A New Method for Selective Extraction of Torularhodin from Red Yeast Using CO2-SFE Technique.

Torularhodin is a dark pink colored carotenoid belonging to the xanthophylls group that can be biologically synthesized by red yeasts, especially by Rhodotorula and Sporobolomyces genera. The growing interest in this molecule is due to its biological activities such as antioxidant, anticholesterolemic, anti-inflammatory, antimicrobial, and anticancer. To satisfy potential commercial markets, numerous methods have been proposed to develop a cost-effective and environmentally friendly downstream process for the purification of torularhodin. However, obtaining high purity products without resorting to the use of toxic solvents, which can leave residues in the final preparations, remains a major challenge. In this context, the present study aimed to develop a new efficient method for the isolation of torularhodin from the red yeast Rhodotorula strain ELP2022 by applying the extraction technique with supercritical CO2 (CO2-SFE) in two sequential steps. In particular, in the first step, the dried lysed biomass of yeast was subjected to the action of CO2 in supercritical conditions (CO2SC) as sole solvent for extraction of apolar carotenoids. In the second step, the residual biomass was subjected to the action of CO2SC using ethanol as a polar co-solvent for the extraction of torularhodin. Both steps were carried out at different operating parameters of temperature (40 and 60 °C) and pressure (from 300 to 500 bar) with a constant CO2 flow of 6 L min-1. Regardless of the operating conditions used, this method allowed to obtain an orange-colored oily extract and a red-colored extract after the first and second step, respectively. In all trials, torularhodin represented no less than 95.2% ± 0.70 of the total carotenoids in the red extracts obtained from the second step. In particular, the best results were obtained by performing both steps at 40 °C and 300 bar, and the maximum percentage of torularhodin achieved was 97.9% ± 0.88. Since there are no data on the selective recovery of torularhodin from red yeast using the SFE technique, this study may be a good starting point to optimize and support the development of industrial production of torularhodin by microbial synthesis. This new method can significantly reduce the environmental impact of torularhodin recovery and can be considered an innovation for which an Italian patent application has been filed. In a circular bioeconomy approach, this method will be validated up to a pilot scale, culturing the strain Rhodotorula spp. ELP2022 on low-cost media derived from agri-food wastes.

Effectiveness of Dunaliella salina Extracts against Bacillus subtilis and Bacterial Plant Pathogens.

Several bacteria pathogens are responsible for plant diseases causing significant economic losses. The antibacterial activity of Dunaliella salina microalgae extracts were investigated in vitro and in vivo. First, biomass composition was chemically characterized and subjected to extraction using polar/non-polar solvents. The highest extraction yield was obtained using chloroform:methanol (1:1 v/v) equal to 170 mg g-1 followed by ethanol (88 mg g-1) and hexane (61 mg g-1). In vitro examination of hexane extracts of Dunaliella salina demonstrated antibacterial activity against all tested bacteria. The hexane extract showed the highest amount of ß-carotene with respect to the others, so it was selected for subsequent analyses. In vivo studies were also carried out using hexane extracts of D. salina against Pseudomonas syringae pv. tomato and Pectobacterium carotovorum subsp. carotovorum on young tomato plants and fruits of tomato and zucchini, respectively. The treated young tomato plants exhibited a reduction of 65.7% incidence and 77.0% severity of bacterial speck spot disease. Similarly, a reduction of soft rot symptoms was observed in treated tomato and zucchini fruits with a disease incidence of 5.3% and 12.6% with respect to 90.6% and 100%, respectively, for the positive control.

Selective Extraction of ω-3 Fatty Acids from Nannochloropsis sp. Using Supercritical CO2 Extraction.

