Aquafaba as an egg white substitute in food foams and emulsions: Protein composition and functional behavior

Food Hydrocolloids 96 (2019) 354–364

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Aquafaba as an egg white substitute in food foams and emulsions: Protein composition and functional behavior

T

Tina F. Buhla,b, Claus H. Christensenb, Marianne Hammershøja,∗ a b

Dept. of Food Science, Aarhus University, Blichers alle 20, DK-8830 Tjele, Denmark NEXUS A/S, Palsgaard A/S, Palsgaardvej 10, DK-7130 Juelsminde, Denmark

A R T I C LE I N FO

A B S T R A C T

Keywords: Aquafaba Foam Emulsion Egg white pH Conductivity

The ability of aquafaba to act as an egg white substitute was tested as effect of change in the level of pH and conductivity of aquafaba. The properties of aquafaba and egg white were analyzed by protein composition, dry matter content and protein concentration. Moreover, the zeta potential of aquafaba was analyzed. Foaming properties were studied by a hand shaken foam test. Emulsions were prepared by mixing either egg white or aquafaba with sunflower oil. Foam produced by either aquafaba or egg white showed distinct properties in foam capacity, foam volume and the liquid ratio in foam as function of time. However, the test of liquid drainage as function of time showed no difference between the two protein sources. Test of emulsifying properties showed more stable emulsions produced by centrifugated aquafaba compared to egg-white based emulsions. The measurements of emulsion stability were based on UV absorbance, emulsion particle size as well as rheological analyses. Aquafaba properties were not affected by increased level of NaCl in the foam and emulsifying tests conducted, however, changes in the level of pH affected all tests with exception of the foam capacity. The stability of emulsions based on aquafaba increased with increasing pH level evaluated by UV-absorbance and emulsion particle size opposite to the yield stress (Pa), which in general decreased with increasing pH level.

1. Introduction Plant-based proteins have in recent years gained increased attention as possible candidates to replace animal-based proteins (Sharif et al., 2017). This interest is spurred by an increased awareness of sustainability and healthiness observed from a modern consumer perspective as well as from the food industry (Boye, Zare, & Pletch, 2010; McClements, Bai, & Chung, 2017; Sharif et al., 2017). The production of pulses and legumes have increased during the last 15 years of e.g. a yield of 12.7 million Mt of chickpeas worldwide in 2016 (Food and Agriculture Organization of the United Nations, 2016). Especially the functional properties of pulses and legumes, which are comparable with proteins from animal and dairy sources, have been a key factor for their increased production (Sharif et al., 2017). The functional properties comprises fat binding, water holding capacity, solubility as well as gelling, foaming and emulsifying properties, which make proteins from pulses and legumes highly relevant as a source of substituting animalbased protein in a broad range of food applications such as e.g. salad dressings and imitation chicken nuggets (Boye et al., 2010; Ma, Boye, & Simpson, 2016; Sharima-Abdullah, Hassan, Norlelawati, & HudaFaujan, 2018). Furthermore, the nutritional benefits of low

∗

allergenicity, together with a sustainable production, low price and high production volumes favor the industrial use of proteins from legumes and pulses (Gumus, Decker, & McClements, 2017; Papalamprou, Doxastakis, & Kiosseoglou, 2010; Sánchez-Vioque, Clemente, Vioque, Bautista, & Millán, 1998). The functional properties of wastewater from the cooking process of chickpeas were discovered few years ago as a search for vegan egg replacements to cook meringues. The wastewater was named aquafaba according to the Latin origin of water (aqua) and beans (faba) and is defined as both the liquid from canned chickpeas as well as the boiling water from chickpea preparations (The Official Aquafaba Website, 2016). Besides the foaming abilities discovered, the wastewater holds emulsifying properties as well (The Official Aquafaba Website, 2016). Based on the foam and emulsifying properties of this plant-based liquid, an increased attention to evaluate the capacity of aquafaba as an egg replacer candidate is observed. However, scientific literature concerning aquafaba is very scarce as only one study on the composition of aquafaba (Mustafa, He, Shim, & Reaney, 2018) and few studies of the functional properties of aquafaba/chickpea cooking water have been published so far and very recently (Damian, Huo, & Serventi, 2018; Mustafa et al., 2018; Stantiall, Dale, Calizo, & Serventi, 2018).

Corresponding author. Department of Food Science, Aarhus University, DK-8830 Tjele, Denmark. E-mail address: [email protected] (M. Hammershøj).

https://doi.org/10.1016/j.foodhyd.2019.05.041 Received 30 October 2018; Received in revised form 21 May 2019; Accepted 23 May 2019 Available online 24 May 2019 0268-005X/ © 2019 Elsevier Ltd. All rights reserved.

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Table 1 Nutritional content per 100 g product and 100 g dry matter (DM) of the canned chickpeas as declared by Salling Group, Brabrand, Denmark, and of egg white protein powder as declared by Sanovo Foods, Odense, Denmark. Canned chickpea

Egg white powder

per 100g

per 100 g DM

per 100g

per 100 g DM

494 2.2 15 6.3 0.65

9.1 62.1 26.1 2.7

1495 < 0.1 4.5 84.1 1.3

0.1 5.0 93.4 1.4

Table 2 Experimental set-up with fixed and variable points for test of pH effect and NaCl (conductivity) effect for aquafaba with egg white as control. The NaCl addition corresponds to 0 mM (conductivity of 16 mS/cm), 106 mM (conductivity of 24 mS/cm), and 212 mM (conductivity of 32 mS/cm). pH level

Energy (kJ) Fat (g) Carbohydrates (g) Protein (g) Salt (g)

Egg white (control) Conductivity effect pH effect

The aim of this study was to elucidate the protein composition of aquafaba from canned chickpeas and test for functional properties in foams and emulsions and the influence of pH and NaCl hereon. Relevant food products for aquafaba could be vegan mayonnaises and salad dressings, which are emulsions at pH 3.5–4.0 and salt contents of ∼4%, and bakery foams as meringues and angel cakes, where pH typically is neutral and salt content is low (Jay, Loessner, & Golden, 2005). Furthermore, the properties of aquafaba were compared to egg white under standardized conditions for foams and emulsions.

