Mucilage Yield, Composition, and Physicochemical Properties of Cultivated Cactus Pear Varieties as Influenced by Irrigation

Abstract

Opuntia spp. plants occupy an important socioeconomic role in arid and semiarid zones where water is scarce. Irrigation increases the productivity of these plants; however, its effect on the yield, composition, and physicochemical properties of the mucilage is unknown. Three irrigation regimens were tested: non-irrigated (rainwater), supplemental irrigation (irrigation between field capacity (0.28 m³ m⁻³) and permanent wilting point (0.14 m³ m⁻³)), and full irrigation (100% of crop evapotranspiration), on the four cactus pear varieties (‘Amarilla Olorosa’ (Opuntia spp.), ‘Cristalina’ (Opuntia albicarpa Scheinvar), ‘Dalia Roja’ (Opuntia spp.), and ‘Roja Lisa’ (O. ficus-indica (L.) Mill)). Irrigation regimens were applied during the dry season (March to June in the northern hemisphere). Composite samples of cladodes per replicate and treatment were collected for mucilage extraction. The mucilage was characterized for yield, color, chemical composition, infrared spectroscopy, viscosity, and molar mass. The combination with the greatest yield was ‘Amarilla Olorosa’ with no irrigation (22.2%), while the least yield was from ‘Cristalina’ undergoing full irrigation (12.2%). In general, non-irrigated plants yielded more mucilage, their color was brighter and less green, and they had more protein and fiber. The viscosity and molar mass were greatest in non-irrigated plants. Total carbohydrate content was similar between non-irrigated and supplementally irrigated plants. Thus, for the cactus pear varieties studied here, either no irrigation or supplemental irrigation could be a feasible strategy to produce mucilage with good characteristics for agro-industrial and pharmaceutical use.

1. Introduction

Cactus pear as a crop is commonly grown under rainfed conditions in areas where rainfall is scarce and erratic. Water is the main limiting factor for its productivity when grown for human consumption (tender cladode and fruit) in arid and semi-arid regions [1]. These conditions induce dehydration of both fruit and cladodes [2]. However, when water is applied using supplemental drip irrigation, both fruit and cladode yields improve significantly [3]. Among other cultural practices, these plants are pruned annually, yielding between 10 and 15 t ha⁻¹ of fresh cladodes. Since 45,320 ha in Mexico are cultivated with cactus pear [4], this vegetative material could be used potentially for agro-industrial or other purposes [5].

Cactus pear cladodes contain a heteropolysaccharide compound called mucilage of great agro-industrial importance [6]. This macromolecule is composed mainly of arabinose, galactose, galacturonic acid, rhamnose, and xylose units [7]. The most important functions of mucilage in cactus pear plants are to maintain the ionic balance in plant cells, water transport and retention, frost tolerance, and carbohydrate storage [8]. Recently, mucilage has gained importance in the agro-industry due to its multiple uses and applications [9]. Nevertheless, the yield and physicochemical properties of the mucilage are influenced by the cactus pear variety, cladode age, and weather conditions [10,11,12]. Nevertheless, studies of the effect of drip irrigation supply on mucilage properties have not been conducted so far, which can have positive implications for their uses in the food and pharmaceutical industry and other industrial applications worldwide. Therefore, this study tested the hypothesis that irrigation would modify the yield, composition, and physicochemical characteristics of mucilage extracted from cladodes of four cactus pear varieties. This study included the color, viscosity, and molar mass of the mucilage because of their importance for much more diverse industrial uses.

2. Materials and Methods

2.1. Experimental Site

The experiment was conducted from 2018 to 2020 at the Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias (INIFAP), Campo Experimental Zacatecas (latitude 22°54′ N, longitude 102°39′ W, elevation 2197 m). The climate of the area is semi-warm, with a mean annual temperature of 14.6 °C, and it receives 416 mm of annual precipitation. The average annual evaporation is 1609 mm. The orchard soil has a loam texture with an organic matter content of 1.73% and a pH of 7.75.

Plant Material and Experimental Process

This research included four cactus pear varieties: ‘Amarilla Olorosa’ (Opuntia spp.), ‘Cristalina’ (Opuntia albicarpa Scheinvar), ‘Dalia Roja’ (Opuntia spp.), and ‘Roja Lisa’ (O. ficus-indica (L.) Mill).

The drip irrigation treatments were: non-irrigated (NI) as a control, supplemental irrigation (SI), and full irrigation (FI). NI plants received only rainfall. SI plants received irrigation at field capacity (CC = 0.28 m³ m⁻³) when the soil water content (θ) was close to the permanent wilting point (PMP = 0.14 m³ m⁻³). The θ was determined weekly, before the next irrigation. FI plants received 100% of the crop evapotranspiration weekly, estimated through a water balance. During the drought period (February to May), the Adcon Telemetry System weather station, located 1.8 km from the experimental site, recorded the weather conditions.

