Composition and Properties of Aquafaba: Water Recovered from Commercially Canned Chickpeas

Summary

Aquafaba is a viscous juice from canned chickpea that, when stirred vigorously, produces a relatively stable white froth or foam. The primary research goal is to identify the components of aquafaba that contribute viscosifying/thickening properties using nuclear magnetic resonance (NMR), ultrafiltration, electrophoresis, and peptide mass fingerprinting.

Abstract

Chickpea and other pulses are commonly sold as canned products packed in a thick solution or a brine. This solution has recently been shown to produce stable foams and emulsions, and can act as a thickener. Recently interest in this product has been enhanced through the internet where it is proposed that this solution, now called aquafaba by a growing community, can be used a replacement for egg and milk protein. As aquafaba is both new and being developed by an internet based community little is known of its composition or properties. Aquafaba was recovered from 10 commercial canned chickpea products and correlations among aquafaba composition, density, viscosity and foaming properties were investigated. Proton NMR was used to characterize aquafaba composition before and after ultrafiltration through membranes with different molecular weight cut offs (MWCOs of 3, 10, or 50 kDa). A protocol for electrophoresis, and peptide mass fingerprinting is also presented. Those methods provided valuable information regarding components responsible for aquafaba functional properties. This information will allow the development of practices to produce standard commercial aquafaba products and may help consumers select products of superior or consistent utility.

Introduction

Increasingly vegetarian products are being developed that mimic the properties of meat, milk, and eggs. The functional properties of pulses are important in their current uses in food applications and their properties are being explored in the development of replacements for animal protein. For example, dairy alternatives sales were $8.80 Billion USD in 2015 and this market is growing rapidly. This market is projected to grow to $35.06 billion by 2024. Moreover, the upward trend in demand for plant-based milk substitutes is, in part, a result of consumer health concerns regarding cholesterol, antibiotics, and growth hormones often used in milk production¹. Similarly, vegetable protein and hydrocolloid egg replacer markets are rapidly expanding and a compound annual growth rate of 5.8% is expected for these materials over the next 8 years with sales of $1.5 billion USD expected in 2026². A growing number of consumers prefer vegan protein sources, allergen reduced diets and reduced carbon footprint for food products. Demand for pulse-based products, especially from lentil, chickpea, and faba bean are steadily growing due to the high protein content, dietary fiber and low saturated fat content of pulses³. Pulses also contain phytochemicals with potentially beneficial biological activity⁴.

Commercial entities, scientists and private individuals have taken different approaches to communicate the quality properties of chickpea based egg and milk replacements. Gugger et al.⁵ produced a milk-like product from high starch grains including adzuki bean and chickpea. In their described methods the proponents attempted to show that their product is unique and different from "aquafaba"⁶. In another commercial approach elucidated by Tetrick et al.⁷ a plant-based egg substitute was developed. Their patent application describes methods of combining pulse flour with known thickeners that emulate the function of egg white in baked materials. Typical formulae include 80-90% pulse flour and 10-20% thickening additives.

Peer reviewed literature also indicates the functionality possible with chickpea and has demonstrated that albumin protein fractions obtained from kabuli and desi chickpea flour have good emulsification properties. They have also found a significant effect of chickpea source on the albumin yield and performance⁸.

After the initial internet report describing "aquafaba" by French chef Joël Roessel, an open source movement is showing the utility of aquafaba as a replacement of egg white and dairy protein in many food applications. There are many highly viewed webpages and YouTube videos showing the incorporation of aquafaba in foods that emulate the qualities of ice cream, meringue, cheese, mayonnaise, scrambled eggs, and whipped cream. Most pioneers providing open source aquafaba applications (recipes) obtain their material by straining canned chickpeas and using the liquid in their recipes. These individuals are mostly not trained scientists. Video comments sections indicate that the respondents have copied the recipes and some have failed to replicate the successes of the aquafaba advocates.

