Impact of Vegetal Protein on the Physicochemical and Microstructural Properties of Microencapsulated Mexican Red Pitaya (Stenocereus thurberi) Juice

Abstract

The effects of different carrier agents—pea protein (PP), rice protein (RP), bean protein (BP), whey protein (WP), and maltodextrin (MT, as a control)—on pitaya juice encapsulation via spray drying were evaluated. Juice and carrier mixtures (30% w/v) were dried at 150 °C, and the resulting powders were analyzed for water activity (aw), hygroscopicity (Hg), water solubility (WSI), bulk density (BD), glass transition temperature (Tg), water absorption (WAI), antioxidant activity (AA), total polyphenol content (TPC), total betalain (TB) content, and TB stability. Vegetable proteins showed promising results, significantly impacting the protein content, Hg content, WAI, WSI, AA, TPC, and TB content and resulting in high Tg values. PP showed the best results, with high betalain retention (>30%), high TPC and AA, high protein levels, and low Hg, similarly to MT. WP had the highest TB, AA, and TPC but the lowest Tg (47.21 °C), thus reducing stability. Encapsulates obtained with plant protein-based wall materials presented high Tg (>58 °C); low aw, WSI, and Hg; high protein contents >40%; and adequate retention of bioactive compounds, with low degradation rate constants and long half-lives. Overall, plant proteins are promising alternatives to traditional carriers, offering improved stability and functionality in encapsulated products.

1. Introduction

The Mexican pitaya (Stenocereus thurberi) is an exotic fruit native to the arid and semidesert regions of Mexico. This mainly red fruit (fruits with white, yellow, and purple pulp are also found) with tiny seeds, can be consumed directly [1,2]. Its coloration is related to the contents of pigments such as anthocyanins and betalains, which are natural pigments present in some fruits [1]. The Mexican pitaya has exceptional sensory qualities along with nutritional, nutraceutical, and medicinal benefits; however, it has not been fully harnessed [1,2]. The main antioxidant characteristics are provided by betalains, which are naturally water-soluble pigments that provide color to flowers and fruits. These compounds are classified into two categories: betacyanins (red) and betaxanthins (yellow) [3,4,5]. Unfortunately, the Mexican pitaya has a very short shelf life because of its rapid deterioration and is consumed near the time of harvest. An alternative method to take advantage of this fruit and preserve its bioactive compounds is through its encapsulation, using preservation techniques such as conventional drying, freeze-drying, and vacuum drying [5]. However, the use of these techniques has disadvantages such as long processing times and high costs. An alternative solution is spray drying, which is considered practical, effective, and applicable because of its economical nature. It requires low maintenance, preserves high bioactivity, and stabilizes bioactive compounds [4,5,6,7]. However, the effectiveness of this technology is linked to the use of appropriate carrier agents to achieve the yields and stability of the bioactive compounds in the food matrix. Maltodextrin is often used as a carrier to effectively stabilize components with good protection during storage [5]. Pui et al. [8] and Utpott et al. [9] studied the preservation of betalains extracted from juice or fruit skins using maltodextrin as a carrier agent. Despite its stabilizing qualities, maltodextrin is highly caloric and rapidly digestible, increases the glycemic index, and has also been associated with a rising incidence of chronic inflammatory conditions. The use of vegetable or animal protein-based carriers, such as whey protein, soy protein, or pumpkin protein, is a suitable alternative owing to their emulsifying activity, film-forming ability, high coverage, and ability to serve as a source of energy and amino acids with high nutritional value, which can maintain thermal stability to minimize the breakdown of bioactive compounds [10,11]. The use of animal proteins such as whey protein in pigment encapsulation has been widely studied; Bavaria and Kumar [12] and Shofinita et al. [13] have studied the stability of betalains extracted from beetroot juice and dragon fruit encapsulated products. However, the use of these wall materials may be unsuitable for people with allergies and lactose intolerance [14].

In recent years, the study of natural resources such as plant proteins has attracted increasing interest in the food industry. This interest is driven by their potential to produce products with high biological and nutritional value as alternatives to animal proteins. Compared with animal proteins, plant proteins have lower allergenicity, better hydrophobic properties, lower toxicity, and lower cost. They are also suitable for encapsulation, offering high-quality and good organoleptic properties [15,16]. Recent studies have highlighted the versatility of plant proteins in food applications. For example, Gomes and Kurozawa [16] successfully microencapsulated linseed oil using rice protein hydrolysate, demonstrating its efficacy in encapsulation technology. Similarly, García-Segovia et al. [4] utilized pea protein to produce beetroot powders with excellent retention of bioactive compounds, demonstrating the effectiveness of plant proteins in preserving nutritional components during processing.

These results underscore the potential of plant proteins to contribute toward preserving the bioactive compounds in food matrices and increasing their stability and properties to favor the development of functional foods and encapsulated products with enhanced nutritional profiles and sensory attributes in the food industry. Additionally, plant proteins represent a promising alternative to replace animal proteins and traditional carriers extensively used in food applications. Reports showing the use of plant proteins as carrier agents and the encapsulation of bioactive compounds or pigments contained in pitaya juice (Stenocereus thurberi) are scarce. The use of vegetable proteins such as rice and peas represents a suitable alternative, as they contain essential amino acids, such as methionine, leucine, and glutamine; are highly digestible and have high biological value; and are gluten-free, lactose-free, vegan, and hypoallergenic [16,17]. In addition to containing bioactive peptides such as prolamin in rice protein, lectin in bean proteins, and globulin in pea proteins, peptides derived from vegetables present improved attributes such as anti-inflammatory, antimicrobial, antioxidant, and enzyme-inhibitory activities [16,18,19]. The use of vegetable proteins is an alternative to the more commonly used milk and soy proteins and presents new opportunities for innovation with legumes and meeting consumer requirements.

