The added value of flavouring extracts from Calabrian chili peppers by-products: characterization of bioactive and volatile compounds comparing green extraction techniques

The added value of flavouring extracts from Calabrian chili peppers by-products: characterization of bioactive and volatile compounds comparing green extraction techniques | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article The added value of flavouring extracts from Calabrian chili peppers by-products: characterization of bioactive and volatile compounds comparing green extraction techniques Samanta Corsetti, Laura Alessandroni, Jean Christophe Coppin, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6549793/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract The industrial demand for natural flavourings is increasing, together with green technologies development and diffusion. This article investigates different extraction strategies for flavouring obtainment from Calabrian chili peppers supply chain by-products. Green solvent mixtures were tested on Soxhlet, Ultrasound-Assisted Extraction (UAE) and Supercritical Fluid Extraction (SFE). Extracts were characterized through bioactive compounds quantification and volatile profile monitoring. Preliminary Soxhlet experiments set the matrix/solvent ratio at 1/25 (g/mL). SFE extracts exhibited the highest concentration of less-polar polyphenols, such as flavonoids, total carotenoids, but also alpha-tocopherol and capsaicinoids. On the other hand, the UAE and Soxhlet processes, involving ethanol/water mixtures, allowed the highest yields, the greatest antioxidant activity and a high content of vitamin C (up to 1.27 mg/g). In conclusion, this work underlines the importance of extraction technique choice and could represent an advancement in industrial flavourings extractions, suggesting sustainable methods to obtain value-added extracts from discarded matrices. Flavourings extraction Supercritical Fluid Extraction chili peppers green extraction bioactive compounds Figures Figure 1 Figure 2 1. INTRODUCTION The flavour represents a crucial element influencing sensory quality of food and drinks (Prescott, 2015 ). Specifically, flavouring additives could be divided according to the origin in synthetic, composed by synthetically prepared compounds, and natural formulations, which must at least include the 95% of product directly obtained from plant, animal or microbiological materials, through appropriate physical, enzymatic or microbiological processes (European Regulation 1334/2008). The interest in industrial production of natural flavours had increased in the last decades due to the higher consumers concern in natural products. For instance, already in 2007, more than 65% of all the flavouring ingredients in USA and EU, were reported as “natural” labelled. Indeed, the natural origin of a food product was considered as important as its palatability (Yasin et al., 2023 ). Chili peppers ( Capsicum annuum L.) are extensively involved as starting matrices in food flavourings industry. The widespread application of these Capsicum fruits extracts extends to a countless number of different food products, including sauces, processed meats, snacks, beverages and cheese (Raju et al., 2010 ). From a botanical standpoint, the Capsicum L. genus belongs to the Solanaceae family and it encompasses more than 30 species of peppers. Among them, five were domesticated with the Capsicum annuum as one of the most used in industrial and home-made food preparation. It includes the Calabrian chili pepper variety, which is furtherly divided into different subvarieties (i.e. Ciliegino, Guglia, Diavulicchio, A Mazzetti, Naso Di Cane, Pizzitano, Sigaretta, Tondo). For their growth, the Calabrian chili peppers require the warm temperatures and sunlight of the mild climates of Calabria region (south of Italy) where they are largely cultivated. According to the obtained prestigious certifications, they must meet rigorous quality criteria, leading to a big amount discarded material (Cicatiello et al., 2016 ). Actually, around 46% of this by-product biomass, including the relevant percentages of items that do not meet the required size and shape, is treated to be sold as dried powder or mixed with other spices (Yasin et al., 2023 ). A growing preference for natural chili pepper flavourings, such as extracts and oleoresins, has been experienced in the last decades, due to their concentrated colour and flavour. Oleoresins are obtained through solid-liquid extraction and subsequent evaporation of the solvent. They may retain the full sensorial characteristics of the spices and could include also the naturally present bioactive substances (Melgar-Lalanne et al., 2017 ). In fact, unlike synthetic additives, natural ones generally offer the added benefit to be source of bioactive compounds with antioxidant and antimicrobial activity. In particular, natural chili pepper flavouring usually contains capsaicinoids, polyphenols, carotenoids and vitamins, derived from the extracted matrix (Hernández-Pérez et al., 2020 ). The traditional natural flavourings obtaining processes, relies on organic solvents, such as hexane, acetone, ethanol or methanol (Madhusankha et al., 2023 ). Among the other solvents involved in hydrophilic flavouring extraction, ethanol is widely used being non-toxic and worldwide authorized in food additives production, moreover, it is classified as green solvent (Madhusankha et al., 2023 ). In particular, for lipophilic extractions, the use of hexane is predominant in the food industry, because it requires a shorter extraction time than other solvents and it is authorized in the USA by the Food and Drugs Administration (FDA). However, the more restrictive European directive allows hexane just within certain residue limits and after specific solvent-removing processes (European Regulation 2009/32). Among the green and sustainable extraction techniques, Supercritical Fluid Extraction (SFE) has emerged as a promising technique providing high extraction yields using CO 2 , which is non-toxic, non-flammable, cheap and easily available in large quantities with a high degree of purity (Duarte et al., 2004 ). In addition, CO 2 is in gas phase at room temperature and consequently there is no need for solvent-removing steps. To increase the polarity, a co-solvent could be added, and, in the context of green and sustainable solutions, water, ethanol and their mixtures are the most used. The main aim of this research work is to propose new green and sustainable strategies to obtain chili peppers flavourings from Calabrian chili peppers which were discarded, due to aspect or sizes requirements. Thus, Soxhlet, Ultrasound-Assisted Extraction (UAE) and SFE were compared in Calabrian chili pepper flavouring extracts production. Green solvents were also used, comparing different ethanol/water percentages, considering their spread and safe use in food flavourings industry. The bioactive compounds extraction power of each method was investigated monitoring polyphenols, flavonoids, antioxidant activity, carotenoids, vitamins C and alpha-tocopherol concentrations, together with their volatile profile composition and level of pungency, that are crucial for the typical chili pepper characteristics. 2. MATERIALS AND METHODS 2.1. Reagents and standards The analytical standards of delphinidin 3-galactoside chloride (absolute purity, C 21 H 21 O 12 Cl) and kaempferol 3-glucoside (absolute purity, C 21 H 20 O 11 ) were purchased from PhytoLab (Vestenbergsgreuth, Germany). Capsaicin (≥ 95%, C 18 H 27 NO 3 ), dihydrocapsaicin (≥ 85%, C 18 H 29 NO 3 ), L-ascorbic acid (99%, C 6 H 8 O 6 ), gallic acid (≥ 97.5%, C 7 H 6 O 5 ) neochlorogenic acid (≥ 98.0%, C 16 H 18 O 9 ), chlorogenic acid (≥ 95%, C 16 H 18 O 9 ), 4-hydroxy benzoic acid (99%, C 7 H 6 O 3 ), caffeic acid (≥ 98.0%, C 9 H 8 O 4 ), vanillic acid (97%, C 8 H 8 O 4 ) p-coumaric acid (≥ 98%, C 9 H 8 O 3 ), ferulic acid (≥ 99%, C 10 H 10 O 4 ), phloridzin (≥ 98.0%, C 21 H 24 O 10 ), hesperidin (≥ 97.0%, C 28 H 34 O 15 ), α-tocopherol (≥ 96% C 29 H 50 O 2 ), 2,2-diphenyl-1-picrylhydrazil (DPPH, C 18 H 12 N 5 O 6 ), 6-hydroxy-2,5,7,8 tetramethylchromane-2-carboxylic acid (≥ 97%, Trolox, C 14 H 18 O 4 ), rutin (≥ 94%, C 27 H 30 O 16 ) and Folin-Ciocalteu phenol reagent were supplied by Sigma–Aldrich (Milan, Italy). HPLC-grade methanol, acetonitrile and isopropanol were purchased from Sigma–Aldrich (Milan, Italy), while HPLC-grade formic acid 99–100% was bought from J.T. Baker B.V. (Deventer, Holland); all the solvents used were analytical grade. A Milli-Q System (Bedford, MA, USA) was used to obtain deionized water (< 8 MΩ cm resistivity). 2.2. Sample Collection Samples were selected among those Calabrian chili peppers (Naso Di Cane subvariety) which were evaluated as not suitable to be sold as fresh, due to the sizes or shape requirements. New Flavours® company (Via dell'Artigianato 7, Monte Santa Tiberina, PG, Italy) provided pre-dried and finely grinded samples, which were stored at -20°C until analyses. 2.3. Extractions procedures 2.3.1 Soxhlet extraction An aliquot of 5 grams of pre-dried chili pepper powder was extracted using a 6-channels Universal Extractor in Soxhlet mode (Büchi E-800) for 4 hours. A preliminary step of matrix/solvent ratio (m/s) optimization was conducted testing m/s of 1:25 (0.04) g/mL, 1:50 (0.02) g/mL and 1:100 (0.01) g/mL. Different ethanol/water solutions were explored, i.e. 100% ethanol (EtOH100), ethanol/water 70:30 v/v (EtOH70), or ethanol/water 50:50 v/v (EtOH50). All extractions experiments were conducted in triplicate. After each extraction, the solvent was evaporated until constant weight. Thus, the extraction yields (EY%) were calculated as the ratio of the dry extract weight and the initial amount of dried matrix. 2.3.2 Ultrasound-assisted extraction The optimized m/s was applied to UAE procedure. The extraction was performed on pre-dried chili pepper powder, the involved instrument was an Ultrasonic Bath (ARGOLAB AU-220), with a frequency of 40 KHz, for 20 minutes at 40°C. The same three Soxhlet solvent mixtures were tested as solvents. All extraction experiments were conducted in triplicate. Then, extracts were dried and EY% were calculated as previously mentioned. 2.3.3 Supercritical Fluid Extraction The instrument used was an SFT-120XW Extractor with a 100-mL extraction vessel, an oven, SFT-10 PID controllers and a Nex10 SCF pump. The extraction parameters were adopted from previously published optimization studies, being 330 bar and 40°C (Yan et al., 2018 ). Given the instrument's attributes, static and dynamic phases were alternated to enhance the extraction yield. In each trial, the extraction started with a 10-min static period followed by a 15-min dynamic mode, this scheme was repeated twice for an extraction time of 50 min. A total of 25 mL of co-solvent was used for the extraction of 5 g of chili pepper powder. The three previously described solvent mixtures were tested. The extracts were collected in glass vials once the CO 2 gas had been fully expelled from the system. All the experiments were performed in triplicate, the EY% and the concentration of analytes were monitored after each repetition. 