Citrus Nutritional and Nutraceutical Importance

#### **Chapter 9**

## Citrus Fruits: Nutritive Value and Value-Added Products

*Abu Saeid and Maruf Ahmed*

#### **Abstract**

Citrus fruits are essential sources of food and energy and play a critical role in supplementing healthy diets. Citrus fruits contain mostly carbohydrates such as sucrose, glucose, and fructose and are good dietary fiber sources, which help prevent gastrointestinal disease and promote high circulating cholesterol. Besides, citrus fruits are also significant sources of vitamin C and various bioactive compounds. It is suggested that these components are of vital importance in improving human health due to their antioxidant properties and being converted to vitamin A. However, citrus fruit is still being used for different purposes like juice, jam, jelly, squash, pies, cake, candies, marmalades, etc. Most citrus waste materials are currently used as animal feed. Innovations are occurring in the conversion of citrus by-products into valuable commodities with the development of innovative technologies. This chapter has put up primary and secondary research findings of citrus fruits, especially lemon and pomelo, their chemical properties, composition, and their use in health and cosmetic needs.

**Keywords:** citrus, lemon, pomelo, nutritional properties, value-added products

#### **1. Introduction**

Citrus is an evergreen shrub that belongs to the Rutaceae family from South Asia, China, India and the Malay Archipelago, which is native to the subtropical and tropical regions of Asian regions [1]. The genus of citrus includes sweet orange (*C. sinensis*: 61.1 % of world citrus production), tangerine (*C. reticulata*: 19.9 %), limon and lime *(C. limon* and *C. aurantifolia*: 12.1 %) and grapefruit (*C. paradisi*: 5%). Minor types of citrus, which constitute much of the remaining 2.0%, include sour orange (*C. quarantium*), shaddocks (*C. grandis*), citrus (*C. medica*), which seem to be promising sources for many beneficial human nuts [2]. Citrus fruit is divided into two sections like peel and flesh (**Figure 1**). Peel is made from epicarp or flavedo (colored peripheral surface) and mesocarp or albedo (white soft middle layer). The peel (60–65%), internal tissues (30–35%), and seeds (0–10%) comprise citrus fruits [3]. Citrus fruits provide carbohydrates, such as sucrose, glucose, and fructose mostly. Fresh citrus fruits are also an immeasurable source of dietary fiber associated with gastrointestinal disease prevention and lowered circulating cholesterol. Citrus fruits also have a distinct aroma and delicious taste along with low protein and fat content.

Citrus fruits also provide the most potent source of vitamins C and B (thiamines, pyridoxines, niacins, riboflavin, pantothenic acids, and folate). The fruit also leads to the use of phytochemicals, such as carotenoids, flavonoids, and limonoids [1].

**Figure 1.** *Structure of the citrus fruit [2].*

Citrus phytochemicals contain antibacterial, antiviral, antifungal, anti-carcinogenic, anti-thrombotic, or anti-inflammatory agents [4]. Several studies have proposed citrus fruit evaluation as a healthy and delicious diet [5]. Prior research suggested that citrus and citrus products are rich sources of vitamins, minerals, and dietary fibers [6]. However, the bioactive and non-nutrient compounds in citrus are appreciated to reduce the risk of various chronic diseases [7].

Citrus fruits are eaten as fresh goods and juice throughout the world. Peel is discarded as waste containing many secondary components with significant antioxidant activity related to other fruit portions [8]. In recent years, flavonoids such as polymethoxy flavones (PMFs), which are present in citrus fruits, have been attracted growing attention by their antioxidants [9] and anti-cancer properties [10]. Various bioactive compounds in citrus peel extract and powder may reduce overall cholesterol, triglycerides, LDL, and glucose levels [11]. Citrus by-products produce a range of value-added products, including essential oils, pectin, enzymes, single-cell collagen, natural antioxidants, ethanol, organic acids, and prebiotics. Orange, lemon, mandarin, and grapefruit contained essential oils show antifungal activity upon the fungi *A. niger*, *A. flavus, P. chrysogenum,* and *P. verrucosum.* The essential oil may be regarded as acceptable for the food industry as alternatives to chemicals [12]. Pectin extracted from *Citrus* peel is used in various industrial food processes as gelling agents, including jam, jellies, and as thickener, texturizer, emulsifier, and stabilizer in dairy products. Pectin is also used to jellify properties in the pharmaceutical, dental, and cosmetic industries [13]. Therefore, this chapter highlighted the nutritional values of major essential nutrients such as Vitamin C, carotenoids and vitamin A, Folate, Dietary fiber, flavonoids, and limonoids, as well as value-added products such as food ingredients, pectin, essential oil, enzymes, a natural antioxidant, and packaging film retrieved from citrus especially lemon and pomelo fruit.

#### **2. Characteristics of citrus fruits (lemon and pomelo) and their chemical compositions**

#### **2.1 Lemon**

Lemon (*Citrus limon L.* from Rutaceae*)* is one of the most common globally and ranks third among the Citrus species globally by 4,200,000 metric tons after orange and mandarin [14]. Lemon fruits typically consist of three parts: pulp, skins (albedo and flavedo), and seeds. It offers an extensive supply of natural compound

*Citrus Fruits: Nutritive Value and Value-Added Products DOI: http://dx.doi.org/10.5772/intechopen.95881*

products such as citric acid, ascorbic acid, minerals, flavonoids and essential oils [15]. Lemon bioactive compounds like flavonoids, vitamins, minerals, dietary fiber (**Table 1**), and essential oils are used in the food, cosmetic, and pharmaceutical industries. Most by-products of the lemon juice industry can provide functional foods with nutritional substances such as non-digestible carbohydrates, dietary fiber and bioactive (flavonoids and ascorbic acid). Lemon fruits can function against photo-oxidamage because carotenoids exist. Lemon fruit, rich in flavonoids, has a significant role in the healthy diet, particularly in preventing diseases such as obesity, diabetes, lowering blood lipids, cardiovascular disease, and some forms of cancer [18]. The citrus fruits used for direct consumption or converted into juices, jam, jelly, molasses, lemoncello beverage and more in addition to the lemon skin are added value products such as pectins, essential oil and functional ingredients [12, 18].

#### **2.2 Pomelo**

Pomelo is one of the most commonly grown and eaten citrus fruits and orange, mandarin, lemon, and grapefruit [19]. Pomelo (**Table 1**) is a promising source of carbohydrates, proteins, fiber, vitamins and minerals originating in warm tropical climates in south-eastern Asia [20]. The presence of bioactive (carotenoids, lycopene,


#### **Table 1.**

*Chemical composition of Citrus fruits as [15–17].*

polyphenols, flavonoids, limonoids, fiber and vitamin C) contributes to their protection against oxidative stress, hyperglycemia, and high blood pressure. Due to its essential health promotion properties, pomelo segments in food products are growing in importance in producing functional foods [21]. Pomelo is eaten fresh or made into juice [19], or pomelo fortified noodles help the diabetic population [21]. On the other hand, researchers have investigated alternative ways of restoring pomelo peels to the advantage of value-added products such as pectin, essential oils, polysaccharides, phytochemicals [19]. Production of juice and consumption of fresh fruit create large quantities of agricultural waste. The main components of wet Pomelo Peel waste, like other citrus fruits, include water, cellulose and hemicellulose, soluble sugars, lipids (mainly D-limonene), and bioactive compounds (i.e., polyphenols, mostly flavonoids).

#### **3. Nutritional values of citrus fruits**

Citrus has many natural plant compounds such as vitamin C, carotenoids (some can convert to vitamin A), folic acid, flavonoids, and fiber. **Table 2** shows the amount of vitamin and mineral consumption in lemon and pomelo fruits.

#### **3.1 Vitamin C (ascorbic acid)**

Citrus is a valuable source of vitamin C. By consuming a moderate amount of citrus fruits each day, an individual can achieve 100 percent Vitamin C level. Vitamin C is an essential water-soluble vitamin essential for the body's defense [22]. It is transmitted through muscle fibers, carnitine biosynthesis, neurotransmitters, collagen, and bones because these particles connect the fibers. The immune system


#### **Table 2.**

*The number of nutrients and the percent of the recommended daily allowance or adequate intake met from the consumption of 100 g of selected citrus fruit [22].*

*Citrus Fruits: Nutritive Value and Value-Added Products DOI: http://dx.doi.org/10.5772/intechopen.95881*

can be effectively stimulated by consuming vitamin C, which boosts white blood cells [23]. When Vitamin C is taken for pregnancy, it can decrease pre-eclampsia risk [24]. Some studies indicate that vitamin C supplementation can reduce the severity of colds symptoms or duration [23]. Anti-oxidants such as Vitamin C could reduce the risk of artery stiffening and cardiovascular diseases [25]. Above 200 mg of vitamin C daily is a healthy intake, and citrus fruits are a huge source of this vitamin. Lemon provides 37 mg of ascorbic acid per 100 g of fruit [16]. Pomelos have 52.3 mg of ascorbic acid in 100 g of the flesh [26].

#### **3.2 Carotenoids and Vitamin A**

There are many types of carotenoids, including terpenes responsible for pigments commonly found in plants, and there are about 600 carotenoids in foods and 50 in human bodies [27, 28]. The highest carotenoid levels, such as lutein, zeaxanthin, lycopene, and vitamin A, are found in fruits and vegetables, including orange and carotene. Benefits of carotenoids in foods include improving immune function, promoting bone formation, promoting eye health, and maintaining visual quality [22]. There is a large amount of data supporting that carotenoids reduce the risk of cancer, macular degeneration, cataracts, skin damage to the sun, and cardiovascular diseases [29]. Higher consumption of β-carotene is linked to a lower breast cancer risk [30]. Beta carotene, lycopene, or lutein may decrease the rate of UV-induced lipid peroxidation in human skin fibroblast cells [30]. Lutein is inversely related to colorectal cancer in both men and women [31]. The levels of lutein, zeaxanthin, β-cryptoxanthin, and β-carotene in the lemon and pomelo, were around 2.95, 0.81, 0.81 and 10.3 (μg/g, db), respectively [32]. The content of carotenoids in pummelos' peel was 0.012-0.015 mg/gdb [33].

#### **3.3 Folate (folic acid)**

Folic acid, which is a water-soluble vitamin, and its derivatives are collectively called folate or folacin. The most notable folate compounds in Citrus are the reduced 5-methyl tetrahydrofolate (monoglutamate) and polyglutamate compounds [34]. Folate plays a vital role in DNA, which is involved in homocysteine regulation and protein production primarily through the methylation transfer reactions [22]. Because there is a high DNA production during pregnancy, a folate deficiency is significantly linked to birth defects such as neural tube defects [35]. Lack of folic acid caused higher levels of homocysteine, raising heart disease and atherosclerosis [22]. Previous studies show that citrus fruits' daily consumption can help improve folate levels, which will subsequently decrease blood homocysteine (tHcy), thus reducing cardiovascular disorder and neural tube defects [36]. Citrus is a parallel source of dietary folate that can help to cover up to 10% to 20% of the recommended daily allowance of adults, children, and infants with a consumption of 100 g of citrus fruits. The consumption of citrus fruit is an easy way to obtain vitamin C and dietary folate, which is vital for absorption in the body. Lemon, a citrus fruits representative, has eleven to sixteen micrograms of Folate in 100 grams [22]. According to El-Otmani and Ait-Oubahou [37] Citrus limon contained 11mg of folic acid per 100 g of citrus.

#### **3.4 Dietary fibre**

The fiber is found in vegetables and fruits cannot be digested and absorb in the small intestine. There are two kinds of dietary fiber; soluble and insoluble fiber. Insoluble fibers are highly fermentable and connected with carbohydrate and lipid metabolism, while soluble fibers contribute to fecal bulk and reduce transit time [34]. Although pectin, cellulose, and hemicellulose comprise the most abundant dietary fiber on the plants, they also contain only trace amounts of lignin. Pectin is citrus' primary fiber, which occurs primarily in citrus peels and rinds. Consumption of citrus fruit can contribute significant quantities of pectin in a diet. Dietary incorporation of pectin appears to affect several metabolic and digestive processes; principal interest affects glucose absorption and cholesterol level [38, 39]. There is a significant benefit in consuming citrus fruit because of its pectin content. Dietary incorporation of pectin appears to have many implications for metabolic, digestive, and health affairs. One way fiber can reduce colon cancer is by diluting and trapping the harmful chemicals in the colon from bile-absorption and bile-excretion [34]. Scientific studies have proven that fiber can help promote laxation and satiety, the uptake and reabsorption of glucose, fat, cholesterol, and bile acids, thereby lessening heart disease risk and possibly enhancing healthy intestinal microbial fermentation [40, 41]. Citrus fruits significantly reduce cholesterol levels depending on the esterification degree of fiber consumption, viscosity, and molecular mass [22]. A fiber-rich diet has a low risk of deadly chronic diseases such as diabetes, heart disease, weight, and cancer and lowers cholesterol levels and blood sugar [42]. Several epidemiological studies reported that citrus peel support reducing plasma liver cholesterol, total serum cholesterol, serum triglyceride levels, and total liver lipids [43].

#### **3.5 Flavonoids and limonoids**

Citrus pulp, peel are rich sources of flavonoids. Toh et al. [44] found that pomelo peeled (356.95 mg/QE) had higher total flavonoid content than pomelo pulp (13.06 mg/QE). Makni et al. [15] found the amount of quercetin in lemon flesh (56.16 mg Eq Quercetin/g dry weight) was higher than in peel (27.50 mg Eq Quercetin/g dry weight). Citrus fruits are also rich in flavonoids such as hesperidin, hesperetin, naringin, naringenin, diosmin, quercetin, rutin, nobiletin, tangeretin, and others [45]. Citrus flavonoids have both antioxidant and anti-inflammatory properties, and because of that, it can increase the antioxidant capacity and effect reducing cholesterol and triglycerides levels and provide more excellent bone health [22]. Preclinical studies and clinical trials demonstrated that flavonoids' effects in the forms of hesperidin and its aglycone hesperetin prevent various types of diseases, including neurological, psychiatric, and cardiovascular disorders [46]. Over the years, naringin and hesperidin are gaining attention for their great antioxidant capacity, contributing sweet flavor to foods and beverages [47]. Some known naturally occurring flavonoids have potency in defending against certain types of RNA and DNA viruses [48].

Limonoids are also known as flavonoids, which are compounds found in citrus fruits. In citrus fruits, there are two groups of limonoids: aglycones and their corresponding glucosides. Bitter taste in citrus results from limonoids present. Limoninin's most important constituents are glycosides called limonin and nomilin [22]. In animal and human cell lines, limonoids slow down the development of aggressive cancers like the pancreas, colon, stomach, and breasts. On the other hand, limonoids are also reported to reduce skin cancer in animal models. Limonoids are known for their medicinal or health beneficial effects like anti-cancer, anti-microbial and antimalarial activities [49]. Limonoids have antibacterial and antiviral effects. Some limonoids are known to stimulate the in vivo production of the detoxifying enzyme glutathione S-transferase in the liver and inhibit the formation of chemically induced tumor cells in the oral cavity, forestomach, small intestine, colon, lung, and skin of

*Citrus Fruits: Nutritive Value and Value-Added Products DOI: http://dx.doi.org/10.5772/intechopen.95881*

#### **Figure 2.**

*Proposed model for signaling pathways leading to growth inhibition by obacunone in estrogen-responsive breast cancer (MCF-7) cells [53].*

animals [50, 51]. Limonoids found in citrus fruits decreased the spread of cancer cells in animal studies [52]. Studies showed that lemon-lime oil in the form of obacunone appeared to prevent breast cancer by inhibiting aromatase enzyme and antiinflammatory pathways [53] (**Figure 2**). Several studies have revealed that limonin and nomilin are found in fruits, pulp, and seeds. Pummelo juice contained 18 ppm of limonin and 29 ppm of total limonoid glucosides [54]. Wattanasiritham et al. [55] reported limonin content of 18 ppm in the juice of pomelo cultivars. Limonin levels in extracted juice from seven pummelo cultivars from Florida ranged from 10.07 to 29.62 ppm. As shown by analysis, mature lemon seeds contain 1300 μg/g of different limonoid glucosides on a fresh weight basis [56]. The mature seeds contain much higher amounts of glucosides than commercialized juice, therefore. Fong et al. [57] documented that the commercial lemon juice had only 82 μg/g of glucosides.

