NaCl were mixed for 4 minutes at 37 ºC. Pressed

6
145 yeast was added in the next phase to be mixed at the same temperature for 30 seconds.

146 Finally, flour was added and mixed with the other ingredients according to a default

147 bread dough mixing program, which provides homogeneous dough. The program

148 system centers on mixing ingredients with random turns of the mixer helix in both

149 directions (550 revolutions/minute) to obtain homogeneous dough. This process was

150 applied for 4.5 minutes at 37 ºC. Then 450 g of dough were placed in the metal mold

151 (8x8x30cm) for fermentation. Height was approximately 1 cm.

152

153 2.2. Characterization of flour mixing dough by a viscometer Rapid Viscosity

154 Analyser (RVA)

155 The pasting properties of the flour mixtures were analysed using the viscosity profile

156 obtained by the viscometer RVA (Rapid Visco Analyser Super 4, Newport Scientific).

157 For this purpose, the method approved by AACC (America Association of Cereal

158 Chemists), whose reference is “General Pasting Method for Wheat or Rye Flour of

159 Starch Using the Rapid Visco Analyser. AACC 2000, number: 76-21”), was used.

160 Samples of 3 g±0.01g were weighed and the amount of water incorporated was 25

161 g±0.01g. The test started at 50 °C and 960 RPM, and was slowed down to 160 RPM at

162 10 s. Temperature was maintained during the first minute. The temperature from 50 °C

163 to 95 °C was increase during the next 4 minutes to reach 95 °C at minute 5 in a second

164 step. The third step involved maintaining a temperature of 95 °C until minute 7.5. The

165 fourth step was to lower the temperature to 50 °C, which was reached at minute 11. The

166 last step was to maintain a temperature of 50 °C until minute 13. Measurements were

167 taken in triplicate.

168

7
169 2.3. Fermentation process and its continuous monitoring

170 The fermentation process was carried out in a chamber with controlled humidity and

171 temperature (KBF720 Binder Tuttlingen, Germany). The conditions were 37 °C and

172 90% of Relative Humidity (RH). Dough growth evolution was monitored by a device

173 installed and calibrated inside the chamber. This device was based on an image analysis

174 of Structured Light (SL), following the method described previously by Ivorra et al.

175 (2014). Only the points between mold edges were analyzed by this method. During the

176 process, one picture per second was captured until the required analysis time according

177 to the phase study. The 3D information calculated from each image during the process

178 focused on maximum height (H: the maximum Z edge value) and transversal area (A:

179 the integration of the Z-values along the X direction of the sample). Data acquisition

180 and data processing were carried out using an own code developed in the Matlab

181 computational environment (The Mathworks. Natick. Massachusetts, USA). The

182 behavior of the dataset average during fermentation was modeled by the SL method,

183 obtained from dough samples per mixture and adapted to the Gompertz prediction

184 model. The Gompertz function is a nonlinear sigmoid growth function which was

185 developed by Gompertz (1825) to calculate the mortality rate of microorganisms. The

186 equation is as follows:

187

𝑉𝑉
188 𝑡𝑡 = 𝐴𝐴 ∙ exp((−exp ( 𝐴𝐴 ∙ (𝐿𝐿𝐿𝐿 − 𝑡𝑡) + 1 )) (1)

189

190 where t is time, A is area during the process, V is the maximum growth rate, and Lt

191 represents the latency time before dough development begins. Model parameters were

8
192 determined by a nonlinear regression procedure and were obtained by minimizing the

193 sum of squares from prediction errors.

194

195 2.4. Study of internal dough structure in the fermentation phase

196 In order to study the chia effect on crumb evolution during the process, dough

197 fermentation was interrupted at 25%, 50%, 75% and 100% compared to its maximum

198 developing time (Ft). Thus the study of maximum fermentation development was firstly

199 analyzed (until dough depletion). In each sample, dough was baked and sliced to be

200 studied by 2D image segmentation. The baking process took place in an oven

201 (Rotisserie, DeLonghi, Italy) at 180ºC for 30 minutes. Once baking finished, six 1 cm-

202 thick slices were obtained from the center of the bread. In order to study crumbs, both

203 sides of each slice were captured in a scanner (Aficio™ MP C300-Ricoh. Tokyo, Japan)

204 to be then analyzed by 2D image segmentation (Verdú et al., 2014; Datta et al. 2007;

205 Esteller et al. 2006). Three bread products of each mixture and fermentation time were

206 examined, which meant that 36 crumb images were obtained. Images were acquired at a

207 resolution of 300 dpi. A black background was used in all the captures to improve

208 contrast and to enhance both cell wall structure and porosity measurements.