2.4 Analysis of bioactive compounds in Calabrian chili pepper flavouring extracts 2.4.1 Spectrophotometric assays For the evaluation of Total Phenolic Content (TPC), the Folin-Ciocalteu method was used (Moreno-Ramírez et al. 2018 ). Dry extracts were firstly dissolved in methanol (2.5 mg/mL) and 0.5 mL of extract solution were added to test tubes along with 2.5 mL of 1:10 water solution of Folin-Ciocalteu phenolic reagent. After 5 minutes, 7 mL of Na 2 CO 3 (7.5% w/v ) solution were added and mixture was kept in the dark for 2 hours at room temperature. The Total Flavonoic Content (TFC) was determined according to Alessandroni et al. ( 2024 ). Thus, 0.5 mL of each resuspended extract were mixed with 0.15 mL of a NaNO 2 0.5 M aqueous solution and then 3.2 mL of methanol/water 30/70 v/v were added. After 5 min, 0.15 mL of a 0.3M AlCl 3 and 1 mL of a 1M NaOH aqueous solutions were added. Then, after vortexing, the mixture was stored in the dark for 30 min at room temperature. The radical-scavenging activity was assessed according to the DPPH method (Alessandroni et al. 2024 ). The procedure involves 0.5 mL of diluted extract, which was mixed with 4.5 mL of a 0.1 mM DPPH (2,2-diphenyl-1-picrylhydrazyl) ethanolic solution. Then, the mixture was stored for 30 min in the dark at room temperature. The Total Carotenoid Content (TCC) was performed resuspending 15 mg of each dry extract in 6 mL of petroleum ether stabilized with 0.2% w/v BHT (butylated hydroxytoluene) and centrifuging for 10 min at 5000 rpm (IEC CL10 Centrifuge, Thermo Fisher Scientific, Waltham, USA). Then, for each assay, the absorbance was measured spectrophotometrically using a Cary 8454 UV-visible spectrophotometer (Agilent Technologies, Woburn, MA, USA). The wavelengths were 765 nm, 506 nm and 517 nm, and the results were expressed in mg of gallic acid equivalents (GAE)/g of dry extract, mg of rutin equivalents (RE)/g of dry extract, mg of Trolox equivalents (TE)/g of dry extract, for TPC, TFC and DPPH respectively. The TCC (expressed as µg/g of dry extract) was calculated through the Eq. 1 , after directly measuring the absorbance at 450 nm (Larocca, et al., 2023 ). \(\:TCC\:(\mu\:g/g)=\:\frac{A\:\times\:V\left(mL\right)\times\:{10}^{4}}{{{A}_{1}}_{cm}^{1\%}\times\:P\left(g\right)}\) Eq. 1. where A is the absorbance of the sample, V (mL) is the total volume of the extract, is the β-carotene extinction coefficient in petroleum ether (2592 mL/g∙cm) and P (g) is the weight of the dried extract (Larocca, et al., 2023 ). Experiments were performed in triplicate for each extract. 2.4.2 Vitamin C quantification through HPLC-DAD An aliquot of 2 mg of each dry extract was dissolved in 1 mL of HPLC-grade methanol, then filtered through a 0.45 µm PTFE filter. The instrument was an HPLC Agilent 1260 Infinity II series (Santa Clara, CA, USA), coupled with a diode array detector (DAD) and equipped with a Synergi Polar-RP C18 (4.6 mm × 150 mm, 4 µm) from Phenomenex (Torrance, CA, USA). The analytical column temperature was set at 40°C, while the injection volume was 10 µL. The mobile phase was made up of MilliQ water with 0.1% formic acid (A) and methanol with 0.1% formic acid (B) and the flow rate was 0.5 mL/min. The elution was performed in gradient mode: 0–6 min 10% B, 6–12 min 90% B. The absorbance was monitored at 245 nm and results were reported as µg/g DE using a Vitamin C analytical standard calibration curve (R 2 > 0.99). The limit of detection (LOD) and limit of quantification (LOQ) were determined as 0.015 µg/g and 0.050 µg/g, respectively. 2.4.3 Capsaicin and dihydrocapsaicin quantification through HPLC-DAD The same HPLC-DAD equipment was used also for the quantification of capsaicin and dihydrocapsaicin, after dissolving 2.5 mg of each dry extract in 1 mL of HPLC-grade acetonitrile and filtering with a 0.45 µm PTFE filter. The separation of analytes was obtained by a Gemini 5u RP-C18 analytical column (150 mm × 4.6 mm × 5 µm) from Phenomenex (California, USA), set at 40°C. The mobile phase was composed of MilliQ water (A), and acetonitrile (B), working isocratic mode with 50% A and 50% B at a flow rate of 1 mL/min. The injection volume was 5 µL. For the detection of capsaicinoids, the 228 nm wavelength absorbance was used. The results are expressed as µg /g dry extracts. A calibration curve of each standard was prepared and showed an excellent linearity (R 2 > 0.99). LOD and LOQ were 0.29 µg/g and 0.95 µg/g for the dihydrocapsaicin and 0.27 µg/g and 0.89 µg/g for capsaicin. 2.4.4 Alpha-tocopherol quantification A total of 2 mg of each sample were redissolved in 1 mL of an acetonitrile–methanol–dichloromethane solution (60:30:10 v/v), then mixed and centrifuged at 5000 rpm for 5 min at room temperature and filtered with 0.45 µm PTFE filters. Alpha-tocopherol was quantified as reported by Nzekoue et al. ( 2022 ), with few modifications. Briefly, the HPLC 1260 equipment was used, coupled with a Fluorescence Detector (FLD). The separation of analytes was obtained by Chromolith RP-18e analytical column (100 × 3 mm I.D., macropore size 2 µm, mesopore size 13 nm) from Merck (Darmstadt, Germany), set at 40°C. The mobile phase was composed of acetonitrile (A), and 2-propanol (B), at a flow rate of 0.8 mL/min. Elution was carried out in gradient mode as follows: 0–5 min 100% A; 5–9 min 80% A and 20% B, and a recondition time of 7 minutes. The injection volume was 10 µL. For quantification, 295 nm and 330 nm as excitation and emission wavelengths, respectively. LOD (0.02–0.05 µg/g) and LOQ (0.05–0.15 µg/g) were previously assessed by Nzekoue et al. ( 2022 ). 2.4.5 Characterization of polyphenols by UHPLC-MS/MS The detection of polyphenols from 1 mg/mL methanolic solution of each dried extract was carried out with an Agilent 1290 Infinity series with a Triple Quadrupole 6420 (Agilent Technology, Santa Clara, CA). Before injection, samples were filtered through a 0.2 µm polyamide filter from Sartorius Stedim (Goettingen, Germany). The used method was previously published by Angeloni et al. ( 2024 ). Briefly, analytes separation was performed on a Synergi Polar–RP C18 analytical column (250 mm × 4.6 mm, 4 µm) from Phenomenex (Chesire, UK) set at 30°C, with a Polar RP security guard cartridge (4 mm × 3 mm ID). The mobile phase consisted of (A) MilliQ water and (B) methanol, both with formic acid 0.1%. The flow rate was 0.8 mL/min with the following elution gradient: 0–1 min, 20% B; 1–25 min, 20–85% B; 25–26 min, 85% B; 26–32 min, 85–20% B. The injection volume was 2 µL. The dynamic-multiple reaction monitoring (dynamic-MRM) mode was chosen for detection and the peak areas were integrated for quantification. 2.5. Analysis of volatile organic compounds using HS-SPME-GC-MS The volatile organic compounds (VOCs) analysis was performed directly on the dried chili pepper matrix and on the obtained dry extracts. A total of 1 g of the dried and finely grinded chili matrix or 100 mg of each dry extract were placed in SPME vials, together with a 1 mg/mL NaCl water solution. A gas chromatography coupled with mass selective detector (GC-MS) with PAL3-Auto Sampler System (Agilent 7890B GC Hardware with an Agilent 5977 Series MSD, Agilent, Santa Clara, CA, USA) and MassHunter GC/MSD Data Aquisition were used. A fiber with a 50/30 µm coating of divinylbenzene/carbon-wide range/polydimethylsiloxane (DVB/C-WR/PDMS, 1 cm length stationary phase) from Supelco (Bellefonte, PA, USA) was selected for this work. The fiber had been conditioned for 10 min at 250°C, then the extraction was performed for 10 min at 60°C. The desorption occurred at 250°C for 2 min, then the fiber was reconditioned at 250°C for 15 min. The column was an HP-5MS UI (30 m x 250 µm x 0.25 µm) (Agilent, 19091S-433UI, Santa Clara, CA, USA). The He flow rate was 1 mL/min in spitless mode. The injector temperature was 260°C. The column temperature was programmed as follows: from 37°C (held for 5 min) to 100°C (4°C per min), immediately from 100°C to 170°C (3°C per min), from 170°C to 230°C (15°C per min) and, lastly, to 250°C (15°C per min), holding this temperature for 3 min; the total run time was 52.417 min. The source was an electron impact ionization (EI) and data were collected in SCAN mode; the transfer line was set at 250°C and the temperature of the ionization source and the mass analyser were set at 230 and 150°C, respectively. Compounds identification was performed by comparison with NIST library (US National Institute of Standards and Technology), and injecting alkane mixes (C 6 -C 22 ) and calculating the retention indices. The results are expressed as relative abundances of each volatile compound (% peak area of the compound/ area of all the peaks). 2.6 Statistical data elaboration and analysis Differences among extracts were evaluated by one-way analysis of variance (ANOVA). Values with p < 0.05 were considered statistically significant. Bioactive compounds values in Soxhlet, UAE and SFE extracts were distributed in a Principal Component Analysis (PCA) performed with XLSTAT software (version 2023.1.4.1408). The entire dataset, including VOCs results, was normalized through log-transformation and Pareto-scaling before Ward hierarchical clustering analysis. This was expressed using a heatmap with dendrograms to provide an intuitive visualization of a data table. 3. RESULTS AND DISCUSSION 3.1. Optimization of matrix/solvent ratio through Soxhlet experiments As first step, different m/s were tested to optimize this parameter, using ethanol and ethanol/water mixtures as solvents. To the best of our knowledge the decrease of solvent volume, which implies lower expenses together with a shorter evaporation time, has not been thoroughly explored in scientific literature for Soxhlet extraction. Several experiments were performed comparing different matrix/solvent ratios (1/25, 1/50 or 1/100 g/mL), within EtOH100, EtOH70 and EtOH50, are reported in Table 1. All the analysis were carried out on dry extracts as it represents the more suitable product from an industrial application standpoint. As expected, high extraction yields (EY%) were reported in the lowest matrix/solvent ratio experiments, and this is in line with previous studies (Ivan et al., 2024). No statistically significant differences emerged for TPC, TFC, antioxidant activity (DPPH), capsaicin and dihydrocapsaicin content, between the different m/s explored. Capsaicin and dihydrocapsaicin are well-known molecules in chili pepper flavorings, since they are the main responsible compound of the pungency. Conversely, among the EtOH100 extracts, statistically significant differences were reported for five specific polyphenols quantified through HPLC-MS-MS, being gallic and chlorogenic acid, phloridzin, 4-hydroxy benzoic acid and kaempferol-3-glucoside. Their concentrations were higher in 1/25 m/s extracts. On the other hand, vanillic acid was more extracted in low m/s samples with statistically significant differences in EtOH100 experiments. Among the EtOH70 group of extracts, higher EY% were reported in 1/50 and 1/100 m/s samples (28.53 % ± 0.42 % and 28.53 % ± 1.67 %, respectively), with a statistically significant difference. This outcome could be explained by a higher effectiveness of a larger solvent volume, which is also heterogeneous in terms of polarity, due to the water presence. The same trend was experienced in TCC results. Similarly, p-coumaric acid, caffeic acid, vanillic acid, and kaempferol-3-glucoside were significantly less extracted using the highest m/s. These antioxidant, anti-cancer, antimicrobial or anti-inflammatory polyphenols were found also in other Capsicum Annuum subvarieties, especially vanillic acid-derivatives were the predominant ones (Barbero et al., 2016). Conversely, gallic acid and neochlorogenic acid where significantly more extracted using high m/s. Among EtOH50 extracts, no statistically significant difference in the monitored parameters was evaluated. Only hesperidin was quantified in small concentrations and just in EtOH50 1/100 m/s sample, probably due to a low affinity for ethanol (Ma et al., 2008). In fact, this flavanone glycoside was also detected in other species of chili pepper, such as in red bell and Balady green peppers extracts, after methanolic extraction (Abdalla et al., 2019), and in habanero pepper by-products , using 80:20 v/v methanol/water solution (Chel-Guerrero et al., 2021). Table 2 reports the results of VOCs detected in the dry matrix and in Soxhlet dry extracts. Many VOCs detected in the matrix were not identified in low m/s Soxhlet extracts, regardless of the extractive solvent. A total of ten compounds where in common between the dry matrix and the 1/25 m/s extracts, being trans-beta-ionone, 5,9-undecadien-2-one,6,10-dimethyl, alpha-curcumene, tetradecanoic acid 12-methyl-methyl ester, 2-methylpentadecane, pentadecane,3-methyl, 2-methylhexadecane and 2-methyl-1-tetradecene. Among these volatile compounds, the 5,9-undecadien-2-one,6,10-dimethyl was already identified both in a fresh and not spicy pepper from C. annuum and in a fresh and spicy pepper from C. chinense (Xu et al., 2020). Qiu et al. (2023) detected this molecule in fermented C. annuum red pepper, as well. The 2-methyl-1-tetradecene was previously reported in different Capsicum fruits by Antonio et al. (2018). In this scenario, a lower m/s can be related both to a longer drying process, when an equal amount of matrix is used, or to an insufficient amount of the material to extract, for an equal volume of solvent used: that can lead to possible losses of volatile molecules. At the same time, just five VOCs were detected only in low m/s samples being hexanoic acid, D-limonene, thymol, tetradecane, nonadecane. In conclusion, considering the emerged results, operating with low m/s value can be related to the advantage of a lower solvent volume consumption, to obtain similar extract characteristic. 