#### **4. Value-added products**

Citrus fruits are known for being highly fragrant, with a tart taste and higher vitamin C content. The world has a wider variety of citrus fruit because of the continued type changes, such as sour oranges, oranges, pummelos, lemons, and limes, among others [22]. Nowadays, citrus pulp/pomace, seed, and peel are used for various commercially valuable products such as food ingredients, pectin, essential oils, enzyme production, a natural antioxidant, and packaging film formation.

#### **4.1 Food ingredient or food products**

The nutritional supplement of pomelo fruit segments has been added to products to be developed like noodles prepared with 30% new segments and 5% dry. These noodles can satisfy those with regular diabetes and the general public [21]. A high dietary fiber food was created by reducing the dietary fiber-rich pomelo peel to a powder that contained nearly 50% of dietary fiber. Lemon fruit is usually eaten fresh, but it is also processed to make juices, jams, jellies, molasses, candies and much more [18]. Lemon juice has been used as a coagulant during the manufacture of wara cheese [58]. Another innovation implemented in the beef burger is to use lemons for "enhancing the cooking properties of the burger" [59]. Lario et al. [60] had reported that the high-fiber lemon powder extract from lemon peel debris by-products is an ideal additive for food products (as meat, dairy, and bakery products).

#### **4.2 Pectin**

Pectin is an agent of gelling, emulsifying, stabilizing, texturizing, which appears as a white to light brown powder broadly accepted as a functional ingredient [61]. Fruit peels are a highly desirable pectin source because they cover up to 20% of the fruit's total Pectin [62]. Pomelo is a highly valued source of natural Pectin. About 20.75% pectin is derived from lemon peel for jams. In the study, the high extraction (36.71%) of Pectin from lemon peel has something to contribute to this industry [63]. Moneim et al. [64] recommended utilizing 20.75% of the lemon peels' total weight in making pumpkin jam. The researchers were added 16.740% of Pectin from pomelo peel to the pressed carrots before storage [65]. Studies by Methacanon et al. [66] have shown that pectin yield was 23.19% for pomelo peel. On the other hand, Roy et al. [65] were found that pomelo peels are a good source of Pectin, and then carrot jam made by extracting Pectin from pomelo peel.

#### **4.3 Essential oil**

Essential oils (Eos) are volatile, complex, natural mixture of aromatic oils obtained from plants [67]. Citrus essential oil is commonly known to produce a good fragrance and has been officially approved for healthy public consumption. All over the world, EOs are used in cosmetics, perfumery, toiletries, flavoring, beverage, pharmaceuticals and other personal hygiene products [68–70]. Lemon oil is often used on the skin because of its antimicrobial and antifungal properties. Pomelo peel (PP) has approximately 299 recognized volatile compounds. A significant number of these volatile compounds is considered as terpenoids (189 volatiles, 63.2%). Various kinds of chemicals released are monoterpenoids, monocyclic monoterpenoids, bicyclic monoterpenoids, diterpenoids, acyclic sesquiterpenoids, monocyclic sesquiterpenoids, bicyclic sesquiterpenoids, and tricyclic sesquiterpenoids. Another major volatile present in PP EO is nonterpenoid alcohols (4.7%), nonterpenoid aldehydes (6.0%), nonterpenoid hydrocarbons (5.7%), and esters (8.7%). The unknown volatiles covered 11.7% (35 volatiles) of total volatile compounds. The structures of widely known terpenoids in Polypropylene EO has shown in **Figure 3a,b**. The most critical monoterpenoids (1 to 16) and sesquiterpenoids (17 to 29) are present in PP EO [19]. According to studies conducted, lemon essential oils retain aroma in foods because of their natural preservative and flavoring properties [71]. The effect of lemon essential oils on the cheesemaking process dramatically reduces microorganisms' population, especially those of the Enterobacteriaceae family [58]. In the food and pharmaceutical industries, citrus

*Citrus Fruits: Nutritive Value and Value-Added Products DOI: http://dx.doi.org/10.5772/intechopen.95881*

#### **Figure 3.**

*(a) Major monoterpenoids in pomelo peel essential oils (1-16); (b) Major sesquiterpenoids in pomelo peel essential oils (17-29).*

EOs can be employed to inhibit mold and fungal growth. Lemon EO (Citrus limon) was used as a possible fungicide to manage the pathogenic fungi attacking grapevines, namely Eutypa sp., Botryospaeria dothidea, and Fomitiporia mediterranea. The antifungal activity was observed for EO against all the three fungi with the highest action against strain Eutypa sp. (82% inhibition) and the lowest tolerance (33.1%) towards F. Mediterranean [72]. These essential oils repress the growth of mold and yeast. The raspberries coated with alginate and lemon EO (0.2 percent) or orange EO (0.1 percent) halted bacteria, yeast, and mold and also reduced the quality deterioration right after harvest [73]. The lemon EOs mixed in the chitosan films can be used to control L. Multicellular pathogens in refrigerated foods Researchers Rahmawati et al. [74] observed that an edible coating applied with lemon essential oil only delayed aging of tofu and fresh strawberry.

#### **4.4 Enzymes**

The most basic usage of citrus peels is to produce the pectinolytic enzyme for beneficial purposes. Larios et al. [75] studied endo-polygalacturonase production by Aspergillus sp. CH-Y-1043 using untreated lemon peel and citrus pectin as carbon sources. Lemon peel being employed as a substrate in submerged cultures to obtain high pectinase titers by A. flavipes FP-500 and A. terreus FP-370 [76].

A. niger produces approximately 2,181.23 U/L pectinases from lemon, peel pomace in a solid-state reactor [77]. Seyis and Aksoz [78] have shown that lemon pomace and peel are suitable substrates for heterotrophic xylanases enzyme production using fungus Trichoderma harzianum. Aspergillus niger LFP-1 was studied in solidstate fermentation (SSF) using pomelo (Citrus grandis) peels as a substrate [79]. Maller, et al. [80] determined that lemon peels are extremely capable of triggering the production of Polygalacturonase in the aspergillus niveus. Pectin lyase yield increased through fungal strain Aspergillus oryzae process derived from lemons peel and used in solid-state fermentations [81]. Studies said that Polemo pericarp powder utilized as a substrate for Aspergillus oryzae JMU316 has Naringinase enzyme [82]. Pectinase enzyme produced from pomelo peels by By Aspergillus niger through Solid State Fermentation [83]. Lemon peels could be a good source of naringin, which could be used as a carbon source in submerged fermentation for naringinase production using Aspergillus niger [84]. Naringinase is essential for the production of sweetener precursors, preparation of prunin, aroma enhancement in winemaking, biotransformation of antibiotics, and rhamnose manufacturing [82].

#### **4.5 Natural antioxidant**

Antioxidants are chemical substances that can reduce or prevent the damage caused by free radicals in the body, thus reducing the risks of cardiovascular disease and cancer [85]. Results of studies showed that lemon peel contained almost 75.9% of antioxidant content. The unique ability of Paneer was derived from compounds found in the Peel of orange, lemon and pomegranate [85]. Peel taken from Tambun White pomelo type contains higher levels of antioxidants and is also a rich source of natural antioxidants [44]. Lemon peel and flesh had the highest antioxidant capacity, and they had a significant impact on the prevention of cardiovascular diseases and other diseases [86].

#### **4.6 Packaging film formation**

More sustainable, biodegradable plastic has gained popularity among environmental scientists. The researcher created plastic films from citrus peels. By applying the peels of citrus, biodegradable packaging material could be made [87]. Wu et al. [88] prepared fruit peel as the edible packaging film with high content biopolymer to form film for packaging. The film was designed to incorporate tea polyphenols, which causes interacting molecules to become more closely crowded. Soy protein with essential oil of lemon peel was used to create a degradable film and create cheese curdorants for preservation [89]. Dias et al. [90] reported that the use of citrus essential oil and its aroma significantly improved consumers' health and significantly increased the acceptance of biscuits' packaging. Das et al. [91] demonstrated that chicken feather keratin combined with pomelo peel pectin to form biodegradable composite film and wrapping of fried fish fillets resulted in less weight loss, hardness value, and reduction in the surface microbial count.

#### **5. Conclusion**

Citrus has positive effects on human health, and it could be an essential raw material to the biotechnological industry. Citrus is a mighty source of vitamins, minerals, and dietary fibers. Bioactive and non-nutrient compounds in citrus are valuable for controlling chronic diseases such as diabetes, cholesterol, obesity, cardiovascular disease, and some forms of cancer. Besides citrus, vitamin C also

#### *Citrus Fruits: Nutritive Value and Value-Added Products DOI: http://dx.doi.org/10.5772/intechopen.95881*

has other benefits, including fighting diseases such as cardiovascular disease, boosting white blood cells, immune function, and symptoms or duration of colds. Peel, flesh/pomace, and seed from the citrus fruit are employed in making different novel foods like noodles, extract pectin, enzyme extracts, and essential oil. Therefore this information might be necessary for the readers because it gives facts about the popular citrus fruit. Also, choosing the best citrus for an edible ingredient can be beneficial for citrus processors.

### **Author details**

Abu Saeid1 and Maruf Ahmed<sup>2</sup> \*

1 Department of Food Engineering, NPI University of Bangladesh, Manikganj, Bangladesh

2 Department of Food Processing and Preservation, Hajee Mohammad Danesh Science and Technology University, Dinajpur, Bangladesh

\*Address all correspondence to: maruf@hstu.ac.bd; maruffpp@gmail.com

© 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.

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#### **Chapter 10**

## Citrus Essential Oils: A Suite of Insecticidal Compounds

*Bulbuli Khanikor, Kamal Adhikari and Bikash Rabha*

#### **Abstract**

Citrus essential oils (CEOs) and their constituent compounds are being reported to have multifarious activities. In this chapter an attempt is made to discuss the insecticidal activities, as well as CEO profile of different vegetative part of *Citrus* species and biocidal potentiality of their constituent compounds against diverse insect pests. It is observed that in most of the CEO constituent profile, limonene is the major constituent compound. Other important constituents present in different percentages in different CEOs are β-citronellal, linalool, pinene, β- caryophyllene, β-myrcene, terpinene, citral etc. These plant EO constituents are reported to have insecticidal effects against diverse insect species. Taking the four peel EOs of *Citrus limon, Citrus paradisi, Citrus medica, Citrus maxima* commonly grown in North Eastern part of India, study on their insecticidal effects against *Dolichoderus affinis* (Hymenoptera: Formicidae) was made and result is presented showing higher fumigant toxicity of *C. medica* and *C. limon* oil against the ant sp. With the increasing awareness for using safe insecticidal products among consumers, the citrus EOs with their attracting terpene compounds having good insecticidal potency bear all attributes to be used as commercial green pesticides in coming days both in indoor and outdoor management of insect pests.

**Keywords:** essential oils, limonene, *Dolichoderus*, *Citrus medica*

#### **1. Introduction**

The genus Citrus has tremendous industrial value all over the globe not only for its nutritive juicy high valued fruits but also for the essential oils present in its different vegetative parts. Thus, both the Citrus fruits and citrus essential oils bear potential to generate livelihood & to boost the country's economy. Citrus essential oils (CEOs) with diverse biologically active compounds of terpene groups with pleasant aroma have already achieved significant positions in flavor, food, cosmetic industries. At the same time, because of their antimicrobial activities as well as anticancer, antioxidant, anti-inflammatory, metabolic disorder alleviating activities etc. these oils and their compounds have been getting importance in pharmaceutical and medical sectors for the last few decades [1]. A good number of studies also reported insecticidal potential of citrus EOs extracted from different citrus sp. and their constituents at different times, a few of which are commercialized to be used by the consumers against insect pests. There are 33 recorded species of citrus worldwide (ThePlantList.org) with many recorded and unexplored varieties present in different parts of the world. The essential oil profile of different citrus species varies although some of the constituent compounds are common but present in different amounts in the total bulk oil. Even the oil profile of different vegetative parts of a single citrus species are not identical. Understanding of essential oil profile of diverse citrus species grown in wild, semi wild and cultivated state across the globe at different seasons is the much-needed task as the quality of the oil, oil yield percentage, consistency of the constituents even varies with seasonal changes, geographical location, harvesting time of the plant parts, soil type etc. however from the existing GC–MS profile of different *Citrus* sp. reported at different times and from different places, it is apparent that two -three dominant compounds are mostly present in most of the Citrus species. The literature revealed that the Citrus EO comprises more than 200 compounds of which 85–99% are volatile and 1–15% nonvolatile compounds. The volatile compounds comprise mostly monoterpenes (predominant limonene), some sesquiterpenes and their oxygenated derivatives [2].

Pest control sector is dominated by synthetic pesticidal products for many decades. At recent times with increasing concern to ecofriendly product, plant essential oils are getting renewed interest as they are not only effective but also comparatively safe and environment friendly in comparison to synthetic counterparts. Essential oils are part of natural plant defense system and many of them are proved effective and some are exploited for integrated management practices of pest and pathogens. As some citrus species are naturally resistant to certain group of pests and or pathogens, it is assumed that certain bioactive compounds may present in the essential oil part of those citrus species. It is already established that citrus essential oils of different citrus species are effective against wide range of pest and pathogens. It is also important to have an insight about the interaction of citrus constituents against its own insect pest and pathogen complex to be used as insecticidal, repellent and bactericidal etc. A few papers highlighted beneficial effects of using citrus essential oil against its own pest and pathogen complex. The added advantage of considering CEO as insecticidal and insect repellent is that the plant is edible therefore safe for residual contamination or toxicity to consumer. At the same time the pleasant aroma offers consumer acceptance.

#### **2. Citrus EO against insect sp**

CEO and extracts have been tried against a wide range of insect pests for assessing their insecticidal as well as repellent properties. In some parts of the world citrus plants have been traditionally used to ward off a insect pests. Some recent reports especially of the last two decades of the insecticidal and repellent effects of different citrus sp. are presented below. Most of the works were carried out on dipteran, lepidopteran, hemipteran and coleopteran insect pests.

Topical toxicity of the essential oil of *Citrus hystrix* with LD50 of 26.748 μL/g and antifeedant properties leading to severe growth inhibition has been reported against tobacco armyworm *Spodoptera litura* [3]. The fumigant toxicity and repellent effect of the n-hexane extract of the plant leaf was documented against stored grain pest *Lasioderma serricorne* [4]. Fumigant toxicity of peel oils of lime, orange, mandarin, tangerine, grapefruit and lemon were reported against three store grain pest species *Callosobruchus maculatus, Sitophilus zeamais and Dermestes maculatus* [5].

The peel essential oil of the plant is reported to possess repellent effect against *Callosobruchus maculatus* [6], *Aedes aegypti* and *Anopheles minimus* [7]. Similarly, the insecticidal and repellent activity of *Citrus reticulata*, *Citrus limon* and *Citrus aurantium* peel oils was demonstrated against *Callosobruchus maculatus* [8]. Insecticidal activity of *Citrus limon* and *Citrus sinensis* against vine mealybug**,** *Planococcus ficus* [9]. The larvicidal and adulticidal effects of *Citrus limon* and

*Citrus Essential Oils: A Suite of Insecticidal Compounds DOI: http://dx.doi.org/10.5772/intechopen.95887*

*Citrus sinensis* are mentioned against *Attagenus fasciatus* and *Lasioderma serricorne* [10]. The seed and peel extracts of *Citrus limon* L. was reported to have the highest larvicidal toxicity (LC50 values of 395.59 ppm for seed; 468.69 ppm for peel) after 24 hours over EOs of *Citrus grandis, Citrus sinensis, Citrus paradise, Citrus reticulate* [11]. Essential oil of *Citrus reticulata* and *Citrus sinensis* was reported effective against the fourth instar larvae and adults of *Tribolium castaneum* with higher potency of *Citrus reticulata* [12].

