Citrus fruits—more precisely, the genus Citrus L., which belongs to the subfamily Aurantioideae in the family Rutaceae—represent the major fruit crops commercially cultivated worldwide ^[1][2][1,2]. These fruits are widely known for their high health-benefiting properties, which is of great importance, since citrus fruits are the most widely consumed fruits globally ^[3][4][5][6][3,4,5,6]. The cultivation of Citrus genus includes the species such as lemon (C. limon (L.) Osbeck), sweet orange (C. sinensis (L.) Osbeck), mandarin (C. reticulata Blanco), grapefruit (C. paradisi Macfad.), pomelo (C. maxima (Burm.) Merr.), citron (C. medica L.), lime (C. aurantiifolia (Christm.) Swingle), and bergamot (C. × bergamia Risso & Poiteau) ^{[4][5][7][8][9]}[4,5,7,8,9]. Although a significant part of the industrially processed citrus fruits is used to produce essential oils and juice, citrus-based candies, jellies, and extracts production also represent key factors for the food industry [5]. A large quantity of waste and by-products yearly produced during citrus processing has become a fundamental concern from both economical and environmental aspects. Citrus processing generates over 15 million tons of residue, mostly in the form of peels, seeds, and membranes ^[10][11][10,11]. Therefore, citrus waste valorization and proper industrial waste management are highly encouraged, being also a high-research priority topic for the scientific community recently. There have been many scientific reports related to the potential and beneficial utilization of industrial by-products, mainly dealing with innovative extraction methods for obtaining extracts rich in bioactive compounds ^{[1][2][3][4][10][11][12]}[1,2,3,4,10,11,12]. These extracts have shown versatile health beneficial activities, due to the high content of biologically active compounds naturally present in the extracts ^[13][14][13,14]. Numerous studies have considered citrus extracts as a natural source of bioactive components exhibiting beneficial activities, including antioxidant ^{[15][16][17][18][19]}[15,16,17,18,19], antibacterial ^{[16][18][19][20][21]}[16,18,19,20,21], antidiabetic ^{[17][22][23][24]}[17,22,23,24], neuroprotective ^{[22][25][26][27]}[22,25,26,27], and anti-inflammatory ^{[28][29][30][31]}[28,29,30,31] activities, as well as antitumor ^{[32][33][34][35]}[32,33,34,35] potential. Also, citrus by-products are considered a valuable source of phytochemicals (such as d-limonene, essential oils, phenolic acids, carotenoids, vitamins, minerals, and flavonoids), which, isolated or in the form of mixtures/extracts, could exhibit versatile biological activities especially beneficial for the food industry ^[36][37][38][36,37,38]. Citrus-based essential oils exhibit significant antimicrobial activity against foodborne bacteria and also antioxidant activity to prevent the effects of oxidation; hence, citrus-based essential oils could act as natural preservatives ^[39][40][41][39,40,41].

