Emulsifying properties of legume proteins at acidic conditions: Effect of protein concentration and ionic strength

Accepted Manuscript Emulsifying properties of legume proteins at acidic conditions: Effect of protein concentration and ionic strength Yakoub Ladjal ettoumi, Mohamed Chibane, Alberto Romero PII: S0023-6438(15)30269-3 DOI: 10.1016/j.lwt.2015.10.051 Reference: YFSTL 5046 To appear in: LWT - Food Science and Technology Received Date: 7 June 2015 Revised Date: 7 August 2015 Accepted Date: 19 October 2015 Please cite this article as: Ladjal ettoumi, Y., Chibane, M., Romero, A., Emulsifying properties of legume proteins at acidic conditions: Effect of protein concentration and ionic strength, LWT - Food Science and Technology (2015), doi: 10.1016/j.lwt.2015.10.051. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. 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MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT Emulsifying properties of legume proteins at acidic conditions: Effect of 1 protein concentration and ionic strength 2 Yakoub LADJAL ETTOUMI a* , Mohamed CHIBANE b , Alberto ROMERO c 3 a Département des Science Alimentaires, FSNV, Université Abderahman Mira, Bejaia, Route 4 Targa Ouzemour, Bejaia 06000, Algérie. 5 b Laboratoire de Gestion et Valorisation des Ressources Naturelles Assurance Qualité, 6 Université Akli Mohand Oulhadj de Bouira, 10000 Bouira, Algérie. 7 c Departamento de Ingeniería Química, Facultad de Química, Universidad de Sevilla, 41012 8 Sevilla, Spain. 9 *Yakoub LADJAL ETTOUMI 10 Département des Science Alimentaires, FSNV, Université Abderahman Mira de Bejaia, Route 11 Targa Ouzemour, Bejaia 06000, Algérie. 12 E-mail: [email protected] 13 Phone:+213 699 44 67 56. 14 Abstract 15 This work is focused on emulsifying properties of pea, chickpea and lentil protein isolates at 16 acidic conditions (pH 3), as affected by protein concentration and ionic strength. Emulsions 17 characteristics and stability (droplet size distribution, flocculation, coalescence and creaming) 18 were determined. The microstructure of selected emulsions was also studied. Results 19 indicated that emulsifying properties (ability and stability) are dependent to protein 20 concentration and highly sensitive to ionic strength. In our conditions, the best emulsifying 21 properties are found around 1.5% of protein, and at less than 50 mM of NaCl. Clearly, ionic 22 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT strength affects emulsions microstructure. Thus, this study indicated that pea, chickpea and 23 lentil proteins would have great potential as emulsifiers in acidic food formulations. 24 Keywords: Acidic emulsions, legume protein, protein concentration, ionic strength, 25 microstructure. 26 1. Introduction 27 In the time being, the consumer demands originated from health concerns, allergenicity, 28 religious limitations (Halal) and rising trend of vegetarianism have increased the interest of 29 food industry in use of functional plant proteins as alternative to animal proteins (Aydemir & 30 Yemenicioglu, 2013; Carbonaro, Maselli, & Nucara, 2014). The production of plant protein 31 isolates is of growing interest to industry because of the increasing applications of plant 32 proteins in food and non-food markets (Zhang, Bo, Wanmeng, & Zhang, 2009). Nevertheless, 33 these applications in the food trade are almost limited to proteins from soybean and wheat, 34 whereas other vegetable proteins are available and less used. Thus, searchers, food 35 manufacturers and consumers are looking for alternative protein sources (Boye et al., 2010; 36 Taherian et al., 2011; Toews & Wang, 2013; Liang & Tang, 2014; Shen & Tang, 2014). 37 Among these are those from dry legumes that are extensively grown in different parts of the 38 world and, in particular, in the Mediterranean region. 