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 code Salt Disodium EDTA Calcium chloride Can lining A + – – White B + – – White C + + – Yellow D – – – Metallic E + + – White F + + – Yellow G + + +/– White H – – – Yellow I + + – Yellow J + + +/– Yellow

Table 1: Additives and can lining.

Brands E and D had the highest (607 g) and lowest (567 g) total mass (chickpeas plus aquafaba). The coefficient of variation (CV) of total mass per can was just 2%. Brand H contained the highest chickpea mass (488 g). Brand J, with the lowest chickpea mass (335 g), was 31% lighter than brand H and had the lowest number of seeds (244) in each can (Table 2). Brand D contained the lowest juice volume in the can (110 mL) while the highest juice volume was observed for brand E (225 mL). The highest viscosity observed, for brand H (114.2 cP), was 4.3 to 20 times greater than observed for other brands (5.7-26.4 cP). A number of significant correlations were observed that might predict the usefulness of canned product for producing aquafaba. Chickpea fresh weight was negatively correlated with juice volume and positively correlated with juice viscosity. Whipping 100 mL of aquafaba uniformly increased the foam by about 5 fold (V f100 = 470) immediately after blending with a CV of just eight percent. Brands D, E and G produced the lowest amount of foam from 100 mL of aquafaba. Aquafaba viscosity was negatively correlated with total foam volume per can (V fcan ) (Table 3). Juice density was the least variable parameter CV of 4.6% while juice viscosity was the most variable 160%. The CV of peas per can was 22%.

Brand code Can content

(g) Chickpea fwt

(g) Seeds/can Juice volume

(mL) Juice density

(kg/m3) Juice viscosity

(cP)1 V f100

(mL)2 V fcan

(mL/can)3 A 588 392 454 170 1067 8.75 ± 0.27bc 500 850 B 581 355 404 200 1100 8.34 ± 0.17abc 500 1000 C 590 389 289 175 1112 10.50 ± 0.18c 470 823 D 567 428 423 110 1180 26.41 ± 1.14e 410 451 E 607 364 300 225 1066 6.21 ± 0.24ab 405 911 F 602 364 304 220 1048 6.32 ± 0.10ab 480 1056 G 598 349 280 220 1103 5.70 ± 0.13a 430 946 H 595 488 429 125 1160 114.2 ± 4.29f 500 625 I 599 385 323 200 1009 16.12 ± 0.64d 500 1000 J 584 335 244 220 1109 5.79 ± 0.30a 510 1122 Mean 591 385 345 186.5 1095.4 20.84 470 878.4 SD 12 44 74.72 41.17 50.83 33.44 40 204.63 CV4 2 12 21.66 22.07 4.64 160.48 8.5 23.29 1Each value is presented as the mean ± SD (n = 3). Values followed by different letters are significantly different (p < 0.05). 2Percent volume increase from whipping 100 mL of chickpea juice. 3Total foam volume per can 4Coefficient of variation (%)

Table 2: Quantitative values of chickpea can.

Can content

(g) Chickpea fwt

(g) Seeds/

can Juice volume

(mL) Juice density

(kg/m3) Juice viscosity

(cP) V fcan

(mL/can)1 Can content (g) 1 Chickpea fwt (g) –0.172 1 Seeds/can –0.442 0.664** 1 Juice volume (mL) 0.600 –0.878** –0.739** 1 Juice density (kg/m3) –0.647** 0.519 0.339 –0.705** 1 Juice viscosity (cP) –0.002 0.890** 0.474 –0.657** 0.512 1 V fcan (mL/can)1 0.483 –0.818** –0.639** 0.927** –0.728** –0.568 1 1Total foam volume from a can of product. Note: There were no significant correlations relating V f100 to other parameters.

Table 3: Correlation coefficient.

The functional properties of aquafaba from these 10 commercial products, especially foam volume and foam stability, varied significantly (Figure 1). The highest V fcan occurred in brands B, F, I and J with volumes over 1,000 mL. The highest juice density and the lowest volume increase ratio were observed in brand D. Despite the uniform yield of foam per 100 mL of aquafaba in all materials the stability of the foam varied greatly. After 1 h liquid had separated from all products except from brand H (Figure 1). After an additional 14 h storage the foam was largely gone from all brands except D and H. Interestingly brands D and H had a number of distinguishing properties including: 1) the highest chickpea mass per can; 2) the lowest aquafaba yield per can; 3) the highest aquafaba density; and 4) the highest aquafaba viscosity. The labels on products D and H indicated that no additives were included other than water and chickpeas.



Figure 1: Aquafaba foam and juice from 10 commercial chickpeas. Please click here to view a larger version of this figure.

Chickpea seeds contained 63.2-69.9% moisture and 45.6-46.5% carbon (Table 4). The highest and lowest protein contents were observed in brands J (22.4%) and B (18.2%), respectively.

