Pectins find many applications in the food and drug industry as a thickening and gelling agents, excipients, and colloidal stabilizers [93].

As it has been already mentioned, the extraction method does affect the structure and the properties of the final product; the traditional acidic water extraction implies a certain degree of hydrolytic deterioration, so that new extraction technologies have been being investigated to improve the quality of the final pectins, that is microwave-assisted extraction (MAE) [94] and ultrasounds assisted extraction (USAE) [35, 95].

#### **2.4 Enzymes**

As it can be easily argued, the CSP cellular system, whose genomic profile has been fully characterized [96], is the site of a complex network of enzymatic activity. Some of the enzymes of CSP have been characterized and employed in many applications.

The acetylesterase (international enzymatic classification: EC <IP_ADDRESS>) from CSP is known since 1947 [97] and was isolated and characterized [98]. The acetylesterase activity of the partially purified enzyme was used for the removal of the acetyl group at the 3 positions of β-lactamic antibiotics **116** [98] (**Figure 7a**). Further, the whole CSP, as well as pomace from the industrial waste of the orange juice production, was successfully employed to catalyze several relevant biotransformations [99] such as the conversion of Geranyl acetate **118** to Geraniol **119** (**Figure 7b**) and the di-acetoxynaphtalene derivative **120** to the vitamin k1 precursor **121** (**Figure 7c**).

*The Orange Peel: An Outstanding Source of Chemical Resources DOI: http://dx.doi.org/10.5772/intechopen.96298*

**Figure 7.** *Chemical reactions biocatalysed by enzymes from* C. sinensis *peels.*

Recently, partial purification and functional characterization of a Uronic acid oxidase from CSP was accomplished [100]; this enzyme promotes the oxidation by O2 of Galacturonic acid **122** to Galactaric acid **123** (**Figure 7d**).

#### **2.5 Miscellaneous**

#### *2.5.1 Highly lipophilic compounds*

The waxy environment of flavedo in CSP does contain several long-chain saturated and unsaturated compounds: alkanes, fatty acids, waxes, higher terpenoids.

Tetracosane, Tetratriacontanoic acid, and Ethyl pentacosanoate were identified in CSP of a Pineapple variety [101]. Further, some carotenoids were identified in the CSP extract obtained with a solvent mixture composed of Ethanol, Ethyl acetate, Petroleum ether 1: 1:1 [102]. This complex blend of carotenoids includes α- and β-Carotene, Phytoene, Phytofluene, (all-E)- and (9Z)-Violoxanthin, (all-E)- Neoxanthin, (13Z)-, (13Z')- and (all-E)-Lutein, (9Z)-Zeaxanthin, (all-E)-Zeaxanthin; the mono and di-esters of violaxanthin, antheroxanthins, Xanthophyll, β-Citraurin with various fatty acids, including Lauraic, Myristic, Oleic, Palmitic, Stearic. The composition of the blend has been correlated with the maturity stage of the fruit.

**Figure 8.**

*Primary structure of cyclic peptide isolated from the* C. sinensis *peels.*

#### *2.5.2 Peptides*

Three cyclic peptides have been isolated from the hot water extract of CSP and were structurally characterized by FAB-MS and 2D-NMR techniques [103]. Their amino-acidic sequences, including a mostly lipophlic heptapeptide **124**, a dihydroxylated heptapeptide **125,** and a Glutamate-rich octapeptide **126**, are reported in **Figure 8**.

#### **3. Conclusions**

The chemical richness of the primary and secondary metabolome of *C. sinesnis* species is undoubtedly impressive. Thousands of different compounds belonging to dozens of structural classes have been isolated and described. The most deeply investigated are sure, on one hand, the mixtures of volatile compounds composing the blend of the essential oil and, on the other hand, polyphenols, especially flavonoids.

The chemical composition of the extract from the exocarp of *C. sinensis* does differ from the composition of juice, or leaf extracts for some aspects [104]: the presence of a higher amount of more lipophilic compounds such as polymethoxyflavonoids, r carotenoids, higher alkanes; a lesser extent of lighter terpenoids, a lower content of glycosylated compounds, the absence of cyanidins and sterols.

It is also a matter of fact that several interesting bioactivities were disclosed in the last years for the *C. sinensis* extracts that have been variously associated with the well-recognized beneficial effects that regular sweet oranges consumption may have on human health. However, a great deal of research work is still needed to clarify the molecular basis and the mechanism of these chemopreventive effects and to relate them with precise chemical entities that can be recognized as valuable nutraceuticals, as it is already the case for the well-established antioxidants Ascorbic acid, Hesperidin, Hesperetin, Quercetin, etc.

#### **Conflict of interest**

The author declares no conflict of interest.

*The Orange Peel: An Outstanding Source of Chemical Resources DOI: http://dx.doi.org/10.5772/intechopen.96298*

### **Author details**

Gianfranco Fontana

Department of Biological, Chemical and Pharmaceutical Sciences and Technologies (STEBICEF), University of Palermo, Palermo, Italy

\*Address all correspondence to<EMAIL_ADDRESS>
© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

### **References**

[1] Food and Agriculture Organization of the United nations: Citrus fruit fresh and processed - Statistical Bulletin 2016 [Internet]. 2017. Available from: http:// www. http://www.fao.org/3/a-i8092e. pdf [Accessed: 2020-12-16].

[2] Tamburino V, Zama DA. I sottoprodotti dell'industria di trasformazione: il pastazzo di agrumi. In: Vacante V, editor. Citrus - Trattato di agrumicoltura. Il Sole 24 Ore-Edagricole; 2009. p. 459–470.

[3] Putnik P, Kovačević DB, Jambrak AR, Barba FJ, Cravotto G, Binello A, Lorenzo JM, Shpigelman A. Innovative "Green" and Novel Strategies for the Extraction of Bioactive Added Value Compounds from CitrusWastes—A Review. Molecules. 2017:22:680–704. DOI: 10.3390/ molecules22050680.

[4] Senit JJ, Velasco D, Gomez Manrique A, Sanchez-Barba M, Toledo LM, Santos VE, Garcia-Ochoa F, Yustos P, Ladero M. Orange peel waste upstream integrated processing to terpenes, phenolics, pectin and monosaccharides: Optimization approaches. Ind. Crops & Prod. 2019:134:370–381. DOI: 10.1016/j. indcrop.2019.03.060.

[5] Lamine M, Gargouri M, Rahali FZ, Mliki A. Recovering and Characterizing Phenolic Compounds From Citrus By-Product: A Way Towards Agriculture of Subsistence and Sustainable Bioeconomy. Waste Biomass Valor. 2020. DOI: 10.1007/s1264 9-020-01306 -9.

[6] Williams CA. Specialized dietary supplements. In: Geor RJ, Harris PA, Coenen M, editors. Equine Applied and Clinical Nutrition. Saunders; 2013. p. 351–366. DOI: 10.1016/C2009-0- 39370-8.

[7] Patel S, Shukla S. Fermentation of Food Wastes for Generation of

Nutraceuticals and Supplements. In: Frias J, Martinez-Villaluenga C, Peñas E, editors. Fermented Foods in Health and Disease Prevention. Academic Press; 2016. p. 707–734. DOI: 10.1016/ C2014-0-01734-0.

[8] Khan N, Mongas M, Urpi-sarda M, Llorach R, Andres-La Cueva C. Contribution of Bioactive Foods and Their Emerging Role in Immunomodulation, Inflammation, and Arthritis. In: Watson RR. Preedy VR, editors. Bioactive Food as Dietary Interventions for Arthritis and Related Inflammatory Diseases. Academic Press; 2013. p. 43–65. DOI: 10.1016/ C2011-0-07467-7.

[9] Rehman Z. Citrus peel extract – A natural source of antioxidant. Food Chem. 2006;99:450–454. DOI: 10.1016/ j.foodchem.2005.07.054.

[10] Abe S, Fan K, Ho CT, Ghai G, Yang K. Chemopreventive Effects of Orange Peel Extract (OPE) II. OPE Inhibits Atypical Hyperplastic Lesions in Rodent Mammary Gland. J. Med. Food 2007;10:18–24; DOI: 10.1089/ jmf.2006.0215.

[11] Lai CS, Li S, Miyauchi Y, Suzawa M, Hob CT, Pan MH. Potent anti-cancer effects of citrus peel flavonoids in human prostate xenograft tumors. Food Funct. 2013;4:944–949; DOI: 10.1039/ c3fo60037h.

[12] Tung YC, Chang WT, Li S, Wu JC, Badmeav V, Ho CT, Pan MH. Citrus peel extracts attenuated obesity and modulated gut 1 microbiota in a high-fat diet-induced obesity mice. Food Funct. 2018;9:3363–3373; 10.1039/ C7FO02066J.

[13] Qiao Y, Jun Xie B, Zhang Y, Zhang Y, Fan G, Yao XL, Pan SY. Characterization of Aroma Active Compounds in Fruit Juice and Peel Oil *The Orange Peel: An Outstanding Source of Chemical Resources DOI: http://dx.doi.org/10.5772/intechopen.96298*

of Jinchen Sweet Orange Fruit (*Citrus sinensis* (L.) Osbeck) by GC-MS and GC-O. Molecules. 2008;13:1333–1344; DOI: 10.3390/molecules13061333.

[14] Dosoky NS, Setzer WN. Biological Activities and Safety of Citrus spp. Essential Oils. Int. J. Mol. Sci. 2018;19: 1966; DOI: 10.3390/ijms19071966.

[15] Ettoumi KY, Zouambia Y, Moulai-Mostefa N. Chemical composition, antimicrobial and antioxidant activities of Algerian *Citrus sinensis* essential oil extracted by hydrodistillation assisted by electromagnetic induction heating. J Food Sci Technol DOI: 10.1007/ s13197-020-04808-5

[16] Leherbauer I, Stappen I. Selected essential oils and their mechanisms for therapeutic use against public health disorders. An overview. Z. Naturforsch. 2020;75: 205–223.

[17] Singh P, Shukla R, Prakash B, Kumar A, Singh S, Mishra PK, Dubey NK. Chemical profile, antifungal, antiaflatoxigenic and antioxidant activity of *Citrus maxima* Burm. and *Citrus sinensis* (L.) Osbeck essential oils and their cyclic monoterpene, DLlimonene. Food Chem. Toxicol. 2010;48: 1734–1740; DOI:10.1016/j. fct.2010.04.001.

[18] O'Bryan CA, Crandall PG, Chalova VI, Ricke SC. Orange Essential Oils Antimicrobial Activities against Salmonella spp. J. Food Sc. 2008;73: M264-M267; DOI: 10.1111/ j.1750-3841.2008.00790.x.

[19] Fisher K, Phillips CA. The effect of lemon, orange and bergamot essential oils and their components on the survival of Campylobacter jejuni, *Escherichia coli* O157, *Listeria monocytogenes*, *Bacillus cereus* and *Staphylococcus aureus* in vitro and in food systems. J. Appl. Microbiol. 2006; 101:1232–1240; DOI:10.1111/ j.1365-2672.2006.03035.x.

[20] Muthaiyan A, Martin EM, Natesan S, Crandall PG, Wilkinson BJ, Ricke SC. Antimicrobial effect and mode of action of terpeneless coldpressed Valencia orange essential oil on methicillin-resistant *Staphylococcus aureus*. J. Appl. Microbiol. 2012;112: 1020–1033; DOI:10.1111/ j.1365-2672.2012.05270.x.

[21] Hung PV, Chi PTL, Phi NTL. Comparison of antifungal activities of Vietnamese citrus essential oils. Nat. Prod. Res. 2013;27:506–508; DOI: 10.1080/14786419.2012.706293.

[22] Najar B, Shortrede JE, Pistelli L, Buhagiar J. Chemical Composition and in Vitro Cytotoxic Screening of Sixteen Commercial Essential Oils on Five Cancer Cell Lines. Chem. Biodiversity 2020;17:e1900478; DOI: 10.1002/ cbdv.201900478.

[23] Faturi CB, Leite JR, Alves PB, Canton AC, Teixeira-Silva F. Anxiolytic-like effect of sweet orange aroma in Wistar rats. Prog. Neuro-Psychoph. 2010;34:605–609; DOI: 10.1016/j.pnpbp.2010.02.020.

[24] Uçar Y. Antioxidant Effect of Nanoemulsions Based on Citrus Peel Essential Oils: Prevention of Lipid Oxidation in Trout. Eur. J. Lipid Sci. Technol. 2020;122:1900405–1900419; DOI: 10.1002/ejlt.201900405..

[25] Ciriminna R, Meneguzzo F, Pagliaro M. Orange Oil. In: Nollet LML, Rathore HS editors. Green Pesticides Handbook – Essential Oils for Pest Control. CRC Press; 2017. p. 215–265.

[26] Lehrner J, Eckersberger C, Walla P, Pötsch G, Deecke L. Ambient odor of orange in a dental office reduces anxiety and improves mood in female patients. Physiology & Behavior 2000; 71:83–86.

[27] Anandakumar P, Kamaraj S, Vanitha MK. D-limonene: A

multifunctional compound with potent therapeutic effects. J. Food Biochem. 2020;45:e13566; DOI: 10.1111/ jfbc.13566.

[28] Li S, Lo CY, Ho CT. Hydroxylated Polymethoxyflavones and Methylated Flavonoids in Sweet Orange (*Citrus sinensis*) Peel. J. Agric. Food Chem. 2006;54:4176–4185. DOI: 0.1021/ jf060234n.

[29] Gao Z, Gao W, Zeng SL, Li P, Liu EH. Chemical structures, bioactivities and molecular mechanisms of citrus polymethoxyflavones. J. Funct. Foods 2018;40:498–509; DOI: 10.1016/j. jff.2017.11.036.

[30] Owis AL. Citrus Polymethoxyflavones: Biofunctional Molecules of Therapeutic Interest. Studies in Nat. Prod. Chem. 2019;59: 509–530. DOI: 10.1016/B978-0- 444-64179-3.00015-3.

[31] Lai CS, Wu JC, Ho CT, Pan MH. Disease chemopreventive effects and molecular mechanisms of hydroxylated polymethoxyflavones. Biofactors 2015; 41:301–313; DOI 10.1002/biof.1236.

[32] Del Río JA, Gómez P, Báidez AG, Arcas MC, Botía JM, Ortuño A. Changes in the Levels of Polymethoxyflavones and Flavanones as Part of the Defense Mechanism of *Citrus sinensis* (Cv. Valencia Late) Fruits against Phytophthora citrophthora. J. Agric. Food Chem. 2004;52:1913–1917; DOI: 10.1021/jf035038k.

[33] Mizuno M, Iinuma M, Ohara M, Tanaka T, Iwamasa M. Chemotaxonomy of the Genus Citrus Based on Polymethoxyflavones. Chem. Pharm. Bull. 1991;39:945–949; DOI: 10.1248/ cpb.39.942.

[34] Kanaze FI, Termentzi A, Gabrieli C, Niopas I, Georgarakis M, Kokkaloua E. The phytochemical analysis and

antioxidant activity assessment of orange peel (*Citrus sinensis*) cultivated in Greece–Crete indicates a new commercial source of hesperidin. Biomed. Chromatogr. 2009;23:239–249. DOI: 10.1002/bmc.1090.

[35] Montero-Calderon A, Cortes C, Zulueta A, Frigola A, Esteve MJ. Green solvents and Ultrasound- Assisted Extraction of bioactive orange (*Citrus sinensis*) peel compounds. Scientific Reports. 2019;9:16120–16128. DOI: 10.1038/s41598-019-52717-1.

[36] Manthey JA. Fractionation of Orange Peel Phenols in Ultrafiltered Molasses and Mass Balance Studies of Their Antioxidant Levels. J. Agric. Food Chem. 2004;52:7586–7592. DOI: 0.1021/ jf049083j.

[37] Anagnostopoulou MA, Kefalas P, Kokkalou E,. Assimopoulou AN, Papageorgiou VP. Analysis of antioxidant compounds in sweet orange peel by HPLC–diode array detection– electrospray ionization mass spectrometry. Biomed. Chromatogr. 2005;19:138–148. DOI: 10.1002/ bmc.430.

