CONVENTIONAL EXTRACTION OF BETALAIN COMPOUNDS FROM BEETROOT PEELS WITH AQUEOUS ETHANOL SOLVENT

Consumer concern over artifi cial food additives has stimulated production of pigments from natural sources. The aim of this study was to examine the eff ects of process variables on the content of colour compounds (betaxanthin and betacyanin) in beetroot peel juice extracted by conventional method. In this research, the extraction processes were carried out according to the central composite design with diff erent process variables. Quantitative measurements of the basic colour compounds in beetroot extract were performed using spectrophotometer. From our experiment, it was found that the most adequate extraction conditions, which gave the highest yield of colour compounds (952.5 mg l–1 of betaxanthin and 1361 mg l–1 of betacyanin), were extraction time 1 h, operating temperature 20 oC, and solvent ratio 0.8 w/v. Being a conventional heating method, it is a simple and cost effi cient process with relatively high yield.

Food safety remains a signifi cant issue especially in developing countries, since the application of food additives continues to increase. However, it is impossible to avoid the application of food colours in food industry, since colour is an important sensory attribute. Natural colorants came into consideration as soon as consumers showed more interest in organic pigments. Nevertheless, their high level of sensitivity to heat, pH, and light, high cost, and low stability are drawbacks for their use.
Common plant pigments, such as betalains, carotenoids, anthocyanins, and carminic acids, are commonly used as natural colourants in medicinal and food applications (S & C , 2004). Among them, betalain, a water soluble compound, is prominent for its biological properties such as antioxidant, anticancer, antiradical, and antimicrobial activities (G et al., 2010). This kind of compound is present in 37 species of 8 genera in the Amaranthaceae family, which is one of the 17 families of the order Caryophyllales (to date) (S et al., 2017; C et al., 2005b) and is noticeably found in the beetroot crops, which represent an important source of bioavailable compounds. Nowadays, red beetroots are receiving signifi cant attention from consumers due to their prominent antioxidant activity and high oxygen radical absorbance capacity (G et al., 2010). Betalain is composed of a nitrogenous core structure, betalamic acid, which combines with amino acid and derives red violet betacyanin (betanin 75-95%) and yellow betaxanthin (vulgaxanthin I ~ 95%) (C et al., 2005a). Owing to their hydrophilic nature, betalain compounds can be extracted with pure water, aqueous methanol, or ethanol. However, the extraction method and choice of solvents aff ect noticeably the content of individual ZIN et al.: EXTRACTION OF BETALAIN COMPOUNDS FROM BEETROOT PEELS Acta Alimentaria 49, 2020 compounds in the extract. In addition, extracted beetroots are susceptible to pH, water activity, temperature, light, oxygen, metal ions, and enzymatic activities (R et al., 2013).
Extraction of valuable compounds has recently been realised by ultrasonic extraction method (S et al., 2009), microwave assisted extraction method (S et al., 2017), and pulse electric fi eld extraction method (L et al., 2009). Undoubtedly, these methods have high effi ciency; however, some of them are rather costly compared to traditional extraction methods.
Many observations have been made for the extraction of betalain compounds and phytochemicals from the whole tuber of beetroot. Moreover, there are some accessible investigations of bioactive antioxidants and colour-giving compounds, which are present in the waste part of beetroot such as peel (S et al., 2017), pomace (K et al., 2018), and stalk (M & P , 2016). Nevertheless, there is still need to do further investigations of important colour compounds extraction from waste parts of beetroots. The aim of this study was to determine the eff ects of process variables (solvent ratio, temperature, and extraction time) on the quantities of betalain compounds in beetroot peel extracts.

Sample acquisition and preparation
Beetroots (Beta vulgaris L., Rhonda type, harvested in 2019) were collected from local supermarket and transported to the laboratory for subsequent analysis. The purchased raw beetroots were cleaned gently with sponge to remove dust and debris. The cleaned beetroots were peeled with a sharp knife about 1 mm thick manually. The peels were then blended in a mixer for 2 min to obtain a pulp.

Analytical procedures
The pulp (50 g) was mixed with (15%) aqueous ethanol in diff erent peel-to-solvent ratios (0.2, 0.6, 1.0 w/v). The extraction processes were carried out with a thermostat water bath maintaining temperature at 20 ºC, 35 ºC, 50 ºC and three diff erent operation times (1 h, 3 h, 5 h) with stirring at 215 r.p.m. After centrifuging the extracted juices for 25 minutes at 6000 r.p.m., the coloured supernatant was collected and kept under refrigeration prior to instrumental analysis.

Spectrophotometry analysis
Spectrophotometric analysis for quantifi cation of two basic colour compounds was done by using DR/2400 UV-visible spectrophotometer. The absorbance values were recorded at 480 nm for betaxanthin compound (K et al., 2018) and 535 nm for betacyanin compound (S et al., 2017). Appropriate dilutions were done for each type of measurements with distilled water. And the concentrations of the respective betalain compounds were calculated according to the following equation as determined by R and co-workers (2013): where A is the absorbance; MW is the molecular weight; DF is the dilution factor; Ԑ is the molar extinction coeffi cient, and L is path length. For quantifi cation of betaxanthin and betacyanin, molar extinction coeffi cients in water and molecular weights were applied as Ԑ=48 000 l mol -1 cm -1 and MW=308 g mol -1 , Ԑ=60 000 l mol -1 cm -1 , and MW=550 g mol -1 , respectively.

