**3. Results**

#### *3.1. AQ Produced from Di*ff*erent Chickpea Cultivars*

AQ prepared from di fferent chickpea cultivars showed significantly di fferent yields and moisture contents (Figure 1). Liquid AQ yields ranged from 70.90 g/100 g seed to 107.44 g/100 g seed, with the highest yield produced by CDC Luna and the lowest by CDC Leader. AQ moisture content ranged from 92.4% to 94.2%, with the highest moisture content in AQ produced by CDC Luna and the lowest by CDC Leader. Commercially, high yield AQ with low moisture content (high dry matter content) would be of greater economic value.

Colour and turbidity of AQ varied with chickpea cultivar (Figure 2). In the current study, CDC Leader, CDC Orion, CDC Luna and Amit are Kabuli class chickpea cultivars which normally have a white to cream–yellow colour seed, while CDC Consul is a Desi class chickpea with brown to fawn colours. AQ produced from CDC Leader and CDC Orion had similar colour and high turbidity. The AQ samples were pale yellow and cloudy liquids. Whereas, AQ produced from Amit was bright yellow and cloudy. Interestingly, AQ produced by CDC Luna had the lowest turbidity, and was nearly translucent and bright yellow. Also remarkable, AQ produced by CDC Consul was a dark brown colour, and had high turbidity. This dark brown colour might arise from tannins in the CDC Consul seed coat that may have migrated to the water during cooking [23]. AQ colour is also influenced by other water-soluble molecules in chickpea seed such as pigments, vitamins and other plant secondary metabolites. Chickpea contains pigments mainly falling into the carotenoids class (β-carotene, cryptoxanthin, lutein and zeaxanthin) as well as small amounts of chlorophyll [24]. Moreover, there are also water-soluble

vitamins in chickpea, such as thiamin, riboflavin, niacin, vitamin B6, folate and ascorbic acid [25]. Additionally, some flavonoid compounds, including anthocyanin, flavonols, isoflavones, flavonol glucosides, phlobaphenes, proanthocyanidin, leucoanthocyanidin and proanthocyanidin in the seed coat might contribute to AQ colour [26].

**Figure 1.** Fresh Aquafaba (AQ) yield (g/100 g seed) and moisture content (%) prepared from different chickpea cultivars from Crop Development Centre (CDC) in Saskatoon, SK, Canada. Means within the same property without a common letter (a–e) are significantly different according to Tukey's test.

**Figure 2.** (**A**) AQ and seed of different chickpea cultivars prepared in jars. From left to right: CDC Consul, CDC Luna, CDC Orion, CDC Leader, and Amit; (**B**) AQ separated from chickpea seed. From left to right: CDC Consul, CDC Luna, CDC Leader, CDC Orion, and Amit.

In general, hemicellulose [27] and cellulose [28] are probably disrupted by cooking for 30 min at 115–118 ◦C and autogenic pressure (70–80 kPa), leading to partial destruction of the chickpea cell wall and breaking bonds between lignin and hemicelluloses [29]. Therefore, AQ turbidity and colour is a result of the disruption of chickpea seed microstructure during cooking, leaching of organic compounds, pigments, proteins, sugars, starch and vitamins into the cooking water [30].

#### *3.2. AQ Emulsification Properties*

#### 3.2.1. AQ Emulsion Capacity

Today, healthier and nutritious foods are demanded by health-conscious consumers. Food oil-in-water emulsions, such as mayonnaise and salad dressing, are often avoided due to their high fat and cholesterol content. Plant-based protein fractions, including soybean and wheat proteins, are ingredients that may be used in replacing egg as emulsifiers in mayonnaise emulsion systems [31,32]. Nikzade et al. (2012) developed a combination of soy milk, gums and other stabilizers to replace egg in low cholesterol-low fat mayonnaise formulas [18]. The application and development of different ingredients in reduced fat/cholesterol salad dressing and mayonnaise have been summarized by Ma and Boye (2013) [33].

*EAI* values of AQ prepared from each of five chickpea cultivars were measured (Figure 3). *EAI* ranged from 1.10 ± 0.04 to 1.30 ± 0.05 m<sup>2</sup>/g with the highest *EAI* observed for AQ prepared from CDC Leader (1.30 ± 0.05 m<sup>2</sup>/g), while the lowest *EAI* occurred with CDC Orion (1.10 ± 0.04 m<sup>2</sup>/g). The *EAI*s of CDC Consul (1.21 ± 0.02 m<sup>2</sup>/g), CDC Luna (1.17 ± 0.07 m<sup>2</sup>/g), and Amit (1.20 ± 0.05 m<sup>2</sup>/g) were not statistically di fferent from each other.