[38] Liew SS, Ho WY, Yeap SK, Bin Sharifudin SA. Phytochemical composition and in vitro antioxidant activities of *Citrus sinensis* peel extracts. PeerJ. 2018;6:e5331. DOI: 10.7717/ peerj.5331.

[39] Guo C, Shan Y, Yang Z, Zhang L, Ling W, Liang Y, Ouyang Z, Zhong B, Zhang J. Chemical composition, antioxidant, antibacterial, and tyrosinase inhibition activity of extracts from Newhall navel orange (*Citrus sinensis* Osbeck cv. Newhall) peel. J Sci Food Agric 2020;100:2664–2674. DOI: 10.1002/jsfa.10297.

[40] Gosslau A, Chen KY, Ho CT, Li S. Anti-inflammatory effects of characterized orange peel extracts enrichedwith bioactive

polymethoxyflavones. Food Sci. Human Well. 2014;3:26–35; DOI:10.1016/j. fshw.2014.02.002.

[41] Hagenlocher Y, Feilhauer K, Schäffer M, Bischoff SC, Lorentz A. Citrus peel polymethoxyflavones nobiletin and tangeretin suppress LPSand IgE-mediated activation of human intestinal mast cells. Eur J Nutr 2017;56: 1609–1620; DOI 10.1007/s00394-016- 1207-z.

[42] Liu L, Xu X, Cheng D, Yao X, Pan S. StructureActivity Relationship of Citrus Polymethoxylated Flavones and Their Inhibitory Effects on Aspergillus niger. J. Agric. Food Chem. 2012;60: 4336–4341. DOI: 10.1021/jf3012163.

[43] Shetty SB, Mahin-Syed-Ismail P, Varghese S, Thomas-George B, Kandathil-Thajuraj P, Baby O, Haleem S, Sreedhar S, Devang-Divakar D. Antimicrobial effects of *Citrus sinensis* peel extracts against dental caries bacteria: An in vitro study. J Clin Exp Dent. 2016;8:e70–7. DOI:10.4317/ jced.52493.

[44] Ortuño A, Báidez A, Gómez P, Arcas MC, Porras I, García-Lidón A, Del Río JA. Citrus paradisi and *Citrus sinensis* flavonoids: Their influence in the defence mechanism against Penicillium digitatum. Food Chem. 2006;98:351– 358; DOI:10.1016/j. foodchem.2005.06.017.

[45] Bagavan A, Rahuman AA, Kamaraj C, Kaushik NK, Mohanakrishnan D, Sahal D. Antiplasmodial activity of botanical extracts against *Plasmodium falciparum*. Parasitol Res 2011;108:1099–1109. DOI: 10.1007/s00436-010-2151-0.

[46] Nakanishi M, Hino M, Yoshimura M, Amakura Y, Nomoto H. Identification of sinensetin and nobiletin as major antitrypanosomal factors in a citrus cultivar. Exp.

Parasitol. 2019;200:24–29; DOI: 10.1016/j.exppara.2019.03.008.

[47] Lara Testai L, Calderone V. Nutraceutical Value of Citrus Flavanones and Their Implications in Cardiovascular Disease. Nutrients. 2017; 9:502–515. DOI:10.3390/nu9050502.

[48] Shalaby NMM, Abd-Alla HI, Ahmed HH, Basoudan N. Protective effect of *Citrus sinensis* and *Citrus aurantifolia* against osteoporosis and their phytochemical constituents. J. Med. Plant. Res. 2011;5:579–588.

[49] Selmi S, Rtibi K, Grami D, Sebai H, Marzouki L. Protective effects of orange (*Citrus sinensis* L.) peel aqueous extract and hesperidin on oxidative stress and peptic ulcer induced by alcohol in rat. Lipids in Health and Disease. 2017;16:152– 164. DOI: 10.1186/s12944-017-0546-y.

[50] Chen PY, Li S, Koh YC, Wu JC, Yang MJ, Ho CT, Pan MH. Oolong Tea Extract and Citrus Peel Polymethoxyflavones Reduce Transformation of L-Carnitine to Trimethylamine-N-Oxide and Decrease Vascular Inflammation in L-Carnitine Feeding Mice. J. Agric. Food Chem. 2019; 67:7869–7879. DOI: 10.1021/acs. jafc.9b03092.

[51] Sathiyabama RG, Gandhia GR, Denadaib M, Sridharanc G, Jothic G, Sasikumard P, Siqueira Quintanse JdS, Narain N, Cuevas LE, Melo Coutinho HD, Barbosa Ramose AG, Quintans-Júniore LJ, Queiroz Gurgela R. Evidence of insulin-dependent signalling mechanisms produced by *Citrus sinensis* (L.) Osbeck fruit peel in an insulin resistant diabetic animal model. Food Chem. Toxicol. 2018;116: 86–99. DOI: 10.1016/j.fct.2018.03.050.

[52] Ahmeda OM, Hassanb MA, Abdel-Twabc SM, Abdel Azeem MN. Navel orange peel hydroethanolic extract, naringin and naringenin have anti-diabetic potentials in type 2 diabetic rats. Biomed.

& Pharmacotherapy 2017;94:197–205. DOI: 10.1016/j.biopha.2017.07.094.

[53] Ahmed OM, Fahim H, Ahmed HY, Al-Muzafar HM, Ahmed RR , Amin KA, El-Nahass ES, Abdelazeem WH. The Preventive Effects and the Mechanisms of Action of Navel Orange Peel Hydroethanolic Extract, Naringin, and Naringenin in N-Acetyl-p-aminophenol-Induced Liver Injury in Wistar Rats. Oxidative Medicine and Cellular Longevity. 2019; DOI: 10.1155/2019/ 2745352.

[54] Kim TW, Lee DR, Choi BK, Kang HK, Jung JY, Lim SW, Hwan Yang S, Suh JW. Hepatoprotective effects of polymethoxyflavones against acute and chronic carbon tetrachloride intoxication. Food Chem. Toxicol. 2016; 91: 91–99; DOI: 10.1016/j.fct.2016.03.004.

[55] Chiu SP, Wu MJ, Chen PY, Ho YR, Tai MH, Ho CT, Yen JH. Neurotrophic action of 5-hydroxylated polymethoxyflavones: 5 demethylnobiletin and gardenin A stimulate neuritogenesis in PC12 cells. J. Agric. Food Chem. 2013;61:9453–9463; DOI: 10.1021/jf4024678.

[56] Lai CS, Ho MH, Tsai ML, Li S, Badmaev V, Ho CT, Pan MH. Suppression of Adipogenesis and Obesity in High-Fat Induced Mouse Model by Hydroxylated Polymethoxyflavones. J. Agric. Food Chem. 2013;61:10320–10328; DOI: 10.1021/jf402257t.

[57] Sergeev IN, Li S, Ho CT, Rawson NE, Dushenkov S. Polymethoxyflavones Activate Ca2+- Dependent Apoptotic Targets in Adipocytes. J. Agric. Food Chem. 2009; 57:5771–5776; DOI:10.1021/jf901071k.

[58] Yu Wang Y, Lee PS, Chen YF, Ho CT, Pan MH. Suppression of Adipogenesis by 5-Hydroxy-3,6,7,8,30,40-Hexamethoxyflavone from Orange Peel in 3T3-L1 Cells. J. Med. Food 2016;19:830–835; DOI: 10.1089/jmf.2016.0060.

[59] Li GJ, Wang J, Cheng YJ, Tan X, Zhai YL, Wang Q, Gao FJ, Liu GL, Xin Zhao X, Hua Wang H. Prophylactic Effects of Polymethoxyflavone-Rich Orange Peel Oil on Nω -Nitro-L-Arginine-Induced Hypertensive Rats. Appl. Sci. 2018;8:752–768; DOI:10.3390/ app8050752.

[60] Miyata Y, Oshitari T, Okuyama Y, Shimada A, Takahashi H, Natsugari H, Kosano H. Polymethoxyflavones as agents that prevent formation of cataract: Nobiletin congeners show potent growth inhibitory effects in human lens epithelial cells. Bioorg. Med. Chem. Lett. 2013;23:183–187; DOI: 10.1016/j.bmcl.2012.10.133.

[61] Li G, Tan F, Zhang Q, Tan A, Cheng Y, Zhou Q, Liu M, Tan X, Huang L, Rouseff R, Wu H, Zhao X, Liang G, Zhao X. Protective effects of polymethoxyflavone-rich cold-pressed orange peel oil against ultraviolet Binduced photoaging on mouse skin. J. Funct. Foods 2020; 67:103834– 103844; DOI: 10.1016/j.jff.2020. 103834.

[62] Zeng SL, Li SZ, Xiao PT, Cai YY, Chu C, Chen BZ, Li P, Li J, Liu EH. Citrus polymethoxyflavones attenuate metabolic syndrome by regulating gut microbiome and amino acid metabolism. Sci. Adv. 2020;6: eaax6208; DOI: 10.1126/sciadv.aax6208.

[63] Bajraktari-Sylejmani G, Weiss J. Potential Risk of Food-Drug Interactions: Citrus Polymethoxyflavones and Flavanones as Inhibitors of the Organic Anion Transporting Polypeptides (OATP) 1B1, 1B3, and 2B1. Eur. J. Drug Metab. Pharm. 2020;45:809–815; DOI: 10.1007/ s13318-020-00634-4.

*The Orange Peel: An Outstanding Source of Chemical Resources DOI: http://dx.doi.org/10.5772/intechopen.96298*

[64] Li S, Wang H, Guo L, Zhao H, Ho CT. Chemistry and bioactivity of nobiletin and its metabolites. J. Funct. Food 2014;6;2–10. DOI: 10.1016/j. jff.2013.12.011.

[65] Li W, Wang X, Niu X, Zhang H, He Z, Wang Y,1 Zhi W, Liu F. Protective Effects of Nobiletin Against Endotoxic Shock in Mice Through Inhibiting TNF-α, IL-6, and HMGB1 and Regulating NF-κB Pathway. Inflammation 2016;39:786–797. DOI: 10.1007/s10753-016-0307-5.

[66] Li W, Wang X, Zhi W, Zhang H, He Z, Wang Y, Liu F, Niu X, Zhang X. The gastroprotective effect of nobiletin against ethanol-induced acute gastric lesions in mice: impact on oxidative stress and inflammation. Immunopharm. Immunot. 2017;39:354– 363; DOI: /10.1080/ 08923973.2017.1379088.

[67] Abe S, Hirose S, Nishitani M, Yoshida I, Tsukayama M, Tsuji A, Yuasa K. Citrus peel polymethoxyflavones, sudachitin and nobiletin, induce distinct cellular responses in human keratinocyte HaCaT cells. Biosci. Biotechnol. Biochem. 2018; 82:2064–2071; DOI: 10.1080/ 09168451.2018.1514246.

[68] Kaneda H, Otomo R, Sasaki N, Omi T, Sato T, Kaneda T. Endotheliumindependent vasodilator effects of nobiletin in rat aorta. J. Pharmacol. Sci. 2019;140:48–53; DOI: 10.1016/j. jphs.2019.04.004.

[69] Wen X, Zhao H, Wang L, Wang L, Du G, Guan W, Liu J, Cao X, Jiang X, Tian J, Wang M, Ho CT, Li S. Nobiletin Attenuates DSS-Induced Intestinal Barrier Damage through the HNF4α-Claudin-7 Signaling Pathway. J. Agric. Food Chem. 2020;68:4641–4649; DOI: 10.1021/acs.jafc.0c01217.

[70] Koolaji N, Shammugasamy B, Schindeler A, Dong Q, Dehghani F, Valtchev *P. citrus* Peel Flavonoids as Potential Cancer Prevention Agents. Current Developments In Nutrition. 2020;4:ID nzaa025;DOI: 10.1093/cdn/ nzaa025.

[71] Tung, YC, Chou YC, Hung WL, Cheng AC, Yu RC, Ho CT, Pan MH. Polymethoxyflavones: Chemistry and Molecular Mechanisms for Cancer Prevention and Treatment. Curr. Pharmacol. Rep. 2019;5:98–113; DOI: 10.1007/s40495-019-00170-z.

[72] Wang L, Wang J, Fang L, Zheng Z, Zhi D, Wang S, Li S, Ho CT, Zhao H. Anticancer Activities of Citrus Peel Polymethoxyflavones Related to Angiogenesis and Others. Biomed. Res. Int. 2014; Article ID 453972; DOI: 10.1155/2014/453972.

[73] Chan EWC, Soo OYM, Tan YH, Wong SK, Chan HT.  J. Chin. Pharm. Sci. 2020;29: 443–454; DOI: 10.5246/ jcps.2020.07.042.

[74] Sergeev IN, Ho CT, Li S, Colby J, Dushenkov S. Apoptosis-inducing activity of hydroxylated polymethoxyflavones and polymethoxyflavones from orange peel in human breast cancer cells. Mol. Nutr. Food Res. 2007;51:1478–1484. DOI: 10.1002/mnfr.200700136.

[75] Rahideh ST, Keramatipour M, Nourbakhsh M, Koohdani F, Hoseini M, Talebi S, Shidfar F. Comparison of the effects of Nobiletin and Letrozole on the activity and expression of aromatase in MCF-7 breast cancer cell line. Biochem. Cell. Biol. 2017;95:468–473;DOI: 10.1139/bcb-2016-0206.

[76] Sergeev IN, Li S, Colby J, Ho CT, Dushenkov S. Polymethoxylated

flavones induce Ca2+�mediated apoptosis in breast cancer cells. Life Sci. 2006;80:245–253; DOI: 10.1016/j. lfs.2006.09.006.

[77] Borah N, Gunawardana S, Torres H, McDonnell S, Van slambrouck S. 5,6,7,30 ,40 ,5'-Hexamethoxyflavone inhibits growth of triple-negative breast cancer cells via suppression of MAPK and Akt signaling pathways and arresting cell cycle. Int. J. Oncol. 2017; 51:1685–1693; DOI: 10.3892/ ijo.2017.4157.

[78] Ademosun AO, Oboh G, Passamonti S, Tramer F, Ziberna L, Augusti Boligon A, Athayde ML. Inhibition of metalloproteinase and proteasome activities in colon cancer cells by citrus peel extracts. J Basic Clin Physiol Pharmacol 2015;26: 471–477. DOI: 10.1515/jbcpp-2013-0127.

[79] Kaur J, Kaur G. An insight into the role of citrus bioactives in modulation of colon cancer. J. Funct. Foods. 2015;13: 239–261;DOI: 10.1016/j.jff.2014.12.043.

[80] Qiu P, Cui Y, Xiao H, Han Z, Ma H, Tang Y, Xu H, Zhang L. 5-Hydroxy polymethoxyflavones inhibit glycosaminoglycan biosynthesis in lung and colon cancer cells. J. Funct. Foods. 2017;30:39–47. DOI: 10.1016/j. jff.2017.01.008.

[81] Silva I, Estrada MF, Pereira CV, Bento da Silva A, Bronze MR, Alves PM,. Duarte CMM, Brito C, Serra AT. Polymethoxylated Flavones from Orange Peels Inhibit Cell Proliferation in a 3D Cell Model of Human Colorectal Cancer. Nutrition and Cancer. 2018;70:257–266. DOI: 10.1080/01635581.2018.1412473.

[82] Charoensinphon N, Qiu P, Dong P, Zheng J, Ngauv P, Cao Y, Li S, Ho CT, Xiao H. 5-Demethyltangeretin inhibits human nonsmall cell lung cancer cell growth by inducing G2/M cell cycle

arrest and apoptosis. Mol. Nutr. Food Res. 2013;57:2103–2111; DOI: 10.1002/ mnfr.201300136.

[83] Xiao H, Yang CS, Li S, Jin H, Ho CT, Patel T. Monodemethylated polymethoxyflavones from sweet orange (*Citrus sinensis*) peel inhibit growth of human lung cancer cells by apoptosis. Mol. Nutr. Food Res. 2009;53: 398–406; DOI 10.1002/ mnfr.200800057.

