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Accepted Manuscript

Impact of Chlorella vulgaris on the rheology of wheat flour dough and bread texture

C. Graça, P. Fradinho, I. Sousa, A. Raymundo

PII: S0023-6438(17)30843-5
DOI: 10.1016/j.lwt.2017.11.024
Reference: YFSTL 6653

To appear in: LWT - Food Science and Technology

Received Date: 20 July 2017


Revised Date: 3 October 2017
Accepted Date: 11 November 2017

Please cite this article as: Graça, C., Fradinho, P., Sousa, I., Raymundo, A., Impact of Chlorella vulgaris
on the rheology of wheat flour dough and bread texture, LWT - Food Science and Technology (2017),
doi: 10.1016/j.lwt.2017.11.024.

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2 Impact of Chlorella vulgaris on the rheology of wheat flour dough and


3 bread texture

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5 Graça, C., Fradinho, P., Sousa, I. and Raymundo, A.*

6 Universidade de Lisboa, Instituto Superior de Agronomia,

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7 LEAF (Linking Landscape Environment Agriculture and Food) Research Center.

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8 Tapada da Ajuda, 1349-017 Lisboa, Portugal

9 *Corresponding author. Tel: 351962943788

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10 E-mail address: anabraymundo@isa.ulisboa.pt
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13 Abstract

14 Modern foods lead to healthier, cheaper and more convenient products to increasingly demanding

15 consumers. Microalgae are an enormous biological resource, representing one of the most

16 promising sources for new products and applications and can be used to enhance the nutritional

17 and technological value of food products. The enrichment of bread with microalgae biomass is a

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18 great challenge resulting from its impact on the development of the gluten structure. The addition

19 of Chlorella vulgaris in a wheat flour dough was studied, to evaluate its influence on the dough

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20 rheology and bread texture. Microalgae contents from 1.0 to 5.0 g per 100 g of wheat flour were

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21 tested and it was observed that up to 3.0 g of microalgae biomass addition, a positive impact on

22 dough rheology and viscoelastic characteristics, with strengthening of the gluten network, was

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23 observed. For higher microalgae content, a negative effect on dough rheology, bread texture and
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24 flavor was noticed and an acceleration on bread aging was relevant. No impact on the kinetics of

25 yeast fermentation, neither on the time required for fermentation, was induced by the biomass
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26 addition.

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28 Key words: Chlorella vulgaris; dough; bread; rheology; texture


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30 1. Introduction

31 The search for new healthy food ingredients is a strategy to overcome the food shortages expected

32 for the next decades. Microalgae are at the bottom of the food chain and can be considered an

33 interesting source of nutrients and bioactive compounds. The production of food products enriched

34 with microalgae has increasingly gaining attention, as people become more aware of the relation

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35 between diet and health (Roohinejad et al., 2017).

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36 Spirulina maxima, Chlorella vulgaris, Haematococcus pluvialis, Diacronema vlkianum, and

37 Isochrysis galbana are some of the most interesting algae with potential bioactive properties,

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38 revealing a well-balanced chemical composition as well as a source of highly valuable molecules,

39 such as polyunsaturated fatty acids, pigments (e.g. carotenoids, chlorophylls), sterols, vitamins,

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40 hydrocolloids and other biologically active compounds (Batista, Gouveia, Bandarra, Franco, &
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41 Raymundo, 2013). All of them present specific functional and technological properties and recent

42 studies also revealed antimicrobial potential against pathogenic and spoilage microorganisms in
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43 food, namely in bread (Pina-Pérez, Rivas, Martínez, & Rodrigo, 2017). Spirulina maxima and

44 Chlorella vulgaris assume special relevance to be used as food ingredients since they are allowed
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45 for that propose by the European Food Safety Authority.


