Senise Red Pepper (Capsicum annuum L.) Wastes as Source of Rich Capsanthin Extracts by Supercritical CO2 Extraction

TL;DR

This study shows how to extract valuable red pigments from waste sweet pepper using an eco-friendly CO2 method.

Contribution

The novel use of supercritical CO2 extraction for recovering high-value carotenoids from Senise pepper waste is demonstrated.

Findings

Supercritical CO2 extraction achieved a high total carotenoid content of 386 mg/kgDW at 450 bar and 60 °C.

Capsanthin was the predominant carotenoid in the extract, with a concentration of 5.88 mg/g.

The green extraction method offers a sustainable way to recover natural pigments from agro-industrial waste.

Abstract

The valorization of agro-industrial by-products represents a key strategy for promoting sustainable resource use and recovering high-value bioactive compounds. This study investigated the extraction of carotenoids from processing residues of Capsicum annuum L. cv Senise, a sweet pepper with Protected Geographical Indication (PGI) status, using supercritical CO2 extraction (SC-CO2). Experiments were conducted under nine pressure–temperature combinations (250–450 bar; 40–60 °C) for 60 min, and the results were compared with those obtained by conventional solvent extraction. The extraction yield ranged from 21.5 to 23.5 g/kgDW, with the highest total carotenoid content (386 mg/kgDW) achieved at 450 bar and 60 °C (SFE9), corresponding to a 70.2% recovery relative to the solvent method. HPLC analysis identified capsanthin (5.88 mg/g) as the predominant carotenoid in this extract, followed by zeaxanthin (2.16 mg/g), lutein (0.71 mg/g), β-cryptoxanthin (0.70 mg/g), β-carotene (0.58 mg/g) and capsorubin (0.38 mg/g). Overall, the results demonstrate that the green extraction of Senise pepper waste offers a sustainable approach to obtaining valuable natural pigments.

Figures

Full text

1. Introduction

Capsicum annuum L. cv Senise, a sweet red pepper cultivated exclusively in the Basilicata region of southern Italy, exemplifies the country’s rich agrobiodiversity. Its distinctive organoleptic properties and strong territorial identity led to its designation as a Protected Geographical Indication (PGI) product under European Regulation (EC) No. 1263/1996, under the name “Peperoni di Senise” [1]. According to the PGI specifications, the production area includes twelve municipalities located between the Sinni and Agri valleys. Cultivation is restricted to three horn-shaped morphotypes—“pointed”, “truncated”, and “hooked”—with the pointed type being predominant. These peppers are grown at altitudes ranging from 250 to 340 m above sea level, in alluvial silty–sandy soils and medium-textured hilly terrains, under a typical Mediterranean climate. Harvesting typically begins in early August, when the fruits develop their characteristic purple-red coloration. A substantial portion of Senise pepper production is intended for drying, a process facilitated by the fruit’s low moisture content and thin pericarp. Traditional drying begins with the peppers being laid out on cloth sheets or nets in shaded, well-ventilated areas for 2–3 days.

The partially dried peppers are then strung into “serte” (necklaces) measuring 1.5–2 m in length and sun-dried for 3 to 7 weeks. Final dehydration is completed indoors in well-ventilated spaces. Once dried, Senise peppers may be fried to produce “cruschi” (crunchy peppers), which are enjoyed as snacks, side dishes, or condiments, or ground into a powder for use as a spice. The growing international demand for dried Senise peppers underscores the need for improvements in production efficiency, sustainability, and quality assurance [2].

Consequently, increased production may lead to greater amounts of waste, such as peppers that cannot be marketed due to imperfections or physiological disorders. However, these by-products could represent a valuable source of bioactive compounds [3]. Indeed, red pepper is among the most nutritionally valuable vegetables, owing to its rich phytochemical profile, which includes high concentrations of ascorbic acid, carotenoids, and phenolic compounds [4,5]. These bioactive constituents, particularly carotenoids, contribute significantly to human health, especially in the prevention of cardiovascular and neurodegenerative disorders [6,7].

