Accepted Manuscript

Effects of enzymatic hydrolysis on conformational and functional properties of

chickpea protein isolate

Abir Mokni Ghribi, Ines Maklouf Gafsi, Assaâd Sila, Christophe Blecker,
Sabine Danthine, Hamadi Attia, Ali Bougatef, Souhail Besbes

PII: S0308-8146(15)00666-4
DOI: http://dx.doi.org/10.1016/j.foodchem.2015.04.109
Reference: FOCH 17510

To appear in: Food Chemistry

Received Date: 19 February 2015

Revised Date: 22 April 2015
Accepted Date: 23 April 2015

Please cite this article as: Ghribi, A.M., Gafsi, I.M., Sila, A., Blecker, C., Danthine, S., Attia, H., Bougatef, A.,
Besbes, S., Effects of enzymatic hydrolysis on conformational and functional properties of chickpea protein isolate,
Food Chemistry (2015), doi: http://dx.doi.org/10.1016/j.foodchem.2015.04.109

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 1 Effects of enzymatic hydrolysis on conformational and functional

2 properties of chickpea protein isolate

4 Abbreviated running title: Functional properties of chickpea protein

6 Abir MOKNI GHRIBI1, Ines MAKLOUF GAFSI1, Assaâd SILA 2,3

, Christophe

7 BLECKER4, Sabine DANTHINE4, Hamadi ATTIA1, Ali BOUGATEF 2, Souhail BESBES1,*.

1
8 Univers0ité de Sfax, Ecole Nationale d’Ingénieurs de Sfax, Laboratoire Analyse,

9 Valorisation et Sécurité des Aliments route de Soukra, 3038 Sfax, Tunisia.

2
10 Université de Sfax, Ecole Nationale d’Ingénieurs de Sfax, Unité Enzymes et Bioconversion,

11 route de Soukra, 3038 Sfax, Tunisia.

3
12 Institut Régional de Recherche en Agroalimentaire et Biotechnologie : Charles Viollette,

13 EA1026, Equipe ProBioGEM, Université Lille 1, France.

4
14 Université de Liège, Gembloux Agro Bio-Tech, Unité de Technologie des Industries Agro-

15 Alimentaires, passage des Déportés 2, 5030 Gembloux, Belgium.

16 (*) Corresponding authors:

17 Tel.: + 216 54174506; fax: + 216 74675761

18 E-mail:

19 besbes.s@voila.fr (S.Besbes).

1
20 Abtract

21 The impact of enzymatic hydrolysis by Alcalase on the conformational and functional

22 properties of chickpea protein isolate (CPI) was investigated. The physicochemical, interfacial

23 tension and surface characteristics of CPI and their hydrolysates (CPH) according to the

24 degree of hydrolysis (DH) were also determined. These parameters were then related to the

25 changes in the emulsification activity (EAI) and stability (ESI). The enzymatic hydrolysis was

26 found to improve protein recovery and solubility, leading to a reduction in the molecular

27 weight bands with a concomitant increase in the intensity and appearance of protein bands

28 having apparent molecular mass below 20 kDa. The interfacial tension decreased from ~66.5

29 mN m−1 for CPI to ~59.1 mN m−1 for CPH. A similar trend was observed for the surface

30 charge which declined from ‒27.55 mV to ‒16.4 mV for the CPI and CPH, respectively.

31 These changes were found to have a detrimental effect on the EAI and ESI values.

32 Keywords: chickpea protein isolates, enzymatic hydrolysis, conformation, functional

33 properties

2
34 1. Introduction

35 The dynamic surface and adsorption of proteins at interfaces are considered to play an

36 important role in the formation and stabilisation of emulsions or foams. Over the past years,

37 there has been interest not only in understanding the interfacial behaviour of adsorbed

38 proteins but also in elucidating the relationship between the interfacial properties and

39 physicochemical properties of emulsions (McClements, 2004). The physicochemical

40 properties of proteins play an important role in determining their emulsifying capacities. For

41 example, surface hydrophobicity influences the ability of the protein to adsorb to the oil side

42 of the interface, where greater integration typically leads to higher emulsion capacities (Kim,

43 Decker, & McClements, 2005). Surface charge has an effect on the protein solubility within

44 the aqueous phase, where high solubility is desired for providing greater diffusion rates to the

45 interface (Can Karaca, Low, & Nickerson, 2011). Once the viscoelastic film is formed,

46 droplets can assume a positive or negative charge, depending on whether the emulsion pH is

47 below or above the protein’s isoelectric point, respectively. In addition, protein size and

48 flexibility influence the emulsion properties. In fact, small molecular weight proteins diffuse

49 rapidly to the interface and give excellent emulsion-forming abilities. Once at the interface,

50 proteins re-align themselves to position their surface hydrophobic amino acids within the oil

51 phase and their hydrophilic amino acids within the aqueous phase.

52 Proteins are increasingly used to improve the functional properties in food formulations.

53 However, near their isoelectric point (pI), the functional properties of proteins are impaired as

54 in the case of most acidic foods. Enzymatic treatment by controlled proteolysis can enhance

55 their functional properties over a wide range of pH and other processing conditions. Besides,

56 due to its higher specificity, easier control of reaction and minimal formation of by-products,

57 enzymatic hydrolysis has been most widely used to improve the functional and nutritional

58 properties of proteins (Tavano, 2013). Nevertheless, in some cases, the extensive enzymatic

3
59 modification may impair some functional properties of food proteins. Hence, the degree of

60 hydrolysis should be controlled to improve the functional properties of proteins whose

61 solubility is one of the most outstanding functional properties that is improved by the limited

62 hydrolysis. While in some plant protein cases, the foaming and emulsifying properties are

63 improved, in other cases, these properties are altered. Indeed, modification by a limited

64 enzymatic hydrolysis is accompanied by (1) a decrease of molecular weight; (2) an increase

65 of the number of ionisable groups; and (3) exposure of previously concealed hydrophobic

66 groups at the interface (Panyam & Kilara, 1996). These effects can well modify the

67 conformation and structure of proteins, thus changing the solubility, surface characteristics

68 and emulsifying properties. Recently, many researchers have been interested in the effect of

69 enzymatic hydrolysis on the functional properties of plant protein isolates, such as sunflower

70 protein (Martinez, Baeza, Millán, & Pilosof, 2005), soy protein (Yang, Yang, Li, Li, & Jiang,

71 2011) and peanut protein (Zhao, Liu, Zhao, Ren, & Yang, 2011).