Brand code Moisture (%) Carbon (%) Protein (%) A 67.3 ± 0.2cd 46.47 ± 0.01f 19.04 ± 0.18b B 66.4 ± 0.4bc 46.24 ± 0.00e 18.24 ± 0.19a C 67.6 ± 0.2d 45.73 ± 0.01b 18.38 ± 0.07a D 69.9 ± 0.7e 46.43 ± 0.02f 21.99 ± 0.23f E 66.8 ± 0.5cd 45.63 ± 0.04a 20.71 ± 0.01d F 67.1 ± 0.2cd 46.24 ± 0.00e 22.05 ± 0.03fg G 63.2 ± 0.4a 46.15 ± 0.01de 19.53 ± 0.08c H 69.3 ± 0.3e 46.49 ± 0.05f 21.53 ± 0.28e I 66.9 ± 1.5cd 46.09 ± 0.09cd 19.82 ± 0.07c J 65.4 ± 0.8b 46.04 ± 0.04c 22.37 ± 0.11g Mean ± SD (CV) 67.0 ± 1.87 (2.79) 46.20 ± 0.29 (0.63) 20.40 ± 1.57 (7.70) Each value is presented as the mean ± SD (n = 3). Values followed by different letters are significantly different (p < 0.05).

Table 4: Composition of chickpea seed.

Brand E had the highest freeze dried mass (17.9 g) (Table 5). Aquafaba obtained from brand D had the highest protein (26.8%) and carbon (39.2%) contents and brand E contained the lowest protein content (23.3%).

Brand code Freeze dried mass (g) Moisture (%) Carbon (%) Protein (%) A 12.4 94.39 ± 0.02bc 35.46 ± 0.59de 24.91 ± 0.55cd B NA 94.06 ± 0.01abc ND ND C 15.7 94.41 ± 1.89bc 35.09 ± 0.06cd 22.65 ± 0.30a D 11 94.07 ± 0.47abc 39.22 ± 0.02g 26.83 ± 0.06e E 17.9 93.59 ± 0.01ab 31.28 ± 0.12a 23.36 ± 0.19b F 15.6 94.28 ± 0.02bc 34.66 ± 0.06c 24.61 ± 0.15cd G 12.7 94.70 ± 0.03bc 33.78 ± 0.13b 22.75 ± 0.19ab H 15.1 92.98 ± 0.00a 37.30 ± 0.10f 24.49 ± 0.26c I 16.3 93.63 ± 0.00ab 35.85 ± 0.00e 23.20 ± 0.02ab J 13.1 95.12 ± 0.00c 34.77 ± 0.05c 25.22 ± 0.44d Each value is presented as the mean ± SD (n = 3) except freeze dried mass. Values followed by different letters are significantly different (p < 0.05). NA = not available. ND =not determined

Table 5: Composition of aquafaba.

Brand D had the highest L value which is associated to seed lightness followed by chickpeas of brand H, on the other hand, brand J had the lowest value which indicates that the seed is darker (Table 6). This could be related to increased values of cooking time and/or seed quality. Additionally, this might be associated with the physical quality of the resulting foam. It is evident that a lighter product is more desirable for use as a thickener.

Brand code L a b A 58.02 ± 0.96cd 8.64 ± 0.95abc 27.23 ± 1.14ab B 57.66 ± 1.92bcd 8.66 ± 0.97abc 24.84 ± 1.64a C 58.45 ± 0.93cde 10.31 ± 0.70d 29.62 ± 2.18b D 60.49 ± 0.55e 7.95 ± 0.69a 27.74 ± 0.87b E 55.65 ± 1.16ab 9.92 ± 0.28cd 27.39 ± 0.87ab F 57.25 ± 0.52bcd 8.51 ± 0.58ab 28.09 ± 1.16b G 59.02 ± 1.20de 7.58 ± 0.34a 28.80 ± 1.58b H 56.63 ± 1.78abc 10.44 ± 0.57d 26.77 ± 0.74ab I 56.25 ± 1.34abc 9.71 ± 1.02bcd 28.87 ± 2.51b J 54.94 ± 0.90a 9.34 ± 0.09bcd 28.29 ± 0.64b Each value is presented as the mean ± SD (n = 3). Values followed by different letters are significantly different (p < 0.05).

Table 6: Color parameters of chickpea seed.

The protein contents in the supernatant increased when MWCO increased (Table 7). When a 3 kDa MWCO membrane was used for aquafaba filtration, no protein was found in the supernatant confirming that proteins present in the chickpea juice had MWs greater than 3 kDa. As membrane MWCO increased, some proteins were observed in the supernatant. The retentate after diafiltration was a gel-like pellet, therefore, it was dissolved in 0.02 M Tris HCl at pH 7.4 or PBS at pH 7.4 buffer.

Fraction MWCO (kDa) 3 10 50 Supernatant 0.05 ± 0.00 g/L 0.17 ± 0.01 g/L 0.89 ± 0.01 g/L Retentate1 0.88 ± 0.01 g/L 1.36 ± 0.00 g/L 0.82 ± 0.02 g/L 1The retentate was dissolved in 0.02 M Tris HCl at pH 7.4. Similar trend and results were obtained when PBS at pH 7.4 was used as solvent.