39 There is increased interest in legume proteins as they can be used as good substitute for 40 animal and soybean proteins (Zhang et al., 2009; Joshi et al., 2012; Liang & Tang, 2014; Shen 41 and Tang, 2014). In this regard, the understanding of factors affecting the functional 42 properties of legume proteins enables better control of these properties, which will facilitate 43 the novel application of these proteins. The functional properties such as solubility, water and 44 oil absorption capacity, gelation, foam and emulsion formation of legume protein isolates 45 were studied to some extent previously (Makri, Papalamprou, & Doxastakis, 2005; Boye et 46 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT al., 2010; Aydemir & Yemenicioglu, 2013). However, there is lack of fundamental knowledge 47 and supporting data on the emulsifying properties of legumes proteins. Particularly, studies on 48 emulsifying properties of legumes protein, taking into account, protein concentration, pH, 49 ionic strength and relationship between structure and functionality are scarce. 50 In general, the emulsifying properties of plant proteins are dependent on the physicochemical 51 properties of proteins, including, composition (e.g. vicilin/legume ratio), solubility, 52 conformational stability and Hydrophobicity (Lestari, Mulder, & Sanders, 2011; Kaushal, 53 Kumar, & Sharma, 2012; Liang & Tang, 2014). Also, environmental conditions, including, 54 pH, ionic strength, protein concentration and oil fraction have an effect (Yu, Ahmedna & 55 GoktepeIpek, 2007; Liang & Tang, 2014). 56 Our previous study (Ladjal E., Boudries, Chibane & Romero, 2015) demonstrated that legume 57 protein isolates have good emulsifying ability and stability at pH 3; suggesting their use in 58 acidic formulation, such as, salad dressing and mayonnaise. Thus, the present study is aimed 59 to investigate the effect of protein concentration and ionic strength on emulsifying properties 60 of protein stabilized emulsions at pH 3 using protein isolates derived from three legumes (pea, 61 chickpea and lentil). The microstructure of the selected emulsions is also studied. 62 2. Material and method 63 2.1. Preparation of legume protein isolates 64 Whole flours of pea (Pisum sativum), chickpea (Cicer arietinum) and lentil (Lens culinaris) 65 were prepared as previously (Ladjal & Chibane, 2015). Legume protein isolates was prepared 66 according to the method of Papalamprou, Doxastakis & Kiosseoglou (2010) with slight 67 modification. In brief, flour (100 g) was mixed with distilled water at a 1:10 ratio (w/v), 68 adjusted to pH 8.0 using 1 M NaOH and stirred at 500 rpm for 45 min at room temperature 69 (20–22 °C). The suspension was then centrifuged at 4,500×g for 20 min at 4 °C to collect the 70 supernatant. The resulting pellet was re-suspended in distilled water at a ratio of 1:5 (w/v), 71 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT adjusted to pH 8.0, stirred for an additional 45 min, followed by centrifugation (4500×g, 20 72 min, 4 °C). Both supernatants were pooled and adjusted to pH=4.0 (pea) or pH=4.5 (chickpea 73 and lentil) using 0.1 M HCl to precipitate the protein. The protein was recovered by 74 centrifugation and collected (Karaca, Low, and Nickerson, 2011). The pH adjustment values 75 are deduced from our previous study on the protein solubility (Ladjal & Chibane, 2015).The 76 obtained precipitate was washed twice with distilled water (4°C) and re-dispersed in distilled 77 water with pH adjusted to pH 7 with 1M NaOH, and freeze-dried (Boye et al., 2010). The 78 protein contents were determined in quadruplicate as %N x 6.25 using a Leco CHNS-932 79 nitrogen micro analyser (Leco Corporation, St. Joseph, MI, USA) (Etheridge, Pesti, & Foster, 80 1998). Their protein contents were 85.7±0.6%, 85.9±0.2% and 84.8±0.1% (fresh matter) for 81 pea protein (PP), chickpea protein (CP) and lentil protein (LP), respectively. 