The experiment was conducted in a randomized complete block design with factorial arrangement in the treatments (three drip irrigation levels × four cactus pear varieties), with three replications. The experimental unit consisted of nine plants. The sampling of reproductive cladodes (one-year-old) consisted of collecting healthy, mechanically undamaged cladodes and was carried out between 15:00 and 16:00 h on 29 May 2019. Cumulative water applied up to cladode sampling was 91, 154, and 196 mm for NI, SI, and FI plants, respectively. The water supply in the NI plants was atypical due to the rainfall recorded during the same experimental period (February to May 2019).

2.2. Mucilage Extraction

The cladodes were manually de-spined and sanitized with chlorinated water at 200 ppm. The chlorenchyma was removed, while the hydrenchyma was cubed into ~1 cm³ pieces. Approximately 100 g tissue cubes were soaked in distilled water (1:10) and mixed in a blender for 45 s. The pH of the mixture was adjusted to 7.00 ± 0.1 with 5 N NaOH, heated at 50 °C with constant stirring for 16 h, and filtered through a sieve. The filtrate was mixed with 96% (v/v) ethanol in a 1:2 ratio. The mixture was stirred for 2 h at room temperature and kept at 4 °C for 24 h until separation. The supernatant was decanted and the precipitate was concentrated in a rotary evaporator (BUCHI R-210, Flawil, Switzerland), lyophilized, and vacuum-packed [13].

2.3. Analysis of pH and Total Soluble Solids Concentration

Solutions of 1% (w/v) freeze-dried mucilage were used to measure pH with a potentiometer (Orion 420ª, Thermo Fisher Scientific Inc., Waltham, MA, USA) [14] and total soluble solids concentration (TSSC) was determined, as degrees Brix (°Brix), with a digital refractometer (NR-151, Selecta group, Barcelona, Spain).

2.4. Mucilage Color

The color of the freeze-dried mucilage was determined with a colorimeter (CR-300 Minolta, Osaka, Japan) using the Hunter Lab color scale. The L* coordinate represents the brightness in a range from 0 (black) to 100 (white). The a* coordinate ranges from green (−a*) to red (+a*). The b* coordinate ranges from yellow (−b*) to blue (+b*). The chromaticity (C*) was estimated with the following equation (Equation (1)):

$$C^{*}={\left(a^{*2}+b^{*2}\right)}^{1/2}$$

(1)

The hue angle (°H) indicates the hue of the sample and was calculated with the following equation (Equation (2)):

$$°H=ta{n}^{-1}\left(b^{*}/a^{*}\right)$$

(2)

2.5. Chemical Composition

The moisture content was determined using the Association of Official Analytical Chemists (AOAC) method 934.06. Bradford’s method was used to determine protein concentration [15]. The amount of ash and total dietary fiber was determined according to the methods 923.03 and 985.29 of the AOAC [16], respectively.

2.5.1. Total Carbohydrates

Approximately 500 mg freeze-dried mucilage was mixed with 2.5 mL H₂SO₄ at 72% (w/v) and placed in a water bath for seven min at 50 °C; the acid in the mixture was diluted with distilled water up to 4% (v/v). After that, the solution was left at 121 °C and 9.806 × 10⁵ Pa pressure for one h. The resulting sample was filtered through a 40 to 90 µm pore plate crucible at a constant weight. The filtrate was neutralized with CaCO₃ and gauged to 75 mL with distilled water. The solutions were paper-filtered (Whatman^® Grade 4) and the recovered liquid was filtered through a 0.2 µM membrane. The solutions were stored at −18 °C until analysis [17].

2.5.2. Neutral Sugars

The determination of neutral sugars was performed as described by Soto et al. [18].

2.5.3. Uronic Acid Content

The determination of uronic acid content was performed as described by Blumenkrantz and Asboe-Hansen [19]. The total carbohydrate content was estimated by adding the neutral sugar content and the uronic acid content.

2.5.4. Monosaccharide Analysis

Monosaccharides were determined by liquid chromatography (Agilent 1260 Infinity HPLC, CA, USA) equipped with a refractive index detector set at 45 °C and an Agilent Hi-Plex H 7.7 × 300 mm column at 35 °C (CA, USA). The mobile phase was five mM H₂SO₄ at a flow rate of 0.5 mL/min. The injection volume was 20 µL hydrolysates with a retention time of 32 min. The identification and quantification of monosaccharides were performed, respectively, with pure standards and previously determined calibration curves.

