Experimental

Chaya leaves were acquired from a local market in Mérida city located in Yucatán state, México. Reagents including HCl, NaOH, sodium borate, diethyl-ethoxymethylenmalonate, Folin-Ciocalteau, Na₂Co₃, gallic acid, polyphenol standards (chlorogenic acid, cyanidin chloride, epicatechin, p-coumaric acid, and vanillic acid), digestive enzymes and bile extracts were purchased from Sigma Aldrich (Missouri, USA).

Leaf protein concentrates (LPC)

Leaves were ground and macerated in an alkaline solution at pH 13. The solution was separated by filtration and acidified using HCl (0.1 N) until reaching a pH of 3.5. Once the protein was precipitated, it was centrifuged at 6000 rpm and 4 °C for 20 min, neutralized, and dried in a vacuum oven at 60 °C until moisture content was below 15 %. [¹⁵]

Proximate analysis

Proximate analyses of chaya leaves and LPC were determined according to the AOAC official methods [¹⁶] for dry matter (925.09), protein (954.01), ash content (923.93), crude fat (920.39), and crude fiber (962.09). Carbohydrates were calculated by difference [100 - (moisture + protein + ash content + crude fat + crude fiber)]. [¹⁷]

Nutritional quality

Protein efficiency ratio (PER)

The protein efficiency ratio (PER) was used for the prediction of protein quality, it was calculated by Eqs. (3)-(5). [²⁰]

Protein bioavailability

In vitro digestion of a control-diet constituted of protein, carbohydrates, and lipids using meat and casein peptone, corn oil, flour, and sugar (~1000 kcal), and a supplemented diet with chaya LPC was carried out. In the stomach, the samples were acidified to pH 2 and kept in continuous stirring. Next, the pepsin enzyme (porcine stomach) was added. Afterward, before adding pancreatic enzymes and bile salts, the pH was adjusted to ~5.5-6. In the end, the final pH was re-adjusted (pH= ~7), and the protein content was determined by the AOAC method 954.01. [¹⁶]

Protein structure

Fourier-Transform Infrared Spectroscopy (FTIR)

The FTIR measurements of LPC were recorded at 400-4000 cm^-1 wavelength with an Agilent Cary 630 spectrophotometer coupled with ATR ZNSe. The absorbance spectrums were analyzed using Microlab PC software (Agilent Technologies Inc., California, USA). [²³]

Differential Scanning Calorimetry (DSC)

For DSC analysis, an amount of 10 mg of protein quantified by the AOAC method 954.01 with 30 % of water (w/v) was placed in hermetically sealed aluminum plate pans. Heat scan was from 20 °C to 90 °C at 10 °C/min using a Q200 calorimeter (TA Instruments, Delaware, USA). Universal Analysis 2000 software (TA Instruments, Delaware, USA) was used to calculate enthalpies. [²⁴]

Total polyphenols content and polyphenolic profile determination

Total phenolics content in three different extracts: chaya leaves, protein precipitation remnant, and pellet from protein extraction (leaves after the whole protein extraction process) were quantified using the Folin Ciocalteau method. Polyphenolic compounds were extracted twice from macerated leaves using methanol-water (80:20) in a ratio of 1:4 (leaves: solvent). Leaves were kept under constant stirring at room temperature for 2 h and centrifuged for 15 min at 4500 rpm and 4 °C; the polyphenol extracts were stored at -20 °C until further use. Folin-Ciocalteau reagent (250 µL, 1 N) was added to 20 µL of polyphenol extract and let rest for 3 min in darkness. Afterward, 1250 µL of Na₂CO₃ 20 % (w/v) and 480 µL of distilled water were added and left in darkness for two h. The final absorbance was measured at 760 nm using a UV-Vis spectrophotometer (Genesys 10S, Thermo Scientific, Massachusetts, USA). A gallic acid calibration curve (0.01 - 0.5 mg/mL) was used as standard.

For polyphenolic profile determination, the samples were filtered through a 0.2 μm nylon syringe filter before chromatographic analysis. A Waters (Massachusetts, USA) LC-MS/MS (liquid chromatography-mass spectrometer) was used for the identification and quantification of polyphenols. An Acquity UPLC BEH C18 (2.1 x 100 mm, 1.7 μm) column was used for compounds separation. Mobile phases were 0.1 % (v/v) formic acid in water (A) and 0.1 % (v/v) formic acid in methanol (B). The column was maintained at 40 °C. A flow rate of 0.45 mL/min and an injection volume of 5 μL were used. Total run time of 14.5 min with 99 % A at 0.5 min, 20 % A at 13.5 min, and 99 % A at 14 min.