3.2. Bioactive compounds in chili pepper extracts from Soxhlet, UAE and SFE Considering the various potentialities of chili pepper extracts, different green solvent mixtures with distinct polarities were tested in this study, with the aim to select the more performative ones for the formulation of value-added flavouring extracts from discarded matrices. Soxhlet, UAE and SFE techniques were investigated using different ethanol/water mixtures, as mentioned in section 2.3. Results from EY%, total content of polyphenols, flavonoids, carotenoids, antioxidant activity, bioactive compounds and polyphenols quantification were grouped in a database which was elaborate by means of a principal component analysis (PCA). The variable projection allowed a clear view on the distribution of the extracts, according to the nine different extraction conditions (three methods and three solvents). The PCA biplot is illustrated in Fig. 1 and numerical data are reported in Table 1S . The high variability percentage (73.94%; 53.40% and 20.54% for F1 and F2, respectively) confirms the reliability of the results and the sample distribution reflects the reproducibility. It is interesting to note that the SFE samples are distributed on the right, while the UAE and Soxhlet ones are partially mixed. This samples distribution is in line with the projections of the less polar analytes on the right part of the graph. In fact, it is well known that the carbon dioxide has high affinity for non-polar compounds and, despite the addition of the polarity-increasing co-solvent (ethanol and ethanol-water mixtures), also analytes with lipophilic properties are extracted. UAE and Soxhlet extracts showed similar characteristics, except in EtOH100 experiments, when UAE revealed its greater capacity in small polar compound extraction. The solvent nature impacts also the EY%, which was significantly higher in UAE and Soxhlet when an EtOH/H 2 O mixture is involved. It has been previously reported that the addition of polar co-solvents, during SFE, can increase the EY% but it cannot reach the values of Soxhlet and UAE (Soldan et al., 2021). Among UAE experiments, EtOH100 EY% were almost 30% lower than those with aqueous solutions. This could be related to the cavitation effect and the mechanical waves propagation in the solvent, which seem to be more effective in presence of water. The Soxhlet samples EY% were higher than those previously reported by both Bae et al. (2012) and Prabakaran et al. (2017), involving different solvents on others chili peppers varieties and ethanolic maceration of red bell peppers ( C. annuum ), respectively. Regarding the monitored variables, clearly TPC, TFC, capsaicin, dihydrocapsaicin and alpha-tocopherol showed higher values in ethanol than in hydroalcoholic solvent. This aspect could be explained by the lower polarity of these compounds and confirmed by the presence of polyphenols and flavonoids with an intermediate lipophilicity, as reported also in other C. annuum varieties (Chilczuk et al., 2021). Pure ethanol was proven to extract higher contents of polyphenols in C. annum species in general, if compared to other solvents (Prabakaran et al., 2017). The extractabilities of capsaicin and dihydrocapsaicin are closely and positively related to each other and negatively related to radical-scavenging compounds. They together represent the 80-90% of all the capsaicinoids in most species of chili peppers (Barbero et al., 2016). Their concentration was around 2 times higher in EtOH100, regardless of the extractive technique chosen. Indeed, comparing the EtOH100 extracts, the non-polar nature of carbon dioxide has raised the total content of capsaicin and dihydrocapsaicin by 21% and 41%, than Soxhlet and UAE, reaching concentrations of 7.61±0.42 mg/g and 3.84±0.22 mg/g respectively. The results clearly highlight that SFE with ethanol as co-solvent is the best option to obtain an extract with high pungency, as it is directly related to these two capsaicinoids concentrations, according to the Scoville Heat Units (SHU) ( Equation 2) (Sweat et al., 2016). The SHU of the SFE EtOH100 dry extract was around 184000, which is very high, if compared to the 50000 of the Calabrian chili pepper fresh fruit (Popelka et al., 2017). In the field of natural flavorings preparation, it is important to take into account the compounds related to the aromatic notes but also the bioactive molecules which could bring added value to the products. Capsaicinoids resulted positively correlated with alpha-tocopherol which was quantified in all the analysed extracts, with higher concentrations in EtOH100 samples. The concentration of alpha-tocopherol varies according to C. annuum varieties and the emerged levels are in accordance with previous studies (Olatunji, 2020). Among the bioactive compounds, the concentration of vitamin C in the extracts is enhanced by the use of water as a co-solvent. This vitamin represents not only an added value for its bioactivity, but also an important compound for the final product preservation, for its antioxidant activity. Regarding polyphenols, p- coumaric acid, 4-hydroxy benzoic acid, ferulic acid, vanillic acid, phloridzin, caffeic acid and gallic acid resulted to be positively correlated each other. This is related to structural similarities, especially within the group of the hydroxycinnamic acid derivatives, which reached more than 2-fold higher concentrations in SFE extracts, compared to Soxhlet or UAE ones. Due to polarity differences, this group of compounds is also clearly negatively correlated with vitamin C. Gallic acid was already reported in SFE extracts from C. annuum var. glabriusculum by Moreno-Ramírez et al. 2018). Hesperidin was detected only in UAE samples as facilitated by the polarity of aqueous solvents and also by cavitation phenomenon (Cheigh et al., 2012). Chlorogenic and neochlorogenic acids showed lower concentrations than other monitored polyphenols, due to their chemical instability and possible degradation during extraction and solvent evaporation processes. These two polyphenols have been reported in other pepper varieties not belonging to the Capsicum genus (Zhang et al., 2021). 3.3. Volatile profile of Soxhlet, UAE and SFE chili pepper flavouring extracts by HS-SPME-GC-MS The volatile profile of the chili pepper flavouring extracts was explored through a HS-SPME-GC-MS approach and the results are resumed in Table 3 . The highest variability of identified volatile compounds, with the 55.56% of the compounds in common with the dry matrix itself, was reported in SFE EtOH100 dry extracts. A similar trend was observed for SFE EtOH70 or EtOH50. A different volatile profile emerges in UAE EtOH70 extracts, with a total of 48 identified VOCs but only the 35.42% of them in common with dry matrix. This phenomenon could be explained by the great extraction capacity of cavitation. Comparing the three extractive techniques, the SFE extracts present higher relative abundances of alkanes, acids, ketones and aldehydes. In fact, CO 2 allowed the extraction of longer aliphatic chains and less/non-polar compounds. Indeed, the SFE procedure is able to retain most of the fragrances and the pungent principles of the matrix. Soxhlet and UAE dry extracts were characterised by several heterocycles and alkenes, mainly characterized by weak “fuel” odours (e.g., 3-hexadecene, 1-tetradecene or 1-octadecene), which were undetected both in SFE samples and in the dry matrix. Many of them may be degradation products of matrix compounds, as treated with high temperature, as Soxhlet, or invasive processes, as UAE (Essien et al., 2020). Aldehydes, mostly deriving from fatty acids carboxylic group reduction, were identified in the extracts but not in the dry matrix. Among them, nonanal and decanal were detected in UAE EtOH50 samples and in SFE extracts. These compounds, linked to citrus notes, had previously been reported in chili pepper extracts (Korkmaz et al. 2017). Safranal, hexanal and benzaldehyde with their spicy, cherry, leafy or tabacco-like notes were identified in the dry matrices but not in the extracts. Among the detected oxygenated monoterpenes, linalool and alpha-terpineol, both characterized by a floral odour, were detected only in EtOH50 UAE and in the SFE extracts. Linalool was also found in green and red chili pepper essential oils by Don et al. (2019). They concluded that its concentration is directly related to the ripeness degree of C. annuum . Conversely, dihydroactinidiolide, a musky or tea-smelling product resulting from the oxidative metabolism of beta-carotene, was identified both in the dry matrix and in all extracts, except for SFE ones. Alkanes are the most abundant class of compounds both in the dry matrix and in the SFE extracts, according with their non-polar characteristics. In particular, 2-methyl tetradecane was the most abundant alkane in dry matrix and in SFE extracts. Conversely, ketones, related to floral, fruity and woody aromas, were mostly abundant in SFE extracts, especially with EtOH50 solvent. Among them, the trans-beta-ionone, identified in the dry matrix and in most of the extracts, was previously investigated as a marker of an initial beta-carotene degradation in the dry matrix (Variyar & Suprasanna, 2024). 3.4 Heatmap and hierarchical clustering of chili pepper extracts analysed features All the obtained results from the analysis of the chili pepper extracts were grouped in a database, data were normalized and involved in a heatmap with hierarchical clustering ( Fig. 2) . The difference in chemical composition between SFE extracts and the others, clearly emerges from the discussed results and is further confirmed by the first level of sample clustering which groups together the Soxhlet and UAE samples, especially when EtOH100 was used. Thus, within SFE group, EtOH70 or EtOH50 extracts resulted similar and not well clustered. The SFE group clustering, among extraction procedures, is mainly due to non-polar nature of the carbon dioxide, regardless of the co-solvent mixture used. Among the emerging features in SFE samples there are: TCC, p-coumaric acid, 4-hydroxy benzoic acid, ferulic acid, vanillic acid, phloridzin, caffeic acid, gallic acid, capsaicin and dihydrocapsaicin concentrations, along with some alkanes (e.g., tetradecane-2-methyl or pentadecane) and alpha-curcumene, which is a volatile sesquiterpene hydrocarbon characterised by a spicy odour. From the clustering, these polyphenols seem to be quite correlated also with capsaicin and dihydrocapsaicin: a biosynthetic pathway actually leads to capsaicinoids and connects their structures with those of these specific polyphenols (Shams et al., 2023). Also the structural analogies of these two capsaicinoids investigated is confirmed by the features dendrogram at first level. On the other hand, the clustering of Soxhlet and UAE samples is also due to the detection of the following features: vitamin C, the antioxidant activity level, the volatile alkenes detection, together with a higher content of hydrophilic compounds, such as chlorogenic acid, neochlorogenic acid and kaempferol-3-glucoside, especially in EtOH70 and EtOH50 samples. As discussed in section 3.3, many alkenes could be fatty acids degradation products deriving from heating or invasive processes, like in Soxhlet extraction and UAE. Moreover, among Soxhlet and UAE extracts, the EtOH100 ones are clustered together due to similarities in capsaicinoids concentration, TPC and TFC results. The antioxidant activity (DPPH) is clustered with EY% and vitamin C in features dendrogram, confirming the important contribution of this vitamin to the radical-scavenging capacity of these extracts. In conclusion, in SFE extract a higher concentration of hydrophobic bioactive compounds was detected, together with a very high amount of capsaicinoids, responsible for the typical pungency of chili peppers. On the opposite the SFE extract is poor in vitamin C, which is more extracted by Soxhlet and UAE systems. 