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Extraction and Stabilization of Betalains from Beetroot (Beta vulgaris) Wastes Using Deep Eutectic Solvents

Deep eutectic solvents (DES) using magnesium chloride hexahydrate [MgCl2·6H2O] and urea [U] proportions (1:1) and (2:1), were prepared for their use as extracting and stabilizer agents for red and violet betalains from beetroot (Beta vulgaris) waste. The synthetized DES [MgCl2·6H2O] [U] showed similar properties to eutectic mixtures, such as, liquid phase, low melting points and conductivity, thermal stability, and variable viscosity. In turn, betalain DES extracts (2:1) exhibited compatibility in the extraction and recovery of betalains from beetroot wastes, showing a betalain content comparable to that of betalain extracts. Betalain stability was determined by degradation tests; the exposure conditions were visible light (12 h), molecular oxygen from atmospheric air and environmental temperature (20–27 °C) for 40 days. The kinetic curves of the betalain degradation of water samples depicted a first-order model, indicating the alteration of a violet colouration of betalains from beetroot waste for 5–7 days. However, betalains from DES extracts were kept under visible light for 150 days, and for 340 days in storage (amber vessels), achieving a stability of 75% in comparison with initial beet extracts.


Introduction
The industrial processing of fruits and vegetables produces waste that is generally disposed of as organic trash, leading to environmental pollution. However, this waste is an important source of phytochemicals and natural pigments that can be recovered and used to develop functional foods, pharmaceutical products, and other valuable compounds for the industry [1,2].
Specifically, beetroot waste contains a great quantity of betalains because they are found throughout the root. This waste includes peel and pulp, and is produced by the industrial manufacture of juice, jam, drinks, and sugar beet. Large amounts of waste with a high content of betalains are also obtained for ethanol fermentation by beetroot extracts.
Betalains are readily available biomolecules used as food colorants; they are known as "beetroot red", covering a gamma of red pigments. Betalains are manufactured as powders (by freeze or spray drying) or extracts (by vacuum concentration of beet juice to 60-65% total solids), containing from 0.3% to 1% of pigment [3]. Nevertheless, the commercialized betalain pigments are highly susceptible to temperature and pH (T > 60 • C, pH > 5), because the aqueous content affects their chemical structure, causing rapid degradation and colour loss [4].
The current situation regarding betalains has led to a plethora of research regarding their stabilizing and colour preservation applications [5]. The inactivation of deleterious enzymes, and the addition of antioxidants and/or chelating additives have been suggested  [21]. In turn, spectra of [U] showed bands at 3340 and 3430 cm −1 , which were linked to -NH2 groups. Additionally, stretching vibrations at 1677 and 1461 cm −1 from C-O and C-N bonds were observed, respectively, while a stretching vibration at 1571 cm −1 was characteristic of the carbonyl group (-C=O).
The FTIR of obtained DES [MgCl2·6H2O] [U] (1:1) confirmed the eutectic mixture, showing characteristic bands of Cl-H hydrogen bonds at 3322-3209 cm −1 . However, a widening of the band at 3430 cm −1 was also observable due to the abundant hydrogen bonds in the DES [18]. The presence of Clfrom MgCl2 was observed in the form of a band at 590 cm −1 ; whereas the -NH2 group from [U] in the DES was also detected by an enlargement (stretching) of the band at 1610 cm −1 , manifesting an increase in the interactions of the hydrogen from -NH2 with Cl − . In addition, the absence of OH − groups from water and the formation of hydrogen bonds between the Cl-H led to a pronunciation of the Cl − group.
The interaction between the hydrogen bond from [U], which was used as a donor (HBD), and the chlorides Cl − from the halogen salt [MgCl2·6H2O], which were used as acceptors (HBA), allowed the melting point of both salts to decrease [22]. This was evidence of the DES formation, which was confirmed in the following Sections.    [21]. In turn, spectra of [U] showed bands at 3340 and 3430 cm −1 , which were linked to -NH 2 groups. Additionally, stretching vibrations at 1677 and 1461 cm −1 from C-O and C-N bonds were observed, respectively, while a stretching vibration at 1571 cm −1 was characteristic of the carbonyl group (-C=O).

