Extraction and characterization of mucilage from Opuntia ficus-indica cultivated on hydroponic system

An interesting component of Opuntia ficus-indica is the mucilage for its properties and industrial uses. However, the great variability of its quantity and quality caused by different growing conditions, the hydroponic system is an alternative. The objective of the present study was cultivating 4 species of Mexican Nopal in a hydroponic system, extract and characterize the mucilage. The characterization consists of pH, ºBrix, colour, proximal analysis, phenols, antioxidant activity, crystallinity, and chemical bonding constituents. ‘Copena F1’ is the best alternative for production of biomass and mucilage. ‘Villanueva’ had high levels of phenols (1,311.83 mg GAE g -1 ), antioxidant capacity ABTS • + (6,301.12 mg TE g -1 ) and FRAP (536.26 mg GAE g -1 ). A large amount of lipids (1.39%), and nitrogen-free extract (49.27%). The functional groups of the mucilage were identified (-OH, -CH, -CH 2 , -CH 3 , C=C, HCH, -CHO) and gypsum, cellulose, SiO 2 CaSO 4 , C 2 H 2 K 2 O 5 , CaCO 3 and CaH 2 by X-ray diffraction. The hydroponic system is a viable alternative for production of nopal and mucilage of high-quality mucilage that can be used in several sectors of the industry. et al ., 2017) and 9.65% (Dick et al ., 2019). Our results are due to the storage of water retained by the solvent trawl for the recovery of the mucilage powder, the variety, quantity of water retention of polymers, matter stage and other factors.


Introduction
In recent years, the interest of the homogeneity of crops has taken a big importance to increase the quantity and quality of food for providing clean and fresh foods for the next generations. One of the main alternatives is to establish crops under systems that allow controlling mainly the quantity of nutrients, increasing populations densities and without the need of large extensions of land. All this, cultivation, propagation, and conservation of several crops like Nopal relies on traditional agro practices techniques. However, these conventional practices did not fulfil the desired needs; as large-scale production (high value, 3 and quantity of produced mucilage by four cultivars. Likewise, to characterize the mucilage produced due to that no of a complete characterization of this material (mucilage) produced under hydroponic conditions.

Materials and Methods
Biological material Four cultivars ('Villanueva','Jalpa,'Ja','Ou' and 'CF1') were provided by the School of Agronomy, Campus Marín, UANL. The material was selected with the similar characteristics (height, thickness, and width) without visual damage.

Establishment of the crop
The hydroponic system (HS) was installed in the same School of Agronomy, Campus Marín, Nuevo León,Mexico (25º 23'N,100º2'O,393 masl). The bench had 1.1 m of wide and 14 m long. The floor and walls are of concrete. Tezontle was used as support (4 months of age). The nutrient solution (NS) composed by macro and microelements (200 N,60 P,250 K,200 Ca,50 Mg,100 S,0.50 Fe,0.25 Mn,0.25 B,0.02 Cu,0.25 Zn and 0.01mg L -1 of Mo) with pre adjusted pH to 5.5 was used according to the proposed by Rodríguez-Fuentes et al. (2011). Four cultivars were placed in the HS with a completely randomized design with a population density of 30 plants m -2 . The irrigation of the crop was performed every 15 days with the NS.

Biomass of Opuntia ficus-indica
To know the accumulation of mucilage inside of cladodes in the period March-April 2018, the sampling of cladodes was taken at 45 days. For each treatment, the number and total fresh weight of the cut cladodes were determined. The cladodes were washed with distilled water and dried in a drying oven (Thermo Fisher Scientific, USA) at 75 ± 2 ºC. The moisture content was calculated according to the reported by Almaguer-Sierra et al. (2014).

Biomass of Opuntia ficus-indica
To know the accumulation of mucilage inside of cladodes in the period March-April 2018, the sampling of cladodes was taken at 45 days. For each treatment, the number and total fresh weight of the cut cladodes were determined. The cladodes were washed with distilled water and dried in a drying oven (Thermo Fisher Scientific, USA) at 75 ± 2 ºC. The moisture content was calculated according to the reported by Almaguer-Sierra et al. (2014).