4. CONCLUSIONS The exploration of different green extraction strategies to obtain Calabrian chili pepper flavourings underlined the specific final product characteristics according to the involved technique. The prior establishment of the matrix/solvent ratio for Soxhlet extraction revealed the less solvent-consuming procedure as the optimal strategy in terms of extract properties and composition. The added value of natural flavourings has been strongly remarked by the results of this study. Soxhlet and UAE emerged as optimal techniques for vitamin C and the most polar antioxidant compounds – rich dry flavourings. Conversely, carotenoid – rich extracts, with high pungency and total polyphenolic content, could be obtained working in SFE. At the same time, the solvent choice is crucial to control the components of the final products. In conclusion, this research may represent an advancement in value-added flavourings extracts preparation to be used as natural food additives, proposing effective, diverse and sustainable techniques and green and safe solvent mixtures. In the meantime, it raises the issue of valorisation of food matrices that are daily discarded from the fresh products supply chain, proposing their alternative use as sources of flavourings extract to be reinvolved in the food industry. Declarations Acknowledgements Authors thank the company New Flavors (Via Dell’Artigianato 7, 06010, Monte Santa Tiberina, Italy) for supporting this research and for Calabrian chili peppers samples supply. Declaration of Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Funding Declaration There was no funding for the realized research. CRediT authorship contribution statement SC : Investigation, Methodology, Validation, Data curation, Writing - original draft. LA : Conceptualization, Visualization, Validation, Data curation, Writing - review & editing. JCC ; VT : Resources; Funding acquisition.: GS : Resources; Funding acquisition; Project administration; Supervision, Writing - review & editing. References Abdalla, M. U. E., Taher, M., Sanad, M. I., & Tadros, L. K. (2019). Chemical properties, phenolic profiles and antioxidant activities of pepper fruits. Journal of Agricultural Chemistry and Biotechnology , 10(7), 133-140. Alessandroni, L., Bellabarba, L., Corsetti, S., Sagratini, G. 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P., Siow, L. F., & Thoo, Y. Y. (2023). Efficacy of green solvents in pungent, aroma, and color extractions of spice oleoresins and impact on phytochemical and antioxidant capacities. Food Bioscience , 56 , 103171. Melgar-Lalanne, G., Hernández-Álvarez, A. J., Jiménez-Fernández, M., & Azuara, E. (2017). Oleoresins from Capsicum spp.: Extraction methods and bioactivity. Food and Bioprocess Technology , 10 , 51-76. Moreno-Ramírez, Y. D. R., Martínez-Ávila, G. C., González-Hernández, V. A., Castro-López, C., & Torres-Castillo, J. A. (2018). Free radical-scavenging capacities, phenolics and capsaicinoids in wild piquin chili ( Capsicum annuum var. glabriusculum ). Molecules , 23(10), 2655. Nzekoue, F. K., Nguefang, M. L. K., Alessandroni, L., Mustafa, A. M., Vittori, S., & Caprioli, G. (2022). Grapevine leaves ( Vitis vinifera ): Chemical characterization of bioactive compounds and antioxidant activity during leave development. Food Bioscience , 50 , 102120. Olatunji, T. L., & Afolayan, A. 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Analysis of Important Volatile Organic Compounds and Genes Produced by Aroma of Pepper Fruit by HS-SPME-GC/MS and RNA Sequencing. Plants , 12 (12), 2246. Raju, P. S., Chauhan, O. P., & Bawa, A. S. (2010). Chili flavor. Handbook of Fruit and Vegetable Flavors , 775-801. Riquelme N. & Matiacevich S. (2017). Characterization and evaluation of some properties of oleoresin from Capsicum annuum var. cacho de cabra, CyTA - Journal of Food , 15:3, 344-351. Shams, M., Yuksel, E. A., Agar, G., Ekinci, M., Kul, R., Turan, M., & Yildirim, E. (2023). Biosynthesis of capsaicinoids in pungent peppers under salinity stress. Physiologia Plantarum , 175(2), e13889. Soldan, A. C. F., Arvelos, S., Watanabe, É. O., & Hori, C. E. (2021). Supercritical fluid extraction of oleoresin from Capsicum annuum industrial waste. Journal of Cleaner Production , 297, 126593. Sweat, K. G., Broatch, J., Borror, C., Hagan, K., & Cahill, T. M. (2016). Variability in capsaicinoid content and Scoville heat ratings of commercially grown Jalapeño, Habanero and Bhut Jolokia peppers. Food chemistry , 210, 606-612. Variyar, P. S., & Suprasanna, P. (2024). Peppers—an Overview on The Bioactives and their Nutritional, Functional Properties and Post-Harvest Processing. Peppers , 1-15. Xu, X., Wu, B., Zhao, W., Pang, X., Lao, F., Liao, X., & Wu, J. (2020). Correlation between autochthonous microbial communities and key odorants during the fermentation of red pepper (Capsicum annuum L.). Food Microbiology , 91 , 103510. Yan, R., Zhao, L., Tao, J., Zou, Y., & Xu, X. (2018). Preparative isolation and purification of capsaicin and dihydrocapsaicin from Capsici Fructus using supercritical fluid extraction combined with high speed countercurrent chromatography. Journal of the Science of Food and Agriculture , 98 (7), 2498-2506. Yasin, M., Li, L., Donovan-Mak, M., Chen, Z. H., & Panchal, S. K. (2023). Capsicum waste as a sustainable source of capsaicinoids for metabolic diseases. Foods , 12 (4), 907. Zhang, D., Sun, X., Battino, M., Wei, X., Shi, J., Zhao, L., ... & Zou, X. (2021). A comparative overview on chili pepper ( capsicum genus) and sichuan pepper ( zanthoxylum genus): From pungent spices to pharma-foods. Trends in Food Science & Technology , 117, 148-162. Tables Tables 1 to 3 are available in the Supplementary Files section. Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6549793","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":455861587,"identity":"60696dc5-0bf0-43f4-a216-364ad736634b","order_by":0,"name":"Samanta Corsetti","email":"","orcid":"","institution":"University of Camerino","correspondingAuthor":false,"prefix":"","firstName":"Samanta","middleName":"","lastName":"Corsetti","suffix":""},{"id":455861588,"identity":"4b970b1d-8e84-486e-9c53-28ae5204a056","order_by":1,"name":"Laura Alessandroni","email":"","orcid":"","institution":"University of Camerino","correspondingAuthor":false,"prefix":"","firstName":"Laura","middleName":"","lastName":"Alessandroni","suffix":""},{"id":455861589,"identity":"25a1888e-f629-46b5-bee7-a55cedcd7ff6","order_by":2,"name":"Jean Christophe Coppin","email":"","orcid":"","institution":"New Flavours, Monte Santa Maria Tiberina, Perugia","correspondingAuthor":false,"prefix":"","firstName":"Jean","middleName":"Christophe","lastName":"Coppin","suffix":""},{"id":455861590,"identity":"4a9f75df-933f-4e21-b9ee-ee4020675dd0","order_by":3,"name":"Virginia Tonanni","email":"","orcid":"","institution":"New Flavours, Monte Santa Maria Tiberina, Perugia","correspondingAuthor":false,"prefix":"","firstName":"Virginia","middleName":"","lastName":"Tonanni","suffix":""},{"id":455861591,"identity":"1282b2a6-de09-45c0-ac3d-39b3c6878647","order_by":4,"name":"Gianni Sagratini","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABEUlEQVRIiWNgGAWjYDACZhBhAOMZMPDwA6kDIDYbUVoOALVINsC04NSDDEBKDQ7AeDi0yLszP3tcUMBgz9+/9uHnDwV3ZIxv5B488KNmGwOffANWLYaH2cyNZxgwJM648dxY4oDBMx6zG3kJB3uO3cbpMMNmBjNpHgOGBIYbxxiAWg4DteQYHGZgw6eF/RtIi738jWPMP0BajGeAtPzDrUWemQdsC+OG821sYFsMJIBaGNtwazFg5ikDapFI3HiDjc3iDFCLxJk3Bgd7+27zsLElYLel//g2aZ4/NvZy548x36j4c9ievz3H+MOPb7fl5JsPYLcFIiwBRGhm8mB3FtCWBhiLH7uZo2AUjIJRMAoYAOuQV3+QxoTwAAAAAElFTkSuQmCC","orcid":"","institution":"University of Camerino","correspondingAuthor":true,"prefix":"","firstName":"Gianni","middleName":"","lastName":"Sagratini","suffix":""}],"badges":[],"createdAt":"2025-04-28 16:53:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6549793/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6549793/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":82708268,"identity":"3a872d0e-d1a8-4fbf-8630-7e7593503da8","added_by":"auto","created_at":"2025-05-14 11:00:02","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":52996,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003ePrincipal Component Analysis biplot of nine Calabrian chili pepper extracts from different extraction methods (Soxhlet, UAE and SFE) and solvents (EtOH100, EtOH70, EtOH50) including EY%, bioactive compounds quantification and main capsaicinoids contents.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6549793/v1/cceb07d4f1cb7dc6e9d8a5c5.jpg"},{"id":82708779,"identity":"b28336d9-b1c1-41df-87e9-b3845cd3e4cf","added_by":"auto","created_at":"2025-05-14 11:08:02","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":126455,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eHierarchical clustering and heatmap graph of the nine Calabrian chili pepper extracts from different extraction methods (Soxhlet, UAE and SFE) and solvents (EtOH100, EtOH70, EtOH50). In this representation, colors are used to visualize the numerical data according to a predetermined color scheme (on the right), with one end of the color scheme representing the higher value data points (dark red), and the other end representing the lower value data points of selected data set (dark blue).\u003c/em\u003e\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6549793/v1/3f946a752dc2713bc523b821.jpg"},{"id":82710505,"identity":"d9f235a2-0fe5-4745-9d04-95fc2ed98ed2","added_by":"auto","created_at":"2025-05-14 11:24:03","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1201953,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6549793/v1/17f199fa-2736-43c0-9968-e6168a58d263.pdf"},{"id":82708272,"identity":"358a3fcd-9ba6-4a4a-9455-a92c1812c342","added_by":"auto","created_at":"2025-05-14 11:00:02","extension":"xlsx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":108803,"visible":true,"origin":"","legend":"","description":"","filename":"Tables.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6549793/v1/56300b24663fa3e7d0938065.xlsx"},{"id":82709533,"identity":"b3dc8348-72f1-41be-a20b-9f85ca73fd23","added_by":"auto","created_at":"2025-05-14 11:16:02","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":13378,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementary.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6549793/v1/6d3681814877fb5f682af2ef.