82 2.3. Emulsions preparation 83 Three protein solutions with a different protein concentration (0.5 - 2%, w/v) were prepared at 84 pH adjusted to 3, stirred using a magnetic stirrer for 2 h at room temperature and, then, stored 85 overnight at 4 °C to allow complete hydration. Each protein solution or dispersion was mixed 86 with sunflower oil at oil fraction (ø) = 0.1, and pre-homogenized using the high-speed 87 dispersing and emulsifying unit (model IKA-ULTRA-TURRAX- T25 basic, IKA Works, 88 Inc., Germany) at 17,000 rpm for 1 min. Then, the pre-homogenized dispersions were further 89 homogenized by a high pressing emulsificator (Emulsiflex-C5, Canada) for one pass at a 90 pressure level of 40 MPa. To investigate the effect of ionic strength, we used the same 91 parameters with protein concentration of 1.5% and the salt concentration (salt dissolved in 92 deionised water) varied from 0 mM to 200 mM of NaCl. The fresh emulsions were stored for 93 various periods of time (e.g., 24 h) prior to further analysis. All other materials and chemicals 94 used were purchased from regular suppliers and were of analytical grade. 95 2.4. Emulsions characterization 96 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT Droplet-size distribution (d 4,3 ), flocculation and coalescence indices (FI and CI) as well as 97 creaming indices of prepared emulsions were evaluated as reported by Liang & Tang (2013). 98 2.5. Emulsion microstructure 99 The microstructure of the selected emulsions was determined by means of confocal scanning 100 laser microscopy a ZEISS LSM 7 DUO (Germany). A small aliquot of freshly prepared 101 emulsion was placed on a microscope slide and covered with a cover slip prior to analysis. 102 Emulsions were colored by Nile bleu. The emission of Nile bleu was detected in the 633 nm). 103 This technique provides images of dark fat droplets and a bright water phase where proteins 104 were found. a 63x objective was used. The microstructure images were analyzed using image 105 analysis software (ZEN_2012SP1_black_SP2_blue). 106 2.6.Statistical analysis 107 Statistical analysis was carried out using analysis of variance (ANOVA) and the significant 108 difference between the samples was determined using LSD test at p < 0.05. 109 3. Result and discussion 110 3.1. Effect of protein concentration on emulsifying properties 111 3.1.1. Droplet size distribution 112 The effects of protein concentration on emulsifying properties were measured at pH 3.0 and 113 an oil fraction of 0.1, at various protein concentrations (0.5, 1, 1.5 and 2%, (w/v)). The d 4,3 of 114 the droplets, diluted in 1% SDS or deionized water, was calculated and summarized in Table 115 1. In general, the smaller the droplet sizes of protein-stabilized emulsions, the better the 116 emulsifying ability of the protein is (Shen & Tang, 2014). 117 Generally, with the exception of PP-stabilized emulsions, CP and LP-stabilized emulsions 118 exhibited the best emulsifying ability (corresponding to the lowest diameter) at 2% 119 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT concentration. But the other concentrations did not display significant difference in their 120 emulsifying ability. This trend was noted both in SDS and water dilutions. For PP emulsion, 121 the smallest droplet size was found at 1.5% in water dilution and at 1% and 1.5% in SDS 122 dilution. From this concentration, there was a slight increase in droplets size indicating an 123 excess of non adsorbing protein which promote droplets flocculation by depletion 124 phenomenon. The high d 4,3 values of the emulsions containing 0.5% protein suggested that 125 protein content may not be enough to cover the oil droplets and form sufficiently dense 126 adsorption layer (Sanchez and Patino, 2005). As a result, protein acted as bridges among the 127 oil droplets and led to droplets aggregation (Sun and Gunasekaran, 2009; Guo & Mu, 2011). 