Statistical evaluations
Empirical design of experiment: Response Surface Methodology was used as a statistical tool to optimize the multivariable process. RSM is one of the statistical tools that are mainly used for the modelling and investigation of multivariable systems in which a response of interest is infl uenced by several variables (S et al., 2017).

Spectrophotometric analysis
The signifi cance test of model fi t was performed by central composite design of Design Expert software version 11.0.3, based on analysis of variance (ANOVA). The cubic model gave false results, whereas the quadratic model was statistically highly signifi cant and so was applied for the next analysis. Based on the small P-value for the quadratic terms, we decided to fi t the second-order model to the yield response. It can be deduced that the models are signifi cant as the F value is high and the Prob>F value is less than 0.0001 (Table 1). Lack of fi t is non-signifi cant, meaning that the model is signifi cant for three variables, whereas the high coeffi cient of determination (R 2 ) account for 0.8758 and 0.9181 for the total variation in betaxanthin and betacyanin extraction, respectively. It was also revealed from the data that the solid-to-liquid ratio was the only signifi cant parameter aff ecting these three variables at linear and quadratic terms. The values of adjusted R 2 and predicted R 2 diff er by less than 0.2, meaning that these two values are in reasonable agreement with one another. The coeffi cients of variation, which measure the residual variability in the data, are expressed as percentage. Twenty treatments (runs) were conducted according to a central composite experimental design ( Table 2). As the results of the diff erent variables, the yield of colour compounds vary from 122.3 mg l -1 to 952.5 mg l -1 for betaxanthin and 148.5 mg l -1 to 1361 mg l -1 for betacyanin, respectively. Even though the eff ects of extraction temperature and time on the colour compound yield were non-signifi cant, lower extraction temperature and time were preferred to give better yield. The possible explanation may be the sensitivity of natural colour compounds to heat. Many phytochemicals are destroyed by high operation time and temperature as reported by H and co-workers (2006); A -J and co-workers (2013); R and co-workers (2013). Figure 1A and B shows the interaction eff ects of temperature and time on colour compounds when solvent ratio is maintained at constant level. As it can be seen in Figure 1A and B, the decrease in time and temperature leads to increase in yield. This correlation can be explained by the fi ndings of H and coworkers (2006), who investigated the impact of heating period on colour compounds.
The signifi cance of the quadratic term for the peel-to-solvent ratio indicates that this variable has impact on the extraction effi ciency. According to Figure 1A, the highest yield of betaxanthin cannot be seen within the investigated time (1-5 h) and temperature (20-50 ºC) ranges, when the solvent ratio is fi xed at centre point. It means that the yield mainly depends on the solvent ratio, which is in agreement with the fi ndings of D A and co-workers (2009). On the contrary, in the case of betacyanin, the highest yield can be seen with the fi xed solvent ratio in Figure 1B. It might be because of the diff erent properties of specifi c colour compounds. The interaction eff ects of temperature and solvent ratio are depicted in Figure 1C and D, when time was fi xed as constant. It was found that the yield increased when solvent ratio increased, with highest yield between 0.6 w/v and 1.0 w/v solvent ratio with low temperature around 20 ºC for betaxanthin compound. In case of betacyanin, the highest yield was obtained around 0.8 w/v of solvent ratio. Conversely, there was a minimal decrease in yield beyond that solvent ratio range for all colour compounds. The possible explanation is that the maximum extraction limit of colour compounds was reached, and further increment in solvent ratio became unnecessary for any driving force to occur with mass transfer. The same situation was observed when the temperature was fi xed at 35 ºC, providing the highest yield after 60 min as shown in Figure 1E and F. The operating conditions were optimised to achieve the highest yields of basic colour compounds: betaxanthin and betacyanin with a desirability of 0.951 ( Figure 2). The regression equations for obtaining betalain concentrations at any given parameters were given as Sqrt (Betaxanthin) (mg l -1 ) = +5.00-0.44A-0.06B+65.99C-42.51C 2 Sqrt (Betacyanin) (mg l -1 ) = +9.50-0.73A-0.10B+84.52C-55.46C 2

Conclusions
Central composite design used in this study suggested that the quadratic model with optimum conditions of extraction time 1 h, operating temperature 20 ºC, and solvent ratio 0.8 w/v gave a yield of 839.115 mg l -1 betaxanthin and 1512.14 mg l -1 betacyanin. Since we only used traditional extraction, our study encourages the use of this reasonably effi cient method with cost effi ciency. * The project is supported by the European Union and co-fi nanced by the European Social Fund (grant agreement no. EFOP-3.6.3-VEKOP-16-2017-00005) and the Tempus Public Foundation under the Stipendium Hungaricum Scholarship Program.