Viscosity Behaviour of DES
The FTIR of obtained DES [MgCl 2 ·6H 2 O] [U] (1:1) confirmed the eutectic mixture, showing characteristic bands of Cl-H hydrogen bonds at 3322-3209 cm −1 . However, a widening of the band at 3430 cm −1 was also observable due to the abundant hydrogen bonds in the DES [18]. The presence of Cl − from MgCl 2 was observed in the form of a band at 590 cm −1 ; whereas the -NH 2 group from [U] in the DES was also detected by an enlargement (stretching) of the band at 1610 cm −1 , manifesting an increase in the interactions of the hydrogen from -NH 2 with Cl − . In addition, the absence of OH − groups from water and the formation of hydrogen bonds between the Cl-H led to a pronunciation of the Cl − group.
The interaction between the hydrogen bond from [U], which was used as a donor (HBD), and the chlorides Cl − from the halogen salt [MgCl 2 ·6H 2 O], which were used as acceptors (HBA), allowed the melting point of both salts to decrease [22]. This was evidence of the DES formation, which was confirmed in the following Sections.  According to the fluid type, DES (1:1) and (2:1) showed non-Newtonian behavi (range 0-60 Pa); specifically, as pseudoplastic and dilatant fluids, respectively, wher the range >60 Pa displayed similar Newtonian behaviour for both proportions.

Viscosity Behaviour of DES
Regarding the viscosity variation as a function of temperature, DES (1:1) and ( exhibited a reduction in viscosity with an increasing temperature, which was caused weak molecular interactions.
DES (1:1) showed most viscosity in the range of 0-30 °C. After the last range (>30 ° both proportions of DES presented an equivalent viscosity and the same behaviour. The viscosity difference in DES was associated with the proportion of the an [MgCl2·6H2O] present. A particularly high concentration of this ion in DES (2:1) inhibi the capacity to form several hydrogen bonds and the availability for acceptance by donor group [U], leading to less viscosity.
The DES proportion 1:1 indicated the most extensive H-bonding network betw [U] and [MgCl2·6H2O], leading to a slightly higher viscosity value.
Similar viscosity values in the >30 °C range were attributed to a comparable wa content in both proportions of DES. The increase in viscosity of DES compared to wa was attributed to enhanced van der Waals forces relative to the hydrogen bonding [ In addition, the DES viscosity was not comparable with individual components beca the holes for the solvents or ions to move into are of different sizes, and the size of the i themselves is different [24].
Regarding the viscosity variation as a function of temperature, DES (1:1) and (2:1) exhibited a reduction in viscosity with an increasing temperature, which was caused by weak molecular interactions.
DES (1:1) showed most viscosity in the range of 0-30 • C. After the last range (>30 • C), both proportions of DES presented an equivalent viscosity and the same behaviour.
The viscosity difference in DES was associated with the proportion of the anion [MgCl 2 ·6H 2 O] present. A particularly high concentration of this ion in DES (2:1) inhibited the capacity to form several hydrogen bonds and the availability for acceptance by the donor group [U], leading to less viscosity.
The DES proportion 1:1 indicated the most extensive H-bonding network between [U] and [MgCl 2 ·6H 2 O], leading to a slightly higher viscosity value.
Similar viscosity values in the >30 • C range were attributed to a comparable water content in both proportions of DES. The increase in viscosity of DES compared to water was attributed to enhanced van der Waals forces relative to the hydrogen bonding [23]. In addition, the DES viscosity was not comparable with individual components because the holes for the solvents or ions to move into are of different sizes, and the size of the ions themselves is different [24].