[84] Wang Y, Chen Y, Zhang H, Chen J, Cao J, Chen Q, Li X, Sun C. Polymethoxyflavones from citrus inhibited gastric cancer cell proliferation through inducing apoptosis by upregulating RARβ, both in vitro and in vivo. Food Chem. Toxicol. 2020;146: 111811–111821; DOI: 10.1016/j. fct.2020.111811.

[85] Li S, Pan MH, Lai CS, Lo CY, Dushenkovc S, Ho CT. Isolation and syntheses of polymethoxyflavones and hydroxylated polymethoxyflavones as inhibitors of HL-60 cell lines. Bioorg. Med. Chem. 2007;15:3381–338. DOI: 10.1016/j.bmc.2007.03.021.

[86] Fan K, Kurihara N, Abe S, Ho CT, Ghai G, Yang K. Chemopreventive Effects of Orange Peel Extract (OPE) I. OPE Inhibits Intestinal Tumor Growth in ApcMin+ Mice. Med. Food 2007;10:11–17. DOI: 10.1089/ jmf.2006.0214.

[87] Tajaldinia M, Samadia F, Khosravib A, Ghasemnejadd A, Asadie J. Polymethoxylated Protective and anticancer effects of orange peel extract and naringin in doxorubicin treated esophageal cancer stem cell xenograft tumor mouse model. Biomed. Pharm. 2020;121:e109594. DOI: 10.1016/j. biopha.2019.109594.

[88] Lia Y, Kandhare AD, Mukherjee AA, Bodhankar SL. Acute *The Orange Peel: An Outstanding Source of Chemical Resources DOI: http://dx.doi.org/10.5772/intechopen.96298*

and sub-chronic oral toxicity studies of hesperidin isolated from orange peel extract in Sprague Dawley rats. Regul. Toxicol Pharm. 2019;105:77–85. DOI: 10.1016/j.yrtph.2019.04.001.

[89] Bellavite P, Donzelli A. Hesperidin and SARS-CoV-2: New Light on the Healthy Function of Citrus Fruits. Antioxidants. 2020;9:742–760. DOI: 10.3390/antiox9080742.

[90] Ganeshpurkar A, Saluja A. The pharmacological potential of hesperidin. Indian J. Biochem. Biophys. 2019;56: 287–300.

[91] Murray RDH. Naturally Occurring Plant Cumarins. In: Herz W, Grisebach H, Kirby GW, editors. Progress in the Chemistry of Organic Natural Products. Springer-Verlag. New York; 1978. p. 199–400.

[92] Mohamed TK. Chemical constituents and antioxidant activity of *Citrus paradisi* (star-ruby red grapefruit) and *Citrus sinensis* (blood sweet orange) Egyptian cultivars. Asian J. Chem. 2004:887815.

[93] Cunha AG, Gandini A. Turning polysaccharides into hydrophobic materials: a critical review. Part 2. Hemicelluloses, chitin/chitosan, starch, pectin and alginates. Cellulose. 2010;17: 1045–1065. DOI: 10.1007/s10570-010- 9435-5.

[94] Sua DL, Li PJ, Quekc SY, Huang ZQ, Yuan YJ, Li GY, Shan Y. Efficient extraction and characterization of pectin from orange peel by a combined surfactant and microwave assisted process. Food Chem. 2019;286:1–7. DOI: 10.1016/j. foodchem.2019.01.200.

[95] Hosseini SS, Khodaiyan F, Kazemi M, Najari Z. Optimization and characterization of pectin extracted from sour orange peel by ultrasound assisted method. Int. J. Biol. Macromol. 2019;125:621–629. DOI: 10.1016/j. ijbiomac.2018.12.096.

[96] Jiao WB, Huang D, Xing F, Hu Y, Deng XX, Xu Q, Chen LL. Genomewide characterization and expression analysis of genetic variants in sweet oranged. The Plant Journal 2013;75:954– 964. DOI: 10.1111/tpj.12254.

[97] Jansen EF, Jang R, Mac Donnel LR. Citrus acetylesterase. Arch Biochem. 1947;15:415–431.

[98] Pasta P, Verga R, Zambianchi F, Daminati M. Acetyl Esterase from Mediterranean Oranges: Partial Purification, Immobilisation and Biotransformations. Biocatal. & Biotrasf. 2004;22:221–224. DOI: 10.1080/10242420410001697089.

[99] Fontana G, Bruno M, Maggio A, Rosselli S. Functional investigation and applications of the acetylesterase activity of the *Citrus sinensis* (L.) Osbeck peel. Nat. Prod. Res. 2020; DOI: 10.1080/14786419.2020.1737055.

[100] Wei Y, Tan YL, Ang FL, Zhao H. Identification and Characterization of Citrus Peel Uronic Acid Oxidase. ChemBioChem 2020;21:797–800. DOI: 10.1002/cbic.201900546.

[101] Rani G, Yadav L, Kalidhar SB. Chemical Examination of *Citrus sinensis* Flavedo Variety Pineapple. Indian J. Pharm. Sci. 2009;71:677–679.

[102] Lux PE, Carle R, Zacarías L, Rodrigo MJ, Schweiggert RM, Steingass CB. Genuine Carotenoid Profiles in Sweet Orange [*Citrus sinensis* (L.) Osbeck cv. Navel] Peel and Pulp at Different Maturity Stages. J. Agric. Food Chem. 2019; 67:13164–13175. DOI: 10.1021/acs.jafc.9b06098.

[103] Matsubara Y, Yusa T, Sawabe A, Itzuka Y, Takekuma S, Yoshida Y. Structures of New Cyclic Peptides in

Young Unshiu (*Citrus unshiu* Marcov.), Orange (*Citrus sinensis* Osbeck.) and Amanatzu (*Citrus natzudaidai*) Peelings. Agric. Biol. Chem. 1991;55:2923–2929.

[104] Favela-Hernández JMJ, González-Santiago O, Ramírez-Cabrera MA. Esquivel-Ferriño PC, Camacho-Corona MdR. Chemistry and Pharmacology of *Citrus sinensis*. Molecules. 2016;21, 247– 271. DOI: 10.3390/molecules21020247.

#### **Chapter 12**

## Physiological Functions Mediated by Yuzu (*Citrus junos*) Seed-Derived Nutrients

*Mayumi Minamisawa*

#### **Abstract**

This section is focused on the physiological functions of yuzu (*Citrus junos*) to improve health. The modern lifestyle involves number of modern lifestyles involve various factors that may increase the production of active oxygen species. Nutritional supplements and medicines are commonly utilized to maintain health. Yuzu seeds contain >100-fold the limonoid content of grapefruit seeds and are rich in polyamines (PAs), including putrescine, spermidine, and spermine. Limonoid components mediate the antioxidant properties of citrus. Limonoids and PAs convey various bioactivities. PAs are closely associated with maintaining the function of the intestinal mucosal barrier, which might be involved in the metabolic processes of indigenous intestinal bacteria and in the health of the host. After ingestion, food is digested and absorbed in the intestinal tract, which is also responsible for immune responses against food antigens and intestinal bacteria. Detailed investigations of the physiological functions of extracted yuzu seed extracts may help to develop new treatment strategies against diseases associated with inflammatory responses.

**Keywords:** Yuzu (*Citrus junos*), limonoids, polyamine, gut microbiota, anti-inflammatory, short-chain fatty acid (SCFA), central neurodegenerative disease

#### **1. Introduction**

In 1997, the World Cancer Research Fund published 14 articles concerning dietary recommendations in addition to smoking cessation for the prevention of cancer in *Food, Nutrition and the Prevention of Cancer: a Global Perspective* (2007 revised edition) to promote international awareness of the relationship between nutrition, diet, and cancer. Articles 1, 4, and 5 strongly recommend the consumption of foods of plant origin, and especially emphasized the importance of fruits and vegetables for the prevention of many types of cancer [1].

There are more than 1000 species of citrus and various varieties account a major part of all fruit production worldwide. In particular, citrus species native to Asia are believed to have originated in the Assam area of India around 30–40 million years ago and were propagated in China, Thailand, Malaysia, Indonesia, and Taiwan before being brought to Japan [2]. The tachibana orange, which is the oldest variety of mandarin orange in Japan, was introduced in Japan from Taiwan via the Korean Peninsula from mainland China, and is listed in the *Manyoshu*, the oldest extant

collection of classical Japanese poetry compiled sometime after 759 AD during the Nara period, as the only citrus fruit that existed in the wild. After that, it is estimated that the daidai, an Asian variety of bitter orange, and other small oranges arrived in Japan around 2 to 300AD. Yuzu (*Citrus junos Sieb*. *ex Tanaka*) originated in China and was introduced to Japan and other countries around the 4th to 8th centuries, as this fruit is mentioned in the *Shyoku-Nihongi*, an imperially-commissioned Japanese history text completed in 797 AD.

The traditional Japanese meal *washoku* was recognized as a UNESCO Intangible Cultural Heritage of Humanity in 2013.The Japanese have the highest life expectancy of any other ethnicity. Therefore, washoku has attracted attention as a healthy diet. Especially, yuzu is an essential ingredient of the Japanese diet in the winter months. A traditional 0sechi dish, including yuzu, to be eaten on New Year's Day is shown in the photo in **Figure 1**.

Yuzu is a commercially important fruit, as compared to other sour citrus fruits, and has become very popular in Japan. Although rarely eaten as a fruit, yuzu is a common ingredient in Japanese cuisine, where the aromatic zest (outer rind) as well as juice are used much in the same way as lemons in other cuisines. The yuzu fruit and juice are traditionally used in making vinegar and seasoning yuzu peel and juice, and along with sudachi, daidai, and other similar citrus fruits, are integral ingredients in the citrus-based sauce ponzu. In addition, yuzu is often used as an ingredient in alcoholic drinks, such as the yuzu sour. Recently, yuzu kosho "yuzu and pepper" has become a very popular spicy Japanese sauce made from the peel (zest) of green or yellow yuzu, combined with green or red chili peppers and salt. Yuzu is also well-known because of its pleasant aroma and essential oil of the outer rind. In fact, in Japan, it has been customary since ancient times to take a bath with yuzu in hot water during the winter solstice. The yuzu peel is particularly high in aromatic compounds and pectin; therefore, the waste peel from juice extraction is sometimes used to produce essential oils and flavorings as well as for medicinal purposes. Similarly, yuzu is industrially used in the production of sweetened beverages, cosmetics, and perfumes, as well as oils for aromatherapy [3]. Only a small portion of produce is used for natural medicine, while satsuma mandarins, oranges, and grapefruits are commonly used for the production of fruit juices.

There is a reason why yuzu is not often eaten as a fruit in Japan because it contain large seeds that convey a bitter taste to the juice. The well-known constituents of citrus fruits include essential oil components, flavonoid glycosides, and other basic substances with biological activities, including limonene, a major component of essential oils found in the juice and rind, polymethoxyflavones, coumarins, carotenoids, which are pigments, vitamins, and terpenoids [4–7]. In the past, the bitterness of citrus juices, skins, and seeds hindered the demand for citrus fruits.

**Figure 1.** *A typical Osechi package for New Year's day in Japan.*

#### *DOI: http://dx.doi.org/10.5772/intechopen.95534 Physiological Functions Mediated by Yuzu (*Citrus junos*) Seed-Derived Nutrients*

Much research has been conducted to produce bitter-free citrus fruits. At the time when there was very little demand for bitter fruit and juice, Hasegawa et al. [8–10] reported that the high physiological activity of limonoids was responsible for the bitter taste in citrus juice. Limonoids are a group of triterpene derivatives found in plants of the Rutaceae and Meliaceae families. So far, more than 300 types of limonoids have been reported, and about 100 types have been isolated from the neem and sendan plants of the family Meliaceae.

Limonoids are characterized by a furan ring at C-17, a lactone ring at C-3 or C-6, and an epoxide between C-14 and C-15 (**Figure 2**). Of the four basic structures of limonoids, reversible opening and closing occurs in A, D lactone rings. For example, in the case of limonin, one of the major limonoids, a closed D-ring creates a bitter taste, while the open D-ring form (limonate A-ring lactone) has no bitter taste. Furthermore, the limonate A-ring lactone at C-17 is converted into the limonoid glycoside 17-β-D-glucopyranoside, which is D-glucose bound with β-glucoside.

Until recently, not much had been known about the metabolism of limonoids found in fruit. In 1991, Hasegawa et al. [11] discovered that limonate A-ring lactone, an open D-ring form of a limonoid aglycone, was metabolized to a glucoside derivative in the late stages of fruit growth and maturation, and suggested that this occurred independently in both the seeds and fruit. It is known that the limonoid aglycone and glycosides accumulate in the seeds [12].

To date, 36 types of aglycones and 17 types of glycosides have been identified mainly in citrus fruits of the family Rutaceae, which is composed of 160 genera and about 2,070 species [13]. The first study of the physiological effects of *Citrus limon*oids reported inhibitory effects on the eating behaviors of armyworms and predatory insects [14]. Strong inhibitory effects on eating were subsequently observed in termites. It has been reported that the ligand activity of the bile acid receptor TGR5 increases the inhibition of tumor formation and the activity of glutathione S-transferase, which is a detoxification enzyme that assists with the excretion of toxic substances by the liver and digestive organs, as well as increasing anti-obesity effects via insulin and increased heat production [15]. The seeds of citrus fruits contain particularly potent limonoids. The metabolic pathway of limonoid biosynthesis in citrus fruits has been nearly elucidated by Hasegawa et al. [16] with the use of 14C-labeled radioisotopes as tracers (**Figure 3**). Within the phloem of the stem, nomilin is synthesized through the metabolism of acetic acid, mevalonic acid,

**Figure 2.** *D lactone ring structures of limonin.*

**Figure 3.** *The limonoid biosynthesis pathway of yuzu seeds.*

and furnesole phosphate. Especially in the stems of seedlings, nomilin synthesis becomes very active [17].

Nomilin is synthesized only in the stem and then transferred to the leaves, fruits, and seeds where it is metabolized into other limonoids. Since metabolism proceeds with the D-ring open form, limonoids exist in the D-ring open form in the stems, fruits, and leaves, and mostly form glycosides. In seeds, metabolism to limonoid aglycone with a closed D-ring also proceeds at the same time, so that aglycone and glycoside are accumulated simultaneously. Therefore, the aglycon in the fruit tissue decreases during maturation, but continues to accumulate in the seeds.

#### **2. Bioactive substances of yuzu seeds**

Minamisawa et al. [18, 19] has been searching for new antioxidants to maximize the original functions of living organisms with the use of waste resources derived from natural products, including yuzu seeds, that can be regenerated as many times as possible in the human life span. In 2011, the oldest original species of yuzu in Japan was discovered in the village of Mizuo, which is located in the north-west of Kyoto city [2]. This yuzu is characterized by "seedlings" grown from seeds in the land associated with Emperor Seiwa (9*th* century) and Emperor Hanazono (15*th* century), and it takes about 20 years to harvest. Most of the citrons cultivated in Japan today originate from the yuzu of Mizuo, which is cultivated mainly by grafting, and the growth is faster than that of seedlings. Since the yuzu of Mizuo is considered to be the finest quality, the fruit is highly demanded by high-end restaurants serving Japanese cuisine in Kyoto. However, yuzu seeds, which are closer to the ancestral citrus, account for 20–30% of the fruit weight, but are discarded as waste after the juice extraction process.

Hence, our team chose to evaluate the this development of active natural resources would encompass the application in nutrition and environmental attributes of yuzu seeds as natural resources with bioactivities [20]. In 2014, we reported the development of a relatively simple technique to simultaneously extract secondary metabolites of yuzu seeds, including expensive limonoids and yuzu seed oil with high total antioxidant capability, from the waste of fully ripe fruits [2]. Yuzu seeds contain higher amounts of fat-soluble limonoid aglycones, water-soluble limonoid glycosides, and oil than other citrus fruits (**Figure 4**).