NaCl (mS/cm)

Fixed

Variable

Fixed

Variable

pH 6 pH 6 -

pH 3, pH 4.5, pH 6, pH 7, pH 8.5

16 16

16, 24, 32

2.2. Sample set-up and preparation The effect of changes in the pH and conductivity level of aquafaba was measured by establishment of fixed and variable set points. The pH 6 and the natural conductivity level of 16 mS/cm, as measured directly in the aquafaba, were chosen as the reference conditions in the further tests of effects of pH and conductivity variations, respectively. The pH range included test of pH 3, pH 4.5, pH 6, pH 7, and pH 8.5 and the conductivity level included test of 16 mS/cm, 24 mS/cm (+50%), and 32 mS/cm (+100%). The control sample of EWP was kept fixed at pH 6 and at the conductivity level of 16 mS/cm. This set-up constitutes the center point of this study (Table 2). Prior to all analyses, the aquafaba (section 2.1) was thawed overnight at 5 °C and centrifuged at 3500×g for 20 min at 4 °C and the supernatant was used for further analysis. The centrifugated aquafaba (CAF) was adjusted with HCl or NaOH to reach the targeted pH-values, respectively, and recorded at a pH meter (PHM 92, Radiometer Copenhagen). A conductivity level of 24 mS/cm was obtained by addition of 0.410 g NaCl, equal to 106 mM, to 66 mL of CAF. Addition of 0.816 g NaCl, equal to 212 mM, to 66 mL of CAF provided a conductivity level of 32 mS/cm. The conductivity level was measured by a SensION™+ EC71 (HACH Co., Loveland, CO, USA) fitted with a 3-pole conductivity cell (CDC536T-6, Radiometer Analytical, HACH Co., Loveland, CO, USA) and calibrated with a standard solution of 11.67 mS/cm at 20 °C. A saline solution of 16 mS/cm was provided by dissolving 0.884 g NaCl in 100 mL Milli-Q water followed by the adjustment to pH 6 with 1 M HCl. A mass of 5.29 g of EWP was gradually dissolved in 50 mL, resulting in a protein concentration of 10 g/L of the prepared NaCl solution and stirred for 1 h to assure a homogenous suspension and an appropriate wetting, hence, the resulting pH of the EWP was measured to be pH 6.1. All samples were aligned to ensure an equal protein concentration of 10 g/L. The samples were dissolved with Milli-Q ultrapure water.

2. Materials and methods 2.1. Materials The aquafaba material used in this study originated from canned chickpeas and was pooled from 114 cans, frozen in aliquots, thawed and centrifugated before use to avoid inter-can variations. The canned chickpeas (Pasciá, Kabuli) were grown in Italy and imported by Salling Group, Brabrand, Denmark. The nutritional profile of the canned chickpea product is shown in Table 1. Hen egg white powder (EWP) was selected as the reference to evaluate the ability of aquafaba as an egg white substitute. Pasteurized and spray dried hen EWP with high whip ability was provided by Sanovo Foods A/S, Odense, Denmark and stored in plastic bags at 5 °C throughout the entire study to assure optimal product quality. Sodium chloride (NaCl) was supplied from Merk Millipore (Burlington, MA, USA). For the SDS-PAGE analysis in section 2.5, a chickpea protein isolate was prepared based on dried chickpeas. Chickpeas (Spaniola, Kabuli) were harvested in North America and imported by System Frugt, Tilst, Denmark. The chickpeas were soaked with Milli-Q ultrapure water (Millipore Corporation, Burlington, MA, USA) at a 1:5 ratio (w/v) for 12 h before the soaking water was discarded and the chickpeas were boiled with Milli-Q water at a 1:5 ratio (w/v) for 1 h. The cooked chickpea seeds were crushed initially in a household, coffee blender and finally sieved through a fine-mesh screen to obtain chickpea flour. Chickpea protein isolate was prepared by the method of Ladjal-Ettoumi, Boudries, Chibane, and Romero (2016) original developed by Papalamprou et al. (2010) with modifications as only 5 g of the chickpea flour was used. Moreover, an additional centrifugation step at 4500×g for 10 min at 4 °C was introduced after both washing steps. A protein suspension of 2 g/L was prepared by mixing 20 mg of the freezedried chickpea protein isolate with 10 mL of Milli-Q water. The suspension was stirred on a magnetic stirrer at 600 rpm for 30 min at room temperature (r.t.). A volume of 2 mL was subjected to an oscillation movement performed at 50 1/s for 5 min in a TissueLyser (Qiagen, Hilden, Germany) to dissolve the protein isolate followed by centrifugation at 1000×g for 1 min and the supernatant was collected for further analysis.

2.3. Dry matter content The dry matter content was measured in a Mettler Toledo moisture analyzer (Greifensee, Switzerland) by heating either 1.7 g of CAF or 0.2 g of EWP at 125 °C until weight equilibria was established and the final water content (%) was measured. The dry matter content (%) was calculated by a subtraction of the water content (%) from a total of 100%. Measurements were conducted in triplicates. 2.4. Protein concentration determination Protein concentration was quantified by the bicinchoninic acid assay (BCA, Thermo Scientific™, USA). The reagent assay containing Cu2+ and BCA was added to a 92-micro well plate together with 25 μL of c = 10 g/l of the protein sample or reference protein, i.e. 250 μg protein, and was incubated for 30 min at 37 °C followed by cooling for 5 min at r.t. before the UV-absorbance was measured at 562 nm in a 355

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volume at 1 min and Vfoam, t = foam volume at time t = 1–90 min.

Synergy™ 2 multi-Mode microplate reader (BioTek Instruments Inc, Winooski VT, USA). The detected UV absorbance values were converted to protein concentrations (g/L) based on a regression curve of 2 g/L bovine serum albumin used as the reference protein. All measurements were performed in triplicates with additional three replicates per sample in the 92- micro well plate.