Mucilage extraction
The extraction was carried out based on previous reports (Espino -Díaz et al., 2010;Rodríguez-González et al., 2014;Sandoval et al., 2019) with some modifications. The cladodes were cut into small pieces, placed in distilled water (1:1 w/v) and mixed in a magnetic stirring plate (Thermo Fisher Scientific SP13163033Q, USA) for 10 min until obtain a homogeneous suspension. After that, was heated (110 ºC/60 min) with constant agitation and centrifuged (Ultracentrifuge 5810/5810 R, Eppendorf, U.S.A.) at 252 x g/20 min. To precipice the mucilage, ethanol (98%) was added to the supernatant in a ratio 2:1 v/v and stored at 4ºC/60 h. Ethanol was evaporated in a convention oven (Model ED 115, Thermo Fisher Scientific, USA) at 70 ºC/2 days. The mucilage precipitated was pulverized and sieved (sieve No. 4, particle size of Ø 1.75 mm, MEQUIM, U.S.A.) to obtain a homogeneous particle size. The samples were stored in an airtight container until use. The yield was calculated according to the equation 1.
Mucilage's characterization Physicochemical analysis One g of mucilage powder was resuspended in 10 mL of water-methanol (1:1.75 v/v). The pH was measured with a calibrated pH meter (Hanna HI 2211, Germany). Total soluble solids (TSS) were determined with a 0-30% refractometer (Optika, DC-HR 130, China). The color was measured with a colorimeter (Minolta, CR-400, USA) and the results expressed based on the CIELAB profile, using the color coordinates L* (luminosity), a* (red/green) and b* (yellow/blue). The determinations were made in triplicate.
Proximal analysis Proximal evaluations were made according to the recommendations of Official Analysis Method (AOAC, 1984). Moisture was carried out with the 934.06 test. Crude protein was analyzed with the Kjeldhal method (Model GL-44, USA) 960.52. Total fat according with the Soxhlet method (Model EV6 All, Gerhardt, Germany) 960.39. Ash content was calculated based on the incineration in a muffle (Model ECO-2L, USA) at 550 ºC following the test 923.03. Crude fiber was evaluated with the enzymatic-gravimetric method (method 985.29). Nitrogen-free extract was analyzed qualitatively with the difference of 100% of the samples. The values obtained in percentages were converted with the angular transformation of Bliss through the results of arcsine in triplicate to be analyzed statistically.
Subsequently, 25 µL of mucilage suspension was mixed with 25 µL of Folin-Ciocalteu reagent, and after 1 min, 25 µL of sodium carbonate (75 g L -1 ) was added. The solution was mixed thoroughly and incubated a 40 ºC/30 min in a water bath. After that, the mixture was adjusted with 200 µL of distilled water and the absorbance was recorded at 750 nm with a microplate reader (Synergy HT Multi-Detection Microplate Reader, BioTek Instruments, USA). The absorbance of the sample was compared with the gallic acid standard curve to estimate the concentration of TPC in the mucilage suspension. The TPC were calculated as mean ± SD and expressed in mg of gallic acid per g of raw material (mg GAE g -1 ).
Antioxidant activities DPPH • radical scavenging activity The antioxidant activity in the mucilage was evaluated as the DPPH • free radical-scavenging activity.
This activity was estimated using the method proposed by Brand-Williams et al. (1995), Castro-López et al. (2017) and Rojas et al. (2018). The hydrogen atom or electron donation abilities of the sample was measured from light-purple colored DPPH methanol solution (60 mM). Subsequently, 5 µL of mucilage solution was added to a 295 µL DPPH • radical solution. After a period of incubation in the dark for 30 min, the absorbance was recorded at 517 nm with the same microplate reader. The ability to inhibit was calculated by the eq. (2), and expressed as percent inhibition of DPPH • radical: Where AControl is the absorbance of the control reaction (containing all reagents except the test compound) and ASample is the absorbance with the mucilage solution. Gallic acid was used as reference. The DPPH • inhibition was expressed as the average of three replications in gallic acid equivalents in mg per gram (mg GAE g -1 ) of raw material. 5 ABTS •+ radical scavenging activity The inhibition of the ABTS •+ radical was made according to the methodology proposed by Hernández et al. (2018) and van den Berg et al.,1(999). The ABTS •+ radical cation was generated by mixing an aqueous solution of potassium persulfate (2.45 mM) and ABTS •+ (7mM). These reagents react stoichiometrically in a ratio of 1:2 respectively and must be kept in the dark at room temperature for 12 h before use. Diluted solutions of ABTS •+ were prepared in ethanol until value of 0.700 ± 0.002 nm absorbance was obtained. Then, 95 µL of the dilute solution of ABTS •+ was mixed with 5 µL of mucilage solution and the absorbance was measured at 734 nm with the same microplate reader. The capacity to inhibit the radical was calculated according to the eq.
(3) and the results were expressed as percent inhibition of the ABTS •+ radical: Where AControl is the absorbance of the control reaction (containing all reagents except the test compound) and ASample is the absorbance with the mucilage solution. The ABTS •+ scavenging ability of mucilage solution was calculated according to the standard curve plotted with Trolox and expressed as Trolox equivalent in mg per gram (mg TE g -1 ) of raw material.
Ferric reducing power assay (FRAP) The ferric ion (Fe 3+ ) reducing power was determined as described by Benzie & Strain, 1996 with a slight modification by Castro-López et al. (2017) and Bautista-Hernández et al. (2021). To 5 µL of a mucilage solution was added 12 µL of phosphate buffer pH 7. After that, it was added to the mixture 22 µL of potassium ferrocyanide 1%, homogenized and incubated in a boiling water bath at 50 ºC/20 min. After cooling, 12 µL of trichloroacetic acid 10% was added. Also, 45 µL of distilled water and 10 µL of ferric chloride 0.1% were added and shaken thoroughly. The absorbance was recorded at 700 nm with the same microplate reader. Finally, the results were reported as gallic acid equivalents in mg per gram (mg GAE g -1 ) of raw material.