128 Our results are in agreement with those reported by Sun and Gunasekaran (2009), and 129 corroborate well with those of Joshi et al. (2012), which suggest that the higher the protein 130 concentration (lentil protein), the greater was the reduction in interfacial tension, displaying 131 stabilized emulsion with high turbidity and small oil droplet. 132 3.1.2. Flocculation in fresh emulsions 133 The flocculated state of oil droplets was evaluated in terms of flocculation index (FI), as 134 shown in Table 1. The FI (0h) varied with type and concentration of protein. Basically, the FI 135 (0h) varied considerably from 0.88 in 1.5% LP emulsion to 3.72 in 1% PP emulsion. PP- 136 stabilized emulsion exhibited the lowest FI (1.19) at 2% concentration. Whereas, 1.5% 137 concentrations displayed the lowest FI in CP and LP emulsions (1.82 and 0.88 respectively). 138 At any concentration (except to 2% PP emulsion), LP-stabilized emulsions exhibited lower 139 flocculation phenomenon than PP and CP emulsions, which might be due to its lower content 140 of SH and SS bounds (Ladjal E. et al., 2015). Thus, SH groups can form bridge and enhance 141 droplets flocculation. In general, FI% progressively decreased to a minimal value, as the 142 concentration increased from 0.5 to 1.5%, while a further increase in concentration (2%) 143 contrarily led to a gradual increase in FI%. An increase in protein concentration enhanced 144 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT protein adsorption and surface coverage of oil droplets, which effectively inhibited droplet 145 aggregation (Sun and Gunasekaran, 2009). However, further increase in protein concentration 146 (up to 1.5% in our case), could promote depletion flocculation. Interestingly, protein 147 stabilized emulsions showed low FI at 1.5% concentration, suggesting that the oil–water 148 interface is saturated by protein molecules. Joshi and collaborators (2012) suggest a 149 concentration of 1% as interface saturation point in lentil protein stabilized emulsion with oil 150 fraction of 10%. According to Liang and Tang (2014), the flocculated state of droplets 151 displayed inverse trend in pea proteins stabilized emulsions, where FI% progressively 152 increased up to a maximal value, as the concentration increased from 0.25 to 1.0 g/100ml, 153 while a further increase in concentration contrarily led to a gradual decrease in FI%. 154 3.1.3. Flocculation and coalescence stability 155 Emulsions are inclined to break down over time through a variety of physicochemical 156 mechanisms, including gravitational separation, flocculation, coalescence and Ostwald 157 ripening. The stability of various emulsions formed at various concentrations upon storage of 158 24 h was evaluated in terms of flocculation (FI) and coalescence indexes (CI). 159 The FI (24h) varied with concentration and type of proteins. PP-stabilized emulsion exhibited 160 high FI at 0.5 and 1.0% concentration, and low FI at 1.5 and 2% concentrations. In the 161 contrary, CP emulsion showed high flocculation index at 2% concentration, but the other 162 concentration displayed low values. In the case of LP emulsion, the FI decreased when 163 concentration increased to reach the minimal value at 2.0% concentration. 164 Regarding coalescence, there was also great variation in the results, depending to the type and 165 concentration of protein. CI% values diverged from 2.98% in 1.5% LP emulsion to 46.51% in 166 2% LP emulsion. PP-stabilized emulsions showed low CI at 0.5% and 2% concentrations, 167 while CI was more or less high at 1% and 1.5% concentrations respectively. In the case of CP 168 emulsions, the lowest CI value was obtained at 1.5% concentration, and the highest value was 169 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT obtained at 0.5% concentration. Differently, LP-stabilized emulsions displayed very low CI at 170 0.5% and 1.5% concentrations, and high values at 1% and 2% concentrations. Basically, 171 protein-stabilized emulsions showed good emulsifying stability at 1.5% and 2% 172 concentrations. 