All three approaches (corporate, scientific and open source) to developing egg and milk replacements have merit but are missing an important dimension. Applied scientists, basic scientists and individuals promulgating pulse-based products have incompletely characterized and standardized their input material. Standardization of a product for a specific use is a normal industrial practice. Chickpea cultivars have not been standardized for aquafaba quality and industrial canning practices are standardized to produce consistent chickpeas not aquafaba.

Based on studies of other commodities, it is predictable that both genotype and environment will contribute to pulse aquafaba quality. It is known that both genotype and environment affect kabuli chickpea canning properties⁹. Typically, genotypic effects are large between related species and smaller within members of a species. Variation in physical and chemical properties can be minimized through identity preservation that allows the selection of cultivars with desired properties. Environmental effects can also be large and are managed by quality evaluation and blending to standard performance in specific tests¹⁰.

There are many genetically distinct cultivars of chickpea in commercial production. For examples, the University of Saskatchewan Crop Development Centre, a major source of commercial chickpea germplasm, has released 23 chickpea cultivars since 1980 of which 6 are currently recommended for cultivation in Canada. While scientific manuscripts often describe the cultivar used in a study, the patents and internet pages that were surveyed did not indicate the cultivar used or the provenance of the chickpeas. The standardization of cultivars and handling could help users increase their success in using chickpea but this information is not available on canned chickpea products.

The objective of this research is to determine the aquafaba components that contribute foaming properties. Here,the rheological properties of aquafaba from commercial chickpea brands were compared and the chemical properties were studied by NMR, electrophoresis, and peptide mass fingerprinting. To our knowledge, this is the first research which describes the chemical composition and the functional properties of the aquafaba viscosifier components.

Protocol

Separation of Aquafaba from Chickpeas

1. Obtain cans of chick peas from local grocers and open with a manual can opener.
2. Label cans from A to J.
3. Separate chickpeas from aquafaba using a stainless steel meshed kitchen strainer and weigh the separated chickpeas and aquafaba.

Obtain a Representative Sample of Chickpeas and Aquafaba for Chemical Analysis.

4. Randomly select ten chickpeas after draining the aquafaba to determine moisture content.
5. Place the selected chickpeas in a drying tin and heat at 100 ± 2 °C for 16-18 h in a drying oven.
6. After drying, grind the chickpeas to a powder for further use (e.g. protein and carbon content analysis).
7. Freeze aquafaba and aquafaba foam from each source to -20 °C and then dry in a freeze dryer. The dried aquafaba will be used for nitrogen and carbon content determination.

Aquafaba Functional Properties

8. Determine aquafaba viscosity at 25 °C using a No. 2 shell cup.
   1. Immerse the shell cup in the aquafaba.
   2. Record the time required to drain the cup¹¹. A timer is started when the cup was lifted from the aquafaba and stopped when the stream leaving the cup stops.
   3. The aquafaba viscosity can be ascertained by a chart supplied with the cup that relates viscosity and drainage time.
9. Foaming capability
   1. Blend the aquafaba solution (100 mL) in a stainless steel bowl with a hand mixer at speed setting of 10 for 2 min.
   2. Record the foam volume immediately after blending 100 mL of the aquafaba solution as V_f100 by placing the foam in a graduated measuring cup.
   3. Allow the foam to stand and dry over time to observe foam stability and obtain a sample of dried foam.

Color Parameters of Chickpea Seed

10. Randomly select chickpea seed from each can for color determination.
11. Calibrate the Hunter lab colorimeter using white, black and reference standards prior to measurements.
12. Color coordinate values are determined by the CIE Lab system¹², including the L (positive represents white and 0 represents dark), a (positive is red and negative is green), and b (positive is yellow and negative is blue) in triplicate with day light 65°.

Protein and Carbon Contents

13. Determine protein and carbon contents by combustion using an elemental analyzer¹³. Protein content is estimated as nitrogen content multiplied by 6.25¹⁴.

