Efficient Extraction of the RuBisCO Enzyme from Spinach Leaves Using Aqueous Solutions of Biocompatible Ionic Liquids

Ribulose-1,5-biphosphate carboxylase/oxygenase (RuBisCO) is the most abundant protein on the planet, being present in plants, algae and various species of bacteria, with application in the pharmaceutical, chemical, cosmetic and food industries. However, current extraction methods of RuBisCO do not allow high yields of extraction. Therefore, the development of an efficient and selective RuBisCOs’ extraction method is required. In this work, aqueous solutions of biocompatible ionic liquids (ILs), i.e., ILs derived from choline and analogues of glycine-betaine, were applied in the RuBisCO’s extraction from spinach leaves. Three commercial imidazolium-based ILs were also investigated for comparison purposes. To optimize RuBisCO’s extraction conditions, response surface methodology was applied. Under optimum extraction conditions, extraction yields of 10.92 and 10.57 mg of RuBisCO/g of biomass were obtained with the ILs cholinium acetate ([Ch][Ac]) and cholinium chloride ([Ch]Cl), respectively. Circular dichroism (CD) spectroscopy results show that the secondary structure of RuBisCO is better preserved in the IL solutions when compared to the commonly used extraction solvent. The obtained results indicate that cholinium-based ILs are a promising and viable alternative for the extraction of RuBisCO from vegetable biomass.

Chlorophylls Extraction from Spinach Leaves Using Aqueous Solutions of Surface-Active Ionic Liquids

Chlorophylls and their derivatives have been extensively studied due to their unique and valuable properties, including their anti-mutagenic and anti-carcinogenic features. Nevertheless, high-purity-level chlorophylls extracted from natural sources are quite expensive because the methods used for their extraction have low selectivity and result in low yields. This study aimed to develop a “greener” and cost-effective technology for the extraction of chlorophylls from biomass using aqueous solutions of ionic liquids (ILs). Several aqueous solutions of ILs, with hydrotropic and surface-active effects were evaluated, demonstrating that aqueous solutions of surface-active ILs are enhanced solvents for the extraction of chlorophylls from spinach leaves. Operating conditions, such as the IL concentration and solid–liquid ratio, were optimized by a response surface methodology. Outstanding extraction yields (0.104 and 0.022 wt.% for chlorophyll a and b, respectively, obtained simultaneously) and selectivity (chlorophyll a/b ratio of 4.79) were obtained with aqueous solutions of hexadecylpyridinium chloride ([C16py]Cl) at moderate conditions of temperature and time. These extraction yields are similar to those obtained with pure ethanol. However, the chlorophyll a/b ratio achieved with the IL aqueous solution is higher than with pure ethanol (3.92), reinforcing the higher selectivity afforded by IL aqueous solutions as viable replacements to volatile organic compounds and allowing the obtainment of more pure compounds. Finally, the recovery and reuse of the solvent were evaluated by using a back-extraction step of chlorophylls using ethyl acetate. The results disclosed here bring new perspectives into the design of new approaches for the selective extraction of chlorophylls from biomass using aqueous solutions of surface-active ILs.

Impact of Plasma-Activated Water Treatment on Quality and Shelf-Life of Fresh Spinach Leaves Evaluated by Comprehensive Metabolomic Analysis

Fresh baby spinach leaves are popular in salads and are sold as chilled and plastic-packed products. They are of high nutritional value but very perishable due to microbial contamination and enzymatic browning resulting from leaf senescence. Therefore, innovative food processing methods such as plasma-activated water (PAW) treatment are being explored regarding their applicability for ensuring food safety. PAW’s impact on food quality and shelf-life extension has, however, not been investigated extensively in vegetables so far. In the present study, a comprehensive metabolomic analysis was performed to determine possible changes in the metabolite contents of spinach leaves stored in a refrigerated state for eight days. Liquid chromatography high-resolution mass spectrometry, followed by stringent biostatistics, was used to compare the metabolomes in control, tap-water-rinsed or PAW-rinsed samples. No significant differences were discernible between the treatment groups at the beginning or end of the storage period. The observed loss of nutrients and activation of catabolic pathways were characteristic of a transition into the senescent state. Nonetheless, the presence of several polyphenolic antioxidants and γ-linolenic acid in the PAW-treated leaves indicated a significant increase in stress resistance and health-promoting antioxidant capacity in the sample. Furthermore, the enhancement of carbohydrate-related metabolisms indicated a delay in the senescence development. These findings demonstrated the potential of PAW to benefit food quality and the shelf-life of fresh spinach leaves.

Chlorfenapyr and Methomyl Deterioration on Spinach Plants and Their Residual Effects in Vitro on Egyptian cotton Leafworm (Spodoptera littoralis)

Field trials conducted to determine the degradation of chlorfenapyr and methomyl insecticides in/on spinach leaves. Spinach plants sprayed with chlorfenapyr (Challenger Super™ 24% SC) and methomyl (Neomyl™ 90% SP) at the rates of 50 cm3/100 L water and 715 g/ ha, respectively. The QuEChERS method used for the extraction and clean-up of the samples. Residue amounts determined at 2 h, 2, 4, 6, 9, 13 and 16 days after application by UHPLC-UV. The mean of recovery percentages was 98.78 and 99.05 % for chlorfenapyr and methomyl, respectively. The initial deposits of chlorfenapyr and methomyl on/in spinach leaves, two hours after a single application of the insecticides were 23.17 and 235.37 mg/kg, respectively. The percentages of dissipation of chlorfenapyr were 37.68, 55.29, 69.45, 84.45 and 96.83% for 2, 4, 6, 9 and 16 days after application. The corresponding dissipation percentages of methomyl were 38.27, 56.01, 71.44, 84.34 and 97.81%. The rates of degradation (k values) were 0.212 and 0.223, while the corresponding half-life times (t0.5) were 3.27 and 3.11 days with chlorfenapyr and methomyl, respectively. It could be recommended that single application of chlorfenapyr on Spinach plants at the early ages followed by single application of methomyl at least 17 days before harvest.

Effect of Vacuum Impregnation with Sucrose and Plant Growth Hormones to Mitigate the Chilling Injury in Spinach Leaves

Vacuum impregnation (VI) has been immensely used in modifying the physicochemical properties, nutritional values and sensory attributes of fruits and vegetables. However, the metabolic consequences of the plant tissue upon impregnation have not been profoundly explored although shelf life is strongly dependent on this factor. In this study, spinach leaves were impregnated with salicylic acid (SA), γ-aminobutyric acid (GABA) and sucrose to improve its quality and storage ability by reducing the chilling injury through the improvement of proline content. The spinach leaves were stored at 4 °C for 7 days and were analyzed at 12 h interval. Upon 1 day of impregnation, the proline content in GABA, sucrose and SA impregnated leaves was increased by 240%, 153% and 103%, respectively, while in non-impregnated leaves, the proline content was decreased by 23.8%. The chlorophyll content of GABA impregnated leaves exhibited the lowest reduction (49%) followed by sucrose (55%) and SA (57%); meanwhile, non-impregnated leaves reduced 80% of chlorophyll content at the end of storage. Sensory evaluation showed that GABA, sucrose and SA impregnated leaves respectively, obtained higher score in terms of freshness, color, texture and overall appearance as compared to non-impregnated leaves.