Introduction

In 2022, Indonesia, the Philippines, and Costa Rica were the world’s top three pineapple producers, with a worldwide production of around 29.4 million metric tons (Global Pineapple Production by Leading Countries 2022 | Statista, 2024). Pineapple (Ananas comosus (L.) Merrill) mainly contains carbohydrates and water, followed by organic acids, vitamins, and minerals (García et al., 2021; Mohd Ali et al., 2020). However, the non-edible pineapple fraction can be as high as 60%, and hence, the pineapple processing industry generates a substantial amount of waste, primarily in the form of pineapple peel (30–42%), followed by the core waste (~ 10%) representing a significant environmental challenge. Unfortunately, these wastes are typically discarded or underutilized for animal feed, disposed of waste in landfills, or burned for energy production (Campos et al., 2020; Roda & Lambri, 2019). Most scientific studies related to the revaluation of pineapple waste have focused mainly on the reuse of proteolytic enzymes such as bromelain (Manzoor et al., 2016), the extraction of pectin from the peel (Selvanathan & Masngut, 2023), and the use of the juice to produce vinegar or bioethanol (Chalchisa & Dereje, 2021). Against the backdrop of escalating concerns regarding food waste and sustainable resource management, there is a growing imperative to explore innovative approaches for the valorization of pineapple waste. The pineapple core represents an attractive substrate for the extraction of bioactive compounds due to its high content of phenolic compounds and carotenoids. The main phenolic compounds identified are gallic acid, catechin, epicatechin, and ferulic acid, in respective amounts of 32, 60, 50, and 20 mg per 100 g of dry extracts (Hamzah et al., 2021). In the study carried out by Sepúlveda et al. (2018), the extraction of pineapple by-products using the autohydrolysis process yielded a product with a high antioxidant power. The potential of pineapple residues as an antioxidant has also been evaluated by Rashad et al. (2015), in which methanolic extracts from fermented pineapple by-products contained high levels of phenolic species, i.e., around to 20 mg of gallic acid/100 g dry by-product.

Regarding the presence of carotenoids in pineapple by-products, Freitas et al. (2015), showed that β-carotene was the major carotenoid found in the core and peel of pineapple, with concentrations of 960 and 2500 µg/100 g dry weight, respectively. Carotenoid lutein (300–1120 µg/100 g dry sample) was also found to be predominant in pineapple peel, with lower contents of α-carotene (89–126 µg/100 g dry sample) (de Moraes Crizel et al., 2016; Freitas et al., 2015). Although the carotenoid profile varies depending on the cultivar, ripening stage, and environmental conditions, pineapple by-products are a rich source of carotenoids, with potential applications as natural pigments with antioxidant properties and pro-vitamin A activity (Meléndez-Martínez et al., 2022). To improve the extraction process, ultrasound-assisted extraction (UAE) is an established technique for the extraction of bioactive compounds from plant materials, offering advantages such as reduced extraction time, lower solvent consumption, and enhanced extraction efficiency. Moreover, UAE can facilitate the extraction of thermolabile compounds without compromising their stability, making it particularly suitable for heat-sensitive bioactive compounds like carotenoids and phenolic compounds (Kumar et al., 2021). Finally, ultrasounds propagate through solids, thus enhancing the extraction efficiency of bioactive compounds from solid substrates.

The literature regarding the application of UAE to the extraction of bioactive compounds from pineapple core residues is scarce. In this sense, Rathnakumar et al. (2017) optimized an ultrasonic extraction method to obtain bioactive compounds from pineapple rind and core by using a Box–Behnken design (BBD). The optimal conditions that allowed them to achieve the highest values of antioxidant capacity were 100% sonotrode power, 10 min sonication time, and ethanol/H2O (28/ 72 v/v) extracting solution. To promote the simultaneous recovery of multiple bioactive compounds from agro-industrial by-products, a cascade approach has been suggested. This concept introduces a sequential extraction method that allows the recovery of multiple bioactive compounds within a single process, also aligning with the concept of zero waste. In this sense, Kehili et al. (2016) effectively implemented a cascade methodology to extract carotenoids, proteins, hemicellulose, and cellulose from tomato pomace. Nevertheless, there is a noticeable absence of research in the literature regarding the ultrasound-assisted cascade extraction of phenolic compounds and carotenoids from pineapple core samples. Moreover, carotenoids, for instance, are liposoluble compounds that have traditionally been extracted using toxic and/or volatile organic solvents such as acetone, hexane, and ethyl acetate (Da Silva et al., 2014). Nowadays, the use of biodegradable surfactants, such as sodium dodecyl sulfate (SDS), has emerged as an alternative tool to extract carotenoids in a more environmentally sustainable way, which can be optimized to obtain the maximum yield (Ferrando et al., 2023). To the best of our knowledge, no studies have been found on the extraction of carotenoids from pineapple or pineapple by-products using UAE.

The aim of the present work was to introduce a novel cascade approach, utilizing UAE, for the valorization of pineapple core wastes through the recovery of phenolic compounds and carotenoids. Notably, this is the first time such an approach has been proposed. Initially, phenolic compound extraction was conducted, followed by a subsequent carotenoid extraction. Using BBD optimization, the ideal operating conditions for maximizing the extraction yield of bioactive compounds from pineapple cores were identified. A multi-response surface (MRS) optimization approach was applied to achieve this. Extraction yields were assessed for the antioxidant fraction by using total polyphenol content (TPC) and antioxidant activity evaluated with 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and ferric-reducing antioxidant power (FRAP) assays. The carotenoids obtained underwent characterization by using high-performance liquid chromatography with a diode array detector (HPLC–DAD) analysis.