Moisture Content

14. Determine seed and aquafaba moisture contents by heating samples at 100 ± 2 °C for 16-18 h in a drying oven¹⁵.

NMR Spectrometry

15. Sample preparation
    1. Prior to spectrometry, centrifuge aquafaba samples at 9200 × g for 10 min. After centrifugation, pass the supernatant through a 0.45 µm syringe filter (25 mm syringe filter with polytetrafluoroethylene (PTFE) membrane). Transfer the aquafaba sample (0.4 mL) into a NMR tube wuth 50 mg added deuterium oxide inside and subject the sample for NMR analysis.
    2. For the dried foam sample previously described, the sample (25 mg) is in deuterium oxide (500 mg) and the solution is subjected to NMR analysis.
    3. Place all samples for ¹³C-NMR and ¹H-NMR in capped 5 mm NMR tubes. Add deuterium oxide and 3-(trimethylsilyl)propionic-2,2,3,3-d₄ acid sodium salt (TMSP) to each NMR tube to provide a solvent lock signal and act as an internal standard, respectively.
16. NMR conditions
    1. Acquire proton NMR spectra with a 500 MHz NMR or similar high field NMR spectrometer) with at least 16 scans per spectrum using a water suppression program such as the double pulse field gradient spin echo proton NMR (DPFGSE-NMR) pulse sequence¹⁶.
    2. Adjust the spectral shift to place the TMSP peak at 0 ppm then use the integration software to determine relative amounts of compounds present.

Electrophoresis

NOTE: For this step, aquafaba that yielded the most stable foam (brand H) was selected. This brand did not contain salt.

17. Sample preparation
    1. Introduce aquafaba to the upper reservoir of three centrifugal regenerated cellulose ultrafiltration devices each with different molecular weight cut offs (MWCOs) of 3, 10, or 50 kDa.
    2. Place the centrifugal filter units in a suitable centrifuge at 4 °C and centrifuge at 3900 × g for 2 h to obtain supernatant and retentate fractions. The supernatant fractions were used for ¹H-NMR analysis of smaller molecules in the absence of higher molecular weight (MW) species. The 3 kDa membrane retentate fraction was used for electrophoretic separation as it was believed that this membrane would retain most proteins.
    3. Estimate the protein contents of both supernatant and retenate using a modified Bradford method using bovine serum albumin as a standard¹⁷.
    4. Place samples of supernatant and retentate fractions in Eppendorf tubes and subject these fractions to shaking at a frequency of 25 per s (10 min) in a suitable shaker¹⁸. Observe the shaken solution to determine if a foam has formed from shaking.
    5. Dissolve the retentate by adding 2.0 mL of distilled water to the ultrafiltration device for a second centrifugation treatment to achieve diafilteration. Subject the ultrafiltration device to a second centrifugation at 4 °C and 3900 × g for 2 h.
    6. Mix the retentate (0.025 g) with 1 mL of 0.02 M Tris-HCl pH 7.4 or phosphate-buffered saline (PBS) pH 7.4 to dissolve protein.
    7. Centrifuge the mixture at 21000 x g for 1 min.
    8. Use the supernatant to determine protein content using the modified Bradford as previously mentioned.
18. Electrophoretic separation
    NOTE: The 3 kDa MWCO membrane retentate (described above) is subjected to electrophoretic separation using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
    1. Prepare polyacrylamide gels using a 15% polyacrylamide resolving gel and a 5% polyacrylamide stacking gel.
    2. Apply a sample of 20 µg protein to one lane of the gel and PageRuler Prestained Protein Ladder with a range of 10-170 kDa to a separate polyacrylamide gel lane.
    3. Subject the gels to an electrical current in a Mini-Protein Tetra Cell system as modified from Laemmli (1970)¹⁹. For electrophoresis operating conditions, gel staining, and destaining follow Ratanapariyanuch et al. (2012)²⁰.

Peptide Mass Fingerprinting

NOTE: Cut bands from the SDS-PAGE gel of 3 kDa retentate (MWs of approximately 8, 10, 13, 14, 15, 20, 22, 31, 37, 55, and 100 kDa) for trypsin digestion according to Ratanapariyanuch et al. (2012)²⁰ and perform mass spectral analysis.