2.8. Emulsion preparation Oil in water emulsions (O/W) were prepared by mixing 6 mL of a 10 g/L protein source and 18 mL of commercial sunflower oil (Ponte Sisto, Nyborg, Denmark) resulting in an oil volume fraction of 0.75. This protein and oil sample was mixed by an Ultra Turrax T25 (IKA Werke GmbH & Co. KG, Staufen, Germany) for 4.5 min at 8000 rpm followed by 4 min at 13,500 rpm according to the procedure by Schmidt, Damgaard, Greve-Poulsen, Bach Larsen, and Hammershøj (2018) with modification. Emulsions for rheological analysis were prepared similarly, only in larger sample volumes.

2.5. SDS-PAGE protein profile A Sodium Dodecyl Sulfate Poly Acrylamide Gel Electrophoresis (SDS-PAGE) protein profile of the samples was elucidated using Criterion™ TGX Strain-Free™ 4–15% precast gels (Bio-Rad, Richmond, CA, USA) and performed essentially as the method described of Laemmli (1970) with modifications. Centrifugated aquafaba, chickpea protein isolate or EWP were mixed 1:1 with sample buffer (20 mM Tris, 2% SDS, 20% glycerol, pyronin Y) and disulfide bond reduction was done with 0.2 M dithioerythritol (DTE) before heated for 3 min at 95 °C. Sample volumes of 15 μL and concentration of 1 g/L for all samples were loaded onto the gel and electrophoresis was run at a constant current at 200 V for 30 min in running buffer (0.25 M Tris, 0.192 M glycine, 0.1% SDS). An additional SDS-PAGE was run with 0.5 g/L of EWP, due to an overload of specifically ovalbumin bands. The molecular masses of protein bands were estimated by known molecular weight (Mw) markers (Thermo Scientific™, Spectra™ Multicolor High Range Protein marker and Thermo Scientific™, Spectra™ Multicolor Broad Range Protein marker) and the Image Lab 5.2.1 estimation computer software (Bio-Rad, Richmond, CA, USA). To visualise some protein bands better, both unstained gels and gels stained with Coomassie Brilliant Blue G-250 were prepared.

2.9. Emulsifying activity and stability The emulsifying activity index (equation (4)) and emulsifying stability index (equation (5)) were evaluated by the method of Cheung, Wanasundara, and Nickerson (2014) originally developed by Pearce and Kinsella (1978). Emulsions were stored at 5 °C in minimum 2 h before dilution in order to stabilize the samples prior to the analysis according to the procedure by (Schmidt et al., 2018). The UV absorbance at λ = 500 nm was analyzed (UV–visible spectrophotometer, Cary 60, Agilent Technologies, CA, USA) with the 0.1% SDS solution as reference (Schmidt et al., 2018). The emulsifying activity index (EAI) and emulsifying stability index (ESI) values were calculated by the following equations.

m2 2 × 2.303 × A0 × N EAI ⎜⎛ ⎟⎞ = c × φ × 10000 ⎝ g ⎠

2.6. Zeta potential

ESI (min) = The zeta potential (mV) of centrifugated aquafaba with a protein concentration of 10 g/L was tested as function of pH by a Stabino® (Particle Metrix, Meerbusch, Germany) coupled with a 400 μm piston. A stable level (mV) was ensured prior to the measurement by an equilibration time of 180 s, where only the endpoint (mV) was recorded. The zeta potential is evaluated by a constant oscillation movement of the piston, which produces a streaming potential. The electrophoretic potential of a zeta potential standard of 0.01 mol/L KCl is used for calibration of the streaming potential. The data were obtained in triplicates with two technical replications per sample.

2.10. Emulsion droplet size and distribution The emulsion droplet size distribution was measured by integrated light scattering using a Master Sizer 2000 (Malvern Instruments Ltd, Malvern, U.K.) fitted with a Hydro 2000S sample unit. Emulsions were diluted with 1% SDS in a 1:2 ratio (v:v) and volume-weighted mean diameter, D 4,3 was obtained. Measurements were conducted in quadruplicates with three technical replications per sample.

Foamability and foam stability were evaluated by the hand shaken foam method as described earlier by Hammershøj, Peters, and Andersen (2004) with minor modifications. Foam was generated by shaking 20 mL of samples with protein concentration of 10 g/L in a 100 mL closed glass cylinder for 45 s at a frequency of 4 Hz. All measurements were conducted in triplicates. Foam capacity (equation (1)) was evaluated as relative foam overrun (Hammershøj et al., 2004) and foam stability as liquid drainage (equation (2)) and as liquid ratio in foam (equation (3)) (Hammershøj, Prins, & Qvist, 1999).

Vfoam,

t = 1 min

Vliquid,

t = 0 min

Liquid drainage =

[L/ L]

Vliquid t = 0 min − DVliquid, t

Liquid ratio in foam =

t

[L/ L]

t = 1min

Vliquid t = 0 min − DVliquid, t Vfoam,

2.11. Rheology of emulsions for yield stress analysis Yield stress (Pa) measurements of the emulsions were performed in an AR G2 rheometer (TA instruments, New Castle, DE, USA) fitted with a 28 mm diameter and 42 mm height four-bladed vane geometry. The four-bladed vane geometry was chosen in order to minimize any wallslip issues and to ensure minimal deformation of the emulsion before analysis. A disadvantage of the vane geometry is however its requirement of a large sample volume (Barnes & Nguyen, 2001). Emulsion samples were kept cool at 5 °C for at least 2 h prior to the analysis and measured directly in the 150 mL glass beakers, in which they were prepared to ensure undisturbed emulsions. The stepped flow test included a 1 min equilibration at the condition step followed by the step flow step, which counted a shear stress ramp range of 1.0–100.0 Pa. In total, 70 sample points were detected at a constant time of 20 s for each shear stress (Pa) value measured. Measurements were conducted in duplicates. The yield stress value (Pa) of the relationship between strain and shear stress was determined by the tangent cross-over method (Steffe, 1996). In principle, this method analyses the point of

(1)

Vliquid t = 0 min − DVliquid,

[L/ L]

(5)

where A0 = absorbance at 0 min and A10 = absorbance at 10 min, N is the dilution factor (500), c is the protein concentration (0.01 g/mL), t = time (min), and φ is the oil volume fraction of the emulsion (0.75). All measurements are performed at r.t. and measured in triplicates.