Materials and Methods

Reagents and Chemicals

Trolox®, potassium persulfate, ethanol, ABTS, TPTZ, hydrochloric acid, gallic acid, Folin-Cicalteu reagent, sodium carbonate, Kjeldahl tablets, methyl orange indicator, mixed indicator (iron(II) sulfate and phenolphthalein), sodium acetate, anhydrous ferric chloride, sodium hydroxide, toluene, sulfuric ácid, glacial acetic acid, sodium chlorite, ferulic acid, epicatechin, succinic acid, p-coumaric acid, and syringic acid were obtained from Sigma-Aldrich (Germany). The carotenoid standards α-carotene and β-carotene were purchased from LGC Standard (Barcelona, Spain). SDS was obtained from Sigma-Aldrich (Steinhim, Germany). Methanol (MeOH), hexane, tert-butyl methyl ether (TBME), THF, and ACN of HPLC grade, and acetone were purchased from Honeywell (Seelze, Germany). HNO3 was provided by Labbox Labware S.L. (Vilassar de Dalt, Barcelona, Spain) and H2O2 was bought from ScharLab S.L. (Sentmenat, Barcelona, Spain). A multielemental stock solution (SCP33MS) purchased from SCP SCIENCE (Clark Graha, Baie D’Urfé, Canada) was used. SCP33MS contained the following analytes: As, Ba, Bi, Cd, Co, Cr, Cu, Fe, La, Li, Mn, Mo, Ni, Pb, Rb, Sn, Sr, Ti, V, and Zn.

Pineapple Samples and Their Preliminary Assessment

Raw pineapple (A. comosus (L.) Merrill) frozen cores were acquired as by-products from a fruit processing company in Valencia, Spain. Prior to their analysis, samples were dried at 40 °C for 48 h by using a Cosori stainless steel food dehydrator to reduce the water content. The following physicochemical properties of the dry pineapple core (DPC) samples were determined: moisture and ash, hot water solubility, soluble compounds in hot alkali solution, solvent extractives, and acid-insoluble lignin according to the Technical Association of Pulp and Paper Industry (TAPPI) standard methodologies (Technical Association of the Pulp & Paper Industry, 2023). The content in holocellulose was determined according to the methodology described by Wise et al. (1946) and the determination of α-cellulose and hemicellulose contents following the approach described by Rowell (2007). Furthermore, the protein, lipid, and carbohydrate composition of pineapple core was assessed following the Official Methods of Analysis outlined by AOAC INTERNATIONAL (Association of Official Analytical Chemists, 2000).

To determine the initial mineral composition, seven samples of the DPC were digested using a microwave digestion system Start D (Milestone, Sorisole, Italy). Approximately 0.5 g (with a precision of 0.1 mg) of the sample was transferred to a digestion vessel and then 7 mL of 65% HNO3 and 1 mL of 30% H2O2 were added. Samples were digested at 200 °C for 30 min, and final digests were transferred to plastic vessels and diluted to 20 g with Milli-Q water. The digested samples were analyzed by means of an ICP-MS Agilent 7700 (Santa Clara, California) using external calibration and on-line addition of internal standards for quantification. A micromist nebulizer (Glass Expansion, Weilburg, Germany) and a thermostatted double-pass spray chamber (Glass Expansion) were used to introduce the samples into the plasma. Table S1 summarizes the instrumental operating conditions.

Optimization of the Sequential Extraction Procedure Based on Ultrasound-Assisted Extraction (UAE)

A sequential extraction method employing UAE was implemented to extract phenolic compounds and carotenoids (see Fig. 1). The extraction was conducted by using a CY-500 ultrasound homogenizer (JP Selecta, Barcelona, Spain) equipped with an ultrasonic transducer (500 W) consisting of a piezoelectric converter with a 1/4 inch titanium alloy probe (5.6 mm diameter and 60 mm height). For the extraction of phenolic compounds and carotenoids, the solid waste mass and extraction solvent volume were set at 1.5 g and 13 mL, respectively. This extraction ratio (1.5 g/13 mL) was chosen based on previous tests to ensure the maximum sample quantity that could be used without causing solid aggregates to form in the flask during the extraction process. This is crucial because, during ultrasound extraction, maximizing the contact surface area between phases is beneficial. This extraction ratio represents the maximum amount that can be properly dispersed in 13 mL of solvent. This volume was chosen based on the capacity of the extraction vial, ensuring that the ultrasound probe is submerged in the solvent without coming into direct contact with the sample. This setup prevents sample degradation while still enhancing the extraction process. The ultrasonic probe was immersed 3 cm into the solution contained in a 50-mL beaker with the sample and extracting solution.