209

210 2.5. Study of the end product

211 To study the effect of chia on several of the most relevant bread properties, various

212 assays were carried out at different times before and after packing to test their evolution.

213 Baking loss (∆Mb) was first determined. In this phase, mass loss during the baking

214 process was concluded by the difference between the pre-baking dough weight and the

9
215 finished bread weight (both bread products were cooled at room temperature for 1 h).

216 Then samples were packed in a low-density polyethylene bag, similar to that used in

217 commercial presentations, and were stored under environmental conditions (23 ºC and

218 72% R.H. approximately). The assay was carried out for 1, 3 and 7 days of storage.

219 Mass loss of whole breads, the texture profile analysis (TPA) and water activity (aw) of

220 crumbs were performed for each time.

221 Mass loss (∆Mt) was analyzed by paying attention to the weight at the beginning and at

222 each sampling time. TPA was performed following the method used by Miñarro et al.

223 (2012), where two 12.5 mm-thick cross-sectional slices were obtained from the center

224 of each bread product. The texture profile analysis was carried out in a TA-TX2 texture

225 analyzer (Stable Micro Systems, Surrey, UK). A 25kg load cell (35 mm diameter) was

226 used. The assay speed was set at 1.7 mm/s to compress the bread crumb center to 50%

227 of its previous height. The time between compressions was 5 s. The studied parameters

228 were: hardness (D), springiness (S), cohesiveness (C), gumminess (G), chewiness (Ch)

229 and resilience (R). Water activity of crumbs (aw) was determined in an Aqualab® dew

230 point hygrometer (DECAGÓN Aqualab CX-2, Pullman, WA, USA).

231

232 2.6 Statistical analysis

233 The results of pasting properties, fermentation parameters (A, V, Ft), bubble size and

234 baking loss were studied by a one-way variance study (ANOVA). A multifactor

235 ANOVA was used to study the main effects and interactions on the evolution of the

236 parameters studied during storage (texture, mass loss and water activity). In those cases

237 where the effect was significant (P-value < 0.05), the average was compared by Fisher’s

238 least significant difference (LSD).

10
239

240 3. Results and Discussion

241

242 3.1. Characterization of flour mixtures by Rapid Viscosity Analysis

243 The results of the pasting properties obtained by RVA and the profiles of the different

244 mixtures samples are shown in Fig. 1. The corresponding pasting parameters are

245 summarized in Table 1. The pasting times of the samples lowered due to degree of chia

246 substitution, and significant differences were observed for the 10% and 15%

247 substitution mixtures. Peak viscosity presented an inverse behavior and became higher

248 with increasing chia substitution, where the 10% and 15% mixtures continued to show

249 significant differences compared to the control and 5% mixture despite the peak time

250 presenting this inverse behavior. Trough, breakdown and setback also showed a clear

251 increment with degree of substitution.

252

253

254 Figure 1. Pasting profile of control wheat flour ─; 5% ─; 10% ─; 15% ─,

255 temperature ─ ─

256

11
257 Initially, reduced water availability due to the presence of chia compounds should make

258 starch gelatinization difficult. Thus the pasting temperature should present the opposite

259 behavior to that observed in the results. One possible explanation is that chia mucilage,

260 in combination with water and heat, produces increased viscosity at a lower temperature

261 compared to starch. Therefore, the increase in viscosity at the beginning of the assay, at

262 the 10% and 15% degrees of substitution could be attributed more to mucilage

263 hydration than to starch gelatinization.