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"The added value of flavouring extracts from Calabrian chili peppers by-products: characterization of bioactive and volatile compounds comparing green extraction techniques","fulltext":[{"header":"1. INTRODUCTION","content":"\u003cp\u003eThe flavour represents a crucial element influencing sensory quality of food and drinks (Prescott, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Specifically, flavouring additives could be divided according to the origin in synthetic, composed by synthetically prepared compounds, and natural formulations, which must at least include the 95% of product directly obtained from plant, animal or microbiological materials, through appropriate physical, enzymatic or microbiological processes (European Regulation 1334/2008). The interest in industrial production of natural flavours had increased in the last decades due to the higher consumers concern in natural products. For instance, already in 2007, more than 65% of all the flavouring ingredients in USA and EU, were reported as \u0026ldquo;natural\u0026rdquo; labelled. Indeed, the natural origin of a food product was considered as important as its palatability (Yasin et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eChili peppers (\u003cem\u003eCapsicum annuum\u003c/em\u003e L.) are extensively involved as starting matrices in food flavourings industry. The widespread application of these \u003cem\u003eCapsicum\u003c/em\u003e fruits extracts extends to a countless number of different food products, including sauces, processed meats, snacks, beverages and cheese (Raju et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). From a botanical standpoint, the \u003cem\u003eCapsicum\u003c/em\u003e L. genus belongs to the Solanaceae family and it encompasses more than 30 species of peppers. Among them, five were domesticated with the \u003cem\u003eCapsicum annuum\u003c/em\u003e as one of the most used in industrial and home-made food preparation. It includes the Calabrian chili pepper variety, which is furtherly divided into different subvarieties (i.e. Ciliegino, Guglia, Diavulicchio, A Mazzetti, Naso Di Cane, Pizzitano, Sigaretta, Tondo). For their growth, the Calabrian chili peppers require the warm temperatures and sunlight of the mild climates of Calabria region (south of Italy) where they are largely cultivated. According to the obtained prestigious certifications, they must meet rigorous quality criteria, leading to a big amount discarded material (Cicatiello et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Actually, around 46% of this by-product biomass, including the relevant percentages of items that do not meet the required size and shape, is treated to be sold as dried powder or mixed with other spices (Yasin et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eA growing preference for natural chili pepper flavourings, such as extracts and oleoresins, has been experienced in the last decades, due to their concentrated colour and flavour. Oleoresins are obtained through solid-liquid extraction and subsequent evaporation of the solvent. They may retain the full sensorial characteristics of the spices and could include also the naturally present bioactive substances (Melgar-Lalanne et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). In fact, unlike synthetic additives, natural ones generally offer the added benefit to be source of bioactive compounds with antioxidant and antimicrobial activity. In particular, natural chili pepper flavouring usually contains capsaicinoids, polyphenols, carotenoids and vitamins, derived from the extracted matrix (Hern\u0026aacute;ndez-P\u0026eacute;rez et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe traditional natural flavourings obtaining processes, relies on organic solvents, such as hexane, acetone, ethanol or methanol (Madhusankha et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Among the other solvents involved in hydrophilic flavouring extraction, ethanol is widely used being non-toxic and worldwide authorized in food additives production, moreover, it is classified as green solvent (Madhusankha et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In particular, for lipophilic extractions, the use of hexane is predominant in the food industry, because it requires a shorter extraction time than other solvents and it is authorized in the USA by the Food and Drugs Administration (FDA). However, the more restrictive European directive allows hexane just within certain residue limits and after specific solvent-removing processes (European Regulation 2009/32).\u003c/p\u003e \u003cp\u003eAmong the green and sustainable extraction techniques, Supercritical Fluid Extraction (SFE) has emerged as a promising technique providing high extraction yields using CO\u003csub\u003e2\u003c/sub\u003e, which is non-toxic, non-flammable, cheap and easily available in large quantities with a high degree of purity (Duarte et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). In addition, CO\u003csub\u003e2\u003c/sub\u003e is in gas phase at room temperature and consequently there is no need for solvent-removing steps. To increase the polarity, a co-solvent could be added, and, in the context of green and sustainable solutions, water, ethanol and their mixtures are the most used.\u003c/p\u003e \u003cp\u003eThe main aim of this research work is to propose new green and sustainable strategies to obtain chili peppers flavourings from Calabrian chili peppers which were discarded, due to aspect or sizes requirements. Thus, Soxhlet, Ultrasound-Assisted Extraction (UAE) and SFE were compared in Calabrian chili pepper flavouring extracts production. Green solvents were also used, comparing different ethanol/water percentages, considering their spread and safe use in food flavourings industry. The bioactive compounds extraction power of each method was investigated monitoring polyphenols, flavonoids, antioxidant activity, carotenoids, vitamins C and alpha-tocopherol concentrations, together with their volatile profile composition and level of pungency, that are crucial for the typical chili pepper characteristics.\u003c/p\u003e"},{"header":"2. MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Reagents and standards\u003c/h2\u003e \u003cp\u003eThe analytical standards of delphinidin 3-galactoside chloride (absolute purity, C\u003csub\u003e21\u003c/sub\u003eH\u003csub\u003e21\u003c/sub\u003eO\u003csub\u003e12\u003c/sub\u003eCl) and kaempferol 3-glucoside (absolute purity, C\u003csub\u003e21\u003c/sub\u003eH\u003csub\u003e20\u003c/sub\u003eO\u003csub\u003e11\u003c/sub\u003e) were purchased from PhytoLab (Vestenbergsgreuth, Germany). Capsaicin (\u0026ge;\u0026thinsp;95%, C\u003csub\u003e18\u003c/sub\u003eH\u003csub\u003e27\u003c/sub\u003eNO\u003csub\u003e3\u003c/sub\u003e), dihydrocapsaicin (\u0026ge;\u0026thinsp;85%, C\u003csub\u003e18\u003c/sub\u003eH\u003csub\u003e29\u003c/sub\u003eNO\u003csub\u003e3\u003c/sub\u003e), L-ascorbic acid (99%, C\u003csub\u003e6\u003c/sub\u003eH\u003csub\u003e8\u003c/sub\u003eO\u003csub\u003e6\u003c/sub\u003e), gallic acid (\u0026ge;\u0026thinsp;97.5%, C\u003csub\u003e7\u003c/sub\u003eH\u003csub\u003e6\u003c/sub\u003eO\u003csub\u003e5\u003c/sub\u003e) neochlorogenic acid (\u0026ge;\u0026thinsp;98.0%, C\u003csub\u003e16\u003c/sub\u003eH\u003csub\u003e18\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e), chlorogenic acid (\u0026ge;\u0026thinsp;95%, C\u003csub\u003e16\u003c/sub\u003eH\u003csub\u003e18\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e), 4-hydroxy benzoic acid (99%, C\u003csub\u003e7\u003c/sub\u003eH\u003csub\u003e6\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e), caffeic acid (\u0026ge;\u0026thinsp;98.0%, C\u003csub\u003e9\u003c/sub\u003eH\u003csub\u003e8\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e), vanillic acid (97%, C\u003csub\u003e8\u003c/sub\u003eH\u003csub\u003e8\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e) p-coumaric acid (\u0026ge;\u0026thinsp;98%, C\u003csub\u003e9\u003c/sub\u003eH\u003csub\u003e8\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e), ferulic acid (\u0026ge;\u0026thinsp;99%, C\u003csub\u003e10\u003c/sub\u003eH\u003csub\u003e10\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e), phloridzin (\u0026ge;\u0026thinsp;98.0%, C\u003csub\u003e21\u003c/sub\u003eH\u003csub\u003e24\u003c/sub\u003eO\u003csub\u003e10\u003c/sub\u003e), hesperidin (\u0026ge;\u0026thinsp;97.0%, C\u003csub\u003e28\u003c/sub\u003eH\u003csub\u003e34\u003c/sub\u003eO\u003csub\u003e15\u003c/sub\u003e), α-tocopherol (\u0026ge;\u0026thinsp;96% C\u003csub\u003e29\u003c/sub\u003eH\u003csub\u003e50\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e), 2,2-diphenyl-1-picrylhydrazil (DPPH, C\u003csub\u003e18\u003c/sub\u003eH\u003csub\u003e12\u003c/sub\u003eN\u003csub\u003e5\u003c/sub\u003eO\u003csub\u003e6\u003c/sub\u003e), 6-hydroxy-2,5,7,8 tetramethylchromane-2-carboxylic acid (\u0026ge;\u0026thinsp;97%, Trolox, C\u003csub\u003e14\u003c/sub\u003eH\u003csub\u003e18\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e), rutin (\u0026ge;\u0026thinsp;94%, C\u003csub\u003e27\u003c/sub\u003eH\u003csub\u003e30\u003c/sub\u003eO\u003csub\u003e16\u003c/sub\u003e) and Folin-Ciocalteu phenol reagent were supplied by Sigma\u0026ndash;Aldrich (Milan, Italy). HPLC-grade methanol, acetonitrile and isopropanol were purchased from Sigma\u0026ndash;Aldrich (Milan, Italy), while HPLC-grade formic acid 99\u0026ndash;100% was bought from J.T. Baker B.V. (Deventer, Holland); all the solvents used were analytical grade. A Milli-Q System (Bedford, MA, USA) was used to obtain deionized water (\u0026lt;\u0026thinsp;8 MΩ cm resistivity).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Sample Collection\u003c/h2\u003e \u003cp\u003eSamples were selected among those Calabrian chili peppers (Naso Di Cane subvariety) which were evaluated as not suitable to be sold as fresh, due to the sizes or shape requirements. New Flavours\u0026reg; company (Via dell'Artigianato 7, Monte Santa Tiberina, PG, Italy) provided pre-dried and finely grinded samples, which were stored at -20\u0026deg;C until analyses.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Extractions procedures\u003c/h2\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003e2.3.1 Soxhlet extraction\u003c/h2\u003e \u003cp\u003eAn aliquot of 5 grams of pre-dried chili pepper powder was extracted using a 6-channels Universal Extractor in Soxhlet mode (B\u0026uuml;chi E-800) for 4 hours. A preliminary step of matrix/solvent ratio (m/s) optimization was conducted testing m/s of 1:25 (0.04) g/mL, 1:50 (0.02) g/mL and 1:100 (0.01) g/mL. Different ethanol/water solutions were explored, i.e. 100% ethanol (EtOH100), ethanol/water 70:30 \u003cem\u003ev/v\u003c/em\u003e (EtOH70), or ethanol/water 50:50 \u003cem\u003ev/v\u003c/em\u003e (EtOH50). All extractions experiments were conducted in triplicate. After each extraction, the solvent was evaporated until constant weight. Thus, the extraction yields (EY%) were calculated as the ratio of the dry extract weight and the initial amount of dried matrix.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e2.3.2 Ultrasound-assisted extraction\u003c/h2\u003e \u003cp\u003eThe optimized m/s was applied to UAE procedure. The extraction was performed on pre-dried chili pepper powder, the involved instrument was an Ultrasonic Bath (ARGOLAB AU-220), with a frequency of 40 KHz, for 20 minutes at 40\u0026deg;C. The same three Soxhlet solvent mixtures were tested as solvents. All extraction experiments were conducted in triplicate. Then, extracts were dried and EY% were calculated as previously mentioned.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e2.3.3 Supercritical Fluid Extraction\u003c/h2\u003e \u003cp\u003eThe instrument used was an SFT-120XW Extractor with a 100-mL extraction vessel, an oven, SFT-10 PID controllers and a Nex10 SCF pump. The extraction parameters were adopted from previously published optimization studies, being 330 bar and 40\u0026deg;C (Yan et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Given the instrument's attributes, static and dynamic phases were alternated to enhance the extraction yield. In each trial, the extraction started with a 10-min static period followed by a 15-min dynamic mode, this scheme was repeated twice for an extraction time of 50 min. A total of 25 mL of co-solvent was used for the extraction of 5 g of chili pepper powder. The three previously described solvent mixtures were tested. The extracts were collected in glass vials once the CO\u003csub\u003e2\u003c/sub\u003e gas had been fully expelled from the system. All the experiments were performed in triplicate, the EY% and the concentration of analytes were monitored after each repetition.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Analysis of bioactive compounds in Calabrian chili pepper flavouring extracts\u003c/h2\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e2.4.1 Spectrophotometric assays\u003c/h2\u003e \u003cp\u003eFor the evaluation of Total Phenolic Content (TPC), the Folin-Ciocalteu method was used (Moreno-Ram\u0026iacute;rez et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Dry extracts were firstly dissolved in methanol (2.5 mg/mL) and 0.5 mL of extract solution were added to test tubes along with 2.5 mL of 1:10 water solution of Folin-Ciocalteu phenolic reagent. After 5 minutes, 7 mL of Na\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e (7.5% \u003cem\u003ew/v\u003c/em\u003e) solution were added and mixture was kept in the dark for 2 hours at room temperature.