This research was supported by the Institute of International Education&#39;s Scholar Rescue Fund (IIE-SRF).

Separation of Aquafaba from Chickpeas
<ol>
	<li>Obtain cans of chick peas from local grocers and open with a manual can opener.</li>
	<li>Label cans from A to J.</li>
	<li>Separate chickpeas from aquafaba using a stainless steel meshed kitchen strainer and weigh the separated chickpeas and aquafaba.</li>
</ol>
Obtain a Representative Sample of Chickpeas and Aquafaba for Chemical Analysis.
<ol start="4">
	<li>Randomly select ten chickpeas after draining the aquafaba to determine moisture content.</li>
	<li>Place the selected chickpeas in a drying tin and heat at 100 &#177; 2 &#176;C for 16-18 h in a drying oven.</li>
	<li>After drying, grind the chickpeas to a powder for further use (e.g. protein and carbon content analysis).</li>
	<li>Freeze aquafaba and aquafaba foam from each source to -20 &#176;C and then dry in a freeze dryer. The dried aquafaba will be used for nitrogen and carbon content determination.</li>
</ol>
Aquafaba Functional Properties
<ol start="8">
	<li>Determine aquafaba viscosity at 25 &#176;C using a No. 2 shell cup.
	<ol>
		<li>Immerse the shell cup in the aquafaba.</li>
		<li>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.</li>
		<li>The aquafaba viscosity can be ascertained by a chart supplied with the cup that relates viscosity and drainage time.</li>
	</ol>
	</li>
	<li>Foaming capability
	<ol>
		<li>Blend the aquafaba solution (100 mL) in a stainless steel bowl with a hand mixer at speed setting of 10 for 2 min.</li>
		<li>Record the foam volume immediately after blending 100 mL of the aquafaba solution as Vf100 by placing the foam in a graduated measuring cup.</li>
		<li>Allow the foam to stand and dry over time to observe foam stability and obtain a sample of dried foam.</li>
	</ol>
	</li>
</ol>
Color Parameters of Chickpea Seed
<ol start="10">
	<li>Randomly select chickpea seed from each can for color determination.</li>
	<li>Calibrate the Hunter lab colorimeter using white, black and reference standards prior to measurements.</li>
	<li>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&#176;.</li>
</ol>
Protein and Carbon Contents
<ol start="13">
	<li>Determine protein and carbon contents by combustion using an elemental analyzer13. Protein content is estimated as nitrogen content multiplied by 6.2514.</li>
</ol>
Moisture Content
<ol start="14">
	<li>Determine seed and aquafaba moisture contents by heating samples at 100 &#177; 2 &#176;C for 16-18 h in a drying oven15.</li>
</ol>
NMR Spectrometry
<ol start="15">
	<li>Sample preparation
	<ol>
		<li>Prior to spectrometry, centrifuge aquafaba samples at 9200 &#215; g for 10 min. After centrifugation, pass the supernatant through a 0.45 &#181;m 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.</li>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>NMR conditions
	<ol>
		<li>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.</li>
		<li>Adjust the spectral shift to place the TMSP peak at 0 ppm then use the integration software to determine relative amounts of compounds present.</li>
	</ol>
	</li>
</ol>
Electrophoresis
NOTE: For this step, aquafaba that yielded the most stable foam (brand H) was selected. This brand did not contain salt.
<ol start="17">
	<li>Sample preparation
	<ol>
		<li>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.</li>
		<li>Place the centrifugal filter units in a suitable centrifuge at 4 &#176;C and centrifuge at 3900 &#215; 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.</li>
		<li>Estimate the protein contents of both supernatant and retenate using a modified Bradford method using bovine serum albumin as a standard17.</li>
		<li>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.</li>
		<li>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 &#176;C and 3900 &#215; g for 2 h.</li>
		<li>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.</li>
		<li>Centrifuge the mixture at 21000 x g for 1 min.</li>
		<li>Use the supernatant to determine protein content using the modified Bradford as previously mentioned.</li>
	</ol>
	</li>
	<li>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).
	<ol>
		<li>Prepare polyacrylamide gels using a 15% polyacrylamide resolving gel and a 5% polyacrylamide stacking gel.</li>
		<li>Apply a sample of 20 &#181;g 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.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>
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.
<ol start="19">
	<li>In-gel digestion
	<ol>
		<li>De-stain the bands twice by immersing in 100 &#181;L of 200 mM ammonium bicarbonate (NH4HCO3) in 50% acetonitrile (ACN) and incubate at 30 &#176;C for 20 min.</li>
		<li>After the completion of de-staining, treat the gel samples with ACN (100 &#181;L) for 10 min and then dry under vacuum, using a centrifugal vacuum evaporator, for 15 min at room temperature.</li>
		<li>Immerse the dried gel samples in 100 &#181;L of 10 mM dithiothreitol (DTT) in 100 mM NH4HCO3 solution and incubate at 56 &#176;C for 1 h.</li>
		<li>Remove excess reducing buffer and replace it with 100 &#181;L alkylating buffer [100 mM iodoacetamide in mass spectrometry (MS) grade water].</li>
		<li>Incubate the gel samples at room temperature in the dark for 30 min.</li>
		<li>Wash gel samples twice with 200 mM NH4HCO3 (100 &#181;L), shrink the gels by immersing them in ACN (100 &#181;L), re-swell the gels with 200 mM NH4HCO3 (100 &#181;L) and again shrink by treating with ACN (100 &#181;L).</li>
		<li>Drain the ACN and dry the gel samples under vacuum in a centrifugal vacuum evaporator for 15 min.</li>
		<li>Re-swell dried gel samples using 20 &#181;L trypsin buffer (50 ng/&#181;L trypsin in 1:1,200 mM NH4HCO3: Trypsin stock solution) followed by addition of 30 &#181;L of 200 mM NH4HCO3 to each sample.</li>
		<li>Incubate samples on a Thermomixer overnight at 30 &#176;C with shaking at 300 rpm.</li>
		<li>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 &#181;L of 0.1% trifluoracetic acid in 60% ACN and dry under vacuum using a centrifugal vacuum evaporator.</li>
		<li>Store tryptic peptides in -80 &#176;C prior to mass spectrometric analysis.</li>
	</ol>
	</li>
	<li>Mass spectrometry
	<ol>
		<li>Reconstitute tryptic peptides by adding 12 &#181;L of MS grade water: ACN:formic acid (97:3:0.1, v/v) and then vortex for 1 to 2 min to dissolve peptides.</li>
		<li>Centrifuge the resulting solution at 18,000 g for 10 min at 4 &#176;C.</li>
		<li>Transfer solution aliquots (10 &#181;L) 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.</li>
		<li>Perform chromatographic peptide separation using a high-capacity HPLC-Chip: consisting of a 360 nL enrichment column and a 75 &#181;m &#215; 150 mm analytical column, both packed with C18-A, 180 &#197;, 3 &#181;m stationary phase.</li>
		<li>Load samples onto the enrichment column with 0.1% formic acid in water at a flow rate of 2.0 &#181;L/min.</li>
		<li>Transfer samples from the enrichment column to the analytical column.</li>
		<li>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 &#181;L/min.</li>
		<li>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 &#176;C with a flow rate of 12.0 L/min.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Protein identification
	<ol>
		<li>Convert spectral data to a mass/charge data format using Agilent MassHunter Qualitative Analysis Software.</li>
		<li>Process data against the UniProt Cicer arietinum (chickpea) database, using SpectrumMill as the database search engine.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