Carotenoids are a diverse group of naturally occurring pigments responsible for the bright colors of many fruits and vegetables. In addition to their visual appeal, they function as potent antioxidants and free radical scavengers, contributing to photoprotection in plants and providing health benefits in humans [8]. Due to their health-promoting properties, carotenoids are widely employed as natural colorants in the food, cosmetic, and pharmaceutical industries [9]. Global interest in carotenoids continues to grow, with market projections estimating an increase from USD 1.5 billion in 2019 to USD 2.0 billion by 2026 [10]. Red pepper contains high levels of capsanthin, along with other bioactive pigments such as capsorubin, β-carotene, zeaxanthin, violaxanthin, β-cryptoxanthin, and lutein [11]. These compounds not only serve as vitamin A precursors but also contribute to cholesterol regulation, thereby reducing the risk of cardiovascular disease [6,12,13,14].

The extraction of carotenoids from natural sources has become a major focus of research, driven by consumer demand for clean-label ingredients and the growing recognition of their nutraceutical potential [15]. Natural carotenoids are generally considered safer and more effective than their synthetic counterparts, which may pose toxicological or carcinogenic risks [16].

Traditional methods for carotenoid extraction typically rely on organic solvents, whereas newer techniques, such as microwave-assisted and ultrasound-assisted extraction, have gained increasing attention. However, these approaches often present limitations, including long processing times, low selectivity, residual solvent contamination, and thermal degradation of sensitive compounds [17,18,19].

A promising alternative could be the use of supercritical fluid extraction (SFE), a green technology that offers enhanced selectivity, reduced environmental impact, and improved preservation of thermolabile compounds. This method allows the recovery of highly pure compounds while eliminating the use of toxic solvents, which can pose risks to both human health and the environment [20,21].

A supercritical fluid is defined as a substance operating above its critical temperature and pressure, where it exhibits hybrid properties between those of gases and liquids [22]. These fluids combine the density and solvating power of liquids with the high diffusivity and low viscosity of gases, making them exceptionally effective in penetrating solid matrices [23]. This results in high-purity extracts and eliminates the need for the lengthy solvent recovery procedures typically associated with conventional methods [24]. Among supercritical fluids, carbon dioxide (CO_2_) is particularly attractive due to its non-toxic, non-flammable, chemically stable, cost-effective, and recyclable nature. Its use aligns with sustainability goals, as CO_2_ capture and reuse in extraction processes may contribute to reducing greenhouse gas emissions [25].

CO_2_ reaches its supercritical state at 31.1 °C and 73.8 bar, making it ideal for extracting nonpolar and weakly polar compounds. To enhance its polarity and broaden its extraction capabilities, a co-solvent such as ethanol can be added. One of the key advantages of carbon dioxide supercritical extraction (SC-CO_2_) lies in its tunability: by adjusting temperature and pressure, it is possible to selectively extract specific thermolabile compounds [26]. To enhance the efficiency of supercritical fluid extraction (SFE), one approach is to increase the solubility of carotenoids in supercritical CO_2_ by raising the operating pressure [27]. For example, it has been reported that increasing the pressure from 300 bar to 500 bar at a constant temperature enhanced carotene extraction from palm oil production residues by 60% to 100% [28].

Several studies have demonstrated the effectiveness of SC-CO_2_ in extracting carotenoids from peppers [29,30,31]. Nonetheless, to the best of our knowledge, there are no data available regarding the optimization of SC-CO_2_ conditions for Senise red peppers, and limited information about the high carotenoid content of this regional variety has been reported [32,33].

The aim of this study was to evaluate the potential of SC-CO_2_ extraction for recovering carotenoids from Senise pepper production waste, which is unsuitable for commercial distribution, offering promising applications in the nutraceutical and pharmaceutical sectors, contributing to a circular and sustainable agri-food model. To this end, trials were conducted to recover carotenoids from peppers unsuitable for commercialization, using various operational parameters with a focus on maximizing extraction yields.