This presents an opportunity for developing natural pigments with minimal losses and preserved functionality. Therefore, this work aimed to evaluate the use of vegetable proteins as carrier agents on the physicochemical and microstructural characteristics of encapsulated red pitaya fruit processed by spray drying.

2. Materials and Methods

2.1. Materials

Mexican red pitaya (Stenocereus thurberi) was collected from Sonora State, Mexico, and frozen immediately after harvesting. The following materials were used as carrier agents: maltodextrin “GLOBE^® 15, Ingredion™” (MT) as a control; whey protein isolate “Hilmar™ 9400” (WP-90% protein); vegan pea protein “Wildmountain^®” (PP-63% protein); and vegan rice protein “Wildmountain^®” (RP-86% protein), which were purchased from a local market in Monterrey, Mexico. The bean protein “Tepary” (BP-61% protein) was extracted from white tepary bean (P. acutifolius) seeds according to López-Ibarra et al. [19].

2.2. Chemical Material

Folin–Ciocalteu phenol (SKU-47641), Trolox (SKU-238813), and DPPH (SKU-D9132) were obtained from Sigma–Aldrich (St. Louis, MO, USA). Sodium carbonate, gallic acid, methanol, and hydrochloric acid were obtained from J.T. Baker (Mexico City, Mexico).

2.3. Juice Extraction

The frozen fruit was thawed in a water bath at 25 °C and then passed through a #30 (600 μm) mesh to separate the seeds and mucilage. The resulting mixture was further filtered through a plastic cloth to remove suspended solids. The juice was stored at −20 °C until further analysis. The total soluble solids (°Brix) were determined using an Abbe refractometer (NAR-1T solid, ATAGO, Tokyo, Japan). Antioxidant activity, total polyphenols, and total betalain were analyzed as described below.

2.4. Protein Hydrolysis

Batches of 40 g/L PP, RP, and BP were hydrolyzed to increase their solubility and improve emulsion stability before spray-drying, thus increasing their functionality for encapsulation [19,20,21]. The hydrolysis was performed as described by López-Ibarra et al. [19], with some modifications. For hydrolysis, 40 g of protein was added to 1 L of 1 N HCl solution, and sonicated in an ultrasonic bath (Branson, 1800) for 15 min at 25 °C. After sonication, the samples were placed in a water bath at 95 °C for 4 h to complete the acid hydrolysis. Once cooled to room temperature, the hydrolysate was neutralized with NaOH-1 N and centrifuged at 3000× g (Sorvall ST 8R, Waltham MA, USA) for 15 min to eliminate the insoluble materials. The supernatant was then collected, dried at 50 °C for 8 h, and stored in plastic bottles until analysis. WP was not hydrolyzed because of its solubility and stability in the medium used for the extraction process through filtration.

2.5. Spray Drying Process

The pitaya juice and carrier agent (MT, WP, PP, RP, or BP) were added individually at a concentration of 30% w/v and subsequently mixed and homogenized in an ultrasonic bath (BRANSON CPX 1800) for 25 min at 25 °C. The different treatments were spray-dried using a lab-scale spray dryer (Yamato ADL311S, Tokyo, Japan). The process conditions were as follows: an inlet temperature of 150 °C, an outlet temperature of 70 °C, a solution feed rate of 10 mL/min, an atomizing pressure of 0.1 MPa, and a spray nozzle of 711 μm. The encapsulated material was stored in hermetically closed plastic jars for the subsequent analysis of its physical and chemical properties. The yield of the spray drying process was evaluated based on the relationship between the amount of powder recovered from the drying chamber and the total solid mass.

2.6. Proximal Characterization

The encapsulated materials were characterized according to the AOAC [22] methods: moisture content (934.06), fat (945.16), ash (942.05), protein (920.152), and crude fiber (962.09). For protein analysis, specific nitrogen conversion was considered for RP (5.95), BP (5.28), WP (6.39), and PP (5.4) as described by Mariotti et al. [23]. The carbohydrate content was determined based on these differences.

2.7. Hygroscopicity (Hg)

The amount of Hg encapsulated was determined according to the methods of Tabio-Garcia [24]. Encapsulated samples of 500 mg were kept in airtight containers containing a saturated NaCl solution at 25 °C for seven days. The encapsulated material was weighed, and the hygroscopicity was expressed as a weight percent. Measurements were carried out in triplicate.

2.8. Water Activity (aw)

The water activity (aw) of the produced powders was assessed via a water activity meter (AquaLab 4TE, Pullman, WA, USA) at 25 °C. Measurements were carried out in duplicate.

2.9. Bulk Density

Bulk density was determined as described by Tze et al. [25]. The encapsulated material was placed in a graduated cylindrical container up to 5 mL. BD was determined by dividing the mass contained within the cylinder by the volume it occupied. The measurements were performed in triplicate, and the results are expressed in kg/m³.