Physicochemical Characteristics of DES
The physicochemical characteristics of prepared DES [MgCl 2 ·6H 2 O][U] (1:1) evidenced the formed DES as eutectic mixtures type IV with a general formula nMgCl 2 + U. However, they were different in appearance. DES (2:1) was a viscous, homogeneous, colourless liquid, whereas DES (1:1) was a whitish, more viscous liquid. Table 1 presents the physicochemical properties of DES (1:1) and (2:1), showing that the rate and hydration of magnesium chloride played an important role in obtaining DES and their properties. This is explained below.
The melting points of both DES were lower than the pure components, [U] (135 • C ± 1) and [MgCl 2 ·6H 2 O] (118 • C ± 0.5), demonstrating the DES formation, because systems with an extreme reduction in the melting point can be recognized as DES [25]. However, herein was observed that the (2:1) proportion was slightly higher than the (1:1) proportion, indicating the influence of the anion proportion [MgCl 2 ·6H 2 O] on the melting points of these DES.
The decrease in melting points in DES has been explained in different ways. According to Sun et al. [26], coulombic interactions between the cation and the anion in DES cause the delocalization of the negative charges through hydrogen bonding, leading to reduced melting points. Consequently, a higher amount of [MgCl 2 ·6H 2 O] in DES could reduce the number of coulombic interactions and increase the melting point of DES (2:1). However, Ashworth et al. [27] revealed that a wide variety of bond types (neutral, ionic, doubly ionic) are present in DES, showing competitive HBA-anion and HBD-cation interactions, which may well promote the reduction in the melting point of DES.
Nevertheless, recent studies of [ChCl][U] by neutron diffraction experiments and simulated tests indicated that charge delocalization is not responsible for lowering the melting point [12]. Consequently, the reduction in the secondary electrostatic cation-anion interactions, and an overall decrease in anion coordination at the cation charge centres could cause changes in the melting point of DES [ChCl][U], but the HOH-Cl − hydrogen bond was not a key interaction in the formation of this DES, because this structure remained unperturbed in the solid salt lattice.
Furthermore, the density of DES [MgCl 2 ·6H 2 O][U] showed imperceptible differences. DES (2:1) was slightly higher than (1:1), because the density decreased when the percentage of salt increased in the DES; however, the increase in (2:1) was almost imperceptible, because the density decreased with the water percentage in the DES (2:1). The analogous carbon number of cations of [U] in both proportions also allowed a little variation.
In comparison with the pure-compound vacancies, DES presented an increment in the density, which was explained because the number of vacancies in the molecular structure of DES had increased.  6 ], and they were similar to water (1.2-1.35 g/mL) [26].
Electrical conductivity from DES presented reduced values compared to individual components. Sequentially, the highest conductivity of DES was observed in proportion (2:1). The difference between both DES proportions was attributed to the high viscosity of DES (1:1), which was observed in Section 3.2. A high viscosity causes the ionic species not to be completely dissociated in the mixture and, thus, their movement is not independent [23]. Therefore, coulombic interactions between cations and anions were reduced. Contrarywise, the increase in anion [MgCl 2 ·6H 2 O] in DES proportion (2:1) reduced the viscosity of DES and, thus, coulombic interactions increased its electrical conductivity.
Reports  (1:1), respectively (maintaining a weight >5%). The discrepancy in Td proportions demonstrated that the mixtures presented different structures. Thus, Td was dependent on the intermolecular interaction and coordinating nature of the ion in the mixtures. In addition, the water content influenced Td, indicating the highest value for DES proportion (1:1). In sequence, the degradation of [U] was similar to the values reported by Chemat et al. [7].
The glass transition temperature (Tg) was detected at −45.8 and −40.1 • C for the (1:1) and (2:1) proportions, respectively; a constant increase in the endothermic process was observed, due to the relaxation of hydrogen bonds from DES [29][30][31]