2. Results

2.1. Oleoresin Yield and Total Carotenoid Content After SC-CO2 Extraction

Figure 1 and Figure 2 illustrate the extraction yield and total carotenoid content obtained from dried Senise red peppers, processed under the operating conditions detailed in Table 1 using supercritical CO_2_.

Under the tested experimental conditions, the extraction yield in oleoresin terms ranged from 21.5 to 23.5 g/kg, with significant differences, and the highest yield was observed at 450 bar and 60 °C.

Regarding the carotenoid content, it ranged from 187 ± 19.51 to 386 ± 22.81 mg/kg of dry pepper biomass, with significant differences, and the highest concentration was observed at 450 bar and 60 °C. These findings underscore the importance of optimizing pressure and temperature conditions to maximize carotenoid recovery.

Figure 3 presents the carotenoid relative yields (%), calculated from comparison with the conventional solvent extraction method (550 ± 13.9 mg/kg). The 3D plot shows a clear trend with increasing pressure across all tested temperatures. In particular, at 250 bar, recovery values ranged from 28.96% to 34.09%, whereas at 350 bar they increased to 60.10–67.70%. The highest recovery was achieved at 450 bar, reaching a maximum of 70.24% at 60 °C. The effect of temperature appears to be pressure-dependent. At the lowest pressure (250 bar), increasing the temperature did not significantly affect carotenoid recovery (%), suggesting a possible reduction in solubility under these conditions.

In contrast, at higher pressures (350 and 450 bar), increasing the temperature moderately enhanced carotenoid recovery, likely due to improved solubility and mass transfer in the supercritical CO_2_ phase.

Overall, the optimal extraction conditions identified in this study were 450 bar and 60 °C, yielding a maximum carotenoid recovery of 70.24%.

2.2. Chromatographic Analysis of Carotenoid Profile by HPLC

HPLC-DAD analysis confirmed the effectiveness of the saponification procedure in hydrolyzing xanthophyll esters. The main peaks observed in the chromatogram (Figure 4) correspond, in order, to the following carotenoids: capsorubin, lutein, capsanthin, zeaxanthin, β-cryptoxanthin, and β-carotene. Table 1 reports the content of the main carotenoids (mg/kg_DW_) extracted from dried Senise peppers using supercritical CO_2_ extraction (SFE) under nine different operating conditions (SFE1–SFE9), compared with conventional organic solvent extraction (OS).

Carotenoid concentration increased progressively across the extraction conditions from SFE1 to SFE9. The capsanthin content ranged from 46.14 ± 3.71 to 138.19 ± 15.89 mg/kg_DW_, while zeaxanthin levels varied between 16.96 ± 2.78 and 50.65 ± 5.56 mg/kg_DW_. Lutein concentrations in mg/kg_DW_ increased from 7.02 ± 3.45 to 16.67 ± 3.63 and β-cryptoxanthin from 8.16 ± 1.74 to 16.56 ± 1.98. β-Carotene contents in mg/kg_DW_ ranged between 11.23 ± 1.85 and 13.55 ± 1.39, whereas capsorubin increased from 2.36 ± 0.19 to 8.89 ± 2.12. The conventional solvent extraction (OS) sample exhibited higher concentrations for all carotenoids compared to the SFE extracts, with values (mg/kg_DW_) as follows: capsorubin 12.70 ± 1.32, lutein 25.24 ± 1.56, capsanthin 191.70 ± 13.21, zeaxanthin 75.14 ± 6.23, β-cryptoxanthin 25.68 ± 2.36, and β-carotene 20.85 ± 2.51. In all samples analyzed, capsanthin was the predominant carotenoid, representing, on average, approximately 35% of the total carotenoid content.