2.10. Glass Transition Temperature (Tg)

The Tg was determined via a calorimeter (TA Q-200; TA Instruments, Crawley, UK) according to Tabio-Garcia [24]. A cylinder of compressed material weighing approximately 20 mg of the different encapsulates was placed in a closed aluminum pan. The scans were conducted under three temperature cycles: an argon atmosphere. The first cycle ranged from 0 °C to 120 °C, the second cycle ranged from 120 °C to −30 °C, and the third cycle ranged from −30 to 120 °C at 10 °C/min. Measurements were carried out in duplicate and were analyzed via Universal Analysis Software (https://www.tainstruments.com/support/software-downloadssupport/accessed on 14 April 2025).

2.11. Chemical Properties

2.11.1. Extract Preparation

The methodology described by Soto-Dagnino et al. [26], with some modifications, was used to extract the TPC and AA. Briefly, 0.2 g of pitaya encapsulates (RP, PP, and BP) were homogenized with 10 mL of methanol, and for encapsulated MT and WP, only water was used. For the extraction of BT (0.2 g), RP, PP, and BP were homogenized with 10 mL of 0.1 N methanol/water/HCl (50:40:10 v/v), and MT and WP were homogenized with 0.1 N water/HCl (50:50 v/v). The homogenates were then subjected to ultrasonication for 30 min in an ultrasonic bath at 25 °C and centrifuged at 3000× g in a Sorvall ST 8R centrifuge (TMO, Waltham, MA, USA) for 10 min. The supernatants were filtered through a 0.45 µm nylon membrane.

2.11.2. Total Polyphenol Content

The TPC was measured using the Folin–Ciocalteu method with modifications. Briefly, 100 µL of extract from each treatment with 50 mL of Folin reagent was added to 3 mL of distilled water. After a 10 min reaction, 400 μL of sodium carbonate (7.5%) was added. A calibration curve was generated using gallic acid. To avoid overestimation of total polyphenol content, the whites of each wall material were determined, and this value was subtracted from the final encapsulation value. The absorbance of the samples was measured at 760 nm using a Lambda 25 spectrophotometer (PerkinElmer, Waltham, MA, USA). The final values were expressed as milligrams of gallic acid per 100 g of sample.

2.11.3. Antioxidant Activity

The antioxidant activity of the encapsulates was assessed via the DPPH method. The reaction was carried out by mixing 0.1 mL of each extract with 3.9 mL of DPPH (100 μM), followed by incubation for 3 h in the dark. The absorbance of each sample was measured at 517 nm via a Lambda 25 spectrophotometer (PerkinElmer, Waltham, MA, USA). The results are expressed as micromoles of Trolox equivalents per 100 g of sample.

2.11.4. Total Betalain Content

The betalain content was determined following the methods of Soto-Dagnino et al. [26]. The betacyanin (BC) and betaxanthin (BX) contents were calculated from the absorption at 535 nm and 483 nm, the maximum absorption wavelengths for betacyanins and betaxanthins, respectively. The results are expressed as the total betalain content in mg/g powder, which is the sum of the BC and BX contents.

2.12. Color Parameters

The color parameters of the encapsulants were measured via a CR-400/410 colorimeter (Minolta, Osaka, Japan), which had been previously calibrated. The samples were placed in a transparent plastic container and filled, and the excess sample was removed via a ruler to homogenize the surface. The parameters L*, a*, and b* were recorded as the average of ten measurements. These color parameters were used to determine the hue angle (H°), which was calculated via Equation (1):

$$H°=atan\left({\displaystyle \frac{b}{a}}\right)$$

(1)

2.13. Stability of Powders During Storage

The stability of the encapsulates was assessed as described by Tabio-Garcia et al. [24] with modifications. The encapsulated material was placed in glass tubes in the dark and kept at 30 °C, 45 °C, or 60 °C. The betacyanin and betaxanthin levels in the samples were recorded every seven days throughout the storage period. The degradation rate constant (k) and half-life (t_1/2) of the betalains were estimated using the following equations:

$$Ln\left({\displaystyle \frac{C_t}{C_0}}\right)=-kt$$

(2)

$$T_{1/2}={\displaystyle \frac{Ln2}{k}}$$

(3)

where C₀ and C_t represent the betacyanin or betaxanthin concentrations at the initial time and time t, respectively.

2.14. FTIR Analysis

The pits were encapsulated following the methods outlined by Neder-Suarez et al. [27] via a PerkinElmer ATR-FTIR instrument (PerkinElmer, Norwalk, CT, USA). The FT-IR spectra were measured in the wavenumber range of 4000–650 cm⁻¹.

2.15. Scanning Electron Microscopy

The pitaya encapsulated particles, with a size smaller than 74 µm, were analyzed via a scanning electron microscope (JSM-5800LV, Akishima, Japan) following the methodology described by Neder-Suarez et al. [24]. The obtained images were processed with ImageJ 1.50i software to evaluate the particle size.

2.16. Experimental Design

A completely randomized univariate design was used, with duplicate measurements. The independent variables were the different carrier agents (MT, WP, RP, PP, and BP) employed for the encapsulation of pitaya juice. The data were examined using ANOVA and Pearson’s correlation in Minitab^® 17.1.0 (Minitab Inc., State College, PA, USA), with the significance level set at 0.05.

