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.

Influence of Phenotypic Dissociation in Bacillus subtilis Strain ET-1 on Iturin A Production.

Iturin A is a very important cyclic lipopeptide produced by several B. subtilis strains and has large commercial and therapeutic application potentials but its production on industrial scale has not been realized yet. In the present study, we have observed that the strain ET-1 of Bacillus subtilis, a producer of Iturin A, can present at least three different colony morphologies, which we arbitrarily called Rough, Smooth, and Mucoid morphotypes (R-, S-, and M-form). Performing HPLC analysis, a significant difference between the amounts of Iturin A produced by the three morphotypes was found. The morphotype R-form showed the highest productivity with yields about 10 and 100 times higher than morphotypes S and M, respectively. The results show that the production of Iturin A by B. subtilis could be strongly influenced by the phenotypic heterogeneity of cells within the inoculum. Indeed, we have observed that, pasteurizing the inoculum before seeding in order to improve the homogeneity removing the phenotypes less able to synthesize the Iturin A, its yields in a bench-scale production could be significantly improved. This can represent an important control factor also at industrial scale to improve the Iturin A yields, the robustness, the replicability, and consequently the cost-effectiveness of fermentation processes.