3. Discussion

In line with a circular bioeconomy approach, red peppers (C. annuum) deemed unsuitable for commercial sale due to substandard quality were repurposed as a raw material for recovering bioactive compounds, with particular emphasis on carotenoids, via supercritical CO_2_ (SC-CO_2_) extraction.

From a thorough literature review, no information was found regarding the application of supercritical fluid extraction techniques for obtaining extracts from the Senise red pepper cultivar. Furthermore, there is limited data available on the qualitative and quantitative composition of carotenoids in extracts of this cultivar. In particular, one study characterized the phytochemical profile of a C. annuum ethanol extract, identifying β-carotene, capsorubin, antheraxanthin, and β-cryptoxanthin as the main carotenoids; however, no quantitative data were reported [33]. Meanwhile, another study reported only the total carotenoid content, distinguishing between the yellow and red fractions, without specifying individual compounds [34].

Therefore, we carried out an experimental design consisting of trials conducted at different pressures (250, 350, and 450 bar) and temperatures (40, 50, and 60 °C). Our results indicate that increasing the pressure significantly affects oleoresin yield and implies a marked increase in the total carotenoid concentration in the extracts. These findings are consistent with those reported by Tepić et al. [35], who observed that increasing pressure did not significantly enhance oleoresin yield, although it reduced the required extraction time. In particular, the authors reported oleoresin yields of 10.6%, 10.6%, and 10.3% after 14, 10, and 6 h of extraction at pressures of 200, 300, and 400 bar, respectively. Our oleoresin yields were lower, ranging from 2.15% to 2.35%, likely due to the shorter extraction times, yet remained consistent with the values reported by Uquiche and Millao [36]. However, this latter study reported extraction yields ranging from 18.5 to 30.3 g/kg, depending on pretreatment and extraction temperature, whereas in our experiments, increasing the temperature did not result in higher oleoresin yields.

In contrast, the effect of temperature on total carotenoid recovery appears to be pressure-dependent; however, its influence is less pronounced than that of pressure, suggesting that the low vapor pressure of carotenoids limits the contribution of temperature during their extraction with supercritical CO_2_ [27].

Better yields were observed at 350 and 450 bar when the temperature was increased from 40 to 60 °C. In contrast, no effect was observed at 250 bar. Most likely, at this lower pressure, the temperature increase significantly reduces the density of CO_2_, counteracting its positive effect on solute solubility [37]. Indeed, some authors report that increasing the extraction temperature from 50 to 80 °C at a pressure of 250 bar leads to a reduction in carotenoid extraction yields from pumpkin (Cucurbita maxima) [38].

Our results show that the total carotenoid content in the extracts ranged from 187 ± 19.51 to 386 ± 22.81 mg/kg DW, whereas the conventional extraction method yielded 550 ± 13.9 mg/kg_DW_. These values are consistent with literature data reported by other independent authors for different cultivars of C. annuum. For example, Kim et al. [39], who analyzed eight red cultivars of C. annuum, reported carotenoid contents ranging from 135.31 to 433.2 mg/kg_DW_. Jang et al. [40] reported that the red pepper cultivar “Long Sweet” had a total carotenoid content of 521 mg/kg_DW_, with capsanthin as the predominant carotenoid (185 mg/kg_DW_). Asrat et al. [41], characterizing the physicochemical properties of three Ethiopian C. annuum cultivars (Marekofana, Melkawaze, and Gebaba), observed that the total carotenoid content varied from 317.84 to 334.85 mg/kg DW, depending on both the cultivar and the growing location.

Regarding carotenoid profiles determined by chromatographic analysis, the elution order reflects the molecular structure diversity of the compounds analyzed. Indeed, as illustrated in Figure 5, where the red dots represent the oxygen atoms, increases in the molecular polarity influenced retention times under the chromatographic conditions employed. Specifically, capsorubin, lutein, capsanthin, and zeaxanthin eluted within the first 7.3 min, followed by β-cryptoxanthin at 12.8 min and β-carotene at 18.2 min.