Crystallinity and chemical bonding constituents
Crystallinity phases of the mucilage were evaluated by X-ray diffraction (XRD) according to the method reported by Ballesteros et al. (2018) using a D8 Discover diffractometer (Brunker, corporation) with the Cu tube (λ= 1.5406 Â). The radiation was generated at 25 mA and 30 kV. The scattering angle of 2θ from 10º to 60º at the step size of 0.04 and 1 s exposure at each step. Chemical groups and bonding arrangements of constituents present in the mucilage were determined by Fourier transform infrared spectroscopy (FTIR) using a Bunker Model FT-IR Vertex 80/80 v spectrometer (Boston, USA) in attenuated total reflectance mode (ATR) with a platinum crystal accessory. The measurements were recorded with a wavenumber range from 4000 to 400 cm-1 and 3 scans per sample.

Statistical analysis
All experiments were analyzed in triplicate (n = 3) and results expressed as mean ± standard deviations. Analysis of variance (ANOVA) was calculated with Tukey's test through the Statistica 21.0 software (IBM Corp., New York, NY, USA) at a 95% confidence level.

Total polyphenols content (TPC)
The quantification of TPC can be an indication of the bioactivity and/or functionality of the crop and the TPC in the cultivars evaluated in this work was high that found by other authors. TPC varied from 565.76 ± 1.5 to 1,311.83 ± 2.3 mg GAE g -1 of dried weight (Table 4). Among the four cultivars studied, significant differences (p ≤0.05) were found. The low values were found in 'Ja' (565.76 ± 1.5 mg GAE g -1 ) and 'CF1' (593.81 ± 2.8 mg GAE g -1 ), whereas 'Va' contained the highest amount (1,311.83 ± 2.3 mg GAE g -1 ).