The seed EOs of *Citrus reticulate* var. kinnow, *Citrus reticulate* var. freutrall, *Citrus sinensis* and *Citrus jambhiri* was tested against *Tribolium castaneum* with promising efficacy in terms of LC50 for *Citrus jambhiri* followed by Citrus reticulate and *Citrus sinensis* [13]. Similarly Oboh et al. [14] recorded insecticidal efficacy of *C. sinensis* peel essential oil against Callosobruchus mamulatus, *Tribolium confusum*, *Sitophilus oryzae* with LC50 value of 21.8, 38.9, 60 μl/l.

Comparative evaluation of toxicity of EOs of *C. limon, C. aurantifolia, C.sinensis* in filter paper impregnation method showed highest toxicity of *C. limon* (95% mortality) followed by *C. aurantifolia* (92.5%) and *C. sinensis* (82.5%) against carpenter ant *Camponotus nearcticus* [15]. But, Guerra et al. [16] comparatively lower topical toxicity (15% mortality) of *C. limon* EO against *Camponotus pennsylvanicus* among the eleven different EOs tested. Essential oils and or extracts of *C. maxima* or *C.grandis* have been reported effective against different mosquito species. In our earlier studies we recorded differential biocidal activities of essential oil extracted from peel and leaf part of *Citrus grandis* grown in Assam against different developmental stages of *Aedes aegypti* and *Culex quinquefasciatus* [17, 18]. EO extracted from leaves was found more effective against egg stage while oil from peel was recorded more effective against larval and adult stages of *A. aegypti* [18]. The leaf and peel oil of the plant was recorded highly effective against egg and larval stage with LC50 value of below 50 ppm but did not found much effective against adult stage of *Culex quinquefasciatus* although having repellent properties with good protection time [19]. In a recent study we observed synergistic larvicidal response of *Citrus grandis* leaf oil with *Allium sativum* bulb oil against *C. quinquefasciatus* [19]. Manorenjitha et al. [20] tested hexane, ethyl acetate, methanol, water and essential oil extract of *C. grandis* peel extract for evaluating oviposition deterrent and repellent properties on *Aedes aegypti*. They observed promising oviposition deterrent activity of ethyl acetate fraction (10 ppm concentration) in breeding plates kept within mosquito cage and effective repellency (94.7%) of 20% essential oil fraction of the peel while offering animal bait in modified tunnel test. A study for toxicity assessment on worker termites *Odontotermes feae*, essential oils of *Citrus grandis* with LC50 value of 273.36 ppm was found to show maximum toxicity out of *Citrus paradisi, Cassia fistula*, *Citrus grandis* EOs [21].

The peel essential oils of *Citrus aurantifolia* has been reported as insecticidal, repellent, and larvicidal against *Aedes aegypti* [22]. In our previous study, we observed the ovicidal, larvicidal and adulticidal effects of leaf and peel essential oil of *Citrus aurantifolia* against *Aedes aegypti* [23].

Promising fumigant toxicity of the peel EO of *Citrus aurantium* and *Citrus sinesis* from the north east Brazil with LC50 value of 5.80 μL/L of air and 3.80 μL/L of air respectively and oviposition deterrent activity at 3.5 and 7.0 μL/L of air against the whitefly *Bemisia tabaci* [24]. The same oil was reported effective against the larval and adult stages of tomato leafminer *Tuta asoluta* (Lepidoptera: Gelechiidae) [25]. The insecticidal activity of *Citrus aurantium* EO against adult housefly *Musca domestica* was reported next to the activity of EO of *Citrus sinensis* [26]. *Citrus aurantium* leaf EO was found as effective fumigant against sawtoothed grain beetle *Oryzaephilus surinamensis*, cigarette beetle *Lasioderma serricorne* and rice weevil *Sitophilus oryzae* with LC50 value of 64.94, 202.49 and 364.25 μL/L of air

respectively [27]. Similarly, Bnina et al. [28] noted fumigant toxicity of peel, leaves and flowers essential oil of *Citrus aurantium* against four stored grain pests namely *Tribolium castaneum, Liposcelis bostrychophila, Sitophilus granarium, Cryptolestes ferrugineus* with LC50 value of 64.78%, 23.11%, 101.50% and 20.62% respectively. They also noted repellent property of the oil against these pests. Yazdgerdian et al. [29] tested contact and residual toxicity of eleven essential oils including *Citrus aurantium*, *Citrus sinensis*, *Citrus limon* against wooly beech aphid*, Phyllaphis fagi* (Hemiptera: Aphididae) and rice weevil, *Sitophilus oryzae* (Coleoptera: Curculionidae) and recorded highest residual toxicity (40%) of *C. aurantium* among the citrus sp. tested against the targeted species. Similarly, without affecting the seed viability of stored cowpea and consumer acceptability, peel EOs of *Citrus nobilis* and *Citrus medica* was reported to show significant reduction of egg laying, egg hatching, and adult emergence percentage of pulse beetle *Callosobruchus maculatus*. Both the oils showed dose dependent repellency with higher effect for EO of *C. nobilis* [30]. Like that of EOs, the nonpolar petroleum ether extract of ripe fresh fruit of *C. aurantium* was reported effective against adults of olive fruit fly *Bactrocera oleae* (Diptera:Tephritidae) in petri dish residual exposure test [31]. The same solvent extract also reported to have good toxicity against medfly *Ceratitis capitata* adults (LC50 value of 70.6 and 147.1 lg/cm2 for male and female respectively at 96 h in Petri dish residual bioassay) [32].

Moravvej et al. [33] tested fumigant toxicity of EOs from four citrus species namely *C. paradisi, C. limonium, C. sinensis* and *C. aurantium* among which *C. paradisi* was the most effective with LC50 value of 125 μl/L and *C.sinensis* is the least effective with LC50 value of 269 μl/L against *Callosobruchus maculatus.* However, fumigant toxicity of *C.sinensis* was reported against *Solenopsis invicta* (Hymenoptera: Formicidae) with 100% mortality at 3 mg/tube after 24 hrs [34]**.** Ethanolic extract of the same plant was found effective against *Anopheles stephensi, Aedes aegypti* and *Culex quinquefasciatus* with larval and adulticidal LC50 value of below 500 ppm along with more than 50% repellency at 150 ppm concentration till 180 min [35].

Ezeonu et al. [36] reported the insecticidal properties of the volatile peel extracts of *Citrus sinensis* and *Citrus aurantifolia* against mosquito, cockroach and housefly and recorded higher insecticidal potency of the peel extract of *Citrus sinensis* with maximum fumigant effect (85% at 60 min) against cockroach.

Zewde and Jembere [37] evaluated the solvent extract and essential oil of *Citrus sinensis* against *Zabrotes subfasciatus* (Coleoptera: Bruchidae) for their repellent, fumigant and protectant properties and recorded no progeny emergence on application of oil at low dose (30 mg of EO), prominent percent mortality at high dose (750 mg of essential oil killed 67% of Z. subfaciatus after 96 hours). Fumigant toxicity of *C. sinensis* EO was also reported effective against second instar larvae of *Musca domestica* (Diptera: Muscidae) with LC50 values of 71.2 μl/L as well as percent inhibition of pupae with 46.4% at 40 μl/L exposure concentration [38].

Orange oil extract was also recorded effective against the subterranean termite *Coptotermes formosanus* (Isoptera: Rhinotermitidae). Application of the oil extract at 5 ppm concentration resulted in 96% and 68% mortality respectively in closed container. At the same time termites did not show tunneling behavior on the 0.2 & 0.4% oil treated soil [39].

Majeed et al. [40] reported the insecticidal activity of the acetone, ethanol and aqueous extracts of seeds, leaves and fruit peels and leaves of *Citrus aurantium* and *Citrus sinensis* and two other plants against mealy bug *Drosicha mangiferae* (Hemiptera; Pseudococcidae) of which ethanol extracts of *C. sinensis* seeds and *C. aurantium* leaves were suggested as considerably toxic against the said insect.

*C. medica* peel essential oil was reported as more effective in filter paper fumigation method against stored grain pest *Tribolium castaneum* (Coleoptera) with LC50 value of 29.5 mg/L air than the leaf EO [41].

Abdel-Kawy et al. [42] showed *Citrus trifoliata* essential oil loaded nanocubosome significantly enhanced insecticidal property of the essential oil against the second instar larvae of *Spodoptera littoralis*.

While working as biocidal and repellents, plant products including EOs and constituent terpene compounds are reported to act on cholinergic system [43], voltage – gated sodium channel of the nerve membrane, glutamate-gated chloride channel [44], GABA-system [45], Octopaminergic system [46], mitochondrial system [47], endocrine system disrupting the endocrinological balance and respiratory system of insect body. However, not much studies yet conducted on detailed study on the mode of action of EOs and their constituent compounds.

#### **3. GC-MS profile of citrus EO**

With the development of GC–MS technique, profiling of essential oil became easier. The composition of different citrus species from different parts of the world have been reported utilizing this technique. Most of the profiling results although detected average 20–50 numbers of compounds, a few compounds mostly occupy the major share of the bulk oil. The dominating compounds in most citrus species is limonene. In some species like *C. hystrix* the major compound is β-citronellal. The other common constituent compounds observe to be present in many GC–MS results of citrus species are linalool, pinene, β- caryophyllene, β-myrcene, terpinene, citral etc. Some of the reported constituent profiles of citrus species are mentioned below.

#### **3.1** *Citrus hystrix*

From the leaf essential oil of *C. hystrix* in Malaysia, 29 compounds were reported, out of which beta-citronellal was the major compound (66.85%) [3]. The other compounds present in more than 1% total compositions were β-citronellol (6.59%), linalool (3.90%), 5,9-dimethyl-1-decanol (4.96%), methyl citronellate (1.90%), geranyl acetate (1.80%), citronellol (1.76%), 3-undecanol (1.04%). The same compound (beta-citronellal) with 86.43% amount was reported in another study from Indonesia along with 11.48% citronellol and 1.65% β-linalool [4].

#### **3.2** *Citrus maxima* **(synonym** *Citrus grandis***)**

In the leaf EO of *C. maxima*, 42 constituent compounds were reported to be present with citronellol as the major compound (28.26%) of the essential oil. The compounds comprised more than 1% of the oil compositions were β- caryophyllene (16.89%), −spathulenol (9.32%), α-caryophyllene (2.48%), γ-cardinol (3.16%), α-cardinol (2.51%), 2n-hexylcyclopentanone (2.22%), caryophyllene oxide (1.03%). Again, 34 compounds were identified from the peel essential oil of the same plant with limonene comprising bulk of the oil constituents (89.04%) [48]. The major compounds identified in more than 1% of the total composition were β-pinene (2.25%), β-myrcene (2.06%), β- copaene (1.76%). Compounds comprising more than 0.3% amount but below 1% were linalool, β-phellandrene, α-pinene, terpinene-4-ol. Earlier 35 compounds with limonene as the major constituent compound (93.2%) was reported from the fruit peel oil [49]. Myrcene comprised 2.9%

and other six compounds namely α-pinene, octyl acetate, germacrene -D, linalool, decanal, geranial comprised above 0.2% but below 1% of the total composition.

#### **3.3** *Citrus aurantium*

Phytochemical profiling of essential oil of *C. aurantium* showed the presence of 25 compounds with limonene occupying 87.52% followed by linalool (3.365%) and β-myrcene (1.628%) as dominant compounds [25]. From Tunisian *Citrus aurantium*, limonene percentage in leaves, flowers and peel EOs were 6.52, 5.03, 73.6% respectively, linalool occupied 37.24, 41.82, 4.8% respectively, linaly acetate occupied 7.87, 13.75, 1.6% respectively and neral share was almost of similar (3.40, 4.80, 3.26% respectively) percentage. β-pinene composition for leaves and flowers were 9.68 and 9.21% respectively and α-thujene composition were 10.65 and 6.15% respectively but both β-pinene and α-thujene present below 0.5% in peel EO of the plant [28].

#### **3.4** *Citrus aurantifolia*

In our recent studies 31 compounds from the leaf oil and 26 compounds from the peel essential oil was recorded from GC–MS analysis of EO of *Citrus aurantifolia* in India. Citral and limonene were noted as the major constituent compound of leaf oil and limonene and palatinol-1C as the major constituent of peel essential oil of the plant [19, 23]. The EO of the plant from Italy reported to comprise limonene (53.8%), γ-terpinene (16.5%), β-pinene (12.6%), β-Bisabolene (1.33%), Geranyl acetate (1.06%), Neryl acetate (1.12%), Geranial (1.84%), sabinene (1.74%), and α-Pinene (1.97%) [50]. Phytochemical analysis of leaf and peel EOs of the plant from Brazil [51] showed limonene as the dominant compound in both leaf (32.7%) and peel (77.5%) part. Other prominent compounds in the leaf EO were linalool (20.1%), citronellal (14.5%), citronellol (14.2%), trans-β-Ocimene (2.7%), geranial (2.6%), neral (2.1%), trans-β-Caryophyllene (2%), myrcene (1.4%) etc. The other major compounds of leaf EO were myrcene (4.4%), linalool (3.5%), citronellal (3.2%), citronellol (2%), β-Bisabolene (1.5%) etc.

#### **3.5** *Citrus sinensis*

Phytochemical analysis of peel essential oil from three varieties of *C. sinensis* from Kenya showed presence of 56 components in Salaustiana variety, 73 in Valencia and 72 in Wshington varirty. Limonene occupied more than 90% in all the essential oil (Salustiana 94.6%, Valencia 92.5% and Washington 90.5%); alpha terpinene occupied 1.5% in Valencia and Washington and 1.7% in Salustiana [52]. Limonene with 90% share and β-myrcene, γ-terpine, linalool with 1.88%, 1.21% and 0.88% share out of 32 identified compounds in peel EO of *C. sinensis* has been reported from China [53]. GC–MS analysis of the plant EO from Argentina showed limonene (92.47%), linalool (1.43%), and β-myrcene (0.88%) as the major constituent compounds along with terpineol (0.28%) in lesser amount [26]. Almost similar constituents were identified from EO of *C. sinensis* with D-limonene (65.28–80.18%), Linalool (0.32–2.20%) and β- pinene (1.71 5.58%) as major part in another study [34].

#### **3.6** *Citrus nobilis*

Phytochemical study on constituents from the peel of *C. nobilis* from China showed D- limonene (12,601 μg/g) as the major constituent compound followed by β-myrcene (1600 μg/g), β-pinene (82.64 μg/g), p-mentha-1,8-dien-3-one (41.33 μg/g), α-pinene (25.41 μg/g), geranial (21.32 μg/g), sabinene (21.18 μg/g), E-β-ocimene (14.97 μg/g), linalool (10.38 μg/g), α-terpineol (6.36 μg/g). Other compounds are present in lower amounts (below 5 μg/g) [54]. In another study from Sri Lanka, 37 compounds were reported from peel part of which D-limonene (45%) was the major one followed by cyclopentane-2-methyl-1-methylene-3- (1-methylethenyl) (3.94%), p-mentha-4,8-diene (3.73%), α-terpinolene (3.03%), methyl-2-(methylamino) benzoate (2.4%), α-fernesene (1.1%). Other compounds were present in below 1% [30].