Carotenoids with a higher number of hydroxyl groups exhibit shorter retention times on the Zorbax RX-C18 reversed-phase column due to their increased polarity. This behavior is consistent with the findings of Turcsi et al. [42], who demonstrated that on octadecyl (C18)-bonded silica phase columns, carotenoids elute in order of increasing polarity, with more polar compounds eluting earlier due to weaker hydrophobic interactions with the stationary phase. However, the identified carotenoids accounted for approximately 63% of the total carotenoid content, while the remaining fraction likely corresponds to unidentified or minor carotenoids that were not quantified in this analysis.

The carotenoid composition of all extracts showed that capsanthin consistently emerged as the predominant carotenoid, accounting for approximately 36% of the total carotenoid content. This observation is consistent with the qualitative profiles reported in the literature, where capsanthin is described as the main red xanthophyll of the red pepper C. annuum [43,44,45]. In a study reporting the qualitative and quantitative differences in the carotenoid composition of yellow and red peppers, it was found that in red peppers, the principal carotenoid was capsanthin (32.6 mg/kg_DW_), followed by lutein (7.5 mg/kg_DW_), β-carotene (5.8 mg/kg_DW_), and violaxanthin (2.7 mg/kg_DW_) [46].

Furthermore, the predominance of capsanthin after extraction aligns with the findings of Alamu et al. [47], who reported that drying and thermal treatments reduce capsorubin more than capsanthin, due to the latter’s greater thermal stability. These authors demonstrated that drying methods significantly influence the carotenoid profile. Specifically, capsorubin and capsanthin exhibited comparable levels in fresh peppers, whereas after drying, capsanthin predominated due to its greater thermal stability.

Some differences in the carotenoid profiles were observed among extracts obtained under different operating conditions. An increase in pressure and temperature enhanced capsanthin recovery, raising it from 46.14 ± 3.71 to 138.19 ± 15.89 mg/kg_DW_. Similar behavior was reported by Kostrzewa et al. [17], who identified 450 bar and 50 °C as the optimal conditions for carotenoid extraction. The importance of temperature, pressure, and their interaction on carotenoid yields was also emphasized by Fornereto Soldan et al. [48], while Uquiche et al. [49] showed that increasing pressure enhances carotenoid solubility in supercritical CO_2_.

Considering that most data reported in the literature express carotenoid concentrations as a function of the weight of the extracted fraction, our results have been recalculated to allow a direct comparison, expressing carotenoid values per gram of extract. Therefore, in the SFE9 extracts, the total carotenoid concentration was 16.43 mg/g, while the concentrations of the identified carotenoids were 0.38, 0.71, 5.88, 2.16, 0.70, and 0.58 mg/g for capsorubin, lutein, capsanthin, zeaxanthin, β-cryptoxanthin, and β-carotene, respectively. Tepić et al. [35], after 6 h of extraction at 400 bar and 40 °C, obtained a total capsanthin content of 9.38 mg/g, considering free, monoester, and diester forms, while our capsanthin concentration of 5.88 mg/g was achieved after only 1 h of extraction. Furthermore, compared to Kostrzewa et al. [50], our capsanthin level is lower than their maximum (SFE5: 18.61 mg/g, 66%), but still within the reported range (3.93–8.61 mg/g). Notably, the SFE9 extract showed a relatively higher zeaxanthin content (2.16 mg/g) and a lower β-carotene content (0.58 mg/g). However, the capsanthin level was much lower than that reported by Jiménez et al. [51], who found a capsanthin content of 109 mg/g in SFE extracts obtained from oven-dried red pepper powder at 60 °C and 250 bar.

Overall, the optimal extraction conditions identified in this study were 450 bar and 60 °C, under which maximum recoveries of 2.35% and 70.24% were achieved for oleoresin yield and carotenoid recovery, respectively. These findings are in line with those of Kostrzewa et al. [52], who, using response surface methodology, reported that supercritical CO_2_ extraction of sweet paprika (C. annuum) under optimal conditions (50 °C, 450 bar, and 56 min) resulted in an 8.5% extract yield with 84% carotenoid recovery.