Beetroot Betalain Extraction by DES
Betalain extraction by DES [MgCl 2 ·6H 2 O][U] at neutral and acidic pH were identified as BED7 and BED3, respectively, while betalain water extracts at a similar pH were described as BEW7 and BEW3.
Data were relative to betalain extraction by the DES [MgCl 2 ·6H 2 O][U] (2:1) proportion, because the high viscosity of DES (1:1) affected the diffusion of betalains negatively, hindering their extraction at 20-25 • C (data of viscosity of proportion 1:1 were presented in Section 3.2). In this case, the anion of DES was a crucial factor for viscosity properties.
As a result, Figure 3 shows UV-Vis spectra from DES and BEW extracts, exhibiting similar bands; however, BEW samples presented absorption bands at 480 nm, which were identified as yellow betaxanthins, while bands at 535-540 nm were recognized as red-violet betacyanin. The difference between betaxanthin and betacyanin is that the substituent of betaxanthin is betalamic acid residue and the substituent of betacyanin is dihydroxyphenylalanine. The substituent of betaxanthins is commonly glutamine (vulgaxanthin) [5].
UV-Vis spectra of BED3 extracts also showed the highest betalain content, due to the pH. The increase in the proton quantity favoured the hydrogen bond augmentation [33].
UV-Vis spectra of BED3 extracts also showed the highest betalain content, due to the pH. The increase in the proton quantity favoured the hydrogen bond augmentation [33].  Table 2 presents data for the betalain content of beetroot extracts and the extraction yield (%). The order of Betacyanin content was observed as BED3 > BED7 > BEW3 > BEW7, whereas the total content of betalain was found in the 3.65-3.99 mg/g range in DES, and BEW extracts showed 3.49-3.55 mg/g. Table 2. Betalain content on fresh weight and betalain yield (%) in beetroot DES (2:1) (BED7 and BED3) and water extracts (BW7 and BEW3). The extraction yield indicated that DES solvent (2:1) exhibited compatibility in the extraction and recovery of betalains from beetroot waste, because its viscosity facilitated the suspension of beet betacyanin. In addition, acid extracts provided the highest yield of betalains; however, DES extracts exhibited a noticeable reduction in betaxanthins. This phenomenon was attributed to betacyanin stability and diffusivity in the extracts, as well as DES selectivity by betacyanin, trapping the molecule [34].

Extraction
The results of the total betalain content from BEW and BED extracts were in accordance with [35], showing 3.80 mg/g on fresh weight. Through an HPLC analysis, it was found that the major constituents of red beet pigments were betalain and isobetalain. The  Table 2 presents data for the betalain content of beetroot extracts and the extraction yield (%). The order of Betacyanin content was observed as BED3 > BED7 > BEW3 > BEW7, whereas the total content of betalain was found in the 3.65-3.99 mg/g range in DES, and BEW extracts showed 3.49-3.55 mg/g. Table 2. Betalain content on fresh weight and betalain yield (%) in beetroot DES (2:1) (BED7 and BED3) and water extracts (BW7 and BEW3).

Extraction Solvent Betacyanin (mg/L) Betaxanthin (mg/L) Total Betalain (mg/g) Betalain Yield (%)
BED7 The extraction yield indicated that DES solvent (2:1) exhibited compatibility in the extraction and recovery of betalains from beetroot waste, because its viscosity facilitated the suspension of beet betacyanin. In addition, acid extracts provided the highest yield of betalains; however, DES extracts exhibited a noticeable reduction in betaxanthins. This phenomenon was attributed to betacyanin stability and diffusivity in the extracts, as well as DES selectivity by betacyanin, trapping the molecule [34].
The results of the total betalain content from BEW and BED extracts were in accordance with [35], showing 3.80 mg/g on fresh weight. Through an HPLC analysis, it was found that the major constituents of red beet pigments were betalain and isobetalain. The results of Singh et al. [36] were also comparable. The authors achieved 2.42 and 4.59 mg/g of betalains, using microwave-assisted extraction and citric acid, as well as ethanolic water dissolutions as solvents, respectively.
The enhancement of betalain extraction with acidification was also previously corroborated by other authors [36,37]. They found that the increase in extraction occurs in a pH range between 3 and 5, using water as an extraction solvent.