Crystallinity and chemical bonding constituents
Cultivars 'Va' and 'CF1' were selected to perform FT-IR and X-ray diffraction considering that they had the best physicochemical properties (Figure 2). The first band which ranged between 3400 and 300 cm -1 represents the -OH region (Cai et al., 2008;Contreras-Padilla et al., 2016;Mejías Díaz et al., 2013;Wang et al., 2007). The second and third bands, ranging between 3000-2800 cm -1 , are representative of the -CH, -CH2 and -CH3 groups. In the bands 1500 cm -1 , the C = C group with low intensity is visible, which coincides with aromatic and alkene vibrations (Rubinson and Rubinson, 2000). The frequencies of the bands > 1000 cm -1 were identified as the HCH-flexed bonds that are isometrically present, possibly at -CHO. Band 8 that oscillates in mucilage's > 500 cm -1 is associated with β-D-glucose. The last FT-IR spectrum of bands 9-10 was attributed to the N-H bond and -OH respectively (Monrroy et al., 2017;Smith, 1998;Zhao et al., 2017). The X-ray diffraction patterns of hydroponic mucilage with and without fiber is shown in Figure 3. In the first found was gypsum (with confirmation on peak four) and CaCO3 in the second peak. The silicon (third peak) and the fourth peak, the calcium sulphate. Followed by potassium oxalate monohydrate and the last peak showed the calcium hydride in the mucilage's with fiber, but the same compound was absent in the mucilage's without fiber.  (Flores-Mendiola, 2012). The use of hydroponic systems is better than traditional or intensive systems due to the use of water with high salt contents that produce highest values of yield, length and diameter with the cultivar 'Va', and with the 'CF1', and 'Ja', less (Ramírez-Tobías, 2012;Vázquez-Alvarado et al., 2009). The difference in biomass production is attributed to the lack of assimilation of nutrients by some cultivars due to the competition for prickly pear production, given that, in shorter period, higher production of cladodes is obtained and the density of population to which the vegetative material established produces more (Rodríguez-Fuentes et al., 2011). Also, air temperature is one factor since these conditions prevail in their maximum production during the spring-summer period. The traditional systems produce up to 90 tons ha -1 yr -1 and hydroponic systems up to 470 tons ha -1 yr -1 . This variability is due to the management systems, the differential genetic potential of the variety, que quality and the quantity of the climatic supply of different state. On the other hand, the optimal nutrients supply according to the cultivar and its phenological stage (Horibe, 2018).
Mucilage yields from 3.8 to 19% has been reported (Matsuhiro et al., 2006;Sepúlveda et al., 2007;Cai et al., 2008;Contreras-Padilla et al., 2016). The yield depends of cultivar type, crop age and vary with the extraction conditions, as well as environment and genotype (Kaur et al., 2018). Also, the temperature (up to 110 °C) interfere with the yield due to the polysaccharides begin to degrade al 90 °C (Huang et al., 2000;Karazhiyan et al., 2011) and the use of alkaline conditions could increase the yield by hydrolyzing soluble and insoluble components (Hong and Ibrahim, 2013). However, the use of controlled conditions (hydroponic 10 culture) allows to increase the concentration of mucilage because it reaches the physiological maturity of the culture in a fast and controlled way (Hooshmand et al., 2019).

