*4.5. Physico-Chemical Composition Analysis*

Protein, fat, ash and moisture contents were determined using AACC International Approved Methods 46-12.01, 30-25.01, 08-01.01 and 44-15.02, respectively. The pH was measured with a HQ30d portable pH Meter (HACK, Germany) on a slurry prepared with 10 g legume flours in 40 mL of boiled, deionized water according to official AOAC procedures (AOAC, 02-52.01). The total acidity value was determined according to Romanian standard method SR 90:2007. Carbohydrate contents of the legume samples were calculated according to the following formulas [92]: carbohydrates, % = 100 − (protein, % + fat, % + ash, % + moisture, % content of legumes).

The Na, Mg, Fe and Zn contents of the vegetable samples were analyzed by flame atomic absorption spectrometry (FAAS) (AA-6300 Shimadzu, Kyoto, Japan) equipped with air-acetylene flame. Hollow cathode lamps of Na, Mg, Fe and Zn were used. In total, 10 g with an accuracy of 10 mg from each sample was used for calcination. The calcination temperature was increased with a maximum speed of 50 ◦C/h up to 450 ◦C. The calcination time was 8 h. Ash digestion was performed using 10 mL 0.1 mol/L nitric acid (HNO3) (Sigma-Aldrich/Merck, Darmstadt, Germany) on a hot plate. After digestion of the ash samples, up to 50 mL was filled with bidistilled and deionized water. Standard solutions of Na, Mg, Fe and Zn (Sigma-Aldrich/Merck, Darmstadt, Germany) were used and diluted as necessary to obtain working standards. In order to eliminate the risk of contamination, all glassware was washed after each use with HNO<sup>3</sup> solution and rinsed with bidistilled and deionized water. The instrumental conditions for determining the mineral content of vegetable samples by FAAS method are shown in Table 3.


**Table 3.** Instrumental conditions for mineral analysis to flame atomic absorption spectrometry (FAAS).

The number of injections of the solutions in the flame was: standard solution—5; blank solution—3; sample solution—3. In order to eliminate analytical errors, each sample was analyzed three times.

#### *4.6. FT-IR Analysis*

In order to highlight the changes in the composition of grains subjected to germination process, FT-IR analysis was also used. This allows the realization of a correlation between the spectra and the physicochemical data obtained previously. To obtain the spectra, a FT-IR spectrometer (Thermo Scientific, Karlsruhe, Dieselstraße, Germany) was used, equipped with the ATR IX option, which allowed the obtention of accurate data, using a detector at 4 cm−<sup>1</sup> . Therefore, an attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR) was used to obtain the spectra. This technique is based on tracking the interaction between infrared radiation and the material under analysis. Fourier transform infrared spectroscopy (FT-IR) contains a source of light with infrared radiation which passes through the sample and this absorbs luminous energy. At the same time, occur vibrational movements due to the chemical bonds inside the molecules. These vibrational movements provide information about the chemical structure of the sample and these are provided as an FT-IR spectrum. FT-IR spectra were recorded in the spectral range situated between 800 and 4000 cm−<sup>1</sup> . This spectral range was suitable for characterizing the chemical compounds present in the seed samples: proteins, carbohydrates, lipids, etc.