2.7. Analysis of foaming properties

Foam overrun =

A0 × t A0 − A10

(4)

(2)

(3)

Vfoam = foam volume at 1 min, Vliquid = initial liquid volume, DVliquid = drained liquid volume at time t = 1–90 min, Vliquid = liquid 356

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intersection, where the two tangents crosses. The first curve defines where the emulsion is below the yield stress value, i.e. has a solid-like behavior, and opposite of the second curve, which indicates a fluid-like behavior of the emulsion, hence, the yield stress value is exceeded (Steffe, 1996).

is assumed to be lipoxygenase (Singh, Wani, Kaur, & Sogi, 2008). Egg white powder showed bands at 63 kDa, 42 kDa, and 14 kDa beyond others, corresponding well with known masses of ovotransferrin, ovalbumin, and lysozyme, respectively (Hammershøj et al., 2004). The surface net charge of centrifugated aquafaba at different pH values was evaluated by a zeta potential analysis (Fig. 2). The proteins carried a net negative charge at pH 8.5 (−2.5 mV) towards pH 4.6 (isoelectric point), where the net charge became 0 mV, followed by an increase in charge to 0.3 mV at pH 3.

2.12. Data analysis Means and standard deviations for all data were calculated with Microsoft excel 2010. The statistical analysis was performed by the statistical software, Minitab version 18 (Minitab Inc, United Kingdom). A minimum of 95 %-level (P < 0.05) was considered as statistically significant with a 0.95% confidence interval and normal distribution of the data were ensured prior to the statistical analysis. One-way analysis (ANOVA) was applied for set-ups, which included one variable i.e. pH, conductivity, or protein type. The Turkey's honest significant difference (HSD) test was used to determine statistically significant differences between means. A two-way analysis (ANOVA) was used in set-ups with two variables, i.e. pH and time; conductivity and time; and protein type and time.

3.2. Foaming properties The foam capacity (L/L) of centrifugated aquafaba evaluated as overrun was affected neither by change in conductivity (Fig. 3A) nor by change in pH (Fig. 3B). A comparison of the foam capacity (L/L) showed significantly (P < 0.05) higher foam capacity (L/L) for EWP than for centrifugated aquafaba at pH 6 and 16 mS/cm (Fig. 3C). Gravity affects foam stability as is cause liquid to drain from the foam. Foam stability was evaluated as the liquid drainage per initially bound liquid after foam production and as liquid ratio in foam (L/L) over time (Fig. 4 and Fig. 5). Liquid drainage of centrifugated aquafaba was not affected by change in conductivity (Fig. 4A), however, there was a significant effect by change in pH (P < 0.05) (Fig. 4B). No significant difference in the liquid drainage pattern was observed between EWP and centrifugated aquafaba at pH 6 and 16 mS/cm (Fig. 4C). Change in time affected all factors (P < 0.05), however, no significant interactions were found (Fig. 4D). It is by the amount of liquid retained in foam possible to determine the degree of foam dryness or wetness. The liquid ratio in foam (L/L) decreased significantly with time (P < 0.05) for all factors (Fig. 5D), i.e. as liquid drained from the foam, the remaining foam structure contained less moisture and hence became more dry in nature. Change in pH of centrifugated aquafaba significantly (P < 0.05) affected the liquid ratio (Fig. 5B), however, no effect was observed by change in conductivity (Fig. 5A). In comparison, centrifugated aquafaba and EWP showed significantly different liquid ratio patterns at pH 6 and 16 mS/ cm (P < 0.05) (Fig. 5C). Centrifugated aquafaba had a higher liquid ratio in the foam (L/L) compared to EWP, i.e. a drier foam for EWP than centrifugated aquafaba. The interaction between time and each factor was in-significant (Fig. 5D).

3. Results 3.1. Protein content and composition Only small, non-significant (P = 0.429), differences in the protein concentration (g/L) of centrifugated aquafaba evaluated at different pH values were observed. The amount of acid/base added to centrifugated aquafaba depended on the molarity (M) of the acid/base and by the pH value's distance to the original pH value of aquafaba (Table 3). The protein composition evaluation of centrifugated aquafaba, chickpea protein isolate and EWP revealed detectable protein bands with an intensively increased band appearance followed by staining (Fig. 1). Proteins of large Mw were observed for EWP at unstained and un-reduced conditions as proteins remained in the well of lane 7 (Fig. 1A) and lane 11 (Fig. 1C). Centrifugated aquafaba, independent of whether the pre-handling conditions were frozen or fresh, showed similar protein composition with bands at 23 kDa, 15 kDa, 12 kDa and 10 kDa detected at unstained conditions (Fig. 1A). In general, no differences between the un-reduced and reduced stage of centrifugated aquafaba were observed. An unspecific polymerization of centrifugated aquafaba was observed after staining as smeared bands in the high-Mw end of the SDS-PAGE gel (Fig. 1B). Chickpea protein isolate revealed beyond the detected bands of centrifugated aquafaba additional bands with higher molecular at 99 kDa, 51 kDa, 39 kDa especially prominent after staining. The subunits of 10 kDa and 12 kDa may be attributable to the subunits of the 2S albumin (Vioque et al., 1999). The 51 kDa-band was assigned to the oligomeric protein, 7S vicilin and the band of 16 kDa might correspond to the γ-subunit of 7S vicilin (Aryee & Boye, 2017; Ladjal-Ettoumi et al., 2016). The subunits of 23 kDa and 39 kDa might be ascribed to the basic and acidic subunits of 11S legumin (Aryee & Boye, 2017; Ladjal-Ettoumi et al., 2016) and the 99 kDa-band