72 Chickpea is a rich source of dietary protein (17–22%) and its global production ranks

73 thirty among pulse crops (FAO, 2014). Various biological activities, including antioxidant

74 activity, antifungal activity, antigenic activity, metal-chelating ability and angiotensin I-

75 converting enzyme (ACE) inhibition, have been reported for chickpea protein hydrolysate

76 (Mokni et al., 2015; Yust, Pedroche, Giron-Call, Alaiz, Millán, & Vioque, 2003). Yust,

77 Pedroche, Millán-Linares, Alcaide-Hidalgo, & Millán (2010) have reported that although the

78 partial hydrolysis of chickpea protein isolate (CPI) with immobilised Alcalase is a helpful

79 strategy to improve some functional properties, such as solubility, oil absorption capacity, and

80 foaming capacity and stability, it has poorer emulsifying properties than CPI. However, to the

81 best of our knowledge, there is a lack of supporting data and fundamental knowledge on the

82 interfacial and emulsification properties of CPH. These interfacial and emulsification

83 properties are essential, particularly when the proteins are intended to be used as emulsifiers.

4
84 Because surface dynamic properties of proteins (adsorption rate and viscoelastic

85 characteristic of the adsorbed film) are of great importance during emulsion formation and

86 stabilisation, the overall goal of this research was to study the effect of enzymatic

87 modification of CPI on some functional properties (solubility and emulsifying properties) and

88 to investigate structure‒function relationships associated with the enzymatic hydrolysis. This

89 paper reports on the added value of chickpea hydrolysates (CPH) through their use in food

90 formulation.

5
 91 2. Materials and methods

92 2.1. Reagents

93 The common chemicals and solvents of analytical grade were obtained from different

94 commercial sources. Water, whose resistivity was approximately 18 MΩ, was obtained from a

95 Culligan system (Model 8003; Culligan Company, Rosemont, IL). All other chemicals and

96 reagents used were of analytical grade.

97 2.2. Enzyme

98 The serine protease Alcalase® (Subtilisin Carlsberg), produced from fermentation of

99 Bacillus licheniformis (Novozymes®, Bagsvaerd, Denmark), was used for the production of

100 hydrolysate. Protease activity was determined according to the method of Kembhavi,

101 Kulkarni, and Pant (1993) using casein as a substrate. One unit of protease activity was

102 defined as the amount of enzyme required to liberate 1 µg of tyrosine per min under the used

103 experimental conditions.

104 2.3. Materials

105 The seeds of chickpea cultivar were dehulled and ground to pass through a 0.5-mm

106 sieve to obtain chickpea flour. The samples were stored in sealed plastic bags at −20 °C until

107 further analyses.

108 2.4. Preparation of chickpea protein isolates (CPI)

109 Chickpea flour was defatted by stirring in hexane (1:3 [w/v], flour: hexane) for 4 h. This

110 procedure was repeated two additional times. Protein isolates were prepared from the defatted

111 flour based on the method of Papalamprou, Doxastakis, Biliaderis, and Kiosseoglou (2009)

112 with slight modifications. In brief, 100 g of defatted flour were mixed with water at a 1:10

113 (w/v) ratio. The pH of the resulting suspension was adjusted to 9.00 using 1.0 N NaOH

114 followed by mechanical stirring at 500 rpm for 2 h at room temperature (25 °C). The mixture

115 was then centrifuged at 5000 g at 4 °C for 20 min to collect the supernatant. The resulting

6
116 pellet was re-suspended in water at a ratio of 1:5 (w/v), adjusted to pH 9.00 using 1.0 N

117 NaOH, stirred for 2 h at room temperature, followed by centrifugation (5000 g, 20 min, 4 °C).

118 Supernatants were pooled and adjusted to pH 4.50 with 0.1 M HCl to precipitate the protein.

119 CPI was washed with water, frozen and then stored at – 30 °C until further analyses.

120 2.5. Preparation of chickpea protein hydrolysate (CPH)

121 CPI (500 g) was first suspended in 500 mL distilled water and then cooked at 80 °C for

122 5 min to inactivate endogenous enzymes. Cooked protein samples were then homogenised for

123 about 2 min. The sample was adjusted to optimal pH and temperature for Alcalase® (pH 8.0;

124 50 °C).

125 The protein solutions were allowed to equilibrate for 30 min before the enzyme

126 addition. After equilibrium was reached, the hydrolysis reaction was started by the addition of

127 the enzyme at a 1:1 (U/mg) enzyme/protein ratio. The reaction was conducted at 50 °C and

128 pH 8.0 for 210 min. During the reaction, the pH of the mixture was maintained at the desired

129 value by the continuous addition of NaOH (4 N). After the required digestion time, the

130 enzymatic hydrolysis was stopped by heating the solutions for 20 min at 80 °C to inactivate

131 the enzyme. Protein hydrolysates were then centrifuged at 5000 g for 20 min. Finally, the

132 soluble fractions were freeze-dried at ‒50 °C.

133 2.6. Determination of the degree of hydrolysis (DH)

134 The degree of hydrolysis (DH), defined as the percent ratio of the number of peptide

135 bonds cleaved (h) to the total number of peptide bonds in the studied substrate (htot) was

136 calculated from the amount of NaOH added to keep the pH constant during hydrolysis (Adler-

137 Nissen, 1986) according to the following equation.

138 h B × Nb 1 1
DH (%) = ×100 = × × ×100
htot MP α htot
139 where B is the amount of NaOH consumed (mL) to keep the pH constant during the

140 proteolysis of the substrate. Nb is the normality of the base, MP is the mass (g) of the protein

7
141 (N × 6.25), and α represents the average degree of dissociation of the α-NH2 groups in the

142 protein substrate expressed as:

143

144 where pH and pK are the values at which the proteolysis was conducted. The total number of

145 peptide bonds (htot) in the protein substrate was assumed to be 7.22 mmol/g (Mokni et al.,

146 2015).

147 2.7. Physicochemical properties and amino acids content of CPI and CPH

148 The dry matter (DM) was calculated according to AOAC method 934.01 (1990). The

149 samples were estimated for their ash, fat and protein (N × 6.25) content using the standard

150 methods of analysis. Carbohydrate content was estimated by the difference of mean values,

151 100 ‒ (sum of percentages of moisture, ash, proteins and lipids) (AOAC, 1995).

152 Amino acids were determined by high-performance liquid chromatography (HPLC) (HP

153 1090; Hewlett Packard, Palo Alto, CA) according to the OJEC standard method (OJEC,

154 1998). Amounts of 100 mg of CPI and CPH samples were hydrolysed with 6 N hydrochloric

155 acid in an ampoule containing 0.1% phenol (for protection of tyrosine) for 24 h at 110 °C.