Analysis of the components of limonoids from yuzu seeds by high-performance liquid chromatography–mass spectrometry identified five limonoid aglycones (deacetylnomilin, limonin, nomilin, obacunone, and ichangensin) and eight

*DOI: http://dx.doi.org/10.5772/intechopen.95534 Physiological Functions Mediated by Yuzu (*Citrus junos*) Seed-Derived Nutrients*

#### **Figure 4.**

*The extracted components from Yuzu seed.*

limonoid glycosides (limonin glucoside, ichangin glucoside, deacetyl nomilinic acid glucoside, deacetylnomilin glucoside, nomilin glucoside, nomilinic acid glucoside, ichangensin glucoside, and obacunone glucoside) (**Figure 4**, **Table 1**). Yuzu seed oil extracts (**Table 2**) contain large amounts of oleic and linoleic acids ([2], in preparation). The contents of limonoids extracted from yuzu seeds compared with the results of previous studies are shown in **Table 1** [21–23].

As compared to other citrus seeds, the concentrations of limonoid aglycones extracted from the seeds of yuzu fruit from Kyoto were two- or three-fold greater than in fruits from Tokushima and California (334 vs. 167 and 0.94 mg/g, respectively). According to Nogata [22], the iyokan fruit (*C*. *iyo*), Valencia orange (*C. sinensis Osbeck*), and hyuganatsu (*C. tamurana Hort. ex Tanaka*) belong to the same family as the *daidai* (*C. aurantium group V*). Hence, the limonoid compositions of these varieties are similar (**Table 1**). Although the amount of nomilin in the Valencia orange is similar to that in the *iyokan* and *hyuganatsu* varieties, the amount of limonin is approximately two-fold greater, while the amount of deacetylnomilin is higher and that of obacunone is significantly lower.

The yuzu and hanayu (*C. hanaju*) varieties are classified to yuzu group VI. However, both the compositions and amounts of the limonoid aglycones differed markedly between these two species in the present study, which may be attributed to differences in the metabolism of the seeds and fruits [24, 25]. For this reason, the ratio of aglycone to glycosides in mature fruit tissues is mostly due to glycosides, whereas the glycoside content in seeds may be the same or lower than that of aglycones (**Table 1**). These findings indicate that limonoids are biosynthesized completely independently of fruit tissues and seeds.

Nogata et al. [22] pointed out that the high amounts of glycosides in seeds of the iyo and shiikuwasha fruits could be due to the high activity of uridine diphosphate-D-glucose transferase, and perhaps in the yuzu seeds as well. The high limonoid content in the seeds of yuzu fruit grown in Kyoto is thought to be related to the seedling cultivation method. Similar to yuzu seeds, the glycosides deacetyl nomilinic acid glucoside and deacetylnomilin glucoside, but not ichangensin glucoside, accumulate in hanayu seeds. Ichangensin is reportedly metabolized from nomilin through the intermediaries deacetylnomilin and deacetyl nomilinic acid [26, 27].

*Citrus - Research, Development and Biotechnology*

**Table 1.** *Limonoids in various citrus seeds.*

*DOI: http://dx.doi.org/10.5772/intechopen.95534 Physiological Functions Mediated by Yuzu (*Citrus junos*) Seed-Derived Nutrients*

**Table 2.**

*The FA content was determined by gas chromatography with the use of a GC-14 gas chromatograph (Shimadzu corporation, Kyoto, Japan) equipped with a DB-1 column (30 m, 0.25 mm, Agilent Technologies, Inc., Santa Clara, CA, USA), which was maintained at a constant temperature of 300°C. The yield of yuzu seed oil was 100 mg/g of dry seeds.*

The hanayu are, therefore, different from other citrus varieties. Although both belong to the same yuzu group, there are differences in characteristics, such as aglycones contents.

Several *in vitro* studies have shown that limonoid components mediate the antioxidant properties of citrus. Reactive oxygen is believed to be a factor in diseases with underlying cellular disorders [28]. Modern lifestyles and diets involve a number of factors that can increase the production of active oxygen species, which can overwhelm the body's self-regulating defense mechanisms [29, 30]. One way to protect oneself from active oxygen species is to consume foods containing antioxidants. One of the main reasons for our interest in antioxidants is the link between active oxygen species and aging.

Limonoids components of *C. junos* are known to possess a vast range of biological activities, including antioxidant functions, protective effects on vascular endothelial cells [31], and anti-carcinogenic activities [15, 20, 32–34].

Study also evaluated the *in vitro* antioxidant activities of yuzu seed aglycones, glycosides, and oil extracts. Notably, the yuzu seed oil, the potential extracts had high antioxidant activities due to the presence of lipophilic aglycones (**Figure 5**, new unpublished data). Yuzu seed oil is a semi-drying oil that contains large amounts of unsaturated fatty acids (FAs), mainly oleic acid and linoleic acid, in addition to a lot of palmitic acid.

Virgin yuzu seed oil, which is obtained by a pressing process without heating, contains about 2% of limonoid aglycones. Pure oil with the composition shown in **Table 2** can be obtained by heating and drying roasted yuzu seeds, followed by extraction with an organic solvent, such as hexane, and purification.

Lipophilic limonoid aglycones, which were extracted from the residual extracts of yuzu seed oil [2], were composed of the following concentrations of limonoids per gram of dry seeds: deacetylnomilin, 105 mg; limonin, 95 mg; nomilin, 115 mg; obacunone, 17 mg; and ichangensin, 2.1 mg.

**Figure 5.**

*Total potential antioxidant activities of various plant seed oils and yuzu aglycones by the total potency of antioxidants that are soluble in oil method [35]. Measurements were performed 4–6 times or more. The inhibition ratio is presented as the average value ± standard deviation (S.D.).*

The total potential antioxidant capacity of yuzu seed oil and lipophilic limonoid aglycones was measured by utilizing the reduction reaction of copper (Cu++/Cu+ ) [35]. Many other plant oils, including olive oil [36], tea tree oil, grape seed oil [37], and neem seed oil [38], which have strong antioxidant activities, were measured at the same time for comparisons. Among all of the tested plant seed oils, limonoid aglycones extracted from yuzu seeds had the highest antioxidant capacity, followed by yuzu seed oil, neem seed, grape seed, tea tree, olive oil, and pure yuzu seed oil. The antioxidant capacity of pure yuzu seed oil was approximately 6–9-fold greater than that of palmitic acid, oleic acid, and linoleic acid.

While water-soluble antioxidants are rapidly excreted through the urine if an excessive amount is ingested, fat-soluble antioxidants are adsorbed onto lipoproteins and cell membrane lipids, and are therefore considered to exhibit a higher activity in the body. For this reason, fat-soluble antioxidants are expected to be beneficial in preventing diseases caused by oxidative stress. Vitamin E, oryzanol, and carotenoids are well-known examples of fat-soluble antioxidants. Neem (*Azadirachta indica*) seed oil, which has the same total antioxidant capacity as yuzu seed oil, contains the triterpene derivative azadirachtin, which is similar to the triterpene limonoids of yuzu seed oil, which is a potent insect repellent [39]. Press-extracted virgin olive oil contains oleocanthal that has a potent anti-inflammatory effect strikingly similar to that of ibuprofen. Both of these molecules inhibit the same cyclooxygenase enzymes in the prostaglandin-biosynthesis pathway [40].

The result in **Figure 5** suggest the presence of other types of fat-soluble antioxidants. Limonoid aglycones also contribute to the high antioxidant capacity of yuzu seed oil.

#### **3. Yuzu seeds contain arginine and polyamines (PAs)**

Atherosclerosis has become a serious health concern worldwide, as one-third of the global population is at risk for diseases associated with arteriosclerosis, which accounts for about half of deaths in developed countries. In particular, cardiovascular disease (CVD), which is a consequence of atherosclerosis, is the leading cause of death in industrialized nations. Besides lifestyle habits, body weight, socio-economic factors, and certain pre-existing conditions, a number of foods seem to play a role in the incidence of CVD [41, 42]. In addition, many studies have suggested the importance of inflammation in atherosclerosis and CVD [43, 44].

Some food components with anti-inflammatory properties can decrease the risk of CVD [44, 45]. Many foods contain wide-ranging concentrations of natural PAs, such as spermidine (Spd) and spermine (Spm), which suppress the synthesis of pro-inflammatory cytokines [46, 47]. In particular, an epidemiological survey of Westerners found that "people who eat cheese or yogurt every day are less likely to have myocardial infarction." [48]. The Japanese consume a lot of traditional fermented foods, mainly soybeans, which are thought to suppress arteriosclerosis [49]. PAs concentrations are relatively high in yogurt, cheese, and traditional Japanese foods. PAs are aliphatic amines that are essential for the growth of all living cells [50]. PAs exist primarily in association with RNA and are involved in promoting the synthesis of specific proteins and overall protein synthesis via the ribosome activation. As shown in **Figure 6**, the PAs comprising Put (NH2 (CH2)4NH2) → Spd (NH2 (CH2)3NH(CH2)4NH2) → Spm (NH2(CH2)3NH(CH2)4NH(CH2)3NH2) are produced from arginine via ornithine or agmatine [51].

PAs have been implicated in the regulation of several growth and development processes in plants, including cell division, morphogenesis, flower initiation, pollen *DOI: http://dx.doi.org/10.5772/intechopen.95534 Physiological Functions Mediated by Yuzu (*Citrus junos*) Seed-Derived Nutrients*

#### **Figure 6.**

*PA synthesis and degradation [50, 51]. AcPAO, acethylpolyamine oxidase; ODC, ornithine decarboxylase; SAMDC, S-adenosylmethionine decarboxylase; SMO, spermine oxidase; SPDS, spermidine synthase; SPMS, spermine synthase; SSAT, spermidine/spermine N1-acetyltransferase.*

#### **Table 3.**

*PAs and arginine contents in several citrus (nmol/g).*

tube growth, and senescence [52]. Analyses of the PA contents of various fruits have mainly been conducted in Europe [53, 54].

Recent studies have indicated that citrus limonoids have antitumor, detoxification, and anti-obesity effects [55, 56], which may indirectly contribute to the suppression of acrolein production, which is a side reaction product of PA metabolism (**Figure 6**). Hence, we measured the PA and arginine contents of yuzu, which produces very high concentrations of limonoids, as well as lemons produced in Japan for comparison. The PA contents, as determined by high-performance liquid chromatography, as well as the arginine and free arginine contents, as determined by automated amino acid analysis, of various citrus fruits are shown in **Table 3**.

As compared with the juice and peel of yuzu and lemons, the seeds contain higher quantities of PAs and arginine. The Put contents are high in all citrus fruits, but the quantities of Spd and Spm in yuzu seeds were 5–23-fold greater than the reference values. The PA contents of yuzu and lemon fruit are not high as compared to legumes [57], but when the limonoid and arginine contents are also considered, these fruits have high levels of functional constituents.

As mentioned earlier, limonoids and PAs have various bioactivities and reportedly have strong anti-inflammatory capabilities. Hence, the potential antioxidant activities (i.e., H2O2-scavenging activity, 2,2-diphenyl-1-picrylhydrazyl radicalscavenging activity, and inhibition of superoxide dismutase [SOD] and antioxidants with SOD-like activities) of PAs (Put, Spd, and Spm) and arginine were investigated (in preparation). The results showed that these compounds have no antioxidant activities or only weak (less than 10%) inhibitory potential. As reported in many studies, the anti-inflammatory activities of PAs and arginine are due to factors other than antioxidant capacity.

#### **4. Yuzu seed limonoids or Spm increased survival of mice with Sandhoff disease**

In our previous study, we investigated the life-extending effect of limonoids (lipophilic limonoid aglycones) and Spm as an exogenous anti-inflammatory component in a mouse model of Sandhoff disease (SD), which is a lysosomal disease [58]. Lysosomal storage disorders are caused by functional defects of proteins that are essential for normal lysosome function, such as enzymes that play critical roles in the intracellular digestion of glycoproteins, glycolipids, glycosaminoglycans, and other macromolecules [59]. SD is an autosomal recessive hereditary disease [60]. The gangliosides GM2 and GA2 accumulate in the nervous system, resulting in severe developmental and neurological disorders, and death, which usually occurs during infancy because of the lack of effective treatment methods. Neurological dysfunction is the major clinical manifestation of GM2 gangliosidosis [61–64].

SD mice present with trembling, startled responses, and decreased motor activities from 11 to 15 weeks of age (105 days) due to damage caused by microglial activation, macrophage infiltration, and oxidation associated with the accumulation of glycolipids. It has been suggested that inflammation may be fatal [51]. The therapeutic effects of enzyme replacement therapy and anti-inflammatory drugs have been reported [65]. Inflammation due to the accumulation of lipids is inhibited by antioxidant and anti-inflammatory treatments, which can delay disease progression, but no cure exists at present.

We consider the degeneration of the nervous system might be rooted in oxidative stress and inflammation. Given that dietary interventions can moderate these phenomena, consuming foods with antioxidants and anti-inflammatory components, such as limonoid aglycons (limonoids) and Spm, could effectively combat or minimize neurological damage. Therefore, the inhibition of SD pathologies could be promoted by factors other than suppressing the storage of gangliosides.

Preventing inflammation appears to be one of the most effective approaches for increasing longevity [66, 67]. To test this hypothesis, the life spans of SD mice treated with limonoids or Spm were assessed. The prognostic outcomes of SD mice, a typical model of abnormal glycolipid metabolism in humans, were observed after administration of limonoids extracted from yuzu seeds and Spm. The treated mice

*DOI: http://dx.doi.org/10.5772/intechopen.95534 Physiological Functions Mediated by Yuzu (*Citrus junos*) Seed-Derived Nutrients*

**Figure 7.**

*H&E-stained thalamus sections of control SD mice (A and C) and SD mice administered yuzu limonoids (B) and Spm (D). Enlarged cells with ganglioside storage are indicated. The numbers of neurons in the thalamus and midbrain of SD mice administered limonoids (B) and control SD mice (A). Data are presented as the mean ± S.D. \*p* ≤ *0.05 (Student's t-test).*

lived significantly longer than untreated littermates (9–10%, *p* < 0.01) and had a slower rate of disease progression (*p* < 0.01) [58]. When limonoid treatment was combined with Spm therapy, synergy resulted in a maximum improvement of 12% in survival (*p* < 0.001) (in preparation). The hematoxylin and eosin (H&E) staining results of thalamus sections of SD mice following administration of limonoids or Spm are shown in **Figure 7**.

H&E staining results of the neural tissues of the SD control mice (A) and(C) correspond to SD mice treated with limonoids (B) and Spm (D), respectively.

Gangliosidosis and inflammatory/autoimmune diseases are characterized by degeneration and the accumulation of fat, granulovacuolar degeneration, rodshaped microglia, and neuronal inflammation in metabolic diseases, as determined by analyses of pathological tissues (Tokyo Metropolitan Institute for Medical Science). The characteristic degeneration was clearly decreased in SD mice treated with limonoids or Spm. The numbers of neurons in the thalamus and midbrains of SD mice treated with limonoids were higher than those in the control SD mice. These results demonstrate that inflammation contributes to disease progression and the anti-inflammatory effects of Spm and limonoid therapies as a potential adjunctive approach to slow the clinical course of inflammatory diseases.

#### **5. Bacterial flora analysis of SD mouse feces by the 16S ribosomal DNA (16S rDNA) terminal restriction fragment length polymorphism (T-RFLP) method**

PAs possess anti-inflammatory activities by inhibiting the synthesis of inflammatory cytokines by macrophages and the regulation of nuclear factor-κB activation, which are closely associated with maintaining the intestinal mucosal barrier function [68]. Bilateral signals between the intestine and brain are involved in the control of nerve, hormone, and immune activities, as well as prolonging longevity [69]. Recent studies have shown that bilateral signals between the brain and intestine are important for maintaining homeostasis and extending the life span [70]. In particular, the functions mediated by PAs may be involved in metabolism by indigenous intestinal bacteria and the health of the host [71].

