---
title: "Ultrasound‑pretreated enzymatic extraction of soluble dietary fiber from orange peel by-products"
id: "plos-one-14-valorization-of-orange-peel-by-products-via-ultrasound-pretreated-enzymatic"
canonical_url: "https://medichelpline.com/clinical-feed/plos-one-14-valorization-of-orange-peel-by-products-via-ultrasound-pretreated-enzymatic"
content_type: "clinical_feed_article"
specialty: "General"
source_name: "PLOS ONE (Medicine)"
source_url: "https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0357376"
published_at: "2026-09-01T14:00:00.000Z"
evidence_level: "Journal Feed"
license: "CC-BY-NC-4.0 / Informational Use"
---
# Ultrasound‑pretreated enzymatic extraction of soluble dietary fiber from orange peel by-products
## Provenance & Clinical Metadata
- **Canonical URL:** https://medichelpline.com/clinical-feed/plos-one-14-valorization-of-orange-peel-by-products-via-ultrasound-pretreated-enzymatic
- **Specialty:** [General](https://medichelpline.com/clinical-feed/general.md)
- **Primary Source:** PLOS ONE (Medicine)
- **Source URL:** [Original Journal Publication](https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0357376)
- **Published At:** 2026-09-01T14:00:00.000Z
- **Evidence Rating:** Journal Feed
## Executive GIST (TL;DR)
- Agro-industrial orange peel by-products (OPB) are abundant and underused sources of bioactive compounds including **soluble dietary fiber (SDF)**, pectin, carotenoids and flavonoids; valorization aligns with circular economy goals and reduces waste. - The study compared four extraction approaches applied to OPB: alkaline extraction (ALE), alkaline extraction with ultrasound pretreatment (UAEAL), enzymatic extraction (EE), and ultrasound‑pretreated enzymatic extraction (UAEE). - **Ultrasound‑pretreated enzymatic extraction (UAEE)** achieved the highest SDF recovery reported here: 26.04 ± 0.81% with roughly 90% purity under a low mass-to-volume ratio (1:25) and temperature‑controlled sonication. - Total phenolic content bound to SDF ranged between 3.23 ± 0.03 and 12.15 ± 0.21 mg GAE g⁻¹ across samples, indicating retention of phenolic compounds in extracted fibers. - Functional properties varied by method: SDF from alkaline extraction exhibited the highest water‑holding capacity (WHC) at 9.16 ± 0.08 g g⁻¹; oil‑holding capacity (OHC) was similar across methods (1.08 ± 0.12 to 1.24 ± 0.05 g g⁻¹). - Enzymatic treatments induced structural modifications and partial depolymerization, decreasing porosity and water affinity in some SDF samples. - UAEE produced SDF with a more amorphous structure, lower crystallinity, greater surface disruption on SEM, and increased thermal stability by TGA compared with other methods. - The UAEE‑recovered SDF served as a substrate for growth of beneficial mammalian intestinal symbionts such as Faecalibaculum rodentium and Bacteroides thetaiotaomicron, supporting biological functionality beyond physicochemical characterization. - Ultrasound pretreatment is presented as a promising, scalable strategy to recover high‑value SDF from OPB, enabling tailored functional ingredients for food applications; details on scale‑up metrics and full experimental parameters were reported in the source but are not reproduced here.
