From Extraction to Stabilization: Employing a 22 Experimental Design in Developing Nutraceutical-Grade Bixin from Bixa orellana L.

Citation: Luna-Finkler, C.L.; Gomes, A.d.C.; de Aguiar Júnior, F.C.A.; Ribeiro, E.; de Melo Barbosa, R.; Severino, P.; Santini, A.; Souto, E.B. From Extraction to Stabilization: Employing a 22Experimental Design in Developing Nutraceutical-Grade Bixin from Bixa orellana L. Foods 2024, 13, 1622. https://doi.org/ 10.3390/foods13111622 Academic Editor: Arun K. Bhunia Received: 2 April 2024 Revised: 16 May 2024 Accepted: 18 May 2024 Published: 23 May 2024 Copyright: © 2024 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). foods Article From Extraction to Stabilization: Employing a 22Experimental Design in Developing Nutraceutical-Grade Bixin from Bixa orellana L. Christine L. Luna-Finkler 1, Aralída C. Gomes 2, Francisco C. A. de Aguiar Júnior 2, Ester Ribeiro 1, Raquel de Melo Barbosa 3,* , Patricia Severino 4,5 , Antonello Santini 6,* and Eliana B. Souto 7 1Department of Antibiotics, Federal University of Pernambuco, Cidade Universitária, Recife 50670-901, PE, Brazil; [email protected] (C.L.L.-F.); ester[email protected] (E.R.) 2Academic Center of Vitória, Federal University of Pernambuco, R. Alto do Reservatório, s/n, Bela Vista, Vitória de Santo Antão 55608-250, PE, Brazil; [email protected] (A.d.C.G.); [email protected] (F.C.A.d.A.J.) 3Department of Pharmacy and Pharmaceutical Technology, School of Pharmacy, University of Seville, C/Professor García González, 2, 41012 Seville, Spain 4 Laboratory of Nanotechnology and Nanomedicine (LNMED), Institute of Technology and Research (ITP), Av. Murilo Dantas, 300, Aracaju 49010-390, PE, Brazil; [email protected] 5Industrial Biotechnology Program, University of Tiradentes (UNIT), Av. Murilo Dantas, 300, Aracaju 49032-490, PE, Brazil 6Department of Pharmacy, University of Napoli Federico II, Via D. Montesano, 49-80131 Napoli, Italy 7 Laboratory of Pharmaceutical Technology, Faculty of Pharmacy, University of Porto, 4050-313 Porto, Portugal; [email protected] *Correspondence: [email protected] (R.d.M.B.); [email protected] (A.S.) Abstract: Bixin is the main carotenoid found in the outer portion of the seeds of Bixa orellana L., commercially known as annatto. This compound is industrially employed in pharmaceutical, cosmetic, and food formulations as a natural dye to replace chemical additives. This study aimed to extract bixin from annatto seeds and obtain encapsulated bixin in a powder form, using freeze-drying encapsulation and maltodextrin as encapsulating agent. Bixin was extracted from annatto seeds employing successive washing with organic solvents, specifically hexane and methanol (1:1 v/v), followed by ethyl acetate and dichloromethane for subsequent washes, to effectively remove impurities and enhance bixin purity, and subsequent purification by crystallization, reaching 1.5 ± 0.2% yield (or approximately 15 mg of bixin per gram of seeds). Bixin was analyzed spectrophotometrically in different organic solvents (ethanol, isopropyl alcohol, dimethylsulfoxide, chloroform, hexane), and the solvents chosen were chloroform (used to solubilize bixin during microencapsulation) and hexane (used for spectrophotometric determination of bixin). Bixin was encapsulated according to a 2 2 experimental design to investigate the influence of the concentration of maltodextrin (20 to 40%) and bixin-to-matrix ratio (1:20 to 1:40) on the encapsulation efficiency (EE%) and solubility of the encapsulated powder. Higher encapsulation efficiency was obtained at a maltodextrin concentration of 40% w/vand a bixin/maltodextrin ratio of 1:20, while higher solubility was observed at a maltodextrin concentration of 20% w/vfor the same bixin/maltodextrin ratio. The encapsulation of this carotenoid by means of freeze-drying is thus recognized as an innovative and promising approach to improve its stability for further processing in pharmaceutical and food applications. Keywords: carotenoid; urucum; Bixa orellana L.; freeze-drying; stability; foodstuff 1. Introduction Bixa orellana L. is a native plant from Tropical America, commonly found between Guianas and the State of Bahia in Brazil, and is a rich source of annatto seeds. Annatto carotenoids are widely used in the food and pharmaceutical industries [ 1 ]. In the food Foods 2024,13, 1622. https://doi.org/10.3390/foods13111622 https://www.mdpi.com/journal/foods Foods 2024,13, 1622 2 of 16 industries, they are used as antioxidants [ 2 ], photoprotective agents [ 3 ], and as natural dyes [ 4 ]. From a medicinal point of view, annatto seeds have expectorant properties, laxative, cardiotonic, hypotensive, antibiotic, anti-inflammatory, and healing activities [ 5 , 6 ]. Bixin is a dicarboxylic