The slightly lower oleoresin yield observed in our study may be attributed to the exclusion of seeds from the samples, which are known to be rich in oils [53]. Therefore, the results of the present study underscore the critical influence of extraction parameters, such as pressure and temperature, on carotenoid yield, and support the assertion that the relative concentration of carotenoids in red peppers depends on multiple factors.

A high variability in total carotenoid levels and profiles has been reported as a function of pepper genotype, ripening stage, harvest time, and cultivation season [54,55,56].

Indeed, Tang et al. [57] reported that during fruit ripening of high pigment pepper varieties, the total carotenoid content increased 168-fold, with a notable accumulation of zeaxanthin, β-cryptoxanthin, and capsanthin.

In conclusion, although conventional solvent extraction is generally conducted under more economically favorable conditions and can sometimes provide slightly higher yields, SFE technology offers a major advantage by producing solvent-free extracts [21]. Moreover, SFE extracts exhibited greater oxidative stability compared to those obtained with conventional solvents [58]. In addition, traditional solvent extraction techniques often require prolonged extraction times, large amounts of solvent, and multiple processing steps [59]. This makes SC-CO_2_ particularly attractive for applications where purity, safety, and the absence of residual solvents are critical, such as food, nutraceuticals, and cosmetics [60,61].

4. Materials and Methods

4.1. Bench-Scale System Description

Feasibility studies of the extraction process were carried out using the bench-scale supercritical CO_2_ extraction system shown in Figure 6. The system is equipped with a high-performance heating furnace capable of reaching temperatures up to 250 °C and a pneumatic pump that compresses CO_2_ to pressures of up to 680 bar. It supports both static and dynamic extraction modes, with temperature precisely controlled via a thermocouple-regulated system.

Pressure monitoring is ensured by two Wika transmitter units (Klingenberg, Germany), enabling precise control both upstream and downstream of the extraction vessel. CO_2_ flow is regulated via a micrometric valve, with a maximum flow rate of 10 L/min and monitored in real time using a flowmeter (LPN/S80 AL G 2.5, Sacofgas, Milan, Italy).

Following extraction, the extract and CO_2_ are directed through a capillary tube and collected into a vial for further processing. All operational parameters are managed using the EasyCom2011 software (version 2.0.5.16, WIKA Alexander Wiegand SE & Co. KG, Miltenberg, Germany), ensuring precise and seamless control throughout the extraction process.

4.2. Drying Process of Senise Red Pepper

Fresh Senise peppers exhibiting visual defects, either of physiological origin or resulting from microbial or insect damage, were selected. These peppers, deemed unsuitable for commercial distribution, were harvested during the 2024 season from agricultural fields in the Senise area, deseeded, and subsequently dried in a forced-air cabinet at 40 °C (Heraeus Instruments, mod. UT 6420, Hanau, Germany). Once the losses in weight were constant, dried peppers were finely ground using a laboratory blender (Waring Commercial, mod. 38BL45, Torrington, CT, USA). The resulting powder, with a moisture of 7.2% ± 0.29, determined by a thermogravimetric balance (Gibertini, mod. Eurotherm, Novate Milanese, Italy), was then stored under vacuum in the dark to preserve its chemical integrity until the extraction experiments were conducted.

4.3. Carotenoids Extraction by Traditional Method

Samples of approximately 0.2 g of the ground material were suspended in 5 mL of a solvent mixture composed of hexane, acetone, and ethyl acetate in a 2:1:1 (v/v) ratio. The suspension was vortexed for 5 min to ensure complete homogenization and then separated using a refrigerated centrifuge (Beckman Coulter Allegra™ 2IR, Brea, CA, USA) for 10 min at 8900 g and 5 °C.