Table 1. Pasting characteristics of different mixtures

Pasting time Peak viscosity Trough Breakdown Final visc Setback Peak time
68.5 ± 0.6 c 2245 ± 15 a 1421 ± 49 a 823 ± 10 a 2660 ± 56 a 1238 ± 10 a 6.1 ± 0.1 c

67.7 ± 0.1 c 2289 ± 98 a 1430 ± 14 a 859 ± 12 a 2738 ± 12 a 1308 ± 21 b 5.8 ± 0.1 b

66.1 ± 0.1 b 2472 ± 26 b 1530 ± 65 b 942 ± 4 b 2965 ± 58 b 1435 ± 70 c 5.5 ± 0.2 a

63.9 ± 0.6 a 2639 ± 91 c 1673 ± 10 c 966 ± 19 b 3173 ± 49 c 1500 ± 15 d 5.3 ± 0.1 a

t letter within columns means significant differences at p ≤ 0.05

264

265 The peak time results were also affected for the same reason, which lowered. This

266 indicates a higher degree of substitution. Yet despite the reduction in peak time, peak

267 viscosity increased with degree of substitution. These behaviors proved that chia

268 components mainly affected the viscosity of mixtures, independently of wheat, because

269 the degree of substitution incremented the viscosity level. Similarly, final viscosity was

270 another parameter that showed considerable changes. This implies that the changes in

271 pasting properties observed in the behaviors of the mixtures could be produced by chia

272 seed mucilage. This component has a high water-holding capacity and hydration

273 features (Inglett et al. 2014) and this phenomenon had no major influence on starch

274 granule gelatinization. However, the rapid formation of hydrocolloids, when they came

12
275 into contact with water, was the main factor responsible for the variations in the

276 viscosity parameters observed.

277

278 3.2. Fermentation process

279 Figure 2A shows the results obtained by the Gompertz fitting model of fermentation

280 curves generated by a 3D device. The parameters obtained from each mixture are

281 presented in Table 2. Maximum dough growth, represented by parameter A, did not

282 present significant differences among samples, except for the 15% mixture, which was

283 significantly higher than the rest.

284

Table 2. Results of fermentation process characterization

Control 5% 10% 15%

A (m2.10-4) 12.9 ± 1.1 a 13.1 ± 1.2 a 13.5 ± 1.5 a 15.8 ± 0.9 b
Curve V (m2.10-4/min) 32.9 ± 3.5 a 45.4 ± 4.1 b 47.5 ± 2.4 b 50.9 ± 1.3 c
parameters
Lt (min) 0.55 ± 0.1 a 1.62 ± 0.1 c 1.70 ± 0.2 c 1.10 ± 0.1 b
Ft (min) 81 ± 3.2 b 77 ± 2.1 b 68 ± 3.1 a 65 ± 2.9 a
T1 (25%) 1.1 ± 1.0 b 1.2 ± 1.3 b 1.8 ± 1.9 b 0.8 ± 0.9 a
Bubble size T2 (50%) 3.7 ± 1.2 b 3.4 ± 1.4 b 5.0 ± 1.9 b 3.4 ± 1.0 a
(m2.10-6 )
T3 (75%) 4.1 ± 2.3 a 4.2 ± 2.0 a 7.1 ± 2.2 b 5.3 ± 1.4 a
T4 (100%) 4.9 ± 1.9 a 5.2 ± 2.0 a 7.7 ± 2.5 a 7.2 ± 1.7 a
T1 (25%) 13.4 ± 1.0 a 13.2 ± 1.3 a 14.4 ± 1.9 b 13.0 ± 0.9 a
Nº of T2 (50%) 5.9 ± 1.2 a 8.2 ± 1.4 b 7.4 ± 1.9 b 10.5 ± 1.0 c
bubbles/m2.10-
4
T3 (75%) 6.4 ± 2.3 b 6.9 ± 2.0 b 5.1 ± 2.2 a 7.5 ± 1.4 b
T4 (100%) 7.4 ± 1.9 a 7.5 ± 2.0 a 6.7 ± 2.5 a 7.5 ± 1.7 a
Time tAc (min) 79.2 60.8 52.8 44.1
parameters
comparing to 23.2 33.3 44.3
control ∆tAc (%) -

Different letters within rows mean significant differences at p ≤ 0.05

285

286 Velocity features (V) were similar in the 5% and 10% mixtures. The control sample

287 gave the lowest value, while the 15% mixture obtained the highest one, and both

13
288 showed significant differences with the 5% and 10% mixtures. Lt (time latency) also

289 presented significant differences between the control and mixtures, with values up to 3-

290 fold lower than the chia mixtures. The control obtained the highest Ft (maximum

291 development time) value, followed by the 5%, 10% and 15% mixtures in ascending

292 order, and the differences among them were significant. The highest values in the 10%

293 and 15% mixtures were for the average bubble size data (Table 2), where these mixtures

294 gave significant differences compared to the other samples at Ft.