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 (Vf100) 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 with lower foam stability. This result differs from that of Behera et al. (2014)22 where it was reported that micellar foam volume was reduced in the presence of salt and that foam collapse rate was slowed by salt. Chickpea aquafaba obtained from samples cooked with added salt also had higher moisture and lower carbon contents compared to aquafaba from chickpea canned with salt. Adding salt prior to cooking chickpea may have limited starch and protein swelling23.
Protein separations by membrane filtration followed by SDS-PAGE and peptide mass fingerprinting showed that aquafaba proteins were largely known heat soluble hydrophilic species. Curiously, there was also evidence of tryptic fragments that suggested this material was contaminated with ascochyta blight. The NMR analysis revealed up to 20 small organic solutes including alcohols, acetate, lactic acid and succinic acid. These results appear to indicate that fermentation products have accumulated in the aquafaba. These compounds may have been produced by microorganisms during steeping of the seed prior to canning. Proton NMR spectra of the foam and juice showed that isoflavones and volatile components were present in the juice while the foam contained mainly polysaccharides, sucrose and protein. Also of interest the 1H-NMR indicates the presence of nucleic acids (possibly DNA).
Clearly processes used in canning affected aquafaba properties and quality. It is possible that a consumer could identify better sources of aquafaba based on solution concentration. Primarily a source could be selected that was canned without added salt or EDTA. After a can is opened the consumer could measure the ratio of the mass of chickpeas and aquafaba to determine the concentration. However, a manufacturer could standardize processing conditions and produce standardized aquafaba as a commercial product.
In this research peptide mass fingerprinting and 1H-NMR were used to analyze the composition of aquafaba. Peptide mass fingerprinting identified aquafaba proteins contributing to foaming properties. The thermostability of the proteins that were identified is well known. The technique was sufficiently discerning to identify the presence of proteins associated with seed fungal organisms. However, various factors might influence the results24. The critical steps are sample preparation, protein purification, separation prior to gel electrophoresis, trypsin digestion and mass search that leads to identification. Software database searches of tryptic fragments consider the presence of many peptide modifications including carbamylated lysine, oxidized methionine, pyroglutamic acid, deamidated asparagine, phosphorylated serine, threonine, and tyrosine&#160;as variable modifications. The validated hits from the two stage analysis are then searched using a semi-trypsin non-specific C-terminus, and semi-trypsin non-specific N-terminus. After the iterative analysis the validations are provided for peptide and proteins with a 1% false discovery rate.
Proton NMR was used to determine the presence of organic small molecules in aquafaba. Several compounds were identified by their spectra. Filtration and centrifugation of aquafaba samples was conducted to eliminate insoluble particles which can interfere with NMR peak shapes and lower sensitivity. In addition, solvent suppression was employed to improve the NMR detector sensitivity for molecules that overlap with the broad baseline caused by the strong water resonance.
Proton NMR is an established tool in quality control of food products. Compared to chromatographic methods such as GC/MS, LC/UV, and LC/MS, 1H-NMR method is typically less sensitive. However, this method requires very little sample preparations and achieves rapid results and broad information in one measurement25. Where sufficient material is present 1H-NMR is also a very reliable method for quantitative analysis and elucidation of chemical structure of unknown compounds.