#### **3.7** *Citrus limon*

Phytochemical analysis of citrus leaf EO from Iran showed presence of 27 compounds of which the major compound was linalool (30.62%). The other compounds present in significant amount were geraniol (15.91%), α-terpineol (14.52%), linalyl acetate (13.76%), geranyl acetate (6.75%), Β-pinene (4.51%), neryl acetate (4.24%), p-Cymene (1.86%), and limonene (1.13%) [55]**.** Chemical composition of EO of *C. limon* grown in Iraq [56] showed presence of 24 compounds with limonene as principal compound with 29.52% share. Other major compounds were β-Pinene (23.89%), α-Pinene (2.25%), Myrcene (1.31%), (Z)-β-Ocimene (2.09%), Linalool (1.41%), (R)-Citronellal (15.10%), α-Citronellal (3.57%), (+)-α-Terpineol (1.57%), Neral (Z-Citral) (1.19%), Geranial (E-Citral) (1.73%), Thymol (9.79%), Citronellyl acetate (1.87%), Caryophyllene (1.36%), Phytol (1.36%). The analysis of the essential oil of *Citrus limon* from North-East India reported presence of 43 constituent compounds of which limonene (55.40%), neral (10.39%) trans-verbenol (6.43%) and decanal (3.25%) were the major constituent compounds [57].

#### **3.8 Citrus paradisi**

Phytochemical analysis result of *C. paradisi* peel EO from Turkey demonstrated presence of 25 constituent compounds of which limonene occupied the highest percentage (88.6%) of the oil. The other major compounds were α-terpinene (1%), and β-pinene (1.2%) [58].

From Nigeria, fifteen phytochemical constituents of the plant oil were reported. Among the compound limonene (94.2%) occupied the major share [59].

#### **3.9** *Citrus medica*

A total of 19 constituent compounds were identified from leaf essential oil of *Citrus medica* from Bangladesh of which erucylamide (28.43%), limonene (18.36%), citral (12.95%), Mehp (8.96%), 2,6-octadien-1ol,3,7-dimethyl-acetate, (Z) (5.23%) were the major compounds. From peel essential oil 43 compounds were reported out of which isolimonene (39.37%), citral (23.12%), limonene (21.78%) were the major constituents. Three other compounds namely β-myrcene, neryl acetate and neryl alcohol were reported to present at around 2% each in total composition and remaining compounds were present in traces amount [60]. In a study carried out by Li et al. [61], all total 28 compounds were reported to present in the fruit essential oil of *Citrus medica* of which limonene (45.36%), γ-terpinene (21.23%), dodecanoic acid (7.52%) were documented as major constituent compounds. Compounds like β-bisabolene, tetradecanoic acid, α-terpineol, terpinene-4-ol, hexadecenoic acid, α-bergamotene, α-pinene, β-pinene comprised between 5–1% range in total composition. In another study fruit peel EO of the plant was reported to comprise limonene (38.7%), γ- terpinene (28%) and ο-cymene (15.2%) as major compounds [41].

#### **4. Citrus EO compounds against insect sp**

Essential oil composition of different citrus sp. across the globe although may vary but some of the compounds are observed as common in most of the oil profile. The most dominating and commonly present compound is limonene. Other common compounds are citronellal, citronellol, linalool, pinene, myrcene, ocimene, terpinene, caryophyllene etc. The bioactivity of EOs is often related to the activity of major compounds present in the crude oil and some of the studies have already established this fact. Individual assessment for insecticidal property of these common constituent compounds have been performed by different researchers and some of them were found active against insect pest. Limonene and other *Citrus limon*oids are reported as insect repellents, feeding deterrents, growth disruptors, and reproduction inhibitors against a wide range of pest complexes. Insecticidal activity of limonene was reported effective against *Tuta asoluta* (Lepidoptera: Gelechiidae) [25]. Yoon et al. [62] revealed repellent property of different citrus oil and its major compound limonene against different species of cockroaches like *Blatella germanica, Periplaneta americana* and *Periplaneta fuliginosa.* However, Karr and Coats [63] did not get significant insecticidal activity of d-limonene against *Blattella germanica, Musca domestica, Sitophilus oryzae* and *Diabrotica virgifera virgifera.* In contrast they reported enhanced growth of nymph of *Blatella germanica* after oral administration of d-limonene.

In another study against cat flea species *Ctenocphalides felis* (Siphonaptera: Pulicidae), d-limonene (LD 50 against larvae, adults 226, 160 μg/cm2 respectively) and d-limonene with piperonyl butoxide (PB) (LD 50 against larvae, adults 157, 49 μg/cm2 respectively) were reported effective against all the life stages except the pupal stage of the flea species [64]. Fumigant toxicity of d-limonene, α- terpineol etc. also reported against honey bee *Apis mellifera* and tracheal mite parasite species *Acarapis woodi* [65].

Fouad and da Camara [66] extracted the essential oil from *Citrus aurantiifolia* and *Citrus reticulate* and analyzed the phytochemical constituents using GC–MS and found limonene as the major constituent compound, 38.9% of the *C. aurantiifolia* oil and 80.2% of the *C. reticulata* oil. They analyzed the enantiomers of limonene against the said insect. They found that *Citrus reticulate* was more toxic than *Citrus aurantifolia* towards the said insects. (R)-limonene was shown to have greater toxicity against *S. zeamais* than the (S)-limonene as found in the ingestion bio assay. Repellent bioassay showed (S)-limonene more susceptible to *S. zeamais* than (R)-limonene.

After identifying limonene as major compound in the EOs of *Citrus aurantiifolia* (38.9%) and *Citrus reticulate* (80.2%) Fouad and da Camara [66] tested enantiomers of limonene against *Sitophilus zeamais* and recorded greater toxicity of (R)-limonene than the (S)-limonene in the ingestion bio assay. But in the repellency test they found more susceptibility of *S. zeamais* towards (S)-limonene than (R)-limonene.

In a recent study, Sowler et al. [67] comparatively evaluated the effect of laboratory grade limonene and a commercial limonene-based insecticide against *Haematobia irritans irritans* in terms of deterrence, mortality, and reproduction. They showed that the egg viability was decreased in both the treatment, however, commercial limonene that caused loss of viability at 5.8% concentration was ovicidal in case of laboratory grade limonene. However, in terms of knockdown effect commercial limonene was better. Interestingly, at a concentration of less than 0.1%, both the commercial and laboratory grade limonene were acted as attractant.

#### *Citrus Essential Oils: A Suite of Insecticidal Compounds DOI: http://dx.doi.org/10.5772/intechopen.95887*

Giatropoulos et al. [68] tested essential oil of *Citrus sinensis, Citrus limon,* and *Citrus paradise* and their constituents and recorded γ- terpinene as the most toxic compound against *Aedes albopictus* larvae. They also reported that the constituent compound tested for repellency were better mosquito repellent than the parent essential oil. Similarly, Luo et al. [41] analyzed composition of leaf and peel essential oil of *C. medica* and tested both crude oil and major compounds viz. limonene, terpinene, o-cymene, β-caryophyllene against *Tribolium castaneum* and recorded γ-terpinene as the most effective insecticidal compound having LC50 value of 4.1 mg/l air and β- caryophyllene as the effective repellent compound. Limonene was reported to have almost similar fumigant toxicity like that of crude EO of *C. aurantium* against *Tribolium castaneum, Sitophilus granarium* and *Cryptolestes ferrugineus* [28]. In our earlier studies we found higher toxicity of citral, that is the major compound of the essential oil of *Citrus aurantifolia* than the crude oil as mosquito larvicidal, ovicidal and adulticidal against *Aedes aegypti* [23]. Plata-Reuda et al. [69] reported the insecticidal activity of citral and geranyl acetate against peanut beetle *Ulomoides dermestoides*. These compounds affected the survivorship, locomotor activity and reduced the respiration rate of the said species.

Nootketone and carvacrol, a phytochemical constituent present in essential oil of Citrus [70] acts as insecticidal compound against *Aedes aegypti* [71]. Pajaro-Castro et al. [72] recorded the neurotoxic effects of linalool and β-pinene on *Tribolium castaneum*. They observed that at low concentration both the compounds were attractant towards the insect and at higher concentration the compounds were repellent**.** Individual treatment of limonene and linalool was found to have fumigant toxicity against two ant species namely *Acromyrmex balzani* and *Atta sexdens* with LC50 values of 5.72 μl/L, 5.38 μl/L and 2.40 μl/L, 5.34 μl/L respectively [73]**.** Similarly individual treatment of β-caryophyllene is reported to have good contact toxicity against these two ant sp. but with low fumigant toxicity [74]. Fumigant toxicity of limonene, linalool and β- pinene were also reported effective against fire ant *Solenopsis invicta* (Hymenoptera: Formicidae) [34].

Linalool, α-terpinene was reported to show 100% fumigant toxicity against adult rice weevil *S. oryzae* at 3.9 mg/L [75].

Muller et al. [76] recorded 85.4%, 71.1%, and 29% repellency of the candles prepared with 5% geraniol, 5% linalool and 5% citronella against mosquitoes on human landing bioassay. They observed similar repellency against sand flies too. 78% repellency of *Culex pipiens pallens* was reported after using 30% citronellal [77]. Progeny deterrent, antifeedant, egg hatching inhibition activity was documented after application of 5–10 μl citronellol against *Callosobruchus analis* [78]. Compounds like limonoids act as synergists to enhance the activities of other biological and or synthetic insecticides [79]. We recorded medium larvicidal and adulticidal potential of limonene against *Aedes aegypti*, but found higher toxicity when combined with diallydisulphide and carvone respectively [80]. So, it is not always the individual compound that act as the most active but appropriate combinations of compounds having synergistic effects would be more fruitful as insecticidal against insect pests.

#### **5. Citrus of North East India**

North East India is enriched with Citrus species having documented 23 species and 68 varieties out of the 27 species of Citrus found in India [81, 82]. It is established that some of the citrus species are endemic and some are in endangered status [83]. According to Hore and Barua [84], there are eight citrus species indigenous to this region scattered in the form of semi-wild, wild state and some in cultivated

state. Some of the species are naturally tolerant to viral and bacterial diseases and also for drought, cold and rainfall. For instance, *Citrus limon* is reported to resist scab, canker and gummosis, *C. indica* resistant to greening disease [84]. Due to diverse ecogeographical conditions the Citrus species and varieties of this region may bear specific traits including its aroma and essential oil constituents which needs to be investigated. However citrus crop and citrus essential oil-based industry is not yet flourishing in this part of the country. The information regarding Citrus essential oils extracted from the Citrus species grown in this particular area is relatively scanty. As the Citrus plants are rich in secondary metabolites to naturally defend an array of pathogens and pest complexes, it is expected that some of the key compounds for controlling insect pest may lie within the secondary metabolite compounds especially in the diverse aromatic essential oil part of the plants at least in the resistant citrus species.

Here we have attempted to evaluate insecticidal properties of essential oil extracted from the fruit peel of four citrus species namely *Citrus limon, Citrus maxima*, *Citrus paradisi* and *Citrus medica* grown in North Eastern part of India against one of the household ants *Dolichoderus affinis* (Hymenoptera: Formicidae)*.* Fruits of *Citrus limon, Citrus paradisi* and *Citrus medica* were collected from Udalguri district, Assam (26.7210 ̊ N, 91.9906 ̊ E) and *Citrus maxima* from a daily market at Guwahati, Assam (26.1445 ̊N, 91.736 ̊E), in September, 2020. Essential oils from fresh peels were extracted by hydro-distillation using Clevenger's apparatus. After 6 hours, essential oils were collected, anhydrous sodium sulfate was added to absorb traces of moisture and were stored at 4 ̊C till its use. The worker ants of *Dolichoderus affinis* from naturally existed colony located in the wooden frame of house wall were considered for the assessment. Fumigant toxicity of these oils were assessed following the method described by Hu et al. [34]. Six different concentrations viz. 0.25 μL, 0.5 μL, 1 μL, 2.5 μL, 5 μL and 7.5 μL. of each EO was individually loaded in 1.5 ml centrifuged tubes, evaporation of EOs was allowed by making five small holes of 1–1.2 mm diameter and placed into 500 ml properly cleaned borosilicate conical flask. Twenty worker ants were taken per flask in a replication and the flask was covered by aluminum foil and bound tightly by rubber band to prevent the loss of volatile compounds. For each concentration three replications were made. Equal number of controls set without oil were placed against each treatment. The environmental temperature range was 21–30°C and relative humidity range 56–99 during the experimental period. The Percent mortality [percent mortality = (Total no. of dead ants/Total no. of treated ants) × 100] data was recorded after 12 h and 24 h of treatment. The ants were considered to be dead if touched with a needle but did not show any movement. Based on the results sublethal concentration was determined using probit analysis with the help of SPSS and Minitab software. As shown in the figure, after treatment with *C. limon*, maximum 71.66% mortality was recorded at 12 h and 91.66% was recorded after 24 h at 7.5 μL treatment. The calculated LC50 for the oil was 2.66 μl / 500 air volume. For the EO of *C.paradisi*, maximum 15% mortality was recorded at 12 h and 66.66% mortality was recorded after 24 h of treatment. LC50 for the oil at 24 h was 7.32 μl / 500 air volume. For the EO of *C. maxima*, not more than 10% mortality was recorded even after 24 h at the highest dose applied and LC50 could not be computed for the oil. While for the EO of *C. medica*, maximum 56.6% mortality was recorded at 5 μl concentration at 12 h and maximum 88.33% mortality was recorded after 24 h at 7.5 μl. LC50 for *C. medica* at 24 h was recorded as 2.09 μl/500 air volume (**Figure 1**, **Table 1**). Highest toxic effect was recorded for *C. medica* followed by *C. limon*. Earlier Adusei-Mensah et al. [15] evaluated insecticidal properties of three citrus species viz. *Citrus aurantifolia*, *Citrus sinensis* and *Citrus limon* against *Camponotus nearcticus* (Formicidae) and recorded highest performance from *C. limon* with 95% mortality.

#### *Citrus Essential Oils: A Suite of Insecticidal Compounds DOI: http://dx.doi.org/10.5772/intechopen.95887*

#### **Figure 1.**

*Relation between concentration of EOs and respective percent mortality.*


#### **Table 1.**

*LC50 values of the individual citrus oils against Dolichoderus affinis at 24 h.*

But Guerra et al. [16] recorded only 15% mortality of *Camponotus pennsylvanicus* (Hymenoptera: Formicidae) on topical application of *C. limon*, the efficacy of which was comparatively lower than other eight EOs tested against the ant species. Not much studies on insecticidal activities of citrus EO against ants have been found to be reported. The findings showed the prospect of using *C. medica* and *C. limon* oil for controlling household ants.

#### **6. Conclusion**

With the increasing awareness of consumers for ecofriendly products and at the same time increasing resistance of insect pests against insecticides, the demand for novel, safe and effective products is increasing. As discussed above, the existing literature revealed presence of a good number of terpene compounds in different *Citrus* species which are present in different ratios although in most cases limonene is the predominant constituent. Both the crude oil as well as individual compounds possess good insecticidal and repellent properties against diverse insect pests, both indoor and outdoor. Our study also showed promising potential against *Dolichoderus affinis* while using four Citrus essential oils with higher efficacy of *Citrus medica* and *Citrus limon* essential oils. It is expected that in near future Citrus plant essential oils with their pleasant aroma and array

of chemical compounds shall take leading space in development of insecticidal and repellent products to be used in both indoor and outdoor pest management practices against insect pests.

#### **Author details**

Bulbuli Khanikor\*, Kamal Adhikari and Bikash Rabha Department of Zoology, Gauhati University, Guwahati, Assam, India

\*Address all correspondence to: khanikorbulbuli@yahoo.co.in

© 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.