3.3. Emulsion properties The emulsion activity index (EAI), which gives the interfacial area stabilized by emulsifier per unit of weight, was 3-fold higher for centrifugated aquafaba based-emulsions than EWP based-emulsions at pH 6 and 16 mS/cm (P < 0.05) (Fig. 6C). The change in conductivity of centrifugated aquafaba did not affect the EAI (Fig. 6A), however, change in pH significantly affected the EAI (P < 0.05) (Fig. 6B). The emulsion stability index (ESI), which is a measurement of the emulsion's stability over time, showed same pattern as observed for the EAI (Fig. 7). Again, the change in conductivity of centrifugated aquafaba did not cause an affect (Fig. 7A). However, a significant effect of change in pH (P < 0.05) on the ESI was observed with increasingly higher ESI at pH > 6 (Fig. 7B). The ESI was higher for centrifugated aquafaba based-emulsions compared to EWP-based emulsions at pH 6 and 16 mS/cm (P < 0.05) (Fig. 7C). The mean emulsion droplet diameter evaluated as volume weighted diameter (D 4,3) differed significantly for centrifugated aquafaba based-emulsions at different pH values, with decreased D 4,3 as pH increased (Fig. 8B). The emulsion droplet size distributions as function of pH are provided in Fig. 9. The change in conductivity of centrifugated aquafaba did not have a significant effect on D 4,3 (Fig. 8A). EWP-based emulsions consisted of larger oil droplets compared to centrifugated aquafaba-based emulsions at pH 6 and 16 mS/cm

Table 3 Protein concentration (g/L) of centrifugated aquafaba at different pH values. The ‘% acid or base’ is the amount of acid or base used for each pH stabilization. Type of acid or base and molar (M) are given, n = 3, values showed as mean ± SD. Sample

Protein concentration (g/ L)

% Acid or base

Acid or base

Molar (M)

pH pH pH pH pH

13.00 13.65 13.26 13.86 13.24

1.00 0.41 0.13 1.13 1.03

HCl HCl HCl NaOH NaOH

6 6 1 1 2

3 4.5 6 7 8.5

± ± ± ± ±

0.23 0.97 0.68 0.37 0.31

± ± ± ± ±

0.12 0.02 0.01 0.22 0.07

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Fig. 1. SDS-PAGE gel of centrifugated aquafaba, chickpea protein isolate and of egg white powder (EWP) at pH 6, as both non-reduced (-) and reduced (+) and as A + C) unstained, and B + D) stained with coomassie Brilliant Blue. Lanes 1 and 2: Fresh centrifugated aquafaba, Lanes 3, 4, 9 and 10: Frozen centrifugated aquafaba; Lanes 5 and 6: Chickpea protein isolate, Lanes 7, 8, 11 and 12: EWP, Mw: Molecular weight markers with masses given in kDa. Note: A + B) protein concentration of all samples = 1 g/L, and C + D) protein concentration of aquafaba = 1 g/l and of EWP = 0.5 g/L.

(Fig. 8C). The yield stress value (Pa) of emulsions was analyzed by a strain/ shear stress relationship and evaluated by the tangent cross-over method (Weiss, 2017). The cross point of the two tangents marks the yield stress value (Pa) (Juszczak, Fortuna, & Kośla, 2003). The first tangent at low stress values, indicates the situation, where the emulsion is below the yield stress level and the second slope, defines where the emulsion has exceeded its level of yield stress (Weiss, 2017). The yield stress values of centrifugated aquafaba-based emulsions were not affected by change in conductivity, however, by change in pH (Table 4). The lowest pH value at pH 3 displayed the highest level of yield stress (> 50 Pa) followed by a significant decline with increased pH value until pH 7.0–8.5, where a steady yield stress was observed. Emulsions based on centrifugated aquafaba showed a significantly higher yield stress value (Pa) compared to the emulsions based on EWP at pH 6 and 16 mS/cm (Table 4) with the rheometer output illustrated (Fig. 10). Fig. 2. Zeta potential (mV) of centrifugated aquafaba as function of pH, c = 10 g/L. Vertical bars indicate ± SD, n = 3.

Fig. 3. Foam capacity as overrun (L/L) of centrifugated aquafaba (CAF), c = 10 g/L as function of A) conductivity (mS/cm) and B) pH and C) comparison of egg white powder (EWP), c = 10 g/L, and CAF at pH 6 and 16 mS/cm. Bars are means ± SD, n = 3, and n = 6 for CAF, and different letters indicate significant differences (P < 0.05), NS = not significant.

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Fig. 4. Foam stability of centrifugated aquafaba (CAF), c = 10 g/L as liquid drainage per initially bond liquid (L/L)as function of time after foam production and of A) conductivity and B) pH, C) comparison of egg white powder (EWP), c = 10 g/L, and CAF at pH 6 and 16 mS/cm, and D) overview of statistical significance levels of the experimental factors in A, B, and C. Vertical bars indicate ± SD, n = 3 and n = 6 for CAF, NS = not significant.

4. Discussion

and 4, respectively. Whether the aquafaba proteins were reduced by DTE or not did not change the band positions on the SDS-PAGE. This unaffected behavior of centrifugated aquafaba indicates proteins with either a denatured and covalent bond ruptured structure, which may be due to the processing of canned chickpeas, or that the proteins contain no intermolecular disulfide bonds (Alajaji & El-Adawy, 2006; Boye et al., 2010). This could be a useful property in a variety of food applications where protein unfolding, and no disulfide bonding is wanted (Ma et al., 2011; Moure, Sineiro, Domínguez, & Parajó, 2006). The protein composition of chickpea protein isolate revealed similar protein composition as centrifugated aquafaba for bands of size ≤24 kDa, which indicates that proteins with larger Mw are retained in the chickpeas during cooking. Functional properties of proteins are dependent on their surface charge (Tang & Sun, 2011), which in this study was evaluated as the zeta potential as function of pH. The pI value of 4.6 detected for centrifugated aquafaba is very close to the value of the isoelectric point found for chickpea protein isolate of pH 4.5 (Ladjal-Ettoumi et al., 2016). The surface charge pattern of centrifugated aquafaba as function of pH changed from a net negative charge towards a positive charge by decreased pH level. Similar patterns are observed for other proteins e.g. protein isolates based on legumes (Gumus et al., 2017; Karaca, Nickerson, & Low, 2011). In general, a low charge (mV) was observed for centrifugated aquafaba, which can be explained by the inherent level of NaCl (Gumus et al., 2017).

