Recovery, physicochemical characterization and thermal properties of lipids from R. opacus PD630

Abstract

Over the past few decades, microbial oils have been considered as a source of novel oils due to their potential commercial applications including nutraceuticals, pharmaceuticals, feed ingredients for aquaculture as well as feedstock for producing biodiesel. Rhodococcus opacus strain PD630 is a genus of aerobic, non-sporulating gram-positive bacteria that has the ability to accumulate large amounts of oil.

An optimized procedure for extraction of microbial lipids from R. opacus PD630 is proposed. