**2. Materials and analytical methods**

#### **2.1. Selection of plant material and fertilization experiment**

The experiment was carried out in 2012 under the controlled conditions of the central research greenhouse at Biotechnical Faculty (46°04′N, 14°31′W; 320 m a.s.l.). The commercial radicchio varieties were included in our research: red ('Treviso', 'Verona', and 'Anivip'), red-spotted ('Castelfranco'), and green ('Monivip'). Photos of individual radicchio variety are shown in **Figures 1** to **3**.

**Figure 1.** Var. 'Anivip' (left) and var. 'Monivip' (right). Photo: D. Žnidarčič.

Lipids are derived from long-chain fatty acids and alcohols or closely related derivatives. They are water-insoluble components of cells that can be extracted by nonpolar solvents. In various parts of the plants, mostly in the cell membranes, are small amounts of lipids (~2%). In higher plants, the predominant fatty acid residues consist of palmitic, oleic, linoleic, and stearic acid.

Fatty acids with <12 and >20 carbon atoms are less common in nature [28]. The most common fatty acids in plants are those containing 16 or 18 carbon atoms. These include saturated palmitic (C16:0) and stearic (C18:0) acids, monounsaturated oleic acid (C18:1n9), polyunsatu‐ rated linoleic acid with two double bonds (C18:2n6), and linolenic acid with three double bonds (C18:3n3) [29]. When the carbon atoms in the hydrocarbon chain of a fatty acid hold their full complement of hydrogen, they are described as saturated. Where two adjoining carbon atoms in the hydrocarbon chain of a fatty acid each lack a hydrogen atom, a double bond forms between them. The fatty acid is then said to be unsaturated. The term polyunsaturated fatty acid (PUFA) is accepted as referring to those fatty acids that contain two or more carbon-carbon double bonds within the hydrocarbon chain [30]. Particular PUFAs, which the human system can employ as building blocks while being unable to synthesize them, have been classed as essential fatty acids. The *n*-3 (ω-3, omega-3) PUFAs found in plants refer to a number of health benefits [31]. The most common and most important PUFA is linolenic acid, which is known as a precursor of the long-chain fatty acids (eicosapentaenoic and docosahexaenoic) [32]. Modern agriculture and food industrialization are associated with large changes in the structure of contemporary Western diets. The intake of *n*-6 fatty acids has enlarged during evolution, and the intake of *n*-3 fatty acids has been reduced. Consequently, the *n*-6/*n*-3 ratio increased from 1 to 10 or, in some places, even up to 20 or even 25. These differences in food

Over the past decade, radicchio has become popular for cultivation and consumption in different regions of the world. Scientific literature has revealed that radicchio plants contain important compounds with biological activity and several vitamins and minerals [4,18,34–36]. The effects of fertilizer managements (organic, mineral) on the phenolic and fatty acid profiles in different radicchio varieties (red, red-spotted, green) are poorly discussed in scientific data. This chapter discusses the effect of fertilizers (organic, mineral, and combination) on the total phenolics, the main phenolic classes, and the fatty acids levels of five *C. intybus* varieties. Highperformance liquid chromatography (HPLC) was used for the analysis of phenolic compounds and their classes and gas chromatography (GC) was used for the determination of fatty acid

The experiment was carried out in 2012 under the controlled conditions of the central research greenhouse at Biotechnical Faculty (46°04′N, 14°31′W; 320 m a.s.l.). The commercial radicchio varieties were included in our research: red ('Treviso', 'Verona', and 'Anivip'), red-spotted

consumption led to increased risk of numerous modern diseases [33].

**2. Materials and analytical methods**

312 Organic Fertilizers - From Basic Concepts to Applied Outcomes

**2.1. Selection of plant material and fertilization experiment**

levels.