4.1. Conditions of pH and conductivity A total of eight combined variations were included in this experimental set-up, which required a center point of analysis with fixed levels of both pH and conductivity. The level of conductivity was given beforehand by the natural content of NaCl in aquafaba and a pH level of pH 6.0 was chosen as the center point of pH to accomplish a value approximating to both the natural pH level of fresh egg white ∼ pH 7 (Hammershøj et al., 1999) and of centrifugated aquafaba (pH 6.13). The EWP functioned as the reference sample to aquafaba. 4.2. Protein composition The protein determination of centrifugated aquafaba showed an average protein concentration of 1.3% w/v. This result is consistent with the findings of Mustafa et al. (2018) and Stantiall et al. (2018), who report a protein concentration of 1.5% and 0.95% of aquafaba, respectively. The dry matter content of centrifugated aquafaba was found to account for 7.89 ± 0.09% w/v of the plant-based liquid. This result is supported by recent literature of aquafaba (Mustafa et al., 2018; Stantiall et al., 2018), which suggest that the dry matter content mainly consists of carbohydrates in form of sugars, water soluble- and insoluble fibers as well as protein. The low Mw (≤24 kDa) bands found for proteins of centrifugated aquafaba are consistent with the result obtained by Mustafa et al. (2018), who suggest that the proteins of aquafaba belong to the group of albumins (Singh et al., 2008). No significant differences were observed between the pre-conditions of centrifugated aquafaba; whether it was fresh or frozen as illustrated in Fig. 1, lanes 1 and 2 versus lanes 3

4.3. Foam properties A significantly higher foam capacity was observed for egg white compared to centrifugated aquafaba. These findings are in accordance with Stantiall et al. (2018), who find that water boiled with chickpeas has significant lower foam capacity than foam prepared by fresh egg 359

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Fig. 5. Foaming stability of centrifugated aquafaba (CAF), c = 10 g/L as liquid ratio in foam (mL/mL) as function of time and of A) conductivity and B) pH, C) comparison of egg white powder (EWP) and CAF at pH 6 and 16 mS/cm and D) overview of statistical significance levels of the experimental factors in A, B, and C. Vertical bars indicate ± SD, n = 3 and n = 6 for CAF, NS = not significant.

As gravity cause liquid to drain from the foam, a measurement of the foam stability as liquid retention is appropriate to categorize the dryness of the foam. Foam produced by centrifugated aquafaba was characterized as more wet compared to foam produced by egg white, as the first had a higher liquid ratio, i.e. more liquid relatively to the foam volume. This difference in the liquid retention could suggest different targeted use, as centrifugated aquafaba could be more relevant in highmoisture-based food applications than egg white. For both of the foam stability measurements, the effect of gravity was considered a significant factor, as all foam produced did change over time. The pH of centrifugated aquafaba also significantly affected both liquid ratio in foam as well as liquid drainage (Figs. 4B & 5B), where aquafaba foam at pH 4.5 (near pI) remained highly stable for up to 1 h. The results indicate that the protein surface charge affect the foam stability, and previous research show an increased foam stability

white. However, we found that the foam capacity of centrifugated aquafaba was neither affected by changes in level of pH nor the content of NaCl within the studied range. Earlier studies of foam capacity of egg white showed an affect by the increase of NaCl (Raikos, Campbell, & Euston, 2007) as well as by change in pH (Hammershøj et al., 1999). The opposing resistance towards changes in chemical environment observed for the two protein sources support previous studies of aquafaba as a relevant egg white replacement candidate in food foams (Mustafa et al., 2018; Stantiall et al., 2018). Liquid drainage from the foam is mainly due to gravity force (Hammershøj et al., 1999; Murray & Ettelaie, 2004). Foam produced by centrifugated aquafaba and egg white were not significantly different to each other, when evaluated for liquid drainage/initially bound liquid as function of time. This result might be blurred by the large standard deviations observed.

Fig. 6. Emulsion activity index (EAI) of centrifugated aquafaba (CAF), c = 10 g/L as function of A) conductivity and B) pH and C) comparison of egg white powder (EWP) and CAF at pH 6 and 16 mS/cm. Vertical bars indicate ± SD, n = 3 and n = 6 for CAF. Values with different letters are significantly different (P < 0.05), NS = not significant.

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Fig. 7. Emulsion stability index (ESI) of centrifugated aquafaba (CAF), c = 10 g/L as function of A) conductivity and B) pH level and C) comparison of egg white powder (EWP) and CAF at pH 6 and 16 mS/cm. Vertical bars indicate ± SD, n = 3 and n = 6 for CAF. Values with different letters are significantly different (P < 0.05), NS = not significant.

for pH samples near the isoelectric region, where the net charge of the proteins are zero and the electrostatic forces are minimal (Hammershøj et al., 1999). At this pH region, hydrophobic interactions and hydrogen bonding between proteins may be enhanced due to shorter distance between proteins, which cause strong interfacial film formation and thereby stabilize the foam (Aluko, Mofolasayo, & Watts, 2009; Hammershøj et al., 1999). The ability to form a film that stabilizes the air/water interface (Zhang, Dalgleish, & Goff, 2004) and lower the surface tension are important for proteins to ensure good foaming properties (Hammershøj et al., 1999). None of the foam stability parameters of centrifugated aquafaba were affected by the applied NaCl-levels. These findings are inconsistent with the suggestion of a higher water retention rate for samples with a high level of NaCl, which is explained by an ability of surfactant molecules to readily adsorb to the air-water interface (Raikos et al., 2007). Fig. 9. Mean volume distribution (%) of emulsion oil droplet sizes (μm) on logscale of centrifugated aquafaba as function of pH values, n = 3.