cis-apocarotenoid monomethyl ester with a high number of conjugated double bonds. It mostly occurs in annatto seeds (Bixa orellana L.) [ 7 ], making up at least 80% of the total carotenoids in the seed [ 8 ]. It has the molecular formula C 25 H 30 O 4 and is present in the outer coating of the grains of the plant [ 9 ]. It is considered a natural dye responsible for shades ranging from yellow to red. A lengthy chain of polyunsaturated hydrocarbons with methyl ester groups and carboxylic acids at either end makes up the bixin structure (Figure 1). Due to the presence of these functional groups in its chemical structure, the molecule is insoluble in water but soluble in polar organic solvents, such as hexane, methanol, and acetone [ 10 ], characterizing it as a fat-soluble compound [ 11 ]. The molecule can absorb light at visible spectrum wavelengths, which are close to 440–489 nm, thanks to changes in the chain. This allows for the creation of hues ranging from yellow to orange-red [ 12 ]. This ability is due to the presence of a conjugated polyene chain, which has a centrally located double-bond system that acts as a chromophore and is essential for multiple functions and actions [ 13 ]. Thus, UV–visible spectroscopy and high-performance liquid chromatography (HPLC) are the most often employed analytical models for the quantification of bixin [9]. Foods 2024, 13, x FOR PEER REVIEW 2 of 17 1. Introduction Bixa orellana L. is a native plant from Tropical America, commonly found between Guianas and the State of Bahia in Brazil, and is a rich source of annatto seeds. Annatto carotenoids are widely used in the food and pharmaceutical industries [1]. In the food industries, they are used as antioxidants [2], photoprotective agents [3], and as natural dyes [4]. From a medicinal point of view, annatto seeds have expectorant properties, laxative, cardiotonic, hypotensive, antibiotic, anti-inflammatory, and healing activities [5,6]. Bixin is a dicarboxylic cis-apocarotenoid monomethyl ester with a high number of conjugated double bonds. It mostly occurs in annatto seeds (Bixa orellana L.) [7], making up at least 80% of the total carotenoids in the seed [8]. It has the molecular formula C25H30O4 and is present in the outer coating of the grains of the plant [9]. It is considered a natural dye responsible for shades ranging from yellow to red. A lengthy chain of polyunsaturated hydrocarbons with methyl ester groups and carboxylic acids at either end makes up the bixin structure (Figure 1). Due to the presence of these functional groups in its chemical structure, the molecule is insoluble in water but soluble in polar organic solvents, such as hexane, methanol, and acetone [10], characterizing it as a fat-soluble compound [11]. The molecule can absorb light at visible spectrum wavelengths, which are close to 440–489 nm, thanks to changes in the chain. This allows for the creation of hues ranging from yellow to orange-red [12]. This ability is due to the presence of a conjugated polyene chain, which has a centrally located doublebond system that acts as a chromophore and is essential for multiple functions and actions [13]. Thus, UV–visible spectroscopy and high-performance liquid chromatography (HPLC) are the most often employed analytical models for the quantification of bixin [9]. Figure 1. Chemical structure of bixin ((2E,4E,6E,8E,10E,12E,14E,16Z,18E)-20-methoxy-4,8,13,17- tetramethyl-20-oxoicosa-2,4,6,8,10,12,14,16,18-nonaenoic acid, IUPAC nomenclature). The presence of a conjugated polyene chain makes carotenoids prone to isomerization and oxidation [13], making them unstable due to factors such as the presence/absence of oxygen, high temperatures, and light [14]. In recent years, the encapsulation of bioactive natural products has become a critical area of research, particularly for enhancing the stability and bioavailability of these compounds for pharmaceutical and nutraceutical applications [15–17]. Encapsulation techniques vary widely but typically involve the use of a matrix or wall material to form a barrier around the active ingredient, thereby protecting it from environmental factors and controlled release [18,19]. Common materials used include lipids, proteins, polysaccharides, and synthetic polymers, each selected based on their biocompatibility, protective properties, and processing characteristics [20,21]. Maltodextrin was selected as the wall material for the encapsulation of bixin, a natural carotenoid derived from Bixa orellana L. This choice was informed by maltodextrin’s superior film-forming capabilities, its capacity to act as a protective barrier against oxidation, and its relatively neutral flavor profile, which is especially beneficial in food applications [21]. Furthermore, the