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 &#34;aquafaba&#34;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 &#34;aquafaba&#34; by French chef Jo&#235;l 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.

<table><tbody><tr><td>&lt;strong&gt;Freeze Dryer&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Stoppering Tray Dryer</td><td>Labconco Inc.</td><td>7948040</td><td></td></tr><tr><td>&lt;strong&gt;Mixer&amp;nbsp;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Stainless steel hand mixer&amp;nbsp;</td><td>Loblaws</td><td>PC2200MR</td><td></td></tr><tr><td>&lt;strong&gt;Viscosity Measurement&amp;nbsp;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Shell cup No. 2&amp;nbsp;</td><td>Norcross Corp.</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Color Measurement&amp;nbsp;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Colorflex HunterLab spectrophotometer&amp;nbsp;</td><td>Hunter Associates Laboratory Inc.</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Protein and Carbon Contents&amp;nbsp;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Elemental analyzer&amp;nbsp;&amp;nbsp;</td><td>LECO Corp.</td><td>CN628</td><td></td></tr><tr><td>&lt;strong&gt;NMR Spectrometry&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Spectrafuge 24D&amp;nbsp;&amp;nbsp;</td><td>Labnet International Inc.</td><td></td><td></td></tr><tr><td>Syringe filters&amp;nbsp;</td><td>VWR International</td><td>CA28145-497</td><td>25 mm, with 0.45 &amp;micro;m PTFE membrane</td></tr><tr><td>Deuterium oxide&amp;nbsp;</td><td>Cambridge Isotope Laboratories Inc.</td><td>7789-20-0</td><td></td></tr><tr><td>3-(trimethylsilyl)propionic-2,2,3,3-d4 acid sodium salt</td><td>Sigma-Aldrich</td><td>169913-1G</td><td></td></tr><tr><td>Bruker Avance 500 MHz NMR spectrometer&amp;nbsp;</td><td>Bruker BioSpin</td><td></td><td></td></tr><tr><td>TopSpin 3.2 software&amp;nbsp;</td><td>Bruker BioSpin GmbH</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Electrophoresis&amp;nbsp;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Regenerated cellulose membrane&amp;nbsp;</td><td>Millipore Corp.</td><td></td><td>3, 10, 50 kDa (MWCO)</td></tr><tr><td>Centrifugal filter unit&amp;nbsp;</td><td>Millipore Corp.</td><td></td><td></td></tr><tr><td>Benchtop centrifuge&amp;nbsp;</td><td>Allegra X-22R, Beckman Coulter Canada Inc.</td><td></td><td></td></tr><tr><td>Mixer Mill MM 300&amp;nbsp; bead mill&amp;nbsp;</td><td>F. Kurt Retsch GmbH &amp;amp; Co. KG</td><td></td><td></td></tr><tr><td>Eppendorf centrifuge 5417C</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>Phosphate buffered saline, pH 7.4</td><td>Sigma-Aldrich</td><td>P3813-10PAK</td><td></td></tr><tr><td>Tris-HCl buffer pH 7.4&amp;nbsp;</td><td>Sigma-Aldrich</td><td>T6789-10PAK</td><td></td></tr><tr><td>PageRuler Prestained Protein Ladder&amp;nbsp;</td><td>Fisher Scientific</td><td></td><td></td></tr><tr><td>Mini-Protein Tetra Cell system</td><td>BioRad</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Peptide Mass Fingerprinting&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Thermo-Savant SpeedVac</td><td>BioSurplus</td><td></td><td>Centrifugal vacuum evaporator&amp;nbsp;</td></tr><tr><td>Trypsin buffer&amp;nbsp;</td><td></td><td></td><td>20 &amp;micro;L trypsin in 1 mM hydrochloric acid and 200 mM NH&lt;sub&gt;4&lt;/sub&gt;HCO&lt;sub&gt;3&lt;/sub&gt;</td></tr><tr><td>Iodoacetamide</td><td>Sigma-Aldrich</td><td>I1149-5 g</td><td></td></tr><tr><td>Trifluoroacetic acid&amp;nbsp;</td><td>Fluka</td><td>BB360P050</td><td></td></tr><tr><td>Acetonitrile</td><td>Fisher Scientific&amp;nbsp;</td><td>L14734</td><td></td></tr><tr><td>Formic acid&amp;nbsp;</td><td>Sigma-Aldrich</td><td>33015-500mL</td><td></td></tr><tr><td>Mass spectrometry vial&amp;nbsp;</td><td>Agilent Technologies Canada Ltd.</td><td></td><td></td></tr><tr><td>Agilent 6550 iFunnel quadrupole time-of-flight mass spectrometer&amp;nbsp;</td><td>Agilent Technologies Canada Ltd.</td><td></td><td>Agilent 1260 series LC instrument and Agilent Chip Cube LC-MS interface</td></tr><tr><td>HPLC-Chip II: G4240-62030 Polaris-HR-Chip_3C18&amp;nbsp;</td><td></td><td></td><td>360 nL enrichment column and 75 &amp;micro;m &amp;times; 150 mm analytical column, both packed with Polaris C18-A, 180&amp;Aring;, 3 &amp;micro;m stationary phase.&amp;nbsp;</td></tr><tr><td>Agilent MassHunter Qualitative Analysis Software</td><td>Agilent Technologies Canada Ltd.</td><td></td><td></td></tr><tr><td>SpectrumMill data extractors</td><td>Agilent Technologies Canada Ltd.</td><td></td><td></td></tr></tbody></table>