2.6. Fourier Transform Infrared Spectroscopy (FTIR)

FTIR spectroscopy of freeze-dried mucilage samples was carried out in a spectrophotometer (Perkin Elmer FT-IR, Perkin Elmer, Inc., Waltham, MA, USA) in the spectral range of 4000–550 cm⁻¹, performing 16 scans per sample.

2.7. Viscosity and Molar Mass Determination

Approximately 500 mg of freeze-dried mucilage was mixed with 20 mL of distilled water and vortexed. The solutions were centrifuged (Sigma 3-18KS, Osterode am Harz, Germany) at 10,000 rpm for 30 min at 25 °C. The supernatant was collected, shaken, and re-centrifuged three times. The solutions were filtered, first through 1.2 µM glass microfiber filters and subsequently through 0.45 µM microfilters. From each mucilage sample, samples at concentrations of 6, 12, 15, 15, 18, and 25 mg/mL (w/v) were prepared using distilled water as solvent. Viscosity was measured directly in a viscometer (MicroViscTM, Rheosense Inc., Biolab A/S, Risskov, Denmark) at 25 °C. Three readings per sample were executed and the viscosity was expressed in mPa s.

The intrinsic viscosity (η) was determined using the viscosity of mucilage solutions (from 6 to 25 mg mL⁻¹; w/v) using the equation [20]

$$\left[\eta \right]=\frac{2{({\eta }_{sp}-\ln {\eta }_r)}^{1/2}}{C}$$

(3)

where η was already defined, ${\eta }_{sp}$ is the specific viscosity, ${\eta }_r$ is the relative viscosity, $\ln$ is the natural logarithm, and $C$ is the sample concentration (g/dL). Likewise, the molar mass was estimated from the intrinsic viscosity using the equation [21]

$$\left[\eta \right]=K\ast M_V{}^{\alpha }$$

(4)

where η was already defined, $K$ is the value 3.81 × 10⁻⁴ dL/g, $M_V$ is the molar mass (g/mol or Da), and α is constant with a dimensionless value of 0.723. The constant values of $K$ and α depend on the polymer shape, the solvent used, and the measurement temperature [22].

2.8. Relative Water Content (RWC)

The RWC was determined simultaneously with cladode collection using the following protocol. Tissue samples were obtained randomly from two cladodes of two plants per treatment replicate and deposited in Eppendorf tubes. The tissue samples were individually punched with a punch (17 mm inner Ø). This was performed between 12:00 and 13:00 h. Once in the laboratory, the samples were individually weighed and fresh mass (FM) was determined. The samples were placed in Eppendorf tubes and hydrated for 3 h to full turgidity and individually reweighted to determine the turgid mass (TM). The samples were oven dried at 65 °C to constant mass to determine the dry mass (DM). This variable was determined with the equation: RWC = (FM-DM/TM-DM) − 100.

2.9. Statistical Analysis

The information was analyzed in a randomized complete block model with factorial arrangement in the treatments. Fisher’s least significant difference test with p ≤ 0.05 was used in the post hoc analysis of treatment means. All calculations were performed in the STATISTICA^® 7.0 system (StatSoft, Inc., Tulsa, OK, USA).

3. Results and Discussion

3.1. Mucilage Yield

The statistical analysis detected significant interaction among the levels of the factors of irrigation and variety for yield, pH, and Brix (

Table 1. Yield, pH, and total soluble solids concentration (TSSC, °Brix) of freeze-dried mucilage from cladodes of Opuntia varieties subjected to irrigation regimes.