## Clinical Analysis & Structured Key Points
Valorization of orange peel by-products via ultrasound-pretreated enzymatic extraction of soluble dietary fiber: Structural and functional characterization | PLOS One Browse Subject Areas ? Click through the PLOS taxonomy to find articles in your field. For more information about PLOS Subject Areas, click here . Article Authors Metrics Comments Media Coverage Peer Review Reader Comments Figures Figures Abstract Agro-industrial by-products valorization has attracted increasing attention due to their potential as sustainable sources of bioactive compounds, particularly dietary fibers. In this study, orange peel by-products were used as a raw material for the recovery of soluble dietary fiber via enzymatic extraction and ultrasound-pretreated enzymatic extraction. Ultrasound-pretreated enzymatic extraction achieved a soluble dietary fiber recovery of 26.04 ± 0.81% with approximately 90% purity. The content of phenolic compounds bound to soluble dietary fiber ranged from 3.23 ± 0.03 to 12.15 ± 0.21 mg GAE g -1 . Soluble dietary fiber obtained by alkaline extraction exhibited higher water-holding capacity (9.16 ± 0.08 g g -1 ). Enzymatic extraction caused structural modifications and partial depolymerization, reducing fiber porosity and water affinity. Oil-holding capacity ranged from 1.08 ± 0.12 to 1.24 ± 0.05 g g -1 , with no differences among extraction methods. Ultrasound-pretreated enzymatic extraction produced soluble dietary fiber with a more amorphous structure, lower crystallinity, greater surface disruption, and higher thermal stability. Moreover, it is a suitable substrate for the growth of mammalian intestinal symbionts such as Faecalibaculum rodentium and Bacteroides thetaiotaomicron , species recognized as beneficial for host health. Ultrasound pretreatment is a promising strategy for recovering high-value soluble dietary fibers from orange peel by-products, thereby enabling the production of tailored functional ingredients for food applications. Citation: Quiñonez-Ensuncho JD, Rosales G, Ortega-Ruiz L, Benítez-Páez A, Valdés-Duque BE, Lima ÁS, et al. (2026) Valorization of orange peel by-products via ultrasound-pretreated enzymatic extraction of soluble dietary fiber: Structural and functional characterization. PLoS One 21(9): e0357376. https://doi.org/10.1371/journal.pone.0357376 Editor: Karthikeyan Venkatachalam, Prince of Songkla University, THAILAND Received: February 12, 2026; Accepted: August 17, 2026; Published: September 1, 2026 Copyright: © 2026 Quiñonez-Ensuncho et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: https://doi.org/10.5281/zenodo.18625111 . Funding: Jhonatan D. Quiñonez-Ensuncho, Beatriz E. Valdés-Duque, and J. Felipe Osorio-Tobón are grateful to the Institución Universitaria Colegio Mayor de Antioquia for funding this work - Convocatoria 2020. Gala Rosales and Álvaro S. Lima thank the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001. Laura Ortega-Ruiz and Alfonso Benítez-Páez thank the Spanish Ministry of Science, Innovation and Universities (PID2023-150523NB-I00) for funding ABP. The Accreditation as Center of Excellence Severo Ochoa CEX2021-001189-S, funded by MICIU/AEI/10.13039/501100011033, is also fully acknowledged. Competing interests: The authors have declared that no competing interests exist. Abbreviations: ALE, Alkali extraction; DF, Dietary fiber; EE, Enzymatic extraction; FTIR, Fourier transform infrared spectroscopy; GAE, Gallic acid equivalents; IDF, Insoluble dietary fiber; OHC, Oil-holding capacity; OPB, Orange peel by-products; SDF, Soluble dietary fiber; SEM, Scanning electron microscopy; TDF, Total dietary fiber; TFC, Total flavonoid content; TGA, Thermogravimetry analysis; TPC, Total phenolic content; UAEAL, Alkali extraction with ultrasound pretreatment; UAEE, Ultrasound-pretreated enzymatic extraction; WHC, Water-holding capacity; XRD, X-ray diffraction. Introduction Orange is one of the most important crops worldwide, with an estimated annual production of 47 million tons in 2024 [ 1 ]. Globally, the management of citrus residues is a critical environmental challenge, as millions of tons of organic waste are generated each year, releasing significant amounts of greenhouse gases, including carbon dioxide and methane, during decomposition [ 2 ]. These by-products, which include peel, albedo, and endocarps, account for up to 55% of the fruit’s weight. In Colombia, where production reached 756 thousand tons in 2023, the common practice of discarding these residues in landfills represents a substantial loss of biomass rich in carotenoids, flavonoids, pectin, and dietary fiber (DF) [ 3 ]. Consequently, in accordance with the principles of the circular economy, it is imperative to develop valorization strategies that mitigate environmental impacts while recovering high-value functional ingredients [ 4 ]. DF is a carbohydrate found in plant foods that is not