295

14
 A
16

14
Ac
12

10

A (m2.10-4)
∆tAc 15%
8
∆tAc 10%
6
∆tAc 5%
4

tAc
0
0 20 40 60 80
t (min)

22

b
20
ab
18
∆Mb %

a
16 a

14

12

10
Control 5% 10% 15%

C
0,98
Day 0
0,975

0,97

0,965
Day 1
0,96
aw

0,955
Day 3
0,95

0,945

0,94

0,935 Day 7

0,93
0,18 0,19 0,2 0,21 0,22 0,23

296 Xw

297 Figure 2.

298 A: Fermentation curves resulted from Gompertz curve-fitting procedure.

299 B: Percentage of mass-loss during baking process (∆Mb). Bars express standard

300 deviation and letters on the columns mean significant differences at p ≤ 0.05.

301 C: Evolution of Xw and aw during the storage of breads

15
302 Colors mixtures correspond as follows: Control ─; 5% ─; 10% ─; 15% ─; control

303 area (Ac) − −; time to reach control area (tAc) − −.

304

305 In order to evaluate the influence of chia in the fermentation phase, the resultant data of

306 the fermentation curves, which took the control curve as a reference, were studied. Thus

307 the time when each mixture reached the same A value of the control was determined

308 (Ac). This time factor was called tAc. Once each mixture tAc was obtained, it was

309 calculated how long it took to reduce in each mixture to reach Ac. This parameter was

310 called ∆tAc. The results showed that tAc reduced according to the increase in the degree

311 of substitution. After analyzing the ∆tAc results, the 5% mixture reached 23.2% Ac

312 faster than the control, 33.3% for the 10% mixture, while the 15% mixture obtained the

313 highest value and reduced the time to 44.3%.

314 Several authors have reported studies about different chia flour properties, which have

315 proven the improvement of gas retention in the dough matrix through variations in their

316 pasting properties; for example, in products prepared with gluten-free flours (Moreira et

317 al., 2013). Some changes in the rheological properties of dough resulted mainly from

318 the polymeric structure of chia mucilage, whose capacity to form hydrocolloids

319 increases water retention, and thus the surface tension of the matrix. This property could

320 improve the volume of dough through the formation of hydrophilic complexes between

321 their ionic groups and gluten proteins to favor the gluten matrix formation and,

322 therefore, to help avoid gas from leaking (Iglesias & Haros, 2013).

323 Therefore, the improvement of gas retention could lead to increased growth velocity,

324 and reaching Ac required less time. The 15% of degree of substitution, and probably

325 higher degrees, could keep enough gas to overcome Ac for the same initial dough mass.

326 Likewise, the effect of this property on bubble size was increased mechanical resistance
16
327 to the matrix walls, which confers more porous matrix to the gluten network, and

328 permits greater stability and further expansion during the fermentation process

329 (Bárcenas & Rosell, 2005). Consequently, the structure coalescence delay took place,

330 followed by collapsing dough.

331 The number of bubbles in the different mixtures was similar to that in the control.

332 However for the highest substitution mixtures, 10% and 15%, bubble size was

333 significantly larger at T4 (100% of dough growth). Those results are in agreement with

334 Iglesias and Haros (2013), whose study did not report differences in bubble size

335 between the wheat flour control and the 5% mixture. However, the significantly larger

336 bubble size in the 10% and 15% mixtures, and the fact that, at the same time, no

337 differences were found in their number of bubbles /m2·10-4, could explain their high A

338 at Ft values in the other samples due to a reduced gas leak. Therefore some changes in

339 crumb structure were observed from degrees of substitution above 5%, which also had a

340 direct effect on A.

341

342 3.3. End product

343 3.3.1. Baking process phase

344 Figure 2B represents the middle increment of mass (∆Mb) in % in relation to the baking

345 process phase of each mixture at tAc. This baking-loss was due mainly to the water loss

346 which resulted from heat treatment. The results show reduced baking loss when

347 incrementing the degree of substitution. The 10% and 15% mixtures presented

348 significant differences to the control, but not to the 5% mixture, and significant

349 differences were found among them. The chia compounds property on water retention,

350 as previously postulated by some authors, was also observed in this phase of the

351 process. In line with this, the studies into chia mucilage by Muñoz et al. (2012) reported

17
352 that it is able to hydrate 27 times its own weight. Likewise, the results obtained by

353 Vázquez-Ovando et al. (2009), but on a fiber-rich fraction of defatted chia, confirmed

354 its good water-holding capacity, among other aspects.