high solubility and low viscosity of maltodextrin Figure 1. Chemical structure of bixin ((2E,4E,6E,8E,10E,12E,14E,16Z,18E)-20-methoxy-4,8,13,17- tetramethyl-20-oxoicosa-2,4,6,8,10,12,14,16,18-nonaenoic acid, IUPAC nomenclature). The presence of a conjugated polyene chain makes carotenoids prone to isomerization and oxidation [ 13 ], making them unstable due to factors such as the presence/absence of oxygen, high temperatures, and light [14]. In recent years, the encapsulation of bioactive natural products has become a critical area of research, particularly for enhancing the stability and bioavailability of these compounds for pharmaceutical and nutraceutical applications [ 15 – 17 ]. Encapsulation techniques vary widely but typically involve the use of a matrix or wall material to form a barrier around the active ingredient, thereby protecting it from environmental factors and controlled release [ 18 , 19 ]. Common materials used include lipids, proteins, polysaccharides, and synthetic polymers, each selected based on their biocompatibility, protective properties, and processing characteristics [20,21]. Maltodextrin was selected as the wall material for the encapsulation of bixin, a natural carotenoid derived from Bixa orellana L. This choice was informed by maltodextrin’s superior film-forming capabilities, its capacity to act as a protective barrier against oxidation, and its relatively neutral flavor profile, which is especially beneficial in food applications [ 21 ]. Furthermore, the high solubility and low viscosity of maltodextrin at elevated concentrations render it particularly suitable for freeze-drying processes, the encapsulation method utilized in this study for bixin [22]. Encapsulation is one method amongst potential alternatives utilized to increase the stability of annatto carotenoids [ 23 , 24 ]. The use of lyophilization as a microencapsulation technique has the advantage of obtaining products with high solubility and oxidative stability. To this end, the drying of the product of interest is carried out by the addition of Foods 2024,13, 1622 3 of 16 an encapsulating agent, generally a polymer, as a wall material. According to the choice of encapsulating agent used in the process, particles of different sizes can be obtained [25]. To document the innovative aspects of this research, we have conducted a survey in Scopus recorded on the 25 March 2024, using a combination of terms, namely, “bixin” and “freeze-drying” or “lyophilization”, which resulted in a total of 205 published papers since ever indexed in the Scopus core collection. VOSviewer software was employed for data analysis, generating the bibliometric map shown in Figure 2[ 26 ]. The outputs were four clusters, covering nano/microencapsulation, animal experimentation, bioactives, and comparative study as the major topics. A total of 117 papers were linked to Agricultural and Biological Sciences, 73 to Chemistry, 54 to Chemical Engineering, 41 to Engineering, 30 to Biochemistry, Genetics, and Molecular Biology, and only 28 were published in the subject area of Pharmacology, Toxicology, and Pharmaceutics which, together with the food sector, is one of the areas of interest for the use of bixin. The novelty of our work relies on the exploitation of strategies to improve the stability of this carotenoid by its encapsulation by freeze-drying, which is for potential use in oral administration (pharmaceutics or foods). Foods 2024, 13, x FOR PEER REVIEW 3 of 17 at elevated concentrations render it particularly suitable for freeze-drying processes, the encapsulation method utilized in this study for bixin [22]. Encapsulation is one method amongst potential alternatives utilized to increase the stability of annatto carotenoids [23,24]. The use of lyophilization as a microencapsulation technique has the advantage of obtaining products with high solubility and oxidative stability. To this end, the drying of the product of interest is carried out by the addition of an encapsulating agent, generally a polymer, as a wall material. According to the choice of encapsulating agent used in the process, particles of different sizes can be obtained [25]. To document the innovative aspects of this research, we have conducted a survey in Scopus recorded on the 25 March 2024, using a combination of terms, namely, “bixin” and “freeze-drying” or “lyophilization”, which resulted in a total of 205 published papers since ever indexed in the Scopus core collection. VOSviewer software was employed for data analysis, generating the bibliometric map shown in Figure 2 [26]. The outputs were four clusters, covering nano/microencapsulation, animal experimentation, bioactives, and comparative study as the major topics. A total of 117 papers were