Industrial Fruits By-Products and Their Antioxidant Profile: Can They Be Exploited for Industrial Food Applications?

foods Article Industrial Fruits By-Products and Their Antioxidant Profile: Can They Be Exploited for Industrial Food Applications? Cássia H. Barbosa 1,† , Mariana A. Andrade 1,2,† , Raquel Séndon 3, Ana Sanches Silva 4,5,* , Fernando Ramos 2,* , Fernanda Vilarinho 1, Khaoula Khwaldia 6and Letricia Barbosa-Pereira 3   Citation: Barbosa, C.H.; Andrade, M.A.; Séndon, R.; Silva, A.S.; Ramos, F.; Vilarinho, F.; Khwaldia, K.; Barbosa-Pereira, L. Industrial Fruits By-Products and Their Antioxidant Profile: Can They Be Exploited for Industrial Food Applications?. Foods 2021,10, 272. https://doi.org/ 10.3390/foods10020272 Academic Editors: Alfredo Cassano and Mohamed Koubaa Received: 31 December 2020 Accepted: 25 January 2021 Published: 29 January 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). 1Department of Food and Nutrition, National Institute of Health Dr. Ricardo Jorge, Av. Padre Cruz, 1649-016 Lisbon, Portugal; [email protected] (C.H.B.); [email protected] (M.A.A.); [email protected] (F.V.) 2Faculty of Pharmacy, University of Coimbra, REQUIMTE/LAQV, Coimbra, Azinhaga de Santa Comba, 3000-548 Coimbra, Portugal 3 Analytical Chemistry, Nutrition and Food Science Department, Pharmacy Faculty, University of Santiago de Compostela, 15782 Santiago de Compostela, Spain; [email protected] (R.S.); [email protected] (L.B.-P.) 4National Institute for Agricultural and Veterinary Research (INIAV), I.P., Rua dos Lagidos, Lugar da Madalena, 4485-655 Vairão, Vila do Conde, Portugal 5Center for Study in Animal Science (CECA), ICETA, University of Oporto, 4051-501 Oporto, Portugal 6Laboratoire des Substances Naturelles, Institut National de Recherche et d’Analyse Physico-Chimique, INRAP, Pôle Technologique de Sidi Thabet, Sidi Thabet 2020, Tunisia; [email protected] *Correspondence: [email protected] or anateress@gmail (A.S.S.); [email protected] (F.R.) † These authors contributed equally to the work. Abstract: Fruit by-products have a low economic value and have proven biological activities, such as antioxidant capacity due to the presence of active compounds. The main objective of this study was to obtain and determine the antioxidant capacity, through DPPH radical assay and β -carotene bleaching assay, of three food grade extracts from apple, lemon, and orange industrial by-products. Furthermore, the extracts were characterized by ultra-high performance liquid chromatography coupled to mass spectrometry (UHPLC-MS/MS). LC with diode array detector (LC-DAD) was used for the quantification of the main polyphenols. Lemon extract presented the highest inhibition percentage of DPPH radical (51.7%) and the highest total phenolics content (43.4 mg GAE/g) from the by-products studied. Orange by-product was that with the higher number of polyphenols while lemon extract was that with the highest content of individual phenolics. The by-product obtained from the lemon was that with higher amounts of hydroxycinnamic acids (407 µ g/g of by-product), mainly chlorogenic acid (386.7 µ g/g), followed by the apple by-product (128.0 µ g/g of by-product), which showed higher amounts of rosmarinic and chlorogenic acids. These industrial by-products have great potential as a source of natural antioxidants to be used directly as food additives or to be incorporated in packaging to produce active food packaging. Keywords: antioxidant capacity; apple; industrial by-products; LC-DAD; lemon; orange; UHPLCESI-MS/MS 1. Introduction Fruitsare often presented to consumers in several forms, suchasjams, juices, concentrates , and pastes. The manufacturing processes of these formulations do not use the fruit entirely, originating a large quantity of fruit by-products that, in turn, have to be discarded in a responsible and sustainable way, which may imply a significant increase in the final price of the product [ 1 , 2 ]. Additionally, fruit by-products have a low economic value and have proven biological activities derived from the presence of phenolic compounds, vitamins, carotenoids, among other active compounds. Phenolic compounds, chemically characterized for having at least one phenol unit, are present in most terrestrial plants, and they are Foods 2021,10, 272. https://doi.org/10.3390/foods10020272 https://www.mdpi.com/journal/foods Foods 2021,10, 272 2 of 16 responsible for the plant’s defense against external stimuli such as radiation, predators and microorganisms [ 3 , 4 ]. The presence of these compounds is directly linked to the occurrence of antioxidant and antimicrobial activities, making these compounds of major interest for the food, cosmetic and pharmaceutical industries. Apple is a well-known fruit of the genus Malus (family Rosaceae) [ 5 ] and one of the most-consumed fruits all over the world [ 6 ]. According to the Food and Agriculture Organization of the United Nations, the global production of apples was over 85 million tons in 2019 [ 7 ]. Although the majority is consumed as a fresh fruit, 25–30% are converted into processed products, with apple juice being the main product [ 8 ]. Apples represent an important source of bioactive compounds like pectins, dietary fibers, vitamins, oligosaccharides, triterpenic acids and phenolic compounds, such as flavonols, monomeric and oligomeric flavanols, dihydrochalcones, anthocyanidins, p-hydroxycinnamic and p-hydroxybenzoic acids [ 5 , 6 ]. Apples with a higher content in phenolic compounds tend to have a higher antioxidant capacity. The content in phenolic compounds varies with edaphoclimatic conditions (such as weather and water availability), cultivation practices, harvesting, storage conditions, and apple cultivars, the apple cultivar being the main factor in determining the content on bioactive compounds [ 6 , 9 ]. Furthermore, differences can also be found among the different parts of the apple, since the peel contains a higher content in phenolic compounds than the flesh [ 5 ]. Apple pomace, the mixture of peel, core, seed, calyx, stem and soft tissue resulting from apple juice production, is the main by-product generated, accounting for close to 25% of the fresh apple weight [ 8 , 10 ] and has approximately 20–30% of dried matter [11]. Lemons and oranges are other well-known fruits, belonging to the genus Citrus, with a production of more than 95 million tones worldwide, in 2019 [ 7 ]. Lemon is mostly consumed as juice, originating a large quantity of lemon by-products, which are a very good source of dietary fiber, pectin, flavonoids, limonoids, coumarins and carotenoids [ 12 ]. Lemon essential oil can be obtained from lemon peels, which has proven antimicrobial activity against Escherichia coli,Staphylococcus aureus and Pseudomonas aeruginosa [ 13 , 14 ]. Oranges are also largely consumed in juice form, leaving a large trail of by-products. The orange peel, similar to lemon peel, is a good source of dietary fiber, pectin, phenolic acids and flavonoids, including polymethoxylated flavones and flavonols [ 15 , 16 ]. Dietary fiber is an important resource used in the prevention of cardiovascular diseases, diabetes, cancer and gastrointestinal disorders [17]. To delay the natural degradation of foods, the food industry resorts to antioxidant and antimicrobial additives, normally from a synthetic origin. The indirect and unaware consumption of these compounds has been associated with the promotion of carcinogenesis and their effects on human health due to long exposure are still unknown [ 2 , 18 – 20 ]. Therefore, it is important to find alternatives to these additives not associated with adverse health effects, such as extracts, and essential oils obtained from fruit by-products. Fruits are a good source of antioxidants, with important health benefits. Their by-products (peel, stems, and seeds) are also an excellent source of antioxidants [ 2 , 21 ]. However, there are not many studies on industrial fruit by-products. The majority of the studies are with specific parts of the by-products, such as peel, stems or seeds. Furthermore, there is not a method that can measure the antioxidant capacity precisely, therefore, different assays should be performed to obtain a more accurate result [22,23]. The main objective of this study was to obtain and determine three food-grade extracts from apple, lemon, and orange by-products and determine their antioxidant capacity. Moreover, the three extracts were chemically characterized, and their main compounds were quantified by UHPLC-ESI-MS/MS. 2. Materials and Methods 2.1. Fruits By-Product Extraction The by-products of lemon, orange and apple were kindly supplied by the Portuguese juice company, Frubaça—Cooperativa de Hortofruticultores. Absolute ethanol was the Foods 2021,10, 272 3 of 16 chosen solvent for the production of the extracts since the main goal of the extract is to be applied directly or indirectly (through an active packaging) in foods. Ethanol is authorized by the Directive 2009/32/EC [ 24 ] in the extractions of bioactive compounds to be applied in foods. The samples were first grinded and freeze-dried, followed by the extraction process. Briefly, 5 g of sample 50 mL of absolute ethanol was added, the mixture was agitated on a compact shaker (Edmund Bühler GmbH model KS-15, Hechingen, Germany) at 450 rpm for 30 min at room temperature (23 ± 1 ◦ C), protected from the light. Then, the mixture was centrifuged (Heraeus Multifuge X3 FR, Thermo Scientific, Langenbold, Germany) at 6000 rpm at 10 ◦ C for 10 min. After that, the supernatant was removed to an amber pear-shaped flask and the ethanol was completely evaporated on a rotary evaporator (Büchi model R-210 Labortechnik, Switzerland) at 35 ◦ C. The extract was removed with an aid of a spatula, held at − 20 ◦ C, protected from the light, until further use. To evaluate the antioxidant capacities of the different extracts, free radical DPPH inhibition and β -carotene bleaching assays were performed. In addition, the Total Phenolic Compounds (TPC) and the Total Flavonoid Content (TFC) were determined. To perform the antioxidant activity assays, the extracts obtained were dissolved in absolute ethanol, at a concentration of 3 mg/mL. 2.2. Antioxidant Activity 2.2.1. Free Radical DPPH Inhibition Assay For the free radical DPPH inhibition assay, the method described by Moure et al. (2001) [ 25 ] and modified by Andrade et al. (2018) [ 4 ], was applied. Briefly, 2 mL of a DPPH methanolic solution (14.2 µ g/mL) were added to 50 µ L of the sample. The mixture was homogenized and kept in the dark for 30 min, at room temperature (23 ± 1 ◦ C). Absorbance was then measured at 515 nm using a spectrophotometer Evolution 300 UV-Vis (ThermoScientific ™ , England). A control assay was performed with the solvent in which the sample was dissolved. The inhibition percentage (IP) of DPPH was calculated according to the following Equation (1): IP(%)=Ac −As Ac ×100 (1) where Ac is the absorbance of the control and As is the absorbance of the sample. Furthermore, a calibration curve using Trolox (6-hydroxy-2,5,7,8-tetramethylchroman2-carboxylic acid) as a standard was drawn with a working range of 10–175 µg/mL. 2.2.2. β-Carotene Bleaching Assay The β -carotene bleaching assay was performed according to the described by Miller (1971) [ 26 ] and modified by Andrade et al. (2018) [ 4 ] Firstly, a solution with 20 mg of linoleic acid, 200 mg of Tween ® 40 and 1 mL of β -carotene in chloroform (0.2 mg/mL) was prepared. The chloroform was evaporated on a rotary evaporator at 40 ◦ C. Then, 50 mL of MilliQ ™ water was added, and vigorously agitated, until an emulsion was formed. Finally, to 200 µ L of the sample, 5 mL of the emulsion was added. Afterwards, the absorbance of the control was measured at 470 nm and the mixtures were subjected to 50 ◦ C for 120 min. The antioxidant activity coefficient (AAC) was calculated according to the Equation (2): AAC =(As120 −Ac120) (Ac0 −Ac120)×1000 (2) where, A s120 is the absorbance of the sample after 120 min, A c120 is the absorbance of the control after 120 min and Ac0 is the absorbance of the control at 0 min. 2.3. Total Phenolic Compounds Content (TPC) The determination of the Total Phenolic Compounds Content was carried out according to the Erkan et al. (2008) [ 27 ] method. According to the method, 7.5 mL of an Foods 2021,10, 272 4 of 16 aqueous solution of Folin-Cioucalteu (10%, v/v) was added to 1 mL of sample. After 5 min, 7.5 mL of an aqueous solution of sodium carbonate (60 mg/mL, w/v) was added. Then, the samples were kept in the dark for 120 min, and the absorbance was measured at 725 nm. Gallic acid was used as a standard for the calibration curve, with a working range between 5–150 µg/mL. 2.4. Total Flavonoid Compounds (TFC) The Total Flavonoid Content method was performed according to the Yoo et al. (2008) [ 28 ] method. To 1 mL of sample, 4 mL of MilliQ water and 0.3 mL of aqueous solution of sodium nitrite (5%, w/v) were added, and the solution was homogenized. After 5 min, 0.6 mL of aqueous solution of aluminum chloride (10%, w/v) were added and the solution was once again homogenized. After 6 min, 2 mL of sodium hydroxide (1 M, w/v) and 2.1 mL of MilliQ ™ water were added. The solution was homogenized and the absorbance was measured at 510 nm. Epicatequin was used as a standard for the calibration curve with a working range between 5–125 µg/mL. 2.5. Identification of the Polyphenolic Compounds by UHPLC-ESI-MS/MS The identification/tentative identification of phenolic compounds in the fruit byproducts extracts was performed with a UHPLC-ESI-MS/MS (Thermo Fisher Scientific, San José, CA, USA), equipped with a degasser, Accela quaternary pump, autosampler, and column oven, coupled to a triple quadrupole mass spectrometer TSQ Quantum Access max. The instrument control and data collection and processing were performed with Xcalibur 2.1 software (Thermo Fisher Scientific, San José, CA, USA). A reverse-phase Kinetex ® EVO C18 100Å column (150 × 3 mm internal diameter, 5 µ m particle size) (Phenomenex, Torrance, CA, USA) was used for phenolic compound separation at 30 ◦ C, according to Andrade et al. [ 21 ] The injection volume was 20 µ L, and the mobile phase flow rate used was 0.6 mL/min. The solvents used as mobile phase were water (solvent A) and methanol (solvent B), both acidified with formic acid at 0.1% (v/v). The gradient elution used was as follows: 95% A; 3 min, 90% A; 10 min, 80% A; 18 min, 70% A; 25 min, 30% A; 33 min, 0% A; 33–40 min, 0% A and 100% B isocratic; and finally, the column was washed and reconditioned with 95% A (40–46 min). The mass spectrometer electrospray ionization source (ESI) operated in both negative and positive mode, according to the nature of the phenolic compound. The optimized MS/MS detector settings were as follows: spray voltage 2500 V; vaporizer and capillary temperatures were set at 340 ◦ C and 350 ◦ C, respectively. Nitrogen (purity > 99.98%) was used as sheath gas (pressure 35 psi) and as auxiliary gas (the pressure set 10 arbitrary units), and Argon as the collision gas (1.5 mTorr). The MS/MS data acquisition was performed in a Single Reaction Monitoring (SRM) mode. After the first screening at MS scan range of 100–800 m/z, tentative identification of polyphenols was accomplished by comparing their precursor ion [M-H] −1 and mass spectrometry fragmentation pattern (MS/MS) with those already described in the literature. The identification of the individual phenolic compounds was accomplished by comparison of the retention time with those obtained by injecting pure standards, when available, under the same chromatographic conditions, and with the molecular ion and product-ions data provided by MS/MS analysis. 2.6. Quantification of the Polyphenolic Compounds by HPLC-DAD/UV The quantification of phenolic compounds was performed with an Agilent HPLC system 1100 (Hewlett-Packard, Waldbronn, Germany), equipped with a quaternary pump, a degassing device, an autosampler, a column thermostat system, coupled to a diode array detector (DAD), and controlled by HP ChemStation software (version B.03.0.1). The column and chromatographic conditions used were the same described above for UHPLC-ESI-MS/MS analysis. DAD spectra acquisition was performed continuously in full scan modality during the run time ranging from 200 to 400 nm. The identification Foods 2021,10, 272 5 of 16 of individual phenolic compounds was achieved by comparing their retention times and the UV spectrum ( λmax ) characteristics of the different family of phenolic compounds or with that obtained with commercial standards injected under the same chromatographic conditions, whenever available. Phenolic compounds were monitored and quantified at 230, 278, 300, 325, and 360 nm. Quantification was carried out by the external-standard method with six-point calibration curves. 2.7. Statistical Analysis All experiments were conducted using a completely randomized design with three replications. Statistical analysis of data was performed through a one-way analysis of variance (ANOVA) using the Software IBM ® SPSS ® Statistics, version 26.0.0.0, and differences among mean values were processed by the Tukey test. All requirements necessary to carry out the ANOVA (namely, normality of data and homogeneity of variances) have been validated. Significance was defined at p< 0.05. Results are expressed as the means of the replicants ±standard deviation. 3. Results and Discussion 3.1. Antioxidant Capacity In this study, four assays were performed for a better characterization of the antioxidant. For antioxidant potential, DPPH radical scavenging capacity and β -carotene bleaching assay were performed. Besides that, TPC and TFC were quantified for antioxidant potential assessment. For all the assays, the extracts were analyzed in the concentration of 3 mg of extract per mL of ethanol. Table 1shows the IP of DPPH and the Trolox Equivalent (TE) for all the extracts. Lemon extract presented the highest IP of DPPH radical (51.67 ± 4.61%) followed by the apple extract (39.92 ± 1.68%) and orange extract (31.20 ± 1.28%). The DPPH radical scavenging capacity assay measures the reducing capacity of antioxidants. Different results were obtained in other studies. Albuquerque et al. [ 29 ] evaluated a water extract obtained from industrial oranges by-products. The authors found lower values (898.9 µ mol Trolox/L fruit by-products water extracts) when compared to the ethanolic extract of the orange by-products. This can be explained by the use of different extraction solvents in the two studies. Guimarães et al. [ 30 ] evaluated the antioxidant capacity of orange and lemon peel essential oils. The authors obtained good EC 50 values for orange (95.67 ± 2.21 mg/mL) and lemon (116.25 ± 10.56 mg/mL). M ' hiri et al. [ 12 ] studied the effects of different drying processes on the antioxidant activity of industrial lemon by-products. The authors concluded that all the drying processes decreased the total content of phenolic compounds, and antioxidant radical scavenging activity, supporting the room temperature extraction procedure of active compounds, such as the method used in the present study [12]. Regarding the β -carotene bleaching assay, orange extract (3 mg/mL) presented the highest AAC (237.21 ± 29.78) (Table 2). The β -carotene bleaching assay is also based on color change. In the absence of antioxidants, the free linoleic acid radical bonds to the β-carotene molecule and the orange color fade. The lemon by-products extract presented the highest TPC (43.38 mg GAE/g ± 0.84 mg GAE/g) from the studied extracts. The TPC of orange and apple were 23.32 ± 0.18 mg GAE/g and 14.02 mg ± 0.13 mg GAE/g, respectively (Table 2). Phenolic compounds are recognized for their contribution as one of the most important antioxidants in the diet [ 29 , 31 ]. Therefore, it is essential to quantify the TPC presented in the food and its by-products. Guimarães et al. [ 30 ] analyzed lemon and orange peel and obtained a higher value of TPC, 87.77 mg/g extract and 79.75 mg/g, respectively. Li et al. [ 32 ] analyzed lemon and orange peel too, and the results presented as fresh matter were 118.75 mg/g and 73.59 mg/g, respectively. M’hiri et al. [ 12 ] analyzed lemon by-products and for TPC the results were 5.52 g/100 g as dry matter. On the other hand, the TPC obtained for apple by-products in this study were higher than the ones of Diñeiro García et al. [ 33 ]. Foods 2021,10, 272 6 of 16 Raudone et al. [ 6 ] quantified the TPC in apple by-products and the result was 31.01 mg/g as dry weight. It is also important to identify the individual phenolic compounds present in the fruits’ by-products. The TFC of lemon, orange, and apple were 20.76 mg ± 0.61 mg ECE/g, 7.29 mg ± 0.32 mg ECE/g, and 24.63 mg ± 1.61 mg ECE/g (Table 2). Apple extract showed the highest TFC and orange extract showed the lowest TFC. Flavonoids are important phytonutrients too. The results obtained in this study for lemon by-products were higher than those from Guimarães et al. [ 30 ] M’hiri et al. [ 12 ] obtained 4.35 g/100 g as dry matter for TFC. For orange by-products, there were studies with higher and lower values than those obtained in this study [ 16 , 30 ]. No studies with TFC for apple by-products were found in the literature. Dissimilarities in all results can be due to different fruits’ variability and their degrees of maturation. External factors such as climate, soil and fertilization applied can also affect the results. Apart from these, the results can be affected by the variability in the solvents used for the extractions and changes in the methods used [34–37]. In general, all three fruit by-products presented a good source of antioxidant compounds, able to be incorporated as dry extracts in food and in food packaging. However, from the studied industrial by-products, lemon extract was revealed to have the greatest potential as an antioxidant extract. Table 1. DPPH radical scavenging capacity of different fruit by-products. The results are expressed as mean of three replicas ±SD. Different letters indicate statistical differences. Fruits By-Products Inhibition Percentage (%) Trolox Equivalent (mg Trolox/g of Extract) Lemon 51.67 ±4.61 a33.17 ±2.94 d Orange 31.20 ±1.28 b20.13 ±0.43 e Apple 39.92 ±1.68 c25.69 ±0.56 f Table 2. Antioxidant capacity and characterization of different fruit by-products. The results are expressed as Mean ±SD. Different letters indicate statistical differences. β-Carotene Bleaching Assay TPC (mg GAE/g) TFC (mg ECE/g) Mean ±SD Mean ±SD Mean ±SD Lemon 67.35 ±1.96 a43.38 ±0.84 c20.76 ±0.61 f Orange 237.21 ±29.78 b23.32 ±0.18 d7.29 ±0.32 g Apple 107.44 ±23.81 a14.02 ±0.13 e24.63 ±1.61 h 3.2. Chromatographic Polyphenolic Profile of the Fruit By-Products The phenolic compounds of fruit by-products identified or tentatively identified by HPLD-DAD and UHPLC-ESI-MS/MS are described in Table 3. The identification of phenolic compounds was based on the elemental composition data determined from accurate mass measurements in negative ionization mode and comparison with the literature and that obtained with the available standards, except for compound 25 (Quercetin), which was measured in the positive mode as described previously by Andrade et al. [ 21 ] Each compound was characterized by its retention time (R t ), maximum absorption wavelengths ( λmax ), structural class, molecular formula, molecular ion, and main MS/MS fragments. The peak names of the Table 3correspond to the peak labels of the chromatograms obtained at 278 nm by HPLC–DAD for each fruit by-product represented in Figure 1. In this work, a total of 26 compounds (19 for orange, 18 for lemon, and 16 for apple by-products) from different classes of polyphenols were identified, including phenolic acids (benzoic acid derivates, hydroxycinnamic acids derivatives, and their glycosides) and flavonoids (flavonols, flavones, flavanones, and dihydrochalcones, as well as their glycosides). The Foods 2021,10, 272 7 of 16 confirmation of the identity of 17 polyphenols was achieved by a comparative analysis of authentic standards based on compounds retention time, the UV–visible spectra, and MS/MS fragmentation patterns. 