*2.7. Total Carotenoid Content*

The total carotenoid content (TC) in the powders and the concentrate was determined spectrophotometrically after an extraction step in sunflower oil. Sample dilutions which had already been prepared (see previous section) in concentrated sugar solution, were consequently mixed with a known volume of sunflower oil (Cargill, Amsterdam, The Netherlands) in a laboratory blender for 90 s. To accelerate phase separation, the viscous mixture was transferred to centrifugal vials and centrifuged for 20 min (rcf = 2800 g, Eppendorf, Wesseling, Germany). After centrifugation the absorbance spectrum of the oil phase was recorded from 380 to 700 nm (Figure 2) and the absorbance at λmax (458 nm) multiplied by the dilution was used to calculate the TC.

Initially, two methods were applied to quantify carotenoids in samples in the range of 0.2–2.2 mg/g powder. The first method was the extraction of the carotenoids from the sugar mixture and measurement in oil. The second method was carried out based upon the extraction procedure described by Sadler et al. [11], with some modifications. Briefly, 1–3 g of the sample was dissolved in 100 mL of distilled water prior to extraction. A total of 10 mL of extraction solvent (Acetone:Ethanol:Hexane, 1:1:2, 0,1% (*w*/*v*) BHT) were added to 2 mL of sample dilution and shaken for 1 min. After phase separation, the hexane phase was washed with 3 mL distilled water and transferred to a 25 mL volumetric flask. The extraction step was repeated at least two times by adding 5 mL of hexane. The hexane phases were combined in the volumetric flask and the volumetric flask was topped off with hexane to the mark and shaken before measuring the absorbance of the hexane phase in the spectrophotometer. Equation (1) was used to calculate the total carotenoid content.

$$\text{TC} \left[ \frac{\text{mg}}{\text{g}} \right] = \frac{\text{A} \times \text{V} \times 10^3}{\frac{1\%}{1 \text{ cm}} \text{E} \times 2 \text{ mL} \times \text{m}} \tag{1}$$

where A = absorbance at λmax, V = the total volume of the extract (mL), 1% 1 cmE = the average extinction coefficient of carotenoids in hexane 2500 [12] and m = the sample weight in g. The results from two methods correlated linearly (*R*<sup>2</sup> = 0.996) as shown in the Appendix A (Figure A1). Therefore, due to its non-toxic and non-volatile properties, oil was selected as a suitable extraction solvent. For calculations, the linear regression was used to calculate the TC in powders from absorbance values obtained from the carotenoid measurement in oil.