The resulting supernatant was carefully collected, while the solid residue underwent two additional extraction cycles under identical conditions to maximize carotenoid recovery. The combined extracts were concentrated using a vacuum rotary evaporator (Steroglass Kentron—Strike 202, Perugia, Italy) at 35 °C. The concentrated extracts were then stored at −20 °C until analysis for total carotenoid content and determination of their profile by high-performance liquid chromatography (HPLC).

4.4. Evaluation of SC-CO2 Technique for Carotenoid Recovery from Red Pepper

SC-CO_2_ extraction of carotenoids from red pepper Senise was evaluated through trials conducted at different temperatures (40, 50, 60 °C) and pressures (250, 350, 450 bar). For this purpose, a full 3 × 3 factorial design was employed to systematically investigate the influence of the selected process variables. Each temperature level was combined with each pressure level, resulting in a total of nine experimental runs (Table 2).

For each extraction trial, approximately 5 g of powder was placed into a 50 mL stainless steel extraction vessel.

To promote uniform sample distribution and enhance mass transfer, the vessel was pre-filled to 60% of its volume with 3 mm glass beads. Additionally, a metal filter with a 5 µm pore size was used to ensure a clean extraction process and prevent sample loss.

The extraction vessel was securely positioned within the apparatus and subsequently filled with carbon dioxide. Once the predefined operating conditions were reached, the dynamic extraction process was initiated and maintained for 60 min, with a constant CO_2_ flow rate of 5 L/min measured at room temperature and atmospheric pressure.

The recovered product was collected into a glass vial, which was placed in a thermostatic bath maintained at 25 °C. Upon completion of the extraction process, the extract was accurately weighed using an analytical balance (Kern, model 870, Vicenza, Italy).

The extraction yield was calculated and expressed as mg/kg_DW_, based on the ratio between the weight of the extract (oleoresin) and the dried weight (DW) of the powdered sample. The vials containing the extracts were subsequently stored at −20 °C to preserve their integrity until further analysis. These preserved extracts were later used for the quantification of total carotenoids and the determination of their main constituents.

4.5. Determination of Total Carotenoids by Spectrophotometric Analysis

For the quantification of total carotenoid (TC) content, all extracts were resuspended in 5 mL of ethanol containing 0.01% BHT (butylated hydroxytoluene) as a stabilizing agent to prevent oxidation. The solutions were analyzed spectrophotometrically at 448 nm using a Thermo Scientific Multiskan GO (Waltham, USA), following the protocol established by Davies [62].

The concentration was calculated using the following formula:

$$[eqn]$$

where A is the absorbance value; V is the total volume of the sample (mL); l is the path length (cm); and $[eqn]$ is the extinction coefficient of β-carotene in ethanol (2620 mL/mg cm).

The carotenoid yield (CY) was expressed in terms of mg/kg and was given following the formula:

$$[eqn]$$

where TC is the total carotenoids (mg) in the extract and PB_DW_ is the dried weight (kg) of pepper biomass.

Furthermore, the effect of operating parameters in SFE-CO_2_ extraction was evaluated by comparing the carotenoid yields with those obtained using an organic solvent. The results were expressed as a percentage of relative yields (RY), calculated using the following formula:

$$[eqn]$$

where CY_SFE_ and CY_CS_ are the total carotenoid yields obtained using the CO_2_-SFE process and conventional solvent, respectively.

These data were plotted on a response surface graph using the free trial version software MODDE^®^ 13.1 (Sartorius, Göttingen, Germany) to evaluate the interaction between temperature and pressure and to determine the ideal value of each influencing factor for the maximum extraction efficiency of carotenoids.

4.6. Carotenoid Profile by HPLC Analysis

The main carotenoids present in the red pepper extracts were determined by HPLC analysis on samples that underwent a saponification procedure to hydrolyze esterified carotenoids, thus ensuring a more accurate identification by a partially modified method indicated by Hong et al. [63].