2. Structural and Chemical Characteristics of Citrus Fruits By-Products

Citrus fruits, like many other agricultural products, are characterized by their agricultural biodiversity ^[42][62]. Their physicochemical characteristics, as well as a diversity of chemical compounds, depend on a variety of factors and environmental conditions, such as soil, fertilization, age, position on the tree, maturity, and others ^{[42][43][44][45]}[62,63,64,65]. It is interesting that all varieties of citrus fruits, by means of microscopic and macroscopic views, have similar structural and anatomical characteristics. A schematic view of the anatomical characteristics and structural compositions of citrus fruits is presented in Figure 1. Citrus fruits are widely consumed due to their nutritional qualities and appealing taste and fragrance, and the products of citrus processing mostly include the production of food-grade (jellies, jams, candies, flavoring agents, etc.) and aromatic/cosmetic (essential oils) products ^[10][46][47][10,66,67]. The generation of significant amounts of waste during citrus fruit processing is a major concerning issue, as the waste represents almost 50% of the fresh fruit mass ^[46][66]. The generated waste includes by-products such as peels (the highest percentage of almost 50%), seeds (20–40%), pomace, and wastewater (the residue of spoiled parts of the fruits) ^[46][47][48][66,67,68]. The outer layer of the citrus fruit consists of the peel, which can be roughly divided into two regions: flavedo (lat. flavus means yellow) and albedo (lat. albus means white) ^[49][50][69,70]. The flavedo region comprises characteristic peel oils and pigments, and the white spongy part of the peel is referred to as the albedo ^[51][71]. Although, it is not uncommon that some literature reports refer to flavedo as epicarp and albedo as mesocarp ^[52][72]. The flavedo region is covered with a thin layer of cuticle, consisting of natural waxes and continuous polymerized materials ^[49][53][60,69]. The role of the cuticle is mainly protective against microorganism attacks, limit vapors, and water loss, regulating also the exchange of oxygen and carbon dioxide ^[54][55][73,74]. From a chemical point of view, natural waxes are characterized by the presence of long-chain alkanes, fatty acids, aldehydes, and alcohols ^[48][52][68,72], while the polymerized material originates from hydroxylated fatty acids ^[56][75]. Below the cuticle and within the flavedo region, pigments and essential oils are present. The citrus pigments are located within the chloroplasts (if green) and in chromoplasts (if yellow, orange, or red color) ^[42][62]. The composition and differences in the carotenoid content determine the color of citrus fruits ^[57][76]. The green or yellowish-green color of immature citrus fruits originates from the accumulation of lutein and a certain content of chloroplastic carotenoids, such as β- and α-carotene, neoxanthin, and zeaxanthin ^[57][76]. However, the orange color formed during the natural ripening of citrus fruits is caused by the increase in the content of colored carotenoids (β,β-xanthophylls) and by a decrease in the lutein concentration ^[58][59][77,78]. The essential oils are found in the oil glands located in the citrus flavedo layers and are defined as fragrant compounds present in the peel. The citrus essential oils consist of volatile compounds in the majority (85–99%) and in lower fractions as non-volatile compounds (1–15%) ^[60][79]. The albedo layer is considered to be a white and relatively porous layer of the citrus peel, consisting of pectic substances, cellulose, starch, and phenolic compounds ^[42][61][62,83]. The pectins are a complex group of compounds defined as non-starch polysaccharides, mostly consisting of conjugates of D-galacturonic acid; acid groups (methoxy esters); and some neutral sugars (rhamnose, glucose, xylose, arabinose, etc.) formed through α-(1-4)-glycosidic bonds ^[62][63][84,85]. Based on the degree of methylation and acetylation—more precisely, by the number of methoxyl and acetyl groups substituted by the carboxylic acid on the D-galacturonic acid chain—high-methoxyl or low-methoxyl pectins can be formed ^[62][84]. Although it is well known that low-methoxyl pectins are used in the food industry due to their gelation characteristics ^[64][86], pectins are also studied for their utilization in pharmacy and medicine (cholesterol reduction, drug delivery, immune modulation, etc.) ^[63][85]. Furthermore, phenolic compounds and flavonoids provide several benefits associated with health-promoting effects ^[45][65][65,90]. The major flavonoids present in citrus peels are hesperidin, narirutin, naringin, and rutin, while the content of each flavonoid depends on the physicochemical characteristics of the cultivated fruit and analyzed citrus variety ^[66][91].

3. Converting Waste into Treasure—Utilization of Citrus By-Products

Recently, waste management has become one of the great concerns globally, and its valorization has created more sustainable and smart waste management solutions. Primarily, waste valorization includes employing different technologies toward obtaining value-added products with a wide spectrum of potential applications. Citrus by-products have been extensively studied due to their rich-bioactive properties, and their valorization enables beneficial gains from both economical and environmental points of view ^[67][68][96,97]. The scientific focus has been placed on innovative extraction methods for obtaining high-quality citrus essential oils ^{[69][70][71][72]}[98,99,100,101] and enriched extracts in general ^{[11][73][74][75]}[11,94,102,103]. Solid citrus waste can be also utilized for the production of animal food. Due to its good nutritional composition containing dietary fibers, lipids, flavonoids, enzymes, vitamins, and carotenoids, citrus waste represents a promising by-product for the production of livestock feeds ^[76][105]. The literature reports that citrus pulp (the main residue after juice extraction), citrus molasses (produced by concentrating on the press liquor of citrus peel residue with a high content of sugars), citrus peel liquor (similar to molasses but not as concentrated), and citrus-activated sludge (produced from liquid waste) could be considered as by-product feedstuffs ^[77][106]. The nutrient content of citrus by-products mainly depends on the source and variety of citrus fruits, as well as on the type of processing ^[78][88]. The main issues in the utilization of citrus by-products for the production of feedstuffs are the low nitrogen content and poor storage, which can lead to the development of mycotoxins ^[78][88]. Another valuable utilization of citrus waste is the production of packaging films that meets all the standards of sustainable and biodegradable forms of biopolymers ^[10][79][10,107]. Conventional packaging films are considered an environmental concern due to their poor biodegradable properties, and therefore, new innovative and sustainable solutions are welcomed ^[79][107]. An important advantage of using biopolymers derived from plant materials is that those raw materials naturally contain significant amounts of bioactive components that exhibit antioxidant and antimicrobial properties. The matrix of the citrus-based package is pectin, which enables solid support for the production of active packaging films [10].  The application of plant-based and phenolic extracts in the food industry is highly limited due to their poor bioaccessibility, low water, and liquid solubility, while it is well known that bioactive phenolic compounds are extremely sensitive to light, oxidants, and changes in pH conditions and temperatures ^[68][80][81][47,97,109]. The main challenge presents as overcoming the limiting incorporation of low water-soluble compounds into aqueous-based foods, which directly limits the proper gastrointestinal bioaccessibility. In order to overcome these issues, encapsulation has been employed more frequently to protect bioactive compounds ^[80][82][83][47,110,111]. The spray-drying and freeze-drying techniques are commonly used methods for obtaining stable encapsulated functional substances, while extrusion methods, coacervation, and emulsification methods have been also applied ^[82][110].