**Figure 3.** Emulsion capacity and stability of AQ prepared from di fferent chickpea cultivars. Means within the same property without a common letter (a–c) are significantly di fferent according to Tukey's test.

#### 3.2.2. AQ Emulsion Stability

Emulsion stability of AQ from cooked whole seed of five chickpea cultivars was also investigated in this study (Figure 3). AQ emulsion stability ranged from 71.9 ± 0.8% to 77.1 ± 0.5%, with the highest emulsion stability also observed from AQ prepared by CDC Leader and Amit, while the lowest emulsion stability for AQ prepared by CDC Luna. The emulsion stability of AQ from CDC Orion (75.6%) and CDC Consul (74.7%) were not statistically di fferent.

As a novel water-soluble emulsifier, AQ can stabilize oil-in-water emulsions to prepare egg-free vegan food oil emulsions. Additionally, our results showed that AQ prepared from Kabuli type (CDC Leader) chickpea exerted the best emulsion capacity and stability compared to the other chickpea cultivars studied in this research. This indicates the potential for selecting CDC Leader to produce an AQ emulsifier. This di fference in emulsification properties between di fferent chickpea cultivars is probably related to the di fference in the physical properties and chemical compositions of chickpea seed which a ffect the mass transfer to the cooking water during cooking.

To better understand the emulsion properties di fferences of AQ prepared from di fferent chickpea cultivars, we studied the physiochemical properties of chickpea seed, and we tested for correlations between these properties and AQ emulsion properties.

#### *3.3. Chickpea Physical Properties*

Seed coat cracking after soaking and during cooking results from splitting of the outer cell wall layers. During chickpea cooking, this seed coat works as a membrane that controls mass transfer, which would a ffect the composition, and therefore, the functional properties of the resulting cooking water (AQ) [8]. Seed coat physical characteristics depend on the genotype and environmental conditions (temperature, soil and moisture) at the time of seed maturity or during storage. Physical characteristics of seed from di fferent chickpea cultivars are shown in Table 1. Significant di fferences were observed in *HSW*, *ED*, *SCI* and *WSA* among chickpea cultivars. *HSW* and *ED* ranged from 22.43 ± 0.08 g to 42.9 ± 0.3 g and 6.89 ± 0.5 mm to 8.49 ± 0.2 mm, respectively. CDC Leader exhibited heavier (42.90 ± 0.3 g/100 seed) and larger (8.49 ± 0.2 mm) seed compared with the other cultivars.

CDC Orion was not significantly (*p* > 0.05) di fferent from CDC Luna, except for *WSA* (10 ± 1 mg/cm2). CDC Consul exhibited the greatest *SCI* (11.2 ± 2%) and *WSA* (15 ± 1 mg/cm2). Amit showed the highest *SSA* (0.668 ± 0.09 mm<sup>2</sup>/mg). These observed values are comparable to results reported previously [19], where three Sicilian chickpea cultivars (Calia, Etna and Principe) were evaluated for their *HSW*, *ED*, *SCI*, *SSA* and *WSA*. In their study, the chickpea *HSW* value ranged from 31.3 g/100 seed to 48.8 g/100 seed. Three Sicilian chickpea cultivars had similar *ED* and *SCI* with an average of 7.8 mm and 5.78%, respectively. The *SSA* value di ffered among chickpea cultivars and ranged from 0.45 mm<sup>2</sup>/mg to 0.6 mm<sup>2</sup>/mg. The *WSA* value showed a wide range from 8.3 to 11.9 mg/cm2. These di fferences in the physical characteristics between di fferent chickpea cultivars will be reflected in the seed coat behavior during soaking and cooking and might explain the variation in AQ properties. CDC Leader exhibited a good seed weight (42.90 ± 0.3 g) with the lowest *SCI* (3.89 ± 0.3%) which might explain why AQ prepared from this variety has the highest dry material (7.6%) and emulsion capacity and stability. *SCI* reflects fiber content, seed coat thickness and compactness, which is correlated with the di ffusion resistance and leaching of soluble solids during soaking and cooking. The di fferences in the cookability of di fferent chickpea genotypes have been reported previously which they attributed to the di fference in the seed characteristics [8,19].



Data are expressed as mean ± SD (*n* = 3). Value within rows followed by the same letter (e.g., a, b, c, d) indicates no significant difference (*p* > 0.05) between varieties by Tukey's test. CDC: Crop Development Centre (Saskatoon, SK, Canada).