**Figure 2.** Var. 'Treviso' (left) and var. 'Castelfranco' (right). Photo: D. Žnidarčič.

**Figure 3.** Var. 'Verona.' Photo: D. Žnidarčič.


**Table 2.** Fertilizer managements used to set up the pot experiment.

The growing experiment in controlled conditions included two mineral fertilizers, two organic fertilizers, a combination of one organic and one mineral fertilizer, and the control (no added fertilizer). In each of the five radicchio varieties, the same six fertilizer managements were applied as presented in **Table 2** in the following design: unfertilized control (CONT), two organic fertilizers (ORG1 and ORG2), two mineral fertilizers (MIN1 and MIN2), and combi‐ nation of organic and mineral fertilizer (ORG1+MIN1). The experiment consisted of 30 plastic pots filled up with 7 L of soil with application of the selected fertilizers. Sowing was performed on 30 January 2012. Then, the pots were placed in the greenhouse and irrigated appropriately. Water-soluble mineral fertilizer (MIN1) was applied through the irrigation solution containing water and MIN1. The sampling of developed leaves was performed on 10 June 2012. A few leaves from each pot were lyophilized and powdered using a ball mill before analysis. The dry matter content of radicchio leaves varied from 6.8% to 14.8%.

### **2.2. Extraction and identification of phenolic compounds**

**Fertilizer treatment Fertilizer name N/P/K Application details Mark** Unfertilized / / Watering CONT Single basal organic Plantella Organik 3/3/2 67.5 g/7 L soil ORG1 Single basal organic Stallatico Pallettato 3/3/3 45 g/7 L soil ORG2 Water soluble mineral Kristalon Blue 19/6/20 Irrigation with 9 g/100 L MIN1 Single basal mineral Entec perfect 14/7/17 7.9 g/7 L soil MIN2

> 3/3/2 + 19/6/20

The growing experiment in controlled conditions included two mineral fertilizers, two organic fertilizers, a combination of one organic and one mineral fertilizer, and the control (no added fertilizer). In each of the five radicchio varieties, the same six fertilizer managements were applied as presented in **Table 2** in the following design: unfertilized control (CONT), two organic fertilizers (ORG1 and ORG2), two mineral fertilizers (MIN1 and MIN2), and combi‐ nation of organic and mineral fertilizer (ORG1+MIN1). The experiment consisted of 30 plastic pots filled up with 7 L of soil with application of the selected fertilizers. Sowing was performed

Plantella Organic 3.5 g /7 L soil + after 1 month irrigation with 3.5 g/L Kristalon Blue

ORG1+MIN1

Combination of organic and mineral fertilizer

**Figure 3.** Var. 'Verona.' Photo: D. Žnidarčič.

314 Organic Fertilizers - From Basic Concepts to Applied Outcomes

Plantella Organik + Kristalon Blue

**Table 2.** Fertilizer managements used to set up the pot experiment.

Radicchio powder was mixed with the solvent 5% formic acid in methanol, which contained flavone as an internal standard. For extraction, an ultrasonic bath at 4°C for 30 min was used. After centrifugation, a 10 μL aliquot of supernatant was injected into the HPLC system. For analysis, reverse-phase HPLC coupled with a diode array detector (DAD) was used. The phenolic compounds were separated on Nucleosil C18 analytical column (250 cm × 4 mm; 3 μm) and eluted using 5% formic acid and HPLC-grade methanol at a constant flow rate. The gradient profile has been flowing to the protocol previously published for the analysis of complex polyphenol mixtures [37].

The DAD was scanning from 250 to 600 nm with four discrete channels. Phenolics were gathered into five classes and monitored at related wavelengths: unknown phenolic com‐ pounds (UPCs; 280 nm), HCAs and flavones (320 nm), flavonols (350 nm), and anthocyanins (540 nm). The quantification of each phenolic compound was carried out using the internal standard manner. The phenolic compounds in the radicchio leaves separated by HPLC are presented in **Table 3**. They were classified based on the absorbance spectra [38] and the comparison to representatives [39]. Chlorogenic and caftaric acids were confirmed by previ‐ ously identified standards [40].



**Table 3.** Phenolic compounds in the radicchio leaves separated by HPLC.

### **2.3. Determination of fatty acid levels**

Fatty acid levels were analyzed using GC with prior prepared fatty acid methyl esters. In the protocol [41], NaOH and BF3 in methanol were used for transesterification and heptadecanoic acid (C17:0) was used as an internal standard for the quantification of fatty acids. The solution of fatty acid methyl esters was quantified on the GC (Agilent 6890N, USA) with flame ionization detector (FID). At the constant flow rate, the separation was performed on a column for analyses of PUFAs as fatty acid methyl esters. The identification and quantification of fatty acids were carried out using a reference standard mixture of methyl esters of greater fatty acids regularly before the samples. The following fatty acids were detected in the radicchio plants: C16:0, C18:0, C18:1n9, C18:2n6, C18:3n3, and C20:0 (**Table 4**).