156 After acid hydrolysis, 30 mL of citrate buffer (pH 2.2) were added and the pH was adjusted

157 between 0.5 and 1 with 7.5 N NaOH and pH 2.2 with 1 N NaOH. The obtained sample was

158 diluted to 100 mL with citrate buffer after adding 1 mL of a norleucine solution (50 µM as an

159 internal standard). The sample was filtered through a 0.2-µm nylon filter before being

160 analysed by HPLC. Sulfur amino acids, cystine and methionine were determined after a pre-

161 hydrolysis oxidation with performic acid (OJEC, 1998). The contents of the different

162 recovered amino acids were expressed as g/100 g protein concentrates. The HPLC system

163 (Biochrom Ltd, Cambridge, UK) was equipped with a UV-Vis detector with two wavelengths,

164 440 nm and 570 nm for proline and other amino acids, respectively, and a cation exchange

165 column (200 × 4.6 mm) (Hypersil C18; Interchim, Montluçon, France).

8
166 2.8. Differential scanning calorimetry (DSC) and colour characteristics

167 The thermal properties of CPH were performed according to the method of Abbes et al.

168 (2015) with slight modifications. The experiments were performed on a TA2920 (TA

169 Instruments New Castle, DE) with refrigerated cooling assessory and modulated capability.

170 The cell was calibrated for temperature and heat flow using indium (Tonset: 156.6 °C, ∆H:

171 28.7 J g−1) and eicosane (Tonset: 36.8 °C, ∆H: 247.4 J g−1). Specific heat capacity (Cp) was

172 calibrated using a sapphire. The analysed sample mass was 5 ± 0.25 mg. The pans were

173 hermetically sealed and heated in the calorimeter from 0 to 200 °C at a rate of 5 °C/min. A

174 sealed empty pan was used as a reference.

175 The CIE Lab coordinates (L*, a*, b*) were directly read with spectrophotocolorimeter

176 Mini Scan XETM (HunterLab Inc., Reston, VA). In this coordinate system, the L* value is a

177 measure of lightness, ranging from 0 (black) to +100 (white); the a* value ranges from ‒100

178 (greenness) to +100 (redness) and the b* value ranges from ‒100 (blueness) to +100

179 (yellowness) (Besbes, Blecker, Deroanne, Drira, & Attia, 2004).

180 2.9. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)

181 The polypeptide profiles of CPI and CPH samples were determined by sodium dodecyl

182 sulfate polyacrylamide gel electrophoresis (SDS-PAGE) according to the Laemmli method,

183 using 15% for separating gel and 4% for stacking gel (Laemmli, 1970) .

184 2.10. Protein solubility

185 Protein dispersions in deionised water (1%, w/v) were stirred magnetically for 30 min,

186 and then the pH was adjusted to the desired value with 0.5 M HCl or 0.5 M NaOH. After 30

187 min of stirring, the pH was readjusted if necessary, and then the dispersions were centrifuged

188 at 12 000 g for 20 min at 20 °C. After appropriate dilution, the protein content of the

189 supernatants was determined by the Kjeldahl method. Protein was calculated using the general

9
190 factor (6.25) (AOAC, 1995). Solubility was expressed as percentage ratio of supernatant

191 protein content to the total protein content.

192 2.11. Surface charge (zeta potential) measurements

193 The surface charges for CPI and CPH materials were measured at pH 7 and a

194 concentration of 0.2% (w/w) at room temperature using a Delsa Nano C Instrument (Malvern

195 Instruments, Westborough, MA).

196 2.12. Dynamic surface tension measurements.

197 The automatic drop volume tensiometer TVT1 (Lauda Dr. R. Wobser GmbH & Co.

198 KG, Lauda-Königshofen, Germany) was employed in dynamic mode for measuring the

199 surface tension time. The method is based on a continuous formation of drops at the capillary

200 tip with a definite diameter. The drop falls when a critical volume is reached and then a new

201 one forms. Thus, it is possible to establish surface tension as a function of the drop time

202 curves [y = f(t)]. A capillary tip of 1.055 mm internal radius was connected to a Lauda syringe

203 (2.5 mL). The drop-forming time was from 0.07 to 0.8 s/µL. The drop formation rate was

204 progressively reduced to 2, 5, and 20 times as the drop volume increased to minimise the

205 hydrodynamic effects of the liquid flow into the detaching drop.

206 2.13. Emulsifying properties

207 The emulsifying activity (EAI) and stability indices (ESI) of CPI and CPH were

208 determined by the method described by Pearce and Kinsella (1978). The emulsions at pH 7.0

209 were prepared by homogenising 50 mL of 0.5% (w/w) protein solution with 2 mL of soybean

210 oil for 1 min at a speed of 13.500 rpm using an Ultra-Turrax T 25 Basic (IKA Werke GmbH

211 & Co., Staufen, Germany). Emulsion (100 µL) sample was immediately taken from the

212 bottom of the tube and diluted in 7.5 mL of 10 mM sodium phosphate buffer (pH 7.0)

213 containing 0.1% sodium dodecyl sulfate (SDS) and this solution was vortexed for 10 s. An

10
214 aliquot of this suspension was taken at 10 min, and the absorbance of the diluted emulsion

215 was measured at 500 nm.

216 EAI and ESI were calculated using the following equations:

  .    
217 EAI (m2/g) =
 φ 



218 ESI (min) = .
∆

219 where, A0 is the absorbance of the diluted emulsion immediately after homogenisation, N is

220 the dilution factor (×150), c is the weight of protein per volume (g/mL), φ is the oil volume

221 fraction of the emulsion, ∆A is the change in the absorbance between 0 and 10 min (A0 – A10)

222 and t is the time interval, 10 min.

223 2.14. Statistical analysis

224 The analytical values were carried out using three independent determinations. The

225 results were expressed as mean values ± standard deviation of three independent

226 determinations.

227 The statistical analyses were determined using SPSS for Windows version 11.0 (SPSS

228 Inc., Chicago, IL). The data were subjected to analysis of variance using the general linear

229 model to determine significant differences between samples (p < 0.05). The post-hoc test used

230 for the analyses was the Duncan test.