Main Effects/Interaction	Yield (% Dry Weight)	pH	TSSC (°Brix)
Irrigation regime
Non-irrigated (NI)	17.91 ± 3.14 a a	7.65 ± 0.35 b	0.25 ± 0.15 a
Supplemental irrigation (SI)	14.36 ± 1.81 b	7.81 ± 0.31 b	0.16 ± 0.07 b
Full irrigation (FI)	13.76 ± 1.94 b	8.29 ± 0.18 a	0.10 ± 0.00 c
LSD	0.96	0.17	0.04
Significance	0.0000	0.0000	0.0000
Variety
‘Roja Lisa’ (RL)	17.11 ± 1.52 a	7.83 ± 0.47 b	0.26 ± 0.16 a
‘Cristalina’ (C)	13.25 ± 1.54 c	8.06 ± 0.30 a	0.10 ± 0.00 c
‘Amarilla Olorosa’ (AO)	16.33 ± 4.58 a	7.67 ± 0.43 b	0.20 ± 0.08 b
‘Dalia Roja’ (DR)	14.68 ± 1.59 b	8.11 ± 0.17 a	0.11 ± 0.04 c
LSD	1.10	0.19	0.05
Significance	0.0000	0.0002	0.0000
Interaction effects
NI × RL	18.57 ± 0.35 a	7.51 ± 0.29 ef	0.45 ± 0.07 a
NI × C	14.45 ± 1.76 de	7.85 ± 0.13 cd	0.10 ± 0.00 d
NI × AO	22.22 ± 0.49 a	7.22 ± 0.03 f	0.30 ± 0.00 b
NI × DR	16.38 ± 0.91 c	8.03 ± 0.02 bc	0.15 ± 0.00 c
SI × RL	16.65 ± 1.50 bc	7.56 ± 0.20 de	0.25 ± 0.07 b
SI × C	13.12 ± 1.46 ef	8.05 ± 0.26 bc	0.10 ± 0.00 d
SI × AO	14.46 ± 0.39 de	7.58 ± 0.05 de	0.20 ± 0.00 c
SI × DR	13.22 ± 1.16 ef	8.06 ± 0.26 bc	0.10 ± 0.10 d
FI × RL	16.12 ± 1.38 cd	8.42 ± 0.04 a	0.10 ± 0.00 d
FI × C	12.18 ± 0.63 f	8.29 ± 0.35 ab	0.10 ± 0.00 d
FI × AO	12.31 ± 1.50 f	8.20 ± 0.10 ab	0.10 ± 0.00 d
FI × DR	14.43 ± 0.56 de	8.26 ± 0.09 ab	0.10 ± 0.00 d
LSD	1.92	0.334	0.007
Significance	0.0000	0.0199	0.0633

^a Within each column, mean values (±standard deviation; n = 3) with different letters indicate statistical differences according to Fisher’s least significant difference (LSD) test at p ≤ 0.05.

). In general, ‘Amarilla Olorosa’ and ‘Roja Lisa’ varieties cultivated under NI produced more mucilage than the other irrigation × variety combinations. The amount of mucilage in the cladodes varied according to the genetic differences of the variety [23]. However, in addition to genetic differences, the main effect of irrigation suggests that mucilage yield and total soluble solids concentration (TSSC, °Brix) may have been associated with a dilution phenomenon [24], measured here as relative water content (RWC), since as the amount of water increased, mucilage yield (r = −0.54; p = 0.001) and CSST (r = −0.40; p = 0.055) decreased (Table 1). As water-deficit stress increases, the amount of mucilage in the hydrenchyma increases [25]. This is possible because mucilage compounds, along with the solutes, retain more water, and thus, increase the plant’s resistance to water deficit [26]. The difference in mucilage yield between SI and NI plants averaged ~3%, but when SI was applied, annual fresh biomass increased up to 10-fold in these plants, suggesting greater mucilage yield in SI plants in a commercial-scale scenario [27].

Mucilage pH was more alkaline in all cactus varieties with FI and decreased proportionally in all varieties in SI and NI plants (Table 1). This could, in part, be associated with the presence of uronic acids in the mucilage, since low pH values (less alkaline) are inversely related (r = −0.08; p = 0.70) to the uronic acid content [6]. Although this was not measured, the increase in pH could also be associated, in part, with increased salts in mucilaginous cells, which were absorbed and transported as water content increased in the cladodes of FI and SI plants (r = 0.29; p = 0.09) [28]. In contrast, mucilage pH values in NI and SI plants also suggest more acidification may be due to water-deficit stress [29]. However, 24-h observations are needed to offer a proper explanation.

3.2. Mucilage Color

The average values of the color parameters L* (82.4), a* (−2.6), b* (15.9), c* (16.2), and °H (98.89) indicate that the mucilage samples were off-white powders with yellow-green tints. These values are consistent with previous reports [30]. There was no significant interaction between the irrigation levels and variety in any color attributes (

Table 2. Color parameters of freeze-dried mucilage from cladodes of Opuntia varieties subjected to irrigation regimes.

	Color Parameters a
Main Effects	L*	a*	b*	c*	°H
Irrigation regime
Non-irrigated	84.13 ± 3.09 a b	−2.00 ± 1.06 a	14.33 ± 3.53 b	14.93 ± 4.81 b	97.70 ± 2.44 b
Supplemental irrigation	82.40 ± 3.50 b	−2.69 ± 1.06 b	16.50 ± 3.65 a	16.00 ± 3.88 b	99.50 ± 2.28 a
Full irrigation	80.92 ± 4.19 c	−2.86 ± 0.91 b	17.08 ± 3.24 a	17.94 ± 3.53 a	99.20 ± 1.56 a
LSD	1.46	0.24	1.6	1.11	1.05
Significance	0.0005	0.0000	0.0000	0.0000	0.0228
Variety
‘Roja Lisa’	85.86 ± 2.26 a	−1.53 ± 0.52 a	11.59 ± 1.51 c	10.81 ± 2.31 d	97.45 ± 2.03 c
‘Cristalina’	81.13 ± 1.93 c	−2.62 ± 0.52 c	18.38 ± 1.35 a	18.40 ± 1.32 b	98.06 ± 1.64 bc
‘Amarilla Olorosa’	83.98 ± 2.60 b	−2.07 ± 0.32 b	14.20 ± 1.85 b	14.56 ± 2.13 c	98.72 ± 1.79 b
‘Dalia Roja’	78.70 ± 3.30 d	−4.07 ± 0.39 d	19.29 ± 2.29 a	20.76 ± 2.04 a	101.50 ± 0.78 a
LSD	1.7	0.28	1.8	1.3	1.21
Significance	0.0000	0.0000	0.0000	0.0000	0.0000