3. Results

3.1. Materials and Encapsulated Materials

The physicochemical properties of red pitaya juice include 12.2% ± 0.14 solids and high levels of bioactive compounds, a total betalain content of 2.51 ± 0.32 mg/g, an antioxidant activity of 22.03 ± 1.36 μmol TE/g, and a total polyphenol content of 1149.72 ± 18.05 mg GAE/100 g. Pasko et al. [28], using the H. costaricensis variety, performed extraction of TPC; the fruit was naturally air dried, extracted with methanol and water for 3 h, and dried by evaporation under reduced pressure at 50 °C, and the dry residues were dissolved in dimethyl sulfoxide, generating values of 2.3 ± 0.1 mg BC/g and 1030 ± 40 mg GAE/100 g for aqueous extracts and 8.4 ± 0.4 mg BC/g and 880 ± 40 mg GAE/100. Vieira et al. [6], for the extraction of compounds from red pitaya H. polyrhizus, peeled and pulped the fruit and lyophilized the material at −80 °C for 48 h; the lyophilized products showed conventional extraction generated values of 1.08 ± 0.1 mg BC/g and 860 ± 20 mg GAE/100 g, while ultrasound-assisted extraction generated values of 1.66 ± 0.1 mg BC/g and 1614 ± 10 mg GAE/100 g. The values generated in this study for BC were significantly different from those reported by Pasko et al. [28] when performing the extraction with methanol and were significantly higher than those reported by Vieira et al. [29]. Further, our TPC values were significantly higher than those reported by Pasko et al. [28] and significantly lower than those reported by Vieira et al. [6] when performing ultrasound-assisted extraction of bioactive compounds. The pigmentation of the fruits, indicated by the betalain content, depends on the proportion in which betacyanins and betaxanthins are combined, which results in hues varying from yellow to purple, orange, and different shades of red. These differences are shown in the pigmentation of the juice, which generated lower values of color parameters than those reported by Vieira et al. [6], with values of 30.75, 57.13, and −3.46 for l, a*, and b*, respectively. The approximate compositions of the encapsulated red pitaya are listed in

Table 1. Proximal analysis and color parameters of pitaya encapsulates.

Treatment	Moisture Content (%)	Protein (%)	Ash (%)	Fat (%)	Crude Fiber (%)	Carbohydrates (%)
RP	1.14 ± 0.08 b	55.19 ± 0.25 a	0.74 ± 0.15 b	0.08 ± 0.02 d	1.31 ± 0.18 ab	38.7
PP	1.37 ± 0.16 b	43.88 ± 0.69 c	2.19 ± 0.13 a	0.69 ± 0.11 b	1.05 ± 0.12 b	43.9
BP	1.29 ± 0.12 b	40.06 ± 0.36 d	2.14 ± 0.09 a	3.81 ± 0.12 a	1.52 ± 0.15 a	43.8
WP	2.08 ± 0.14 a	49.22 ± 0.87 b	2.18 ± 0.11 a	0.63 ± 0.09 b	ND	46.9
MT	1.54 ± 0.22 ab	0.11 ± 0.01 e	1.77 ± 0.18 a	0.34 ± 0.08 c	ND	96.2

Values are the average of triplicate measurements ± standard deviation. Average values in each column with different letters represent a significant difference according to the Tukey test (α = 0.05). RP = rice protein, PP = pea protein, BP = bean protein, WP = whey protein, MT = maltodextrin.

. The moisture content was approximately 1.14 to 2.08%; the highest moisture value was generated in the WP treatment, with significant differences (p > 0.05) compared to those of the treatments with protein-based encapsulates. The protein content of vegetable proteins ranged from 40–55%, with the highest protein content in the RP treatment (55.19%), which was significantly different from that in the PP and BP treatments. Our protein content was higher than that reported by Chen et al. [29] when pea water was dried, with a value of 34%, and similar to that reported by Wang et al. [30], which was 58%, when bayberry juice was dried via WPI. The ash content was between 0.74% and 2.18%, without significant differences except for the RP treatment, and the fat content was between 0.08% and 3.81%, generating the highest values for the BP treatment; similar results were reported by López-Ibarra et al. [19]. The highest fiber contents were generated in the BP and RP treatments, but the difference was not significant. The WP and MT treatments did not generate a crude fiber content because they have high solubilities in water (>94%) (

Table 2. Physicochemical properties of red pitaya encapsulates.

Treatment	Aw	Hg	YE	WSI	WAI	BD	TG	TPC	AA	BT	BTC	BTX
RP	0.171 ± 0.01 b	12.17 ± 0.18 c	52.85 ± 4.44 b	22.87 ± 0.37 e	3.16 ± 0.14 a	0.277 ± 0.004 c	59.56 ± 0.72 b	218.60 ± 19.90 d	2.95 ± 0.16 e	0.665 ± 0.012 c	0.247 ± 0.005 d	0.417 ± 0.006 c
PP	0.151 ± 0.01 c	17.91 ± 0.18 b	54.81 ± 2.56 b	37.83 ± 0.31 c	3.06 ± 0.11 a	0.350 ± 0.008 b	59.91 ± 0.90 b	639.71 ± 8.94 b	10.71 ± 0.25 b	0.772 ± 0.093 b	0.282 ± 0.010 c	0.490 ± 0.004 b
BP	0.125 ± 0.01 e	18.01 ± 0.41 b	44.52 ± 6.25 c	28.74 ± 0.62 d	3.07 ± 0.10 a	0.345 ± 0.006 b	58.13 ± 0.86 b	327.52 ± 13.48 c	7.75 ± 0.10 c	0.673 ± 0.025 c	0.268 ± 0.015 cd	0.408 ± 0.010 c
WP	0.251 ± 0.02 a	20.84 ± 0.22 a	60.45 ± 4.36 a	94.58 ± 0.02 b	0.42 ± 0.01 b	0.271 ± 0.002 c	47.21 ± 0.69 c	680.09 ± 13.48 a	13.53 ± 0.31 a	1.229 ± 0.079 a	0.439 ± 0.005 a	0.792 ± 0.006 a
MT	0.144 ± 0.01 d	21.10 ± 0.06 a	63.82 ± 5.89 a	99.38 ± 0.83 a	0.33 ± 0.02 b	0.463 ± 0.002 a	64.83 ± 0.53 a	292.84 ± 16.29 c	5.64 ± 0.21 d	0.803 ± 0.013 b	0.335 ± 0.013 b	0.488 ± 0.026 b