linked to Agricultural and Biological Sciences, 73 to Chemistry, 54 to Chemical Engineering, 41 to Engineering, 30 to Biochemistry, Genetics, and Molecular Biology, and only 28 were published in the subject area of Pharmacology, Toxicology, and Pharmaceutics which, together with the food sector, is one of the areas of interest for the use of bixin. The novelty of our work relies on the exploitation of strategies to improve the stability of this carotenoid by its encapsulation by freeze-drying, which is for potential use in oral administration (pharmaceutics or foods). Figure 2. Bibliometric map obtained by VOSviewer software version 1.6.16 (https://www.vosviewer.com, accessed on 25 March 2024), using “bixin” and “freeze-drying” or “lyophilization” as keywords, recorded from Scopus database (recorded on the 25 March 2024). Figure 2. Bibliometric map obtained by VOSviewer software version 1.6.16 (https://www.vosviewer. com, accessed on 25 March 2024), using “bixin” and “freeze-drying” or “lyophilization” as keywords, recorded from Scopus database (recorded on the 25 March 2024). This work aims to encapsulate by lyophilization the carotenoid bixin, using maltodextrin as a wall material. The encapsulation was carried out following a 2 2 experimental design, having as independent variables the concentration of maltodextrin and the proportion of bixin in relation to the encapsulating material, while the response variables were the encapsulation efficiency and the solubility of the microencapsulated powder. Foods 2024,13, 1622 4 of 16 2. Materials and Methods 2.1. Materials Annatto seeds (Bixa orellana L.) were purchased from Beleza da Terra company (Space Green FE0444, Curitiba, PR, Brazil), being stored in hermetically sealed polyethylene bags in a refrigerator at 8–10 ◦ C in the Bioprocess Laboratory of the Federal University of Pernambuco, Vitoria Academic Centre (PE, Brazil). The seeds were characterized in terms of protein, lipid, ash, and moisture content, as described below. All used organic solvents with HPLC grade and standard bixin were purchased from Sigma-Aldrich (St. Louis, MO, USA). All other solvents and chemicals were of analytical grade. 2.2. Bixin Extraction Each bixin extraction was performed from 200 g of annatto seeds previously ground and passed through a sieve filter with a size of 48 mesh. Initially, two successive washings of the seeds were carried out with a hexane/methanol solution (1:1) for 15 min under stirring. Then, the seeds were washed twice with ethyl acetate for 15 min under stirring. In this step, the removal of undesirable compounds that affect the purity of the final product occurs, which, in most cases, are composed of lipids and polar compounds. After washing the seeds, they were drained and dried at 30 ◦ C in an oven. The flowchart of the interferents removing process is presented in Figure 3. Foods 2024, 13, x FOR PEER REVIEW 4 of 17 This work aims to encapsulate by lyophilization the carotenoid bixin, using maltodextrin as a wall material. The encapsulation was carried out following a 2 2 experimental design, having as independent variables the concentration of maltodextrin and the proportion of bixin in relation to the encapsulating material, while the response variables were the encapsulation efficiency and the solubility of the microencapsulated powder. 2. Materials and Methods 2.1. Materials Annatto seeds (Bixa orellana L.) were purchased from Beleza da Terra company (Space Green FE0444, Curitiba, PR, Brazil), being stored in hermetically sealed polyethylene bags in a refrigerator at 8–10 °C in the Bioprocess Laboratory of the Federal University of Pernambuco, Vitoria Academic Centre (PE, Brazil). The seeds were characterized in terms of protein, lipid, ash, and moisture content, as described below. All used organic solvents with HPLC grade and standard bixin were purchased from Sigma- Aldrich (St. Louis, MO, USA). All other solvents and chemicals were of analytical grade. 2.2. Bixin Extraction Each bixin extraction was performed from 200 g of annatto seeds previously ground and passed through a sieve filter with a size of 48 mesh. Initially, two successive washings of the seeds were carried out with a hexane/methanol solution (1:1) for 15 min under stirring. Then, the seeds were washed twice with ethyl acetate for 15 min under stirring. In this step, the removal of undesirable compounds that affect the purity of the final product occurs, which, in most cases, are composed of lipids and polar compounds. After washing the seeds, they were drained and dried at 30 °C in an oven. The flowchart of the interferents removing process is presented in Figure 3. Figure 3. Schematic representation of bixin extraction and washing out of interferences. After removing