173 This divergence between protein emulsification behaviors might be due to the difference in 174 physicochemical properties, such as, molecular weight, hydrophobicity, free SH and SS 175 bounds as determined previously (Ladjal E. et al., 2015). Clearly, LP-stabilized emulsions are 176 the most stable comparing to the others. According to our results, the underlying mechanism 177 for emulsion instability was by means of droplet flocculation and coalescence. Stable 178 emulsions can be prepared when the interface is completely covered with particles. However, 179 upon insufficient loading of the interface, droplet coalescence cannot be fully prevented, and 180 partial coalescence may take place (Scholten, Moschakis, & Biliaderis, 2014). The thickness 181 of the interfacial layer determines the magnitude and range of the steric repulsion between 182 droplets. Droplet flocculation can be inhibited if the range of the steric repulsion is longer 183 than the range of any attractive interactions, such as van der Waals or hydrophobic (Chung & 184 McClements, 2014). Hence, emulsion stability at high protein concentration can be attributed 185 to the formation of multilayered protein shell around oil droplet which successfully prevents 186 the coalescence of oil droplets Joshi et al. (2012). 187 3.1.4. Creaming stability 188 The creaming index of the three protein emulsions at different protein concentrations, upon 189 quiescent storage up to 7 days was investigated. As expected, various emulsions showed 190 different creaming behaviors, depending on the type of proteins and the applied concentration. 191 Clearly, for any emulsion at 0.5% concentration, the creaming index was the highest. It 192 increased with storage to reach the maximum values on the 7 th day. It should be noted that 193 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT This study confirms that, at pH 3, protein concentration and ionic strength (sodium chloride) 343 have a major influence on the characteristics of legume protein-stabilized emulsions, 344 including, droplet size distribution, flocculation, coalescence creaming and microstructure. 345 Basically, it was found that 1.5 % (w/v) of protein and 0 mM to < 50 mM of NaCl are the best 346 conditions to produce stable emulsions with legume proteins. It should be noted that high 347 ionic strength (≥50 mM) promotes flocculation and accelerates destabilization of legume 348 protein emulsions, whilst, high protein concentration improves their stability. Our results 349 suggest the use of these proteins as emulsifiers in acid food formulation, such as, mayonnaise 350 and salad dressing. 351 Acknowledgments 352 The authors wish to thank Béjaia University and the Universidad de Seville for the valorous 353 technical support. The authors acknowledge also to the Microscopy Service (CITIUS- 354 Universidad de Sevilla) for providing full access and assistance. 355 References 356 Aydemir, L. Y., & Yemenicioglu, A. (2013). Potential of Turkish Kabuli type chickpea and 357 green and red lentil cultivars as source of soy and animal origin functional protein 358 alternatives. LWT - Food Science and Technology, 50, 686-694. 359 Boye, J.I., Aksay, S., Roufik, S., Ribéreau, S., Mondor, M., Farnworth, E., & Rajamohamed 360 S.H. (2010). Comparison of the functional properties of pea, chickpea and lentil protein 361 concentrates processed using ultrafiltration and isoelectric precipitation techniques. 362 Food Research International, 43(2), 537-546. 363 Carbonaro, M., Maselli, P., & Nucara, A. (2014). Structural aspects of legume proteins and 364 nutraceutical properties. Food Research International. 365 http://dx.doi.org/10.1016/j.foodres.2014.11.007. 