*Citrus Essential Oils: A Suite of Insecticidal Compounds DOI: http://dx.doi.org/10.5772/intechopen.95887*

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#### **Chapter 11**

## The Orange Peel: An Outstanding Source of Chemical Resources

*Gianfranco Fontana*

#### **Abstract**

*Citrus sinensis* (L.) Osbeck is a very common cultivar belonging to the *Rutaceae* family. It is largely diffused in several areas of the world characterized by mild to warm climate conditions. Its abundant worldwide production (up to 10<sup>7</sup> Tons. per year) and consumption both as the edible part of the fruit and as several types of derivative products imply the production of a huge amount of waste, such as the fruit pomace. Several ways of recycling this material have been developed in recent years: employment as fertilizer, fodder ingredient, and even cloth material. However, the chemical added value of *Citrus sinensis* peel has been underestimated despite the diversified and significant content of useful chemicals, such as polyphenols, polymethoxylated phenols, glycosylated flavonoids, volatile and non-volatile terpenoids, pectins, enzymes, etc. This work aims to highlight the outstanding chemical potential of *Citrus sinensis* peel.

**Keywords:** biological activity, *Citrus sinensis*, essential oil, flavonoids, orange peels, polymethoxyphenols

#### **1. Introduction**

*Citrus sinensis* (CS) (L.) Osbeck is a perennial species growing in warm climate areas of the world and largely employed as food in form of fresh fruit, with a global production of ca. 6.7X10<sup>7</sup> tons. per year (TPY) in 2016 [1], or as a processed derivative (ca. 1.85x10<sup>7</sup> TPY) such as juice, marmalade, flavor, fragrance and coloring additive, pectin.

CS is an evergreen tree, 3 to 9 mts. high with sparingly barbed branches, alternate leaves with toothed blades differently shaped, oval or elliptical, connected to the stem by winged-petioles. Axillary flowers are present singly or in whorls of 6 and possess 5 white petals and up to about 25 yellow colored stamens. The pericarp of CS has a spherical or oval shape of 6–10 cm diameter with the color changing from green to yellow-orange during the ripening; the endocarp containing juice sac glands is enclosed within a wrinkled epicarp or exocarp or flavedo containing a great number of essential oil glands protected by a waxy epidermis. Below the flavedo is the albedo, also called mesocarp, a white filamentary tissue composed of tubular-like cells.

The principal industrial application of CS is the production of frozen concentrated juice. The procedure of juice extraction eventually accompanied by the extraction of the essential oil, implies the generation of a major "by-product" constituted by a pomace, mainly containing peels, accounting for up to around 60% w/w of the original fruit mass processed [2]. This huge amount of biomass does pose serious environmental concerns because of its high level of total organic carbon (TOC) and biological oxygen demand (BOD) that make disposal procedures rather complex and demanding from both the legal and industrial points of view. This is because there is an increasing trend to modify the way of approaching this problem by reconsidering the post-production orange pomace more like a by-product rather than a waste. In the last years, many producers have subjected this material to processings involving partial acidic fermentation, drying, and packaging to biologically and chemically stabilize the biomass before its application as animal feed in zootechnics, soil conditioners in agriculture, or the manufacturing of compost and biogas [2].

Beyond the standard workup of the *Citrus sinensis* peel (CSP) waste, new perspectives have been being opened in the context of the high chemical added value of the CSP [3–5] also by the complete knowledge of the rich metabolomics profile of this species. The use of CS peel has been proposed for a variety of purposes that include the production of antioxidant-enriched dietary supplements in veterinary [6], the preparation of human dietary supplements, and nutraceuticals such as citric acid [7] and flavonoids [8, 9]. The extract of CS peel is the source of a huge variety of phytochemicals and has been investigated on several applications including its chemotherapeutic and chemopreventive potential for several relevant human pathologies, such as cancer [10, 11] and obesity [12]. The extraction procedures vary in function of the main components that have to be obtained: from the simple cold pressing of pomace and the extraction with water to obtain highly hydroxylated compounds to the employment of mixtures organic protic solvent/water and finally low polar organic solvents such as Chloroform and Ethyl acetate to obtain polymethoxylated phenols (PMF, see below). New extraction technologies such as ultrasounds and microwaves may help to obtain better extraction yields.

In the following sections, the chemical structures and the biological effects of these compounds will be discussed.

#### **2. The chemistry of** *Citrus sinensis* **peel**

#### **2.1 Essential oils**

The essential oil (EO) is mainly obtained from the CS peel as a major by-product of the juice production process by a cold-pressing method that can provide the intact blend of compounds without losing the lighter, more volatile, components of the complex mixture that can be lost in the standard EO extraction procedure that is the hydrodistillation. The last one is mainly used in small scale applications, for example in research laboratories.

The chemical composition of CSP EO [13–15] is reported in **Table 1**. As it can be seen, the major component is D-Limonene, accompanied by several minor components belonging to the classes of monoterpene alkenes, oxygenated monoterpenes including alcohol aldehydes and esters, sesquiterpenes as well as linear alkanes and aldehydes. This rather complex blend accounts for the numerous deal of biological activities reported for the CSP EO [14–16], which include anthelmintic, antiaflatoxigenic [17], antibacterial [18–20], anticarcinogenic, antifungal [21], antioxidant [17], anti-tumor [22], anxiolytic [23], food preservative [24], hepatocarcinogenesis suppressant, insecticidal and larvicidal [25], pain relief and relaxant [26]. It can be argued that the main effects are due to the presence of the major component Limonene that showed several bioactivities when tested as pure compound [27]. However, it is possible that synergistic effects due to the combination of Limonene with other minor components may be speculated and should have to be demonstrated.


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

#### **Table 1.**

*Composition of* C. sinensis *essential oil obtained from peels.*

#### **2.2 Polyphenols**

#### *2.2.1 Flavanoids*

Polyphenols extracted from the CS peel belongs to the general structural categories of flavanones (**Figure 1a**), flavones (**Figure 1b**), flavonols (**Figure 1b**), with and without sugar moieties attached to one or more of the hydroxyl groups [28]. It is worthy of particular mention the rarely occurring class of C-glycolflavones (**Figure 1b**, compounds **63**–**65**, **85**, **86**).

These compounds are produced *in vivo* from the biogenetic mixed pathway of the Acetate and Shikimate that implies the enantiospecific formation of the basic aromatic bicyclic framework of the flavanone, from which a huge number of flavonoids originate employing selective enzymatic hydroxylations, methylations, and glycosylation steps. As can be seen from the structures shown in **Figure 1**, most of the chemical entities found in the peel extract contain several methoxy fragments that decorate the carbon skeleton. This characteristic makes those molecules to get a rather apolar character that explains their presence in the hydrophobic environment of the waxy peel. On the contrary, compounds containing a major number of hydroxyl groups are less present in the peel and are instead more significantly concentrated in the juice of the pericarp. However, some glycosylated compounds are present in the peel. In these molecules, the aglicone bears a monosaccharide unit (mainly glucose) or a disaccharide, in most of the cases being

Rutinose (**91**) – Rhamnosyl (α1 ! 6) glucose – or Neohesperidose (**92**)- Rhamnosyl (α1 ! 2) glucose (**Figure 2**).

The composition of the peel extracts described in the literature may slightly vary depending on the cultivar and the region of harvesting but some general points are



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


**Figure 1.** *Chemical structures of flavonoids from* C. sinensis *peels.*

common, that is the presence of the high amount of bioactive polymethoxyflavonoids [29, 30](PMF) some of which are rather ubiquitous, e.g. Nobiletin **53**, Sinensetin **51**, 3<sup>0</sup> ,4<sup>0</sup> ,3,5,6,7,8-Heptamethoxyflavone **55**; some other compounds

**Figure 2.**

*Chemical structures of the disaccharides most commonly bound to flavonoids of* C. sinensis *peel.*

containing one to six methoxy groups in place of the hydroxyl groups are present at variable amounts. The presence of one or more residual hydroxy groups in the molecule may result in a higher bioavailability and in other general differences in their mechanism of biological and therapeutic actions [30, 31].

The biological role of these secondary metabolites in the plant is still matter of debate. It has been proposed their involvement in the mechanism of defense of the fruits exposed to the attack of phytopathogens, such as *Phytophthora citrophthora* [32].

Further, the composition of the PMF blend can be employed for the chemiotaxonomic characterization of the *Citrus* genus [33].

However, it needs to be stressed that in many cases the reported compounds were recognized by mass spectrometry and electronic spectroscopy. It is not always a matter of simplicity to discern the exact structure of a given PMF and to discriminate between different regioisomers, normally quite similar in terms of mass and electronic spectra, if an isolation procedure is not conducted and followed by a complete bi-dimensional NMR characterization. Significant differences in the extract composition do arise also in consequence of the extraction method; nonpolar solvents such as Methanol, Chloroform Ethyl acetate let to obtain PMFs-rich extracts while, on the other hand, hydroalcoholic and aqueous extracts do contain a low concentration of PMFs and a higher concentration of un-methylated polyphenols as well as glycosylated compounds.

The biological activities disclosed for the flavonoids extracted from CSP are variegated. They include antioxidant [9, 34–39], anti-inflammatory [40, 41], antimicrobial [39, 42–44], antimalarial [45], antitrypanosomal [46], cardioprotective [47], anti-osteoporosis [48], anti-ulcer [49], vascular protective [50], anti-diabetes [51, 52], hepatoprotective [53, 54], neurotrophic [55], antiadipogenesis and anti-obesity [56–58], anti-hypertensive [59], cataract prevention [60], sun protection [61], metabolic syndrome control [62]. Further, it has been demonstrated [63] that while both flavonoid set **40**, **42**, **43** and the PMFs **51**–**53** were able to inhibit the anion transportin polypeptide OATP2B1 in HEK293 cells, only the PMF group displayed this inhibitory activity also for the OATP1B1 and OATP1B3 carriers.

The most abundant PMF occurring in CSP, Nobiletin **53**, was proven to possess sevral bioactivities, such as antioxidant, anti-inflammatory, cancer preventive [64] and also a significant protective effect *in vivo* against the endotoxic shock [65] and ethanol-induced acute gastric lesions [66] in mice. Further, compound **53** demonstrated the capacity to induce autophagy in human keratinocyte HaCaT cells [67], vasodilatator effect in the rat aorta [68] and to protect the intestinal barrier from the demages induced by dextran sulfate sodium [69].

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

PMFs can be considered as especially promising lead compounds for cancer therapy as asignificant cytotoxic activity has been demonstrated toward a number of cancer cells [70, 71] with several mechanisms of action [72, 73]; the cytotypes investigated include MCF-7 [73–76], Hs578T triple-negative breast cancer [73, 77]; colon cancer cells CaCo-2 [19], LoVo [78], HTC-116 [79, 80] and HT-29 [79, 81]; lung cancer cells A549 [80, 82], H460 [82, 83], H1299 [82, 83]; gastric cancer cell lines AGS, BGC-823, and SGC-7901 [84]; leukemia cells HL-60 [85]. However, data regarding a possible antitumor activity *in vivo* are still rather uncommon. An interesting example is the case of the significant reduction of the intestinal tumor mass in ApcMin/+ mice treated with a CSP extract containing various PMF [86]. Further, CSP extract and pure Naringin **47** were tested for their efficacy against a YM1 esophageal cancer in an animal model [87].

Given the development of pharmacological applications of CSP extract components, further investigations are needed to better understand the bioavailability, safety, and efficacy of these compounds in humans. Most of the data reported concern *in vitro* experimentations or animal model tests. For example, the toxicity of Hesperidin **40** was evaluated [88] in Sprague Dawley rats showing a 50% lethal dose (LD50) of about 5 g/Kg body weight (BW) and a lowest-observed-adverseeffect level (LOAEL) of ca. 1 g/Kg BW.

In general, it should be emphasized as the body of evidence concerning the actual efficacy of sweet orange-derived compounds in human health is still far to be exhaustive. For example, while this work is under typewriting, a severe acute respiratory syndrome pandemic due to a COVID-19 virus is in act and a big deal of research has been being directed toward antiviral remedies and therapies. Research on nutraceuticals is not an exception and in particular some authors have shown by computational and molecular docking methods how Hesperidin **40**, the most abundant polyphenol obtained from *C. sinensis*, would be able to bind the spyke protein of this virus thus inhibiting its activity [89]. Despite their undoubted interest, these results need to be further investigated with different experimental approaches.

The pharmacological potential of pure Hesperidin **40** was also investigated for several relevant human morbidity, such as cancer, hypertension, and ulcer [90].

#### *2.2.2 Hydroxy-acids*

Several hydroxylated carboxylic acids belonging to several structural sub-classes are present foremostly in the extract obtained with mixed hydro-organic solvents, such as MeOH/water and EtOH / water [37, 38, 51, 78]; these include the aliphatic Ascorbic, Citric, Kojic, Lactic, and L-Malic acids; the aromatic 4-Hydroxybenzoic, Protocatechulic, and Gallic acids. Further, the cinnamyl compounds (**Figure 3**) Cinnamic (**93**), p-Cumaric (**94**), Caffeic (**95**), Ferulic (**96**), Sinapinic (**97**) acids, and Artepillin (**98**) were identified in some CSP extracts that showed relevant biological activities, such as antioxidant [34, 37, 38] and antidiabetes [51].

These organic acids are mainly found in free form but in some cases, they are esterified with a variety of alcoholic compounds, such as Ethanol in Ethyl gallate **99** [51], 2-Phenylethanol in Phenylethyl ester of Caffeic acid **100** [51] and ()-Quinic acid in Chlorogenic acid **101** [51]. An interesting ester derivative (**102**) in which the anomeric hydroxyl of Glucose is esterified with a O-Caffeylsinapoyl acid unit was found in the methanolic extract of a Greek cultivar of *C. sinensis* [34].

It was shown [38] that the antioxidant properties of a CSP extract better correlated with the total phenols content (TPC) of the sample rather than with its total flavonoid content (TFC), as it can be expected from the known relevant antioxidant character of hydroxycynamic derivatives.

**Figure 3.** *Chemical structures of cinnamic acids extracted from* C. sinensis *peels.*

#### *2.2.3 Coumarins*

Coumarins are aromatic compounds biogenetically related to the o-hydroxysubstituted cynamic acids from which originate by the intramolecular condensation between the carboxylic and the o-hydroxy groups. These compounds are most commonly encountered in other species of *Citrus* taxa [91], such as *C. aurantium* (bitter orange), *C. limon*, (lemon), *C. limetta* (lime), *C. paradisi* (grapefruit) and only a few molecules of this class were Isolated from extracts of CSP endowed with activity against osteoporosis [48] and antioxidant [92]; these compounds are shown in **Figure 4**. As coumarins are relatively less common in *C. sinensis* cultivars compared to other species of the *Citrus* taxa, their rarity can be considered as a chemotaxonomic marker for *C. sinensis*.

#### *2.2.4 Catechins*

The NADPH dependent bioreduction of flavanols is the biogenetic origin of this class of compounds, present as minor constituents in CSP extract possessing significant antioxidant activity [38]; they are the two enantiomeric forms Catechin **113** and Epicatechin **114**, together with Epigallocatechin **115** (**Figure 5**).

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

**Figure 4.**

*Chemical structure of coumarins extracted from* C. sinensis *peels.*

#### **2.3 Pectins**

Pectins [93] are chemically definable as complex mixtures of polyglyconic acids in which a linear polymeric backbone is structured by a series of α (1 ! 4) linkages (**Figure 6**). The main sugar monomer is always Galacturonic acid with the presence

**Figure 5.** *Chemical structure of catechins from* C. sinensis *peels.*

**Figure 6.**

*Minimal representation of a Homopolygalacturonic acid domain of the linear primary pectin structure with a 1/3 Mol. /Mol. Esterification degree.*

of possible heterogeneous domains of other sugars such as Xylogalacturonan and Rhamnogalacturonan-I. A variable amount of the free carboxy functions may be esterified with methyl groups, while the hydroxy groups at C-2 and C-3 positions of the sugar monomers may be acetylated. Even though the primary structure of the main chain is linear, a possible degree of ramification, depending on the pectin source, may also be found. The differences in the pectins composition and structures, depending on their natural source, do confer them different physio-chemical properties, such as water solubility, sol–gel concentrations, etc. On the ground of the degree of methylation of the acid moieties, pectins are classified as "low methoxyl" (LMP, -COOMe/-COOH <50% mol.) or as "high methoxyl" (> 50% mol). A simplified representation of pectin structure is given in **Figure 6**.