^a L* = luminosity, a* = red (+) or green (−) axis, b* = yellow (+) or blue (−) axis, c* = chromaticity, °H = Hue angle. ^b Within each column, mean values (±standard deviation; n = 3) with different letters indicate statistical differences according to Fisher’s least significant difference (LSD) test at p ≤ 0.05.

). NI plants produced, on average, a mucilage powder with higher L*, less green (a*), and yellow (b*) than mucilage powder from plants receiving SI or FI. The latter was corroborated by the increase in c* and °H in mucilage powder from plants that received SI or FI (Table 2). The latter behavior may be associated with high ash contents in the mucilage samples [31], which coincided with the high ash concentration in the mucilage powder in plants undergoing SI or FI (

Table 3. Chemical composition of freeze-dried mucilage extracted from cladodes of Opuntia varieties subjected to irrigation regimes.

Main Effects	Moisture (%)	Protein	Ashes	Total Fiber	Total Carbohydrates
Non-irrigated	4.07 ± 0.41 c a	0.92 ± 0.30 a	14.30 ± 3.26 c	65.80 ± 4.11 a	75.45 ± 6.47 a
Supplemental irrigation	4.66 ± 0.50 b	0.81 ± 0.23 b	16.09 ± 3.71 b	61.88 ± 4.42 b	69.96 ± 8.70 b
Full irrigation	5.69 ± 0.21 a	0.69 ± 0.22 c	18.03 ± 4.36 a	59.81 ± 5.27 c	68.64 ± 7.28 b
LSD	0.36	0.06	0.87	1.30	3.2
Significance	0.0000	0.0000	0.0000	0.0014	0.0000
Varieties
‘Roja Lisa’	4.92 ± 0.56 ab	0.44 ± 0.06 d	11.13 ± 1.29 c	68.57 ± 2.14 a	78.10 ± 3.90 a
‘Cristalina’	4.69 ± 0.75 bc	0.96 ± 0.18 b	19.46 ± 1.98 a	60.20 ± 4.04 c	64.58 ± 3.53 b
‘Amarilla Olorosa’	4.49 ± 0.99 c	0.73 ± 0.08 c	14.69 ± 1.22 b	63.51 ± 2.86 b	77.97 ± 3.60 a
‘Dalia Roja’	5.12 ± 0.80 a	1.04 ± 0.13 a	19.28 ± 2.68 a	57.72 ± 3.36 d	64.76 ± 5.07 b
LSD	0.42	0.08	1.00	1.50	3.67
Significance	0.0320	0.0000	0.0000	0.0000	0.0014

^a Within each column, mean values (±standard deviation; n = 3) with different letters indicate statistical differences according to Fisher’s least significant difference (LSD) test at p ≤ 0.05.

). The main effect of variety was that, on average, mucilage powder from ‘Roja Lisa’ plants had the highest L*, with lower values of a* and b* (less green and yellow) than the other varieties included in this study. The above was consistent with the average values of c* and °H for mucilage powder from ‘Roja Lisa’ plants. Therefore, mucilage powder from this variety can produce solutions with colorations that, in theory, will not interfere with the color of the product when used as an additive [11]. The genetic component of color properties differed significantly among varieties, which had not been documented previously. Such properties can be exploited by the industry. Nonetheless, this topic deserves further study.

3.3. Chemical Composition

There was no significant interaction between irrigation level and the variety on the chemical composition of mucilage. The moisture and ash contents of mucilage from plants receiving FI were, on average, greater than in the mucilage from plants receiving SI or NI (Table 3). The increased moisture and ash (minerals, primarily Mg, P, K, and Ca) can be explained, in part, by a direct effect of increased water absorbed by the root systems of plants receiving FI. The opposite occurs in temperate fruit trees exposed to water deficit [32] or induced stress due to salts [33]. In this study, the association between RWC and moisture content was low (r = 0.47; p = 0.02) and very low with ash content (r = 0.19; p = 0.36). In contrast, NI plants produced, on average, the most protein, total fiber, and total carbohydrates (Table 3). The increased protein in these plants may be associated with de novo synthesis of protective proteins triggered by abiotic stress [34], which may occur in NI plants. The high concentration of carbohydrates (stored energy) in NI plants may be associated with a lack of growth in sink tissues [35] due to the water deficit imposed on NI plants. Cactus pear cladodes collected in the dry season also had more carbohydrates [36], which can be used potentially as an energy source.