\u003c/p\u003e \u003cp\u003eThe Total Flavonoic Content (TFC) was determined according to Alessandroni et al. (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Thus, 0.5 mL of each resuspended extract were mixed with 0.15 mL of a NaNO\u003csub\u003e2\u003c/sub\u003e 0.5 M aqueous solution and then 3.2 mL of methanol/water 30/70 \u003cem\u003ev/v\u003c/em\u003e were added. After 5 min, 0.15 mL of a 0.3M AlCl\u003csub\u003e3\u003c/sub\u003e and 1 mL of a 1M NaOH aqueous solutions were added. Then, after vortexing, the mixture was stored in the dark for 30 min at room temperature.\u003c/p\u003e \u003cp\u003eThe radical-scavenging activity was assessed according to the DPPH method (Alessandroni et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The procedure involves 0.5 mL of diluted extract, which was mixed with 4.5 mL of a 0.1 mM DPPH (2,2-diphenyl-1-picrylhydrazyl) ethanolic solution. Then, the mixture was stored for 30 min in the dark at room temperature.\u003c/p\u003e \u003cp\u003eThe Total Carotenoid Content (TCC) was performed resuspending 15 mg of each dry extract in 6 mL of petroleum ether stabilized with 0.2% \u003cem\u003ew/v\u003c/em\u003e BHT (butylated hydroxytoluene) and centrifuging for 10 min at 5000 rpm (IEC CL10 Centrifuge, Thermo Fisher Scientific, Waltham, USA).\u003c/p\u003e \u003cp\u003eThen, for each assay, the absorbance was measured spectrophotometrically using a Cary 8454 UV-visible spectrophotometer (Agilent Technologies, Woburn, MA, USA). The wavelengths were 765 nm, 506 nm and 517 nm, and the results were expressed in mg of gallic acid equivalents (GAE)/g of dry extract, mg of rutin equivalents (RE)/g of dry extract, mg of Trolox equivalents (TE)/g of dry extract, for TPC, TFC and DPPH respectively. The TCC (expressed as \u0026micro;g/g of dry extract) was calculated through the \u003cb\u003eEq.\u0026nbsp;1\u003c/b\u003e, after directly measuring the absorbance at 450 nm (Larocca, et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cspan class=\"InlineEquation\"\u003e \u003cspan class=\"mathinline\"\u003e\\(\\:TCC\\:(\\mu\\:g/g)=\\:\\frac{A\\:\\times\\:V\\left(mL\\right)\\times\\:{10}^{4}}{{{A}_{1}}_{cm}^{1\\%}\\times\\:P\\left(g\\right)}\\)\u003c/span\u003e \u003c/span\u003e \u003cb\u003eEq.\u0026nbsp;1.\u003c/b\u003e\u003c/p\u003e \u003cp\u003ewhere A is the absorbance of the sample, V (mL) is the total volume of the extract, is the β-carotene extinction coefficient in petroleum ether (2592 mL/g∙cm) and P (g) is the weight of the dried extract (Larocca, et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Experiments were performed in triplicate for each extract.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003e2.4.2 Vitamin C quantification through HPLC-DAD\u003c/h2\u003e \u003cp\u003eAn aliquot of 2 mg of each dry extract was dissolved in 1 mL of HPLC-grade methanol, then filtered through a 0.45 \u0026micro;m PTFE filter. The instrument was an HPLC Agilent 1260 Infinity II series (Santa Clara, CA, USA), coupled with a diode array detector (DAD) and equipped with a Synergi Polar-RP C18 (4.6 mm \u0026times; 150 mm, 4 \u0026micro;m) from Phenomenex (Torrance, CA, USA). The analytical column temperature was set at 40\u0026deg;C, while the injection volume was 10 \u0026micro;L. The mobile phase was made up of MilliQ water with 0.1% formic acid (A) and methanol with 0.1% formic acid (B) and the flow rate was 0.5 mL/min. The elution was performed in gradient mode: 0\u0026ndash;6 min 10% B, 6\u0026ndash;12 min 90% B. The absorbance was monitored at 245 nm and results were reported as \u0026micro;g/g DE using a Vitamin C analytical standard calibration curve (R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.99). The limit of detection (LOD) and limit of quantification (LOQ) were determined as 0.015 \u0026micro;g/g and 0.050 \u0026micro;g/g, respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003e2.4.3 Capsaicin and dihydrocapsaicin quantification through HPLC-DAD\u003c/h2\u003e \u003cp\u003eThe same HPLC-DAD equipment was used also for the quantification of capsaicin and dihydrocapsaicin, after dissolving 2.5 mg of each dry extract in 1 mL of HPLC-grade acetonitrile and filtering with a 0.45 \u0026micro;m PTFE filter. The separation of analytes was obtained by a Gemini 5u RP-C18 analytical column (150 mm \u0026times; 4.6 mm \u0026times; 5 \u0026micro;m) from Phenomenex (California, USA), set at 40\u0026deg;C. The mobile phase was composed of MilliQ water (A), and acetonitrile (B), working isocratic mode with 50% A and 50% B at a flow rate of 1 mL/min. The injection volume was 5 \u0026micro;L. For the detection of capsaicinoids, the 228 nm wavelength absorbance was used. The results are expressed as \u0026micro;g /g dry extracts. A calibration curve of each standard was prepared and showed an excellent linearity (R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.99). LOD and LOQ were 0.29 \u0026micro;g/g and 0.95 \u0026micro;g/g for the dihydrocapsaicin and 0.27 \u0026micro;g/g and 0.89 \u0026micro;g/g for capsaicin.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e \u003ch2\u003e2.4.4 Alpha-tocopherol quantification\u003c/h2\u003e \u003cp\u003eA total of 2 mg of each sample were redissolved in 1 mL of an acetonitrile\u0026ndash;methanol\u0026ndash;dichloromethane solution (60:30:10 v/v), then mixed and centrifuged at 5000 rpm for 5 min at room temperature and filtered with 0.45 \u0026micro;m PTFE filters. Alpha-tocopherol was quantified as reported by Nzekoue et al. (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), with few modifications. Briefly, the HPLC 1260 equipment was used, coupled with a Fluorescence Detector (FLD). The separation of analytes was obtained by Chromolith RP-18e analytical column (100 \u0026times; 3 mm I.D., macropore size 2 \u0026micro;m, mesopore size 13 nm) from Merck (Darmstadt, Germany), set at 40\u0026deg;C. The mobile phase was composed of acetonitrile (A), and 2-propanol (B), at a flow rate of 0.8 mL/min. Elution was carried out in gradient mode as follows: 0\u0026ndash;5 min 100% A; 5\u0026ndash;9 min 80% A and 20% B, and a recondition time of 7 minutes. The injection volume was 10 \u0026micro;L. For quantification, 295 nm and 330 nm as excitation and emission wavelengths, respectively. LOD (0.02\u0026ndash;0.05 \u0026micro;g/g) and LOQ (0.05\u0026ndash;0.15 \u0026micro;g/g) were previously assessed by Nzekoue et al. (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section3\"\u003e \u003ch2\u003e2.4.5 Characterization of polyphenols by UHPLC-MS/MS\u003c/h2\u003e \u003cp\u003eThe detection of polyphenols from 1 mg/mL methanolic solution of each dried extract was carried out with an Agilent 1290 Infinity series with a Triple Quadrupole 6420 (Agilent Technology, Santa Clara, CA). Before injection, samples were filtered through a 0.2 \u0026micro;m polyamide filter from Sartorius Stedim (Goettingen, Germany). The used method was previously published by Angeloni et al. (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Briefly, analytes separation was performed on a Synergi Polar\u0026ndash;RP C18 analytical column (250 mm \u0026times; 4.6 mm, 4 \u0026micro;m) from Phenomenex (Chesire, UK) set at 30\u0026deg;C, with a Polar RP security guard cartridge (4 mm \u0026times; 3 mm ID). The mobile phase consisted of (A) MilliQ water and (B) methanol, both with formic acid 0.1%. The flow rate was 0.8 mL/min with the following elution gradient: 0\u0026ndash;1 min, 20% B; 1\u0026ndash;25 min, 20\u0026ndash;85% B; 25\u0026ndash;26 min, 85% B; 26\u0026ndash;32 min, 85\u0026ndash;20% B. The injection volume was 2 \u0026micro;L. The dynamic-multiple reaction monitoring (dynamic-MRM) mode was chosen for detection and the peak areas were integrated for quantification.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Analysis of volatile organic compounds using HS-SPME-GC-MS\u003c/h2\u003e \u003cp\u003eThe volatile organic compounds (VOCs) analysis was performed directly on the dried chili pepper matrix and on the obtained dry extracts. A total of 1 g of the dried and finely grinded chili matrix or 100 mg of each dry extract were placed in SPME vials, together with a 1 mg/mL NaCl water solution. A gas chromatography coupled with mass selective detector (GC-MS) with PAL3-Auto Sampler System (Agilent 7890B GC Hardware with an Agilent 5977 Series MSD, Agilent, Santa Clara, CA, USA) and MassHunter GC/MSD Data Aquisition were used. A fiber with a 50/30 \u0026micro;m coating of divinylbenzene/carbon-wide range/polydimethylsiloxane (DVB/C-WR/PDMS, 1 cm length stationary phase) from Supelco (Bellefonte, PA, USA) was selected for this work. The fiber had been conditioned for 10 min at 250\u0026deg;C, then the extraction was performed for 10 min at 60\u0026deg;C. The desorption occurred at 250\u0026deg;C for 2 min, then the fiber was reconditioned at 250\u0026deg;C for 15 min. The column was an HP-5MS UI (30 m x 250 \u0026micro;m x 0.25 \u0026micro;m) (Agilent, 19091S-433UI, Santa Clara, CA, USA). The He flow rate was 1 mL/min in spitless mode. The injector temperature was 260\u0026deg;C. The column temperature was programmed as follows: from 37\u0026deg;C (held for 5 min) to 100\u0026deg;C (4\u0026deg;C per min), immediately from 100\u0026deg;C to 170\u0026deg;C (3\u0026deg;C per min), from 170\u0026deg;C to 230\u0026deg;C (15\u0026deg;C per min) and, lastly, to 250\u0026deg;C (15\u0026deg;C per min), holding this temperature for 3 min; the total run time was 52.417 min. The source was an electron impact ionization (EI) and data were collected in SCAN mode; the transfer line was set at 250\u0026deg;C and the temperature of the ionization source and the mass analyser were set at 230 and 150\u0026deg;C, respectively. Compounds identification was performed by comparison with NIST library (US National Institute of Standards and Technology), and injecting alkane mixes (C\u003csub\u003e6\u003c/sub\u003e-C\u003csub\u003e22\u003c/sub\u003e) and calculating the retention indices. The results are expressed as relative abundances of each volatile compound (% peak area of the compound/ area of all the peaks).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Statistical data elaboration and analysis\u003c/h2\u003e \u003cp\u003eDifferences among extracts were evaluated by one-way analysis of variance (ANOVA). Values with \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e were considered statistically significant. Bioactive compounds values in Soxhlet, UAE and SFE extracts were distributed in a Principal Component Analysis (PCA) performed with XLSTAT software (version 2023.1.4.1408). The entire dataset, including VOCs results, was normalized through log-transformation and Pareto-scaling before Ward hierarchical clustering analysis. This was expressed using a heatmap with dendrograms to provide an intuitive visualization of a data table.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. RESULTS AND DISCUSSION","content":"\u003ch2\u003e3.1. Optimization of matrix/solvent ratio through Soxhlet experiments\u0026nbsp;\u003c/h2\u003e\n\u003cp\u003eAs first step, different m/s were tested to optimize this parameter, using ethanol and ethanol/water mixtures as solvents. To the best of our knowledge the decrease of solvent volume, which implies lower expenses together with a shorter evaporation time, has not been thoroughly explored in scientific literature for Soxhlet extraction. Several experiments were performed comparing different matrix/solvent ratios (1/25, 1/50 or 1/100 g/mL), within EtOH100, EtOH70 and EtOH50, are reported in \u003cstrong\u003eTable 1.\u003c/strong\u003e All the analysis were carried out on dry extracts as it represents the more suitable product from an industrial application standpoint. As expected, high extraction yields (EY%) were reported in the lowest matrix/solvent ratio experiments, and this is in line with previous studies (Ivan et al., 2024).\u003c/p\u003e\n\u003cp\u003eNo statistically significant differences emerged for TPC, TFC, antioxidant activity (DPPH), capsaicin and dihydrocapsaicin content, between the different m/s explored. Capsaicin and dihydrocapsaicin are well-known molecules in chili pepper flavorings, since they are the main responsible compound of the pungency.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eConversely, among the EtOH100 extracts, statistically significant differences were reported for five specific polyphenols quantified through HPLC-MS-MS, being \u0026nbsp;gallic and chlorogenic acid, phloridzin, 4-hydroxy benzoic acid and kaempferol-3-glucoside. Their concentrations were \u0026nbsp;higher in 1/25 m/s extracts. On the other hand, vanillic acid was more extracted in low m/s samples with statistically significant differences in EtOH100 experiments.