In this article, microalgae Nannochloropsis sp. was used for fatty acid (FA) extraction, using a supercritical fluid-carbon dioxide (SF-CO2) extraction method. This study investigated the influence of different pre-treatment conditions by varying the grinding speed (200-600 rpm), pre-treatment time (2.5-10 min), and mixing ratio of diatomaceous earth (DE) and Nannochloropsis sp. biomass (0.5-2.0 DE/biomass) on FAs extraction. In addition, the effect of different operating conditions, such as pressure (100-550 bar), temperature (50-75 °C), and CO2 flow rate (7.24 and 14.48 g/min) on eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) recovery, was analyzed. Experimental data evidenced that, keeping constant the extraction conditions, the pre-treatment step enhanced the FAs extraction yield up to 3.4 fold, thereby the maximum extracted amount of FAs (61.19 mg/g) was attained with the pre-treatment with a ratio of DE/biomass of 1 at 600 rpm for 5 min. Moreover, by increasing both SF-CO2 pressure and temperature, the selectivity towards EPA was enhanced, while intermediate pressure and lower pressure promoted DHA recovery. The highest amount of extracted EPA, i.e., 5.69 mg/g, corresponding to 15.59%, was obtained at 75 °C and 550 bar with a CO2 flow rate of 14.48 g/min, while the maximum amount of extracted DHA, i.e., ~0.12 mg/g, equal to 79.63%, was registered at 50 °C and 400 bar with a CO2 flow rate of 14.48 g/min. Moreover, the increased CO2 flow rate from 7.24 to 14.48 g/min enhanced both EPA and DHA recovery.

Supercritical Carbon Dioxide Extraction of Astaxanthin, Lutein, and Fatty Acids from Haematococcus pluvialis Microalgae.

Haematococcus pluvialis microalgae in the red phase can produce significant amounts of astaxanthin, lutein, and fatty acids (FAs), which are valuable antioxidants in nutraceutics and cosmetics. Extraction of astaxanthin, lutein, and FAs from disrupted biomass of the H. pluvialis red phase using carbon dioxide (CO2) in supercritical fluid extraction (SFE) conditions was investigated using a bench-scale reactor in a semi-batch configuration. In particular, the effect of extraction time (20, 40, 60, 80, and 120 min), CO2 flow rate (3.62 and 14.48 g/min) temperature (50, 65, and 80 °C), and pressure (100, 400, and 550 bar.) was explored. The results show the maximum recovery of astaxanthin and lutein achieved were 98.6% and 52.3%, respectively, at 50 °C and 550 bars, while the maximum recovery of FAs attained was 93.2% at 65 °C and 550 bars.

Chemical Profile and Antioxidant Properties of Extracts and Essential Oils from Citrus × limon (L.) Burm. cv. Femminello Comune.

Citrus × limon cv. Femminello Comune (Rutaceae) from Rocca Imperiale (Italy), one of the six Protected Geographical Indication (PGI) Italian lemon crops, has been recently received renewed interest. In this work, fresh and dried peels and leaves were extracted by hydrodistillation, supercritical fluid extraction (SFE), and Soxhlet apparatus. Chemical profile was assessed by gas chromatography (GC) and gas chromatography/mass spectrometry (GC/MS). Except for leaves extracts obtained by Soxhlet apparatus, the monoterpene hydrocarbons fraction dominated. Limonene, γ-terpinene, and ß-pinene were the main identified compounds. The antioxidant activity was investigated using different in vitro assays namely 2,2-diphenyl-1-picrylhydrazyl (DPPH), ABTS, ferric reducing ability power (FRAP), and ß-carotene bleaching test. In DPPH test, the essential oil obtained by hydrodistillation of fresh peel exhibited the highest activity (IC50 of 1.17 mg/ml). Leaves extracted by SFE showed a good activity in both DPPH and ß-carotene bleaching test with IC50 values of 2.20 and 6.66 mg/ml, respectively. Monoterpene hydrocarbons fraction exhibited a positive Pearson's correlation coefficient with all antioxidant assays. Leaves, often considered waste material, should be considered from a different point because they represent a matrix of indisputable interest.