The difference in the chemical composition of the cladodes is mainly attributed to their genetic differences [37,38]. The latter was confirmed by the analysis of the main effect of varieties (Table 3). The mucilage (on a dry weight basis) of ‘Dalia Roja’ plants, on average, had a greater percent of moisture, protein, and ash, but also presented the least total fiber and carbohydrates of the tested varieties (Table 3). Compared with other studies on cladode chemical composition [37,38], the mucilage protein content of our four cactus varieties was low, because it was determined only in mucilaginous cells, which are known to have little or no protein [7]. Protein content did not correlate with the RWC (r = −0.38; p = 0.07). However, the low mucilage protein content, here observed, shows the efficacy of our extraction protocol, because high protein contents would show polysaccharide contamination [39].

In general, there was more carbohydrate in the mucilage of NI plants, while the SI and FI treatments had similar amounts (p > 0.05). This is consistent with previous findings that cactus pear cladodes collected during the dry season had more carbohydrates than those collected during the rainy season (Ribeiro et al. [36]). Little plant growth is observed during the dry season; therefore, cladodes are serving as carbohydrates storage. In this study, the association of RWC with fiber (r = −0.24; p = 0.25) and total carbohydrate (r = −0.24; p = 0.26) content was low, which deserves further study.

Sugar and Uronic Acid Composition

Monosaccharide analysis revealed the presence of glucose (29.98 to 61.36%), xylose (11.77 to 42.99%), arabinose (13.51 to 27.02%), and uronic acids (4.30 to 9.32%) in the mucilages of the four cactus pear varieties (

Table 4. Concentrations of sugars and uronic acids (% molar) in freeze-dried mucilage from cladodes of Opuntia varieties subjected to irrigation regimes.

Main Effects/Interaction	Glucose	Xylose	Arabinose	Uronic Acids
Irrigation system
Non-irrigated (NI)	52.00 ± 8.68 a a	16.52 ± 6.80 c	23.26 ± 3.08 a	8.19 ± 1.02 a
Supplemental irrigation (SI)	47.57 ± 9.85 b	25.59 ± 7.45 b	20.38 ± 4.24 b	6.44 ± 1.21 b
Full irrigation (FI)	45.42 ± 11.03 c	30.87 ± 8.67 a	17.31 ± 3.02 c	6.38 ± 1.77 b
LSD	1.6	1.30	1.3	0.78
Significance	0.0000	0.0000	0.0000	0.0004
Variety
‘Roja Lisa’ (RL)	60.08 ± 1.42 a	17.54 ± 4.65 d	15.60 ± 3.01 b	6.76 ± 0.57 b
‘Cristalina’ (C)	34.42 ± 4.18 c	35.78 ± 7.12 a	22.35 ± 1.71 a	7.44 ± 1.60 b
‘Amarilla Olorosa’ (AO)	49.19 ± 3.25 b	23.54 ± 9.03 b	21.78 ± 4.95 a	5.47 ± 1.55 c
‘Dalia Roja’ (DR)	49.64 ± 4.13 b	20.45 ± 5.56 c	21.53 ± 2.69 a	8.36 ± 0.82 a
LSD	1.83	1.46	1.47	0.91
Significance	0.0000	0.0000	0.0000	0.0001
Interaction effects
NI × RL	61.36 ± 1.04 a	11.77 ± 0.43 f	19.39 ± 0.76 de	7.48 ± 0.15 a
NI × C	39.08 ± 1.00 a	27.32 ± 1.73 d	24.28 ± 0.06 b	9.32 ± 0.79 a
NI × AO	52.93 ± 1.00 a	12.78 ± 0.77 f	27.02 ± 0.06 a	7.27 ± 0.73 a
NI × DR	54.66 ± 0.48 a	14.25 ± 1.95 f	22.36 ± 2.04 bc	8.72 ± 0.57 a
SI × RL	60.14 ± 0.54 a	19.58 ± 1.86 e	13.91 ± 1.32 gh	6.36 ± 0.00 a
SI × C	34.21 ± 0.25 a	37.03 ± 0.01 b	21.85 ± 0.74 bcd	6.89 ± 1.00 a
SI × AO	47.84 ± 0.25 a	25.13 ± 1.02 d	22.18 ± 1.85 bc	4.84 ± 0.80 a
SI × DR	48.12 ± 2.33 a	20.62 ± 0.10 e	23.56 ± 2.43 b	7.70 ± 0.00 a
FI × RL	58.74 ± 1.39 a	21.29 ± 1.31 e	13.51 ± 0.12 h	6.45 ± 0.20 a
FI × C	29.98 ± 1.88 a	42.99 ± 0.71 a	20.91 ± 1.43 cde	6.12 ± 0.26 a
FI × AO	46.82 ± 1.88 a	32.72 ± 1.29 c	16.15 ± 0.94 fg	4.30 ± 0.94 a
FI × DR	46.82 ± 1.88 a	26.51 ± 0.90 d	18.66 ± 0.20 ef	8.67 ± 1.32 a
LSD	3.2	2.53	2.56	1.57
Significance	0.0846	0.0013	0.0026	0.1098