Values are the average of triplicate measurements ± standard deviation. Average values in each column with different letters represent a significant difference according to the Tukey test (α = 0.05). RP = rice protein, PP = pea protein, BP = bean protein, WP = whey protein, MT = maltodextrin, MC = moisture content, AW = water activity, Hg = hygroscopicity, Y_E = yield of encapsulation, WSI = water solubility index, WAI = water absorption index, BD = bulk density (g/mL), Tg = glass transition temperature (°C), TPC = total polyphenol content (mg of gallic acid equivalent (GAE)/100 g of powder), AA = antioxidant activity (µmol of Trolox equivalent (TE)/g), BT = total betalain content (mg/g), BTC = total betacyanin content (mg/g), BTX = total betaxanthin content (mg/g).

). The carbohydrate content of the different treatments was less than 47%, except for MT, whose percentage was greater than 96%.

3.2. (Hg) and Water Activity (aw)

The hygroscopicity of the encapsulated red pitaya ranged from 12.17–21.10 (Table 2), and was significantly affected by the carrier agent. Compared with the traditional carrier agents WP and maltodextrin, the use of vegetal proteins as wall materials decreased hygroscopicity. The lowest Hg value was generated in the RP treatment, which was significantly different from that in the PP and BP treatments (Table 2). This decrease in Hg is due to the composition of RP, mainly gluten, which has greater surface hydrophobicity than that of the protein fractions of albumin and globulin contained in PP and BP, respectively [29,31], resulting in lower solubility, which reduces the interaction between molecules, yielding structures that do not hold water, making it less hygroscopic [4,15].

The encapsulants of the WP and MT treatments generated the highest values of Hg, which were significantly different (p < 0.05) from those processed with vegetable proteins. This difference was attributed to the good solubility of MD in water [31]. The values generated for WP and MT were similar to those reported by Tabio-Garcia et al. [24], Bazaria and Kumar [12], and Pui et al. [8]. Water activity is an essential parameter for spray-dried powders because of its influence on product shelf-life. Water activities less than 0.3 are considered safe from both microbiological and chemical perspectives [7,24]. The water activities of the encapsulants ranged from 0.12 to 0.25 (Table 2), indicating that these encapsulants are suitable for long-term preservation.

Bulk density is fundamental to storage, processing, packaging, and distribution conditions in the food industry. The carrier agents had a significant effect (p < 0.05) on the bulk density (BD). Our findings indicated that the greatest significance followed the order of MT > PP > BP > RP > WP (Table 2). The highest values were observed when maltodextrin was used as a carrier agent, which was attributed to the arrangement of particles with minimal spaces between them [25]. Bazaria and Kumar [12] and Tze et al. [25] have reported similar values for encapsulated pitaya using maltodextrin. Conversely, the lowest BD values were recorded for protein-based encapsulates. Tontul et al. [7] and Mirlohi, Manickavasagan, and Ali. [32] reported that encapsulated products produced with carbohydrates have higher densities than those of protein-based products because of the molecular weight of carrier materials. García-Segovia et al. [4] and Tontul et al. [7] reported that increasing the protein concentration results in a lower bulk density in beetroot and that tomato encapsulates using pea and cosucra protein.

3.3. Water Absorption Indices (WAI) and Water Solubility Indices (WSI)

The WAI and WSI ranged from 0.33–3.16% and 22.87–99.38%, respectively. Treatments with vegetal proteins as carrier agents generated the highest WAI values, which were significantly different (p < 0.05) from those of MT and WP. The dry spray process with proteins significantly increases the WAI owing to the increase in the amount of water immobilized by the samples, which is trapped by the protein matrix and reduces the soluble solids present in the product [4,15]. García-Segovia et al. [4] reported an increase in the WAI at higher protein concentrations in beet microencapsulation using pea protein. The WSI is associated with the quantity of soluble solids in the product, which depends on the solubilization of starches, sugars, proteins, fibers, and maltodextrin. The treatments with the highest WSI values were MT and WP, which showed a significant effect (p < 0.05) because of their high solubility in water [7,30]. The WSI values generated for the plant protein-based carriers were significantly lower than those generated for the MT and WP treatments. However, their solubility was greater than that of a native protein, as these proteins have low solubility in water [30]. This increase in WSI is due to the hydrolysis process, which reduces the molecular size of the proteins, thereby increasing nitrogen solubility [32].