Stabilization Analysis of Betalains in DES Extracts by Environmental Conditions of Light and Oxygen
DES and BEW extracts displayed initial purpura-red tonality from the beetroot; however, water extracts exhibited an alteration in the colouration and loss of pigment because environmental conditions of light and oxygen exhibited a negative impact on the betalain stability, increasing molecular reactivity during the oxidation of polyphenol oxidases [38].
Conversely, the highest colouration of betalains was well preserved in extracts BED7 and BED3 (2:1). A reduced quantity of water and oxygen contained in the samples extended the betalains' life. The principal betalain reduction was detected after 27 days. However, after this time, the betalains were sustained, even with exposure to light. Figure 4 exhibits the linear kinetic curves of betalain degradation from beet extracts BED3, BED7, BEW3, and BEW7. The kinetics confirmed a fast reduction in betalains in water extracts BEW7 and BEW3, as well as a colour stability in BED3 and BED7. The data here were relative to changes in betalain concentrations during the test, owing to the exposure of samples to environmental conditions for 40 days of visible light (12 h), and molecular oxygen from atmospheric air and environmental temperature (20-27 • C).
results of Singh et al. [36] were also comparable. The authors achieved 2.42 and 4.59 mg/g of betalains, using microwave-assisted extraction and citric acid, as well as ethanolic water dissolutions as solvents, respectively.
The enhancement of betalain extraction with acidification was also previously corroborated by other authors [36,37]. They found that the increase in extraction occurs in a pH range between 3 and 5, using water as an extraction solvent.

Stabilization Analysis of Betalains in DES Extracts by Environmental Conditions of Light and Oxygen
DES and BEW extracts displayed initial purpura-red tonality from the beetroot; however, water extracts exhibited an alteration in the colouration and loss of pigment because environmental conditions of light and oxygen exhibited a negative impact on the betalain stability, increasing molecular reactivity during the oxidation of polyphenol oxidases [38].
Conversely, the highest colouration of betalains was well preserved in extracts BED7 and BED3 (2:1). A reduced quantity of water and oxygen contained in the samples extended the betalains' life. The principal betalain reduction was detected after 27 days. However, after this time, the betalains were sustained, even with exposure to light. Figure 4 exhibits the linear kinetic curves of betalain degradation from beet extracts BED3, BED7, BEW3, and BEW7. The kinetics confirmed a fast reduction in betalains in water extracts BEW7 and BEW3, as well as a colour stability in BED3 and BED7. The data here were relative to changes in betalain concentrations during the test, owing to the exposure of samples to environmental conditions for 40 days of visible light (12 h), and molecular oxygen from atmospheric air and environmental temperature (20-27 °C). According to final colouration, a possible hydrolysis of the aldimine bond of betalains from water extracts resulted in the formation of betalamic acid (yellow colour) and the colourless cyclo-Dopa-O-ß-glucoside [38]. Hydrolytic reactions were linked to the interaction of oxygen with light, inducing the fast degradation of betaxanthins and betacyanin [5,6].
The probable dehydrogenation of phyllocactin and hylocerenin, forming other yellow-coloured degradation products, could also provide evidence of the degradation of betalains and the final colouration in water extracts [39]. In addition, the free sugars and nitrogenous compounds caused fermentation, probably contributing to the reduction in the violet colouration.
The pH also started the degradation of BEW3, because the increase in the interaction of H+ bonds with the protonation of the aromatic characteristic betacyanin group caused According to final colouration, a possible hydrolysis of the aldimine bond of betalains from water extracts resulted in the formation of betalamic acid (yellow colour) and the colourless cyclo-Dopa-O-ß-glucoside [38]. Hydrolytic reactions were linked to the interaction of oxygen with light, inducing the fast degradation of betaxanthins and betacyanin [5,6].
The probable dehydrogenation of phyllocactin and hylocerenin, forming other yellowcoloured degradation products, could also provide evidence of the degradation of betalains and the final colouration in water extracts [39]. In addition, the free sugars and nitrogenous compounds caused fermentation, probably contributing to the reduction in the violet colouration.
The pH also started the degradation of BEW3, because the increase in the interaction of H+ bonds with the protonation of the aromatic characteristic betacyanin group caused a rise in betalamic acid; consequently, a reduction in the pigment stability, and less colouration.
In turn, beet DES extracts BED7 and BED3 increased the stability of the betalains because the total betalain content was higher than water extracts, resulting in a 75% increase in beet water extracts. The red-violet colouration was also maintained during test degradation, exposing the condensation of betalamic acid to cyclo-Dopa (betacyanins) to obtain a maximum stability of betalains.
Furthermore, betalains from DES extracts were well preserved under visible light for 150 days, and 340 days in storage (amber vessels). Consequently, DES resulted selective in terms of betalains from beets, and the viscosity of (2:1) proportion was an additional factor to keep the betalain diffusion in the samples, because a high viscosity reduces the mass transfer of oxygen, preventing betalain degradation. Additionally, the probable conservation of amino compounds from betalains in DES extracts allowed the maintenance of the violet colouration because they are generally coloured.
According to results, Equation (1) describes the betalain degradation of DES extracts, expressing a first-order kinetic model. The general solution of Equation (1) is expressed in Equation (2), where Kb is the degradation rate constant of betalains, B Ct is the betalains concentration in extracts at a definite time (day), BC 0 is the betalain concentration at initial time zero, and t is the time of the test (0-40 day). Table 3 exhibits the values of the K b constant (line slope) and correlation coefficient (R 2 ) by the linear regression of the linear kinetic model of betalain degradation from beetroot extracts. The high values of R 2 long confirmed a first-order reaction of betalain degradation from beetroot extracts by light and oxygen. In addition, the high values of K b from extracts BEW3 and BEW7 validated the fast degradation of betalains in these extracts, while the K b values from BED7 and BED3 indicated a slow degradation. Therefore, the presence of DES [MgCl 2 ·6H 2 O][U] in extracts reduced the instability of betalains in the presence of light and oxygen.