Fig. 1
figure 1

Sequential extraction scheme of phenolic compounds and carotenoids using ultrasound-assisted methodology. A, amplitude; B, cycles; C, extraction time

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The extraction of phenolic compounds was carried out by using a mixture of EtOH/water (50 wt.%) as a solvent, with the optimization of the amplitude (A, 20–70%), cycles (B, 2–7), and extraction time (C, 5–15 min) conducted through a BBD. In relation to the percentage of ethanol and water, studies have shown that using hydroalcoholic solvents, rather than just water or alcohol alone, is an effective method for extracting most polyphenols from plant matrices. The ethanol concentration plays a crucial role in optimizing the extraction yield of polyphenols. Based on both literature and the authors’ expertise, excessively high or low ethanol concentrations in the solvent mixture can significantly reduce the amount of bioactive compounds in the extract (Zhang et al., 2011). Most studies indicate that a 50% ethanol concentration in the solvent is optimal for extracting phenolic compounds from plant materials. (Carpentieri et al., 2021; Hosseini et al., 2018; Jovanović et al., 2017). The proposed BBD consisted of 17 experiments carried out in randomized order, including five central points. To ensure the preservation of the target compounds, the temperature was monitored at the end of all experimental conditions to verify that it remained below 45 °C. The obtained extracts were centrifuged for 15 min at 4500 rpm using a Digicen 21R centrifuge (MEDIFRIGER-BLT 230 V 50HZ, Selecta, Ortoalresa, Madrid, Spain), and the recovered solid was used in the following extraction process to obtain the carotenoids. In the liquid phase, the response derived from the BBD was assessed through antioxidant activity measurements using FRAP and ABTS methods, along with the values obtained for TPC.

The green extraction of carotenoids from pineapple byproducts was optimized by using the BBD employing different conditions of extraction time (1, 3, and 5 min), cycles (2, 5, and 7), and wave amplitude (20, 45, and 70%) of the ultrasonic probe. To do that, the residue from the phenolic compounds extraction (RE) was subjected to another extraction process by using sodium dodecyl sulfate (SDS) according to the method described by (Ferrando et al., 2023) with some modifications. Briefly, samples (1.5 g) were mixed with 13 mL of 0.3% SDS, and the pH was adjusted to ~ 7. Then, the mixture was sonicated and shaken for 1 h to promote the solubilization of the chromoplasts in SDS. After that, the vegetal material was removed by centrifugation at 3000 rpm for 15 min. Finally, the supernatant containing the chromoplasts was subjected to ultracentrifugation (14,000 rpm, 45 min), and the precipitate was recovered as the carotenoid-rich fraction. The evaluation of the BBD’s response was conducted determining the levels of β-carotene. To compare the efficiency of two different solvents used under optimal extraction conditions, β-carotene extraction was also performed using hexane as the solvent for both DPC and RE samples. Furthermore, β-carotene extraction using sodium dodecyl sulfate (SDS) was conducted on the DPC sample, and the results were compared with those obtained from the RE sample. This analysis aimed to assess the impact of prior phenolic compound extraction on the final yield of β-carotene when SDS is used as the extraction solvent.

For carotenoids quantification, the carotenoid-rich fractions isolated in the seventeen trials were mixed with 1 mL of MeOH/TBME (1:1, v:v) solution, and then, samples were evaporated in the speed vacuum concentrator (Concentrator Plus, Eppendorf, Germany) and dissolved in 400 µL of MeOH/TBME (1:1, v:v), sonicated for a few seconds, filtered by 0.22 µm, Ø 13 mm, PTFE (Agilent Technologies, Waldbronn, Germany), and injected into the HPLC–DAD system.

The experimental conditions (amplitude, cycles, and extraction time) for sequential extraction of phenolic compounds and carotenoids using ultrasound-assisted methodology are shown in Table S2.

In both BBDs, the adequacy of the fitted models was evaluated using the lack of fit value, and the coefficient of determination (R2) obtained from the analysis of variance. Model parameter significance was determined at α = 0.05. Further confirmation experiments (in triplicate) were carried out under optimal conditions to validate the models.

Analysis of Total Polyphenol Content (TPC) and Antioxidant Activity Assays

All the extractions were performed in triplicate. In the ethanolic extract, the TPC and the antioxidant capacity were assessed. TPC and antioxidant capacity were also determined on the residue resulting from the extraction of the phenolic compounds fraction (RE). In the RE case, the extraction of antioxidants was carried out with a mixture of EtOH/water (50 wt.%) according to a previously published methodology (García et al., 2021). In total, 1.0 ± 0.1 g of the RE was weighed in a polyethylene test tube, and 4 mL of the extraction mixture was added. The mixture was vortexed for 1 min and then left to stand for 16 h in the fridge. Afterward, the tubes were vortexed again for 1 min and then centrifuged at 5000 rpm for 10 min. The supernatant was collected and passed to a new tube with a Pasteur pipette. The extraction process was repeated twice but without leaving the sample in contact with the extractant overnight. The combined extracts were stored in the freezer at − 18 °C until analysis. The Trolox equivalent antioxidant capacity (TEAC) was determined using the ABTS+ discoloration assay, as described previously, with slight modifications (Sanahuja et al., 2019). For the TEAC assay, 200 µL of the extracts were mixed with 3 mL of diluted ABTS solution in a polystyrene disposable cuvette, which was then homogenized in a vortex for approximately 5 s. The reaction mixture was then incubated at 25 ± 2 °C for 30 min, followed by measurement of absorbance at 734 nm.

The FRAP assay employed was based on the methodology developed by (Benzie & Strain, 1996), with slight modifications optimized in previous studies (Valdés et al., 2015). Here, 200 µL of the extract was mixed with 3 mL of FRAP reagent and incubated for 30 min at room temperature in darkness. Measurements were taken using a biomate-3 UV–VIS spectrophotometer (Thermo Spectronic, USA) at 593 nm. The results of FRAP and ABTS were expressed as mg Trolox equivalents/100 g sample. The TPC assay was conducted following the methodology outlined in prior studies with certain modifications (Valdés et al., 2015). Specifically, 200 µL of the ethanolic extract was combined with 500 µL of distilled water and 500 µL of a 7.5% sodium carbonate solution and left for 5 min. Then, 100 µL of Folin and Ciocalteu reagent (2 N) were added to the solutions, and it was allowed to incubate at room temperature for 30 min in darkness. The absorbance of mixtures was then measured at 760 nm. Results were expressed as mg of gallic acid equivalents (GA)/100 g sample.