3.4. Glass Transition Temperature (Tg)

The Tg is an indicator of the stability of the encapsulated material during storage [16,32]. The carrier agents had a significant effect (p < 0.05). The highest Tg values were obtained for encapsulation with MT (64.8 °C), which resulted in better stability of the dried powder, resulting in low deterioration of the bioactive components due to interactions such as dipole–dipole interactions and hydrogen bonding with molecules such as betalains and water. These interactions disrupt polymeric aggregations, altering the size of the wall material molecular aggregates [33]. Similar results were reported by Sandate-Flores et al. [34] and Tabio-Garcia et al. [24] for beetroot and amaranth extract powders, respectively, when maltodextrin was used as a carrier agent. The encapsulation of vegetable proteins ranged from 58.1–59.9 °C. These high Tg values may be attributed to the existence of hydrophilic-hydrophobic sites and the presence of sulfhydryl groups and disulfide-sulfhydryl bonds [35]. Similar values have been reported by Fang and Bhandari [36] in the spray drying of plant proteins, with higher values than those reported by Kurek and Pratap-Singh [37] in the microencapsulation of hempseed oil using rice protein hydrolysate. Furthermore, the highest transition temperature value was observed. The lowest Tg value was generated for the WP treatment (47.21 °C), due to the increase in the mobility of protein molecules, resulting in structural changes such as the formation of intermolecular covalent bonds and noncovalent interactions [38].

3.5. Color

Color is the primary property of encapsulated compounds and their content of bioactive compounds. The color parameters of red pitaya encapsulated with pitaya juice are shown in

Table 3. Color parameters of red pitaya encapsulates.

Treatment	L*	a*	b*	°Hue	Color
RP	56.97 ± 0.29 b	30.17 ± 0.30 e	29.89 ± 0.33 c	44.73 ± 0.09 a
PP	44.31 ± 0.16 d	37.32 ± 0.23 b	30.52 ± 0.10 b	39.27 ± 0.06 d
BP	39.82 ± 0.22 e	37.16 ± 0.26 b	26.56 ± 0.25 d	35.55 ± 0.09 e
WP	55.74 ± 0.83 c	35.42 ± 0.31 c	31.21 ± 0.35 a	41.38 ± 0.06 b
MT	59.29 ± 0.34 a	34.89 ± 0.16 d	30.33 ± 0.18 bc	41.00 ± 0.10 c
Pitaya juice	29.45 ± 0.09 f	45.93 ± 0.12 a	10.03 ± 0.14 e	12.34 ± 0.08 f

Values are the average of triplicate measurements ± standard deviation. Average values in each column with different letters represent a significant difference according to the Tukey test (α = 0.05). RP = rice protein, PP = pea protein, BP= bean protein, WP= whey protein, MT = maltodextrin.

. After processing, the parameter a* values were lower than those of pitaya juice, which could be due to the degradation of betalains at elevated temperatures during processing. The use of carriers significantly affected (p < 0.05) the color parameters of the encapsulated pitaya. In general, changes in the color parameters of the encapsulated protein depend on the type of carrier; the use of vegetable proteins such as PP and BP significantly decreases the luminosity and hue parameters (p < 0.05) and increases the a* parameter of the encapsulated protein, tending toward reddish pigmentation of the powder, possibly owing to the intrinsic characteristics of proteins such as protein purity and pigment contamination [38], as globulin and phaseolin are the most abundant amino acids in pea and tepary bean proteins [18,19]. Compared with the TT treatment, the RC treatment generated an encapsulation with greater luminosity and lower a* parameter; these differences in pigmentation are possibly attributed to rice proteins containing mainly the glutelin fraction, which generates this pigmentation. The highest luminosity value was observed with MT; this increase could be due to the white color of maltodextrin, which tends to lighten the powders, resulting in a light red color. Tze et al. [25] and Wang et al. [30] reported similar values for pitaya and black mulberry juices dried with maltodextrin as the carrier. However, when WP is used, the encapsulation tends to be similar to that of MT, increasing the luminosity and b and °hue parameters and decreasing the a* parameter; this is possibly because of the formation of a film layer that can protect against the damage caused by oxidation, solid surfactant properties, and the high luminosity naturally possessed by the carrier [13].

3.6. Chemical Properties

3.6.1. Total Betalain Content

The color of the product encapsulates is one of the primary quality characteristics after drying. Betalain is unstable when exposed to environmental and processing factors, such as fluctuations in temperature, water activity, and the presence of light or oxygen. High drying temperatures can degrade betalain, leading to the breakdown of thermosensitive compounds, and efficient retention of bioactive compounds depends on the type and amount of wall material used [6]. After drying, the TB content was significantly (p < 0.05) affected by the carrier agent, resulting in a decrease compared with that of unprocessed pitaya juice (materials and encapsulated section). The highest values of TB content were generated in encapsulated WPs, showing significant differences (p < 0.05) compared with those in the other treatments, reaching values of 1.22 mg/g and preserving 49% of the initial content (Table 2). These high values were attributed to the fact that WP mainly consists of β-lactoglobulin and α-lactalbumin, which exhibit strong binding affinities mainly by alterations in their secondary structure [39] and, during spray drying, form a film around the particle because of electrostatic interactions or hydrogen bonding, resulting in smaller particles with more spaces to hold the betalains [10,11,40,41]. Moreover, MT treatment generated a TB content retention of 32%, This behavior in response to the cationic features of betalains promotes betalain-polymer interactions owing to electrostatic interactions or hydrogen bonding and dipole-dipole interactions [28,33]. The betalain retention was similar to that reported by Duong, Tran, and Hoang [41] and Tabio-Garcia et al. [24] in amaranth extracts using maltodextrin as a carrier. Furthermore, the use of vegetable proteins resulted in good retention of TB content, especially the protein from peas, which generated values similar to those obtained for MT, above 30%, without showing a significant difference. This considerable retention of bioactive compounds is possibly attributed to albumins present, which showed strong binding affinities with several bioactive compounds including betalains through hydrophobic interactions and hydrogen bonding, as well as structural modifications, which allow the stability and solubility of proteins in water [42]. Finally, the lowest values were obtained with vegetable proteins BP and RP in the encapsulates; this low retention of compounds may be attributed to the fact that capsule formation was not achieved during the process, which generated inadequate preservation of bioactive compounds sensitive to temperature, thus reducing their content and generating less stable pigment complexes.