the interferents, the seeds were subjected to the extraction process itself in order to obtain the bixin crystals. This step promotes the extraction and purification of annatto extract through crystallization. The dried seeds were washed with dichloromethane and absolute ethanol (in a ratio of 1:4 by volume). Then, the material was taken to an ice bath for 5 min and then frozen. Then, the mixture was filtered, washed with absolute ethanol, and dried at 29 °C in an oven under air circulation for 24 h. The bixin crystals were then kept in an amber flask wrapped in aluminum foil and frozen at −8 °C. Figure 3. Schematic representation of bixin extraction and washing out of interferences. After removing the interferents, the seeds were subjected to the extraction process itself in order to obtain the bixin crystals. This step promotes the extraction and purification of annatto extract through crystallization. The dried seeds were washed with dichloromethane and absolute ethanol (in a ratio of 1:4 by volume). Then, the material was taken to an ice bath for 5 min and then frozen. Then, the mixture was filtered, washed with absolute ethanol, and dried at 29 ◦ C in an oven under air circulation for 24 h. The bixin crystals were then kept in an amber flask wrapped in aluminum foil and frozen at −8◦C. Ten extraction experiments were carried out in order to obtain crystals with a standardized content of bixin and in sufficient quantity to carry out the microencapsulation tests. The bixin content of the extract was evaluated by HPLC. The extraction of bixin crystals has its yield (Y%) calculated by the ratio between the mass of bixin crystals obtained after extraction and the initial mass of seeds (200 g). Thus, the yield was calculated according to Equation (1): Yield (%) = mass of cystals mass of seeds ×100% (1) Foods 2024,13, 1622 5 of 16 2.3. Spectrophotometric Analysis of Bixin in Different Solvents For the bixin scanning tests in different solvents, a mass of 25 mg of bixin was added to 30 mL of the solvent under study. After vortexing for 1 min and resting at 30 ◦ C, the samples were centrifuged at 5000 rpm for 5 min. Subsequently, the supernatants were submitted to spectrophotometric analysis in a UV–Vis spectrophotometer (Thermoscientific Genesys 10S, Thermo Fisher Scientific, Waltham, MA, USA) in a wavelength range from 300 to 800 nm. Scanning assays were performed in the following solvents: isopropyl alcohol, ethanol, dimethyl sulfoxide (DMSO), chloroform, and hexane. 2.4. Experimental Factorial Design for the Encapsulation of Bixin by Lyophilization A type 2 2 experimental statistical design was carried out, with two variables at two levels and quintuplicated at the central point, accounting for a total of nine experiments. The independent variables were the concentration of maltodextrin and the proportion of bixin in relation to the encapsulating material (Mb/Me), while the response variable was the encapsulation efficiency (EE%) and the solubility of the microencapsulated powder. Table 1shows the values of the variables that were investigated in the experimental design. The complete planning matrix is shown in Table 2. The experiments were performed randomly, and data analysis was performed using the Statistica®program (version 7.0). Table 1. Variables investigated in the experimental design. Variable −1 0 +1 Maltodextrin concentration (%) 20 30 40 Bixin/maltodextrin ratio (Mb/Me) * 1:20 1:30 1:40 * Mb—bixin mass; Me—encapsulating mass (maltodextrin). Table 2. Central composite planning matrix 22with five central points (level 0). Essay Maltodextrin Concentration (%) Bixin/Maltodextrin Ratio (Mb/Me) * 1−1−1 2−1 +1 3 +1 −1 4 +1 +1 5 0 0 6 0 0 7 0 0 8 0 0 9 0 0 * Mb—bixin mass; Me—encapsulating mass (maltodextrin). Firstly, the mass of maltodextrin indicated by each condition of the experimental statistical design was previously dissolved in water heated to 60 ◦ C to form an aqueous suspension. To facilitate emulsion formation, Tween 80 (0.1%) was added to each maltodextrin aqueous emulsion. The bixin crystals were dissolved in 5 mL of chloroform, and the mixture was added to the aqueous suspension of maltodextrin. The suspension was vigorously stirred on a magnetic stirrer for 30 min, according to work by Spada et al. (2012) [ 27 ], with modifications. Chloroform was chosen (relatively polar solvent, dipole moment 1.04 D) because it has a melting point of − 63 ◦ C (1 atm), which allows the samples to be solidified at the temperature used for freezing them (−80 ◦C) before the lyophilization procedure. Furthermore, despite being a relatively polar solvent, it is able to solubilize bixin crystals. The samples were distributed in penicillin-type glass vials with a rubber stopper Foods 2024,13, 1622 6 of 16 for lyophilization and later frozen at − 80 ◦ C overnight. Then, the samples were lyophilized using a lyophilizer (FTS systems Model LYOACC3P1 Cleveland, OH, USA). The drying process was carried out for up to 48 h at − 84 ◦ C, with a vacuum at 188 mT. The vials containing the powders were sealed and stored protected from light in a refrigerator for subsequent analyses. 