4. Bioactivities of the Individual Groups of Compounds Present in Citrus By-Products

4.1. Waxes and Carotenoids

Cuticular wax plays an important role in fruit preservation and proper storage, and it is well known that it acts as a natural barrier that protects plants from biological and non-biological stress ^[54][55][73,74]. Also, the structural characteristics, content, and composition of cuticular wax have been found to affect the postharvest storage quality against fruit water loss and softening and could be responsible for the resistance to fruit diseases. Waxes are comprised of long-chain fatty acids and their derivates, esters, aldehydes, ketones, primary and secondary alcohols, and triterpenoids ^[84][115]. Most of the studies related to the topic of citrus cuticular waxes focused on the synthesis and transcriptional regulation of cuticular wax in citrus fruits. The investigation of the carotenoid content separated from C. reticulata by-products and its influence on the immuneoxidative status of broiler chickens was carried out by Mavrommatis et al. ^[85][118]. The carotenoid-rich extract was prepared, and the chickens were fed a supplemented diet consisting of a freeze-dried formulation containing carotenoid extract and soluble starch. It was demonstrated that carotenoid-supplemented feed exerted inhibitory activity against Gram-positive (Staphylococcus (S.) aureus), as well as Gram-negative (Klebsiella (K.) oxytoca, Escherichia (E.) coli, and Salmonella (S.) typhimurium), bacteria. The implementation of the carotenoid content in the supplementation led to alanine aminotransferase and breast muscle malondialdehyde, and the activity of superoxide dismutase increased. Also, several parameters were downregulated, such as catalase, NADPH oxidase 2, interleukin 1β, and tumor necrosis factor. 

4.2. Aromatic Compounds—Essential Oils

Citrus essential oils are known as a fragrant mixture of chemical compounds exhibiting versatile activities used in the food, cosmetical, and pharmaceutical industries, as well as in aromatherapy ^[60][79]. The involvement of nanotechnology has provided new solutions for developing essential oil-based nanosystems with the aim of bioaccessibility enhancement. Interestingly, the formulation of C. lemon essential oil in nanohexosomes was prepared by the group of authors Sedeek et al. ^[86][120] for the purpose of antifungal activity investigation. Firstly, C. lemon, C. aurantifolia, C. maxima, and C. sinensis essential oils were extracted using hydrodistillation in a Clevenger’s apparatus from powdered peels. The hexosomal dispersions loaded with oils were prepared by the hot emulsification method reported by Abdel-Bar et al. ^[87][121]. Furthermore, the antimicrobial efficacy of citrus-based essential oils against foodborne pathogen Listeria monocytogenes (L. monocytogenes) was demonstrated in the study by Guo et al. ^[88][126]. Gram-positive bacteria L. monocytogenes is a highly adaptable pathogen that causes listeriosis, a life-threatening infection, and it is especially dangerous if the central nervous system is affected ^[89][141]. The essential oil from the C. Changshan-huyou Y.B. Chang (Huyou) species was extracted from peels by the steam distillation procedure using water as the solvent.  The results of the antimicrobial activity of Huyou essential oil against L. monocytogenes showed dose-dependant antimicrobial activity when comparing treatments of pathogens with the 1xMIC (minimum inhibitory concentration), 0.25xMIC, and 0.125 MIC. The study also discussed the changes in the physical morphology of L. monocytogenes biofilms when treated with 1xMIC for 8, 16, and 24 h by using scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) analyses. 