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46 Several traditional products, such as pasta (Fradique et al., 2010), biscuits (Gouveia, Batista,

47 Miranda, Empis, & Raymundo, 2007; Gouveia, et al., 2008b), puddings/gelled desserts (Batista,
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48 Gouveia, Nunes, Franco, & Raymundo, 2008) and mayonnaises/salad dressings (Raymundo,
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49 Gouveia, Batista, Empis, & Sousa, 2005 ; Gouveia, Batista, Raymundo, Sousa, & Empis, 2006)
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50 have been developed with the incorporation of microalgae in their formulation, and expectations

51 about this practice are promising for the food industry (Pina-Pérez, Rivas, Martínez, & Rodrigo,

52 2017). However, few studies have been done on the incorporation of microalgae in bread, despite

53 its high interest as a staple food with great importance in human nutrition. It is a source of

54 carbohydrates, proteins, dietary fibre, vitamins, micronutrients and antioxidants, being appreciated

55 for its taste, versatility, convenience, texture and appearance (Rubel, Pérez, Manrique, &

56 Genovese, 2015).

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57 In fact, the incorporation of new ingredients into a dough matrix is always a significant

58 technological challenge. The unique bread-making properties of wheat flour can be mainly

59 attributed to the ability of its gluten proteins to form a viscoelastic network when mixed with water.

60 Several studies suggested that the weakening of this network, due to the presence of foreign

61 proteins on wheat flour dough, was the result of a dilution of the gluten structure, that resulted in

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62 lower loaf volume and subsequently a negative effect on other quality attributes, such as crumb

63 grain and tenderness (Belitz, Kieffer, Seilmeir, & Wieser, 1986; Dervas, Doxastakis, Hadjisavva-

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64 Zinoviadi, & Triantafillakos, 1999; Mohammed, Ahmed, & Senge, 2012). A similar effect was also

65 reported for the addition of soluble dietary fibre (inulin, pectin and maltodextrins) to wheat flour

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66 dough for bread-making (Arufe et al., 2017; Sirbu & Arghire, 2017; Noort, Mattila, Katina, & Willem

67 van der Kamp, 2017), where the negative impact of fibre addition on dough rheology and bread

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texture was evidenced and a critical dosage should be establish to ensure the acceptable dough
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69 rheological behaviour and consequent quality of the enriched bread.
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70 The negative effect resulted from the incorporation of non-traditional ingredients to the dough

71 matrix, related to the disturbance of the dough structure. Dough has a foam-like structure,
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72 consisting of a dispersion of discrete gas cells in a continuous starch–protein matrix that are
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73 incorporated by the occlusion of air during mixing, and later expand as carbon dioxide (CO2) is

74 produced by yeast fermentation (Gan, Ellis, & Schofield, 1995). Bread dough shows viscoelastic
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75 behaviour, which is mainly attributed to gluten, the major protein source in wheat flour

76 (Mirsaeedghazi, Emam-Djomeh, & Mousavi, 2008) and has a major impact on bread quality.
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77 Disturbing the gluten network by adding other ingredients such as protein or fibres will affect
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78 physical and rheological properties of the wheat flour dough and its final products. A similar effect

79 is expected for the microalgae addition, considering their composition, namely the high contents of

80 protein (Batista et al., 2013).

81 Physical dough testing devices have been extensively applied to evaluate the bread making

82 potential and performance characteristics of formulations enriched with different types of

83 ingredients (Arufe et al., 2017; Rebellato et al., 2017; Sirbu & Arghire, 2017). Information about

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84 rheological properties of dough will be useful for predicting the potential application of the

85 microalgae and also the quality of the produced bread. The quality of wheat flour bread is

86 determined by the rheological behaviour of dough (Serpil, Kevser, Bengihan, & Hamit, 2008) and is

87 strongly related to bread characteristics and consumer acceptance (Sirbu & Arghire, 2017).

88 The farinograph is one of the rheological tests that was invented in the 1930s, as the first of the

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89 physical dough testing instruments (Janssen, Van Vliet, & Vereijken, 1996) and it is the most

90 popular of the empirical rheological instruments. It is widely used in baking industry to determine

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91 flour quality and relates to the desired end product and its manufacturing process. Another reliable

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92 empirical method, suitable for wheat flour quality testing, is the alveograph, where a biaxial

93 extension is applied during dough bubble inflation, from the process of dough stretching. The

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94 alveograph has been used to evaluate bread making quality of wheat flours (Duyvejonck, Lagrain,
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95 Dornez, Delcour, & Courtin, 2012) through the extensibility (L) and tenacity (P) or resistance to

96 extensibility parameters. From the side of the fundamental rheology testing dynamic oscillatory
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97 measurements with small amplitude are commonly useful to obtain information on the viscoelastic

98 properties of the food materials. Nevertheless, few work reported on the use of these
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99 methodologies to evaluate the dough rheology (Lee & Campanella, 2013; Verheyen, Jekle, &
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100 Becker, 2014).