Physicochemical and proximal analysis
The obtained data on the pH of mucilage from Opuntia ficus-indica are scare in the available literature. However, similar values have been reported from 5.5 to 6.0 (León-Martínez et al., 2011;Contreras-Padilla et al., 2016;Monroy-Gutiérrez et al., 2017). The difference is attributed to the accumulation of citric and malic acid in the cladodes before extracting the polymer, due to the presence of Ca2 (due to the hydroponic culture system) increased the pH (Du Toit et al., 2019;Trachtenberg and Mayer, 1980) and the regeneration of carbohydrates and accumulation of acids or different precursors of the enzyme phosphoenol-pyruvate could generate a positive increase in the acidity of the mucilage's at the time of being extracted. These values obtained for TSS are higher than that reported on literature and is due to the specie, hydroponic culture system and stage of maturity of the nopal (Madera-Santana et al., 2018), which allows adequate physiological development due to the ease of nutrient uptake. The positive values of a* in the mucilage 'Va' and 'Ou' indicate a slight brown coloration that is attributed to alterations due to the heating process during the extraction of the mucilage. This caused a change of olive-green color to brown in the samples, due to the formation of pheophytin (Du Toit et al., 2019). According to determination of color of the mucilage's, it is suggested that these might contain pigments like carotenoid, which means that they have an attractive antioxidant power for use in the industry (Hong and Ibrahim, 2013).  -Santana et al., 2018;Sepúlveda et al., 2007). There is great variation on the protein content in the mucilage, however, this variation is due to the vegetative material and maturity (Martin et al., 2017;Dick et al., 2019). Likewise, this is due to the water retention in nopal during nutrition using the hydroponic system, variability of the genetic material and the mucilage's can acquire nitrogen from various sources, such as nucleic acids, nitrates and nitrites that were reserved by the nopal cactus (Madera-Santana et al., 2018). Furthermore, the high values of ashes in the cladodes can be attributed to the high salinity of the soil and to the mineral bioavailability in hydroponic systems (Rodríguez-Fuentes, 2009). The mucilage's extracted from the nopal are highly rich in dietary fiber, this variation occurs at higher concentration as the nopal matures, since the fibers vary in their structure and size and therefore in the not solubilization or solubilization in the aqueous medium. Mucilage's have organic molecules that are considered hydrophobic and hydrophilic because their covalent bonds of hydrogen and carbon are not polarized, which distances them from polarized water molecules. This same property makes them insoluble in water or totally soluble (Rodríguez-Fuentes, 2009;Espino-Díaz et al., 2010;Contreras-Padilla et al., 2016). In general, these differences revealed the behavior of the mucilage cells present in the cactus cultivars and the accumulation of evaluated components, as the cladode matures, the nutritional content increases, since it reserves energy.
Total polyphenols content (TPC) Reported values varied from 0.77 to 25.54 mg GAE g -1 (Jaramillo-Flores et al., 2003;Kim et al., 2013;Sangeethapriya and Siddhuraju, 2014). This variability is due to the generation of ascorbic acid present in the nopal mucilage's for the generation of phenolic compounds by providing hydrogen atoms to the phenoxy 11 radicals, which are formed when the phenols are oxidized (Keshani-Dokht et al., 2018). Besides, interaction of this vitamin with polyphenoloxidase causes irreversible inhibition of the tendency (Zheng and Wang, 2001;Wojdyło et al., 2007). Additionally, it was observed that the concentrations studied depend on the state of the polymeric material, variety, stage of maturity and the hydroponic system allows the formation of phenolic compounds because the crop grows in optimal conditions compared to a traditional system (Mattson and Lieth, 2019;Xu et al., 2019).

Antioxidant activity
Several reports inform about the antioxidant capacity of mucilage but not at these levels (Kim et al., 2013;Motiwala et al., 2015). This is due to the chemical composition of each species, the culture system and the concentration of the natural antioxidants that can have a synergist effect between the bioactive compounds that make up the mucilage for each cultivar. The differences between the results obtained between methods has already been described, as in the work of Floegel et al. (2011) whose research focused on comparing these methods and showed that the antioxidant capacity was better reflected by ABTS •+ than by DPPH • in a wide variety of foods. The DPPH • method is more selective because it does not react with flavonoids lacking hydroxyl groups in the B ring, nor with aromatic acids containing a single hydroxyl group.