## *3.4. Hydration Kinetics*

Water absorption capacity during soaking is generally related to the physical properties of the seed. Hydration of the testa and swelling of the cotyledons soften the cell walls and change tissue permeability, reduce the cooking time, and affect mass transfer to the cooking water. The relationship between soaking time and cumulative values of water uptake (Figure 4) was described by a nonlinear iterative regression method with an exponential relationship (Equation (8)). The applied model fitted the experimental data with an R<sup>2</sup> that was greater than 0.94 for all cultivars. Therefore, a single curve for water uptake was used in further analysis with all data combined. Soaking processes achieve rapid water uptake (*Hrate* = 0.38 g H2O g/min), and after soaking for 6 h, water absorbed reached 90% of seed dry weight. Subsequently, the water absorption rate declined until the hydrated seed weight was 2.06-fold greater than before hydration, where total hydration reached saturation at 1.06 g H2O g/dw (*Hmax*). Similar trends for chickpea seed hydration were described by several authors [19,34,35]. However, water content of chickpea seed exceeded 90% of total water imbibition after4h[19].

**Figure 4.** Chickpea seed water absorption kinetics. A common curve fitted all data (different chickpea cultivars and hydration solutions).

Statistical analysis revealed no significant differences among *Hmax*, except Amit seed which absorbed more water than the other chickpea cultivars (1.20 g H2O g/dw) in all soaking solutions (Tables 1 and 2). The modeled hydration rate (*Hrate*) of these five chickpea cultivars were mostly similar and ranged from 0.328 to 0.417 g H2O g/dw. Interestingly, the *Hmax* and *Hrate* of chickpea seed soaking in NaCl solution was slightly lower compared with that obtained in deionized water. However, seed of CDC Leader, exhibited similar *Hrate* in both deionized water and NaCl solution. Conversely, soaking seed in NaHCO3 solution increased *Hmax* for CDC Luna and *Hrate* for CDC Consul. These results are partially in agreemen<sup>t</sup> with previous reports that the presence of salt in soaking solution results in slowed seed hydration [36], but contrast with the findings of Avola and Patanè (2010) and Clemente et al. (1998), where no effect was observed on the hydration of chickpea seed soaking in salt solutions [19,34].

There were two possible explanations for increased *Hmax* results in NaHCO3 solution: (1) the osmotic pressure gradient across membranes of cotyledon cells was decreased [37], (2) there was an interaction of carbonate ions with biopolymers in cotyledon cells which might produce molecular unfolding with a possible exposure of new sites for water binding [38].


**Table 2.** Kinetic constants of the nonlinear regression analysis for chickpea seed hydration.

*Hmax*, Max hydration capacity; *Hrate*, initial hydration rate.

#### *3.5. Chickpea Chemical Properties*

The main chemical constituents (moisture, carbohydrate, protein, fat, ash and fibre content) of different cultivars of chickpea are summarized in Table 1. The moisture content of raw chickpea seed showed significant difference among chickpea cultivars (5.29 ± 0.01% to 10.7 ± 0.1%), with the highest moisture content for CDC Consul and the lowest for Amit. Carbohydrate was the main component in all of the samples, while protein was the second most abundant component. CDC Luna had the highest carbohydrate content (67.4 ± 0.7 g 100 g/dw), followed by Amit (66.8 ± 1 g 100 g/dw) and CDC Leader (65.4 ± 2 g 100 g/dw). It is important to note that CDC Orion had the lowest carbohydrate content, which might be correlated with the lowest emulsion properties observed for AQ prepared from this cultivar.

Protein content ranged from 18.3 ± 0.3 (CDC Luna) to 23.6 ± 0.08 g 100 g/dw (CDC Orion). The former also contained more fat (7.24 g ± 0.5 g 100 g/dw) than other chickpea cultivars, while Amit had the lowest fat content (4.10 ± 0.5 g 100 g/dw). Chickpea ash content did not differ with genotype. The mean value of ash content was 3.0 g 100 g/dw. Crude fibre content ranged from 4.32 ± 1 g 100 g/dw to 8.59 ± 0.6 g 100 g/dw. These observations are in agreemen<sup>t</sup> with previous studies by Xu et al. (2014), Özer et al. (2010), and de Almeida Costa et al. (2006) for chemical composition of raw chickpea seed from different chickpea cultivars [39–41]. In addition, Khattak et al. (2006) analyzed protein and ash content of seven Kabuli chickpea cultivars, which ranged from 18.08 to 19.22% and 2.45% to 2.94% [42], and thus, was similar to the values in this study.