Quantitative Analysis of Key Phenolic Acids via High-Performance Liquid Chromatography Coupled with Mass Spectrometry (HPLC–MS) Methodology

The identification and quantification of individual key phenolic acids in the ethanolic extract obtained from pineapple core samples were conducted using an Agilent 1290 Infinity UHPLC (ultra-high-performance liquid chromatography) system coupled with an Agilent 6490 triple quadrupole mass spectrometer (Agilent Technologies, Waldbronn, Germany). The system was equipped with an Agilent Jet Stream ion source operating in negative ionization (NI) mode, except for epicatechin, which required positive ionization (PI) mode. Separation of analytes was achieved using an Agilent Poroshell 120 EC-C18 column (3 × 100 mm, 2.7 µm particle size) maintained at 25 °C. Optimal separation conditions were established with a mobile phase consisting of solvent A (0.1% formic acid in water) and solvent B (0.1% formic acid in acetonitrile), employing a gradient elution program as follows: initial conditions 50% A (5 min), 80% A (11.5 min), 10% A (7 min), with a constant flow rate of 0.4 mL min−1. A 1 µL injection volume was used for all samples.

The analysis was conducted in multiple reaction monitoring (MRM) mode to track transitions from precursor ions to dominant product ions. Source parameters were optimized as follows: gas curtain temperature 275 °C, gas flow 11 L min−1, cell acceleration voltage 4 V, nebulizer pressure 40 psi, capillary voltage 3500 V in the positive mode and 2800 V in the negative one, fragmentor voltage 380 V, with a resolution of 0.7 for both the first and second quadrupole, and a DPCell time of 10–40 ms depending on the compound to be determined.

Specific transitions were utilized to identify each compound, with optimized collision energy applied to maximize intensity. The MassHunter Workstation (version B.07.01) facilitated data acquisition, while MassHunter Qualitative Analysis (version B.07.00) and Quantitative Analysis Software (version B.07.00) were employed for data processing. Abundant MRM transitions were selected as quantifiers, with other transitions serving as qualifier ions. The calibration curves were performed in the concentration range of 50 to 0.0055 mg kg−1.

Thermogravimetric Analysis (TGA) of Samples

The lyophilized extract rich in phenolic compounds underwent TGA analyses. These analyses were further performed on the residual material obtained post-extraction of the phenolic compounds fraction (RE). TGA analysis of samples was conducted using a TGA/SDTA 851 Mettler Toledo thermal analyzer (Schwarzenbach, Switzerland). Four milligrams of the samples were heated from 30 to 700 °C at a rate of 10 °C min−1 under a nitrogen atmosphere (50 mL min−1). The initial degradation temperature (Tini), calculated at 1% weight loss, and the temperature of maximum decomposition rate (Tmax) were determined. The analyses were performed in triplicate.

Quantification of Individual Carotenoids by High-Performance Liquid Chromatography with Diode Array Detection (HPLC–DAD)

Carotenoids were analyzed in an Agilent 1200 high-performance liquid chromatography with a diode array detector (HPLC–DAD) system, fitted with a quaternary pump, a degasser, a thermostatted column support, and an autosampler (Agilent Technologies, Waldbronn, Germany), according to Böhm (2001) with some modifications. Briefly, the chromatography separation was carried out using a C30 column 250 mm × 4.6 mm, 5 µm i.d. (Análisis Vínicos S.L., Villarobledo, Spain), at 17 °C, using as mobile phases tert-butyl methyl ether (A) and methanol (B) at a flow of 1 mL min−1. The gradient started with 2% A to reach 35% A at 35 min, 50% A at 45 min, 60% A at 55 min, and returning to the initial conditions for 5 min before the next injection.

Carotenoids were identified according to their UV spectra and retention times by chromatographic comparisons with standards. The β-carotene and total carotenoids were quantified at 450 nm. The results were expressed as µg carotenoids/100 g RE.

Statistical Analysis

In relation to the results obtained from the BBDs, the analysis of the fitted models was conducted by using the StatGraphics Centurion XV software (Statistical Graphics Corporation, Rockville, MD, USA). For ANOVA and Tukey’s test at a significance level of p ≤ 0.05, commercial software SPSS version 15.0 (Chicago, IL, USA) was employed to determine differences between values.

Results and Discussion

Chemical Characterization of Pineapple Core Samples

Dry pineapple core (DPC) samples contained 1.20 ± 0.05; 5.3 ± 0.4 and 88.6 ± 0.5% (w/w) of proteins, lipids, and carbohydrates, respectively. This nutritional composition demonstrated the DPC suitability for applications in the food and nutrition sectors, among others (Sengar et al., 2022). Santos et al. (2021) previously reported DPC protein and carbohydrate contents of 2.7 ± 0.2% and 87 ± 3%, respectively. These values were in accordance with the results presented in the present work.

The moisture content was remarkably low (c.a., 2.74 ± 0.03%), confirming that the studied core samples had undergone a significant drying process as mentioned above since, according to the literature, the moisture content of the raw pineapple core reaches values close to 85.2 ± 1.0% (Sengar et al., 2022). This level of dehydration significantly reduced microbial activity and extended the shelf life of the product.