101 The aim of the present work was to evaluate the impact of the Chlorella vulgaris addition on wheat
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102 flour dough and the respective impact on bread texture and aging kinetics. Different contents of

103 microalgae were added to wheat flour and the impact on dough rheology was measured by the
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104 farinograph and alveograph as well as using small amplitude oscillatory measurements. The
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105 breads with the same levels of microalgae incorporation were prepared and its initial texture,

106 texture decay over time, and appearance was evaluated to establish storage time and the

107 maximum content of Chlorella vulgaris with potential application in market terms.

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109

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110 2. Experimental

111 2.1. Raw materials

112 Bread was prepared using commercial wheat flour - WF (Espiga T65) with 9.1 g/100 g protein and

113 1.6 g/100 g ash. Commercial white crystalline saccharose (Sidul), sea salt, fresh yeast (Levital),

114 SSL-E481- sodium stearoyl-2 lactylate (Puratos). Chlorella vulgaris (Cv) powder (Iswari by Allma -

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115 Portugal) was used, with the following nutritional composition: 60.7 g/100 g protein, 12.4 g/100g

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116 fiber, 2.3 g/100 g lipids, 13.8 g/100 g carbohydrates, and 0.2 g/100 g salt. Presence of vitamin

117 B12, 220 µg/100 g (Cobalamina), iron, 120 mg/100 g and chlorophyll, 2183 mg/100 g was also

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118 referred in the information available on package label.

119 2.2. Bread doughs preparation

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120 A control dough, without Chlorella vulgaris addition, was prepared using the following composition

121 for 100 g of dough: 59.3 g wheat flour, 1.0 g salt, 0.3 g SSL (E481), 2.35 g of fresh yeast, 0.6 g of
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122 sugar and 36.45 g of water, a quantity that was determined according to the farinograph tests. The

123 breads enriched with Cv, were prepared considering incorporations of 1.0, 2.0, 3.0, 4.0 and 5.0 g
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124 Cv /100 g WF of the wheat flour. As before, water content was adjusted according to the
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125 farinograph measurements.

126 Six formulations were prepared by mixing the solid ingredients with water, following the current
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127 procedure for bread production: Bread dough preparation was performed in a thermo-processor

128 (Bimby-Vorwerk, Cloyes-sur-le-Loir, France). First, the fresh yeast was activated in warm water on
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129 the processor cup, during 30 s at position 3. The rest of the solid ingredients were added and
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130 homogenized during 60 s at position 6, and the kneading was carried out during 120 s. Bread

131 dough was placed in a rectangular bread container (5.0 cm x 20.0 cm x 8.5 cm), previously oiled

132 and floured, following up the fermentation during 60 min at 37 °C (optimal yeast activity

133 time/temperature, previously optimized), in an electric oven (Ariana). Breads were then baked in

134 the oven at 160 °C during 30 min. After cooling, the breads were stored in a plastic bag, at room

135 temperature, in a cupboard protected from light.

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136 2.3. Dough rheological characteristics

137 Farinograph characteristics were determined according to AACC International Method 54-21.02.

138 The following parameters were determined in a farinograph equipped with a 300 g mixing bowl

139 (Brabender, Duisburg, Germany): water absorption, i.e. percentage of water required to yield

140 dough consistency of 500 BU (Brabender Units), dough development time (DDT) or time to reach

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141 maximum consistency, stability (time at a consistency of 500 BU) and degree of softening

142 (difference, in BU, between 500 and the medium line of the curve 12 min after development time).

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143 For each sample three measurements were performed.

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144 Alveograph tests were performed using AlveoLab (Chopin Technologies, Villeneuve-La-Garenne,

145 France) according to the AACC International Method 54-30.02. The following alveograph

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146 parameters were automatically recorded by the respective computer software program: P - tenacity
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147 or resistance to extension (the pressure inside the dough bubble in mmH2O) as the height of the

148 peak in the alveogram and L - dough extensibility (in mm), as the width of the curve. The curve
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149 configuration ratio (P/L), that express the equilibrium between the tenacity and the extensibility. For

150 each sample five measurements were performed.


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151 Viscoelastic behaviour of dough during fermentation was accessed using a controlled stress
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152 rheometer (Haake Mars III – Thermo Scientific, Karlsruhe, Germany), with an UTC - Peltier system

153 to control temperature, using a serrated parallel-plate sensor system (PP20) and 2 mm gap
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154 between plates, to overcome the slip effect. The gap was previously studied and fixed at 2 mm to
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155 reduce the interference of normal stresses during the fermentation process, in agreement with the
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156 conditions previously optimised by Verheyen, Jektl, & Becker (2014).