#### **Figure 8.**

*Estimated ratios (%) of the taxonomic categories of the bacterial flora at the order level were identified by T-RFLP analysis of 16S rRNA in the feces of SD mice at 12 weeks of age in the control, Spm, and limonoids groups (n = 9/group). Values are presented as the mean ± S.D. data of the treatment groups are plotted against those of the control group. \*p* ≤ *0.05, \*\*p* ≤ *0.001, and \*\*\*p* ≤ *0.0001 vs. the untreated control SD mice or each (dashed line----). All experiments were performed at least three times.*

At 12 weeks of age for which the survival period was extended by limonoids or Spm, T-RFLP analysis of 16S rRNA was performed to classify the intestinal microbiota at the order level for each mouse group (**Figure 8**). The results showed that the taxonomic groups of the bacterial flora in feces after administration of limonoids or Spm were completely different from those of the SD control mice.

The bacterial flora in feces after administration of limonoids or Spm had increased proportions of *Bacteroidales* and *Clostridiales*. However, *Lactobacillus* was remarkably prevalent in feces of the SD control mice. The abundance of *Clostridiales* were significantly increased in the feces of SD mice treated with Spm, whereas *Bacteroidales* were increased in the feces of SD mice treated with limonoids. The administration of Spm or limonoids slightly increased the proportion of *Erysipelotrichales*.

It is generally known that the abundance of *Erysipelotrichaceae* is increased due to fat accumulation in mice [72]. In this case, it was possible that the bacterial flora of the SD control mice caused dysbiosis [73]. In the SD control mice, dysbiosis may have been due to suppressed absorption of dietary fats and other nutrients. Even more interesting was the significant appearance of *Verrucomicrobiaceae* in feces after the administration of limonoids or Spm, which were not found in feces of the control SD mice. *Verrucomicrobiaceae* include mucin-degrading bacteria that are also present in the human intestine, and especially *Akkermansia*, which promote the suppression of obesity, diabetes, and inflammation [74, 75].

It will be necessary to investigate the specific bacteria involved in more detail. Unfortunately, the T-RFLP method made it difficult to analyze the bacterial flora in more detail, and it was not possible to identify particular species. We are currently preparing a report of the findings of next-generation sequencing that allowed for more detailed classification.

#### **6. Short-chain fatty acid (SCFA) production in SD mouse feces**

There have been many reports of the relationships between chronic inflammatory diseases and the intestinal bacterial flora that have helped to clarify the balance

#### *DOI: http://dx.doi.org/10.5772/intechopen.95534 Physiological Functions Mediated by Yuzu (*Citrus junos*) Seed-Derived Nutrients*

between the intestinal ecosystem and diseases related to the intestinal tract. For example, genetic abnormalities and the breakdown of the intestinal ecosystem have been detected in inflammatory bowel disease [76]. In particular, members of the genus *Clostridium* promote the production of butyric acid, induce an immune response in the intestinal mucosa, and promote the differentiation of regulatory T cells (Tregs) that contribute to suppression. Thus, changes in intestinal *Clostridium* are considered to be closely related to the onset of inflammatory bowel disease [77, 78]. It has been reported that the SCFAs produced by intestinal bacteria may function as bio-modifying factors. Hence, the SCFA composition of feces from the same mice at 12 weeks were determined (**Figure 9**).

The production levels of SCFAs comprising acetic acid, propionic acid, and butyric acid were increased in mice administered limonoids or Spm as compared to SD control mice. In particular, the production levels of all SCFAs were higher in SD mice following administration of limonoids or Spm. The experimental results demonstrated differences between the fecal microflora composition and these metabolites after administration of limonoids or Spm. Butyric acid is a SCFA that is produced by clostridia [78].

As shown by the results presented in **Figures 8** and **9**, the addition of Spm to the diet clearly increased the proportion of *Clostridiales* and butyric acid in feces. Previous metabolomic analyses have shown that butyric acid contributes to the induction of Treg differentiation in the colonic mucosa. Thus, butyric acid functions as a histone deacetylase inhibitor and as an immunomodulator responsible for inducing Treg differentiation in the colonic mucosa, as well as the activation of dendritic cells [79]. Acetic acid produced by intestinal bacteria suppressed colitis in a mouse model by promoting apoptosis via the GPR43 receptor expressed by neutrophils and plays a central role in the inflammatory reaction [80, 81]. Furthermore, the addition of limonoids seems to contribute to the production of acetic acid and propionic acid as well as butyric acid.

Acetate, butyrate, and propionate are produced by members of the intestinal microbial community through fermentation of dietary fibers and starches, which are unable to be broken down by host metabolism [82]. In turn, these metabolites are sensed by host cells through various G-protein coupled receptors, known as free

#### **Figure 9.**

*SCFA contents (μmol/g) in feces of SD mice in the control, Spm, and limonoid groups. SD mice were treated at 12 weeks of age (n = 9/group). Values are presented as the mean ± S.D. data of the treatment groups are plotted against those of the control group. \*p* ≤ *0.05, \*\*p* ≤ *0.001, and \*\*\*p* ≤ *0.0001 vs. the untreated control SD mice or each (dashed line----). All experiments were performed at least three times.*

fatty acid receptors, and intracellular peroxisome proliferator-activated receptor gamma. Furthermore, SCFAs can also regulate cellular responses through inhibition of histone deacetylases. Examples of the effects of SCFAs on the host include differentiation of Tregs and macrophages, and downregulation of pro-inflammatory mediators. These effects underline the fine balance that SCFAs help to maintain between intestinal immunity and inflammation [82–84].

These results suggest that yuzu conveys anti-inflammatory and lipid metabolism-promoting activities in mice following administration of limonoid aglycones and Spm. Thus, the metabolites of intestinal bacteria may be indirectly involved in suppressing the inflammatory mechanism to directly enhance the health of the host. Furthermore, administration of limonoids or Spm improved the proportions of beneficial bacterial in the intestinal flora and associated metabolites. In the healthy intestinal tract, the microbiota and gut-associated immune system are assumed to be at a dynamic homeostatic equilibrium [85], but the inflammation process may undermine this balance. We consider that the human lifespan can be extended by inhibiting inflammation via control of the intestinal microbiota.

However, it was not possible to elucidate the mechanisms underlying the effects of limonoid aglycones and Spm on the extended life span of SD mice. Thus, in order to clarify the anti-inflammatory effects of yuzu seed extract, limonoids, and Spm, as well as to widely apply yuzu to promote health and enhance longevity, it will be necessary to determine the composition of the bacterial flora based on detailed metagenomic analyses of 16S rRNA. Furthermore, it will be necessary to analyze the anti-inflammatory effects of limonoids and Spm in yuzu seed extracts at the gene level.

PAs quantities have reference values. The reference values for the PAs contents of legumes are also shown in **Table 3**. The values for Japanese produced yuzu and lemons are shown, as well as the reference values for other citrus fruits. No previous studies reported the quantities of arginine in citrus fruits, so only the compared PA quantities are indicated by reference values. The reference values for the PA contents of legumes are also shown.

#### **7. Conclusions**

Yuzu is a natural and renewable resource of limonoids, arginine, and PAs. The results of the present study suggest that yuzu limonoids and Spm improved the proportions of beneficial bacteria and their metabolites in the intestinal flora. Thus, the ingestion of fruits that contain high concentrations of specific ingredients may be a simple method to suppress inflammation, thereby enhancing immune function, improving intestinal health, and increasing lifespan. In other words, our this study demonstrated the possibility that bilateral signals between the brain and intestine are important for maintaining homeostasis and extending lifespan. However, it was not possible to examine the physiological effects of limonoids and Spm. Thus, future studies are needed to evaluate the effects of limonoids and Spm on metabolism and the immune response, and to explore the potential of these molecules as natural antioxidants/antibiotics for lysosomal diseases, such as SD.

#### **Acknowledgements**

The authors wish to thank Kyoto Mizuo Community Council for providing the yuzu samples. We are grateful to Dr. Shoichiro Yoshida and the students of the Environmental Science Laboratory of the Department of Biotechnology (Tokyo

*Physiological Functions Mediated by Yuzu (*Citrus junos*) Seed-Derived Nutrients DOI: http://dx.doi.org/10.5772/intechopen.95534*

College of Medico-Pharmaco Technology), Prof. Gota Kawai (Department of Life Science, Faculty of Advanced Engineering, Chiba Institute of Technology, Japan), Dr. Kyoko Suzuki, Mr. M. Kawashima, Dr. Akira Yamaguchi, and Dr. Shoji Yamanaka (Department of Pathology, Yokohama City University, Japan) for providing the SD mice, and to Shimadzu Co., Ltd. (Kyoto, Japan), Techno Suruga Laboratory Co., Ltd., and i-on Co., Ltd. for their help with the pathological experiments on mice, gut microbiota testing by NGS, and SCFA analyses. Financial support from Chiba Institute of Technology is gratefully acknowledged.

### **Conflict of interest**

The authors declare no conflict of interest.

### **Author details**

#### Mayumi Minamisawa

Faculty of Advanced Engineering, Chiba Institute of Technology, Graduate School of Engineering, Chiba Institute of Technology, Narashino, Chiba, Japan

\*Address all correspondence to<EMAIL_ADDRESS>
© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

### **References**

[1] World Cancer Research Fund/ American Institute for Cancer Research, 1997.

[2] Minamisawa M, Yoshida S, Uzawa A. The functional evaluation of waste yuzu (Citrus junos) seeds.Food and Function. 2014;5:330-336. DOI: 10.1039/ c3fo60440c.

[3] Sawamura *M. citrus* JUNOS SIEB. Ex TANAKA (YUZU), FURUIT. In: Ramadane, ed. Fuits:Growth, nutrition, and quality. Helsinki: WFL Publisher; 2005. p. 1-24.

[4] Sawamura M, Bando A, Nobuyuki O, Kusunose H. Japan food industry. 1986:33:566.

[5] Sawamura M, Kuroiwa N, Kuriyama T, Kusunose H. Japan food industry 1986;33:773.

[6] Lan Phi NT, Sawamura M. Food Science and Technology Research. 2008;14:359-366. DOI: 10.3136/ fstr.14.359.

[7] Lan Phi NT, Nishiyama C, Choi HS, Sawamura M. Evaluation of characteristic aroma compounds of Citrus natsudaidai Hayata (Natsudaidai) cold-pressed peel oil. Bioscience, Biotechnology, and Biochemistry. 2006;70:1832-1838. DOI: 10.1271/ bbb.50705.

[8] Hasegawa S. Quality factors of fruits and vegetables. In: Jen JJ, ACS, ed. Symp. American Chemical Society; 1990. p. 84.

[9] Hasegawa S,, Pelton VAW. American Chemical Society. Journal of Agricultural and Food Chemistry. Jan/ Feb 1983;31:178-180.

[10] Hsu WJ, Berhow M, Robertson GH, Hasegawa S. Limonoids and flavonoids in juices of Oroblanco and Melogold

grapefruit hybrids. Journal of Food Science. 1998;63:57-60. DOI: 10.1111/ j.1365-2621.1998.tb15675.x.

[11] Hasegawa S, Ou P, Fong CH, Herman Z, Coggins CW, Atkin DR. Changes in the limonoate A-ring lactone and limonin 17-β-D-glucopyranoside content of navel oranges during fruit growth and maturation.Journal of Agricultural and Food Chemistry. 1991;39:262-265. DOI: 10.1021/ jf00002a008.

[12] Hasegawa S, Bennett RD, Herman Z, Fong CH, Ou P. Limonoid glucosides in citrus. Phytochemistry. 1989;28:1717-1720. DOI: 10.1016/ S0031-9422(00)97831-2.

[13] Hasegawa S, Citrus Limonoids. American Chemical Society; 2000. 9-30.

[14] Hasegawa S, Ifuku Y. Biochemistry of citrus limonoids. Metabolism and Biological Functions. Nippon Shokuhin Kogyo Gakkaishi. 1994;41:372-380. DOI: 10.3136/nskkk1962.41.372.

[15] Ono E, Inoue J, Hashidume T, Shimizu M, Sato R. Anti-obesity and anti-hyperglycemic effects of the dietary citrus limonoid nomilin in mice fed a high-fat diet. Biochemical and Biophysical Research Communications. 2011;410:677-681. DOI: 10.1016/j. bbrc.2011.06.055.

[16] Hasegawa S Jen JJ, editors.Quality factors of Fruit and Vegetables Chemistry and Technology . Washington DC: American Chemical Society; 1989. p. 84.

[17] Ou P, Hasegawa S, Herman Z, Fong CH. Phytochemistry. 1988;27:115-118. DOI: 10.1016/0031-9422(88)80600-9.

[18] Minamisawa M, Yoshida S, Uzawa A. The pyrolytic synthesis of *Physiological Functions Mediated by Yuzu (*Citrus junos*) Seed-Derived Nutrients DOI: http://dx.doi.org/10.5772/intechopen.95534*

calcium phosphate compounds from carbonized fowl droppings. Powder Technology. 2012;230:20-28. DOI: 10.1016/j.powtec.2012.06.034.

[19] Minamisawa M, Minamisawa H, Yoshida S, Takai N. Adsorption behavior of heavy metals on biomaterials. Journal of Agricultural and Food Chemistry. 2004;52:5606-5611. DOI: 10.1021/ jf0496402.

[20] Roy A, Saraf S. Limonoids: overview of significant bioactive triterpenes distributed in plants kingdom. Biological and Pharmaceutical Bulletin. 2006;29:191-201. DOI: 10.1248/bpb.29.191.

[21] Hashinaga F, Herman Z, Hashegawa S. Limonoids in seeds of yuza (Citrus junos. Sieb. ex Tanaka), Nippon Shokuhin Kogyo Gakkaishi. 1990; 37: 380-382

[22] Nogata. National Agricultural Research Organization (NAR) Western Region Agricultural Research Center report. 2005;5:19-84.

[23] Ozaki Y, Fong CH, Herman Z, Maeda H, Miyake M, Ifuku Y, Hasegawa S. Limonoid glucosides in citrus seeds. Agricultural and Biological Chemistry. 1991;55:137-141. DOI: 10.3390/molecules21111530

[24] Ozaki Y, Miyake M, Maeda H, Ifuku Y, Bennett RD, Herman Z, Fong CH, Hasegawa S. Ichangensin glucoside in Citrus junos, Citrus sudachi and Citrus sphaerocarpa. Phytochemistry. 1991;30:2659-2661. DOI: 10.1016/0031-9422(91)85118-J.

[25] Herman Z, Hasegawa S, Fong CH, Ou P. Limonoids in Citrus ichangensis. Journal of Agricultural and Food Chemistry. 1989;37:850-851. DOI: 10.1021/jf00088a003.

[26] Fong CH, Hasegawa S, Herman Z, Ou P. Biosynthesis of limonoid glucosides in lemon (Citrus limon). Journal of the Science of Food and Agriculture. 1991;54:393-398. DOI: 10.1002/ jsfa.2740540310.

[27] Hasegawa S, Herman Z, Orme E, Ou P. Biosynthesis of limonoids in Citrus: sites and translocation. Phytochemistry. 1986;25:2783-2785. DOI: 10.1016/ S0031-9422(00)83741-3.

 In: Berhow MA, Hasegawa S, Manners GD, (Eds.), *Citrus limon*oids: functional chemicals in agriculture and foods. Washington, DC: American Chemical Society; 2000. p. 164-174.

[29] Straface E, Matarrese P, Gambardella L, Vona R, Sgadari A, Silveri MC, Malorni W. Oxidative imbalance and cathepsin D changes as peripheral blood biomarkers of Alzheimer disease: A pilot study. FEBS Letters. 2005;579:2759-2766. DOI: 10.1016/j.febslet.2005.03.094.

[30] Pregel P, Bollo E, Cannizzo FT, Biolatti B, Contato E, Biolatti PG. Antioxidant capacity as a reliable marker of stress in dairy calves transported by road. Veterinary Record. 2005;156:53-54. DOI: 10.1136/ vr.156.2.53.

[31] Yoon J, Park M, Lee Jh, Min BS, Ryoo S. Endothelial nitric oxide synthase activation through obacunonedependent arginase inhibition restored impaired endothelial function in ApoEnull mice. Vascular Pharmacology. 2014;60:102-109. DOI: 10.1016/j. vph.2014.01.006.