3.6.2. Total Phenolic Content (TPC) and Antioxidant Activity (AA)

After drying, the TPC was significantly (p < 0.05) affected by the carrier agent, resulting in a decrease compared with that of unprocessed pitaya juice. The highest values of TPC were generated in encapsulated WP, with significant differences (p < 0.05), reaching values of mg GAE/100 g d.b. These high values are attributed not only to the protection that WPs provide to betalain but also to the potential presence or breakdown of other substances with antioxidant activity. WPs may undergo the Maillard reaction, which usually occurs during processing at high temperatures, and the resulting generated compounds have antioxidative properties [13,30,36]. The use of vegetable proteins such as BP and PP generated values of 218.60 to 639.71 mg GAE/100 g d.b., which were significantly higher than those of MT (Table 2); this can be attributed to protein-phenolic complexes, especially covalently linked complexes, which increase the stability of phenolic compounds and maximize the retention of bioactive ingredients by increasing the TPC [10]. Similar results were reported by Wang et al. [30] when mulberry juice was encapsulated since the use of pea protein increased the TPC. The lowest TPC values were generated in the RP and MT treatments, possibly because of the reaction of betalain pigments with Folin reagent. The antioxidant activity could be attributed to the combined effects of various compounds, including phenolics, peptides, amino acids, Maillard reaction products, and other minor components [24]. AA was significantly (p < 0.05) affected by the carrier agent. The spray drying method reduced the antioxidant activity of the powders (Table 2) because of the loss of bioactive compounds such as betalains and polyphenols, which are sensitive to high temperatures [30]. A similar TPC behavior in AA demonstrated a positive correlation (R = 0.950; p = 0.01). The highest values of AA were generated in WP, with a value of 13.53 µmol (TE)/g. This could be attributed to protein denaturation because of the drying process, which releases bioactive peptides, thereby increasing AA [10]. Despite this, high activity was generated in the BP and PP treatments, indicating that, compared with traditional carriers such as maltodextrin, the use of plant proteins improved the antioxidant activity of the encapsulated pitaya by almost double the activity for PP and 37% for BP. Wang et al. [30] reported greater AA concentrations when black mulberry juice was encapsulated with pea protein than when it was encapsulated with maltodextrin.

3.7. Stability of Encapsulated Materials During Storage

The carrier agent affected the stability of betalains (p < 0.05). First-order kinetics were determined for different storage temperatures (Figure 1); the degradation parameters are shown in

Table 4. Betanin degradation rate constants of encapsulates during storage at different temperatures.

Treatment	Temperature	k (days−1)	Correlation Coefficient (R2)	Half-Life Period t1/2 (days)
RP	30	0.0022	0.98	315.06 ± 32.67 bc
	45	0.0095	0.90	72.96 ± 15.40 e
	60	0.0123	0.92	56.35 ± 4.55 e
PP	30	0.0035	0.98	198.04 ± 26.02 d
	45	0.0143	0.91	48.44 ± 3.58 e
	60	0.0197	0.97	35.18 ± 4.64 e
BP	30	0.0037	0.94	187.33 ± 23.31 d
	45	0.0197	0.91	35.18 ± 6.35 e
	60	0.0224	0.82	30.94 ± 10.90 e
WP	30	0.0031	0.97	223.59 ± 41.92 d
	45	0.0125	0.97	55.45 ± 4.56 e
	60	0.0499	0.63	13.89 ± 0.77 e
MT	30	0.0008	0.92	818.28 ± 68.07 a
	45	0.0018	0.97	386.27 ± 30.34 b
	60	0.0027	0.91	256.72 ± 41.25 cd

Mean ± standard deviation of treatments in duplicate. Values with different letter in each column for every parameter indicate significant difference (p < 0.05) according to the Tukey test. RP = rice protein, PP = pea protein, BP = bean protein, WP = whey protein, MT = maltodextrin.