Table 7: Protein content (g/L) of supernatant from filtering brand H juice using different MWCO.

Aquafaba from 10 commercially available chickpea products was analyzed using DPFSE-1H-NMR. A typical annotated aquafaba 1H-NMR spectrum is depicted in Figure 2. The resonances of constituents were assigned according to the previous publications21. A total of 20 compounds were identified including alcohols (isopropanol, ethanol, methanol), organic acids (lactic acid, acetic acid, succinic acid, citrate, formate, malate), sugars (glucose, sucrose), amino acids (alanine), and nucleosides (inosine, adenosine).



Figure 2: Representative 1H-NMR Spectrum of the total region of aquafaba. 1, Isopropanol; 2, ethanol; 3, lactic acid; 4, alanine; 5, acetic acid; 6, glutamine; 7, succinic acid; 8, citrate; 9, malate; 10, choline; 11, phosphocholine; 12, methanol; 13, sucrose; 14, glucose; 15, β-glucose; 16, α-glucose; 17, sucrose; 18, inosine; 19, adenosine; 20, formate. Please click here to view a larger version of this figure.

Most of the 1H-NMR spectra of aquafaba from commercial samples (except brands E, G and J) showed a triplet at 1.2 ppm from ethanol methyl group (Figure 3). Acetic acid fermentation in aquafaba can also be verified through acetate signal at 1.95 ppm. Aquafaba from brand F show high level of lactic acid (1.35 ppm), while aquafaba from A show high level of lactic acid and succinic acid (2.48 ppm). The singlet at 3.2 ppm in the 1H-NMR spectra of all aquafaba investigated indicates the presence of choline. It is probable that ethanol, acetic acid and lactic acid are produced during steeping of the chickpeas prior to canning. Sucrose, choline and other molecules might arise from the chickpeas as normal metabolites.



Figure 3: 1H-NMR Spectrum of aquafaba from different commercial product. 2, Ethanol; 3, lactic acid; 5, acetic acid; 8, citrate; 9, malate; 10, choline; and 11, phosphocholine. Please click here to view a larger version of this figure.

To understand the foam composition, the 1H-NMR spectrum of the liquid separated from the foam after 12 h from brand A was compared with the spectrum of the foam (Figure 4). Proton signals in the region (5.0-5.4 ppm) were highly enriched in the foam spectrum. The sucrose concentration (3.63 ppm) was greater in the foam than in the liquid. Volatile components such as methanol (3.40 ppm), ethanol (1.20 ppm), lactic acid (1.35 ppm), acetic acid (1.95 ppm) and succinic acid (2.48 ppm) decreased in the foam layer, likely due to evaporation. Proton signals of proteins (0.5-3.0 ppm, the protons of aliphatic amino acid side chain) are present in the foam.



Figure 4: 1H-NMR Spectrum of aquafaba foam and juice from brand A. 1, Isopropanol; 2, ethanol; 3, lactic acid; 4, alanine; 5, acetic acid; 6, glutamine; 7, succinic acid; 8, citrate; 9, malate; 10, choline; 11, phosphocholine; 12, methanol; 13, sucrose; 14, glucose; 15, β-glucose; 16, α-glucose; 17, sucrose; 18, inosine; 19, adenosine; 20, formate; and *, polysaccharide. Please click here to view a larger version of this figure.

The aquafaba juice from brand H was subjected to ultrafiltration using three different MWCO membranes and the 1H spectra were compared (Figure 5). The 10 kDa membrane separated an apparent polynucleotide (6.5-8.5 ppm), while polysaccharides, contributing peaks of 5.0-5.2 ppm, were passed by the 50 kDa membrane. Peptide signals (0.5-2.5 ppm) were detected in the 3 kDa filtrate.



Figure 5: 1H-NMR Spectrum of aquafaba subjected to membrane filtration. 16, α-Glucose; 17, sucrose; *, polysaccharide. Please click here to view a larger version of this figure.

Enrichment of proteins from product H, the one yielding the most stable foam by membrane filtration followed by SDS-PAGE of the retentate revealed clear bands of heat soluble proteins with MWs greater than 3 kDa (Figure 6A). Curiously, five identified protein bands appeared to contain proteins from the chickpea pathogenic fungus Didymella rabiei. Most of the other proteins belonged to known heat soluble proteins such as late embryogenesis abundant proteins and dehydrins (Figure 6B). Identified proteins also included heat shock protein, defensin, histone, non-specific lipid transfer protein and superoxide dismutase. Major storage proteins provicillin and leguminin were also present.



Figure 6: (A) SDS-PAGE Separation of chickpea juice protein; (B) Potential protein identification per band. Five identified protein bands are highlighted. LEAP = Late embryogenesis abundant protein, HSP = Heat shock protein. Please click here to view a larger version of this figure.