digestible in the human small intestine and is used as an energy source by the microbiota in the large intestine. DF is considered a functional ingredient due to its beneficial properties. For example, DF is recognized for reducing the risk of cardiovascular and diabetes diseases, as well as its positive effects on the intestine, promoting bowel movement and relieving constipation [ 5 ]. Based on water solubility, DF is classified as soluble dietary fiber (SDF) and insoluble dietary fiber (IDF). SDF increases viscosity, decelerates gastric emptying, and reduces blood glucose and cholesterol levels. Moreover, SDF stabilizes insulin, improves macronutrient absorption, and reduces the risk of diseases such as cancer and obesity, thereby benefiting metabolism and modulating the microbiota [ 6 ]. IDF presents a slow and incomplete fermentation process, which is directly related to intestinal peristalsis, decreased gastrointestinal transit time, and increased fecal volume, facilitating defecation and alleviating constipation [ 7 ]. DF can be extracted using biological, chemical, and physical techniques, or their combinations (e.g., physical and biological). Traditional chemical extraction methods (acidic or alkaline) are common due to low cost but often require high temperatures and pose environmental challenges [ 8 ]. In contrast, ultrasound-assisted extraction (UAE) is a sustainable “green” technology that operates at lower temperatures with reduced solvent requirements [ 9 ]. The core mechanism is acoustic cavitation: sound waves (typically> 20 kHz) create microbubbles that grow and then collapse, releasing high-intensity energy. This generates hydrodynamic shear forces that induce cell wall disruption. This structural modification increases the surface area and improves mass transfer, allowing solvents and reagents to penetrate the internal structure more efficiently, thereby increasing the recovery of DF fractions [ 10 ]. On the other hand, the enzymatic extraction (EE) method is considered an effective approach for removing impurities from DF, such as proteins and starch, through enzymatic hydrolysis. This process generally requires protease, α-amylase, and amyloglucosidase. This approach to extraction is suitable for obtaining high-quality dietary fiber, as enzymatic action modifies fiber structure and redistributes its composition [ 11 ]. The primary advantages of this method include high extraction efficiency, mild processing conditions, and reduced environmental impact due to minimal hazardous by-product formation [ 7 ]. The integration of EE and ultrasound enables the production of fibers with interesting properties at enhanced yields [ 12 ]. Conventional techniques (such as acid, alkaline, and hot-water extraction) are often limited by low extraction yields and the risk of polysaccharide degradation from harsh reagents and high temperatures. For example, Bakr et al. [ 13 ] reported a modest SDF yield of 5.61% from quince using alkaline extraction. In contrast, green technologies such as ultrasound-assisted enzymatic extraction have demonstrated higher efficiency. Panwar et al. [ 12 ] achieved an SDF yield of 12.19% from sweet lime pomace using this approach. These alternative methods represent a significant innovation in fiber purity and preserving structural integrity while adhering to environmentally sustainable practices. Despite these advances, limiting solid-liquid ratios is often employed, restricting the scalability of these technologies for industrial DF recovery. Another critical bottleneck is temperature control during ultrasound-assisted enzymatic extraction. Because the enzymatic process is carried out at temperatures higher than those recommended by the ultrasound equipment supplier, the simultaneous application of ultrasound and enzymatic hydrolysis is limited. Thus, the application of ultrasound as a pretreatment is a valuable alternative. Although previous studies have reported that extraction conditions significantly influence DF quality and properties, there remains a gap in research on the application of ultrasound-pretreated enzymatic extraction of SDF from orange peel by-products (OPB). Moreover, evidence on the application of ultrasound as a pretreatment for SDF recovery from orange residues at low mass-to-volume ratios, such as 1:25, and under temperature-controlled sonication conditions remains limited. This mass-to-volume ratio offers a critical advantage by reducing solvent consumption and minimizing waste generation, thereby facilitating process scalability and industrial implementation. Beyond these operational advantages, the innovative contribution of this work lies in transcending the conventional physicochemical characterization by validating the biological functionality of the recovered SDF. This study aims to evaluate the effects of different extraction methods (alkaline extraction, alkaline extraction with ultrasonic pretreatment, enzymatic extraction, and enzymatic extraction with ultrasonic pretreatment) on the extraction yield of dietary fiber fractions from OPB. The influence of these treatments on the techno-functional and structural properties of SFD was also assessed. Additionally, its potential as a carbon source for the growth of Faecalibaculum rodentium ABL288 was evaluated. Materials and methods Materials OPB were kindly provided by Fruti Paisa, Medellín, Colombia. The pre-treatment was carried out as described by Ye et al. [ 14 ] with some modifications. Briefly, the OPB were first washed with distilled water to remove inorganic residues. They were then submerged in water at 70 °C for 20 min to eliminate low-molecular-weight sugars, organic acids, and inactive enzymes. The OPB were dried in an oven at 50 °C for 48 hours (Memmert, model UN110, Schwabach, Germany). After drying, the peels were milled and sieved using a 50-mesh sieve (0,297 mm). Processed OPB were stored in hermetically sealed bags (Alico S.A., Flex Up, Medellín, Colombia) and protected from light to prevent degradation. Samples were stored at room temperature until fiber extraction. Thermostable α-amylase (12000 U g -1 ) and amyloglucosidase (104000 U g -1 ) were purchased from Proenzymas S.A.S. (Cali, Colombia), and protease (2.4 U g -1 ) was purchased from Sigma-Aldrich. All reagents used in this study were of analytical grade. Extraction of dietary fiber from orange peel by-products The extraction of soluble and insoluble dietary fibers was evaluated using methods based on those described by Kaur et al. [ 15 ], with some modifications. Fig 1 presents the flowsheet of the experimental extraction protocol and fiber characterization. Download: PNG larger image TIFF original image Fig 1. Flowsheet of the experimental protocol for extracting the SDF from OPB. https://doi.org/10.1371/journal.pone.0357376.g001 Alkali extraction For alkali extraction (ALE), 5 g of OPB powder was mixed with 1% (w/w) NaOH at a Solid-to-Liquid solvent-to-feed ratio of 25. The mixture was agitated at 500 rpm for 30 min at room temperature (25 °C). It was then incubated at 50 °C for 30 min in a thermostatic bath (VWR, WB05, Radnor, USA). Subsequently, the mixture was centrifuged (Hermle, Z326K, Wehingen, Germany) at 3620 g for 30 min to separate the residues from the supernatant. IDF was recovered from the wet pellet and dried in an oven at 45 °C for 24 hours. SDF was recovered from the supernatant by adding four volumes of 96% ethanol and incubating the mixture for 2 hours. Then, the residues were rinsed with 100% ethanol and dried in an oven under the same conditions as previously described. The extraction yield was calculated using Equation (1) . (1) Where Y denotes the yield of the recovered dietary fiber fraction (SDF or IDF), W f denotes the dry weight of the recovered fiber, and Weight of OPB denotes the dry weight of the orange residues. TDF was calculated as the sum of the SDF and IDF weights. Enzymatic extraction Enzymatic extraction was performed following the methodology described by Kaur et al. [ 15 ], with some modifications. 5 g of powdered OPB was weighed and suspended in 125 mL of 0.08 M phosphate buffer (pH 6.0). Subsequently, 100 µL of 1 M calcium chloride (CaCl 2 ) was added, and the pH was adjusted to 6.0. Then, 0.25 mL of thermostable alpha-amylase (12000 U g -1 ) was added, and the mixture was incubated in a water bath at 90°C for 15 min. After the mixture was brought to room temperature, the pH was adjusted to 7.5 using NaOH, and 0.50 mL of protease (2.4 U g -1 ) was added, followed by incubation at 60°C for 30 min. The mixture was brought to room temperature, and the pH was adjusted to 4.5 with HCl. Subsequently, 1 mL of amyloglucosidase (104000 U g -1 ) was added. The mixture was incubated again at 60°C for 30 min. Finally, the mixture was cooled to room temperature, and the pH was adjusted to 6.5–7.0 to stop enzymatic activity. The sample was centrifuged under the same conditions used for alkaline treatment to recover SDF and IDF, as described earlier. Ultrasound pretreatment A 750-W ultrasonic homogenizer (Cole-Parmer, 750W, Vernon Hills, USA) was used for ultrasound pretreatment. Five grams of orange peel powder were mixed with the appropriate solvent. After preliminary trials, ultrasound pretreatment conditions were set to pulse mode (2 seconds on, 2 seconds off) for 15 min at 60% amplitude and room temperature (25 °C). SDF and IDF were recovered using the previously described methodology for alkali and enzymatic extractions. UAEAL corresponds to alkali extraction with ultrasound pretreatment, whereas UAEE corresponds to the enzymatic extraction with ultrasound pretreatment. Determination of total dietary fiber content Total dietary fiber content was determined using a total dietary fiber assay kit (TDF100A, Sigma-Aldrich) according to the AOAC 991.43 method. This method is based on a combination of enzymatic digestion and gravimetric determination. Dried samples were gelatinized with alpha-amylase, then digested with protease and amyloglucosidase to remove proteins and starches. Ethanol was then added to precipitate the SDF fraction. The resulting residue was filtered and washed sequentially with ethanol and acetone. After drying, the residue was weighed. Half of the samples were analyzed for protein content using the Kjeldahl method, while the remaining half were incinerated. The total dietary fiber content was calculated as the residue weight minus protein and ash weights. Functional and bioactive properties Extraction of bioactive compounds from SDF was performed as described by Sheng et al. [ 16 ], with modifications. One gram of sample was suspended in 50 mL of water and placed in a thermostatic bath (Quimis, Q226M1, Diadema, Brazil) with constant agitation at 45 °C for one hour. The extraction solution was then separated from the insoluble residues by centrifugation (Quimis, Q222T108, Diadema, Brazil) at 3000 g for 5 min. Determination of total phenolic content Total phenolic content was determined by the Folin–Ciocalteu method, using gallic acid as the standard, following the procedure of Ferreira-Anta et al. [ 17 ] with minor modifications. Briefly, 0.5 mL of the sample was mixed with 4 mL of deionized water and 0.5 mL of the Folin-Ciocalteu reagent. After 3 min of incubation, 0.5 mL of sodium carbonate solution (prepared by dissolving 1g of Na 2 CO 3 in 3.5 mL of water and incubating for 30 min at 37°C) was added. Absorbance was measured at 660 nm using a spectrophotometer (Shimadzu UV-3600 Plus, Shimadzu, Kyoto, Japan). Results were expressed as milligrams of gallic acid equivalents per gram of biomass (mg GAE g -1 ). Determination of total flavonoid content Total flavonoid content was determined via the aluminum chloride assay with catechin as the standard [ 18 ]. A 1 mL sample aliquot was mixed with 5 mL of ethanol (80% v/v) and 1 mL of NaNO 2 solution (5% w/v) and allowed to react for 6 min. Subsequently, 1 mL of AlCl 3 solution (10% w/v) was added and mixed thoroughly. After an additional 6 min, 10 mL of NaOH solution (1 M) was added. The mixture was then allowed to stand for 15 min before measuring the absorbance at 510 nm. The values were expressed as milligrams of catechin equivalents per gram of biomass (mg CE g -1 ). Water and oil-holding capacities Water and oil-holding capacities were determined following the method described by Panswar et al. [ 12 ], with modifications. Water-holding capacity (WHC) was prepared by hydrating the powdered sample (0.5 g) with distilled water (10 mL) and vigorously mixing for 5 min. Then, it was allowed to rest for 30 min at room temperature; furthermore, it was centrifuged at 3620 g for 20 min. Finally, the supernatant was discarded, and the water holding capacity was calculated using Equation (2) . (2) Where W 1 is the weight of the sample, and W 2 is the final weight of the sample. Oil holding capacity (OHC) was determined by vigorously mixing a sample (0.5 g) with olive oil (10 mL) and whisking for 5 min. The mixture was kept at room temperature for 30 min, then centrifuged at 3620 g for 20 min. The supernatant was discarded, and the OHC was determined using equation (3) : (3) Where O 1 is the weight of the sample; O 2 is the final weight of the sample. Fourier transform infrared (FTIR) spectroscopy Infrared spectra were obtained using a spectrophotometer (PerkinElmer, Spectrum 400, Shelton, USA). Analytical-grade KBr pellets were used for measurements, with a resolution of 4 cm -1 , a scanning range of 4000–600 cm -1 , and 20 scan cycles. X-ray diffraction The crystallinity of SDF was analyzed by X-ray diffraction using a diffractometer (Shimadzu, XRD-6000, Tokyo, Japan) equipped with a monochromatic graphite source and CuKα radiation, operating at 40 kV and 30 mA, with a scanning speed of 4°·min -1 and a 2θ scanning range of 10° to 80°. The degree of crystallinity was evaluated using the crystallinity index, calculated according to equation (4) described by de Sun et al. [ 19 ]. (4) where I 002 is the diffraction peak intensity of the crystalline material near 2θ = 20°, due to the nature of the soluble fiber, and I am is the diffraction peak intensity of the amorphous material near 2θ = 15°. The values obtained from the crystallinity index are relative measures of molecular ordering in the polymer matrix. Scanning electron microscopy Scanning electron microscopy was performed using a Hitachi S-3400N (Tokyo, Japan). Samples were mounted on carbon tape and coated with platinum. Images were obtained at an accelerating voltage of 10 kV and a magnification of ×500. Thermogravimetry analysis The thermogravimetric analysis was performed using a thermogravimetric analyzer (Shimadzu, DTG-60H, Tokyo, Japan). 10 mg of the sample was weighed and placed in an alumina tray under a nitrogen
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