355 In order to study the observed effect and the results based on the literature in detail,

356 diverse parameters were calculated for the different compositional fractions of each

357 mixture and bread product, and how the process affected them. Table 3 summarizes the

358 calculated parameters and ratios. Firstly, although fat influenced several dough

359 properties, the final dry matter without fat (DMf) of each mixture was calculated

360 following the assays on matter balances by Vázquez-Ovando et al. (2009) as proteins,

361 starch and other carbohydrates, such as fiber, are the main influential components of

362 flour in terms of the interactions of dough in water (Wilhelm et al. 2005). In order to

363 obtain an overview of the variations in this parameter with degree of substitution, the

364 decrease in %DMf was calculated compared to the control flour.

365

Table 3. Parameters of dry matter of flour mixtures and their effect on baking process.

g Water of bread / %DMf %Xw %gWater / g DMf

bstitution DMf Xw of bread
g DMf reduced increment increment

Control 0.84 0.20 ± 0.01 a 0.10 ± 0.01 a - - -

5% 0.83 0.21 ± 0.02 ab 0.12 ± 0.02 b 1.3 5.8 26.4
10% 0.82 0.22 ± 0.01 bc 0.16 ± 0.01 cd 2.5 13.5 64.7
15% 0.81 0.24 ± 0.00 c 0.18 ± 0.01 d 3.8 18.1 80.9

DMf : Fraction of dry matter without fat of each flour mixture.

Xw of bread: moisture of bread after baking process.

g Water of bread / g DMf : ratio between grams of water in bread per each gram of dry matter without fat of flour mixture.

%DMf reduced: Reduction of dry matter without fat by the increase of % substitution compared to control flour.

%Xw increment: Increment of moisture in bread after baking in each flour mixture compared to control flour.

% g Water / g DMf increment: Increment of grams of water in bread per each gram of dry matter without fat of flour mixture

compared to control flour.

-Different letters within columns mean significant differences at p ≤ 0.05.

366

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367 Thus the results showed that DMf lowered when degree of substitution increased and,

368 therefore, the decrease in %DMf varied from 1.3% for the 5% mixture to 3.8% in the

369 15% mixture. Those variations were produced because chia flour has more than 10

370 times the amount of lipids compared to wheat flour and, consequently, due to a lower

371 proportion of the remaining dry matter. Secondly, moisture of bread products was

372 calculated (Xw). It was observed how bread moisture augmented with an increasing

373 degree of substitution. To link these two parameters, the grams of water contained in

374 each bread product per gram of DMf (g Water of bread / g DMf) were calculated. The

375 results indicated that although there was less dry matter in highly substituted flour,

376 water retention per gram rose when the degree of substitution was higher. Thus this last

377 parameter was estimated as a % in relation to the control (%gWater / g DMf increment).

378 The 15% mixture contained 80.9% ore water per gram of dry matter of flour mixture

379 than the control flour. It was, followed by the 10% mixture with 64.7% and the 5%

380 mixture with 26.4%. The contribution of chia to water retention in the matrix allowed

381 baking loss to lower with the same g of water/ g of dry matter ratio of the control

382 sample.

383

384 3.3.2 Texture, mass loss and water activity of bread

385 End product package evolution over time was analyzed. All bread products were firstly

386 fermented until tAc to evaluate differences with the control at the same degree of dough

387 development. Table 4 shows the results of the mass loss and texture parameters of TPA

388 at four different time points: days 0, 1, 3 and 7.

389 On day 0, hardness did not present any important difference between the control and the

390 chia mixtures, nor on day 1. On day 3, high levels of hardness were observed for them

391 all (more than 1400 g), where the highest value went to the control compared to the chia

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392 mixtures. After seven days, the hardness of the control increased to reach values of

393 around 2600g, while the values of the chia mixtures were maintained from day 3 with

394 no statistical differences. Significant differences were found for springiness between the

395 15% mixture and the other mixtures on day 3, between the 5% mixture and the other

396 mixtures on day 0, and for the 5% and 15% mixtures on day 1. Cohesiveness presented

397 significant differences for the 10% and 15% mixtures, principally on day 0. The

398 chewiness of the 5% mixture obtained significantly lower values compared to the

399 control for days 3 and 7, and the 10% and 15% mixtures presented a significantly high

400 value on day 1 compared to the control. However, significant differences with lower

401 values compared to the control on days 5 and 7 were observed. Obviously the fact that

402 all the samples presented textural properties on day 3 is unfavorable for consumption

403 from an organoleptic point of view. However, the observation made until day 7 allowed

404 us to note the effect of chia under extreme conditions.