19. In-gel digestion
    1. De-stain the bands twice by immersing in 100 µL of 200 mM ammonium bicarbonate (NH₄HCO₃) in 50% acetonitrile (ACN) and incubate at 30 °C for 20 min.
    2. After the completion of de-staining, treat the gel samples with ACN (100 µL) for 10 min and then dry under vacuum, using a centrifugal vacuum evaporator, for 15 min at room temperature.
    3. Immerse the dried gel samples in 100 µL of 10 mM dithiothreitol (DTT) in 100 mM NH₄HCO₃ solution and incubate at 56 °C for 1 h.
    4. Remove excess reducing buffer and replace it with 100 µL alkylating buffer [100 mM iodoacetamide in mass spectrometry (MS) grade water].
    5. Incubate the gel samples at room temperature in the dark for 30 min.
    6. Wash gel samples twice with 200 mM NH₄HCO₃ (100 µL), shrink the gels by immersing them in ACN (100 µL), re-swell the gels with 200 mM NH₄HCO₃ (100 µL) and again shrink by treating with ACN (100 µL).
    7. Drain the ACN and dry the gel samples under vacuum in a centrifugal vacuum evaporator for 15 min.
    8. Re-swell dried gel samples using 20 µL trypsin buffer (50 ng/µL trypsin in 1:1,200 mM NH₄HCO₃: Trypsin stock solution) followed by addition of 30 µL of 200 mM NH₄HCO₃ to each sample.
    9. Incubate samples on a Thermomixer overnight at 30 °C with shaking at 300 rpm.
    10. Stop the trypsin digestion reaction by adding 1/10 of the final volume (volume of mixture after step 8) 1% trifluoracetic acid and extract the tryptic peptides from gel samples using 100 µL of 0.1% trifluoracetic acid in 60% ACN and dry under vacuum using a centrifugal vacuum evaporator.
    11. Store tryptic peptides in -80 °C prior to mass spectrometric analysis.
20. Mass spectrometry
    1. Reconstitute tryptic peptides by adding 12 µL of MS grade water: ACN:formic acid (97:3:0.1, v/v) and then vortex for 1 to 2 min to dissolve peptides.
    2. Centrifuge the resulting solution at 18,000 g for 10 min at 4 °C.
    3. Transfer solution aliquots (10 µL) to MS vials for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis on a quadrupole time-of-flight (QTOF) mass spectrometer equipped with a liquid chromatography instrument and a LC-MS interface.
    4. Perform chromatographic peptide separation using a high-capacity HPLC-Chip: consisting of a 360 nL enrichment column and a 75 µm × 150 mm analytical column, both packed with C18-A, 180 Å, 3 µm stationary phase.
    5. Load samples onto the enrichment column with 0.1% formic acid in water at a flow rate of 2.0 µL/min.
    6. Transfer samples from the enrichment column to the analytical column.
    7. Peptide separation conditions are: a linear gradient solvent system consisting of solvent A (0.1% formic acid in water) and solvent B (0.1% formic acid in ACN). The linear gradient begins with 3% solvent B that increases to 25% solvent B over 50 min. Subsequently solvent B increases from 25 to 90% over 10 min at a flow rate of 0.3 µL/min.
    8. Acquire positive-ion electrospray mass spectral data using a capillary voltage set at 1900 V, the fragment ion set at 360 V, and the drying gas (nitrogen) set at 225 °C with a flow rate of 12.0 L/min.
    9. Collect spectral results over a mass range of 250-1700 (mass/charge; m/z) at a scan rate of 8 spectra/s. Collect MS/MS data over a range of 50-1700 m/z and a set isolation width of 1.3 atomic mass units. Select the top 20 most intense precursor ions for each MS scan for tandem MS with a 0.25 min active exclusion.
21. Protein identification
    1. Convert spectral data to a mass/charge data format using Agilent MassHunter Qualitative Analysis Software.
    2. Process data against the UniProt Cicer arietinum (chickpea) database, using SpectrumMill as the database search engine.
    3. Include search parameters such as a fragment mass error of 50 ppm, a parent mass error of 20 ppm, trypsin cleavage specificity, and carbamidomethyl as a fixed modification of cysteine.