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 3.1.1.6) 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: gianfranco.fontana@unipa.it

© 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.

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#### **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: minamisawa.mayumi@p.chibakoudai.jp

© 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.

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#### **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�<sup>1</sup> <sup>m</sup>�<sup>2</sup> Hz�<sup>1</sup> ]) 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**

In Vis–NIR spectra of fresh fruit, the common spectrum is often described by smooth mounds and soft depressions. This means that adjacent wavelengths can be highly correlated. Therefore, in order to reduce redundancy of information provided by neighbour features *xi* ½ � , *xi*þ1, *xi*þ2, … (also called co-linearity), sometimes it is beneficial to restrict the number of used input variables. This is often called dimensionality reduction and is very important for the right operation of certain calibration models. The simplest way to deal with this problem is by using subsampling, where a certain number of spectral features are discarded, e.g. every 3rd or 5th point in the spectra. To deal with this problem of dimensionality reduction, some 'clever' algorithms were introduced, the most common being the Principal Component Analysis (PCA) algorithm, the Hierarchical Clustering Analysis (HCA) and K-Means. the latest two methods can be used for classification tasks (mapping a cluster to a class) and for outlier detection as well. If samples are too far apart from the defined clusters (according to some metric such as the Euclidean distance or the Mahalanobis distance), then this suggests that it might be an outlier.

#### **3.3 Classification and regression models**

As we mentioned earlier, depending on the problem at hand, we might need to implement a classification or a regression model for our data. Multiple Linear Regression (MLR) is perhaps one of the most straight forward methods to implement. It expands the application of simple linear regression to the multivariate case by linearly combining them. Due to its simplicity, this method has some drawbacks, namely an inefficient applicability in the cases of high co-linearity in the data, and when the number of features in the data set is higher than the number of samples. This is often the case of fresh fruit Vis–NIRS datasets and hence its applicability has been limited. One way of overcoming these limitations, is to use a dimensionality reduction method, such as PCA and then perform MLR on these lower dimensional components. This workflow is known as Principal Component Regression (PCR). Partial Least Square Regression (PLS) is without question the most widely used method to create calibration models to predict the most QA of fresh fruit (**Tables 2**–**4**). As opposed to PCA, the PLS algorithm takes into account the covariance between input *xi* and target *Yi* variables. In the same spirit as PCA, PLS also projects the data into a latent space, but this time the components are defined along the direction of maximum variance between *xi* and *Yi*. These components are called latent variables (also named factors by some researchers), are built in order to model the target variable, and their number is what defines the quality of the PLS model. In general, a low number of latent variables usually lead to more robust predictions, but that might not always be the case. A variant of PLS named PLS Discriminant Analysis (PLS-DA) can be used to deal with classification scenarios when the target variables *Yi* are not continuous (e.g. 0, 1 for fruits without and with defects). The models mentioned so far are can be described as linear because they rely on a linear combination of multivariate solutions. Besides the easiness of implementation, they are also classically appreciated in Chemometrics because they are easy to interpret in terms of feature importance, i.e., after fitting the model to the data we can back-trace some parameters (e.g. regression coefficients) and find what wavelengths or spectral bands better contributed to the prediction. In turn, this allows inferring information about the chemical concentrations and can be used to identify biological and metabolic behaviors.

In the last couple of decades, non-linear models imported from other areas of Machine Learning have begun to permeate Chemometrics, and given its high use case in the literature, Support Vector Machines (SVM) is one of the most popular. The strategy of this model consists in searching for boundaries that separate two cluster or classes. The algorithm tries to find the best boundary between classes by maximizing a distance margin between neighbor samples. It has the advantage that it can use kernel tricks to transform the data points into another mathematical space, where these boundaries are easier to establish. SVMs were initially used for classification tasks, but have been extended to deal with regression problems as well (SVR). SVR has been used successfully for many datasets, and the most often mentioned drawback is the complexity of its optimization task. Another popular type of non-linear models that is often used for classification and regression problems is Neural Networks (NN). These represent a wide class of algorithms with many types of architectures and are derived from the field of Artificial Intelligence. In recent years, classical NN architectures such as the Multi-Layer Perceptron has


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


*b This value corresponds the accuracy of the classification.*

#### **Table 2.**

*Overview of applications of Vis-NIRS to measure the quality attributes of citrus fruit by benchtop devices. List of symbols in Table 5.*



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

*c*

*Cross validation. d Combinations of cal. and val. Sets.*

*e Calibration.*

*f Combinations of cal. and val. sets (harvest day).*

*g Combinations of cal. and val. sets (orchard location).*

*h Combinations of cal. and val. sets (season).*

#### **Table 3.**

*Overview of applications of Vis-NIRS to measure the QA of citrus fruit by portable spectrometers/systems under laboratory conditions. List of symbols in Table 5.*

been increasingly substituted by more modern architectures developed for Deep Learning. The most promising of these NN are the so called Convolutional Neural Networks that have been very successful in image recognition tasks.

#### **3.4 The quality of a calibration model**

Independent of the type of model that is used for prediction or classification, the important thing is to find how well it performs on the desired data. To assess the quality of the predictions made by the calibration models, several metrics are often used. In a recent review by [38], the author makes a case for the uniformization of the report of error metrics in future publications. In what follows these recommendations are highligted. The partitioning of the data for model development is very important. The data set should always be split into two sub-sets, called train and test sets. The train set, as its name suggests, is used for the calibration model development and, once the main hyper-parameters have been established, the model is used to predict the test set and assess its performance. Model development can be done with the full train set using a cross-validation strategy or by further splitting it into calibration and tuning (or assessment or validation) sets. As a note of caution, it is important to mention that for different areas of Machine Learning the names given to these data splits can vary and that can lead to some confusion. Otherwise, the test set should be derived from a different distribution from that of the train, in which case it is named as external validation set [45]. For example, data from two


#### **Table 4.**

*Overview of applications of Vis-NIRS to measure the quality attributes of citrus fruit by portable systems on-tree. List of symbols in Table 5.*

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


#### **Table 5.**

*List of abbreviatures used in Tables 2-4.*

consecutive harvest seasons are used as train set, while the test set uses data from a third season. A similar situation can be envisaged by using a train set collected from different orchards or origins, than that used for validation. Yet, given the large amount of time invested in acquiring this type of data, multi-seasonal or multiorchards test data sets are not often found in the literature. In contrast, laboratory models with homogeneous fruit sets are abundant (**Tables 2**–**4**). Currently, the most common procedure when constructing and validating Vis-NIRS models for the various fruit QA is to separate a fraction of the available samples as train/ calibration set (usually 80%), and the remaining as test/validation set (usually 20%). Furthermore, the validation samples are typically chosen as the best possible representation of the whole set and within the variation range of the train set. This has been applied even when the models comprehend several species and/or cultivars, orchard locations and harvest years, which are mixed in the calibration and validation sets [90]. All studies included in **Tables 2**–**4** that used this approach, were labeled as internal in the validation column. Internal validation does not ensure the success of a continuous monitoring application, which is a dynamic and open process, particularly, if one aims to use the Vis-NIRS in real world applications, being in inline grading systems or handheld devices. Once the model is applied to the test set and a final prediction is made, one can assess how well the model performs by computing several metrics. If the model was developed for regression, the most used are the root mean squared error (RMSE), bias, the slope,

the coefficient of determination (R<sup>2</sup> ), and the standard deviation ratio (SDR) or ratio of performance to deviation (RPD) or residual standard deviation (RSD). If the model is developed for classification, the advised metrics are accuracy (ACC), F1 score and receiver operating characteristic (ROC) curve. For completeness, these metrics are often computed not only for the test set, but also for the calibration, and tuning sets as well. The comparison between calibration (C), tuning (CV) and test set (P) error metrics allows to understand how well the model generalizes, i.e. how the information learned by the model during training transposes to the final external validation dataset.

#### **4. Prediction of quality attributes**

Vis-NIRS combined with various chemometric methods has produced calibration models to predict simultaneously multiple QA of various citrus species and varieties, which are presented in **Tables 2**–**4**. These attributes range from fruit size, weight and color [90] to SSC, MI, external and internal defects, several compounds such as sugars, acids, pigments and antioxidants. As expected, these models address predominantly several varieties of orange and mandarin, but also grapefruit, lime, pomelo, sweet lemon and tangelo. The spectral ranges used cover the whole Vis-NIRS range. The majority of the Vis-NIRS calibration models were obtained from samples collected and assessed under controlled conditions in the laboratory, after fruit temperature equilibration, either with benchtop or handheld devices (**Tables 2** and **3**). Despite the large market availability of the latter, presenting different levels of portability, spectral ranges, sizes, and prices, only a few studies have focused on its application to assess the quality and ripening of oranges and mandarins on-tree (**Table 4**), perhaps due to the complexities involved under field conditions, and the performance deterioration of calibration models, in spite of the spectral range used [86–90]. Nevertheless, the QA assessed on-tree (**Table 4**) comprise fruit mass and size, color parameters [86, 89, 90], pericarp thickness, SSC, TA, firmness, MI, juice pH and mass, and BrimA index, which measures the balance between sweetness and acidity as described by [12]. Noteworthy, the majority of the calibration models exhibited R<sup>2</sup> <0*:*8, despite the range used, and did not include external validation, except for [56, 88].

Grading lines equipped with Vis-NIR sensors are now commercially available from various companies, to assess both the EQA and IQA of citrus fruit [38–40]. Unfortunately, the scientific evidence about the accuracy of these systems is very scarce, due to the 'industrial secrecy'. Nevertheless, there are a few cases of partnership among the industrial sector and the research groups to know the real applicability and their performance in assessing citrus fruit QA by such equipment [91], in most cases still in the prototype stage, as reported by [92, 93]. Valero [91] evaluated the performance of a customized NIR equipment installed underneath the fruit conveyor to sort oranges and mandarins in a Spanish packinghouse. This system working in a transmittance mode in the 650–970 nm spectral range, only provided calibration models that could discriminate between low and high values of SSC for both mandarin (R = 0.76–0.86; SEP 0.9 °Brix; RPD 0.74) and orange (R = 0.87; SEP 0.7 °Brix; RPD < 1.5). No acceptable models were obtained for TA in neither species. Miller and Zude [92] evaluated the performance of a Vis-NIRS system to assess the SSC of 'Indian River' red grapefruit and 'Honey' tangerine from Florida in a sorting inline prototype, with R<sup>2</sup> ranging from 0.15 to 0.67. The prototype percent correct classification averaged 85% for SSC at 10 °Brix and 79% for an 11 °Brix setpoint in the second-year of tests. Otherwise, [93] reported on the development and laboratory testing of the nondestructive citrus fruit quality monitoring

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

prototype system, which consisted of a light detection and ranging (LIDAR) and Vis-NIRS sensors installed on an inclined conveyor for mimicking real-time fruit size and SSC measurement respectively, during harvest. Laboratory tests in 'Valencia' orange revealed that the system was applicable for instantaneous fruit size (R2 = 0.91) and SSC (R<sup>2</sup> = 0.677, SEP = 0.48 °Brix) determination.

The various Vis-NIRS calibration models presented in this chapter, show different levels of accuracy, prediction and robustness for the various attributes, SSC being the most successfully IQA assessed at all spectral ranges, independently of the devices used (**Tables 2**–**4**). Both juice pH and vitamin C also seem easily assessed by devices operating in the Vis-SWNIRS range devices [78, 94, 95], but TA has been shown to require wavelengths range > 1000 nm [13, 96]. Additionally, calibration models for firmness have been difficult to obtain, although there are a few exceptions reported for several orange varieties, in the reflectance mode and in the ranges 500–1690 nm [89], and 1000–2500 nm [73]. Of course, the calibration models for specific compounds, such as sugars, acids or antioxidants, require in most cases the longer NIR spectral range [12, 54, 69].

Both external and internal defects in citrus have been successfully predicted by Vis-NIRS, such is the case of the section drying in tangerine (transmittance; 780 and 960 nm) [10], oleocellosis in 'Trovita' orange (reflectance; 400–1000 nm) [71], the freeze damage in sweet lemon (half-transmittance; 400–110 nm) [13], the rind pitting in 'Marsh' grapefruit (reflectance; 400–2500) [12] or the granulation in 'Shantian' pomelo (transmittance; 400–700) [74]. However, [97] was not able to predict the external disorder RBD in 'Nules Clementine' mandarin (interactance; range 450–1000 nm).

Among the chemometrics methods used to construct the calibration models, PLS is still the main linear regression technique used, and the one to produce the best models for the widest number of QA (**Tables 2**–**4**). However, there are some exceptions, regarding the use of non-linear techniques, which were shown to deliver calibration models with equivalent or even better prediction and accuracy for several QA than PLS. Among these, there is the WT-LSSVR, BP-NN and LS-SMV that provided models with higher prediction capacity for SS in 'Gannan' orange [81], SSC, TA and vitamin C in Nanfeng mandarin [78] and in 'Newhall' orange [96], respectively. The LOCAL algorithm has also shown to produce better models than MPLS for firmness and juice mass in 'Powell Summer Navel' orange [89], and in 'Clemenvilla' mandarin [90]. SIMCA, SVM and particularly PCA-ANN, also allowed to assess with a total accuracy >98% the freeze damage in sweet lemon [13] and PCA-GRNN allowed the assessment of the granulation in 'Honey' pomelo at a classification accuracy (CA) >95% [14].

Independently of the chemometrics technique used, for the majority of the models presented in **Tables 2**–**4**, the train and test fruit sets were chosen from the same batch, orchards or seasons. Even when the whole data set comprised fruit from several orchards, harvest season or citrus varieties, the usual approach was to randomly choose 80% of the whole data set to construct the calibration model and 20% to validate it, as reported by [90, 97]. A truly stringent external validation is thus required to have a realistic idea of the models' performance in orchard and/or cargo batch monitoring. External validation means validation through a dataset with a different origin (spatial or temporal) relatively to the datasets used in calibration. Nevertheless, there are some clear examples of this approach, such as previously reported for mandarin [76, 79, 97–99], orange [12, 56, 70, 72, 73, 79, 88, 97], and grapefruit [12, 70]. Without the effective external validation, it is not possible to know exactly how well these models would work in real conditions due to the large variability within the trees, orchards, sites and harvest seasons. Yet, a certain degree of deterioration of the initial model prediction is expected, which

would warrant further attention. This has been reported by [56, 77, 88]. Yet, there is space and potential for improvement beyond the 'proof of concept', if one aims to use these devices on the daily routines of the orchards' management. This has been suggested by [56, 78] through model recalibration using a few fruits from the new harvest season/orchard, or by achieving a strong degree of robustness by constructing a multi-seasonal and multi-orchard model as reported by [89] for 'Newhall' orange, which will be much more advantageous when assessing the ripening of fruit on-tree.