Additionally, the extractives comprised well-known antioxidant species (Association of Official Analytical Chemists, 2000) such as phenolic compounds and flavonoids, thus suggesting that pineapple core could be a valuable source of natural antioxidants, which are in demand in the food and cosmetic industries because of their health benefits and preservative properties.

The structural components of the pineapple core detailed in Table 1 are crucial for applications such as biofuel production, paper manufacturing, and even dietary fiber supplements (Sukruansuwan & Napathorn, 2018). The pineapple core showed a high solubility in hot water, Table 1, which may be attributed to the presence of hydrophilic compounds such as natural sugars and acids, being particularly relevant for applications requiring the incorporation of natural flavors and nutrients derived from pineapple into hot-processed foods and beverages. The determined DPC ash content was 2.1 ± 0.6%. This value may vary due to factors such as genotypes, soil nutrient levels, harvest timing, and climatic conditions (Lu et al., 2014). On this subject, pineapple could be considered a significant source of essential minerals, including elements such as K, Ca, and Mg (Ancos et al., 2016; BEDCA Database, 2024; USDA Database, 2024; Lu et al., 2014; Mohd Ali et al., 2020; Salomé et al., 2011; Sengar et al., 2022). Table 2 summarizes the multi-elemental concentration of DPC.

Table 1 Initial characterization of DPC samples [mean value (n = 3) ± \(\frac{t\cdot s}{\sqrt{n}}\)]; 95%
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Table 2 Initial mineral composition of DPC samples [mean value (n = 3) ± \(\frac{t\cdot s}{\sqrt{n}}\)]; 95%
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According to Table 2, K was the most abundant mineral in DPC, in accordance with previous studies (Ekpete et al., 2013; Nisha & Radhamany, 2020). Additional principal elemental constituents identified in pineapple core samples included Ca, Mg, Na, Fe, and Mn. The levels of magnesium, calcium, sodium, and manganese agreed with those previously reported (BEDCA Database, 2024; USDA Database, 2024; Ekpete et al., 2013; Nisha & Radhamany, 2020). Meanwhile, the Zn and Cu contents surpassed the values found in previous research (BEDCA Database, 2024; USDA Database, 2024; Ekpete et al., 2013; Nisha & Radhamany, 2020). No heavy metals such as cadmium, arsenic, or lead were detected. This indicated that the DPC samples analyzed in this study were free from contamination by these harmful species.

Optimization of the Ultrasound-Assisted Extraction Through a Box–Behnken Design

Model Fitting: Effect of Variables and Optimal Conditions

The operating conditions of the sequential extraction of phenolic compounds and carotenoids were optimized using an ultrasound-assisted method and BBD. The optimized response variables were achieved by adjusting the levels of amplitude, number of cycles, and extraction time to maximize the response within the specified region. Firstly, a BBD was conducted to maximize the extraction of phenolic compounds from the DPC samples, focusing on enhancing the FRAP, ABTS, and TPC assay responses.

The TPC results indicated that there was room for improvement of the model, (R-squared = 63.4686%) for capturing the variability in terms of phenolic content, because it did not include significant modeling parameters. In this sense, as revealed in Table 3, the actual phenolic content measured was 6% lower than the predicted one. In the case of the FRAP assay, both the amplitude (A) and quadratic amplitude (AA) were significant variables, indicating that these parameters influenced the antioxidant activity measured by this method (Table 3, Fig. 2). The model explained a substantial amount of variability (R-squared = 80.9881%), and the actual FRAP value was slightly higher than predicted (814 ± 25 vs. 779.729 mg Trolox/100 g DPC; Table 3), which could be attributed to the positive effects of quadratic interactions of amplitude testing conditions enhancing the antioxidant potential accordingly with previous studies (Rathnakumar et al., 2017). The ABTS assay showed a good fit for the quadratic amplitude (AA) with an R-squared value of 82.2524%, indicating a reliable predictive model for this parameter. As in the case of FRAP, the actual ABTS value was significantly higher than the predicted (Table 3). Figure 2 shows the Pareto charts to help identify the most influential factors in each assay. These charts illustrate the relative influence of three independent variables—A (amplitude, %), B (cycles), and C (extraction time, min)—on the responses from three different assays used to evaluate antioxidant capacity: FRAP (1), TPC (2), and ABTS (3). The height of the bars indicates the significance of each factor on the respective response, based on the BBD for optimizing the extraction of polyphenol-rich compounds. In addition, the response surface plot (4) depicts the interaction between the independent variables (amplitude, cycles, and extraction time) and their combined effect on maximizing the overall desirability function. The desirability function is a numerical representation that combines the individual responses into a single score, aiming to find the optimal set of conditions that simultaneously maximize the outcomes of all responses. The plot visualizes the optimal region for obtaining the extract with the highest polyphenol content based on the experimental factors tested.