[32] Manners GD. Citrus limonoids: analysis, bioactivity, and biomedical prospects. Journal of Agricultural and Food Chemistry. 2007;55:8285-8294. DOI: 10.1021/jf071797h.

[33] Chidambara Murthy KN, Jayaprakasha GK, Patil BS. Obacunone and obacunone glucoside inhibit human colon cancer (SW480) cells by the induction of apoptosis. Food and Chemical Toxicology . 2011;49:1616- 1625. DOI: 10.1016/j.fct.2011.04.014.

[34] Poulose SM, Harris ED, Patil BS. Antiproliferative effects of citrus limonoids against human neuroblastoma and colonic adenocarcinoma cells. Nutrition and Cancer. 2006;56:103-112. DOI: 10.1207/ s15327914nc5601\_14.

[35] Advanced protocol oxidative stress II; 594 Sakai K, Kino S, Takeuchi M, Ochi T, Da Cruz G, Tomita I, Armstrong D, ed.; 2010. p. 241-250.

[36] Reboredo-Rodríguez P, Figueiredo-González M, González-Barreiro C, Simal-Gándara J, Salvador MD, Cancho-Grande B, Fregapane G. State of the art on functional virgin olive oils enriched with bioactive compounds and their properties. International Journal of Molecular Sciences. 2017;18:121- 126. -DOI: https://doi.org/10.1016/j. foodchem.2008.03.045.

[37] Sahin NO, Berköz M, Eker ED, Pomierny B, Przejczowska K. Cytotoxic and antioxidant effects of grape seed oil on the treatment of leukemia with methotrexate. European Journal of Chemistry. 2012;3:147-151. DOI: 10.5155/eurjchem.3.2.147-151.569.

[38] Biswas K, Chattopadhyay I, Banerjee RK, Bandyopadhyay U. Biological activities and medicinal properties of neem (Azadirachta indica). Current Science. 2002;82:1336-1345.

[39] Kraus W. The neem tree: source of unique natural products for integrated pest management, medicine, industry and other. In: Purposes, (ed.

Schmutterer, H.). VCH, Wenheim; 1995:1-696.; 1995. p. 35-88.

[40] Beauchamp GK, Keast RSJ, Morel D, Lin J, Pika J, Han Q, Lee C-H, Smith AB, Breslin PAS. Phytochemistry: Ibuprofen-like activity in extra-virgin olive oil. Nature. 2005;437:45-46. DOI: 10.1038/437045a.

[41] ML Daviglus ML, Stamler J, Greenland P. Letter to the editor. New England Journal of Medicine. 1997;337:498-499.

[42] Louis J Ignarro LJ, Balestrieri ML, Napoli C. Nutrition, physical activity, and cardiovascular disease. Cardiovascular Research. 2007;15:326-340.

[43] Dariush Mozaffarian D, Katan MB, Ascherio A, Stampfer MJ, Willett WC. Trans fatty acids and cardiovascular disease. New England Journal of Medicine. 2006;354:1601-1613. DOI: 10.1056/NEJMra054035.

[44] Jennifer A Nettleton JA, Steffen LM, Mayer-Davis EJ, Jenny NS, Jiang R, Herrington DM, Jacobs DR. Dietary patterns are associated with biochemical markers of inflammation and endothelial activation in the Multi-Ethnic Study of Atherosclerosis(MESA). American Journal of Clinical Nutrition. 2006;83:1369-1379. DOI: 10.1093/ ajcn/83.6.1369.

[45] Mozaffarian D. Trans fatty acids - effects on systemic inflammation and endothelial function. Atherosclerosis. Supplements. 2006;7:29-32. DOI: 10.1016/j. atherosclerosissup.2006.04.007.

[46] Nettleton JA, Steffen LM, Mayer-Davis EJ, Jenny NS, Jiang R, Herrington DM, Jacobs DR Jr. Dietary patterns are associated with biochemical markers of inflammation and endothelial activation in the Multi-Ethnic Study of Atherosclerosis (MESA). American Journal of Clinical *Physiological Functions Mediated by Yuzu (*Citrus junos*) Seed-Derived Nutrients DOI: http://dx.doi.org/10.5772/intechopen.95534*

Nutrition. 2006;83:1369-1379. DOI: 10.1093/ajcn/83.6.1369.

[47] Zhang M, Caragine T, Wang H, Cohen PS, Botchkina G, Soda K, Bianchi M, Ulrich P, Cerami A, Sherry B, Tracey KJ. Spermine inhibits proinflammatory cytokine synthesis in human mononuclear cells: a counterregulatory mechanism that restrains the immune response. Journal of Experimental Medicine. 1997;185:1759-1768. DOI: 10.1084/jem.185.10.1759.

[48] Soda K, Kano Y, Chiba F. Food polyamine and cardiovascular disease-An epidemiological study-global journal of health science. Global Journal of Health Science. 2012;4:170-178. DOI: 10.5539/gjhs.v4n6p170.

[49] Clarkson TB. Soy, soy phytoestrogens and cardiovascular disease. Journal of Nutrition. 2002;132:566S–569S. DOI: 10.1093/ jn/132.3.566S.

[50] Igarashi K, Kashiwagi K. Modulation of cellular function by polyamines. The International Journal of Biochemistry and Cell Biology. 2010;42:39-51. DOI: 10.1016/j. biocel.2009.07.009.

[51] Liu T, Kim DW, Niitsu M, Maeda S, Watanabe M, Kamio Y, Berberich T, Kusano T. Oryza sativa polyamine oxidase 7 catalyzes terminal catabolism reaction and specifically expressed in anthers. Plant and Cell Physiology. 2014;55:1110-1122.

[52] Bagni N, Tassoni A. Biosynthesis, oxidation and conjugation of aliphatic polyamines in higher plants. Amino Acids. 2001;20:301-317. DOI: 10.1007/ s007260170046.

[53] Chiancone B, Tassoni A, Bagni N, Germanà MA. Effect of polyamines on in vitro anther culture of Citrus clementina Hort. Ex Tan. Plant Cell, Tissue and Organ Culture.

2006;87:145-153. DOI: 10.1007/ s11240-006-9149-4.

[54] Atiya Ali M, Poortvliet E, Strömberg R, Yngve A. Polyamines in foods:development of a food database. Food and Nutrition Research. 2011;55:5572. DOI: 10.3402/fnr. v55i0.5572.

[55] Horiba T, Katsukawa M, Mita M, Sato R. Dietary obacunone supplementation stimulates muscle hypertrophy, and suppresses hyperglycemia and obesity through the TGR5 and PPARγ pathway. Biochemical and Biophysical Research Communications. 2015;463:846-852. DOI: 10.1016/j.bbrc.2015.06.022.

[56] Sato R. Nomilin as an anti-obesity and anti-hyperglycemic agent. Vitamins and Hormones. 2013;91:425-439. DOI: 10.1016/B978-0-12-407766-9.00018-3.

[57] Soda K, Dobashi Y, Kano Y, Tsujinaka S, Konishi F. Polyaminerich food decreases age-associated pathology and mortality in aged mice. Experimental Gerontology. 2009;44:727-732. DOI: 10.1016/j. exger.2009.08.013.

[58] Minamisawa M, Suzuki K, Kawai G, Yamaguchi A, Yamanaka S. Functional evaluation of yuzu (Citrus junos) extracts containing limonoids and polyamine for life extension. Journal of Functional Foods. 2017;38:591-600. DOI: 10.1016/j.jff.2017.09.043.

[59] Neufeld EF. Lysosomal storage diseases. Annual Review of Biochemistry. 1991;60:257-280. DOI: 10.1146/annurev.bi.60.070191.001353.

[60] Platt FM. Sphingolipid lysosomal storage disorders. Nature. 2014;510:68- 75. DOI: 10.1038/nature13476.

[61] Huang JQ, Trasler JM, Igdoura S, Michaud J, Hanal N, Gravel RA. Apoptotic cell death in mouse models of GM2 gangliosidosis and observations on human Tay-Sachs and Sandhoff diseases. Human Molecular Genetics. 1997;6:1879-1885. DOI: 10.1093/ hmg/6.11.1879.

[62] Abo-Ouf H, Hooper AW, White EJ, Janse van Rensburg HJ, Trigatti BL, Igdoura SA. Deletion of tumor necrosis factor-a ameliorates neurodegeneration in Sandhoff disease mice. Human Molecular Genetics. 2013;22:3960-3975. DOI: 10.1093/hmg/ddt250

[63] Wu YP, Proia RL. Deletion of macrophage-inflammatory protein 1 alpha retards neurodegeneration in Sandhoff disease mice. Proceedings of the National Academy of Sciences of the United States of America. 2004;101:8425-8430. DOI: 10.1073/ pnas.0400625101.

[64] Wada R, Tifft CJ, Proia RL. Microglial activation precedes acute neurodegeneration in Sandhoff disease and is suppressed by bone marrow transplantation. Proceedings of the National Academy of Sciences of the United States of America. 2000;97:10954-10959. DOI: 10.1073/ pnas.97.20.10954.

[65] Jeyakumar M, Smith DA, Williams IM, Borja MC, Neville DC, Butters TD, Dwek RA, Platt FM. NSAIDs increase survival in the Sandhoff disease mouse:synergy with N-butyldeoxynojirimycin. Annals of Neurology. 2004;56:642-649. DOI: 10.1002/ana.20242.

[66] Eisenberg T, Knauer H, Schauer A, Büttner S, Ruckenstuhl C, Carmona-Gutierrez D, Ring J, Schroeder S, Magnes C, Antonacci L, Fussi H, Deszcz L, Hartl R, Schraml E, Criollo A, Megalou E, Weiskopf D, Laun P, Heeren G, Breitenbach M, Grubeck-Loebenstein B, Herker E, Fahrenkrog B, Fröhlich KU, Sinner F, Tavernarakis N, Minois N, Kroemer G, Madeo F. Induction of autophagy by spermidine promotes

longevity. Nature Cell Biology. 2009;11:1305-1314. DOI: 10.1038/ ncb1975.

[67] Li L, Rao JN, Bass BL, Wang JY. NF-jB activation and susceptibility to apoptosis after polyamine depletion in intestinal epithelial cells. American Journal of Physiology-Gastrointestinal and Liver Physiology. 2001;280:G992-G1004.

[68] Matsumoto M, Kurihara S, Kibe R, Ashida H, Benno Y. Longevity in mice is promoted by probiotic-induced suppression of colonic senescence dependent on upregulation of gut bacterial polyamine production. PLOS ONE. 2011;6:e23652. DOI: 10.1371/ journal.pone.0023652.

[69] Franceschi C, Capri M, Monti D, Giunta S, Olivieri F, Sevini F, Panourgia MP, Invidia L, Celani L, Scurti M, Cevenini E, Castellani GC, Salvioli S. Inflammaging and antiinflammaging: A systemic perspective on aging and longevity emerged from studies in humans. Mechanisms of Ageing and Development. 2007;128:92-105. DOI: 10.1016/j.mad.2006.11.016.

[70] García-Villalba R, Giménez-Bastida JA, García-Conesa MT, Tomás-Barberán FA, Carlos Espín J, Larrosa M. Alternative method for gas chromatography-mass spectrometry analysis of short-chain fatty acids in faecal samples. Journal of Separation Science. 2012;35:1906-1913. DOI: 10.1002/jssc.201101121.

[71] Kurihara S, Suzuki H, Oshida M, Benno Y. A novel putrescine importer required for type 1 pili-driven surface motility induced by extracellular putrescine in Escherichia coli K-12. Journal of Biological Chemistry. 2011;286:10185-10192. DOI: 10.1074/jbc. M110.176032.

[72] Liou AP, Paziuk M, Luevano JM, Machineni S, Turnbaugh PJ, Kaplan LM. *Physiological Functions Mediated by Yuzu (*Citrus junos*) Seed-Derived Nutrients DOI: http://dx.doi.org/10.5772/intechopen.95534*

Conserved shifts in the gut microbiota due to gastric bypass reduce host weight and adiposity. Science Translational Medicine. 2013;5:178ra41. DOI: 10.1126/ scitranslmed.3005687.

[73] Honda K. Clostridia and the mucosal immune system. Journal of Intestinal Microbiology. 2013;27:187-196.

[74] Dao MC, Everard A, Aron-Wisnewsky J, Sokolovska N, Prifti E, Verger EO, Kayser BD, Levenez F, Chilloux J, Hoyles L, MICRO-Obes Consortium, Dumas ME, Rizkalla SW, Doré J, Cani PD, Clément K "Akkermansia muciniphila and improved metabolic health during a dietary intervention in obesity: relationship with gut microbiome richness and ecology". Gut. 2015;65:426-436. DOI: 10.1136/ gutjnl-2014-308778. ISSN 1468-3288. PMID 26100928.

[75] Derrien M, Belzer C, de Vos WM.. Akkermansia muciniphila and its role in regulating host functions. Microbial Pathogenesis. 2016;106: 1096-1208. DOI:10.1016/j.micpath.2016.02.005. PMID 26875998.

[76] Barrett JC, Hansoul S, Nicolae DL, Cho JH, Duerr RH, Rioux JD, Brant SR, Silverberg MS, Taylor KD, Barmada MM, Bitton A, Dassopoulos T, Datta LW, Green T, Griffiths AM, Kistner EO, Murtha MT, Regueiro MD, Rotter JI, Schumm LP, Steinhart AH, Targan SR, Xavier RJ, NIDDK IBD Genetics Consortium, Libioulle C, Sandor C, Lathrop M, Belaiche J, Dewit O, Gut I, Heath S, Laukens D, Mni M, Rutgeerts P, Van Gossum A, Zelenika D, Franchimont D, Hugot JP, de Vos M, Vermeire S, Louis E, Belgian-French IBD Consortium, Wellcome Trust Case Control Consortium, Cardon LR, Anderson CA, Drummond H, Nimmo E, Ahmad T, Prescott NJ, Onnie CM, Fisher SA, Marchini J, Ghori J, Bumpstead S, Gwilliam R, Tremelling M, Deloukas P, Mansfield J, Jewell D,

Satsangi J, Mathew CG, Parkes M, Georges M, Daly MJ. Genome-wide association defines more than 30 distinct susceptibility loci for Crohn's disease. Nature Genetics. 2008;40:955- 962. DOI: 10.1038/ng.175.

[77] Atarashi K, Tanoue T, Shima T, Imaoka A, Kuwahara T, Momose Y, Cheng G, Yamasaki S, Saito T, Ohba Y, Taniguchi T, Takeda K, Hori S, Ivanov II, Umesaki Y, Itoh K, Honda K. Induction of colonic regulatory T cells by indigenous Clostridium species. Science. 2011;331:337-341. DOI: 10.1126/ science.1198469.

[78] Atarashi K, Tanoue T, Oshima K, Suda W, Nagano Y, Nishikawa H, Fukuda S, Saito T, Narushima S, Hase K, Kim S, Fritz JV, Wilmes P, Ueha S, Matsushima K, Ohno H, Olle B, Sakaguchi S, Taniguchi T, Morita H, Hattori M, Honda K. Treg induction by a rationally selected mixture of Clostridia strains from the human microbiota. Nature. 2013;500:232-236. DOI: 10.1038/ nature12331.

[79] Arpaia N, Campbell C, Fan X, Dikiy S, van der Veeken J, deRoos P, Liu H, Cross JR, Pfeffer K, Coffer PJ, Rudensky AY. Metabolites produced by commensal bacteria promote peripheral regulatory T-cell generation. Nature. 2013;504:451-455. DOI: 10.1038/ nature12726.

[80] Fukuda S, Toh H, Hase K, Oshima K, Nakanishi Y, Yoshimura K, Tobe T, Clarke JM, Topping DL, Suzuki T, Taylor TD, Itoh K, Kikuchi J, Morita H, Hattori M, Ohno H. Bifidobacteria can protect from enteropathogenic infection through production of acetate. Nature. 2011;469:543-547. DOI: 10.1038/ nature09646.