composition and properties of aquafaba: water recovered from commercially canned chickpeas

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.

Watch this Scientific Journal Video about Composition and Properties of Aquafaba: Water Recovered from Commercially Canned Chickpeas at JoVE.com

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

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.

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 ...

composition-properties-aquafaba-water-recovered-from-commercially

Nanyang Technological University

Research

JoVE Journal

Environment

33.3K Views.  University of Saskatchewan. 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. 

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

Preparation of Keratin Hydrolysate from Chicken Feathers and Its Application in Cosmetics

Keratin hydrolysates (KHs) are established standard components in hair cosmetics. Understanding the moisturizing effects of KH is advantageous for skin-care cosmetics. The goals of the protocol are: (1) to process chicken feathers into KH by alkaline-enzymatic hydrolysis and purify it by dialysis, and (2) to test if adding KH into an ointment base (OB) increases hydration of the skin and improves skin barrier function by diminishing transepidermal water loss (TEWL). During alkaline-enzymatic hydrolysis feathers are first incubated at a higher temperature in an alkaline environment and then, under mild conditions, hydrolyzed with proteolytic enzyme. The solution of KH is dialyzed, vacuum dried, and milled to a fine powder. Cosmetic formulations comprising from oil in water emulsion (O/W) containing 2, 4, and 6 weight% of KH (based on the weight of the OB) are prepared. Testing the moisturizing properties of KH is carried out on 10 men and 10 women at time intervals of 1, 2, 3, 4, 24, and 48 h. Tested formulations are spread at degreased volar forearm sites. The skin hydration of stratum corneum (SC) is assessed by measuring capacitance of the skin, which is one of the most world-wide used and simple methods. TEWL is based on measuring the quantity of water transported per a defined area and period of time from the skin. Both methods are fully non-invasive. KH makes for an excellent occlusive; depending on the addition of KH into OB, it brings about a 30% reduction in TEWL after application. KH also functions as a humectant, as it binds water from the lower layers of the epidermis to the SC; at the optimum KH addition in the OB, up to 19% rise in hydration in men and 22% rise in women occurs.

