**2.3 Biochemical analysis**

When the total soluble solids (TSS) reached about 12–14%, five cluster samples were taken from each of the three replicates of treatments on July 1, 2019. After removing from the clusters, stored berries at −20°C before the phytochemical analysis were analyzed in the Department of Genetic and Bio-Engineering, Faculty of Engineering, University of Yeditepe.

#### *2.3.1 Mineral elements*

Macro and micronutrient element analyses were carried out using samples of berries. Phosphorus (P) was determined vanadomolibdo phosphoric acid yellow color method as reported by Bremner [50]. Potassium (K), calcium (Ca), magnesium (Mg), iron (Fe), zinc (Zn), copper (Cu), and manganese (Mn) concentrations of the berries were analyzed by atomic absorption spectrophotometer [51].

#### *2.3.2 Amino acids*

1 g fresh sample was treated with 0.1 N HCl, homogenized with ultra turrax, and incubated at 4°C for 12 hours. Supernatants were filtered through 0.22-m filters after *The Effects of Different Substrates with Chemical and Organic Fertilizer Applications… DOI: http://dx.doi.org/10.5772/intechopen.102345*


#### **Table 2.**

*The amount of nutrients given per plant by different nutrient solutions in 2 years.*

samples were centrifuged at 1200 rpm for 50 minutes (Millex Millipore). The supernatants were then transferred to a vial, and the amino acids were analyzed in HPLC as described by Antoine et al. [52] and Kitir et al. [53]. Readings from Zorbax Eclipse-AAA 4.6150 mm and 3.5 m columns (Agilent 1200 HPLC) were taken at 254 nm, and the amino acids were identified by comparing them to standards of O-phthaldialdehyde (OPA), fluorenylmethyl-chloroformate (FMOC), and 0.4 N borate. The following solutions were used in the mobile phase chromatography system: Phase A: 40 mM NaH2PO4 (pH: 7.8) and Phase B: acetonitrile/methanol/water (45/45/10 v/v/v), after a 26-minute derivation process in HPLC, aspartate, glutamate, asparagine, serine, glutamine, histidine, glycine, arginine, alanine, tyrosine, cysteine, valine, methionine, tryptophan, phenylalanine, isoleucine, leucine, lysine, thionine, and proline.

A 50 mg frozen berry sample was crushed using liquid nitrogen and extracted with 4.5 mL of 3-sulfosalicylic acid, and then filtered through a Whatman filter paper (#2) for proline measurement. In a test tube, 2 mL of the filtrate were mixed with 2 mL acid-ninhydrin and 2 mL glacial acetic acid for 1 hour at 100°C, stopped the reaction with an ice bath, and the filtrates were analyzed. The concentration of proline was measured spectrophotometrically at 520 nm [54].