Quattro Premier XE triple quadrupole mass spectrometer fitted with electrospray ionization (ESI) was used in positive mode for cyanidin and epicatechin. In contrast, the negative mode was used for chlorogenic acid, ρ-coumaric acid, rutin, and vanillic acid. Mass spectrometer optimization was conducted by direct infusion of 50 mg/L stock standard solutions to select the ionization mode and precursor and ions production. The calibration curves of the above-mentioned polyphenols standards were used for quantification.

Proximate analysis

The proximal composition of chaya leaves and chaya LPC is shown in Table 1. Chaya leaves showed higher protein content (32.09 ± 0.48 %) compared with chaya (Cnidoscolus aconitifolius) leaves at different ages of regrowth (~27 %), [²⁵] and other leafy vegetables like lettuce, spinach, and moringa (19 %, 23 %, and 18 % respectively). [²⁶, ²⁷] Ash content, which represents mineral content, was slightly lower than Cnidoscolus aconitifolius (~10 %). Both ash and fiber content were similar to the reported values for Eruca vesicaria leaves (9.3 % of ash content and 6 % of fiber); however, the chemical composition is different in other leafy vegetables, for example, amaranth (Amaranthus cruentus) and spinach (Spinacia oleracea) have approximately 16 - 18 % of ash content and 14 - 16 % of fiber, considerably higher than the results found in chaya leaves. [²⁸, ²⁹, ³⁰] It is well known that several factors can influence the composition of crops, such as climate, age of growth, and harvesting conditions, among others.

In the case of LPC, the content of protein compared to other LPCs obtained from leaves of different crops was much higher (~62 %) in comparison to LPCs from Diplazium esculentum, Albizia lebbeck, and Amaranthus hybridus (~34 %, 37 %, and 39 % respectively). [³⁰, ³², ³³] However, alfalfa (Medicago sativa) and moringa (Moringa oleifera) LPCs showed similar content of protein (60 - 70 %) than in our study. [³⁴, ³⁵]

Nutritional quality

The amino acid profile, ET, and PER values for chaya leaves, and LPC are shown in Table 2. The results for leaves were similar to previously reported values for C. chayamansa. [³⁶] Chaya LPC showed a lower amount of essential amino acids, such as Lys, Leu, and Ile. Still, a higher quantity of Trp and Val in comparison to LPCs produced with Amaranthus hybridus leaves [³¹], Amaranthus spp, eggplant [³⁷], Gliricidia sepium, Leucaena leucocephala [³⁸], and Medicago sativa L. [³⁴]; however, no limiting amino acids were found. The LPC meets the required amounts for children above five years old, adolescents, and adults recommended by FAO [¹⁹] (Table 3). According to the PER value, Chaya leaves can be considered high-quality because the value was higher than 2 (2.87); however, in LPC, these values slightly decreased (2.54). [³⁹] It has been reported that the extraction process of protein could negatively affect its quality due to some amino acids may be partially degraded or destroyed by thermal or acid/alkali processing. [⁴⁰] On the other hand, in vitro digestibility studies showed that chaya LPC was more bioavailable at both the stomach (1.35 g/100 ml) and small intestine (0.94 g/100 mL) in in vitro conditions compared to the protein present in the control diet (1.08 and 0.78 g/100 mL, respectively). These results suggest that chaya LPC could be considered as a good alternative for animal protein substitution in human consumption. There is no information related to the digestibility of leaf proteins in humans. Nevertheless, studies performed on meals using protein from different sources showed higher digestibility for spirulina meal than pork, soybean, and chickpea meals. [⁴¹]

Protein structure

SDS-PAGE gel electrophoresis

Green leaves are constituted primarily by two sorts of protein fractions, the proteins complexed with pigments (chlorophyll) present in the thylakoid membrane (known as green protein) and the soluble proteins (white protein). This white protein is an enzyme known by its short name RuBisCO (Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase), this soluble protein and some chloroplastic proteins were observed in SDS-page gel electrophoresis. The subunit bands (small and large) of the soluble protein (RuBisCO) were detected at molecular weights of 50 kDa and 10 kDa (Fig. 1). Identical bands have been reported in alfalfa and spinach LPCs. [⁴²,⁴³]

Fig. 1 Gel electrophoresis of chaya leaf protein concentrate (LPC) for protein detection. 