The goal of the protocol is to prepare keratin hydrolysate from chicken feathers by alkaline-enzymatic hydrolysis and to test whether adding keratin hydrolysate into a cosmetics ointment base improves skin barrier function (heightening hydration and diminishing transepidermal water loss). Tests are conducted on men and woman volunteers.

Keratin hydrolysates (KHs) are established standard components in hair cosmetics. Understanding the moisturizing effects of KH is advantageous for ...

Biochemistry

Preparation of Biopolymer Aerogels Using Green Solvents

Although the first reports on aerogels made by Kistler1 in the 1930s dealt with aerogels from both inorganic oxides (silica and others) and biopolymers (gelatin, agar, cellulose), only recently have biomasses been recognized as an abundant source of chemically diverse macromolecules for functional aerogel materials. Biopolymer aerogels (pectin, alginate, chitosan, cellulose, etc.) exhibit both specific inheritable functions of starting biopolymers and distinctive features of aerogels (80-99% porosity and specific surface up to 800 m2/g). This synergy of properties makes biopolymer aerogels promising candidates for a wide gamut of applications such as thermal insulation, tissue engineering and regenerative medicine, drug delivery systems, functional foods, catalysts, adsorbents and sensors. This work demonstrates the use of pressurized carbon dioxide (5 MPa) for the ionic cross linking of amidated pectin into hydrogels. Initially a biopolymer/salt dispersion is prepared in water. Under pressurized CO2 conditions, the pH of the biopolymer solution is lowered to 3 which releases the crosslinking cations from the salt to bind with the biopolymer yielding hydrogels. Solvent exchange to ethanol and further supercritical CO2 drying (10 - 12 MPa) yield aerogels. Obtained aerogels are ultra-porous with low density (as low as 0.02 g/cm3), high specific surface area (350 - 500 m2/g) and pore volume (3 - 7 cm3/g for pore sizes less than 150 nm).

A new way for the production of biopolymer-based aerogels by carbon dioxide (CO2) induced gelation is shown. The technique utilizes pressurized carbon dioxide (5 MPa) for the production of biopolymer hydrogels and supercritical CO2 (12 MPa) to convert gels into aerogels. The only solvents needed besides CO2 are water and ethanol.

Although the first reports on aerogels made by Kistler1 in the 1930s dealt with aerogels from both inorganic oxides (silica and others) and biopolymers ...

Chemistry

A Protocol for the Production of Gliadin-cyanoacrylate Nanoparticles for Hydrophilic Coating

This article presents a protocol for the production of protein-based nanoparticles that changes the hydrophobic surface to hydrophilic by a simple spray coating. These nanoparticles are produced by the polymerization reaction of alkyl cyanoacrylate on the surface of cereal protein (gliadin) molecules. Alkyl cyanoacrylate is a monomer that instantly polymerizes at RT when it is applied to the surface of materials. Its polymerization reaction is initiated by the trace amounts of weakly basic or nucleophilic species on the surface, including moisture. Once polymerized, the polymerized alkyl cyanoacrylates show a strong affinity with the object materials because nitrile groups are in the backbone of poly (alkyl cyanoacrylate). Proteins also work as initiator for this polymerization because they contain amine groups that can initiate the polymerization of cyanoacrylate. If aggregated protein is used as an initiator, protein aggregate is surrounded by the hydrophobic poly(alkyl cyanoacrylate) chains after the polymerization reaction of alkyl cyanoacrylate. By controlling the experimental condition, particles in the nanometer range are produced. The produced nanoparticles readily adsorb to the surface of most materials including glass, metals, plastics, wood, leather, and fabrics. When the surface of a material is sprayed with the produced nanoparticle suspension and rinsed with water, the micellar structure of nanoparticle changes its conformation, and the hydrophilic proteins are exposed to the air. As a result, the nanoparticle-coated surface changes to hydrophilic.

This article presents a protocol for the production of protein-based nanoparticles that changes the hydrophobic surface to hydrophilic. The produced nanoparticle is an assembly of gliadin-cyanoacrylate diblock copolymers. Spray coating with the produced nanoparticle changes the surface of target material to a hydrophilic surface.

This article presents a protocol for the production of protein-based nanoparticles that changes the hydrophobic surface to hydrophilic by a simple spray ...