4.4. Emulsion properties

Table 4 Yield stress (Pa) of centrifugated aquafaba (CAF) as function of conductivity, pH and a comparison of egg white powder (EWP) and CAF at pH 6 and 16 mS/ cm, c = 10 g/L, values shown as mean ± SD.

Analysis of the emulsifying properties by the emulsifying activity index (EAI) and the emulsifying stability index (ESI) showed similar results. The emulsifying activity index defines the emulsifier's ability to cover the interfacial area of oil and water (Motta-romero, Zhang, Nguyen, Schlegel, & Zhang, 2017) and the emulsifying stability index reflects the emulsion's stability over time and its ability to resist changes in its microstructure (Boye et al., 2010). The centrifugated aquafaba-based emulsions showed a significantly higher emulsifying activity index and stability index compared to emulsions prepared by egg white. This indicated a faster and greater ability and a higher stability of proteins of centrifugated aquafaba to adsorb at the interface between oil and water and to resist changes in the microstructure compared to the proteins of egg white. Changes in the content of NaCl did not affect neither the emulsifying stability index nor the emulsifying activity index of centrifugated aquafaba. However, for cruciferin

NaCl

Yield stress

mS/cm

Pa

16 24 32

48.75 ± 0.37a 49.18 ± 0.04a 49.49 ± 0.47a

pH

Yield stress

Protein type

Pa 3.0 4.5 6.0 7.0 8.5

51.50 48.97 47.88 45.82 46.51

Yield stress Pa

± ± ± ± ±

0.15a 0.13b 0.19c 0.34d 0.14d

EWP CAF

38.79 ± 1.60b 48.32 ± 0.56a

a-d

values within a column with different superscript letters differ significantly (P < 0.05).

Fig. 8. Mean emulsion droplet diameter given as volume-weighted diameter D 4,3 (μm) of centrifugated aquafaba (CAF), c = 10 g/L as function of A) conductivity and B) pH level and C) comparison of egg white powder (EWP) and CAF at pH 6 and 16 mS/cm. Vertical bars indicate ± SD, n = 4 for all samples, n = 8 for CAF. Values with different letters are significantly different (P < 0.05), NS = not significant.

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there was a tendency to low stability and larger droplets at pH 3.0–4.5 and vice versa with higher stability indexes and smaller droplets at pH ≥ 6. Other parameters such as how homogeneous the emulsion droplet sizes are, the viscosity of the emulsion, and density difference between water phase and oil phase will also affect the emulsion stability according to the Stokes equation on velocity of settling (Yi, Li, Zhong, & Yokoyama, 2014), which are parameters relevant for further studies on aquafaba as emulsifier. Changes in conductivity had no effect on the volume-weighted diameter, D 4,3 of centrifugated aquafaba-based emulsions. Similar findings were observed by Gumus et al. (2017), who found no effect on the surface weighted particle size diameter, D 3,2 of lentil protein concentrate-stabilized emulsions as function of NaCl content ranging from 0 to 500 mM. In comparison, the centrifugated aquafaba-based emulsions showed significant lower volume-weighted diameter, D 4,3 than the emulsions based on egg white. These findings indicate a higher ability of the plant-based emulsion to form particles of smaller diameter, hence a higher likelihood of greater stability at all other conditions equal, which can explain the significantly higher ESI of centrifugated aquafaba emulsions compared with EWP emulsions (McClements, 2007; Yi et al., 2014). The viscoelastic behavior of the emulsions was analyzed by the yield stress value (Pa). Yield stress is a key parameter in the food industry regarding a variety of quality elements (Ma et al., 2016), and more important, a parameter concerning consumer acceptance (Juszczak et al., 2003). A food system shows different behavior dependent on whether the stress put upon it is positioned below or above its yield stress level, defined as the force per area required to deform a system in a constant manner (Barnes & Nguyen, 2001; Ma et al., 2016). The yield stress (Pa) was not affected by change in conductivity of centrifugated aquafaba, however change in pH was inversely correlated to the yield stress values. Based on these results, it is possible to deduce that, at neutral and basic pH values, centrifugated aquafaba-based emulsions requires less force to exceed its yield stress compared to emulsions at acidic pH values, i.e. may indicate less stable and/or less firm emulsions. These findings are however inconsistent with the results of the mean particle size, evaluated as the volume weighted diameter, D 4,3 of centrifugated aquafaba, and the results on emulsion stability index as for both parameters, higher stability (higher ESI, smaller droplets) was observed for emulsions at basic pH conditions. As for all other analysis of the emulsion properties, a significant difference in the yield stress (Pa) and the strain/shear stress relationship between emulsions based on egg white and emulsions based on centrifugated aquafaba were observed. Egg white-based emulsion showed a sudden appointment of its yield stress (Pa) in contrast to the slowly progress in the strain/shear stress relationship for aquafaba based emulsion. One parameter, which was not analysed or accounted for here, was the amount of water soluble carbohydrates (WSC) in the aquafaba studied. We could expect a certain amount of WSC in aquafaba as referred to earlier (Mustafa et al., 2018; Stantiall et al., 2018), and which has been found to correlate positively to emulsifying activity for legume soaking water used in bread baking (Huang et al., 2018). Further studies into aquafaba functional properties would benefit from analyzing carbohydrate content, composition, and pKa values in order to evaluate the emulsifying properties as function of the pH conditions. Besides the presently analysed functional properties, also gelling, water holding capacity and texture of aquafaba in applications of e.g. meat replacement will be relevant (Sharima-Abdullah et al., 2018). Attention should also be drawn to some potentially less favourable issues in using aquafaba or cooking water from pulses, where the phenolic compounds and saponins may interact with protein and thereby affect their functional properties, in addition to their sensory attributes of astringency and bitterness (Damian et al., 2018). Overall, the protein composition of the aquafaba and EWP were, expectedly, very different based on the SDS-PAGE patterns, due to their very different origin. Nevertheless, the proteins in both sources are

Fig. 10. Example of strain (-)/shear stress (Pa) curves for yields stress calculation of emulsions of egg white powder (white) and centrifugated aquafaba (black), c = 10 g/L. The insert shows the curves with strain on a log-axis for clarity of the behavior at low strain values.