. The first-order results adequately fit a first-order model (R² > 0.90) for most storage conditions except for BP and WP at a storage temperature of 60 °C. Notably, pigment degradation was observed under the evaluated conditions, indicating that the wall materials used had a significant effect on betacyanin retention during storage. Using maltodextrin as a carrier agent improved the stability of betalains during storage [9]. To estimate half-life, the rate constants for the initial values of betalain degradation were used for MT (98%, 92%, and 88%), RT (91%, 66%, and 55%), PP (86%, 58%, and 41%), BP (84%, 42%, and 30%), and WP (88%, 54%, and 5%) at 30 °C, 45 °C, and 60 °C, respectively. The results of the color stability analysis of the samples encapsulated during storage at different temperatures are presented in Figure S1 (Supplementary Material); the most notable effects on color changes were generated at temperatures of 45 °C and 60 °C, mainly in the PP, BP, and WP treatments. In most cases, with increasing storage temperature, the L* values decreased. Furthermore, the decreases in the a* and b* values with increasing storage temperature, as shown in Table 3, indicate that the redness of the encapsulated material decreased and generated dark brown coloration, because of the Maillard reaction [11,43]. According to Carmo et al. [11], at higher temperatures, the degradation of betacyanins is accompanied by a color change due to the formation of yellow degradation products, including balsamic acid, neotheracyanins, and betaxanthins. The highest half-life was generated by the MT treatment, which was significantly different from those of the other carriers. This behavior may be explained by the high Tg of the carrier agent, which makes it very stable at the storage temperatures it is subjected to. Despite this, the use of plant proteins such as RPs generated a considerable half-life of 315 days, and the use of PP and BP generated a half-life of 187–198 days, similar to that generated by WP. Similar half-life results were reported by Tabio-Garcia et al. [24], with values of 180–221 and 220–291 days for betacyanins and betaxanthins, respectively, when amaranth extracts were encapsulated with maltodextrin and cactus mucilage as carrier agents. These results indicate the suitability of using plant proteins to encapsulate betaine for storage.

3.8. SEM

Scanning electron microscopy images indicating the influence of the carrier agents of the encapsulated red pitaya powder are shown in Figure 2. The MT treatment (Figure 2a) resulted in capsules with a round shape with collapsed and wrinkled surface formations between particles with an average size of 8.30 μm. This phenomenon has been associated with the shrinkage of microcapsules during the drying process caused by the rapid loss of moisture and sudden cooling [3,9,40]. Further, no cracks were observed on the surface of the microcapsules, which prevents oxygen entry into the microcapsules, improving the stability of the bioactive compounds [24,40]. Similar results were reported by García-Lucas et al. [44] and Duong et al. [41] for the encapsulation of pitaya pigments using maltodextrin. WP treatment (Figure 2b) resulted in particles with smooth surfaces and no surface cracks, with spherical to oval and complete capsules, with an average particle size of 4.70 μm. This finding was also reported by do Carmo et al. [11] and Fang et al. [36] because proteins migrate to the surface of the particles and cover the powder particle surface with a thin film of non-sticky protein, generating more efficient encapsulation and good powder recovery. The results of PP treatment (Figure 2c) revealed that the agglomerated particles were spherical, solid, and regular in shape, with an average size of 5.79 μm. These particle agglomerations are associated with electrostatic effects and van der Waals forces [3,40], which could be attributed to the high surface hydrophobicity value of glutelin, resulting in more significant levels of surfactant and excellent foaming and emulsifying capacity [30]. Cui et al. [18] reported changes in morphological properties, such as less roughness but more agglomeration because of changes in the strength of hydrogen bonding, hydrophobic interactions, and electrostatic interactions. Similar morphologies have been obtained in microcapsules using pea protein in beet microencapsulation [4]. In contrast, the BP and RP treatments (Figure 2d,e) resulted in capsular agglomerations and crystalline forms, with particle sizes of 26.5 and 35.73 μm, respectively; this agglomeration was attributed to the presence of a liquid interface between the particles, which prevents the formation of capsules during the drying process [11]. Grace et al. [45] have reported that particles with fractured or non-encapsulated surfaces could lead to increased oxygen permeability during storage and, consequently, increased degradation of the core material; this is more evident with increasing storage temperature, which causes betanin decarboxylation and results in the formation of neobetanin; this alters the color owing to the creation of a less stable aglycon resulting in brown coloration [11,44]. Similar trends were observed in the PP and BP treatments, which induced changes in pigmentation (Figure S1). Notably, Laokuldilok and Kanha [46] reported that powders with a smaller size and smoother surface can protect against oxidative degradation during storage; however, parameters such as Tg and Hg play an important role in increasing or decreasing the storage time of the encapsulated products.

3.9. Fourier Transform Infra-Red (FT-IR) Spectrometry

Figure 3 shows the FT-IR spectra of pitaya juice encapsulated with different wall materials where functional groups present in betalain are observed. The main absorbances observed in the microcapsules treated with PP, RC, BP, and WP presented strong amide-type I peaks at 1600–1700 cm⁻¹, amide II peaks, C-N peaks at 1530–1550 cm⁻¹, C-O stretching, and C-O-H bending at 1065 cm⁻¹ [5,41]. Furthermore, all treatments resulted in significant vibrations in the betalain region in all samples at 1424 cm-1 and 2924 cm⁻¹, which were related to the C=C bonds in the components of carboxylic acids and betalain pigment groups, and those in 2916–1940 cm⁻¹ were attributed to the C-H bonds belonging to the alkanes of betacyanins [3,14,41].

4. Conclusions

The effects of using different plant protein-based wall materials on the physicochemical properties of red pitaya juice and its contrast with the traditional carrier agents WP and maltodextrin were evaluated. Compared with the control, the use of vegetable proteins, mainly PP, was found adequate for spray-dried products with low hygroscopicity, high protein content above 47%, high betalain retention (>30%), and TPC and AA values, with low degradation rate constants and long half-life times (>180 days) at 30 °C. These results suggest that the use of vegetable proteins as a carrier agent is a good alternative to improve the pigment preservation of pitaya juice, with possible applications in the development of food products with antioxidant properties.