Table 3 Significant parameters (A, amplitude; B, cycles; AA, quadratic amplitude); lack of fit (p-value) and R-squared values for the response variables for each BBD. Predicted and actual values (mean value ± SD, n = 3) under the optimal experimental conditions for the response variables for each BBD*
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Fig. 2
figure 2

Pareto charts, where A, B, and C represent amplitude (%), cycles, and extraction time (min), respectively, for FRAP (1), TPC (2), and ABTS (3), in relation to the experimental BBD for obtaining the polyphenol-rich extract. The response surface plot (4) shows the combination of factor levels that maximize the desirability function within the indicated region

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For the three responses (TPC, FRAP, and ABTS), a significant value of lack-of-fit (p > 0.05) was obtained, indicating a reliable model that explained sufficiently the response despite the variations that could occur within the system. The three studied response variables were simultaneously optimized by using a desirability function to maximize them. The equations of the fitted model for each response variable are the following:

$$\text{FRAP}=618.0-2.2263*A+33.9271*B+9.9124*C+0.0574*A2-0.1139*AB-0.1284*AC-3.2459*B2-0.4581*BC-0.1042*C2$$
$$\text{TP}C=546.507-8.1416*A+2.1526*B-13.8120*C+0.0737*A2-0.1757*AB+0.2178*AC-1.1513*B2+1.8981*BC-0.1499*C2$$
$$AB\text{TS}=616.933-3.8498*A-8.6383*B-2.3911*C+0.0501*A2-0.0152*AB-0.0347*AC+0.2816*B2+0.6592*BC+0.0679*C2$$

Based on these results, the optimal conditions for extracting phenolic compounds were determined to be 70% amplitude, 5 min of extraction time, and 2 extraction cycles. In this study, the optimum extraction time was three times shorter than those reported in other studies applying ultrasound-assisted extraction to pineapple core samples (Rathnakumar et al., 2017). In addition, the optimized desirability value was 81.2%, and under the optimal conditions, the yield of production for the antioxidant extract rich in phenolic compounds was 45.1%. The extraction yield was calculated according to the following equation:

$$\text{Extraction yield }\left({\%}\right)\text=\left({W}_{1}/{W}_{0}\right)\times 100$$

where W1 is the weight of the final dry extract, and W0 represents the weight of the initial sample. This calculation provides the percentage yield of the extraction process based on the mass of the sample before and after extraction.

After establishing the optimal conditions for obtaining the extract rich in phenolic compounds from the DPC, an optimization of carotenoid recovery from the extraction residue (RE) (solid waste) generated from the initial extraction of the pineapple by-product was conducted (see Fig. 1 in Materials and Methods). Carotenoids are lipophilic bioactive compounds that have been traditionally extracted using organic solvents (including hexane, diethyl ether and ethanol-petroleum ether, among others). The use of UAE has been suggested to enhance the extraction yield of carotenoids (Song et al., 2018).

As preliminary tests, the β-carotene extraction efficiency from the core of pineapple utilizing hexane and SDS as solvents was investigated. Additionally, the effect of prior polyphenol extraction on β-carotene yield was assessed. The findings reveal significant differences in β-carotene content based on the solvent used and the pre-treatment applied. When β-carotene was extracted directly from the pineapple core without the preliminary removal of polyphenols, the use of hexane as the extraction solvent resulted in an average β-carotene concentration of approximately 35 ± 2 µg/100 g DPC. In contrast, utilizing SDS under the same conditions yielded a significantly lower average β-carotene content, measuring around 1.5 ± 0.2 µg/100 g of DPC. Despite the lower extraction efficiency of SDS compared to hexane, it is important to highlight that SDS is a much more sustainable and environmentally friendly solvent. The use of hexane implies significant environmental and health risks, including being a contributor to air pollution and having potential toxic effects. In contrast, SDS is a biodegradable and less hazardous alternative, making it a preferable choice for sustainable extraction practices. The use of SDS aligns with the growing demand for greener and more sustainable extraction methods in the food and pharmaceutical industries, where minimizing environmental impact is increasingly prioritized.

After the removal of polyphenols, the subsequent extraction of β-carotene yielded the following results by using hexane and SDS, respectively, 5.9 ± 0.9 µg/100 g of RE and 1.41 ± 0.04 µg/100 g of RE. These findings suggest that the β-carotene content extracted using SDS remains largely unchanged regardless of whether polyphenols have been removed beforehand. This supports the sequence in the cascade extraction approach, where polyphenols are extracted first, followed by β-carotene. The consistency in β-carotene yield using SDS, both before and after polyphenol extraction, indicates that the removal of polyphenols does not significantly impact the efficiency of SDS in extracting β-carotene.

In this study, the UAE time was 3 min, and sodium dodecyl sulfate (SDS) was selected as the extracting solution, thus enabling the eco-friendly extraction of carotenoids. Other green methods have shown longer extraction times for the UAE of carotenoids from other fruits, such as sea buckthorn berries, with fatty acid ethyl esters (i.e., 45 min) (Staicu et al., 2024). In the case of by-products, such as tomato peels, 10 min have been necessary to perform UAE with ethyl acetate (Szabo et al., 2022). Furthermore, no previous studies have been identified in the literature in which the extraction of antioxidant compounds from pineapple by-products has been conducted using a cascade process. The process applied in the present work involved first extracting the phenolic compounds from DPC and then using the extraction residue (RE) for a second extraction of the β-carotene.

Figure 3 shows the Pareto chart (1) that highlights the significance of cycles and their quadratic effects in β-carotene extraction via ultrasound. The response surface plot (2) shows the optimal interaction between amplitude and cycles for maximizing β-carotene content at a constant extraction time of 3 min. Results showed that the number of extraction cycles (B) was statistically significant for the contents of β-carotene in the RE samples, with an R-squared of 69.27 (Table 3. Figure 3). The negative sign of this variable indicated that the use of a higher number of cycles would be significantly associated with a lower extraction of β-carotene. This is possibly because the ultrasound cycles would increase the degradation of this compound. In general, carotenoids oxidation may be induced by an increase in the temperature caused by a prolonged extraction time, an increased amplitude (%), and a higher number of UAE extraction cycles (Song et al., 2018; Staicu et al.,. 2024).