366 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT Chantrapornchai, W., and McClements, D. J. (2002). Influence of NaCl on optical properties, 367 large strain rheology and water holding capacity of heat induced whey protein isolates 368 gels. Food Hydrocolloids, 16, 467-476. 369 Chung, C. & McClements, D.J. (2014). Structure-function relationships in food emulsions: 370 Improving food quality and sensory perception. Food Structure, 1, 106-126. 371 Etheridge, R. D, Pesti, G. M., & Foster, E. H. (1998). A comparison of nitrogen values 372 obtained utilizing the Kjeldahl and Dumas combustion methodologies (Leco CNS) on 373 samples typical of an animal nutrition analytical laboratory. Animal Feed Science and 374 Technology, 73, 21-23. 375 Fisicaro, E., Compari, C., & Braibanti, A. (2011). Hydrophobic hydration processes: thermal 376 and chemical denaturation of proteins. Biophysical Chemistry, 156,51-67. 377 Guo, Q., & Mu, T.H. (2011). Emulsifying properties of sweet potato protein: Effect of protein 378 concentration and oil volume fraction. Food Hydrocolloids, 25, 98-106. 379 Hamada, H., Arakawa, T., & Shiraki, K. (2009). Effect of Additives on Protein Aggregation. 380 Current Pharmaceutical Biotechnology, 10, 400-407. 381 Joshi, M., Adhikari, B., Aldred, P., Panozzo, J.F., Kasapis, S., & Barrow, C.J. (2012). 382 Interfacial and emulsifying properties of lentil protein isolate. Food Chemistry, 134, 383 1343–1353. 384 Joye, I. J., & McClements, D. J. (2014). Emulsifying and Emulsion-Stabilizing Properties of 385 Gluten Hydrolysates. Journal of Agricultural and Food Chemistry, 62, 2623-2630 386 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT Karaca, A. C., Low, N., & Nickerson, M. (2011). Emulsifying properties of chickpea, faba 387 bean, lentil and pea proteins produced by isoelectric precipitation and salt extraction. 388 Food Research International, 44, 2742–2750. 389 Kaushal, P., Kumar, V., & Sharma, H.K. (2012). Comparative study of physicochemical, 390 functional, antinutritional and pasting properties of taro (Colocasiaesculenta), rice 391 (Oryza sativa) flour, pigeonpea (Cajanuscajan) flour and their blends. LWT - Food 392 Science and Technology, 48 (1), 59-68. 393 Ladjal, E. Y., & Chibane, M. (2015). Some physicochemical and functional properties of pea, 394 chickpea and lentil whole flours. International food research journal, 22(3), 987-996. 395 Ladjal, E. Y., Boudries, H., Chibane, M. & Romero, A. (2015). Pea, chickpea and lentil 396 protein isolates: Physicochemical characterization and emulsifying properties. Food 397 Biophysics. Accepted manuscript. http://dx.doi.org/10.1007/s11483-015-9411-6. 398 Lestari, D., Mulderb, W. J., & Sanders, J. P.M. (2011). Jatropha seed protein functional 399 properties for technical applications. Biochemical Engineering Journal, 53, 297-304. 400 Liang, H.-N., & Tang, C.-H. (2013). pH-dependent emulsifying properties of pea 401 [Pisumsativum (L.)] proteins. Food Hydrocolloids, 33, 309-319. 402 Liang, H.-N., & Tang, C.-H. (2014). Pea protein exhibits a novel Pickering stabilization for 403 oil-in-water emulsions at pH 3.0. LWT - Food Science and Technology, 58, 463-469. 404 Makri, E., Papalamprou, E., & Doxastakis, G. (2005). Study of functional properties of seed 405 storage proteins from indigenous European legume crops (lupin, pea, broad bean) in 406 admixture with polysaccharides. Food Hydrocolloids, 19, 583–594. 407 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT Molina-Bolivar, J.A., Galisteo-Gonzalez, F., & Hidalgo-Alvarez R. (2001). Specific cation 408 adsorption on protein-covered particles and its influence on colloidal stability. Colloids 409 and Surfaces B: Biointerfaces, 21, 125–135. 410 Papalamprou, E.M., Doxastakis, G. I., & Kiosseoglou, V. (2010). Chickpea protein isolates 411 obtained by wet extraction as emulsifying agents. Journal of the Science of Food and 412 Agriculture, 90(2), 304–313. 