−dBc
Additionally, values from acid extracts BEW7 and BED7 presented higher K b values than BED3 and BEW3, exhibiting more betalain degradation, which was attributed to the pH.
Similar behaviour was found in previous reports of betalain degradation by temperature [40]. In this test, a first-order kinetic model was also described, indicating an important degradation in the temperature range of 50-120 • C.
Other alternatives to control betalain stability have verified that encapsulation conserves their colouration. Particularly, Ravichandran et al. [41] and Antigo et al. [42] observed an increase in the stability of red beetroot pigments, and betalain microcapsules showed the lowest degradation constant and the longest half-life. However, betalain degradation was also observed after 7 days of storage at 30 • C.
The 5kg of beetroot (Beta vulgaris) waste used in this research was provided by a Mexican beetroot juice manufacturer (México City, México). The beetroot waste included peel and pulp from var. red cloud. The water content of the beetroot waste was determined as 550 ± 17 mg/g. The waste was distributed and stored in plastic bags of 100 g at 0 • C for later use. The loss of water from the thawed waste was found to be 533 ± 17 mg/g, indicating a 3.2% loss of water.
The mixture was placed in a 500 mL closed flask for each molar ratio. The mixture was stirred at a controlled temperature between 50 and 60 • C until a homogeneous liquid was obtained.
The obtained products were identified as DES [MgCl 2 ·6H 2 O][U] (2:1) and (1:1), according to the proportions. Characteristics such as density, viscosity, electrical conductivity, structure by functional groups, and thermal behaviour were determined for both DES. An Anton Paar RheolabQC (Graz. Austria) rotational viscosimeter was used to measure the density (mg/mL) and viscosity (Pa.s) of DES. Both parameters were determined in the temperature range of 0-90 • C and shear stress of 0-180 Pa. Electrical conductivity was measured at 25 • C using a Hanna conductivity meter (Woonsocket, RI, USA). Functional groups were analysed and identified by Fourier Transform Infrared Spectroscopy (FTIR-ATR) Spectrum Varian 640-IR (Agilent, Santa Clara, CA, USA). The spectra conditions were 25 • C, 64 scans, 500-4000 cm −1 and 2 cm −1 resolutions, with respect to the appropriate background spectra. The thermal decomposition and thermodynamic transition properties of DES were determined by thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC), using a PerkinElmer Simultaneous Thermal Analysis model 8000 (TA Instruments, New Castle, DE, USA), with a heating ramp of 10 • C for 60s, in a nitrogen atmosphere, at a flow rate of 0.7 mL for 60 s. Samples were heated from room temperature to 350 • C. The crucibles of aluminium with a 5 mm diameter were used in this test. Savitzky-Golay smoothing algorithm was employed for TGA curves.