Fig. 3
figure 3

Pareto chart (1) showing the standardized effects of amplitude (A), cycles (B), extraction time (C), and their interactions on β-carotene extraction efficiency. Response surface plot (2) illustrates the interaction between amplitude and cycles at a constant extraction time of 3.0 min, highlighting the conditions that maximize the β-carotene content

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In the present work, the optimal conditions for extraction of β-carotene were 20% amplitude, 3 min of extraction time, and 2 extraction cycles. The equation of the fitted model is the following:

$$\upbeta -\text{carotene}=1.20932-0.006108*A-0.129411*B+0.067241*C+0.000014*A2+0.000791*AB+0.000049*AC+0.008081*B2+0.001285*BC-0.013067*C2$$

where A, B, and C represent amplitude (%), cycles, and extraction time (min), respectively.

Results from the evaluation of β-carotene with the optimal conditions showed higher contents (1.41 ± 0.04 µg/100 g RE) (actual value) compared to the predicted value (0.994 µg/100 g RE). To the best of our knowledge, no published studies have been found that optimize carotenoid extraction from pineapple by-products using UAE in combination with green solvents.

Analysis of the Phenolic Lyophilized Extract (PLE)

Once the extract rich in phenolic compounds was obtained, several analyses were conducted beyond the determination of TPC and antioxidant activity. They included quantifying the major phenolic compounds (Table 4), profiling the mineral content (Table 5), and assessing the extract’s thermal stability by using TGA.

Table 4 Phenolic acids and TPC, FRAP, and ABTS values (mean value (n = 3) ± SD) found in the lyophilized extract rich in phenolic compounds
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Table 5 Mineral composition of PLE samples [mean value (n = 3) ± \(\frac{t\cdot s}{\sqrt{n}}\)]; 95%
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Rathnakumar et al. (2017) optimized a UAE method to extract bioactive compounds from the bark and core of pineapple obtaining TPC values in the extract ranging from 9340.26 to 24678.7 µg GA g−1. The optimal conditions that allowed them to achieve the highest antioxidant activity involved using a sonotrode power of 100% for 16 min and employing an ethanol/water (36/64 v/v) solvent mixture. The TPC values recorded were like those observed in the current study (Table 4). Possible deviations in TPC could be attributed to several factors including the cultivar, state of maturation, and differences in the optimal conditions for UAE utilized in the experiments.

In relation to the antioxidant activity, further analysis was carried out by Santos et al. (2021) in which the ultraturrax extraction was used for determining antioxidant values provided FRAP values between 640.74 and 698.31 µmol Fe (II) g−1 and ABTS values between 1036.95 and 1055.97 mg Trolox/100 g of DPC samples. The FRAP value reported in the mentioned study was lower than those obtained in the present study (Table 4) using an optimized UAE procedure. Following this line, in the research conducted by Lasunon et al. (2022), the extraction of phenolic compounds from pineapple core was optimized by using microwave-assisted extraction (MAE) techniques. Their findings indicated an ABTS radical scavenging activity of 307 ± 1 mg Trolox/100 g PLE. Comparatively, our study revealed a higher ABTS value of 1310 ± 30 mg Trolox/100 g PLE, highlighting the suitability of UAE for the extraction of antioxidant phenolic compounds from pineapple core samples. These differences in the ABTS values can be also attributed to different factors such as genotypes, soil nutrient levels, harvest timing, and climatic conditions. as mentioned above.

Five major phenolic compounds were identified in the lyophilized extract: succinic, gallic, syringic, coumaric, and ferulic acid. These compounds exhibit antioxidant activity by donating electrons or hydrogen atoms to neutralize free radicals. Hydroxybenzoic acids, including gallic and syringic acids. as well as hydroxycinnamic acids, like ferulic and coumaric acids, contain hydroxyl groups that can donate hydrogen atoms, thereby stabilizing free radicals via resonance.

In the by-products of pineapple, the primary phenolic compounds identified included gallic acid, catechin, epicatechin, and ferulic acid, in approximate concentrations of 31.7, 58.5, 50.2, and 19.5 mg per 100 g of dry extracts, respectively (Hamzah et al., 2021). Rashad et al. (2015) further evaluated the antioxidant activity of pineapple residues, finding that the methanolic extract from the fermented pineapple by-products contained high levels of phenolic compounds, approximately 20 mg of gallic acid per 100 g of dry by-product. The concentrations determined are presented in Table 4.

The primary compounds for PLE were succinic acid, ferulic acid, and syringic acid. In this sense, the most abundant phenolic acid in DPC, i.e., succinic acid, plays a vital role in plant metabolism. This compound is a key intermediate in the citric acid cycle (Krebs cycle), promoting the conversion of carbohydrates, fats, and proteins into usable energy. In terms of properties, succinic acid has a notable antioxidant activity and helps to protect the cells from oxidative stress by neutralizing free radicals. Succinic acid is also associated with several attributes of the pineapple since it contributes to the tartness and overall flavor complexity of the pineapple. In general terms, the differences observed between the phenolic compound concentration values reported in prior studies, and those obtained in this research may be attributed to variations in the pineapple fraction, extraction methodologies employed, or differences in the sample varieties used. Such variations can significantly impact the yield and composition of extracted phenolic compounds.