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAmong the EtOH70 group of extracts, higher EY% were reported in 1/50 and 1/100 m/s samples (28.53 % \u0026plusmn; 0.42 % and 28.53 % \u0026plusmn; 1.67 %, respectively), with a statistically significant difference. This outcome could be explained by a higher effectiveness of a larger solvent volume, which is also heterogeneous in terms of polarity, due to the water presence. The same trend was experienced in TCC results. Similarly, p-coumaric acid, caffeic acid, vanillic acid, and kaempferol-3-glucoside were significantly less extracted using the highest m/s. These antioxidant, anti-cancer, antimicrobial or anti-inflammatory polyphenols were found also in other \u003cem\u003eCapsicum Annuum\u003c/em\u003e subvarieties, especially vanillic acid-derivatives were the predominant ones (Barbero et al., 2016).\u003c/p\u003e\n\u003cp\u003eConversely, gallic acid and neochlorogenic acid where significantly more extracted using high m/s.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAmong EtOH50 extracts, no statistically significant difference in the monitored parameters was evaluated. Only hesperidin was quantified in small concentrations and just in EtOH50 1/100 m/s sample, probably due to a low affinity for ethanol (Ma et al., 2008). In fact, this flavanone glycoside was also detected in other species of chili pepper, such as in red bell and Balady green peppers extracts, after methanolic extraction (Abdalla et al., 2019), and in habanero pepper by-products , using 80:20 v/v methanol/water solution (Chel-Guerrero et al., 2021).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2\u003c/strong\u003e reports the results of VOCs detected in the dry matrix and in Soxhlet dry extracts. Many VOCs detected in the matrix were not identified in low m/s Soxhlet extracts, regardless of the extractive solvent. A total of ten compounds where in common between the dry matrix and the 1/25 m/s extracts, being trans-beta-ionone, 5,9-undecadien-2-one,6,10-dimethyl, alpha-curcumene, tetradecanoic acid 12-methyl-methyl ester, 2-methylpentadecane, pentadecane,3-methyl, 2-methylhexadecane and 2-methyl-1-tetradecene. Among these volatile compounds, the 5,9-undecadien-2-one,6,10-dimethyl was already identified both in a fresh and not spicy pepper from \u003cem\u003eC. annuum\u003c/em\u003e and in a fresh and spicy pepper from \u003cem\u003eC. chinense\u003c/em\u003e (Xu et al., 2020). Qiu et al. (2023) detected this molecule in fermented \u003cem\u003eC. annuum\u003c/em\u003e red pepper, as well. The 2-methyl-1-tetradecene was previously reported in different \u003cem\u003eCapsicum\u003c/em\u003e fruits by Antonio et al. (2018).\u003c/p\u003e\n\u003cp\u003eIn this scenario, a lower m/s can be related both to a longer drying process, when an equal amount of matrix is used, or to an insufficient amount of the material to extract, for an equal volume of solvent used: that can lead to possible losses of volatile molecules. At the same time, just five VOCs were detected only in low m/s samples being hexanoic acid, D-limonene, thymol, tetradecane, nonadecane.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn conclusion, considering the emerged results, operating with low m/s value can be related to the advantage of a lower solvent volume consumption, to obtain similar extract characteristic.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003e3.2. Bioactive compounds in chili pepper extracts from Soxhlet, UAE and SFE\u003c/h2\u003e\n\u003cp\u003eConsidering the various potentialities of chili pepper extracts, different green solvent mixtures with distinct polarities were tested in this study, with the aim to select the more performative ones for the formulation of value-added flavouring extracts from discarded matrices. Soxhlet, UAE and SFE techniques were investigated using different ethanol/water mixtures, as mentioned in section 2.3.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eResults from EY%, total content of polyphenols, flavonoids, carotenoids, antioxidant activity, bioactive compounds and polyphenols quantification were grouped in a database which was elaborate by means of a principal component analysis (PCA). The variable projection allowed a clear view on the distribution of the extracts, according to the nine different extraction conditions (three methods and three solvents). The PCA biplot is illustrated in \u003cstrong\u003eFig. 1\u0026nbsp;\u003c/strong\u003eand numerical data are reported in\u003cstrong\u003e\u0026nbsp;Table 1S\u003c/strong\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe high variability percentage (73.94%; 53.40% and 20.54% for F1 and F2, respectively) confirms the reliability of the results and the sample distribution reflects the reproducibility. It is interesting to note that the SFE samples are distributed on the right, while the UAE and Soxhlet ones are partially mixed. This samples distribution is in line with the projections of the less polar analytes on the right part of the graph. In fact, it is well known that the carbon dioxide has high affinity for non-polar compounds and, despite the addition of the polarity-increasing co-solvent (ethanol and ethanol-water mixtures), also analytes with lipophilic properties are extracted. \u0026nbsp;UAE and Soxhlet extracts showed similar characteristics, except in EtOH100 experiments, when UAE revealed its greater capacity in small polar compound extraction.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe solvent nature impacts also the EY%, which was significantly higher in UAE and Soxhlet when an EtOH/H\u003csub\u003e2\u003c/sub\u003eO mixture is involved. It has been previously reported that the addition of polar co-solvents, during SFE, can increase the EY% but it cannot reach the values of Soxhlet and UAE (Soldan et al., 2021).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAmong UAE experiments, EtOH100 EY% were almost 30% lower than those with aqueous solutions. This could be related to the cavitation effect and the mechanical waves propagation in the solvent, which seem to be more effective in presence of water. \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe Soxhlet samples EY% were higher than those previously reported by both\u0026nbsp;Bae et al. (2012)\u0026nbsp;and\u0026nbsp;Prabakaran et al. (2017),\u0026nbsp;involving different\u0026nbsp;solvents\u0026nbsp;on others chili peppers varieties and ethanolic maceration of red bell peppers (\u003cem\u003eC. annuum\u003c/em\u003e), respectively.\u003c/p\u003e\n\u003cp\u003eRegarding the monitored variables, clearly TPC, TFC, capsaicin, dihydrocapsaicin and alpha-tocopherol showed higher values in ethanol than in hydroalcoholic solvent. This aspect could be explained by the lower polarity of these compounds and confirmed by the presence of polyphenols and flavonoids with an intermediate lipophilicity, as reported also in other \u003cem\u003eC. annuum\u003c/em\u003e varieties (Chilczuk et al., 2021). Pure ethanol was proven to extract higher contents of polyphenols in \u003cem\u003eC. annum\u003c/em\u003e species in general, if compared to other solvents (Prabakaran et al., 2017).\u003c/p\u003e\n\u003cp\u003eThe extractabilities of capsaicin and dihydrocapsaicin are closely and positively related to each other and negatively related to radical-scavenging compounds. They together represent the 80-90% of all the capsaicinoids in most species of chili peppers (Barbero et al., 2016). Their concentration was around 2 times higher in EtOH100, regardless of the extractive technique chosen. Indeed, comparing the EtOH100 extracts, the non-polar nature of carbon dioxide has raised the total content of capsaicin and dihydrocapsaicin \u0026nbsp;by 21% and 41%, than Soxhlet and UAE, reaching concentrations of 7.61\u0026plusmn;0.42 mg/g and 3.84\u0026plusmn;0.22 mg/g respectively. The results clearly highlight that SFE with ethanol as co-solvent is the best option to obtain an extract with high pungency, as it is directly related to these two capsaicinoids concentrations, according to the Scoville Heat Units (SHU) (\u003cstrong\u003eEquation 2)\u0026nbsp;\u003c/strong\u003e(Sweat et al., 2016).\u003c/p\u003e\n\u003cp\u003e\u003cimg 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\" style=\"width: 648px; height: 27.0728px;\" width=\"648\" height=\"27.0728\"\u003e\u003c/p\u003e\n\u003cp\u003eThe SHU of the SFE EtOH100 dry extract was around 184000, which is very high, if compared to the 50000 of the Calabrian chili pepper fresh fruit (Popelka et al., 2017).\u003c/p\u003e\n\u003cp\u003eIn the field of \u0026nbsp;natural flavorings preparation, it is important to take into account the compounds related to the aromatic notes but also the bioactive molecules which could bring added value to the products. \u0026nbsp;Capsaicinoids resulted positively correlated with alpha-tocopherol which was quantified in all the analysed extracts, with higher concentrations in EtOH100 samples. The concentration of alpha-tocopherol varies according to \u0026nbsp;\u003cem\u003eC. annuum\u003c/em\u003e varieties and the emerged levels are in accordance with previous studies (Olatunji, 2020).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAmong the bioactive compounds, the concentration of vitamin C in the extracts is enhanced by the use of water as a co-solvent. This vitamin represents not only an added value for its bioactivity, but also an important compound for the final product preservation, for its antioxidant activity.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eRegarding polyphenols, \u003cem\u003ep-\u003c/em\u003ecoumaric acid, 4-hydroxy benzoic acid, ferulic acid, vanillic acid, phloridzin, caffeic acid and gallic acid resulted to be positively correlated each other. This is related to structural similarities, especially within the group of the hydroxycinnamic acid derivatives, which reached more than 2-fold higher concentrations in SFE extracts, compared to Soxhlet or UAE ones. Due to polarity differences, this group of compounds is also clearly negatively correlated with vitamin C. Gallic acid was already reported in SFE extracts from \u003cem\u003eC. annuum\u003c/em\u003e var. \u003cem\u003eglabriusculum\u0026nbsp;\u003c/em\u003eby Moreno-Ram\u0026iacute;rez et al. 2018). Hesperidin was detected only in UAE samples as facilitated by the polarity of aqueous solvents and also by cavitation phenomenon (Cheigh et al., 2012). Chlorogenic and neochlorogenic acids showed lower concentrations than other monitored polyphenols, due to their chemical instability and possible degradation during extraction and solvent evaporation processes. These two polyphenols have been reported in other pepper varieties not belonging to the \u003cem\u003eCapsicum\u003c/em\u003e genus (Zhang et al., 2021).\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003e3.3. Volatile profile of Soxhlet, UAE and SFE chili pepper flavouring extracts by HS-SPME-GC-MS\u003c/h2\u003e\n\u003cp\u003eThe volatile profile of the chili pepper flavouring extracts was explored through a HS-SPME-GC-MS approach and the results are resumed in \u003cstrong\u003eTable 3\u003c/strong\u003e. The highest variability of \u0026nbsp;identified volatile compounds, with the 55.56% of the compounds in common with the dry matrix itself, was reported in SFE EtOH100 dry extracts. A similar trend was observed for SFE EtOH70 or EtOH50. A different volatile profile emerges in UAE EtOH70 extracts, with a total of 48 identified VOCs but only the 35.42% of them in common with dry matrix. This phenomenon could be explained by the great extraction capacity of cavitation.\u003c/p\u003e\n\u003cp\u003eComparing the three extractive techniques, the SFE extracts present higher relative abundances of alkanes, acids, ketones and aldehydes. In fact, CO\u003csub\u003e2\u003c/sub\u003e allowed the extraction of longer aliphatic chains and less/non-polar compounds. Indeed, the SFE procedure is able to retain most of the fragrances and the pungent principles of the matrix.