157 Dough fermentation in situ, on the rheometer plates, was carried out to evaluate the impact of Cv

158 addition on fermentation time, by applying a time sweep according to the following conditions: initial

159 temperature of the rheometer at 5 ºC, during 120 s, to avoid the start of the fermentation before the

160 desired time, increasing the temperature up to 37 ºC (optimal temperature of the yeast), and follow

161 up of the fermentation, by the change on storage modulus (G’), at 1 Hz of frequency and constant

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162 shear stress, within the linear viscoelastic region, previously determined by a stress sweep test.

163 This test was performed during 5400 s to obtain an equilibrium of the viscoelastic functions.

164 Frequency sweep, to evaluate the effect of the Cv added on dough structure and the evolution of

165 the dough after fermentation time, the storage (G´) and loss (G´´) moduli were obtained ranging the

166 frequency from 0.001 Hz to 10.0 Hz, at a constant shear stress within the linear viscoelastic region

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167 of each sample. All determinations were repeated at least two times to ensure that reproducible

168 results were obtained.

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169 2.4. Bread Texture

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170 Texture characterisation of bread was performed using a texturometer TA-XTplus (Stable

171 MicroSystems, Surrey, UK), applying a texture profile analysis (TPA) in penetration mode, which

172

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consists of compressing a piece of food two times in a reciprocating motion that imitates the action
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173 of the jaw (Bourne, 2002). Each bread sample was cut into slices 20 mm high of a 120 x 100 mm

174 rectangular shape and rested for 15 min before testing. An acrylic cylindrical probe with 10 mm
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175 diameter pierced 5 mm of the sample at 1 mm / s of crosshead speed, with a load cell of 5 kg.

176 Comparison of bread texture with different Chlorella vulgaris content was performed in terms of
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177 firmness, which was considered the most representative parameter from the texture profile.
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178 Firmness (N) of the bread crumb was considered as the maximum resistance to the penetration of

179 the probe and was calculated as the height of the first force peak. The evaluation of firmness
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180 during the storage time was also performed and the aging kinetic of the bread was described as a
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181 function of biomass incorporation. To discuss the variation of bread texture, the bread moisture

182 was also determined, according to the standard method (AACC 44-15.02).
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183 2.5. Statistical Analysis

184 The experimental results were statistically analyzed by determining the average value, standard

185 deviation, and the significance level was set at 95 %, for each parameter evaluated.

186 Statistical analysis was made by applying variance analysis, the one factor (ANOVA), and multiple

187 comparisons (Tukey test).

188
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189 3. Results and discussion
190 3.1 Effect of Chlorella vulgaris content on farinograph parameters

191 The effect of Cv addition on the farinographic characteristics of the wheat flour dough can be seen

192 in Fig. 1. Water absorption (Fig. 1A) steadily increased up to 3.0 g Cv /100 g WF addition due to

193 the presence of extra-protein from the microalgae. After this concentration, further addition of Cv

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194 results in a slight, but significant (p < 0.05) reduction of water absorption. These results suggest

195 that the Cv cells, together with the gluten proteins, up to a certain level, i.e. 3.0 g Cv /100g WF,

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196 required more water to achieve the standard dough consistency (500 BU). For additions above this

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197 value, aggregation between Cv cells should reduce the water absorption capacity. Similar results

198 were observed by Dervas, Doxastakis, Hadjisavva-Zinoviadi, & Triantafillakos (1999), Mohammed,

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199 Ahmed, & Senge (2012) and Turfani, Narducci, Durazzo, Galli & Carcea (2017), with addition of
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200 legume proteins to wheat flour. An increase in water absorption resulting from the incorporation of

201 vegetable protein or other protein sources, concentrates or isolates, to wheat flour, has been
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202 attributed to the water absorbing capacity of these proteins, and their ability to compete for water

203 with other constituents in the dough system, resulting in doughs which exhibit increased
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204 farinograph water absorption (Dervas, Doxastakis, Hadjisavva-Zinoviadi, & Triantafillakos, 1999;
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205 Doxastakis, Zafiriadis, Irakli, & Tananaki, 2002).The quantity of added water is considered to be