^a Within each column, mean values (±standard deviation; n = 3) with different letters indicate statistical differences according to Fisher’s least significant difference (LSD) test at p ≤ 0.05.

). The latter suggests that the structure of the mucilage is a xyloglucan skeleton of the XXGG type (Xyl, Xyl, Glc, Glc) with arabinose branches joined to xylose residues. However, the branching pattern (side chains) of xyloglucans may change depending on the variety [40], as suggested in this study.

The statistical analysis detected an interaction between irrigation level and variety for xylose and arabinose content only. According to the main effect of irrigation, NI plants had the most glucose and uronic acids (Table 4).

This was consistent with findings in other cactus cladodes collected in the dry season, which had more uronic acid [36]. This is explained, in part, by the association of RWC with uronic acid concentration (r = −0.52; p = 0.01), because as soil water content was greater in SI and FI plants, uronic acid content decreased.

The glucose content behaved similarly to the uronic acid content; however, the association with RWC was weak and not significant (r = −0.26; p = 0.22). As glucose is the basic substrate for the synthesis of starch, cellulose, sucrose, and other carbohydrates, its reduction in SI and FI plants could be associated with catabolism toward simpler compounds needed at points of demand [41]. Among varieties, ‘Roja Lisa’ mucilage had the most glucose, while ‘Cristalina’ mucilage had the most arabinose, xylose, and uronic acids. These differences could be explained by epi and/or genetic differences among these Opuntia species [42].

Xylose and arabinose are the main monosaccharide components of the primary cell wall, considered important in the linkage between pectic polysaccharides, hemicellulose, and cellulose [43]. However, the concentration of these sugars depends, among other factors, on the phenological stage of the cladode [44] and water availability in the soil profile [36]. It is probable that in cactus pear, drought activates enzymes involved in monosaccharide synthesis, leading to changes in polysaccharide composition. The cladodes used in this study were mature and reproductive (>1 year) and their molar concentrations were associated with RWC (xylose, r = 0.56; p = 0.004 and arabinose, r = −0.46; p = 0.022). The interaction, for these monosaccharides, was a function of genetic differences among varieties (Table 4).

The composition, structure, and conformational diversity of the polysaccharide are responsible for some remarkable characteristics of mucilages such as water-holding capacity, emulsification capacity, and rheological properties [45,46,47]. The composition and structure of Opuntia heteropolysaccharides, such as mucilage, are related to their availability as a carbon source and their prebiotic potential [42]. Thus, mucilage properties are fundamental to their use in the agro-industry.

3.4. Fourier-Transform Infrared Spectroscopy (FTIR)

The FTIR of all mucilages showed signals characteristic of a polysaccharide (Figure 1). The bands in the region between 3500 and 3200 cm⁻¹ indicate the presence of hydroxyl groups (-OH) related to the intermolecular bonds of alcohols and carboxylic acids, while the absorption bands between 3000 and 1750 cm⁻¹ are representative of the -CH and CH₂ radicals. The absorption bands between 2920 and 2830 cm⁻¹ are attributed to the stretching vibration of the CH radical. These absorption bands are attributed to functional groups of neutral polysaccharide components of the mucilage, such as arabinose and xylose. The bands in the region between 1800 and 1500 cm⁻¹ indicate the C=C and C=O vibration bonds. The band at 1600 cm⁻¹ corresponds to the stretching vibration of the C=C bonds of the carboxyl group (-COOH).

The absorption band between 1700 and 1600 cm⁻¹ indicates the stretching of the C=O bond of the amide group, indicating the presence of proteins. The absorption band at 1407 cm⁻¹ probably corresponds to the -OH radical of polyphenolic compounds. The absorption bands between 1320 and 1210 cm⁻¹ correspond to the stretching of the C-O bond of carboxylic acids and indicate the presence of uronic acid. The absorption band between 1085 and 1045 cm⁻¹ is attributed to monosaccharides such as glucose and the absorption band at 895 cm⁻¹ corresponds to anomeric β-carbon, indicating the presence of β-type glycosidic bonds in mucilages [48].