Crystallinity and chemical bonding constituents
The hydroxyl group (3400 and 300 cm -1 ) is characteristic of the accumulation of malic acid in the chlorenchyma and parenchyma, some of the most abundant acids and that are easily metabolizable by the microorganisms that attack the cactus. In addition, the -OH group is generated by chemical synthesis for the extraction of mucilage through precipitation (band 1). The presence of -CH, CH2 and -CH3 groups is explained by the carboxyl and xylane groups stored in lignocellulic layers of the mucilage before being recovered (Contreras-Padilla et al., 2016;Monrroy et al., 2017). The C = C, HCH-and -COH possibly by the formation of arabinose and dispersed carbohydrate groups such as mannose and glucose (Cai et al., 2008;Mejías Díaz et al., 2013;Monrroy et al., 2017;Madera-Santana et al., 2018). The results indicated that the maturation and the hydroponic culture system of the mucilage's does not significantly infer the intensity of the bands of the functional groups.
The X-ray diffraction patterns of hydroponic mucilage with and without fiber had the longest peaks that could be due to the larger particle size that the lens could inhibit the perception of crystallized compounds (Contreras-Padilla et al., 2016;Marin et al., 2018). The variation is due to the type of cultivar and culture system where the polymers were extracted, the maturity and the fiber accumulation in these. Calcium is available in the cactus nopal unless they have high concentrations of oxalic acid or dietary fiber since they limit their formation when they are sequestered as oxalates, preventing their absorption by the metabolism (Valenzuela et al., 2018). The silicon (third peak) was absorbed in the mucilage's formed by nopal through nutrition in the form of monosilicic acid by the assimilation of nutrients captured during the hydroponic crop cycle. In the fourth peak, the calcium sulphate that has been reported in the structure of parenchyma nopal, calcium oxalate crystals are formed. These are classified into four types by their morphology: rafidia, drusen, styloid and prisms (Bárcenas et al., 2011;Madera-Santana et al., 2018). Hydrogen oxalate hydrate of potassium is considered an antinutrient that is in the form of calcium crystals or free form and change depending on the maturity of the mucilage (Nerkar and Gattani, 2012;Contreras-Padilla et al., 2016). The calcium carbonate in the fibrous mucilage's can occur in three anhydrous crystalline polymorphs such as calcite, aragonite and vaterite ordered by their thermodynamic stability (Payne et al., 2007). The last peak showed the calcium hydride in the mucilage's with fiber, but the same compound was absent in the mucilage's without fiber. This could be due to the expression of oxalates crystallized directly in the fibrillar tubes of the nopal tissues where the mucilage is stored (Trachtenberg and Mayer, 1982).
12 Conclusions A hydroponic system to produce mucilage is more efficient in quantity and quality. However, 'Copena F1' at culture conditions of 30 plants m -2 is the best alternative to produce biomass and mucilage. 'Villanueva' allows to obtain higher concentrations of TPC (1,311.83 ± 2.36mg GAE g -1 ) with a high antioxidant capacity ABTS •+ (6,301.12 ± 0.61 mg TE g -1 ), FRAP (536.26 ± 0.84 mg GAE g -1 ) and DPPH • (358.26 ± 0.53 mg GAE g -1 ) with a large amount of lipids (1.39 ± 0.01%), nitrogen-free extract (49.27 ± 0.04%) and energy content (4,417.00 ± 1.73 Kcal/kg). The identification of the characteristic functional groups of the mucilage (-OH, -CH, -CH2, -CH3, C=C, HCH, -CHO) and gypsum, cellulose, SiO2 CaSO4, C2H2K2O5, CaCO3 and CaH2 by X-ray diffraction was also done. This study provides a novel, cost-effective and applicable technology in any region without the need for large tracts of land to produce high quality cactus and mucilage with bioactive properties. Ethical approval (for researches involving animals or humans) Not applicable.