206 very important for the distribution of the dough materials, their hydration and the gluten protein
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207 network development, as well as to increase the production yield. The time required for the dough

208 development (DDT), or time necessary to reach 500 BU of dough consistency, was almost
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209 constant until 3.0 g Cv /100 g WF and slightly increased (p < 0.05) for higher concentrations of Cv
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210 (Fig. 1B). This was followed by the dough stability index (Fig. 1B) which, over 3.0 g Cv /100g WF

211 addition showed a sudden significant (p < 0.05) decay to about half of the initial value (from 8.7 to

212 4.6). This means that from 3.0 g Cv /100g WF over of microalgae incorporation, the structure of the

213 wheat dough is somehow disrupted, which resulted in lower dough strength and loaf volume with a

214 negative effect on the quality attributes of bread (Fig. 2), such as less alveoli on crumb grain, and

215 on the crust shape on the top of the loaf, as previously reported for the addition of other protein

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216 sources to bread (Knorr & Betschart, 1978; Mi´s, Grundas, Dziki, & Laskowski, 2012). Similar

217 results have been previously reported by other authors, for the addition of vegetable proteins

218 (Mohammed, Ahmed, & Senge, 2012; Lazo-Velez, Chuck-Hernandez,& Serna-Saldívar, 2015) or

219 hydrocolloids (Doxastakis, Zafiriadis, Irakli, & Tananaki, 2002) on bread formulations. Dough

220 softening degree (Fig. 1C) shows the same pattern, i.e., a sudden increase (p < 0.05) up to twice

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221 the initial value at over 3.0 g Cv /100g WF addition. Similar changes of this parameter were

222 reported for blends with cowpea flour and chickpea flour by Fernandez & Berry (1989), and

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223 Sharma, Bajwa, & Nagi (1999).

224 Wheat flour doughs with different Cv concentrations from 0 to 5.0 g/100g of wheat flour were

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225 formulated, using the water absorption values from the farinographic analyses, to be baked and the

226 resulting breads can be seen on Fig. 2. The impact of the Chlorella vulgaris addition on the bread

227
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volume, as well as on the alveoli structure of the crumb and crust color, are noticeable. The Cv
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228 bread internal structure revealed an increase on the size of the gas cells, compared to the control

229 bread, indicating that the incorporation of microalgae disturbs the dough internal structures. Again,
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230 for additions of over 3.0 g Cv /100g WF, the most relevant effect is observed on the crumb
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231 structure, with a coalescence of alveoli in a less homogeneous structure with big holes. This
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232 important feature, induced by the microalgae addition, can be attributed to the decrease of dough

233 stability, due to the dilution effect of the gluten matrix and phase separation of the excess of Cv
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234 particles with possible disruption of the gluten network. Dervas, Doxastakis, Hadjisavva-Zinoviadi,

235 & Triantafillakos (1999) and Doxastakis, Zafiriadis, Irakli, & Tananaki (2002), also reported a loss
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236 on bread crumb structure with increasing levels of lupin or soy flour and attributed this decrease to
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237 the dilution of the wheat gluten by the legume proteins. According to Mohammed, Ahmed, & Senge

238 (2012) this decrease in bread crumb quality was justified by the combined effects of gluten dilution

239 and mechanical disruption of the gluten network structure by the chickpea proteins.

240 It is also important to note that the incorporation of Cv transmited a marine taste, more intense for

241 higher concentrations, that can be considered a depreciative attribute as it was found in a previous

242 work about the incorporation of Cv on pasta (Fradique et al., 2010).

243

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244 3.2 Influence of Chlorella vulgaris on the alveograph characteristics

245 The effect of Cv addition on the alveograph parameters of wheat flour dough is shown in Fig.. 3.

246 Dough resistance to deformation - tenacity (P) is a predictor of the ability of the dough to retain gas

247 (Rosel, Rojas, & Barber, 2001). This parameter shows a tendency to decrease with the microalgae

248 addition, being similar to the control for 1 g Cv /100 g of WF, slightly reduced for the 2 and 3 g

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249 Cv/100 g WF and subsequently reduced for 4 and 5 g Cv/100 g WF additions, as can be seen from

250 Fig. 3. An opposite effect was observed on the extensibility (L), an indicator of the handling

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251 characteristics of the dough, that was greatly increased with Cv addition, to almost double of the

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252 control dough extensibility. Similar results were observed by Mis, Grundas, Dziki, & Laskowski

253 (2012), with additions of the carob fiber and oat wholemeal to wheat flour. Wheat proteins include

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254 albumins, globulins, gliadins and glutenins but only the last two participate in the formation of a
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255 continuous viscoelastic network, i.e., the gluten network (Shewry, Halforf, Belton, & Tatham, 2002).