3.2.1. Benzoic Acid Derivates The benzoic acid derivates identified in the fruit by-products analyzed were protocatechuic acid (compound 1) and hydroxybenzoic acid (compound 2) detected at UV λmax 293 and 255 nm, respectively. The identification was performed by a comparison of their retention times and MS/MS fragmentation patterns with standards. The hydroxybenzoic acids are widely distributed in nature and have been identified by other authors in citrus and apple fruits and products [ 38 – 40 ]. On the other hand, protocatechuic acid has been described in apple fruits and less in citrus. Indeed, this study identifies for the first time protocatechuic acid in orange by-products. 3.2.2. Hydroxycinnamic Acids and Their Glycosides Several compounds from the group of hydroxycinnamic acids were identified in the fruit by-products by comparison of their retention times, UV–visible typical spectra at λmax 325 nm, and MS/MS fragmentation patterns with standards. Caffeic and p-coumaric acids (compounds 3 and 8, respectively) were identified in all fruit by-products analyzed. Chlorogenic acid (compound 4) was found in lemon and apple by-products, while ferulic acid was determined just in orange by-products. Rosmarinic acid was found for the first time in orange by-products besides apple by-products [ 39 ]. Compound 5, with [M-H] − ion at m/z355 and the MS/MS fragment 193 m/zfrom ferulic acid, was tentatively identified as ferulic acid-O-hexoside. Additionally, compound 7, with [M-H] − ion at m/z385 and the MS/MS fragment 223 m/zfrom sinapic acid, was identified as sinapic acid-O-hexoside. These hydroxycinnamic acid glycosides were already described for orange pulp and juices by De Ancos et al. (2017) [ 41 ] Both compounds were considered for the first time for orange and lemon by-products in this work. 3.2.3. Flavanones Glycosides Together with hydroxycinnamic acids, flavanone glycosides were the main group of phenolic compounds present in the by-products analyzed in this study, mainly in those obtained from citrus fruits. Compound 24, with [M-H] − ion at m/z271, MS/MS fragment 151 m/z, and the UV–visible spectra typical at λmax 290 nm, was identified as Naringenin in all by-products analyzed (orange, lemon, and apple). Besides, compound 11, with [M-H] − ion at m/z595 that displayed the same fragmentation pattern in negative ionization mode that results in the fragment 151 m/z, was identified as eriodyctiol-O-rutinoside (eriocitrin). For this compound, the UV–visible spectrum showed two λmax at 290 and 330 nm, which are characteristic of flavanone glycosides and are usual to the following compounds identified in this group of phenolics. Naringenin-7-O-rutinoside (narirutin) (compound 13), with [M-H]− ion at m/z579, and naringenin-7-O-glucoside (prunin) (compound 20), with [M-H]− ion at m/z433, showed the same fragmentation pattern that results in the fragment 271 m/zof naringenin [40]. The MS/MS fragmentation of hesperidin (compound 15) and neohesperidin (compound 18), with the identical [M-H] − ion at m/z609, results in the same fragment 286 m/z and the UV–visible spectra λmax at 290 and 355. On the other hand, compound 23 was tentatively identified as isosakuranetin-7-O-rutinoside (dydimin), with [M-H] − ion at m/z593, and MS/MS fragment 285 m/z, as described by De Ancos et al. 2017 [41] for orange. The identity of compounds 11, 15, and 24 (eriocitrin, hesperidin, and naringenin, respectively ) was confirmed by comparison with the retention time and fragmentation pattern of commercial standards. Naringenin and prunin were identified in all by-products analyzed, while neohesperidin and dydimin were found just in orange by-products. Eriocitrin , narirutin, and hesperidin were identified in citrus by-products (orange and lemon). Foods 2021,10, 272 8 of 16 The identification of phenolics, for which standards were not available, was supported by recent studies found in the literature on these groups of compounds described for citrus peels (orange and lemon) [ 41 – 43 ], besides some studies for apple products including peels [38,40,44]. 