Title : Optimization of ultrasound assisted extraction of chlorophylls from spinach by products using response surface methodology and stabilization of the extracted pigments by encapsulation
Chlorophylls are tetrapyrrole derivatives with coplanar system of conjugated double bonds which form an aromatic structure with delocalized electron density at the orbitals level. Typically found in higher plants are chlorophyll-a and chlorophyll-b, in various proportions depending on the maturity and species of the vegetal material and also on the light exposure, stress etc. Being widely available in nature, the natural pigment can represent a solution of environmentally friendly textile dyeing option. However, its easily degradable nature stands in the way of utilizing chlorophyll at its maximum potential. The aim of this study was to develop a mathematical model for the optimization of chlorophyll extraction from fresh spinach leaves using ultrasound-assisted extraction (UAE) and ethanol 96% as extractive solvent. The UAE equipment comprises in a Hielscher UP200H Ultrasonic Processor, a jacketed reactor, heating plate equipped with a temperature control and magnetic stirring units. In order to decrease the number of experiments and, consequently, the volume of used solvent, the developed method was optimized with the use of Design of Experiments approach. The optimization of the extraction method was carried out by employing the Box-Behnken response surface methodology (RSM) and its application with selected input variables such as solid/solvent ratio, ultrasound (US) power and stirring rate to the determination of the optimal conditions to maximize the concentration of chlorophyll extraction. The JMP software (Cary, N.C., USA) was used to design the experiment.
The extracts were spectrophotometrically analysed in triplicate and the concentrations of chlorophyll-a and chlorophyll- b were calculated using molar extinction coefficients.
The answers evaluated in this experimental set were the concentrations of chlorophyll-a and chlorophyll-b. The levels of the evaluated factors were determined. Optimization results showed good agreement between predicted and experimental values of chlorophyll-a and chlorophyll-b yields with a high coefficient of determination (R2 = 0.93 and R2 = 0.93, respectively). The optimum solvent/solid ratio, US power and stirring rate, which lead to achieving maximum concentrations of chlorophyll-a and chlorophyll-b (148.8 mg/L and 67.7 mg/L, respectively) were 4.3:1, 120 W, and 1000 rot/min, respectively. Due to the light and heat sensitive nature of the extract, it was further stabilized. The encapsulation strategy involved the silanization of the chlorophyll extract by reaction with 3- aminopropyltriethoxysilane (APTES), followed by a hydrolysis step in the presence of tetraethyl orthosilicate (TEOS) and cetyltrimethylammonium bromide (CTAB) (as stabilizing agent) for the generation of silica. The degradation studies showed an improved stability to storage conditions of the encapsulated chlorophyll compared with the pure compound.
Acknowledgement: This paper was supported by a University Politehnica of Bucharest grant “Inginer in Europa” in online system registered at ME under no. 140/GP/19.04.2021, by using the special situation financing fund which cannot be integrated in the form of financing for higher state education institutions.
This work was supported by a grant of the Ministry of Research, Innovation and Digitization, CNCS/CCCDI– UEFISCDI, project number PN-III-P1-1.1-TE-2019-1387 (contract number TE141/2020), within PNCDI III.
- Advantages of the Design of Experiments studies which imply fewer experimental runs, a better process understanding in a shorter time period and overall, lessened research and development cycles.
- A method of chlorophyll stabilization through encapsulation, which enhances the possible uses of this natural pigment.
- To reconsider the utilization of some natural products in various domains of interest.