413 Piorkowski, D. T., & McClements, D. J. (2013). Beverage emulsions: Recent developments in 414 formulation, production, and applications: Review. Food Hydrocolloids, 1-37. 415 http://dx.doi.org/10.1016/j.foodhyd.2013.07.009. 416 Rao, J.-J., Chen, Z.-M., & Chen, B.-C. (2009). Modulation and stabilization of silk fibroin- 417 coated oil-in-water emulsions. Food Technology and Biotechnology, 47 (4), 413–420. 418 Salminen, H., & Weiss, J. (2014). Electrostatic adsorption and stability of whey protein-pectin 419 complexes on emulsion interfaces. Food Hydrocolloids, 35, 410-419. 420 Sanchez, C. C., & Patino, J. M. R. (2005). Interfacial, foaming and emulsifying characteristics 421 of sodium caseinate as influenced by protein concentration in solution. Food 422 Hydrocolloids, 19, 407-416. 423 Scholten, E., Moschakis, T., & Biliaderis, C. G. (2014). Biopolymer composites for 424 engineering food structures to control product functionality. Food Structure, 1, 39-54. 425 Shao, Y., & Tang, C.-H. (2014). Characteristics and oxidative stability of soy protein- 426 stabilized oil-in-water emulsions: Influence of ionic strength and heat pretreatment. 427 Food Hydrocolloids, 37, 149-158. 428 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT Shen, L., & Tang, C.-H. (2014). Emulsifying properties of vicilins: Dependence on the 429 protein type and concentration. Food Hydrocolloids, 36, 278-286. 430 Sun, C., & Gunasekaran, S. (2009). Effects of protein concentration and oil-phase volume 431 fraction on the stability and rheology of menhaden oil-in-water emulsions stabilized by 432 whey protein isolate with xanthan gum. Food Hydrocolloids, 23, 165–174. 433 Taherian, A. R., Mondor, M., Labranche, J., Drolet, H., Ippersiel, D., & Lamarche, F. (2011). 434 Comparative study of functional properties of commercial and membrane processed 435 yellow pea protein isolates. Food Research International, 44, 2505–2514. 436 Toews, R., & Wang, N. (2013). Physicochemical and functional properties of protein 437 concentrates from pulses. Food Research International, 52, 445-451. 438 Ye, A., & Singh, H. (2000). Influence of calcium chloride addition on the properties of 439 emulsions stabilized by whey protein concentrate. Food Hydrocolloids, 14, 337–346. 440 Yu, J., Ahmedna, M., & Goktepe, I. (2007). Peanut protein concentrate: Production and 441 functional properties as affected by processing. Food Chemistry, 103, 121-129. 442 Yuliana, M., Truong, C. T., Huynh, L. H., Ho, Q. P., & Ju Y.-H. (2014). Isolation and 443 characterization of protein isolated from defatted cashew nut shell: Influence of pH and 444 NaCl on solubility and functional properties. LWT - Food Science and Technology, 55, 445 621-626. 446 Zhang, T., Bo, J., Wanmeng, M., & Zhang, W. (2009). Emulsifying properties of chickpea 447 protein isolates: Influence of pH and NaCl. Food Hydrocolloids, 23, 146-152. 448 449 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT Table 1 Emulsion characteristics, including volume-mean droplet size (d 4,3 ), flocculation and coalescence indices (FI and CI) of legume protein-stabilized emulsions at various protein concentration, freshly prepared or after a storage of 24 h. FI and CI are calculated using mean values of droplet size (d 4,3 ). Emulsion Protein % (w /v) d 4,3 (µm) Indices 0 h 24 h FI CI (%) Water SDS Water SDS 0 h 24h PP emulsions 0.5 9.10±0.56 a 2.69±0.07 b 9.94±0.69 a 3.00±0.13 b 2.38 2.31 11.52 1.0 8.12±0.24 b 1.72±0.01 d 9.02±0.81 ab 1.83±0.01 d 3.72 3.40 19.18 1.5 6.14±0.70 c 2.06±0.14 c 6.22±0.67 c 2.61±0.15 c 1.98 1.37 27.02 2.0 8.15±0.27 b 3.72±0.18 a 8.48±0.04 b 4.23±0.03 a 1.19 1.00 13.70 CP emulsions 0.5 8.48±0.08 a 2.51±0.14 a 8.98±0.7 a 3.64±0.00 a 2.37 1.46 45.01 1.0 8.00±1.85 ab 2.75±0.03 a 7.50±2.07 a 3.56±0.07 a 1.91 1.10 29.45 1.5 7.41±0.14 ab 2.62±0.29 a 8.15±0.25 a 2.69±0.17 b 1.82 1.92 6.48 2.0 6.83±0.31 