231 3. Results and discussion

232 3.1. Physicochemical properties and amino acid contents of CPI and CPH

233 The chemical composition of CPI and CPH is shown in Table 1.The proximate

234 composition of CPI shows that it is characterised by a high protein content (78.53%). The ash

235 and lipid contents of CPI were 4.53% and 4.5%, respectively. All CPH contained significantly

236 higher protein content than CPI, ranging between 79.21% and 83.75%. The protein content

237 increased progressively with the increase in the extent of hydrolysis. This indicates that the

238 proteolytic splitting of peptide bonds results in a progressive reduction of the insoluble protein

11
239 fraction and a higher yield of soluble protein. Alcalase® hydrolyses proteins with broad

240 specificity for peptide bonds, and a preference for a large uncharged residue in P1. Increasing

241 the DH to values above 8.6% did not further increase amounts of free amino groups and this

242 could be due to either inhibition of the enzyme or a lack of further accessible cleavage sites in

243 the protein substrate. These results are in accordance with those of Achouri, Zhang, and

244 Shiying (1998) who found that the enzymatic hydrolysis of soy protein hydrolysis to a DH

245 less than 8% caused an increase in protein content. In that study, a DH higher than 8% did not

246 further increase the amounts of free amino groups. All CPH have low moisture and lipids

247 contents, which might significantly contribute to their stability during storage (Table 1). The

248 degree of ash formation that ranged from 4.53% to 6.14% was higher in the CPH, which

249 could be due to the addition of NaOH for keeping the pH constant during hydrolysis (Table

250 1). The decrease in the lipid content of protein hydrolysates as compared to untreated CPI

251 might enhance the stability of formulated products using CPH by decreasing effects of lipid

252 oxidation.

253 Colour influences the acceptability of food products. The L*, a*, b* values were

254 significantly different for all the samples (p < 0.05) (Table 1). These changes indicate that the

255 colour of CPI powder is positively influenced by the enzymatic treatment with Alcalase. DH

256 = 14.67% sample was the darkest (L* = 29.4) and most yellowish (b* = 37.06), whereas DH =

257 4% sample was the lightest (L* = 72.87) and least yellowish (b* = 31.14). In fact, during

258 hydrolysis, CPI were subjected to different chemical changes including non-enzymatic

259 browning reactions (formation of Maillard products), which occurred spontaneously under the

260 heating conditions, through the conjugation of a reducing carbohydrate with the amino groups

261 of protein, resulting in the reduction in the luminosity and giving a darker appearance to the

262 sample at high DH (Wasswa, Tang, Hong Gu, & Qing Yuan, 2007). Khaur and Singh (2007)

12
263 reported the L*, a*, b* values of 61.33, 1.88, 24.91, respectively for Kabuli chickpea proteins

264 isolates.

265 The thermal properties of CPI and its hydrolysates were evaluated by differential

266 scanning calorimetry (DSC), whose characteristics are summarised in Table 1. The ∆H

267 represents the extent of the ordered structure of a protein. In this study, we notice a decrease

268 in ∆H values of CPH with the increase in the degree of hydrolysis. At above DH 8.6%, there

269 was significant difference (p < 0.05) among ∆H values for various hydrolysates. It is possible

270 to speculate that CPI is composed of sulfhydryl or hydrophobic groups normally localised at

271 the interior of the structure. These groups are affected by Alcalase hydrolysis and heat

272 treatment. In addition, the number of exposed sulfhydryl bonds decreased and many of the

273 intermolecular bonds were disrupted with the increase in DH (Surówka, Zmudzinski, &

274 Surówka, 2004) and thus the compact structure characteristic of CPI was lost and the energy

275 required for the complete denaturation continued decreasing. These results agree well with the

276 findings of Zhao, Liu, Zhao, Ren, and Yang (2011) on peanut protein isolates and their

277 hydrolysates. We can conclude that the structural transitions of protein emanating from

278 cleaving peptide bonds at the interior of the polypeptide chain during enzymatic hydrolysis

279 may be responsible for the changes observed in the DSC characteristics.

280 The amino acid profiles of CPI and CPH were characterised by a higher content in the

281 essential amino acids compared to the suggested pattern of FAO/WHO requirement for

282 children (FAO/WHO, 2007), except for the levels of sulfur-containing amino acids (Met and

283 Cys) (Table 1).

284 The amino acid compositions (g/100 g of protein) of CPI and CPH have revealed that

285 they are rich in Glu, Asp, Arg, Lys and Leu. The high content of flavour-related amino acids

286 such as Glu and Asp could be responsible for the delicious taste of CPH products as

287 determined by the flavour of hydrolysates (Laohakunjit, Selamassakul, & Kerdchoechuen,

13
288 2014). A previous study has suggested that enzymatic hydrolysis frequently results in a bitter

289 taste (Lovšin-Kukman, Zelenik-Blatnik, & Abram, 1996). Decreasing the proportion of bitter-

290 taste peptides may be helpful for increasing the acceptability of CPH as bioactive peptides.

291 After hydrolysis, the amino acid compositions of CPH changed slightly compared to CPI. In

292 fact, the total content of hydrophobic and sulfur-containing amino acids of CPH was higher

293 than CPI. This result has confirmed that hydrolysis increases the availability of a greater

294 number of hydrophobic and sulfur-containing amino acids.

295 3.2. Effect of DH on proteins molecular weight

296 The SDS-PAGE analysis of raw CPI and its hydrolysate samples is shown in Figure 1.

297 CPI (lane 1) reveals several major bands: ~45–66kDa, ~34-45 kDa, ~24-34 kDa, ~24kDa and

298 ~20 kDa. This result is in accordance with that reported by Arcan and Yemenicioğlu (2007)

299 who found that the molecular weight profiles obtained from SDS-PAGE of most of the

300 chickpea proteins range between 15 and 25 kDa, and 30 and 40 kDa.

301 As the % DH of the CPH samples increased from ~4 to ~14% (lanes 3–6), a significant

302 reduction in the ~ 45–66 kDa and ~34-45 kDa molecular weight bands with a concomitant

303 increase in the intensity of protein bands having a molecular weight less than 20 kDa were

304 observed. The results have demonstrated that significant changes occurred to the protein

305 structure of CPI upon treatment with Alcalase to produce CPH. This endopeptidase enzyme

306 could influence the quaternary and tertiary conformations of proteins by cleaving peptide

307 bonds within individual or aggregated proteins to produce smaller protein sub-units and/or

308 smaller peptides. In addition, only minor differences were proven by SDS-PAGE amongst the

309 CPH (% DH 4–14). The disappearing sequence of CPI subunit bands suggests that these

310 bands are the most susceptible to Alcalase hydrolysis.

311

312 3.3. Effect of DH on protein solubility

14
313 The improvement of protein solubility, which is function of hydrophilicity and

314 electrostatic repulsions, is the most notable effect on protein functional properties after the

315 enzymatic hydrolysis process (Panyam & Kilara 1996). Figure 2. shows the protein solubility

316 (PS) profiles of CPI and their hydrolysates obtained at DHs 4%, 8.6%, 10.7% and 14.67%, ,

317 as a function of pH. The PS of CPI was minimum at pH 4.0–5.0 and increased gradually

318 below pH 4.0 and above pH 5.0. The PS profile of CPI is consistent with previous reports

319 (Yust, Pedroche, Millán-Linares, Alcaide-Hidalgo, & Millán, 2010). The enzymatic

320 hydrolysis remarkably improved the PS of CPI at all tested pH values (pH 2–10), especially in

321 acidic aqueous solutions, which are typical for many food formulations At above pH 7.0, the

322 PS of various hydrolysates was over 70%, significantly higher than that of raw CPI (p < 0.05).

323 This effect has been described in the partial hydrolysis of other legume proteins, such as pea

324 (Periago et al., 1998) and soy (Molina Ortiz, & Wagner, 2002).