protein isolate, Cheung et al. (2014) report that the emulsifying activity index and the emulsifying stability index are significantly affected by change in the NaCl-content of emulsions. In general, the EAI and ESI decreased with the addition of NaCl with concentrations at 0 mM, 50 and 100 mM tested at pH 3, 5 and 7. These NaCl levels correspond to the presently tested level of 24 mS/cm, where 106 mM NaCl was added to the aquafaba. The EAI correlates positively with surface hydrophobicity, as high hydrophobicity tend to lead to a better integration and alignment of the proteins into the interface of oil and water, leading to a higher EAI (Cheung et al., 2014). However, the emulsifying activity index and emulsifying stability index were affected by change in the pH level of centrifugated aquafaba. These results agree with those reported by Zhang, Jiang, Mu, and Wang (2009), who find a significant lower emulsifying activity index of samples near the isoelectric point ∼ pH 5.0 of chickpea proteins isolates, where we observed low values at pH 3.0 and pH 4.5 and the highest EAI and ESI at pH ≥ 6 for centrifugated aquafaba. The significant lower EAI and ESI at pH 4.5 for centrifugated aquafaba may be explained by the zero net charge at pI 4.6 giving weak attractive forces between the adsorbed proteins on the interfacial coated protein area (Zhang et al., 2009). In a recent study on dried chickpea protein isolates, the EAI is studied at pH ranging from pH 2 to pH 10, and lowest EAI appears at pH 6, while at pH 4 it was significantly increased (Tontul, Kasimoglu, Asik, Atbakan, & Topuz, 2018). The reason for this contradiction to our results on emulsion properties may be material differences in composition between whole chickpea protein isolate and the aquafaba. Furthermore, drying at high temperatures can cause denaturation of proteins and reduce the solubility, which can affect the molecular interactions in the O/W interface. Protein solubility of chickpea isolate is previously observed to be very low at pH 4 - pH 7 (Tontul et al., 2018), which was not the case for aquafaba (own observations, data not shown). The present observation of both a low EAI and low ESI at pH 3 could be suggested to be caused by other parameters affecting the emulsifying activity and stability of the proteins, such as the internal factors of conformation stability and surface hydrophobicity (Cheung et al., 2014; Moure et al., 2006; Tang & Sun, 2010). As observed for the emulsifying activity and stability index, the change in pH of centrifugated aquafaba also affected the mean particle diameter, D 4,3 as a decreased volume weighted diameter was observed with increased pH value. It is well known that emulsions with particles of small diameter tend to have a higher stability compared to emulsions of larger particle diameter (McClements, 2007; McClements et al., 2017). Significant instability correlating with large particles in samples near pI was not found for emulsions based on centrifugated aquafaba, whereas McClements et al. (2017) report of a significant effect of the isoelectric point of emulsion based on lentil or pea protein. However, 362

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responsible for the functional properties and will be affected by different physicochemical conditions, such as salting in/salting out and surface charge at different pH, which is of high relevance for foods. A direct correlation of the responsible proteins for specific functional properties will require more pure model systems to reveal such effects. However, in food systems the complexity in composition is high, hence our study aimed at the complete EWP and aquafaba materials behavior under realistic food conditions.

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5. Conclusion In summary, aquafaba showed food functional properties as this plant-based liquid had the ability to function both as a foaming agent as well as an emulsifier under food relevant conditions of pH and NaCl concentrations. As a plant-based alternative to egg white, aquafaba appears to be a relevant candidate as protein ingredient in foods where air-and oil-binding properties are important. More research is however needed to understand the aquafaba in details and to elucidate its properties. Especially the change in pH, which affected the emulsifying properties of aquafaba must be further analyzed as the mechanisms observed for other plant protein isolate studies do not correlate highly to aquafaba behavior. An effect of change in the content of NaCl was observed for other studies of emulsifying properties of plant protein but not in our study of aquafaba. This may be due to the inherent level of NaCl in the canned chickpea product. Furthermore, the conditions of pressure and high temperature processing of canned chickpeas also needs further research to correlate and optimize processing conditions to the aquafaba properties. Acknowledgements The authors would like to thank Sandra Beyer Gregersen and Jesper Malling Schmidt at Aarhus University for advice on laboratory analysis. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.foodhyd.2019.05.041. Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Declarations of interest None. References Alajaji, S. A., & El-Adawy, T. A. (2006). Nutritional composition of chickpea (Cicer arietinum L.) as affected by microwave cooking and other traditional cooking methods. Journal of Food Composition and Analysis, 19(8), 806–812. https://doi.org/10.1016/j. jfca.2006.03.015. Aluko, R. E., Mofolasayo, O. A., & Watts, B. M. (2009). Emulsifying and foaming properties of commercial yellow pea (Pisum sativum L.) seed flours. Journal of Agricultural and Food Chemistry, 57, 9793–9800. https://doi.org/10.1021/jf902199x. Aryee, A. N. A., & Boye, J. I. (2017). Comparative study of the effects of processing on the nutritional, physicochemical and functional properties of lentil. Journal of Food Processing and Preservation, 41(1), 24–30. https://doi.org/10.1111/jfpp.12824. Barnes, H. A., & Nguyen, Q. D. (2001). Rotating vane rheometry — a review. Journal of Non-newtonian Fluid Mechanics, 98(1), 1–14. https://doi.org/10.1016/S03770257(01)00095-7. Boye, J. I., Aksay, S., Roufik, S., Ribéreau, S., Mondor, M., Farnworth, E., et al. (2010). Comparison of the functional properties of pea, chickpea and lentil protein concentrates processed using ultrafiltration and isoelectric precipitation techniques. Food Research International, 43, 537–546. https://doi.org/10.1016/j.foodres.2009.07.021. Boye, J., Zare, F., & Pletch, A. (2010). Pulse proteins: Processing, characterization, functional properties and applications in food and feed. Food Research International, 43(2), 414–431. https://doi.org/10.1016/j.foodres.2009.09.003.

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