Beet Betalain Extraction by DES
Betalains extraction from beetroot waste was carried out using the prepared DES [MgCl 2 ·6H 2 O][U] (2:1) and (1:1) as extraction and stabilizing solvents. To measure the effect of pH on extraction efficiency, the DES were previously adjusted to pH 7 ± 0.3 and pH 3 ± 0.2.
Fresh beet pieces (0.5 g) were mixed with the DES solvent at a solid-to-liquid ratio of 1:30 g/mL (15 cm 3 ) in a blender. After, ultrasonic assisted extraction of betalains was applied using an ultrasonic batch (Bransonic CPX1800H, Emerson, México) at 25 • C for 3 h with subsequent vortex agitation for 900 s. The liquid was separated from the beetroot mass by filtration, using Whatman filter paper No.1. The liquid was identified as DES extract from beetroot waste. Water extracts of beetroot waste were also obtained as blank; they were used for comparing DES extracts with conventional isolates. Water betalain extracts were obtained as DES extracts.
The extracts were characterized according to total betalain content (mg/g) and betalain yield (%). The methodology of Castellar et al. [43] and Equation (3) were used to calculate the total betalain content (mg/mL). Betalain colouration was measured in a PerkinElmer Lambda 25 UV-Vis (Waltham, MA, USA), considering betaxanthins and betacyanin as principal structures in betalain extracts.
where A is the absorbance value of the extract at 535 nm for betacyanin and 480 nm for betaxanthin. DF is the dilution factor of the extract, and L is the path length of cuvette (cm). MW represents the molecular weight for betacyanin, 550 g·mol and 308 g·mol for betaxanthin. The extinction coefficients for betacyanin 60,000 M −1 ·cm −1 and 48,000 M −1 ·cm −1 for betaxanthin were relative to ε. A conversion factor of 1000 was used to convert g to mg.

Stabilization Analysis of Betalains in DES by Environmental Conditions of Light and Oxygen
Stabilization analysis of betalain extracts by DES and distilled water were studied by kinetic behaviour, monitoring the total betalain content at time zero (control) to 1, until 40 days of extract storage. The degradation was measured in exposed extracts under environmental conditions of visible light (12 h), molecular oxygen from air in the atmosphere and environmental temperature (25 • C). Betalain content was determined according to Equation (1), using the procedure indicated in Section 2.3. In addition, functional groups were also analysed by FTIR at time zero and after 30 days.

Statistical Analysis
All experiments and analytical determinations were conducted in triplicate. Data were processed with statistical methods in the OriginPro 2016 software 93.
In accordance with viscosity, DES (2:1) was used to extract betalains from beetroot waste, displaying a similar betalain content to water extracts. However, the kinetic curves of betalain degradation by exposing water samples to light and atmospheric oxygen described the alteration of the violet colouration from beetroot pigments. Betalain water extracts were degraded for 5-7 days, whereas betalains from DES extracts were well preserved under visible light for 150 days, and 340 days in storage (amber vessels), achieving a stability of 75% of the red violet colouration.
Based on the aforementioned data, DES [MgCl 2 ·6H 2 O][U] were an effective extraction and stabilizing agent for betalain removal from beetroot wastes. This demonstrated betalain stability, and a suitable level of moisture, as well as miscibility with betalains.
The results obtained in the present study contribute to the field of DES research, and the novel separation techniques for application as natural pigments. However, the direct use of DES extracts is limited in food areas because the presence of DES affect's organoleptic properties. Nevertheless, DES extracts could also be used as antifungal or antibacterial pigments. Therefore, more studies are necessary to suggest a global proposal of application. In addition, further research on the recovery and purification of betalains and DES recovery would provide more information to establish with certainty its usage in the food industry or other areas.