4.3. Pectins

The importance of pectins in the food industry is widely known; however, this group of polysaccharides has found their place in a variety of human applications, such as in the pharmaceuticals, cosmetics, drug delivery, and biomedical fields ^[64][89][90][86,141,142]. The versatile application of pectic biopolymers is enabled due to their structural diversity and chemical complexities, as well as the possibility of structural modifications ^[91][143]. In addition, Zhang et al. ^[92][145] obtained pectin oligosaccharide fractions by the controlled degradation of citrus peel pectin. Three different oligosaccharides were prepared by adjusting the concentration of trifluoroacetic acid or H₂O₂ at the appropriate pH value, producing pectin oligosaccharides of variable molecular weight ranging from <2000 Da, 2000 to 3000 Da, and 3000 to 4000 Da. The results demonstrated a high prebiotic activity (pectic oligosaccharides obtained by H₂O₂ oxidation; 3543 Da) for Bifidobacterium (B.) bifidum and moderate activity against the Lactobacillus (L.) paracasei bacterial species. Theis study showed the enormous prebiotic potential of citrus-based pectic oligosaccharides; however, the greatest challenge remains to be overcome, as the human gastrointestinal tract includes complex pH-dependent processes and the presence of different enzymes that could affect in vivo digestion and bioaccessibility.

4.4. Phenolic Compounds

Natural phenolic compounds have been studied extensively for their essential role in plant protection, as well as for their beneficial effects on human health. It is well known that citrus by-products contain substantial contents of different phenolic compounds in the forms of acids and flavonoids, which have recently become the great subject of studies as natural antioxidants ^[93][94][156,157]. The representative bioactive compounds for the citrus family are flavanone aglycones (hesperetin, naringenin, and eriodictyol); flavone and flavonol aglycones (kaempferol, quercetin, apigenin, and diosmetin); flavanone-7-O-glycosides (eriocitrin, hesperidin, naringin, narirutin, poncirin, and didymin); and polymethoxyflavones (PMFs; nobiletin, tangeretin, and sinensetin) ^[42][66][95][62,91,158].

5. Citrus By-Products Formulations with Enhanced Bioactivities

The biomass-derived compounds are known for their health-promoting properties, and there is a rising trend in waste and by-product valorization to obtain value-added products with a wide spectrum of applications ^{[2][11][96][97]}[2,11,194,195]. The beneficial effects of different citrus by-products on human health are not disputable; however, poor bioaccessibility is a crucial and limiting factor for successful in vivo applications. Therefore, new and innovative ideas with the implementation of nanotechnology brought about some new solutions in bioaccessibility and bioactivity enhancements. The preparation of silver nanoparticles (AgNPs) from citrus (C. tangerina, C. sinensis, and C. limon) peel extract was reported by Niluxsshun et al. ^[98][196]. Firstly, citrus peel extracts were prepared by boiling peels in hot water, and afterward, a solution of AgNO₃ was added to the flask when a golden colloidal suspension was formed. The structural and morphological analyses confirmed the presence of AgNPs in sizes of 10–70 nm, containing different morphological characteristics of nanoparticles. The presence of natural antioxidants, flavonoids, phenolic acids, and other phenolic compounds could act as a reducing agent, leading to the formation of silver nanoparticles. The AgNPs were investigated for antimicrobial activity against the Gram-negative bacteria E. coli and the Gram-positive bacteria S. aureus, and the results showed the superior antimicrobial activity of orange-based AgNPs on both bacteria strains. Also, it is expected that the bioactivity of nanoparticles is dose- and size-dependent ^[99][197], and it is assumed that silver potentially interacts with thiol groups of proteins on cell membranes, causing respiration blocking, which leads to cell death. Another example of AgNP synthesis by using citrus by-products for the purposes of antimicrobial investigation was reported by Alkhulaifi et al. ^[100][198]. In theis study, C. limon peels were used for the synthesis of AgNPs, which were formed by the addition of a AgNO₃ solution. Again, a possible explanation for the AgNPs formation was the reduction of Ag⁺ ions to silver nanoparticles in the presence of phenolic compounds, and the AgNPs demonstrated spherical- and rod-like-shaped morphologies. The antimicrobial activity investigation was carried out on Acinetobacter (A.) baumannii, S. typhimurium, E. coli, Pseudomonas (P.) aeruginosa, S. aureus, and Proteus (P.) vulgaris human pathogenic bacteria. The results indicated the good performance of AgNPs against the Gram-negative (E. coli, S. typhimurium, and P. aeruginosa) and Gram-positive (S. aureus) bacteria.