[81] Maslowski KM, Vieira AT, Ng A, Kranich J, Sierro F, Yu D, Schilter HC, Rolph MS, Mackay F, Artis D, Xavier RJ, Teixeira MM, Mackay CR. Regulation of inflammatory responses by gut

microbiota and chemoattractant receptor GPR43. Nature. 2009;461:1282- 1286. DOI: 10.1038/nature08530.

[82] Koh A, De Vadder F, Kovatcheva-Datchary P, Bäckhed F. From dietary fiber to host physiology: short-chain fatty acids as key bacterial metabolites. Cell. 2016;165:1332-1345. DOI: 10.1016/j.cell.2016.05.041.

[83] Schulthess J, Pandey S, Capitani M, Rue-Albrecht KC, Arnold I, Franchini F, Chomka A, Ilott NE, Johnston DGW, Pires E, McCullagh J, Sansom SN, Arancibia-Cárcamo CV, Uhlig HH, Powrie F. The short chain fatty acid butyrate imprints an antimicrobial program in macrophages. Immunity. 2019;50:432-445.e7. DOI: 10.1016/j. immuni.2018.12.018.

[84] Sun, Wu W, Chen L, Yang W, Huang X, Ma C, Chen F, Xiao Y, Zhao Y, Ma C, Yao S, Carpio VH, Dann SM, Zhao Q, Liu Z, Cong Y. Microbiotaderived short-chain fatty acids promote Th1 cell IL-10 production to maintain intestinal homeostasis. Nature Communications. 2018;9:3555. DOI: 10.1038/s41467-018-05901-2.

[85] Qin J, Li R, Raes J, Arumugam M, Burgdorf KS, Manichanh C, Nielsen T, Pons N, Levenez F, Yamada T, Mende DR, Li J, Xu J, Li S, Li D, Cao J, Wang B, Liang H, Zheng H, Xie Y, Tap J, Lepage P, Bertalan M, Batto JM, Hansen T, Le Paslier D, Linneberg A, Nielsen HB, Pelletier E, Renault P, Sicheritz-Ponten T, Turner K, Zhu H, Yu C, Li S, Jian M, Zhou Y, Li Y, Zhang X, Li S, Qin N, Yang H, Wang J, Brunak S, Doré J, Guarner F, Kristiansen K, Pedersen O, Parkhill J, Weissenbach J, MetaHIT Consortium, Bork P, Ehrlich SD, Wang J. A human gut microbial gene catalogue established by metagenomic sequencing. Nature. 2010;464:59-65. DOI: 10.1038/ nature08821.

#### **Chapter 13**

## Nondestructive Assessment of Citrus Fruit Quality and Ripening by Visible–Near Infrared Reflectance Spectroscopy

*Ana M. Cavaco, Dário Passos, Rosa M. Pires, Maria D. Antunes and Rui Guerra*

#### **Abstract**

As non-climacteric, citrus fruit are only harvested at their optimal edible ripening stage. The usual approach followed by producers and packinghouses to establish the internal quality and ripening of citrus fruit is to collect fruit sets throughout ripening and use them to determine the quality attributes (QA) by standard and, in many cases, destructive and time-consuming methods. However, due to the large variability within and between orchards, the number of measured fruits is seldom statistically representative of the batch, resulting in a fallible assessment of their internal QA (IQA) and a weak traceability in the citrus supply chain. Visible/near-infrared reflectance spectroscopy (Vis–NIRS) is a nondestructive method that addresses this problem, and has proved to predict many IQA of a wide number of fruit including citrus. Yet, its application on a daily basis is not straightforward, and there are still several questions to address by researchers in order to implement it routinely in the crop supply chain. This chapter reviews the application of Vis–NIRS in the assessment of the quality and ripening of citrus fruit, and makes a critical evaluation on the technique's limiting issues that need further attention by researchers.

**Keywords:** nondestructive, Vis–NIRS, citrus fruit, quality, ripening

#### **1. Introduction**

Citrus fruit are grown commercially in more than 50 countries around the world and are major commodities in the international trade [1, 2]. In Europe, the exceptional characteristics met by some of these produces have granted them the Protected Geographical Indication (PGI), such as the lemons (*Citrus limon* (L.) Osbeck) of Menton in France, Sorrento, Amalfi and Syracuse, and the Sicilian blood orange (*Citrus sinensis*) in Italy, the "Algarve Citrus" in Portugal, or the "Valencianos Citrus" in Spain.

As non-climacteric, citrus fruit are only harvested at their optimal edible ripening stage, and are required to meet the expectations of the current consumer who demands for fruit not only with the best appearance, flavor, and nutritional

properties, but that also comply with safety, traceability, and the sustainability of the cultural practices used. Like any other commodity, citrus fruit are subjected to worldwide standard specifications within the value chain [3] on their quality attributes (QA). Additionally, there are also adjustments to these requirements on quality and commercial ripening indices, that arise from the respective PGI normative of each commodity, growing regions and destination markets [4]. The main external quality attributes (EQA) accounted for citrus fruit are general appearance, size, weight, and color. Among the internal quality attributes (IQA), soluble solids content (SSC), titratable acidity (TA), juiciness, maturity index (MI; MI = SSC/ TA), and the absence of internal defects are the most relevant. Although firmness is not defined quantitatively, it represents an important IQA, since it is a limiting factor regarding postharvest handling, transport and shelf-life, fruit being expected to maintain a good consistency through the whole supply chain.

Once fruit attain the expected IQA, additional factors will condition the harvest of citrus fruit: orchard yield and size, ripening variability, harvest cost, storage conditions, market prices and consumers' demand. Although dependent on the country, producers are provided with three options to handle these constrains: (i) immediate harvest and marketing; (ii) immediate harvest and cold-storage; or (iii) delayed fruit harvest. Opting for immediate harvest may result in minimal organoleptic quality and low prices, whereas postponing it until favorable market conditions, risks fruit drop, decay and spoilage caused by extreme weather events, pests and diseases [5]. To prevent some of of these consequences, producers resort to the regular use of pesticides, which increase the production costs and impact negatively the environment [6, 7]. Cold-storage is used in some of the major citrus producing countries, such as Spain or South Africa, and require very strict conditions to avoid fruit loss caused by chilling and/or freezing injury [8]. Both, cold-storage and harvest delay may lead to adverse alterations in the citrus-like flavor, and thus fruit quality deterioration, even if MI or SSC remains acceptable for marketing [9]. In all cases, fruit become more susceptible to the occurrence of physiological disorders that cause internal and/or external defects. Among the most typical physiological disorders registered through the supply chain of citrus fruit, there is the section drying, the rind breaking disorder (RBD), the rind pitting disorder (RP), freezing damage, and granulation, as reported for tangerine (*Citrus tangerine* Tanaka [10], 'Nules Clementine' mandarin (*Citrus clementina*) [11], 'Marsh' grapefruit (*Citrus paradisi* Macfad.) [12], sweet lemons (*Citrus limettioides* Tan.) [13], and 'Honey' pomelo (*Citrus maxima* Merr.) [14], respectively. These disorders are difficult to sort out by visual inspection at harvest, but lead to posterior fruit deterioration, limiting their quality, shelf-life, price and acceptance by consumers. In fact, there are strict standards for fruit sorting and grading, which require the detection of some of these disorders, throughout the supply chain, as established by the California Department of Food and Agriculture (CDFA). For exemple, it is not permitted to sell oranges (*Citrus sinensis* (L.) Osbeck) if, generally, more than 15% of fruits per batch have considerable freezing damage [15].

Therefore, the ripening of citrus fruit at harvest is a major determinant of their final quality after the whole postharvest handling processes, the occurrence of storage disorders, and the produce shelf-life span [16]. It also affects the rate of fruit loss between the tree and the consumers' home. Thus, the management and the decision capacity of the optimal harvest date (OHD) is a critical step in the supply chain. The current approach followed by producers and packinghouses to establish it and therefore, to decide on the harvest, is to collect small fruit sets from the various orchards by the beginning of each variety harvest season, and to use them to determine QA through standard methods, that in most cases are destructive, subjective and very time-consuming.

*Nondestructive Assessment of Citrus Fruit Quality and Ripening by Visible–Near… DOI: http://dx.doi.org/10.5772/intechopen.95970*

However, all QA vary greatly inside the same orchard, either in terms of absolute values and/or in terms of spatial and temporal distribution, and even in the same tree. This has been shown in citrus orchards of 'Shiranuhi' mandarin (*C. unshiu C. sinensis*) *C. reticulata* [17], 'Ortanique' (*Citrus reticulata* Blanco x *Citrus sinensis* (L) Osbeck) [18], mandarin (*Citrus reticulata* Blanco) [19], and 'Newhall' and 'Valencia Late' orange [20]. Multiple factors, such as the level of sunlight exposure and the associated fruit temperature on the tree, fruit yield and size, tree vigor and age, rootstocks, site-specific nutritional requirements and micro topographies within the orchard, are reportedly associated to this variability [21–24]. Furthermore, the location of the orchards and their edaphoclimatic conditions, as well as the cultural practices also induce variability on the fruit maturation process, leading to different levels of QA and different ripening rates observed for the same cultivar at different sites [20, 21]. Consequently, the number of tested fruits with the standard methods is seldom statistically representative of the orchard, leading to the sub-representation of the effective ripening stage of the fruit within and between orchards, which results in a limited assessment of their ripening, heterogeneous fruit quality, a deficient OHD management and a weak traceability in the citrus supply chain [25–27].

Overall, there is the need to upgrade the management and the sustainability of citrus fruit supply chain with smart and nondestructive technologies that allow a fast, objective, accurate and extensive assessment of fruit QA and ripening on-tree and in the following postharvest, to replace conventional methods. Their aim would be to deliver the best produce to the markets, and contribute to reduce the current level of food loss around the globe, that involves a large portion of fruit and vegetables [28–30]. Considering how much of the world's population lacks food security, and the importance of these commodities in the provision of essential nutrients and vitamins, which could prevent malnutrition, that kind of technologies would comply with the sustainable development goals (SDGs) proposed by the Food and Agriculture Organization (FAO), International Fund for Agricultural Development (IFAD), and the World Food Programme (WFP), in the 2030 Sustainable Development Agenda, which supports a global commitment to end poverty, hunger and malnutrition by 2030, creating a #ZeroHunger world [31, 32].

The large number of reports published in the past two decades, show an active, and highly motivated research concerning the development of various nondestructive technologies for the assessment of quality and ripening parameters of a wide variety of fruit, including citrus [16, 33–37]. These techniques are used on inline sorting systems, on the bench or in the field and come in many forms, prices and commercial brands. Among them, the visible–near infrared reflectance spectroscopy (Vis–NIRS), is conceivably one of the most suitable and advanced nondestructive technologies currently used to monitoring several horticultural produces. It has been implemented in applications ranging from the inline automated grading systems, assessing up to 10–12 fruit per second, to handheld units suitable for field use, operating in full sunlight and varying ambient temperature [38, 39]. Additionally, it continues to grow stronger as a major investigation topic worldwide, with a major potential for improvement and contribution to the state of the art of precision agriculture and agronomic systems management [40].

This chapter comprises a brief explanation of Vis-NIRS fundamentals and a review of the various reports on its application published since 2012. Reports published before 2012 were already covered in the last review by [41] and will not be repeated here, with a few exceptions that represent relevant breakthroughs in the area. It will further attempt a critical evaluation on the limiting issues that need further research, to implement it as an effective nondestructive method to assess these commodities' quality and optimal ripening.

The authors invite the reader to complement this chapter with some of the most outstanding reviews published throughout the years, by the main researchers working on the subject (but not only in citrus). These reviews comprise the principles of the technique, its various methods and the listing of fruit and the respective QA for which it has provided calibration models [41–45], the overview on the publications and main research groups in the field [40], various recommendations for future research activity in the area regarding the adequate experimental design and the reporting requirements [38], as well as the current real-life applications available on the market that seem to comply with the warranted robustness for the technology to be integrated in the supply chain of many crops, including citrus [38, 39].

#### **2. Fundamentals of visible–near infrared reflectance spectroscopy (Vis–NIRS)**

In this review we will adopt the most common definition that Vis–NIRS covers the wavelength range 400–2500 nm of the electromagnetic spectrum. The lower limit is consensual, since it is the onset of the visible range, but the upper limit is mainly defined by the spectral response of the most common spectrometers. It comprises the visible (Vis) region (400–750 nm), the more penetrative short wave NIR (SWNIR), or Herschel region (750–1100 nm), and the near infrared region (750–2500 nm) of the spectrum [38, 39, 45]. The NIR radiation was discovered by Friedrich Wilhelm Herschel in 1800, and was first used in agricultural applications to measure the moisture in grain in the late 1960s [45]. The first Vis–NIRS application was commercialized in Japan in 1989 to sort peaches based on SSC in an automated grading line, but the research on its principles, applications and on the development of new customized systems, have only followed some decades later, being quite active nowadays [38–40].

#### **2.1 Interaction of radiation with the fruit**

When a light beam from the sun or a tungsten lamp, hits a fruit or any other sample, the incident radiation may be specularly reflected, absorbed or transmitted, and the relative contribution of each phenomenon depends on the chemical constitution and physical parameters of the sample (**Figure 1**) [46]. The spectral distribution of the radiation that penetrates the product change through wavelength dependent scattering and absorption processes. The photons that enter the fruit may emerge through multiple scattering in the tissue. Light emerging on the same side of incidence is described as *diffuse reflection*, while light emerging on the opposite side is described as *diffuse transmition*. Both diffuse modes may be understood in a general sense as 'transmitted', according to the initial description. The emerging diffuse light is collected by a spectrometer, originating the term *diffuse reflection spectroscopy*. The spectral features depend on the chemical composition of the product, as well as on its light scattering properties which are related to the sample microstructure. Fruit and vegetables are turbid media, in which scattering events dominate over absorption in the visible (400–750 nm), and particularly in the SWNIR and NIR ranges of the electromagnetic spectrum (750–2500 nm) [44] (see **Figure 1**).

In thin rind fruit most of light interaction takes place on the flesh and the skin has mainly a modulation effect upon the spectra. In most citrus, however, most of light interaction occurs in the thick rind and few photons probe the flesh. Thus, the *Nondestructive Assessment of Citrus Fruit Quality and Ripening by Visible–Near… DOI: http://dx.doi.org/10.5772/intechopen.95970*

**Figure 1.**

*When light hits a fruit, the photons may be reflected at the fruit surface (specular reflection) or enter the fruit tissue. In the latter case, a succession of scattering events takes place, where the photons change direction. Some of them reemerge, originating diffuse reflection (or transmission, not represented), and the other are eventually absorbed.*

assessment of IQA depends on the interplay between pulp and skin biochemistry and their optical properties [47–49].

Absolute quantification of diffuse reflected light (for example, as spectral radiance [W�sr�1 m�2 Hz�1 ]) is of little use, because it depends obviously on the characteristics of the light source. The calculation of reflectance avoids that subjectivity, since it normalizes the absolute measurement of the sample's reflection by that of a reference material, usually a near perfect reflector ('white') in the wavelength range under study. It should be stressed, however, that even the reflectance depends on the collection geometry (solid angle of collection, viewed area, etc.). Common choices for the reference material include Spectralon or Teflon, with nearly 100% reflection in the Vis-NIR. Reflectance *R*ð Þ*λ* is calculated according to the Eq. (1) presented below.

$$R(\lambda) = \frac{\mathcal{S}(\lambda) - D(\lambda)}{\text{Ref}(\lambda) - D(\lambda)} \times \mathbf{100},\tag{1}$$

where *S* stands for the Sample counts, *D* for the Dark counts, *Ref* for the Reference counts and *λ* is the wavelength. Here, *counts* refer to the digitized output of the spectrometer, which are proportional to the spectral radiance measured in a specific geometry. The dark counts are obtained with the spectrometer closed and represent the electronic noise, which must be subtracted from the sample and reference measurements.