Thermal stability and secondary protein structure (DSC and FTIR)

The thermal stability and protein footprint of chaya LPC are shown in Fig. 2. The enthalpic peaks during the heating ramp of LPC evinced the presence of protein (Fig. 2(A)). The denaturation temperature of chaya LPC was ~57 °C. These concentrates showed slightly lower thermal stability than RuBisCO proteins (63 °C - 75 °C) obtained from different vegetable sources such as alfalfa, spinach, and Diplazium esculentum. [²⁴,³³,⁴³]

Fig. 2 (A) Differential Scanning Calorimetry (LPC DSC) and (B) Fourier Transform Infrared Spectroscopy (LPC FTIR) in chaya leaf protein concentrate (LPC). 

In the FTIR spectra, the typical protein footprint represented mainly by the Amide A (3300 cm^-1), Amide I (1600 - 1690 cm^-1), and Amide II (1480 - 1575 cm^-1) regions was observed (Fig. 2(B)), Amide I band is related to C=O stretching in the peptide bonds. This absorbance peak is often used to predict the content of secondary structural elements such as α-helixes, β-sheets, and random coils. On the other side, Amide A indicates the stretching of NH bonds, and Amide II represents CN stretching and NH bending of the protein molecule. Another region of interest is between 2800 - 2900 cm^-1, which measures the CH₂ and CH₃ asymmetric stretching of fatty acids; the low absorbance in the LPC might indicate a small proportion of lipids in the samples. Moreover, the presence of peaks around 900 - 1200 cm^-1 is commonly related to the stretching of hydroxyl groups of carbohydrate moieties; therefore, some soluble carbohydrates may be present in the LPC. [⁴⁴-⁴⁷]

Polyphenol content

The content of polyphenols in chaya leaves and the remnant solution from protein precipitation was similar (3.0 and 3.5 mg GAE/g, respectively) and significantly decreased in the pellet from protein extraction (0.8 mg GAE/g). The values in the leaves and remnant were according to a previous study of C. chayamansa, Ramos-Gomez et al. [⁴⁸], who found a total polyphenol content of 3.35 mg GAE/g, respectively. Nevertheless, other studies with the same crop (C. chayamansa) have shown higher polyphenols content (6.34 - 71.3 mg GAE/g), however, the extraction conditions were different to this study (distilled water at 90 °C). It is well known that high temperatures increase the extraction rate of different compounds such as polyphenols. [⁹,⁴⁹,⁵⁰] On the other side, the highest concentration (71.3 mg GAE/g) found by Loarca-Piña et al. [⁹] was obtained using an extensive extraction of 5 days at room temperature with hexane-acetone, and a second extraction with methanol was also applied.The differences observed in the total polyphenols could be due to different reasons such as the type of solvent used for extraction (acetone, ethanol, methanol, or water), total extraction time, harvesting conditions, crop age state [⁹,⁴⁹], and the type of standard used for the calibration curve. The fact that similar amounts of polyphenols were present after protein extraction could indicate that protein-polyphenol conjugates were not formed during protein precipitation. From an added value and nutritional point of view, chaya leaves could be used as a source of these two nutritional compounds without compromising protein quality caused often by non-proteinaceous elements which can cause off-flavors and a decrease in digestibility.

Polyphenol profile

The identified and quantified polyphenols in chaya leaves are presented in Table 4. The obtained profile consisted of 14.8 % anthocyanidins (cyanidin), 61.0 % hydroxycinnamic acids (3-chlorogenic acid and p-coumaric acid), 6.9 % hydroxybenzoic acids (vanillic acid), and 17.2 % flavonols (epicatechin and rutin). All these polyphenols have been previously reported in chaya. [⁴⁸,⁵⁰] A quercetin concentration of 0.040-0.045 mg/g was reported in C. chayamansa raw leaves [⁵⁰], which is similar to the rutin (quercetin-3-O-rutinoside) content found in this work (0.043 mg/g). Chaya leaves extracts have been studied for their potential health benefits, including antimutagenic, antidiabetic, and antioxidant activities [⁹, ⁴⁸] showing promising nutritional applications for this plant extract.