Results

Each can of chick peas is labeled to indicate the ingredients added during canning. Ingredients included water, chick peas, salt, and disodium ethylenediamine tetraacetic acid (EDTA). In addition, two cans were labeled as "may contain calcium chloride". Three distinct lining colours were observed; white, clear yellow and metallic (Table 1).

Brand c...

Discussion

In this research, we have found that chickpea aquafaba from different commercial sources produces foams that vary in both properties (volume and stability of foam) and chemical composition. There was a positive correlation between aquafaba viscosity and moisture content. Foam volume increase (V_f100) was not related to these parameters. Additives such as salt and disodium EDTA might suppress viscosity and foam stability as aquafaba from chickpea canned with these additives had lower viscosity and produced foams...

Materials

Name	Company	Catalog Number	Comments
Freeze Dryer
Stoppering Tray Dryer	Labconco Inc.	7948040
Mixer
Stainless steel hand mixer	Loblaws	PC2200MR
Viscosity Measurement
Shell cup No. 2	Norcross Corp.
Color Measurement
Colorflex HunterLab spectrophotometer	Hunter Associates Laboratory Inc.
Protein and Carbon Contents
Elemental analyzer	LECO Corp.	CN628
NMR Spectrometry
Spectrafuge 24D	Labnet International Inc.
Syringe filters	VWR International	CA28145-497	25 mm, with 0.45 µm PTFE membrane
Deuterium oxide	Cambridge Isotope Laboratories Inc.	7789-20-0
3-(trimethylsilyl)propionic-2,2,3,3-d4 acid sodium salt	Sigma-Aldrich	169913-1G
Bruker Avance 500 MHz NMR spectrometer	Bruker BioSpin
TopSpin 3.2 software	Bruker BioSpin GmbH
Electrophoresis
Regenerated cellulose membrane	Millipore Corp.		3, 10, 50 kDa (MWCO)
Centrifugal filter unit	Millipore Corp.
Benchtop centrifuge	Allegra X-22R, Beckman Coulter Canada Inc.
Mixer Mill MM 300  bead mill	F. Kurt Retsch GmbH & Co. KG
Eppendorf centrifuge 5417C	Eppendorf
Phosphate buffered saline, pH 7.4	Sigma-Aldrich	P3813-10PAK
Tris-HCl buffer pH 7.4	Sigma-Aldrich	T6789-10PAK
PageRuler Prestained Protein Ladder	Fisher Scientific
Mini-Protein Tetra Cell system	BioRad
Peptide Mass Fingerprinting
Thermo-Savant SpeedVac	BioSurplus		Centrifugal vacuum evaporator
Trypsin buffer			20 µL trypsin in 1 mM hydrochloric acid and 200 mM NH4HCO3
Iodoacetamide	Sigma-Aldrich	I1149-5 g
Trifluoroacetic acid	Fluka	BB360P050
Acetonitrile	Fisher Scientific	L14734
Formic acid	Sigma-Aldrich	33015-500mL
Mass spectrometry vial	Agilent Technologies Canada Ltd.
Agilent 6550 iFunnel quadrupole time-of-flight mass spectrometer	Agilent Technologies Canada Ltd.		Agilent 1260 series LC instrument and Agilent Chip Cube LC-MS interface
HPLC-Chip II: G4240-62030 Polaris-HR-Chip_3C18			360 nL enrichment column and 75 µm × 150 mm analytical column, both packed with Polaris C18-A, 180Å, 3 µm stationary phase.
Agilent MassHunter Qualitative Analysis Software	Agilent Technologies Canada Ltd.
SpectrumMill data extractors	Agilent Technologies Canada Ltd.