\u003c/p\u003e\n\u003cp\u003eSoxhlet and UAE dry extracts were characterised by several heterocycles and alkenes, mainly characterized by weak \u0026ldquo;fuel\u0026rdquo; odours (e.g., 3-hexadecene, 1-tetradecene or 1-octadecene), which were undetected both in SFE samples and in the dry matrix.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMany of them may be degradation products of matrix compounds, as treated with high temperature, as Soxhlet, or invasive processes, as UAE (Essien et al., 2020).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAldehydes, mostly deriving from fatty acids carboxylic group reduction, were identified in the extracts but not in the dry matrix. Among them, nonanal and decanal were detected in UAE EtOH50 samples and in SFE extracts. These compounds, linked to citrus notes, had previously been reported in chili pepper extracts (Korkmaz et al. 2017). Safranal, hexanal and benzaldehyde with their spicy, cherry, leafy or tabacco-like notes were identified in the dry matrices but not in the extracts.\u003c/p\u003e\n\u003cp\u003eAmong the detected oxygenated monoterpenes, linalool and alpha-terpineol, both characterized by a floral odour, were detected only in EtOH50 UAE and in the SFE extracts. Linalool was also found in green and red chili pepper essential oils by Don et al. (2019). They concluded that its concentration is directly related to the ripeness degree of \u003cem\u003eC. annuum\u003c/em\u003e. Conversely, dihydroactinidiolide, a musky or tea-smelling product resulting from the oxidative metabolism of beta-carotene, was identified both in the dry matrix and in all extracts, except for SFE ones.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAlkanes are the most abundant class of compounds both in the dry matrix and in the SFE extracts, according with their non-polar characteristics. In particular, 2-methyl tetradecane was the most abundant alkane in dry matrix and in SFE extracts.\u003c/p\u003e\n\u003cp\u003eConversely, ketones, related to floral, fruity and woody aromas, were mostly abundant in SFE extracts, especially with EtOH50 solvent. Among them, the trans-beta-ionone, identified in the dry matrix and in most of the extracts, was previously investigated as a marker of an initial beta-carotene degradation in the dry matrix (Variyar \u0026amp; Suprasanna, 2024).\u003c/p\u003e\n\u003ch2\u003e3.4 Heatmap and hierarchical clustering of chili pepper extracts analysed features\u003c/h2\u003e\n\u003cp\u003eAll the obtained results from the analysis of the chili pepper extracts were grouped in a database, data were normalized and involved in a heatmap with hierarchical clustering (\u003cstrong\u003eFig. 2)\u003c/strong\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe difference in chemical composition between SFE extracts and the others, clearly emerges from the discussed results and is further confirmed by the first level of sample clustering which groups together the Soxhlet and UAE samples, especially when EtOH100 was used. Thus, within SFE group, EtOH70 or EtOH50 extracts resulted similar and not well clustered.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe SFE group clustering, among extraction procedures, is mainly due to non-polar nature of the carbon dioxide, regardless of the co-solvent mixture used. Among the emerging features in SFE samples there are: TCC, p-coumaric acid, 4-hydroxy benzoic acid, ferulic acid, vanillic acid, phloridzin, caffeic acid, gallic acid, capsaicin and dihydrocapsaicin concentrations, along with some alkanes (e.g., tetradecane-2-methyl or pentadecane) and alpha-curcumene, which is a volatile sesquiterpene hydrocarbon characterised by a spicy odour. From the clustering, these polyphenols seem to be quite correlated also with capsaicin and dihydrocapsaicin: a biosynthetic pathway actually leads to capsaicinoids and connects their structures with those of these specific polyphenols (Shams et al., 2023). Also the structural analogies of these two capsaicinoids investigated is confirmed by the features dendrogram at first level.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOn the other hand, the clustering of Soxhlet and UAE samples is also due to the detection of the following features: vitamin C, the antioxidant activity level, the volatile alkenes detection, together with a higher content of hydrophilic compounds, such as chlorogenic acid, neochlorogenic acid and kaempferol-3-glucoside, especially in EtOH70 and EtOH50 samples. As discussed in section 3.3, many alkenes could be fatty acids degradation products deriving from heating or invasive processes, like in Soxhlet extraction and UAE. Moreover, among Soxhlet and UAE extracts, the EtOH100 ones are clustered together due to similarities in capsaicinoids concentration, TPC and TFC results. The antioxidant activity (DPPH) is clustered with EY% and vitamin C in features dendrogram, confirming the important contribution of this vitamin to the radical-scavenging capacity of these extracts. In conclusion, in SFE extract a higher concentration of hydrophobic bioactive compounds was detected, together with a very high amount of capsaicinoids, responsible for the typical pungency of chili peppers. On the opposite the SFE extract is poor in vitamin C, which is more extracted by Soxhlet and UAE systems.\u0026nbsp;\u003c/p\u003e"},{"header":"4. CONCLUSIONS","content":"\u003cp\u003eThe exploration of different green extraction strategies to obtain Calabrian chili pepper flavourings underlined the specific final product characteristics according to the involved technique. The prior establishment of the matrix/solvent ratio for Soxhlet extraction revealed the less solvent-consuming procedure as the optimal strategy in terms of extract properties and composition. The added value of natural flavourings has been strongly remarked by the results of this study. Soxhlet and UAE emerged as optimal techniques for vitamin C and the most polar antioxidant compounds \u0026ndash; rich dry flavourings. Conversely, carotenoid \u0026ndash; rich extracts, with high pungency and total polyphenolic content, could be obtained working in SFE. At the same time, the solvent choice is crucial to control the components of the final products.\u003c/p\u003e \u003cp\u003eIn conclusion, this research may represent an advancement in value-added flavourings extracts preparation to be used as natural food additives, proposing effective, diverse and sustainable techniques and green and safe solvent mixtures. In the meantime, it raises the issue of valorisation of food matrices that are daily discarded from the fresh products supply chain, proposing their alternative use as sources of flavourings extract to be reinvolved in the food industry.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAcknowledgements\u0026nbsp;\u003c/h2\u003e\n\u003cp\u003eAuthors thank the company New Flavors (Via Dell\u0026rsquo;Artigianato 7, 06010, Monte Santa Tiberina, Italy) for supporting this research and for Calabrian chili peppers samples supply.\u003c/p\u003e\n\u003ch2\u003eDeclaration of Interest\u003c/h2\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Declaration\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThere was no funding for the realized research.\u003c/p\u003e\n\u003ch2\u003eCRediT authorship contribution statement\u003c/h2\u003e\n\u003cp\u003e\u003cstrong\u003eSC\u003c/strong\u003e: Investigation, Methodology, Validation, Data curation, Writing - original draft. \u003cstrong\u003eLA\u003c/strong\u003e: Conceptualization, Visualization, Validation, Data curation, Writing - review \u0026amp; editing. \u003cstrong\u003eJCC\u003c/strong\u003e\u003cstrong\u003e; VT\u003c/strong\u003e: Resources; Funding acquisition.: \u003cstrong\u003eGS\u003c/strong\u003e: Resources; Funding acquisition; Project administration; Supervision, Writing - review \u0026amp; editing.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAbdalla, M. 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A comparative overview on chili pepper (\u003cem\u003ecapsicum\u003c/em\u003e genus) and sichuan pepper (\u003cem\u003ezanthoxylum\u003c/em\u003e genus): From pungent spices to pharma-foods. \u003cem\u003eTrends in Food Science \u0026amp; Technology\u003c/em\u003e, 117, 148-162.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 to 3 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"european-food-research-and-technology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [European Food Research and Technology](https://link.springer.com/journal/217)","snPcode":"217","submissionUrl":"https://submission.springernature.com/new-submission/217/3","title":"European Food Research and Technology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Flavourings extraction, Supercritical Fluid Extraction, chili peppers, green extraction, bioactive compounds","lastPublishedDoi":"10.21203/rs.3.rs-6549793/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6549793/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe industrial demand for natural flavourings is increasing, together with green technologies development and diffusion. This article investigates different extraction strategies for flavouring obtainment from Calabrian chili peppers supply chain by-products. Green solvent mixtures were tested on Soxhlet, Ultrasound-Assisted Extraction (UAE) and Supercritical Fluid Extraction (SFE). Extracts were characterized through bioactive compounds quantification and volatile profile monitoring. Preliminary Soxhlet experiments set the matrix/solvent ratio at 1/25 (g/mL). SFE extracts exhibited the highest concentration of less-polar polyphenols, such as flavonoids, total carotenoids, but also alpha-tocopherol and capsaicinoids. On the other hand, the UAE and Soxhlet processes, involving ethanol/water mixtures, allowed the highest yields, the greatest antioxidant activity and a high content of vitamin C (up to 1.27 mg/g). In conclusion, this work underlines the importance of extraction technique choice and could represent an advancement in industrial flavourings extractions, suggesting sustainable methods to obtain value-added extracts from discarded matrices.\u003c/p\u003e","manuscriptTitle":"The added value of flavouring extracts from Calabrian chili peppers by-products: characterization of bioactive and volatile compounds comparing green extraction techniques","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-14 10:59:58","doi":"10.21203/rs.3.rs-6549793/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"267721422342252679068931495857214762104","date":"2025-05-14T09:25:26+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"296087531300968113225620667543326463890","date":"2025-05-13T09:33:06+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"156613035891358061712245927613893117787","date":"2025-05-12T07:02:17+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"337506635620723082783189612905041813352","date":"2025-05-12T06:18:18+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"67301030861429619528983310131105411025","date":"2025-05-12T06:01:06+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-05-12T05:24:00+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-05-06T10:23:34+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-05-06T10:19:09+00:00","index":"","fulltext":""},{"type":"submitted","content":"European Food Research and Technology","date":"2025-04-28T16:50:13+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"european-food-research-and-technology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [European Food Research and Technology](https://link.springer.com/journal/217)","snPcode":"217","submissionUrl":"https://submission.springernature.com/new-submission/217/3","title":"European Food Research and Technology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"df84c4e7-6815-42c9-b89b-10fb9acc84e3","owner":[],"postedDate":"May 14th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-08-12T06:38:12+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-14 10:59:58","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6549793","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6549793","identity":"rs-6549793","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}