#### **5. Future research and perspectives**

Vis-NIRS has been incorporated by a large number of companies in commercial applications to be used on inline, benchtop and handheld systems. However, there are several topics regarding the full potential and limitations of this technology that require attention and further research in order to provide the consistency warranted by the daily basis routines of the citrus supply chain when assessing fruit quality and ripening. Firstly, all researchers engaged in this area should report their results in a uniform way, particularly in what respects the obtained models' metrics [38]. This would allow a better understanding on the effective advances and contributions made by each study. Other models' metrics parameters, such as the prediction gain, may also be useful, as reported by [100]. Secondly, the calibration models' robustness must be addressed and solved through a stringent multi-year, multicultivar and multi-orchard validation, such as previously reported by [56]. The usual approach of validating calibration models with a random fraction of the total available data set, even when the models comprehend several varieties, orchard sites and harvest years does not ensure the success of a continuous monitoring application and delivers unrealistic performance metrics [76, 77]. The usual recalibration and spiking approaches used to improve the initial calibration models with a few fruits from independent data sets that will be then assessed, assume that those fruits used to recalibrate/update the model constitute a faithful representation of the new population and are common techniques in various commercial devices, for inline and benchtop systems. However, this becomes quite difficult to apply if one aims to monitor the on-tree fruit ripening evolution through time, for the fruit sampled in the first weeks cannot represent those to be measured in the last weeks of the harvest season. Thirdly, there is a large potential for models' improvement, by using the non-linear techniques of machine learning, and those of deep learning. Fourthly, there is much to understand on the effect of the rind in the assessment of the pulp IQA in citrus fruit, since the NIR radiation hardly gets to the fruit pulp, and both biochemical and optical properties have a major role to play in the spectral data acquired [47, 49, 95, 101, 102]. Fifthly, the calibration models should be able to predict attributes that are closer to the organoleptic evaluation of the fruit. It is the case of BrimA index, a better indicator of fruit sweetness that the SSC, which was satisfactorily predicted in orange, grapefruit and mandarin [12, 87]. Finally, the handheld devices must really be tested under field conditions, if one aims to assess the fruit on-tree, which is essential for the OHD decision.

#### **6. Conclusions**

The usefulness of Vis-NIRS combined with different chemometric techniques in the supply chain of citrus fruit is already quite extensive and growing, similarly to many other commodities. In this chapter the authors only addressed the classic

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

spectral 'point' measurements, but it is quite clear that both inline, benchtop and handheld devices are used to assess nondestructively multiple QA in various citrus species and cultivars, with a clear predominance of orange and mandarin. Among these attributes, there are both EQA and IQA, as well as defects caused by various factors, such as physiological disorders. The devices available on the market are from various brands, operate in various ranges and present a wide variety of prices. Aside from the "proof of concept" made by many studies, that the authors tried to comprise as much as possible in this chapter, there are several issues that still need to be addressed by researchers, a major one being the need for a stringent external validation of the calibration models, in order to assure robustness and to fulfill with the essential requirements to include this technology in the daily routines of these crop supply chain. This is of the utmost importance when considering the assessment of fruit ripening on-tree to determine the optimal harvest date for each orchard, or sections of the orchard. This is highly significant based on the determinant effect of producing and harvesting the fruit at its best ripening stage, thus assuring the best quality throughout the whole postharvest and shelf-life. As a concluding remark, it is very important to add that these devices are of medium and high cost, and that are not the kind of technology to 'set and forget', as reiterated by [39], which demands not only for a budget to acquire the systems, but also to maintain them, and to keep the continuous update and improvement of the calibration models, that in most cases need the selling company assistance. Thus, there must be a cost–benefit that both the producers and packinghouses have to meet through the added commercial value to citrus fruit by these systems, and the consumer willingness to pay for fresh fruit graded in terms of IQA such as sugar content, acidity and nutraceutical properties.

#### **Acknowledgements**

The authors acknowledge FCT - Fundação para a Ciência e a Tecnologia, Portugal, for funding CEOT project UIDB/00631/2020 CEOT BASE and UIDP/ 00631/2020 CEOT PROGRAMATICO, and MED- Project UIDB/05183/2020. Dário Passos was funded by project OtiCalFrut (ALG-01-0247-FEDER-033652). Rosa Pires was funded through a BI fellowship from project NIBAP (ALG-01-0247- FEDER-037303).

#### **Conflict of interest**

There are no conflicts of interest.

#### **Author details**

Ana M. Cavaco<sup>1</sup> \*†, Dário Passos1†, Rosa M. Pires1,3, Maria D. Antunes2† and Rui Guerra1†

1 CEOT—Center for Electronics, Optoelectronics and Telecommunications, University of Algarve, Faro, Portugal

2 MED—Mediterranean Institute for Agriculture, Environment and Development, University of Algarve, Faro, Portugal

3 Siglas - Formação, Consultoria and Investigação, Lda., Faro, Faro, Portugal

\*Address all correspondence to: acavaco@ualg.pt

† These authors contributed equally.

© 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.

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

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#### **Chapter 14**

Electrochemical Applications for the Antioxidant Sensing in Food Samples Such as Citrus and Its Derivatives, Soft Drinks, Supplementary Food and Nutrients

*Ersin Demir, Hülya Silah and Nida Aydogdu*

#### **Abstract**

Although there are many definitions of antioxidants, the most general description; antioxidants are carried a phenolic function in their structure and prevent the formation of free radicals or intercept from damage to the cell by scavenging existing radicals. Moreover, they are one of the most effective substances that contain essential nutrients for healthy individuals. The importance of these antioxidants, which have an incredible effect on the body and increase the body's resistance, is increasing day by day for healthy individuals. Numerous studies have been carried out for antioxidants with excellent properties and however new, reliable, selective, sensitive and green analytical methods are sought for their determination at trace levels in food samples. Along with the latest developments, electrochemical methods are of great interest in the world of science because they are fast, reliable, sensitive and environmentally friendly. Electrochemical methods have been frequently applied to analyze antioxidant capacity in many nutrients samples found in different forms such as solid, liquid without any pretreatment applications in the last decade. Furthermore, these methods are preferred because of the short analysis time, the ability to lower detection limits, reduction in a solvent, high sensitivity, portability, low sample consumption, wide working range, and more economical than existing other traditional analytical methods. The antioxidant sensing applications by modern electrochemical methods such as cyclic, square wave, differential pulse, and combined with stripping voltammetric techniques were used to deduce antioxidant capacity (AC) in critical nutrients. Moreover, this chapter includes a description of the classification of electrochemical methods according to the working electrode type, dynamic working range, limit of determination (LOD), limit of quantification (LOQ), sample type, and using standard analyte and so forth for each voltammetric methods. While many articles applied for the determination of antioxidant sensing by electrochemistry have gained momentum in the last two decades, we focused on the studies conducted over the last 4 years in this chapter.

**Keywords:** antioxidant determination, electrochemistry, voltammetric methods, potentiometry, amperometry

#### **1. Introduction**

Free radicals occur when an atom or molecule contains one or more unpaired electrons in its outermost orbitals [1]. Basically, three main factors play a role in the formation of free radicals. i) The atoms or molecules can become radical as a result of the fragmentation of covalently bonded molecules exposed to high-energy electromagnetic waves or high temperatures. ii) A molecule that does not have a radical feature experience an electron loss and radicals are formed by leaving unpaired electrons in its outer orbital. iii) A radical is formed when a molecule that does not have a radical property receives an electron from outside and has an unpaired electron in its outer orbital [1, 2]. These unshared electrons as known radicals are highly unstable, transforming them into high-energy and very efficient chemical species. The most active free radicals in biological systems are those based on oxygen and are commonly referred to as reactive oxygen species (ROS) with pathological [3]. This family group includes superoxide radical (O2˙), singlet oxygen, nitroxide (NO), hydroxyl radical (OH˙), and hydrogen peroxide (H2O2) which is not itself radical but causes the formation of radical [1]. Besides, we can classify the causes of free radicals in two groups as endogenous or exogenous [1, 2]. Cigarettes, air pollution, alcohol, radiation, heavy organic solvents and pesticides are among exogenous sources, while enzymes, proteins, oxidative stressors, and heavy metals are endogenous sources [1, 4].

Free radicals cause the greatest damage to human health on basic cellular components such as lipids, proteins and nucleic acids [1, 5]. Therefore, these radicals lead to immune deficiency, hypertension and even important diseases such as cancer, neurodegenerative diseases, heart disease, and atherosclerosis [1, 2]. Also, studies are revealing that radicals disturb the homeostatic balance [6]. To scavenge these drawbacks effects of radicals, which are extremely important for human health, the human body needs antioxidants obtained from the body or nutrition to fulfill biological activities such as survival and healthy life. Antioxidants can be defined as molecules that usually contain phenolic functional groups in their structure and prevent the formation of free radicals that damage the cell or by scavenging existing radicals [3]. The functional task of antioxidants is that they act as shields in the body and neutralize them by donating their electrons with the s-free radicals. Thus, radicals found in a rather unstable structure do not become a threat to human health by transforming into a more stable structure reacted with antioxidants. Moreover, many different equivalent antioxidant expressions are used in antioxidant quantification in food samples. The leading ones are the expressions of "total antioxidant capacity (TAC)", "antioxidant activity (AA)", and "antioxidant capacity (AC)". The total amount of antioxidants is expressed by measurement units such as equivalent trolox, rutin, ascorbic acid, and quercetin, etc.

Antioxidants are mainly obtained via natural and synthetic [7]. The first of these, natural antioxidants, are molecules synthesized by the organism or obtained from food sources. Natural antioxidants produced by the organism are the most important source for human health. Many factors affect the production process of this natural antioxidant. The most important of these is the age of the person. As a person gets older, the amount of natural antioxidants produced by his organism decreases day by day. For this reason, there is a greater need for the natural antioxidants found in foods for older people. The importance of healthy food sources, especially organic-based foods, is increasing day by day. Also, such nutrients should be accessible to all segments of society.

Important dietary flavonoid sources are fruits especially citrus fruits such as oranges, apples, grapes, mandarins, berries lemons, limes and their derived products as well as juices [8]. In general, citrus fruits contain pectin, sugar, carotenoid

pigments, vitamins (A, B1, and C), and; organic acids such as ascorbic acid and citric acid, minerals and a number of active phytochemicals such as flavonoids and coumarins, as naringenin, naringin, hesperidin, neohesperidin, hesperetin, rutin, narirutin and tangeretin [9]. For example; polyphenol antioxidants such as flavanols (epicatechin, catechin), phenolic acids (caffeic acid and gallic acid), anthocyanins (e.g., malvidin-3-glucoside), oligomeric and polymeric proanthocyanidins, flavonols (myricetin, quercetin, and their glycosides), and many others polyphenols exist in wine, especially in red wine [10]. Flavonoids have an important role in scavenging reactive oxygen species, which can counteract lipid oxidation, decrease peroxide formation in vivo, and improve activity of the body's antioxidant enzyme. Citrus flavonoids such as naringin, naringenin, and hesperidin have antioxidant activity [11]. Naringenin is a flavonoid, particularly a flavanone, found in citrus fruits especially oranges and grape fruits and in vegetable's such as tomatoes and their preparations. The pharmacological and biological properties of phytoestrogen naringenin and its derivatives include, anticancer, anti-inflammatory, antiulcer, antifibrotic, diastolic, antioxidant and skin protective effects [8]. Also, citrus species are a rich source of flavanone glycosides such as hesperidin and narirutin, which have anticancer, antioxidant, antiobesity and anti-inflammatory activities [12].

Secondly, the antioxidant group is synthetic, that is a molecule that is obtained as a result of chemical reactions and is generally used as food preservatives [13]. Synthetic antioxidants such as butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), and tertiary butylhydroxyquinone (TBHQ) also extend the shelf life of foods [14]. However, natural antioxidants that can be taken from foods are less risky in terms of human health since synthetic antioxidants can have toxicity even if they are very little, they require high costs and have less capacity than natural antioxidants. Due to this reason, the investigations of foods types that can contain high levels of antioxidants in different types of endemic, organic and traditional food samples have been remarkably increased recently.

For antioxidant content and amount analyzes, oxygen radical absorbance capacity (ORAC) and radical-arrest antioxidant parameter (TRAP), ferric thiocyanate (FTC), Trolox equivalent antioxidant capacity (ABTS/TEAC), cupric ion (Cu2+) reduction antioxidant capacity (CUPRAC), iron ion reducing antioxidant capacity (FRAP), DPPH radical scavenging activity determination and Folin–Ciocalteu methods are the most widely preferred as analytical methods [15–17]. Furthermore, to evaluate and characterize the antioxidant substances in food samples, various analytical methods such as high-pressure liquid chromatography (HPLC) combined with different detection, gas chromatography, micellar electrokinetic capillary chromatography, capillary electrophoresis includes different detection systems and UV–visible spectrophotometry have been used [18–20]. However, these classical methods have great shortcomings for fully validated analyzes such as long pretreatment, need for too much solvent, expensive equipment, long analysis time. They do not provide the necessary procedures for green chemistry, especially due to the use of too much solvent and too much waste in antioxidant analyses. For these reasons, scientists have turned to alternative methods for antioxidant quantification in food samples. Especially in recent years, they have focused on electrochemical techniques which are fast, inexpensive, reliable, non-pre-treatment, and environmentally friendly in the analysis of drugs, pesticides, metal ions and organic molecules such as antioxidants, vitamins and nucleic acid [21–23].

In this chapter, the applicability, sensitivity and reliable maintenance of electrochemical methods, which have attracted great attention in food and food samples, have been examined for the analysis of antioxidants. Moreover, which types of electrochemical methods are used and what advantages they provide have been

investigated for the antioxidant sensing in food samples. It also describes the classification of each used in electrochemical methods by working electrode type, dynamic operating range, the limit of detection (LOD), measurement limit (LOQ), sample type, and standard analyte, etc. While many articles referenced for determining antioxidants by electrochemistry have gained momentum in the literature in the last two decades, we focused our study on the studies conducted in the last 4 years.

#### **2. Electrochemistry**

Electrochemistry is the branch of science which is investigating the physical and chemical changes coming from the interaction of the material with electrical factors such as current, potential, and electron charge. Electroanalytical chemistry is based on measuring the electrical properties of solutions containing analytes and switching to quantification using measured electrical signals a collection of electrochemical methods. Moreover, electroanalytical measurement methods are based on two basic points: potentiometric (static methods) and potentiostatic (dynamic methods). Electrode systems in both methods are immersed in the solution containing the analyte, called the electrochemical cell. Potentiostatic methods are widely used for routine analysis because they are less costly, high sensitive, and selective and have wider potential application areas than other electroanalytical methods. The basic principle of these methods is to measure the current that occurs during the oxidation or reduction of the analyte in the chemical reaction.

Electrochemical methods began with the Czech chemist Jaroslav Heyrovsky, discovering the basis of polarography in 1922 and took an important place among the analytical methods. Especially, since the 1980s, it has been possible to develop electrodes that have been modified mechanically or chemically with improved technology. In modification processes, polymers, organic ligands, inorganic clays, phthalocyanines and nanoparticles have been commonly used for the detection of electroactive substances in very small volume complex samples such as biological, environmental and human bodies. In the last twenty years, even very small quantities of substances that are electroactive have been additionally analyzed at high precision, selective by electrochemical methods by carbon-based or modified electrodes have wonderful properties. Electroanalytical methods have also an important place in quantification as well as in obtaining details such as determination, adsorption, reaction rate and equilibrium constants of the number of electrons transferred in the reduction or oxidation electrode reactions. In short, electroanalytical methods provide details on direct or indirect quantitative and qualitative analysis of electroactive species such as antioxidants, drugs, pesticides, etc.

#### **2.1 Voltammetric application for the determination of antioxidant capacity**

Voltammetry is a potentiostatic assay based on the recording of the peak current at controlled potential variation by the oxidation or reduction which enables qualitative and quantitative analysis by means in electrochemical reactions. Over the last two decades compared to other electroanalytical techniques, voltammetry has been intensely curious in all the electroanalytical methods due to their are used to analyze numerous compounds by anodic or cathodic scanning and to investigate their conceptual basis of electro-mechanism. There are four voltammetric techniques including cyclic (CV), linear (LSV), differential (DPV), and square (SWV) are commonly used to determination of antioxidant-type compounds.