Quantification of Humic and Fulvic Acids in Humate Ores, DOC, Humified Materials and Humic Substance-Containing Commercial Products

The purpose of this method is to provide an accurate and precise concentration of humic (HA) and/or fulvic acids (FA) in soft coals, humic ores and shales, peats, composts and humic substance-containing commercial products. The method is based on the alkaline extraction of test materials, using 0.1 N NaOH as an extractant, and separation of the alkaline soluble humic substances (HS) from nonsoluble products by centrifugation. The pH of the centrifuged alkaline extract is then adjusted to pH 1 with conc. HCl, which results in precipitation of the HA. The precipitated HA are separated from the fulvic fraction (FF) (the fraction of HS that remains in solution,) by centrifugation. The HA is then oven or freeze dried and the ash content of the dried HA determined. The weight of the pure (i.e., ash-free) HA is then divided by the weight of the sample and the resulting fraction multiplied by 100 to determine the % HA in the sample. To determine the FA content, the FF is loaded onto a hydrophobic DAX-8 resin, which adsorbs the FA fraction also referred to as the hydrophobic fulvic acid (HFA). The remaining non-fulvic acid fraction, also called the hydrophilic fulvic fraction (HyFF) is then removed by washing the resin with deionized H2O until all nonabsorbed material is completely removed. The FA is then desorbed with 0.1 N NaOH. The resulting Na-fulvate is then protonated by passing it over a strong H+-exchange resin. The resulting FA is oven or freeze dried, the ash content determined and the concentration in the sample calculated as described above for HA.

This method provides a gravimetric quantification of humic substances (e.g., humic and fulvic acids) on an ash-free basis, in dry and liquid materials from soft coals (i.e., oxidized and non-oxidized lignite and sub-bituminous coal), humate ores and shales, peats, composts and commercial fertilizers and soil amendments.

The purpose of this method is to provide an accurate and precise concentration of humic (HA) and/or fulvic acids (FA) in soft coals, humic ores and ...

Transformation of Organic Household Leftovers into a Peat Substitute

A two-step procedure is described for the synthesis of a carbon material with a similar composition and properties as peat. The produced hydrochar is made suitable for agricultural applications by removing plant growing inhibitory substances. Wet household waste such as fruit peel, coffee grounds, inedible vegetable parts, or wet lignocellulosic material in general, are treated in presence of water at 215 &#x00B0;C and 21 bar in an autoclave, i.e., by hydrothermal carbonization. All these leftovers have a considerable water content of up to 90 weight % (wt%). Adding water extends the procedure to drier materials such as nutshells or even garden prunings and compostable polymers, i.e., the plastic bag for collection of the leftovers.
Usually, the resulting carbon material, called hydrochar, produces a negative effect on plant growth when added to soil. It is supposed that this effect is caused by adsorbed phytotoxic compounds. A simple post-treatment under inert atmosphere (absence of oxygen) at 275 &#x00B0;C removes these substances. Therefore, the raw hydrochar is placed on a glass frit of a vertical tubular quartz reactor. A nitrogen gas flow is applied in down-flow direction. The tube is heated to the desired temperature by means of a heating mantle for up to one hour.
The success of the thermal treatment is easily quantified by thermogravimetry (TG), carried out in air. A weight loss is determined when the temperature of 275 &#x00B0;C is reached, since volatile content is desorbed. Its amount is reduced in the final material, in comparison to the untreated hydrochar.
The two-step treatment converts household leftovers, including compostable bags employed for their collection, into a carbon material that may serve as plant growth promoter and, at the same time, as a carbon sink for climate change mitigation.

A protocol for hydrothermal carbonization of vegetable food waste in an autoclave is presented, with subsequent dry thermal treatment at 275 &#x00B0;C in a continuous flow reactor desorbing volatile organic substances. The aim is to produce a carbon material suitable as soil amendment product or substrate component.

A two-step procedure is described for the synthesis of a carbon material with a similar composition and properties as peat. The produced hydrochar is made ...

Qualitative Characterization of the Aqueous Fraction from Hydrothermal Liquefaction of Algae Using 2D Gas Chromatography with Time-of-flight Mass Spectrometry

Two-dimensional gas chromatography coupled with time-of-flight mass spectrometry is a powerful tool for identifying and quantifying chemical components in complex mixtures. It is often used to analyze gasoline, jet fuel, diesel, bio-diesel and the organic fraction of bio-crude/bio-oil. In most of those analyses, the first dimension of separation is non-polar, followed by a polar separation. The aqueous fractions of bio-crude and other aqueous samples from biofuels production have been examined with similar column combinations. However, sample preparation techniques such as derivatization, solvent extraction, and solid-phase extraction were necessary prior to analysis. In this study, aqueous fractions obtained from the hydrothermal liquefaction of algae were characterized by two-dimensional gas chromatography coupled with time-of-flight mass spectrometry without prior sample preparation techniques using a polar separation in the first dimension followed by a non-polar separation in the second. Two-dimensional plots from this analysis were compared with those obtained from the more traditional column configuration. Results from qualitative characterization of the aqueous fractions of algal bio-crude are discussed in detail. The advantages of using a polar separation followed by a non-polar separation for characterization of organics in aqueous samples by two-dimensional gas chromatography coupled with time-of-flight mass spectrometry are highlighted.