#### **2.2 'Point' and imaging measurements**

Vis-NIRS is most commonly applied on specific 'points' of the fruit, by observing a small area, which produce an average spectrum for that specific site. This is called *point measurements*. But Vis-NIRS can also be applied on extensive sections across the fruit, through *multispectral* and *hyperspectral* measurements, which create an image of the measured sections for each wavelength band [44]. The main difference between multi- and hyperspectral modes are the number of wavebands used. Multispectral imaging uses a set of filters and a common digital camera to deliver typically no more than ten bands, while hyperspectral cameras merge imaging and spectral separation in the optical hardware to produce hundreds of

contiguous wavebands. Another way to look into hyperspectral images is to think that it yields the reflectance spectrum for each spatial position of a sample (*i.e.*, for each pixel of the image) [44]. Both techniques, although costly, have been shown to successfully assess several IQA, diseases and defects in several fruit, including citrus fruit [50, 51]. Yet, extensive investigation is needed to allow both the acquisition and image processing software to be implemented in real-time systems. Thus, this chapter will only address the systems that perform 'point' measurements, based on their much wider spread, cost-effective and friendly use through the supply chain, and particularly under field conditions. Further information on the principles and applications of multispectral and hyperspectral Vis-NIRS technologies could be found in the reviews by [44, 50].

#### **2.3 Instrumentation and measurement setup**

There are currently, a large variety of both commercial and lab-made customized Vis-NIRS systems, with various shapes, sizes, and prices, that operate in many spectral ranges, and have reportedly allowed the assessment of several QA, the content of critical compounds and the diagnostic and/or prediction of disorders in a wide sort of fruits, including citrus (**Tables 2**–**4**). Nevertheless, most of the current commercial fruit applications of Vis-NIRS are based on the use of silicon-based spectrophotometers comprising the Vis-SWNIR region (400– 1100 nm), because of their accessible prices and the larger light penetration depth in this band, in comparison to the significantly more expensive InGaAs-based devices (900–2500 nm), that do not add too much value to the quality assessment procedure [39].

From handheld, benchtop, to inline automated grading system, all Vis-NIRS devices comprehend the following fundamental components: an optical spectrometer, a light source (usually a tungsten halogen light bulb) and collection optics (optical fibers, lenses, integration spheres, dedicated probes). The current spectrometers typically include a connection for an optical fiber, an entrance slit (that defines the spectral resolution), a diffraction grating to separate the light into its spectral components, mirrors for collimation and focusing, and a light sensing device that is usually a one-dimensional CCD (Charge-Coupled Device) or CMOS (Complementary Metal-Oxide Semiconductor).

The Vis-NIR spectra may be acquired according to three principal geometrical configurations, as depicted in **Figure 2**: the reflectance mode (a), the transmittance mode (b), and in the interactance mode (c). The reflectance mode is susceptible to receive specularly reflected light, which may be a disadvantage, since only a fraction of the collected photons probes the fruit interior. In the transmittance mode the photons probe necessarily the fruit interior; however, the optical signal may be weak and noisy. The interactance mode is a tradeoff between the two previous modes: by using a contact probe it avoids specularly reflected photons and receives only those traveling through the fruit flesh. Also, the distance between light injection and collection is small, insuring a good optical signal. However, this is also a disadvantage, since the probing depth into the fruit pulp is shallow.

The choice of the geometry is thus of the utmost importance for obtaining good results, and should account for the fruit and the assessed QA. The penetration of NIR radiation into fruit tissue decreases exponentially with the depth, which is quite critical in thick rind fruit such as citrus [50]. Furthermore, the choice of the detection mode might be influenced by the spectral range used, as report by [48], in which both interactance and reflectance modes produced similar models to assess the SSC of 'Sunkist' navel oranges in the Vis-SWNIR range, but the participation of Vis region degraded this assessment in the transmittance mode. in general, to detect *Nondestructive Assessment of Citrus Fruit Quality and Ripening by Visible–Near… DOI: http://dx.doi.org/10.5772/intechopen.95970*

**Figure 2.**

*Setup for the acquisition of Vis-NIR reflectance spectra in (A) reflectance, (B) transmittance, and (C) interactance modes. Based on [45].*

the internal defects, the transmittance mode should be chosen, while the other two modes are quite reliable regarding other IQA (**Tables 2**–**4**).

#### **2.4 Typical Vis-NIRS spectrum and its interpretation**

All studies on the use of Vis-NIRS to assess the fruit QA start by acquiring the reflectance (R) spectra, which is then converted to the respective absorbance log (1/R) spectra (**Figure 3**). The main spectral differences observed in a wide variety of fruit are in the visible region, namely in the 400–750 nm range. This is due to changes in the pigments' content through ripening, namely chlorophylls, carotenoids and anthocyanins present on the fruit rind [38]. In fruit that change from green

#### **Figure 3.**

*(a) Average reflectance spectra of a set of 255 'Valencia Late' oranges and 239 'Rocha' pears acquired in the Vis/NIR range in the interactance mode; (b) Average absorbance spectra of the same set of fruit. The nominal positions of the most important absorption bands are indicated in the curves. The number is the order of the transition, ν stands for stretching vibration, δ for bending vibrations and the sum indicates combination bands (for example,* 3*ν+δ(O–H) represents the combination band of the second overtone of stretching with the fundamental bending in O–H); (c) Savitzky–Golay [52] filter of second derivative order applied to the absorbances. The bands are again indicated; (d) to (f) Same as in (a) to (c) but in the NIR range, with the spectra acquired in reflectance mode.*

to yellow/orange/red colors through ripening, the spectral information on the pigments' absorption range, may provide accessory indirect correlations with IQA such as firmness, as found in 'Rocha' pear (*Pyrus communis* L.) [53]. This, however, is not so clear in citrus fruit because their color change do not correlate with their maturity and depends on the orchards' location climate [6]. Otherwise, the pattern of the absorption spectra in the NIR range is quite similar among the various fruit species, although position and magnitude of the peaks are fruit specific, even among citrus fruit varieties [41]. The magnitude of the peaks and minima are also dependent on the acquisition mode used, but in general the same features are present and the landscape of the spectra is similar among the same fruit as reported for 'Sunkist' oranges [48].

The spectra in the NIR range convey information mainly related with vibrational bands (stretching and bending) of the relevant functional organic groups, such as O–H, C–H, C–O and C=O. The compilation of the main wavebands present in citrus fruit, namely, O-H and C-H vibration absorptions are presented graphically in **Figure 3**. These groups exist in all fruit organic molecules, but variations associated with water and storage reserves may induce slight changes in the spectra that may be related with the IQA. Vibration states are quantized and the transitions between states are said to be fundamental or overtones. The fundamental transitions (corresponding to a fundamental band) refer to the transition from the ground state to the first excited state, and take place mainly in the infrared range, that is above 2500 nm. In this range, the absorption peaks are distinguishable and correlate directly to specific compounds, allowing a better assessment of organic compounds such as vitamin C, citric acid or sucrose, as reported in 'Valencia' orange [54]. In contrast, the overtone bands correspond to transitions to higher excited states, with a large number falling in the NIR range. For example, the first overtone band corresponds to the transition from the ground state to the second excited state. A very crude approximation is that the n-th overtone frequency is close to (n-1) times the fundamental frequency. Thus, a general rule is that overtones have higher frequencies and lower amplitudes than the fundamental. For example, the fundamental frequency for O–H stretching (1*ν*) is around 2700 nm, which means that is beyond the range of the most common NIR dispersive-type spectrometers. However, the overtones are within their instrumental range: 2*ν* at 1420 nm (first overtone, strong intensity band), 3*ν* at 970 nm (2nd overtone, medium intensity) and 4*ν* at 750 nm (3rd overtone, low/very low intensity band). The quoted values are only indicative of a typical band central value. Indeed, the vibrations are dependent on the chemical environment, which results in a frequency spread of the bands. Finally, it is important to refer the combination bands. These correspond to the superposition of vibration motions. For example, the fundamental bending mode (1*δ*) of water at 6300 nm (infrared range) may combine with the fundamental stretching mode at 2700 nm (1*ν*), to generate a combination band around 1900 nm [*ν*+*δ* (O–H)]. Having in mind that the fruit tissue is composed by many different organic molecules, it is easy to understand that the spectral landscape of fruit NIR reflectance is a continuum, due to band superposition, as previously shown by [54], when comparing NIR and medium infrared spectroscopies (MIR), to assess several compounds in 'Valencia' oranges. Summarizing, the NIR spectra of a fruit contains mainly overtones and combination bands of stretching and bending vibrations of the main functional organic groups of relevant organic compounds regarding the fruit IQA, such as O–H and C–H. The large number of possible vibrations and corresponding bands originates a spectral landscape with very broad and unspecific features, from which it is nevertheless possible to retrieve useful information. For instance, [54] obtained better prediction of fructose and reducing sugars when using NIRS than MIRS.

#### *Nondestructive Assessment of Citrus Fruit Quality and Ripening by Visible–Near… DOI: http://dx.doi.org/10.5772/intechopen.95970*

The typical Vis–NIR and NIR reflectance and absorption spectra of 'Valencia Late' orange and 'Rocha' pear are depicted in **Figure 3** [55]. Both fruit spectra were relatively flat from 700 to 910, followed by strong water absorption peaks around 970, 1450 and 1940 nm. However, the C-H bands may distort slightly the water peaks, and the analysis of this distortion conveys more information than the main peaks alone. It is from these patterns associated with the OH and CH vibrations that it is possible to retrieve the information about sugars. Even the water bands by themselves may convey information about the sugars, because the concentration of sugars and water are interdependent [41, 56–58].

In the Vis–NIR range the most prominent feature is the 3*ν*(O–H) peak at 975 nm. This peak is reported in the literature in the range 960–980 nm [57], but the actual location depends on multiple factors: (i) the degree of OH bonding; (ii) the temperature; (iii) the presence of other close bands. In other words, within different chemical environments, the OH group will peak at different wavelengths. Effect of (iii) is more clearly observed in **Figure 3c**. Indeed, the smooth 975 nm peak observed in absorbance has a more fine structure disclosed upon derivation. Thus, the peak 4*ν*(CH2) at 930 nm is actually coalescing with the main water peak, causing a depression in the 2nd derivative left positive peak. The form of this overlap is a source of information about all organic compounds content, namely sugars, acids, proteins, etc.

A minor feature, but consistently observed in most fruit, is the slight inflection around 840 nm, which is caused by the band 3*ν*+*δ*(O–H). In this case it is more clearly observable in the reflectance spectrum, **Figure 3a**. In this discussion is important to have in mind that second derivation of a symmetric peaks yields a negative peak at the same position, with two lateral smaller positive peaks. This is clearly observed for the 840 nm feature in the 2nd derivative plot, with the negative peak coinciding with the 3*ν*+*δ*(O–H) absorption wavelength. On the contrary, the peaks of the 3*ν*(O–H) features do not coincide in the absorbance and 2nd derivative plots, which clearly indicates spectral overlap.

Similar curves are observed in other cultivars. For example, in 'Newhall' orange [56] the same structure for the second derivative plot was observed, although a different technique was used, namely the Norris derivative [59].

Concerning the NIR spectra, those from the oranges show three main peaks at 1190, 1450 and 1940 nm, whose origin may be traced to the 3*ν*(C–H), 2*ν*(O–H) and *ν*+*δ*(O–H) bands, respectively. However, satellite bands overlap, as in the Vis/NIR case. The most 'pure' peak is the first, around 1200 nm, corresponding to the 3*ν*(C– H) band. As is the 840 nm band, absorbance and 2nd derivative peaks coincide. The other two main peaks are more complex blends of two or more bands. For example, the second peak around 1450 nm, although dominated by the stronger 2*ν*(O–H) band, has contributions from 2*ν*+*δ*(C–H) and 2*ν*+2*δ*(C–H). Consequently, the 2nd derivative feature associated with this mix is more complex. The same could be said about the third peak.

Furthermore, due to several causes, the various peaks, even when they coincide among different fruit, may present different levels of importance, signified by their infrared values, with the various IQA. For instance, the combination band of OH reported at 839 nm correlated highly with SSC in 'Rocha' pear samples, but not in 'Valencia Late' oranges [55].

#### **3. Chemometrics**

As it has been mentioned earlier, Vis–NIR spectra of fresh fruits tend to be composed by a large superposition of absorption bands. The presence of a

substantial amount of water in fresh fruit has a big impact in the spectra, dominating most of the spectral landscape. Therefore, the signals corresponding to the absorption bands of key chemical compounds such as sugars and acids, become masked by water and are only discernible as weak fluctuations in the spectrum. Given the complex interplay between the multiple absorption bands and their weak amplitudes, most of the times the linear relation between chemical compound concentration and absorption (Lambert–Beer law) is almost lost. In order to be able to extract information about chemical concentrations from this type of spectrum, we have to look for relationships' patterns between different wavelengths. This is done resorting to multivariate statistical techniques, that when applied in the field of analytical chemistry is called Chemometrics. This research field can be considered as a subset of the broader area of Machine Learning, and pursues the same goals, i.e., infer critical information from high dimensional data. There is a vast literature on this subject for those who wish to learn more about Chemometrics. Here are some suggestions for introductory and advanced levels [60–64]. In this section, it is presented a brief introduction to the scientific language used in this area, with the sole objective of familiarize the reader with the main concepts that are often presented in the literature. In Vis-NIRS of fresh fruit, the input data are spectra, i.e. one-dimensional arrays of values, each one corresponding to the intensity of light (diffusively reflected or absorbed) at a specific wavelength. Each spectrum *Xn* (*n* ¼ 1, … number of samples) represents a measurement or sample and each point *xi* (*i* ¼ 1, … , number of variables) of the spectrum is usually referred to as input variables, spectral features or simply as 'wavelengths'. The macroscopic properties or QA features that are obtained through laboratory testing (e.g. SSC, firmness, etc.) are commonly defined as target variables *Yn* or simply attributes. Chemometrics consists on the application of mathematical/statistical methods that allow mapping the spectral features *xi* into the target variables *Y*. These methods can be subdivided into two broad subcategories: unsupervised and supervised. In the former type of method, only the input variables *xi* are used and, the main purpose is to find, for example, trends within the data, clusters that can be used for classification or other general characteristics of the data set. On the other hand, supervised methods use both input and target variables and can be used for classification tasks (e.g. discriminate between different fruit sub-species or origins) or for quantitative (regression) prediction of attributes that have continuous distributions (e.g. SSC, firmness, TA, etc.).

#### **3.1 Spectral pre-processing and outlier detection**

In order to implement the best calibration model possible to predict the expected QA, often the spectral data has to be preprocessed before being used. Preprocessing techniques are used to remove irrelevant information (noise, systematic errors and faulty samples) that can degrade the performance of the numerical algorithm used to develop the calibration model. Several preprocessing methods have been created for this purpose and reviews, such as [61, 65], present a wider scope of these techniques. A brief summary of the most commonly used methods is presented in **Table 1**. For Vis-NIRS, the most common forms of spectral preprocessing can be stacked into two groups: scatter corrections (SC) and derivative techniques (DT). Scatter-corrective methods are used to remove the influence of scattered light that can contaminate the diffuse reflectance spectra. The rationale behind SC techniques is to remove effects that are unrelated to the chemical composition of the samples and that just depend on the measurement geometry or samples morphology. On the other hand, DT are designed to improve signal to noise ratios, eliminate systematic baseline biases and enhance spectral variations. Another type of preprocessing

*Nondestructive Assessment of Citrus Fruit Quality and Ripening by Visible–Near… DOI: http://dx.doi.org/10.5772/intechopen.95970*

#### **Table 1.**

*Most common spectral preprocessing techniques.*

commonly mentioned in the literature is outlier detection. This process consists in identifying and removing from the data set, samples that are very different from the rest of the samples. These outliers can be for example, reflectance spectra that were defectively acquired or fruit with odd properties. The idea is to remove these samples from the data set, using some pre-defined metric in order to feed the model only with the most representative samples in the data set that lead to a correct mapping of the attribute being predicted by the calibration model constructed.

#### **3.2 Clustering**