405 Mass loss also differed between mixtures and control. From day 1 onward, the control

406 lost the largest amount of water than the chia mixtures. Thus the control presented a

407 mass loss of 2% on day 7 compared to the mass on day 0, while the chia 5%, 10% and

408 15% mixtures lost 0.7%, 0.6% and 0.5% respectively. A significant effect for the

409 “storage day” main effect was observed for all the variables, except springiness. The

410 “degree of substitution” main effect also had a significant effect, but only for hardness

411 and aw. The interactions between the main effects with a significant effect were

412 observed in hardness, cohesiveness and aw, although with a lower F-ratio compared to

413 the main effects.

414

415 This effect could also derive from water-retention capacity of chia mucilage. Water in

416 the bread matrix was better maintained in the mucilage structures, which made its

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417 outward leak difficult (Capitani et al. 2012). Furthermore, these high levels of moisture

418 could allow to make water available to delay starch retrogadation and could, therefore,

419 influence texture features. This could explain the differences among hardness,

420 gumminess and chewiness throughout storage, and would agree with Bárcenas et al.

421 (2005), whose results concluded that the incorporation of hydrocolloids into bread

422 dough improves the crumb texture profile by reducing crumb hardness.

423 The moisture retention property could also be counterproductive from the standpoint of

424 conservation features. So a decision was made to study this water availability with a

425 water activity analysis. This parameter was studied because the sole determination of

426 moisture content does not inform about the nature of water; that is, whether it is bound,

427 free, inert or occluded (Mathlouthi, 2001). This is a critical factor that affects a

428 product’s shelf life, which controls food behavior during processing and storage (Anese

429 et al. 1996; Yang & Paulson. 2000). Figure 2C shows the tendencies of the evolution of

430 aw and Xw during the storage time of mixtures.

431 From day 0 to 7, the aw values showed marked differences between the control and chia

432 mixtures. On day 0, the control had a higher aw than the rest of the samples, which had

433 between 5.8% and 13.1% less Xw (Table 3). The reduction of aw for the control between

434 the study time (days) was also more intense. The chia mixtures underwent some

435 considerably slighter changes in this parameter throughout storage time. Finally,

436 moisture reduction in the control brought about a sharper drop in the aw values than the

437 chia mixtures.

438 The most interesting phenomenon noted during storage was the property of chia to

439 maintain the aw levels close to an initial value despite the large amount of water they

440 retained in the matrix. The results agree with previously reported studies by Muñoz et

441 al. (2012), who observed the chia mucilage properties of increasing the linked water in

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442 matrix to further reduce the availability of it being used by other reactions, which could

443 prolong storage time and, therefore, the shelf life.

444

445 4. Conclusions

446 The parameters of the different bread-making phases studied herein were modified by

447 substituting wheat flour with chia seed flour. Those modifications can be associated

448 with the effect of the hydrophilic compounds of chia, which has good water-holding

449 capacity, and can develop stable hydrocolloids and improve the gluten net. These

450 properties imply increased viscosity, mainly for 10% and 15% mixtures, and can amend

451 the other pasting properties of flours. In the same way, the kinetics of dough

452 fermentation was modified to cut the time to reach the control’s maximum volume to

453 40%. Bread properties also were modified and less water was lost during both the

454 baking process and storage. Chia also had effect on degree of water activity, which

455 remained the same despite containing a larger amount of crumb moisture. Texture

456 presented the least influenced properties, and retardation of hardness at prolonged times

457 was mainly observed. The results concluded that the degrees of substitution, up to 15%,

458 could be used to improve not only nutritional provision, but also the technological

459 properties of wheat flours. More studies are needed to evaluate consumers’ acceptance

460 of these degrees of substitution and the properties of each fraction of chia seed

461 components.

462 5. Acknowledgements

463 We wish to thank the Polytechnic University of Valencia and the Generalitat Valenciana

464 for the financial support they provided through the Projects PAID-05-011-2870 and

465 GVPRE/2008/170, respectively.

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