2.5. Encapsulation Efficiency The encapsulation efficiency (EE%) was determined according to Equation (2): EE%=Ce Ci ×100 (2) where EE% is the encapsulation efficiency, Ci is the initial bixin concentration in the suspension, and Ce is the bixin concentration in the encapsulated sample. The bixin concentration in the encapsulated sample was determined according to Equation (3): Ce =Ci −Cs (3) where Cs is the surface bixin concentration (unencapsulated sample). The determination of bixin concentrations in the initial (Ci) and surface (Cs) samples was performed based on the fact that this carotenoid is lipophilic and soluble in hexane and that the matrix (maltodextrin) is, on the other hand, soluble in water and insoluble in hexane. The hexane solvent was chosen because it is non-polar (dipole moment 0.00 D), i.e., bixin crystals are soluble in this solvent. The surface bixin concentration was determined by washing 50 mg aliquots of the lyophilized powder with 20 mL of hexane in a centrifuge tube for 15 s at 100 rpm. Then, the sample was centrifuged (Hettich Routina 420R, Tuttlingen, Germany) at 528×gfor 1 min, and the absorbance of the supernatant was measured at 455 nm in a spectrophotometer (Thermoscientific Genesys 10S, Thermo Fisher Scientific, Waltham, MA, USA) [27]. A mass of 50 mg of the lyophilized sample was dispersed in 2.5 mL of water and 20 mL of hexane to set up the initial concentration of bixin. The sample was shaken in a centrifuge (Hettich Routina 420R, Tuttlingen, Germany) at 2500 rpm for 20 min, and aliquots of the organic phase were taken for spectrophotometric analysis at 455 nm. Bixin quantifications by spectrophotometry were performed at 455 nm, the wavelength of maximum absorption of bixin when solubilized in hexane. A standard curve of absorbance × concentration was plotted for the quantification of bixin in the samples, resulting in a linear calibration curve Y=2883.7x+0.0008; R2=1. 2.6. Solubility in Water The water solubility of the microencapsulated powder was evaluated following the methodology described by Eastman and Moore (1984) [ 28 ], with adaptations. A mass corresponding to 0.5 g of the sample was diluted in 50 mL of deionized water. Following five minutes of magnetic stirring to homogenize the suspension, the sample was centrifuged for five minutes at 4500 rpm. An aliquot of 12.5 mL of the supernatant was placed in an oven at 105 ◦ C until constant weight, and the solubility (S) was calculated, in percentage, by the weight difference, using Equation (4). The analysis was performed in triplicate. S%=MS Mt ×100 (4) where MSis the mass of powder in the supernatant and Mtis the total powder mass. 2.7. Moisture The water content of the microencapsulated powder was determined from the mass loss of 5 g of lyophilized sample submitted to heating at 105 ◦ C until constant weight was obtained. Foods 2024,13, 1622 7 of 16 2.8. HPLC Analysis The analysis of the bixin obtained in the extraction step and a sample of standard bixin (Sigma Aldrich Brazil LTDA, St. Louis, MO, USA) with purity ≥ 90% were performed. Samples containing a mass of 0.01 g were prepared and dissolved in the mobile phase methanol/0.5% acetic acid (70:30 v/v), according to Rahmalia et al. (2015) [ 29 ]. The samples were vacuum filtered (Prismatec, Sao Paulo, Brazil) on a Millipore 0.22 µ m membrane (Chromafil ® Xtra PVDF -20/25, Macherey-Nagel, Düren, Germany) before being injected into the chromatograph. A C18 chromatography column (ShimParck CLC ODS (M), Shimadzu, Kyoto, Japan, 25 cm) was used, maintained at 29 ◦ C, mobile phase under isocratic conditions, and flow rate of 1.0 mL/min. The chromatographic analyses were performed on a Shimadzu chromatograph (CGM model—20A, Columbia, MD, USA). The chromatograms were recorded at a wavelength of 455 nm, with a chromatographic run duration of 10 min, and the spectra were obtained between 250 and 600 nm. Table 3shows the chromatogram area and bixin concentration results that were obtained for the injection of volumes of 20, 30, 40, and 50 µ L of the bixin sample obtained by extraction. The concentration values were calculated based on the calibration curve of the bixin standard obtained by HPLC ( Y= 1306.4 x ; R2= 0.0079), considering the area values within the range of the standard curve (Table 4). Table 3. Results of the bixin sample obtained by extraction and quantified using high-performance liquid chromatography. RT * (min) Area Volume (µL) Concentration (g/L) 6.05 3527.4 20 0.1350 5.67 4224.3 30 0.1078 5.79 6514.8 40 0.1247 5.68 7018.6 50 0.1074 Average ±s.d. 0.12 ±0.01 * RT—retention time; s.d.