325 The improvement in PS by enzymatic hydrolysis is attributed not only to the reduction

326 in the molecular weight, but also the increase of soluble peptides from insoluble aggregates or

327 precipitates, as well as the corresponding increase in the ionisable amino and carboxyl groups

328 (Tavano, 2013).

329 3.4. Effect of DH on the surface charge

330 Zeta potential provides a measure of the net surface charge and potential charge

331 distribution at the interface (Avramenko, Low, & Nickerson, 2013). The surface charge of all

332 samples, as indicated by their zeta potentials, has shown a net negative charge at pH 7, which

333 is readily explained by the fact that this pH value was well above the isoelectric point (pI = 4)

334 of CPI (Yust, Pedroche, Millán-Linares, Alcaide-Hidalgo, & Millán, 2010). As shown in Fig.

335 3(A), the sample surface charge which was found to change from ~−27 mV for CPI to ~−16

336 mV for the CPH (DH 14%), was significantly different (p <0.05). Can Karaca, Low, and

337 Nickerson (2011) have reported a zeta potential of −22 mV for an unheated and unhydrolysed

15
338 CPI at pH 7.0. Avramenko, Low, and Nickerson (2013) have reported a zeta potential of ~−35

339 mV for raw lentil protein isolate and ~−37 mV for its hydrolysates.

340 The functional properties of proteins are impaired near their isoelectric point; as is the

341 case in most acidic foods. The enzymatic modification of food proteins can enhance their

342 functional properties over a wide pH range (Panyam & Kilara 1996).Fig. 3(B) shows the

343 charge of CPI and their hydrolysates as a function of pH. CPI was found to carry a negative

344 net charge at solution pHs above the isoelectric point, and a positive charge below. CPH was

345 found to carry a negative charge over the majority of the pH range until pH 2, leading to

346 electrostatic repulsions between polypeptide chains. CPIs are the least soluble at their pI,

347 which limits their use in highly acidic foods. The net charge of the hydrolysates is probably

348 due to the increase in the number of the exposed ionisable amino and carboxyl groups, which

349 enhance intra and inter-molecular electrostatic repulsion and promote the unfolding of

350 protein, reducing protein‒protein aggregation and increasing protein‒water interactions

351 (Achouri, Zhang, & Shiying, 1998).

352 3.5. Effect of DH on the de surface tension

353 The ability of CPI and CPH (2%, w/w; pH 7.0) to lower the interfacial tension between

354 two phases was investigated (Fig. 4(A)). CPI and CPH were able to decrease interfacial

355 tension relative to water (72 mN/ m), indicating that CPI and CPH were surface-active. CPIs

356 takes a longer time to reach their equilibrium interfacial tension values. This interfacial

357 behaviour of CPI may be attributed to its large molecular size, which can reduce the

358 molecular flexibility of native chickpea protein and its susceptibility to conformational

359 changes compared to its hydrolysates (Rodríguez Patino, Rodríguez Niño, Carrera Sánchez,

360 Molina Ortiz, & Añón, 2005).

361 The changes of the interfacial tension of CPI as a function of % DH are shown in Fig.

362 4(B). The statistical analyses have revealed that the interfacial tension for the first drop of CPI

16
363 (~66.5mN m−1) was significantly greater than that of CPH (~59.4 mN m−1) (p < 0.05);

364 however, no differences were observed in equilibrium tension (Fig. 4(B)). As can be seen

365 from this figure, the value of the first drop decreases with the increase in DH, possibly

366 because the reduction of CPI molecular size, the increase of solubility in the aqueous phase

367 and that of the surface activity allow for greater rates of diffusion to the interface. However,

368 the increase in the DH value led to a reduction in the surface activity by a decrease in the first

369 drop value. This can be explained by the fact that hydrolysis leads to the release of small

370 peptides into solution. These small peptides, with the same charge, showed an antagonist

371 behaviour and can compete to reach the air‒water interface, which ultimately results in the

372 slow diffusion into interface. This competition effectively reduces the diffusion to the

373 interface. Therefore, the rate of diffusion depends not only on the molecular size of proteins

374 but also on the modified conformation of the protein with DH due to the action of the enzyme

375 on the native CPI. This result is in agreement with previous results of other authors (Miñones

376 Conde, & Rodríguez Patino, 2007; Miñones Conde, Yust, Pedroche, Millàn, & Rodríguez

377 Patino, 2005).

378 The equilibrium surface tension of CPI and its hydrolysates reached a constant value

379 (Fig. 4B). The tension of equilibrium in the presence of CPI and CPH is the same. This could

380 be explained by the fact that the hydrolysis of CPH leads to the formation of a continuous film

381 at the interface via intermolecular interactions. Moreover, an increased molecular flexibility,

382 as a result of enzymatic hydrolysis, could facilitate protein unfolding and interactions among

383 the adsorbed segments (Perez, Sánchez, Patino, Rubiolo & Santiago, 2012; Miñones Conde &

384 Rodríguez Patino, 2007).

385

386 3.6. Effect of DH on the emulsifying properties

17
387 Proteins are known to be effective emulsifiers and hence are commonly used in food

388 emulsions. The formation and stability of the protein-based emulsions are principally related

389 to the surface activity of proteins at interfaces. (Lam & Nickerson, 2013). Hydrolysis causes

390 changes in the molecular structure of protein (surface hydrophobicity and molecular weight),

391 which are the origin of specific surface characteristics and functional properties. Fig. 5 shows

392 EAI as a function of the degree of hydrolysis of chickpea protein. The EAI was increased by

393 protein hydrolysis, mainly at the lowest DH (4%). This could be due to the increase of

394 solubility, molecular flexibility of polypeptides and the exposure of hydrophobic areas.