b 1.97±0.02 b 6.96±0.31 a 2.24±0.13 c 2.46 2.11 13.51 LP emulsions 0.5 6.69±0.25 a 2.85±0.08 a 8.94±0.97 a 2.98±0.12 b 1.34 2.00 4.31 1.0 6.38±0.83 a 2.66±0.12 b 8.11±0.34 a 3.65±0.11 a 1.39 1.21 37.46 1.5 5.26±0.20 b 2.79±0.13 ab 5.59±0.19 b 2.87±0.17 b 0.88 0.94 2.98 2.0 5.19±0.12 b 2.05±0.03 c 5.62±0.62 b 3.01±0.2 b 1.52 0.86 46.51 Values expressed are mean ± standard deviation. Means in the column (in the same protein emulsion) with different superscript are significantly different at p< 0.05. PP: Pea protein; CP: Chickpea protein; LP: Lentil protein. Table 2 Emulsion characteristics, including volume-mean droplet size (d 4,3 ), flocculation and coalescence indices (FI and CI) of legume protein-stabilized emulsions at various ionic strength, freshly prepared or after a storage of 24 h. FI and CI are calculated using mean values of droplet size (d 4,3 ). Emulsion Ionic strength (mM NaCl) d 4,3 (µm) Indices 0 h 24 h FI CI (%) Water SDS Water SDS 0 h 24 h PP emulsions 0 6.14±0.70 c 2.06±0.14 c 6.22±0.67 c 2.61±0.15 b 1.98 1.37 27.02 50 6.21±0.18 c 2.33±0.10 c 7.06±0.36 c 2.53±0.09 b 1.65 1.78 8.55 100 10.48±0.23 b 3.58±0.52 a 14.86±1.06 b 4.20±0.72 a 1.82 2.33 20.16 150 13.91±0.62 a 2.70±0.04 bc 16.44±1.21 ab 3.44±0.10 ab 4.14 3.77 27.17 200 14.05±0.73 a 3.32±0.67 ab 17.19±0.98 a 4.72±1.50 a 3.06 2.63 36.56 CP emulsions 0 7.41±0.14 d 2.46±0.12 a 8.49±0.33 c 2.71±0.15 b 2.01 2.13 10.16 50 7.67±0.15 cd 1.56±0.11 c 8.47±1.23 c 2.04±0.09 c 3.91 3.14 30.70 100 8.11±0.09 c 1.68±0.01 c 10.27±2.31 bc 2.01±0.07 c 3.81 4.10 19.36 150 10.64±0.56 b 1.90±0.00 b 13.03±3.34 b 2.26±0.26 c 4.60 4.76 18.94 200 12.70±0.37 a 1.57±0.01 c 17.83±1.61 a 4.03±0.43 a 7.09 3.42 156.68 LP emul 0 5.26±0.20 d 2.79±0.13 b 5.59±0.19 d 2.87±0.17 cd 0.88 0.94 2.86 50 5.61±0.32 d 2.95±0.22 b 6.78±0.29 d 3.41±0.21 b 0.89 0.98 15.55 MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT 100 9.35±0.62 c 4.28±0.10 a 11.24±0.71 c 5.07±0.20 a 1.18 1.21 18.35 150 12.49±0.59 b 2.05±0.0 c 20.43±1.78 b 2.55±0.18 d 5.09 7.00 24.55 200 17.13±2.34 a 2.31±0.08 c 25.09±1.95 a 3.23±0.24 bc 6.41 6.76 39.82 Values expressed are mean ± standard deviation. Means in the column (in the same protein emulsion) with different superscript are significantly different at p< 0.05. PP: Pea protein; CP: Chickpea protein; LP: Lentil protein. Table 3 Creaming index of various legume protein emulsions formed at various ionic strengths, upon storage up to 7 days. Each data is means of at least duplicate measurements. Values expressed are mean ± standard deviation. PP: pea protein, CP: chickpea protein and LP: lentil protein. ±: Slight creaming not objectively evaluated; +: clear creaming not objectively evaluated; ND: not determined. Emulsion Ionic strength (mMNaCl) Creaming index 1 day 4 days 7 days PP 0 0.00 24.50±1.50 29 .00±1.00 50 13.33±5.77 25.22±1.34 27.27±2.36 100 26.69±7.84 34.67±2.27 35.47±2.82 150 26.21±3.35 32.20±5.78 33.00±6.45 200 31.92±2.98 37.01±5.92 37.54±5.48 CP 0 ± ± + 50 + 29.00±0.00 34.00±0.00 100 29.00±0.00 29.00±0.00 29.00±0.00 150 30.00±0.00 31.00±0.00 32.00±0.00 200 22.72±3.85 23.63±5.14 23.63±5.14 LP 0 0.0±0.0 0.0±0.0 ND 50 0.0±0.0 0.0±0.0 ND 100 11.87±0.88 16.87±2.65 ND 150 16.31±0.85 20.76±1.08 ND 200 29.86±10.80 36.38±7.45 ND MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT Figure list PP-0 mM-emulsion CP-0 mM-emulsion LP-0 mM-emulsion PP-50 mM-emulsion CP-50 mM-emulsion LP-50 mM-emulsion Fig. 1. CLSM images of legume protein-stabilized emulsions at 0 and 50 mM NaCl. PP: Pea protein, CP: Chickpea protein and LP: Lentil protein. MANUS CRIP T ACCEP TED ACCEPTED MANUSCRIPT Highlights • Emulsifying properties of pea, chickpea and lentil protein isolates at pH 3; • Effect of protein concentration and ionic strength on emulsifying properties of legume proteins at acidic conditions (pH 3). • Effect of salt on the microstructure of acidic emulsions stabilized by legume proteins.