—standard deviation. Table 4. Bixin standard quantified by high-performance liquid chromatography. Bixin Mass (µg) Area RT * (min) 0.00 0.00 0.00 0.64 525.60 5.63 1.28 1281.30 5.63 3.84 4952.60 5.67 5.12 6821.40 5.67 5.76 7570.80 5.69 * RT—retention time. 3. Results and Discussion Table 5shows the approximate composition of bixin seeds. According to Scotter (2009) [ 4 ], annatto seeds typically include 1 to 6.3% bixin, 5.4 to 6.9% ash, 9.6 to 13.3% moisture, 2.0 to 4.8% lipids, 12.1 to 17.0% protein, and 70% of total carbs. Similar values were also obtained for annatto seeds in natura variety Piave by Albuquerque and Meireles (2012) [ 30 ], namely 4.9% ± 0.2% bixin, 6.2% ± 0.1% ash, 12.3% ± 0.1% moisture, 3.7% ±0.0% lipids, 12.1% ±0.2% protein, and 65.7% total carbohydrates. Foods 2024,13, 1622 8 of 16 Table 5. Physicochemical characterization of annatto seeds. Component Concentration (%) (Mean ±s.d. *) Ash 9.73 ±0.45 Proteins 12.93 ±0.15 Lipids 2.12 ±0.20 Moisture 10.18 ±0.68 * s.d.—standard deviation. For the integer annatto seeds in natura, the following values have been reported: moisture 14.0 ± 0.1%, lipids 4.0 ± 0.3%, bixin 2.0 ± 0.2%, and other material up to 80% [ 31 ]. Thus, considering the variability of cultivars, soil, and storage conditions, the values found here differ from the mentioned data. The yield of the bixin extraction process from annatto seeds is a determining factor for the economic viability of this carotenoid in the industry. The extraction of bixin crystals has its yield calculated by the ratio between the amount of powder produced at the end of the extraction and the initial amount of seeds. Using the methodology adopted in this work, a yield of 1.5 ± 0.2% was obtained. Figure 4shows the visual appearance of the annatto seeds in natura and the bixin crystals extracted from the seeds. Foods 2024, 13, x FOR PEER REVIEW 8 of 17 3. Results and Discussion Table 5 shows the approximate composition of bixin seeds. According to Scotter (2009) [4], annatto seeds typically include 1 to 6.3% bixin, 5.4 to 6.9% ash, 9.6 to 13.3% moisture, 2.0 to 4.8% lipids, 12.1 to 17.0% protein, and 70% of total carbs. Similar values were also obtained for annatto seeds in natura variety Piave by Albuquerque and Meireles (2012) [30], namely 4.9% ± 0.2% bixin, 6.2% ± 0.1% ash, 12.3% ± 0.1% moisture, 3.7% ± 0.0% lipids, 12.1% ± 0.2% protein, and 65.7% total carbohydrates. Table 5. Physicochemical characterization of annatto seeds. Component Concentration (%) (mean ± s.d. *) Ash 9.73 ± 0.45 Proteins 12.93 ± 0.15 Lipids 2.12 ± 0.20 Moisture 10.18 ± 0.68 * s.d.—standard deviation. For the integer annatto seeds in natura, the following values have been reported: moisture 14.0 ± 0.1%, lipids 4.0 ± 0.3%, bixin 2.0 ± 0.2%, and other material up to 80% [31]. Thus, considering the variability of cultivars, soil, and storage conditions, the values found here differ from the mentioned data. The yield of the bixin extraction process from annatto seeds is a determining factor for the economic viability of this carotenoid in the industry. The extraction of bixin crystals has its yield calculated by the ratio between the amount of powder produced at the end of the extraction and the initial amount of seeds. Using the methodology adopted in this work, a yield of 1.5 ± 0.2% was obtained. Figure 4 shows the visual appearance of the annatto seeds in natura and the bixin crystals extracted from the seeds. (A) (B) Figure 4. Visual appearance of the annatto seeds in natura (A) and the bixin crystals extracted from the seeds (B). After harvesting and especially during storage, annatto seeds are exposed to some environmental factors, such as water activity, high temperatures, exposure to air, and high relative humidity. Variations in these factors also end up interfering with the yield of pigment production. The content of these compounds present in annatto seeds varies according to the variety of the crop, soil, climate, and cultural practices, and seeds with less than 1% and others with up to 6% of bixin can be found [32]. According to Carvalho et al. (2010) [33], the extraction techniques mostly aim to produce an intense red powder in which bixin, which is most responsible for the color, is in low concentration. Figure 4. Visual appearance of the annatto seeds in natura (A) and the bixin crystals extracted from the seeds (B). After harvesting and especially during storage, annatto seeds are exposed to some environmental factors, such as water activity, high temperatures, exposure to air, and