The mineral profile of the phenolic compound-rich extract derived from the pineapple core was assessed (Table 5). There was a marked decrease in the concentrations of Ba, Ca, Fe, and Na in the extracts with respect to those in DPC samples (compare Tables 2 and 5), which can be attributed to losses during the extraction. Conversely, the concentrations of B, K, Mg, Mn, and Zn increased, suggesting a preconcentration effect. Importantly, as with fresh pineapple, no heavy metals were detected in the extract, This underscores the safety of the extract, confirming that the extraction process neither introduces nor concentrates toxic elements.

The thermogravimetric analysis (TGA) data for the PLE reveals critical parameters related to its thermal stability. Notably, the onset temperature at which a 1% weight loss occurs is measured at 92.1 ± 1.8 °C. This relatively low onset temperature suggested that the extract contained volatile components or moisture that started to evaporate or decompose at this temperature. The maximum degradation temperature was 194 ± 3 °C, which corresponded to the temperature at which the rate of weight loss was highest. This temperature provided insight into the thermal stability of the major constituents of the extract. The fact that significant degradation occurred at this temperature implied that the main components of the extract decomposed or undergone substantial thermal breakdown at around 195 °C. These thermal properties were crucial for understanding the behavior of the extract during processing and storage. Overall, these data points highlight the need for controlled temperature conditions to preserve the quality and efficacy of the pineapple extract during its use in different applications.

Determination of Carotenoids in the Lyophilized Extract (CE) by High-Performance Liquid Chromatography with Diode-Array Detection

Few works have been found regarding the carotenoid profile of pineapple by-products. Furthermore, as far as we know, no studies have been carried out to optimize the extraction of carotenoids from pineapple by-products using green chemistry. Our findings indicated that β-carotene (1.41 µg/100 g RE) was the predominant carotenoid in the extraction residue (RE) derived from the first (poly)phenol extraction. The carotenoid α-carotene was also found in the samples (1.19 µg/100 g RE) in accordance with Rodriguez-Amaya et al. (2008) who described that in fruits and vegetables containing β-carotene. α-carotene was also found at lower levels. In addition, three other individual carotenoids were quantified as “other carotenoids” (1.2 µg/100 g RE) because of their characteristic UV spectrum (max 440 and 472 nm), but their identification was not possible as no similar compounds with these retention times were found in the literature.

Freitas et al. (2015) also reported β-carotene (0.9 mg/100 g dry weight) as the predominant carotenoid in pineapple core when using hexane as an extraction solvent, also lutein and α-carotene identified in the samples. In another study reported by Sengar et al. (2022), the total carotenoid content (0.35 µg/g dry weight) was also determined in the pineapple core by spectrophotometry and by organic solvent extraction.

The present work demonstrated the potential for the extraction of carotenoids from pineapple by-products using SDS as a biodegradable solvent in a relatively short time (3 min) compared to the literature. Most of the published works described the content of phenolic compounds in pineapple by-products (Campos et al., 2020; Lasunon et al., 2022; Lourenço et al., 2021; Nath et al., 2023), but few reports have been found regarding the presence of carotenoids, which are also natural pigments that stand out for their antioxidant activity and pro-vitamin A activity, among other biological activities (Anand et al., 2022). Further experiments are needed to enhance the extraction yield of carotenoids from agro-food byproducts using environmental-friendly methods.

Conclusions

The study successfully characterized dry pineapple core (DPC) samples, highlighting their nutritional composition, low moisture content, and significant extractive content. The thermal properties of the extracts indicated the need for controlled temperature conditions to maintain their quality during processing. Optimizing phenolic and carotenoid extraction using ultrasound-assisted extraction (UAE) and Box–Behnken design (BBD) proved effective, significantly enhancing the yield and efficiency of the extraction processes.

The cascade extraction method, as demonstrated in this study, provides several benefits for the efficient extraction of phenolic compounds and carotenoids from pineapple by-products. This method optimizes the use of raw materials and enhances the yield of valuable bioactive compounds through sequential extraction processes. Cascade extraction allows for the complete utilization of the raw material by sequentially targeting different groups of bioactive compounds. For instance, after extracting phenolic compounds, the remaining biomass can be used to extract carotenoids, ensuring minimal waste. By tailoring each step to specific compounds, cascade extraction can improve the efficiency and yield of each target compound. For example, specific solvents and conditions can be optimized for phenolic compounds in the first step, followed by adjustments for carotenoids in the subsequent step. In addition, this method supports green chemistry principles by potentially reducing the need for harsh chemicals and extensive solvent use. The use of environmentally friendly solvents and conditions aligns with sustainable practices and reduces the ecological footprint of the extraction process. This research underscores the value of pineapple by-products as sources of bioactive compounds and supports sustainable practices through efficient extraction methods.

Looking ahead, future research should focus on optimizing the cascade extraction process to enhance the recovery of additional high-value bioactive compounds from pineapple by-products. In addition, developing scalable models for industrial applications is also crucial for broader implementation. Further studies on the biological activities of phenolic and carotenoid-rich extracts, such as their antimicrobial properties, would strengthen their potential applications in food, pharmaceutical, and cosmetic industries. Additionally, investigating the synergy between bioactive compounds could unlock enhanced functional properties in formulations.

Overall, this research highlights the potential of pineapple by-products as rich sources of bioactive compounds and supports the shift toward sustainable and efficient extraction methods. Future studies in this area could open new opportunities in both industrial and scientific fields.