For this procedure, 1 mL of the extract suspension was combined with 0.2 mL of NaCl solution (10 g/L) and 0.3 mL of KOH solution (600 g/L) in a 15 mL Falcon tube. The mixture was vortexed thoroughly and then incubated in a water bath at 75 °C for 45 min.

Subsequently, 1 mL of each sample was filtered through a 0.22 µm syringe filter and transferred into HPLC vials. Following sample preparation, 20 µL aliquots were injected into an Agilent 1200 Series HPLC system.

The HPLC system (Agilent Technologies, Santa Clara, CA, USA) was configured with the following components: a degasser (G1379B), a binary pump (G1312B), an autosampler (G1367B), a column compartment (G1316A), a UV-Vis detector (G1314B), and a diode array detector (DAD, G1315A).

Chromatographic separation was performed on a reversed-phase C18 analytical column (Zorbax RX-C18, 4.6 × 250 mm, 5 μm, Santa Clara, CA, USA) using acetone (solvent A) and water (solvent B) as mobile phases. The system was operated at a flow rate of 1 mL/min, with the column temperature maintained at 25 °C to ensure optimal resolution and reproducibility.

The gradient elution program was as follows: an initial composition of 75% A; from 0 to 10 min, the proportion of A was increased from 75% to 95%; from 10 to 17 min, held at 95% A; from 17 to 20 min, increased from 95% to 100% A; from 20 to 30 min, maintained at 100% A; and finally, from 30 to 35 min, a linear gradient returned to the initial conditions of 75% A. This gradient profile was designed to achieve efficient separation of carotenoid compounds with varying polarities.

Absorbance detection was performed at the characteristic wavelength of β-carotene (453 nm), while spectra were recorded over the range of 350–650 nm.

The major peaks were identified by comparing the recorded spectra with the literature data [64]. Specifically, the wavelengths corresponding to the maximum absorbance (λmax) and the spectral fine structure were analyzed for the UV-Vis spectrum of each peak. To assess the spectral fine structure for each main peak, the percentage ratio (%III/II) between the relative intensity of the third absorption peak compared to the second was calculated, as reported by Lu et al. [65]. The height of the signal was normalized against the baseline, defined as the valley between the two peaks, i.e., (λ_III_ − λ_baseline_)/(λ_II_ − λ_baseline_).

For a better explanation, Figure 7 provides a carotenoid spectrum along with the relevant data for calculating the %III/II ratio.

Furthermore, the amounts of individual carotenoids were expressed as β-carotene equivalents. For this purpose, synthetic β-carotene (Sigma-Aldrich, PHR1239-1G, Burlington, VT, USA) was first dissolved in hexane and then diluted in ethanol containing 0.01% BHT to prepare stock standard solutions. A calibration curve was constructed with concentrations of 0.024, 0.012, 0.006, 0.0012, and 0.0006 µg/mL, enabling accurate quantification of carotenoids in the extracts.

Data acquisition and analysis were performed using OpenLAB CDS ChemStation Edition Rev. C.01.10(201).

4.7. Statistical Analysis

All the extractions were carried out in triplicate. All data are expressed as the mean ± standard deviation. Two-way ANOVA and Tukey’s honestly significant difference (HSD) test were used to consider the difference between the amount of oleoresin and carotenoids extracted from Senise red pepper under different conditions. Differences with a p-value < 0.05 were considered significant. Analyses were carried out using the software R version 4.5.1.

5. Conclusions

Supercritical CO_2_ extraction proved to be an efficient and environmentally sustainable method for obtaining carotenoid-rich extracts from Senise red pepper wastes. Under optimal conditions (450 bar and 60 °C), the highest carotenoid recovery was achieved, with capsanthin identified as the predominant compound. These findings underscore the potential of this technology to valorise agri-food by-products, contributing to a circular and low-impact production model. Overall, the results provide valuable insights for researchers and industry stakeholders within the Senise pepper supply chain, highlighting the feasibility of using waste materials as a renewable source of natural colorants and nutraceutical ingredients.