high relative humidity. Variations in these factors also end up interfering with the yield of pigment production. The content of these compounds present in annatto seeds varies according to the variety of the crop, soil, climate, and cultural practices, and seeds with less than 1% and others with up to 6% of bixin can be found [ 32 ]. According to Carvalho et al. (2010) [ 33 ], the extraction techniques mostly aim to produce an intense red powder in which bixin, which is most responsible for the color, is in low concentration. Rahmalia et al. (2014) [ 9 ] analyzed the concentration of bixin in annatto seeds using an extraction method with cyclohexane/acetone solvents in different proportions. The authors observed that the amount of carotenoid in their samples was 27.90 ±0.02 mg bixin/g (2.79 ± 0.02% by weight). The average bixin concentration was reported to range from 12 to 23 mg/g in the seeds, depending on the climatic conditions of planting (such as temperature, lighting, rainfall, and soil) and genetic factors (cultivars). Chistéet al. (2011) [ 34 ] employed intact annatto seeds with water, ethanol, ethyl acetate, ethanol/water (1:1, v/v), and ethanol/ethyl acetate (1:1, v/v). On an orbital shaker, solvents were introduced at a mass/solvent ratio of 1:2 (m/v). The highest bixin content Foods 2024,13, 1622 9 of 16 was found in the extracts made with ethanol, ethyl acetate, and ethanol/ethyl acetate. Bixin has the property of being lipid soluble and, therefore, subject to extraction with several organic solvents, among which the most used are chloroform, acetone, propylene glycol, and ethanol [9]. Factors such as choice of solvent, proportion between solvents, extraction temperature, and the amount of water present in the grains influence the final amount of bixin obtained [35]. The difficulties in comparing the results of bixin content from different origins and varieties may be related to the lack of uniformity in the methods of analysis. The methodologies used range from the use of different solutions for extracting the pigments to the inappropriate use of absorption coefficients for quantification [33]. Figure 5shows the chromatogram of the bixin standard (>90% purity—Figure 5a) and the bixin sample obtained after extraction (Figure 5b). Foods 2024, 13, x FOR PEER REVIEW 9 of 17 Rahmalia et al. (2014) [9] analyzed the concentration of bixin in annatto seeds using an extraction method with cyclohexane/acetone solvents in different proportions. The authors observed that the amount of carotenoid in their samples was 27.90 ± 0.02 mg bixin/g (2.79 ± 0.02% by weight). The average bixin concentration was reported to range from 12 to 23 mg/g in the seeds, depending on the climatic conditions of planting (such as temperature, lighting, rainfall, and soil) and genetic factors (cultivars). Chisté et al. (2011) [34] employed intact annatto seeds with water, ethanol, ethyl acetate, ethanol/water (1:1, v/v), and ethanol/ethyl acetate (1:1, v/v). On an orbital shaker, solvents were introduced at a mass/solvent ratio of 1:2 (m/v). The highest bixin content was found in the extracts made with ethanol, ethyl acetate, and ethanol/ethyl acetate. Bixin has the property of being lipid soluble and, therefore, subject to extraction with several organic solvents, among which the most used are chloroform, acetone, propylene glycol, and ethanol [9]. Factors such as choice of solvent, proportion between solvents, extraction temperature, and the amount of water present in the grains influence the final amount of bixin obtained [35]. The difficulties in comparing the results of bixin content from different origins and varieties may be related to the lack of uniformity in the methods of analysis. The methodologies used range from the use of different solutions for extracting the pigments to the inappropriate use of absorption coefficients for quantification [33]. Figure 5 shows the chromatogram of the bixin standard (>90% purity—Figure 5a) and the bixin sample obtained after extraction (Figure 5b). (a) Figure 5. Chromatogram of the bixin standard (>90% purity) (a) and the bixin sample obtained after extraction (b). Figure 5shows that, under the experimental conditions used, the chromatographic separation indicates the appearance of a peak referring to the bixin pattern corresponding to a retention time of about 5.65 s, which overlaps with the peak of the extract obtained Foods 2024,13, 1622 16 of 16 34. Chisté, R.C.; Yamashita, F.; Gozzo, F.C.; Mercadante, A.Z. Simultaneous extraction and analysis by high performance liquid chromatography coupled to diode array and mass spectrometric detectors of bixin and phenolic compounds from annatto seeds. J. Chromatogr. 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