A two-dimensional gas chromatography-time-of-flight mass spectrometry method is described for characterization of the aqueous fraction of bio-crude produced from hydrothermal liquefaction of algae. This protocol can also be employed to analyze the aqueous fraction of liquid products from fast pyrolysis, catalytic fast pyrolysis, catalytic deoxygenation and hydro-treating.

Two-dimensional gas chromatography coupled with time-of-flight mass spectrometry is a powerful tool for identifying and quantifying chemical components in ...

Bioengineering

Film Extrusion of Crambe abyssinica/Wheat Gluten Blends

Crambe abyssinica is a plant with potential for use in industrial (non-food) plant oil production. The side stream from this oil production is a high-protein crambe meal that has limited value, as it is not fit for food or feed use. However, it contains proteins that could potentially make it a suitable raw material for higher-value products. The purpose of this study was to find methods of making this side stream into extruded films, showing that products with a higher value can be produced. The study mainly considered the development of material compositions and methods of preparing and extruding the material. Wheat gluten was added as a supportive protein matrix material, together with glycerol as a plasticizer and urea as a denaturant. The extrudate was evaluated with respect to mechanical (tensile testing) and oxygen barrier properties, and the extrudate structure was revealed visually and by scanning electron microscopy. A denser, more homogeneous material had a lower oxygen transmission rate, higher strength, and higher extensibility. The most homogeneous films were made at an extruder die temperature of 125-130 &#176;C. It is shown here that a film can be extruded with promising mechanical and oxygen barrier properties, the latter especially after a final compression molding step.

Film Extrusion of Crambe abyssinica/Wheat Gluten Blends

The side stream from plant oil production of Crambe abyssinica has limited value. The purpose of this study was to find methods for extruding materials based on this side stream, demonstrating that products with a higher value can be produced. The extrudates were found to have promising properties.

Crambe abyssinica is a plant with potential for use in industrial (non-food) plant oil production. The side stream from this oil production is a ...

Preparation of Expanded Chitin Foams and their Use in the Removal of Aqueous Copper

Chitin is an underexploited, naturally abundant, mechanically robust, and chemically resistant biopolymer. These qualities are desirable in an adsorbent, but chitin lacks the necessary specific surface area, and its modification involves specialized techniques and equipment. Herein is described a novel chemical procedure for expanding chitin flakes, derived from shrimp shell waste, into foams with higher surface area. The process relies on the evolution of H2 gas from the reaction of water with NaH trapped in a chitin gel. The preparation method requires no specialized equipment. Powder X-ray diffraction and N2-physisorption indicate that the crystallite size decreases from 6.6 nm to 4.4 nm and the specific surface area increases from 12.6 &#177; 2.1 m2/g to 73.9 &#177; 0.2 m2/g. However, infrared spectroscopy and thermogravimetric analysis indicate that the process does not change the chemical identity of the chitin. The specific Cu adsorption capacity of the expanded chitin increases in proportion to specific surface area from 13.8 &#177; 2.9 mg/g to 73.1 &#177; 2.0 mg/g. However, the Cu adsorption capacity as a surface density remains relatively constant at an average of 10.1 &#177; 0.8 atom/nm2, which again suggests no change in the chemical identity of the chitin. This method offers the means to transform chitin into a higher surface area material without sacrificing its desirable properties. Although the chitin foam is described here as an adsorbent, it can be envisioned as a catalyst support, thermal insulator, and structural material.

This study describes a method to expand chitin into a foam by chemical techniques that require no specialized equipment.

Chitin is an underexploited, naturally abundant, mechanically robust, and chemically resistant biopolymer. These qualities are desirable in an adsorbent, ...