256 Glutenin polymer chains provide strength and elasticity to bread dough development, whereas
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257 globular proteins, such as gliadins, contribute to the viscosity of dough. Gliadins interact with the

258 glutenin polymers via non-covalent hydrophobic interactions and hydrogen bonding being the
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259 structure of both proteins crucial to the breadmaking process (Sivam, Sun-Waterhouse, Perera, &
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260 Waterhouse, 2012). The incorporation of Cv in wheat flour seems to favor the action of gliadin,

261 contributing to more extensible dough (L), as it can see in Fig. 3A. In contrast, it appears to have
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262 an adverse effect on the glutenin action, verifying a significant decrease in the dough tenacity (P)

263 at over 3 g Cv/100 g WF. Therefore, it can be considered that the Cv proteins disturb the gluten
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264 matrix of the bread dough.


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265 As a result of the microalgae action on both dough resistance and dough extensibility, the P/L ratio,

266 which gives information about the elastic resistance and extensibility balance of a wheat flour

267 dough, was reduced in all doughs (Fig. 3). Similar results were observed by Lazo-Velez, Chuck-

268 Hernandez,& Serna-Saldívar (2015), with soybean proteins in yeast-leavened breads. An ideal P/L

269 ratio for baking varies between 0.5 and 1.20 (Arozanena, Iguaz, Noriega, Bobo & Virseda, 2012).

270 The control dough has a 1.04 P/L ratio, which is classified as balanced and suitable wheat flour for

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271 bread production. According to the results obtained (Fig. 3B), the blends within this range of P/L

272 are the ones with 1.0, 2.0 and 3.0 g Cv /100 g WF. Blends with over 3.0 g Cv /100 g WF showed a

273 reduction on P/L ratio and are not suitable for bread making. Nevertheless, these mixtures can be

274 used for other higher valued applications, such as cookies and biscuits.

275 Comparing these findings with the works performed by Dervas, Doxastakis, Hadjisavva-Zinoviadi,

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276 & Triantafillakos (1999), Rosel, Rojas, & Barber (2001) and Mohammed, Ahmed, & Senge (2012),

277 related to the addition of chickpea and lupin, a similar effect was observed, and the reduction of

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278 P/L was attributed to the reduction of gluten strength to a dilution effect of the gluten proteins.

279

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280 3.3 . Fermentation behaviour of doughs containing Chlorella vulgaris

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281 The changes of dough viscoelastic properties during fermentation time were monitored by a
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282 controlled stress rheometer, following the storage modulus variation (G’). From Fig. 4 (A), a

283 considerable decrease of G’ up to 3600 s, was observed for all the doughs tested. After this time,
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284 the viscoelastic parameter remained almost constant in all cases. The first stage, about 1h,

285 corresponds to the high fermentation activity, with CO2 production, which impacts on the dough
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286 structure, with deformation induced by the growing of the gas cells. On the next stage, the
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287 fermentation process reached a steady state. From these results, it can also be said that the Cv

288 addition does not markedly influence the fermentation kinetics, neither the time required for
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289 fermentation.

290 The frequency sweep performed after in-situ rheometer fermentation was performed to evaluate
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291 the impact of Cv microalgae addition on dough structure after fermentation. The mechanical
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292 spectra presented in Fig. 4 (B and C), suggest that the microalgae have different effects on dough

293 properties, according to the Cv content:

294 i) until 3.0 g Cv /100 g WF, higher G´ values, are an indication of a possible strengthening

295 effect of the dough structure, probably due to a reinforcement of the protein matrix,

296 resulted from the addition of the microalgae with a high protein content;

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297 ii) from 3.0 g Cv /100 g WF over of microalgae, the strengthening effect induced by

298 microalgae is replaced by a destructuring effect probably due to a phase separation of

299 the added biomass with disruption of the gluten matrix.