The mucilage spectra of plants that were not irrigated had more intense bands, indicating higher contents of carboxylic acids, proteins, and uronic acids. This is consistent with our findings of increased proteins, some monosaccharides, and uronic acids in the mucilage from NI plants. This suggests that these compounds are synthesized by cactus pear plants as a possible defense mechanism against drought, either for water retention or energy storage. In response to a severe drought, maize plants showed changes in the infrared bands corresponding to proteins and carbohydrates [49].

3.5. Viscosity and Molar Mass

Viscosity measures a substance’s resistance to shear or tensile stresses. Cladode mucilage viscosity was greater in cactus pear plants grown in low-rainfall areas [50]. We found association of RWC with mucilage viscosity (r = −0.65; p = 0.001) and molecular weight (r = −0.77; p = 0.000). The interaction between irrigation level and variety confirmed these associations (Figure 2 and Figure 3). The viscosity of the mucilage solutions was 192.91% greater in NI plants. The mucilage solutions of ‘Amarilla Olorosa’ cladodes from NI plants showed the greatest viscosity, and the viscosity decreased proportionally when plants were exposed to SI and FI. This decrease with irrigation was consistent across varieties (Figure 2). The average mucilage viscosity of ‘Amarilla Olorosa’ suggests that this variety is very sensitive to water supply, while the mucilage viscosity of ‘Roja Lisa’ and ‘Cristalina’ was similar in plants with SI or FI. In contrast, the average mucilage viscosity of ‘Dalia Roja’ varied markedly due to irrigation treatment (Figure 2).

Interestingly, the mucilage from all NI treatments had the most uronic acids and arabinose for their variety. This may be related to the increased viscosity, since the carboxyl groups of uronic acids can interact with water molecules or with certain cations, such as calcium, increasing the viscosity of the solution [6,12]. Also, mucilage with more arabinose (as detected in all SI treatments) can generate suspensions with greater viscosity because its functional groups are more willing to interact intermolecularly [12].

The molar mass of mucilage ranged from 58 to 214 kDa. The significant interaction between irrigation level and variety followed the same pattern observed for viscosity (Figure 2 and Figure 3). The greater molar mass of NI mucilages may reflect differences in their composition, structure, and number of functional groups. These differences are important because the molar mass of polysaccharides can affect rheological behavior [51] and biological activities, including antioxidant, hypolipidemic, antiviral [52], and prebiotic potential [42]. For example, high molecular weight polysaccharides form many intermolecular associations, creating greater cohesive strength and increasing the thickening effects and viscosity of the solution [51]. These polysaccharides could form gels and protect intestinal cells when consumed [53]. Low molar mass polysaccharides have greater antioxidant activity because they contain more free hydroxyl groups to accept and scavenge hydrogen radicals [54].

4. Conclusions

We hypothesized that irrigation would modify some physicochemical characteristics of mucilage extracted from cladodes of different cactus pear varieties. Statistical evidence did not reject this hypothesis because, regardless of the cactus pear variety, irrigation modified mucilage yield, composition, and physicochemical properties studied here. Also, the mucilage of non-irrigated plants was more luminous, with less green tone and yellow tone than mucilage from plants that received supplemental or full irrigation. Furthermore, mucilage from non-irrigated plants not only had the least ash, but also more protein, total fiber, total carbohydrates, glucose, arabinose, and uronic acids, greater viscosity, and increased molar mass.

The genetic differences among varieties influenced the physicochemical characteristics of the mucilage. ‘Amarilla Olorosa’ and ‘Roja Lisa’ plants receiving no irrigation produced more mucilage than ‘Cristalina’ and ‘Dalia Roja’ plants. Mucilage from ‘Roja Lisa’ cladodes had the highest L* and lower a* and b* shades than the other varieties. The mucilage from ‘Amarilla Olorosa’ plants with no irrigation was the most viscous. Mucilage from ‘Dalia Roja’ plants had the most protein and ash, but the least total fiber and total carbohydrate.

Restricting the amount of water to cactus pear varieties studied here could be a feasible strategy to obtain high-quality mucilage in regions with low rainfall and limited water availability for irrigation. In supplementally irrigated cactus pear orchards, cladodes can be collected at pruning, because irrigation is suspended at fruit harvest and cladode yield can be almost threefold higher than non-irrigated plants, and therefore, mucilage yield increases. The mucilage characteristics observed here suggest a much more diversified potential use not only for the food and pharmaceutical industries, but also for other industrial applications.