Synthesis of Black Seed Oil-loaded-, Alginate-based, pH-sensitive Beads Using Electrospraying Technique

Black seed oil (BSO), derived from the seeds of the Nigella sativa plant, has garnered attention for its potential anti-cancer properties, particularly in the context of colon cancer. Its active compound, thymoquinone, may help inhibit cancer cell growth and induce apoptosis in colon cancer cells. Additionally, black seed oil's anti-inflammatory and antioxidant effects could contribute to a healthier gut environment, potentially reducing cancer risk. Therefore, this study synthesized pH-sensitive alginate beads to deliver BSO into the colon in a controlled-release manner without releasing the drug at pH 1.2 (stomach), thus providing a well-defined release pattern at pH 6.8. The use of electrospray technology improves process performance by making it easier to formulate small, homogeneous beads with a higher rate of swelling and diffusion in the gastrointestinal medium. 
The formulated beads were characterized by an ex-vivo mucoadhesive strength test, bead size, sphericity factor (SF), encapsulation efficiency (EE), scanning electron microscope (SEM), in vitro swelling behavior (SB), and in vitro drug release in acidic and buffer media. All these manufactured beads demonstrated modest sizes of 0.58 &plusmn; 0.01 mm and spherical shape of 0.03 &plusmn; 0.00 mm in this testing. The formulation showed promising floating and releasing properties in vitro. With a very low cumulative percentage of beads, the oil EE of 90.13% &plusmn; 0.93% was high, and the release study demonstrated more than 90% in pH 6.8 with good floating nature in the stomach. Additionally, the beads were evenly spaced throughout the intestine. The electrospraying approach used in this protocol can be reproducible, yielding consistent outcomes. Therefore, this protocol can be used for large-scale production for commercialization purposes.

A technique employing high electric voltage and a targeted, active ingredient-loaded emulsion to fabricate pH-responsive, uniform microbeads is presented.

Black seed oil (BSO), derived from the seeds of the Nigella sativa plant, has garnered attention for its potential anti-cancer properties, particularly in ...

Dry Root Rot Disease Assays in Chickpea: a Detailed Methodology

Dry root rot (DRR) disease is an emerging biotic stress threat to chickpea cultivation around the world. It is caused by a soil-borne fungal pathogen, Rhizoctonia bataticola. In the literature, comprehensive and detailed step-by-step protocols on disease assays are sparse. This article provides complete details on the steps involved in setting up a blotting paper technique for quickly screening genotypes for resistance to DRR. The blotting paper technique is easy and less expensive. Another method, based on the sick pot approach, is a mimic of natural infection and can be applied to study the interacting components&#8212;plant, pathogen, and environment&#8212;involved in the disease triangle.
Moreover, in nature, DRR occurs mostly in rainfed chickpea cultivation areas, where soil moisture recedes as crop growth advances. Drought stress is known to predispose chickpea plants to DRR disease. Pathomorphological and molecular understanding of plant-pathogen interaction under drought stress can pave the way for the identification of elite DRR-resistant varieties from the chickpea germplasm pool. This article provides a stepwise methodology for the preparation of a sick pot and subsequent disease assay. Overall, the information presented herein will help researchers prepare R. bataticola fungal inoculum, maintain this pathogen, set up the blotting paper technique, prepare sick culture and sick pot, and assess pathogen infection in chickpea plants.

This study presents methodologies to study the pathomorphological and molecular mechanisms underlying chickpea&#8211;Rhizoctonia bataticola interaction. The blotting paper method is useful to rapidly study chickpea genotype responses, while the sick pot-based method can be used to simultaneously impose drought and R. bataticola infection and screen for tolerant genotypes.

Dry root rot (DRR) disease is an emerging biotic stress threat to chickpea cultivation around the world. It is caused by a soil-borne fungal pathogen, ...

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

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Rozdziały w tym wideo

Preparation of Keratin Hydrolysate from Chicken Feathers and Its Application in Cosmetics

Preparation of Biopolymer Aerogels Using Green Solvents

A Protocol for the Production of Gliadin-cyanoacrylate Nanoparticles for Hydrophilic Coating

Quantification of Humic and Fulvic Acids in Humate Ores, DOC, Humified Materials and Humic Substance-Containing Commercial Products

Transformation of Organic Household Leftovers into a Peat Substitute

Qualitative Characterization of the Aqueous Fraction from Hydrothermal Liquefaction of Algae Using 2D Gas Chromatography with Time-of-flight Mass Spectrometry

Film Extrusion of Crambe abyssinica/Wheat Gluten Blends

Preparation of Expanded Chitin Foams and their Use in the Removal of Aqueous Copper

Synthesis of Black Seed Oil-loaded-, Alginate-based, pH-sensitive Beads Using Electrospraying Technique

Dry Root Rot Disease Assays in Chickpea: a Detailed Methodology