395 Moreover, at lower DH value that corresponds to a lower protein concentration (Table1), the

396 area occupied per protein molecule is high, which facilitates the rearrangement and adsorption

397 of molecules and results in ameliorative emulsifying capacities. However, with increasing of

398 protein hydrolysis, the emulsifying capacities of the hydrolysates decrease. In addition, at

399 higher DH value, complete unfolding or re-orientation does not occur, due to the

400 overcrowding of protein molecules at the interface (Fainerman & Miller, 1998). This is in

401 accordance with previous results (Wasswa, Tang, Hong Gu, & Qing Yuan, 2007), showing

402 that a low DH of protein is sufficient to improve EAI. As can be seen from Figure 5, the

403 emulsion stability value for DH = 4% and 8.6% compared to CPI shows no significant

404 difference nor enhances the emulsion stability. A DH higher than 8.6% alters emulsion

405 stability. This can be explained by the decrease of the ability of smaller peptides resulting

406 from hydrolysis to interact at the interface and thus decreasing the viscoelasticity of the film.

407 Furthermore, due to the charge repulsions, peptides with low molecular weight can neither

408 unfold nor re-orient at the interface (Severin & Xia, 2006).

409

410 Conclusions

18
411 During hydrolysis, surface properties such as dynamic interfacial tension and surface

412 charge affect the emulsifying properties of CPI. CPH was found to have better ability in

413 lowering surface tension and higher surface charge compared to that of chickpea protein

414 isolate. A limited enzymatic treatment with Alcalase could enhance emulsifying properties of

415 chickpea protein. A small degree of CP hydrolysis (DH = 4%) enhanced both emulsion

416 capacity and emulsion stability. Choosing the right degree of hydrolysis is crucial for

417 enhancing the functional properties of proteins. CPH, with their high solubility over a wide

418 range of pH, may be better suited for many food formulations, especially in acidic foods.

419 Overall, the results indicated that CPH could create new opportunities for the development of

420 effective techno-functional additives for use in a wide range of food, cosmetic and

421 pharmaceutical formulations.

422 Conflict of Interests

423 The authors declare that there is no conflict of interests regarding the publication of this

424 paper.

425 Acknowledgment

426 This work was funded by the Ministry of Higher Education and Scientific Research-Tunisia

427 References

428 Abbes, F., Masmoudi, M., Kchaou, W., Danthine, S., Blecker, C., Attia, H., & Besbes,

429 S. (2015). Effect of enzymatic treatment on rheological properties, glass temperature

430 transition and microstructure of date syrup. LWT -Food Science and Technology, 60(1), 339-

431 345.

432 Achouri, A., Zhang, W., & Shiying, X. (1998). Enzymatic hydrolysis of soy protein

433 isolate and effect of succinylation on the functional properties of resulting protein

434 hydrolysates. Food Research International, 31(9), 617-623.

19
435 Adler-Nissen, J. (1986). Enzymic hydrolysis of food proteins. Elsevier Applied Science

436 Publishers. Barking. UK.

437 AOAC. (1990). Official Methods of Analysis of the Association of Official Analytical

438 Chemists, 15th Ed., (K. Helrich, ed.), Association of Official Analytical Chemists, Inc.,

439 Arlington, Virginia, VA.

440 AOAC. (1995). Official Methods of Analysis. 15th edn. Association of Official

441 Analytical Chemists.

442 Arcan, I., & Yemenicioğlu, A. (2007). Antioxidant activity of protein extracts from heat-

443 treated or thermally processed chickpeas and white beans. Food Chemistry, 103 (2), 301–312.

444 Avramenko, N.A., Low, N.H., & Nickerson, M.T. (2013). The effects of limited

445 enzymatic hydrolysis on the physico-chemical and emulsifying properties of a lentil protein

446 isolate. Food Research International, 51(1), 162-169.

447 Besbes, S., Blecker, C., Deroanne, C., Drira, N.E., & Attia, H. (2004). Date seeds:

448 chemical composition and characteristic profiles of the lipid fraction. Food Chemistry, 8(2),

449 75-80.

450 Can Karaca, A. C., Low, N., & Nickerson, M. (2011). Emulsifying properties of

451 chickpea, faba bean, lentil and pea proteins produced by isoelectric precipitation and salt

452 extraction. Food Research International, 44(9), 2742-2750.

453 Fainerman, V. B., & Miller, R. (1998). Adsorption and interfacial tension isotherm for

454 proteins. In D. Mobius & R. Miller (Eds.), Proteins at liquid interface (pp. 52–102).

455 Amsterdam: Elsevier Science B.V.

456 FAO (2014). Statistical Database. Food and Agriculture Organization of the United

457 Nations, Rome, Italy (http//www.apps.fao.org).

20
458 FAO/WHO (2007). Protein and amino acid requirements in human nutrition. Report

459 of the Joint FAO/WHO Expert Consultation. Rome: Food and Agriculture Organisation of the

460 United Nations.

461 Kembhavi, A. A., Kulkarni, A., & Pant, A. (1993). Salt-tolerant and thermostable

462 alkaline protease from Bacillus subtilis NCIM No. 64. Applied Biochemistry and

463 Biotechnology, 38, 83-92.

464 Khaur, M., & Singh, N. (2007). Characterization of protein isolates from different

465 Indian chickpea (Cicer arietinum L.) cultivars. Food Chemistry, 102, 366–374.

466 Kim, H. J., Decker, E. A., & McClements, D. J. (2005). Influence of protein

467 concentration and order of addition on the thermal stability of beta lactoglobulin stabilized n-

468 hexadecane oil-in-water emulsions at neutral pH. Langmuir, 21, 134–139.

469 Laemli, U. K. (1970). Cleavage of structural proteins during assembly of the head

470 bacteriophage T4. Nature, 227, 680–685.

471 Lam, R.S.H., & Nickerson, M.T. (2013). Food proteins: A review on their emulsifying

472 properties using a structure–function approach. Food Chemistry, 141, 975–984.

473 Laohakunjit, N., Selamassakul, O., Kerdchoechuen, O. (2014). Seafood-like flavour

474 obtained from the enzymatic hydrolysis of the protein by-products of seaweed (Gracilaria

475 sp.). Food Chemistry, 158, 162–170.

476 Lovšin-Kukman, I., Zelenik-Blatnik, M., & Abram, V. (1996). Bitterness intensity of

477 soybean protein hydrolysates , chemical and organoleptic characterization. European Food

478 Research & Technology, 203, 272-276.

479 Martinez, K.D., Baeza R. I., Millán, F., & Pilosof, A. M. R. (2005). Effect of limited

480 hydrolysis of sunflower protein on the interactions with polysaccharides in foams. Food

481 Hydrocolloids, 19(3), 361-369.

482

21
483 McClements, D.J. (2004). Protein-stabilized emulsions. Current Opinion in Colloid &

484 Interface Science. 9, 305–313.

485 Miñones Conde, J., & Rodríguez Patino, J. M. (2007). The effect of enzymatic

486 treatment of a sunflower protein isolate on the rate of adsorption at the air–water interface.