300 All the results obtained, i.e., from the farinograph, alveograph and rheometer are in agreement and

301 a protein network model, dependent on microalgae concentration, can be proposed: up to 3.0 g Cv

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302 /100 g WF an increase of the viscoelastic parameters is observed and this can be attributed to the

303 reinforcement of the viscoelastic protein network with the Cv particles evenly distributed within the

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304 gluten matrix, according to the swollen network model earlier proposed by Morris (1998).

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305 The effect of Chlorella vulgaris addition over 3.0 g/100 g WF on dough structure characteristics can

306 be attributed to the depletion flocculation, or phase separation phenomena (Morris, 1998), as it

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307 was also previously reported by Nunes, Raymundo, & Sousa (2006) for other complex gelling
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308 systems, where the particles segregate each other and phase separate with an antagonistic effect

309 in the dough network structure.


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311 3.4. Bread texture and aging kinetic


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312 The texture of the bread produced with different contents of Chlorella vulgaris was evaluated
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313 through a texture profile analysis (TPA) and it was verified that firmness was the parameter that
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314 efficiently discriminate the different formulations, since the other TPA parameters were not able to

315 discriminate the different formulations, showing no significant differences for all the samples. The
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316 evaluation of firmness during the storage time (72 h) was also performed, to observe the effect of
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317 microalgae addition on the kinetic of bread aging (Fig. 5). A similar procedure was followed by

318 Noort, Mattila, Katina, & Willem van der Kamp (2017), to study the impact of different processing

319 conditions on bread quality.

320 From Fig. 5 it can be observed that, for all the microalgae content, a positive linear relation is

321 observed between bread firmness at storage time, according to a linear equation (R > 0.9):

322 Firmness = A * time + B Eq. (1)

323 where A can be considered the aging velocity and B the initial firmness.

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324 It can be noticed that for the time zero, initial firmness (B) is not substantially affected by microalga

325 concentration (p > 0.05). Nevertheless, the results follow the reverse tendency of the dough water

326 absorption (Fig. 1 A), i.e., less firm with more absorbed water. In terms of bread moisture, a

327 significant (p<0.05) increase was observed: 37 up to 46 g water /100 g bread, when Chlorella

328 content increased from 2 up to 4 g/100 g flour. However, a noticeable increase of the bread aging

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329 velocity (A) with Cv content was observed – from 0.0894 (N/h) to 0.1761 (N/h), representing an

330 increase of 97 %. Nevertheless, for 1.0 and 2.0 g Cv/100 g wheat flour, only a slight variation of

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331 aging velocity was observed. This behavior has an important impact for the commercial purpose

332 and it should be explained by changes in the moisture distribution in the system, as it was early

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333 reported by Noort, Mattila, Katina, & Willem van der Kamp (2017). As it was observed in Fig. 1, a

334 markedly increase of water absorption with biomass addition was observed for 4.0 and 5.0 g

335
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Cv/100g WF, this competition for water absorption by the Cv components should cause an
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336 acceleration in the aging of bread, expressed in terms of firmness increase within short times.

337
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338 4. Conclusions
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339 Given the present results, based on empirical and fundamental experiments, it was found that the

340 addition of Chlorella vulgaris had an impact on the rheological features of the bread dough,
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341 depending on the concentration level: up to 3.0 g Cv/100 g WF, a positive impact on viscoelastic

characteristics, with strengthening of the gluten network was observed. This behavior was also
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343 supported by the increase of farinograph water absorption, presumably due to greater water
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344 requirement to hydrate Cv proteins. At this concentration range, the P/L ratios are within the range
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345 of values appropriate for baking. No impact on the kinetics of yeast fermentation, neither on the

346 time required for fermentation was induced by the biomass addition. For this range of Cv content,

347 the bread firmness is close to the reference wheat bread, and a similar aging kinetic was observed.

348 From the commercial purpose it is also important to refer that overall, up to 3.0 g Cv/100 g WF

349 addition, resulted in breads with an interesting appearance, but with higher Cv contents a markedly

350 depreciation of the global aspect was noticed.

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351 The use of Chlorella vulgaris as a food ingredient is a promising way to enrich bread in bioactive

352 compounds, resulting from its well-known composition. A technological limitation for biomass

353 incorporations higher than 3.0 g Cv/100 g WF was noticed. The addition of microalgae at

354 concentrations above 3.0 g Cv/100 g WF originated doughs with lower strength, lower tenacity

355 (elasticity) and more extensible and consequently with limiting P/L ratios for bread application.