Voltammetric techniques are an alternative analytical method, proved to have an excellent correlation compare with another conventional analytical process, for a while to study the AC in various food and beverage samples. They can be a benefit to characterize which species compounds have a greater contribution to the antioxidant capacity present for the real samples in terms of quantitative and qualitative by controlled the half-wave peak potential, peak current and the electron transfer number in reaction. The antioxidant capacity is related to the peak currents of oxidation species caused by hydroxyl groups (–OH) and antioxidant species contains many hydroxyl groups. They commonly give an electro-oxidation broad peak at a range of 400 mV- 600 mV depend on pH. So that, almost all antioxidant substances have electro-activity compounds and their peak current and peak potential provide quantitative and qualitative details, respectively. Further, the voltammetric techniques allow investigating the electrochemical behavior of antioxidant agents and interaction with oxygenated species.

Voltammetric methods have gained an important place among determinations of the antioxidant capacity in the last decade. Moreover, due to their great superiority, the use of complex samples such as food and beverages they have become widespread and widely found in the literature. Among these electroanalytical methods, square wave stripping, different pulse stripping, and cyclic voltammetric techniques are the most commonly preferred for the analysis of antioxidants by accuracy and precision. From past to these days, the compounds used as standard agents for the evolution of the AC by studies electrochemical methods are apigenin, ascorbic acid, caffeine, catechin, chlorogenic chrysin, p-coumarin acid, eugenol, fisetin, gallic acid, kaempferol, luteolin, morin, quercetin, rutin, t-resveratrol, Trolox and Malvidin-3-glucoside. As far as we have examined the literature, scientists have however preferred ascorbic acid, caffeic acid, gallic acid, catechin, rutin and quercetin which are often used as antioxidant standard substances due to excessive availability of these substances in food and drink. The chemical structures of some antioxidant molecules are given in **Figure 1**.

**Figure 1.** *Molecular formulas of commonly used antioxidants.*

#### *2.1.1 Cyclic voltammetric technique*

Cyclic voltammetry (CV) is usually the first experiment in the electrochemical operation of a compound in biological materials as nature samples to get in details about the electro-behaviors. In particular, to study the thermodynamics, kinetic, electron transfer, substance transfer type, and as well as quantitative determinations of oxidation or reduction processes can be carried out by cyclic voltammetric technique. In addition to taking a single measurement with CV, sequential multiple measurements can be taken. The most common applications of cyclic voltammetry are additionally electro-polymerization, electrochemical characterization, and the design of modified electroanalytical systems. Two types of cyclic voltammograms can be obtained as irreversible or reversible, depending on the chemical components of the target molecules. In reversible voltammetry, there is a difference of about 59 mV between the reduction and oxidation peak potentials (**Figure 2**).

During the past years, cyclic voltammetry has been used as an alternative to existing methods to evaluate the antioxidant sensing in natural samples such as teas, biological fluids, beverage juices plants, foods and beverage juices on different working electrodes. The most using parameter is peak current because of its proportional to the concentration of the antioxidants. Peak current heights also provide quantitative information about the amount of antioxidant capacity in food samples. The carbonbased working electrodes such as glassy carbon electrode (GCE), carbon paste electrode (CPE), screen printed carbon electrode (SPCE), and modified electrodes (Nanoparticle/GCE, Nanoparticle/CPE, Fe3O4/GCE) have been widely preferred in electrochemical measurements for the analysis of total antioxidant capacity (TAC). Peak current and peak potential values of standard substances such as ascorbic acid, caffeic acid, catechin, coumarin, gallic acid, morin, quercetin and rutin were commonly taken care of for the evaluation of TAC. The amount of antioxidants in food samples is generally given as equivalent gallic acid, equivalent value quercetin, etc.

Even though the CV method raises doubts about sensitivity, it also has great advantages. Quick, simple, low detection limit, cheaper and easier application are

**Figure 2.** *Potential-excitation signal and voltammograms for the cyclic voltammetry in details.*

summarized as great advantages. Interferences effect on antioxidant capacity by a nonantioxidant agent to reducing TAC and non-selective to a family of molecules between carotenoids and polyphenols unless the electrode is modified are drawbacks properties. Despite all of these disadvantages, CV attracts a great deal of attention among analytical methods, and a large number of studies deal with CV are also being undertaken. A large part of the work done up to day time to determine the antioxidant capacity by the CV method is summarized in **Table 1**. **Table 1** includes the type of working electrode, working range, the limit of determination (LOD), the limit of quantification (LOQ), measurement parameter, standard compound and food sample.

#### *2.1.2 Square wave voltammetric technique*

Square wave voltammetry (SWV) can be used to perform a faster experiment than other voltammetric techniques. Commonly when the scanning speeds of other techniques are of 1–10 mV/second or more, in the square wave voltammetry a scanning speed is used at 1 V/second. Thus, the target molecule can be analyzed more quickly by SWS. The square wave voltammetry can combine with the stripping technique. Thus, a stripping voltammetric technique was developed to determine electroactive substances at high sensitive enables in ultra-trace concentration levels. Especially, ultra-trace target substances in complex samples can be analyzed by combining the technique with the enrichment stripping process. The working principle of the stripping technique is the same as square wave voltammetry and only two new parameters are more applied as the accumulation time and the accumulation potential (**Figure 3**).

Nowadays SWV and square wave stripping voltammetry (SWSV) are frequently applied to deduce compounds such as drugs, heavy metals, pesticides and antioxidants, etc. in numerous specimen types because they have excellent analytical sensitivity and selectivity. Furthermore, SWV and its derivate combined technique can be applied for simultaneous determination of compounds which are close oxidation or reduction peak potentials like paracetamol, ascorbic acid, uric acid and dopamine. In the last decade, SWV and SWSV have been more effective in determining antioxidant substances in the complex matrix samples and are superior compared with analytical methods especially spectrophotometric to evaluate quantification and qualification. It is one of the most important electroanalytical methods for the determination of antioxidants since it is a wide working range, low detection limits, easy to apply, cheap and non-pretreatment. Furthermore, they have been successfully analyzed the phenols in food samples which is called a type of important antioxidant such as o-phenylenediamine, p-chlorophenol, paminophenol hydroquinone, pyrocatechol and phenol, etc. At the same time, various antioxidant substances such as gallate, gallic acid, quercetin and caffeine were easily studied in food or beverage samples at high precision, accuracy and selective on the carbon-based electrode. Besides, at nM concentration of antioxidant substances comparable to chromatographic techniques have been determined by modified electrodes which are increasing conductivity accurately and selectively in tea samples. Evaluation of antioxidant capacity by SWV or SWSV techniques in the last 4 years are summarized in **Table 2** according to the type of working electrode, working range, the limit of detection (LOD), quantity limit (LOQ), measurement parameter, standard composition and food sample.

#### *2.1.3 Differential pulse voltammetric technique*

Differential pulse voltammetric technique (DPV) is one of the most widely used for the analysis of both organic and inorganic species. Pulse voltammetry





**Figure 3.**

*Potential-excitation signal and voltammogram for the square wave stripping voltammetry in details.*

techniques were proposed by Baker and Jenkin in 1952 as a more sensitive measurement electroanalytical method. Differential pulse voltammetry techniques can be used to determine up to 10<sup>8</sup> M concentration of the target agents. The peak current (Ip) is a function of the concentration for the electroactive species and is linear as I*p* = *f* (C). Also, it is possible to analyze substances not only quantitative analysis but also qualitative analysis with pulse technique. The peak currents are related to the concentration of the substance whereas the peak potential values are related to the selectivity. Thus, simultaneous determinations of the substances have been studied by DPV on bare or modified electrodes (**Figure 4**).

Nowadays, quite a lot of DPV studies can be found in the literature for the very sensitive detection of heavy metal, drug, pesticide, antioxidant agent and inorganic/ organic species on numerous bare and modified working electrodes. Besides, DPV is one of the most important candidates to determine the trace amount of target agents in analytical methods due to its high sensitivity and selective. Also, it can be applied to complex samples as biological and food samples such as blood and serum, beverages. Especially, DPV has an important place among antioxidant determination methods because of these advantages and the availability of low concentration.

In recent years, DPV has been used frequently in determining the total antioxidant capacity without any pretreatment of solid and liquid food samples. The complex matrix such as biological and food samples contain very dense different types of substances. For this reason, despite it is indeed very difficult to selectively and precisely determine the antioxidant capacity in some complex matrixes; DPV is the most applicable method for such species. There are also plenty of studies were published which deal with chlorogenic acid, caffeic acid, p-coumaric acid, quercetin, gallic acid and ferulic acid, etc. as illustrating the antioxidant properties were determined by DPV on bare or modified electrodes based on carbon nanomaterials. Several applications, based commonly on the used as a determination of antioxidant capacity are given in **Table 3**.

In amperometric techniques, the current produced during the reduction or oxidation of an electroactive species at a constant potential value that is applied between a working electrode and reference electrode is measured, in this way providing specific quantitative electroanalytical knowledge for the target analyte. Especially, amperometric, which is based on electrical current analysis, is commonly utilized in microchip electrophoresis applications owing to its high sensitivity, it also lets for the determination of electroanalytical active species without derivatization, accomplishing adjustable versatility and selectivity (**Table 4**).

Ganesh et al., synthesized zinc oxide nanoparticles using mechanochemical synthesis technique. New ZnO nanoparticle as hexagonal prism was investigated by scanning electron microscopy, X-ray diffraction, particle size distribution, ultraviolet– visible spectroscopy, and energy-dispersive X-ray spectroscopic methods. Electrochemical properties of the newly prepared electrode were characterized by using an amperometric method and cyclic voltammetry technique. The prepared electrode has




**Table 2.** *Evaluation of antioxidant capacity by SWV or SWSV.*

**Figure 4.**

*Potential-excitation signal and voltammogram for the differential pulse stripping voltammetry in details.*

a wide working linear range between 0.1–130 μM with a detection limit of 0.02 μM. Obtained results showed that the prepared electrode has numerous active surface sites, good electronic activity, and surface area. They applied the proposed electrode to the determination of gallic acid in samples as wine successfully [40].

Kumar and coworkers successfully synthesized NiO nanoparticles from natural fruit using an efficient, simple, and low-cost technique. The obtained NiO nanoparticles were investigated with various methods such as FTIR, XRD, TEM, SEM, UV, and PL. XRD studies showed that NiO nanoparticles have cubic geometry. The band of Ni-O bond was shown at 430 cm<sup>1</sup> . Photocatalytic properties of the obtained NiO nanoparticles were applied to photodegrade the methylene blue dye. They used the prepared electrode to the determination of dopamine with the LOD of 11 μM [93].

Koçak et al. prepared a new composite electrode using carbon nanotube and polyl-methionine onto the glassy carbon electrode. Electrochemical properties and surface structure of the prepared electrode were studied using electrochemical impedance spectroscopy and scanning electron microscopy. Electrochemical properties of gallic acid with the proposed electrode were investigated in various techniques such as differential pulse voltammetry, cyclic voltammetry and amperometry. The obtained results of electrochemical studies exhibited that the prepared electrode shows a suitable method of determination for gallic acid in pH 2.2 BR buffer solution. The prepared sensor has a wide working linear range with two linear segments between 4 nM-1.1 μM and 1.7–20.0 μM with LOD of 3.1 nM. They used the prepared new sensor for the detection of gallic acid in various samples as black tea, green tea and wine samples. The experimental results showed that the proposed sensor exhibit high selectivity, reproducibility, stability and catalytic effect [88].

Potentiometry is an electrochemical technique based on measuring the potential difference between two electrodes called working and reference electrodes. The working basis of the potentiometry technique is the potential difference based on the concentration of an analyte in the sample solution relative to a reference electrode (**Table 5**).

Brainina and coworkers developed a new, simple, reliable and fast potentiometric method for the determination of plant total antioxidant activity. Plant micro suspension and extracts were analyzed by the proposed method. The experimental conditions for acquiring plant extracts were selected for the highest antioxidant activity as extraction time 20 min at +80°C. The characterization of plant micro suspensions reduces the duration of plant total antioxidant activity evaluation. Comparison of the obtained results of antioxidant activity of green tea and black tea micro suspensions samples with the results of the investigations of extracts prepared by a certified method showed no difference [95] (**Tables 6** and **7**).


#### *Citrus - Research, Development and Biotechnology*



#### *Citrus - Research, Development and Biotechnology*






*Evaluation of antioxidant capacity by DPV or DPSV.*

**Table4.**

 *technique.*



*Evaluation of antioxidant capacity by potentiometric technique.*


> **Table 7.** *Evaluation*

 *of antioxidant*

 *capacity by other techniques.*

#### **3. Conclusion**

Electrochemistry is a powerful and versatile analytical technique for the determination of numerous substances such as drugs, pesticides, inorganic, antioxidanttype compounds and electroactive compounds by rapidly possible applications in a lot of fields. Electroanalytical methods besides providing details on quantitative and qualitative of analyte that offer validation parameters such as sensitivity, accuracy and precision, selective and linear working range. Moreover, it is superior to determine the target analyte by electroanalytical methods lack of interferences effect especially in a complex matrix such as biological and food samples contain countless substances. The improvement of simultaneous determination of analytes considerably has been carried out to be applied in biological and environmental systems by the sensitive and selective electrochemistry methods. Because of this, the use of many areas of electrochemistry is widespread.

Nowadays, electrochemical methods, especially voltammetry from medicine to the determination of antioxidants, have made an important place especially in the world of science. Not only analytical chemists but also biology, food engineering and all people who are engaged in food have been used electrochemical methods to determine the antioxidant capacity in plants, tea, beverages, carbonated beverages and solid food samples, etc. Compounds such as ascorbic acid, caffeic, catechin, ascorbic acid, quercetin, gallic acid and coumarin have been widely used as reference standard agents to an evaluation of antioxidant capacity by electrochemical methods have been carried out until today. Due to advances in electronics and computer science have provided significant benefits in terms of electrochemical instrumentation such as accuracy, sensitivity and easy application, the electro-analysis of antioxidant compounds is successfully applied by stripping voltammetric techniques at nM concentration level. The purpose of this review is to show that electroanalytical methods for commonly used antioxidant types may be the best analytical method for the quantitative and qualitative analyte and that they can successfully compete with more conventional methods especially spectrometric methods. Consequently, voltammetric techniques supply that even at low concentrations, the antioxidant capacities of food samples can be determined to be very fast, simple, non-pretreatment and highly sensitive compared to conventional analytical methods. The review presented that the antioxidant capacity of various food samples can be carried out by voltammetric techniques in the estimation in real samples.

#### **Author details**

Ersin Demir<sup>1</sup> \*, Hülya Silah<sup>2</sup> and Nida Aydogdu1

1 Department of Analytical Chemistry, Faculty of Pharmacy, Afyonkarahisar Health Sciences University, Afyonkarahisar, Turkey

2 Department of Chemistry, Faculty of Art and Science, Bilecik Şeyh Edebali University, Bilecik, Turkey

\*Address all correspondence to: dr.ersindemir@yahoo.com

© 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.

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## *Edited by Muhammad Sarwar Khan and Iqrar Ahmad Khan*

Citrus is an extensively produced fruit crop and is cultivated predominantly in tropical and subtropical regions of the world. The *Citrus* genus consists of a variable number of species due to the admixture of wide morphological diversity, intra- and interspecific sexual compatibility, apomixis, and spontaneous mutations. Citrus fruits are highly nutritious and beneficial for health due to the presence of bioactive compounds that have antioxidant, antitumor, anti-inflammatory, and blood clot-inhibiting characteristics. This book describes the citrus plant and its nutrients, nutritional value, and nutraceutical applications, as well as related biotic and abiotic challenges in its cultivation. Chapters cover such topics as citrus genealogy, production, and crop management; milestones achieved in citrus improvement; importance of weather conditions in cropping systems; effects of changing climate on citrus; and much more.

Published in London, UK © 2021 IntechOpen © scaliger / iStock

Citrus - Research, Development and Biotechnology

Citrus

Research, Development and Biotechnology

*Edited by Muhammad Sarwar Khan* 

*and Iqrar Ahmad Khan*