487 Journal of Food Engineering, 78(3), 1001-1009.

488 Miñones Conde, J., Yust, M. M., Pedroche, J. J., Millàn, F. R., & Rodríguez Patino, J.

489 M. (2005). The effect of enzymatic treatment of extracted sunflower proteins on solubility,

490 amino-acid composition and surface activity. Journal of Agricultural and Food Chemistry,

491 53, 8038–8045.

492 Mokni, A., Sila, A., Przybylski, R., Nedjar-Arroume, N., Makhlouf, I., Blecker, C.,

493 Attia,H., Dhulster, P., Bougatef, A., & Besbes S. (2015). Purification and identification of

494 novel antioxidant peptides from enzymatic hydrolysate of chickpea (Cicer arietinum L.)

495 protein concentrate. Journal of Functional Foods, 12, 516–525.

496 Molina Ortiz, S.E., & Wagner. J.R. (2002). Hydrolysates of native and modified soy

497 protein isolates: structural characteristics, solubility and foaming properties. Food Research

498 International, 35(6), 511-518.

499 Official Journal of the European Communities (1998). Determination of amino acids in

500 feed by HPLC . Development of an optimal hydrolysis and extraction procedure by the EU

501 commission DGXII in three International collaborative Studies. 257, 14-28.

502 Panyam, D., & Kilara, A.(1996). Enhancing the functionality of food proteins by

503 enzymatic Modification. Trends in Food Science & Technology, 7(4), 120-125.

504 Papalamprou, E.M., Doxastakis, G.I., Biliaderis, C.G., & Kiosseoglou. V. (2009).

505 Influence of preparation methods on physicochemical and gelation properties of chickpea

506 protein isolates . Food Hydrocolloids, 23(2), 337-343.

22
507 Pearce, K. N., & Kinsella, J. E. (1978). Emulsifying properties of proteins: Evaluation

508 of a turbidimetric technique. Journal of Agricultural and Food Chemistry, 26, 716–723.

509 Perez, A.A., Sánchez, C.C., Patino, J.M.R., Rubiolo, A. C., & Santiago, L.G. (2012).

510 Foaming characteristics of β-lactoglobulin as affected by enzymatic hydrolysis and

511 polysaccharide addition: Relationships with the bulk and interfacial properties. Journal of

512 Food Engineering, 113(1), 53-60.

513 Periago, M.J., Vidal, M.L., Ros, G., Rincón, F., Martínez, C., López, G., Rodrigo, J., &

514 Martínez, I. (1998). Influence of enzymatic treatment on the nutritional and functional

515 properties of pea flour. Food Chemistry, 63(1), 71-78.

516 Rodríguez Patino, J.M., Rodríguez Niño, M.R., Carrera Sánchez, C., Molina Ortiz, S.E.,

517 & Añón, M.C. (2005). Dilatational properties of soy globulin adsorbed films at the air–water

518 interface from acidic solutions. Journal of Food Engineering, 68(4), 429-437.

519 Severin, S., & Xia, W. S. (2006). Enzymatic hydrolysis of whey proteins by two

520 different proteases and their effect on the functional properties of resulting protein

521 hydrolysates. Journal of Food Biochemistry, 30, 77–97.

522 Surówka, K., Źmudziński, D., & Surówka, J. (2004). Enzymic modification of extruded

523 soy protein concentrates as a method of obtaining new functional food components. Trends in

524 Food Science & Technology, 15, 153–160.

525 Tavano, O.L. (2013). Protein hydrolysis using proteases: An important tool for food

526 biotechnology. Journal of Molecular Catalysis B: Enzymatic, 90, 1– 11.

527 Wasswa, J., Tang, J., Hong Gu, X., & Qing Yuan, X. (2007). Influence of the extent of

528 enzymatic hydrolysis on the functional properties of protein hydrolysate from grass carp

529 (Ctenopharyngodon idella) skin. Food Chemistry, 104(4), 1698-1704.

23
530 Yang, B., Yang, H., Li, J., Li, Z., & Jiang, Y. (2011). Amino acid composition,

531 molecular weight distribution and antioxidant activity of protein hydrolysates of soy sauce

532 lees. Food Chemistry, 124(2), 551-555.

533 Yust, M. M., Pedroche, J., Girón-Calle, J., Alaiz, M., Millán, F., & Vioque, J. (2003).

534 Production of ace inhibitory peptides by digestion of chickpea legumin with alcalase. Food

535 Chemistry, 81, 363–369.

536 Yust, M.M., Pedroche, J., Millán-Linares, M. D. C., Alcaide-Hidalgo, J. M., & Millán,

537 F. (2010). Improvement of functional properties of chickpea proteins by hydrolysis with

538 immobilised Alcalase. Food Chemistry, 122(4), 1212-1217.

539 Zhao, G., Liu, Y., Zhao, M., Ren, J., & Yang, B. (2011). Enzymatic hydrolysis and their

540 effects on conformational and functional properties of peanut protein isolate. Food Chemistry,

541 127(4), 1438-1443.

542

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543 Figure caption

544 Fig. 1 : Sodium dodecyl sulfate–polyacrylamide gel electrophoresis profiles of CPI, and its

545 hydrolysates with different DH in the presence of ß-mercaptoethanol. Lane M on the gels

546 represents the marker proteins; Lane 1: CPI; Lane 2: 4% DH; Lane 3: 8.6% DH; Lane 4:

547 10.7% DH; Lane 5: 13 % DH; Lane 6: 14.67 % DH. Standard protein marker: serum albumin

548 (66,200 Da), ovalbumin (45,000 Da), carbonic anhydrase (31,000 Da), trypsin inhibitor

549 (21,500 Da), lysozyme (14,400 Da).

550 Fig 2. Protein solubility profiles as a function of pH of CPI and CPHs obtained with Alcalase.

551 ( ) CPI; ( ) CPH 4%; ( ) CPH 8.6%; ( ) CPH 10.7%; and ( ) CPH

552 14.67%.

553 Fig 3. (A) Zeta potential for CPI and CPH as a function of degree of hydrolysis (%) at pH 7.

554 (B) Zeta potential (ZP) for CPI and CPHs as a function of pH. ( ) CPI; ( ) CPH 4%;

555 ( ) CPH 8.6%; ( ) CPH 10.7%; and ( ) CPH 14.67%.

556 Fig 4. (A) Comparative evolution of surface tension vs. time for 2.0% (w/v) of CPI, and its

557 hydrolysates with different DH. ( ) CPI; ( ) CPH 4%; ( ) CPH 8.6%; ( )

558 CPH 10.7%; and ( ) CPH 14.67%. (B) First drop ( ) and tension of equilibrium (

559 ) of CPI, and its hydrolysates with different DH

560 Fig 5. Emulsifying activity index and emulsion stability index of CPI and CPHs: :EAI

561 (m2/g): Emulsion activity index and ESI (min): emulsion stability index. Results are means

562 and standard deviations of triplicate determinations. Different letters above the bars indicate

563 significant differences by Duncan test (p ≤ 0.05).