3.2.4. Flavonols and Flavonol Glycosides The MS1 scan spectra, the UV–visible spectra typical at λmax 270 nm and 360 nm, and the same fragmentation pattern in negative ionization mode that results in the fragment 301 m/zin negative ionization mode compared with those of authentic standards determined that compounds 17, 19, and 22 are flavonols glycosides. Quercetin-3-O-rutinoside (rutin) (compound 19) and quercitrin (compound 22) standards allowed the identification of these flavonols in all by-products analyzed (orange, lemon, and apple). These phenolics have been described in other studies for orange, lemon, and apple products/byproducts [40]. Despite isoquercetin (compound 17) being described in apple fruit by Sommella et al. (2015) [ 44 ], in this study, it was just found in orange by-product and confirmed by the standard of reference. The identification of the aglycone quercetin (compound 25) in all fruit by-products was achieved by comparing the data with that obtained from the authentic standard. On the other hand, compound 14, with a [M-H] − ion at m/z593, was tentatively identified as kaempferol-3-O-rutinoside in orange and apple by-products based on the MS/MS fragment 285 m/z, the UV–visible spectra typical at λmax 356 nm and supported by literature where this flavonol glycoside was described before for apple fruit [40]. 3.2.5. Others (Flavones and Glycosides, Dihydrochalcone Glycosides and Flavan-3-ols) Compound 10 showed UV–visible spectra typical of flavones, and the MS1 spectra revealed a high intensity [M-H] − ion at m/z593. Moreover, the comparison of the relative absorbance at 270 and 340 nm allowed the identification flavone nature of the phenolic compound. Additionally, the fragment ion at m/z473 described in the literature for di-C-glucoside flavanone confirmed the identification of this compound as apigenin-6,8di-C-glucoside, more commonly identified as Vicenin-2. Apigenin-6,8-di-C-glucoside was previously identified in pulp and juices of orange and mandarins by De Ancos et al. (2017) [ 41 ], but was described for the first time in this study for orange and lemon byproducts. Compound 26, with [M-H] − ion at m/z269, was identified as another flavone, the aglycone apigenin, that followed the same fragmentation pattern of the reference standard that results in the fragment 117 m/zin negative mode. This compound was described before for orange products such as pulp, juice, and peels [ 40 , 41 ]. Still, in this study, apigenin was detected for the by-products obtained from lemon and apple fruits. Phenolic compounds 12 and 16, with [M-H] − ion at m/z567 and [M-H] − ion at m/z435, respectively, followed the same fragmentation pattern that results in the fragment 273 m/z, were tentatively identified as dihydrochalcone glycosides. Compound 12 was, tentatively, identified as phloretin-O-apiofuranosyl-glucopyranoside, and compound 16 was identified as phloretin-2 0 -O-beta-glucoside (phlorizin) by comparison with the reference standard. These compounds have been described in the literature for apple pomace and were detected in the apple by-product analyzed in this study [40,45]. Finally, also exclusive for apple by-product, phenolic compound 6 with the [M-H] − ion at m/z289, the fragment ions at m/z245 and 203, and the UV–visible spectra typical of flavan-3-ols at λmax 278 nm, was identified as epicatechin and its identity confirmed with the commercial standard. Epicatechin was also already described in the literature for apple products [40,44]. Foods 2021,10, 272 9 of 16 Foods 2021, 10, x FOR PEER REVIEW 9 of 17 These compounds have been described in the literature for apple pomace and were detected in the apple by-product analyzed in this study [40,45]. Finally, also exclusive for apple by-product, phenolic compound 6 with the [M-H]− ion at m/z 289, the fragment ions at m/z 245 and 203, and the UV–visible spectra typical of flavan-3-ols at λmax 278 nm, was identified as epicatechin and its identity confirmed with the commercial standard. Epicatechin was also already described in the literature for apple products [40,44]. Figure 1. HPLC-DAD chromatograms of fruit by-products (a) orange, (b) lemon, and (c) apple, recorded at 278 nm. Figure 1. HPLC-DAD chromatograms of fruit by-products ( a ) orange, ( b ) lemon, and ( c ) apple, recorded at 278 nm. Foods 2021,10, 272 16 of 16 27. Erkan, N.; Ayranci, G.; Ayranci, E. 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