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356 However, in such cases the blends can still be exploited for the production of other bakery

357 products such as biscuits, which requires lower P/L values.

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360

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361 Acknowledgment

362 This work was supported by Portuguese Foundation for Science and Technology (FCT) through
363 the research unit UID/AGR/04129/2013 (LEAF).

364

365

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366

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367 References

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476 Turfani, V., Narducci, V., Durazzo,A., Galli, V., & Carcea, M. (2017). Technological, nutritional and
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478 and Technology, 78, 361-366.
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481 Figure Captions
482
483 Fig. 1. Effect of Chlorella vulgaris addition on the farinograph characteristics of the wheat dough: A

484 - Water absorption; B - Time required for the dough development (DDT) and Dough stability; C -

485 Dough softening degree.

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486

487 Fig. 2. Variation of the bread dough volume with different concentrations of Chlorella vulgaris, after

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488 1 hour of fermentation at 37 ºC (A) and farinograms obtained by farinograph analysis for each flour

489 blend in study: Control Bread (without Cv) and bread produced with 1, 2, 3, 4 and 5 g Cv/100 g WF

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490 (B).

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492 Fig.3. Effects of Chlorella vulgaris addition on wheat flour on the alveograph parameters: A) P -
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493 dough tenacity; L - dough extensibility and B) P/L ratio;

494 0 g/100 g WF; 1 g/100 g WF; 2 g/100 g WF; 3 g/100 g WF; 4 g/100 g WF;
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495 5 g/100 g WF; The black horizontal bars indicate the range of P/L for breadmaking. Different
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496 letters mean statistically different values (p < 0.05).

497
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498 Fig. 4. A) Changes in storage moduli (G`) on bread dough, during fermentations: 0 g/100 g

499 WF; 1 g/100 g WF; 2 g/100 g WF; - 3 g/100 g WF ; 4 g/100 g WF; 5 g/100 g WF; and
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500 B) mechanical spectra at 5 ºC - variation of storage (G´- full symbols) and loss (G” – open symbols)

moduli measured by after fermentation stage, comparing the control (without Cv) with 1 , 2 and 3 g
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502 Cv/100 g WF addition: ; 0 g/100 g WF, ; 1 g/100 g WF, ; 2 g/100 g WF, ;


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503 3 g/100 g WF; and C) comparing the control with 4 and 5 g Cv/100 g WF addition: ; 4 g/100

504 g WF, ; 5 g/100 g WF.

505 Fig.5. Variation of firmness (N) over time, for wheat flour breads prepared with different contents of

506 Cv addition and respective linear equations: 0 g/100 g WF; 1 g/100 g WF;

507 2 g/100 g WF; 3 g/100 g WF; 4 g/100 g WF; 5 g/100 g WF.

508

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Figure 1

B
64 10 Stability
A c c a a a a
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Water absortion (g/100g)

63 Development time
d d 8
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Time (min)
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61 b
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58 1

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Chlorella vurgaris ( g/ 100g WF) Chlorella vurgaris ( g/ 100g WF)

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Figure 2

0 g/100 g 1 g/ 100 g 2 g/ 100 g 3 g/100 g 4 g/100 g 5 g/100 g

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0 g/ 100 g

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60

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P - Tenacity (mmH2O)
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1.E+05

1.E+04
G´ (Pa)

1.E+03

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A

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500 1500 2500 3500 4500 5500
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Figure 5

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Firmness (N)

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Firmness (N)

Cv content (g / 100g)
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0 g / 100 g 1 g / 100 g 2 g / 100 g 3 g / 100 g 4 g / 100 g 5 g / 100 g

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F = 0.0894t + 7.4552 F = 0.0702t + 5.9219 F = 0.0947t + 5.1021 F = 0.1616t + 5.4431 F = 0.1541t+ 6.8729 F = 0.1761t + 7.2543
R² = 0.938 R² = 0.9345 R² = 0.997 R² = 0.9588 R² = 0.9493 R² = 0.9684
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Highlights

- Addition of Cv to wheat flour modified the rheological features of the bread dough.

- More than 3 g of Cv / 100 g of WF has a negative impact on bread quality.

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- Up to 3g / 100 g of Cv induces a gluten network reinforcement.

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- Cv can be used to enrich bread with bioactive compounds.

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- The incorporation of Cv induces changes in the aging kinetic of the bread.
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