564

565

566

567

25
568 Fig. 1

569
M 1 2 3 4 5 6
570

66 kDa
45 kDa

31 kDa
21 kDa

14 kDa
571

572

573

574

575

576

577

578

26
579 Fig. 2

100

90

80

70
Protein solubility (%)

60

50

40

30

20

10

0
0 2 4 6 8 10 12 14
pH

580
581

27
582 Fig. 3 (A)

-30
-28
-26
-24
Zeta potentiel (mV)

-22
-20
-18
-16
-14
-12
-10
0 2 4 6 8 10 12 14 16

Degree of hydrolysis(%)
583

584

585 Fig. 3(B)

19

14

9
zeta potentiel (mV)

4 pI=4.1(CPI)

-1 0 1 2 3 4 5 6

-6 pI <2 (CPH)

-11

586 -16 pH

587

588

28
589

590 Fig. 4 (A)

591
68

66
Surface Ten sion (mN/m)

64

62

60

58

56

54
0 10 20 30 40 50 60 70
Time (min)
592

593 Fig. 4 (B)

594

70

68

66

64
Surface tension (mN/m)

62

60

58

56

54

52

50
0 2 4 6 8 10 12 14 16
Degree of hydrolysis (%)
595

596

29
597 Fig 5.

350
b
300 a
c
250 d
200
e
150 EAI(m2/g)
a a a
ESI(min)
100 b
c
50

0
0 4 8.6 10.7 14.67

Degree of hydrolysis (%)

598

599

600

601

30
Table 1: Physicochemical and amino acids content of chickpea protein isolate and its hydrolysates (DH=4, 8.6, 10.7 and 14.67%)
obtained after treatment with Alcalase.

CPI DH=4% DH=8.6% DH=10.7% DH=14.67%

Dry matter 93.75±0.10A 94.25±0.29 A
92.47±0.37 B
93.25±0.45A 95.13±0.13C
Fat (%) 4.5±0.5A 2.23±0.10B 1.89±0.20C 1.78±0.16C 1.14±0.12D
Carbohydrates (%) 6.2±0.46A 6.18±0.16 A
5.33±0.66 B
3.14±0.28C 4.1±0.25D
Ash (%) 4.53±0.23A 4.03±0.12A 5.1±0.24B 5.7±0.13C 6.14±0.56C
Protein (%) 78.53±0.15A 79.21±0.23B 80.15±0.25C 83.63±0.10D 83.75±0.32D
Amino Acids Amino Acids Content (g/100g protein)
Asp+Asn 13.36 ± 0.20A 11.33±0.14B 11.5±0.16B 11.36±0.07B 10.7±0.12C

Glu+Gln 19.23 ± 0.22A 16.53±0.04B 16.71±0.02B 16.5±0.25B 15.3±0.09C

Ser 6.65 ± 0.04A 6.04±0.07B 6.2±0.03B 5.92±0.08B 5.71±0.30B
Gly 4.03 ± 0.21A 3.84±0.18A 3.52±0.29A 3.75±0.11A 3.71±0.18A
His 3.03 ± 0.08A 3.56±0.06B 3.5±0.02B 3.35±0.13C 3.32±0.04C

Arg 9.57 ± 0.31A 9.6±0.05A 9.64±0.03A 9.14±0.18B 8.86±0.31B

Thr 3.73 ± 0.03A 3.64±0.04A 3.9±0.37A 3.64±0.08A 3.64±0.07A

Ala 4.00 ± 0.28A 4.03±0.14A 4.3±0.09A 4.04±0.04A 4.04±0.03A

Pro 3.09 ± 0.44A 4.51±0.04B 4.5±0.18B 4.43±0.29B 4.2±0.17B

Tyr 2.36 ± 0.03A 3.1±0.37B 3.32±0.07B 3.07±0.02B 3.15±0.05B

VAl 3.94 ± 0.80A 4.68±0.02B 4.61±0.05B 4.71±0.06B 4.61±0.23B

Met 1.23 ± 0.01A 1.71±0.09B 1.8±0.30B 1.7±0.07B 1.73±0.10B

IlE 2.76 ± 0.05A 4.58±0.01B 4.8±0.02C 4.51±0.09B 4.46±0.03B

 Leu 7.43 ± 0.15A 7.52±0.06B 8.01±0.23A 7.7±0.18A 7.55±0.32A

Phe 5.19 ± 0.07A 6.5±0.10B 7.43±0.12C 6.56±0.04B 6.52±0.02B

Lys 7.88 ± 0.25A 7.43±0.03B 6.92±0.31C 7.02±0.02C 6.82±0.13C

essential amino acids 35.19 39.62 40.97 39.19 38.65

sulfur containing amino acids 1.23 1.71 1.8 1.7 1.73

aromatic amino acids 7.55 9.6 10.75 9.63 9.67

hydrophobic amino acids 22.45 27.03 28.32 27.09 26.59

Colour
L* 64.28±0.34A 52,87±0,05B 49.68±0.11C 38.86±0.27D 29.4±0.05E
a* 4.2±0.02A 4.45±0.02 B
5.87±0.04C 5.35±0.01D 6.56±0.02E
b* 30.35±0.09A 32.14±0,07B 34.42±0.09C 34.25±0.04D 37.06±0.09E
Thermal properties

T0(°C) 115.20 ± 1.13A 50.92 ± 0.66B 35.29 ± 1.2C 50.32 ± 0.96B 31.04±1.34D

TD(°C) 133.68 ± 0.68A 60.97 ± 0.37B 49.25 ± 0.23C 56.23 ± 0.7D 43.78±1.22E

ΔH(J/g) 75.93 ± 2.75A 16.13 ± 0.31B 11.03 ± 1.03C 10.82 ± 0.74C 8.83±1.5C

All the data are expressed as mean ± SD and are the mean of three replicates within the same line denote that are significantly different (p < 0.05).
T0 : temperature onset, TD: denaturation temperature, ΔH :enthalpy of denaturation.
602

603 Highlight

604 • Enzymatic hydrolysis of chickpea protein isolate (CPI) improves protein recovery.

605 • Chickpea protein hydrolysate showed higher solubility than CPI, especially at pH near

606 to the isoelectric point

607 • Enzymatic hydrolysis of CPI decreased interfacial tension and surface charge.

608 • A small degree of hydrolysis (DH = 4%) enhanced emulsification activity and

609 stability.

610 • An increase of DH did not improve emulsifying properties.

611

31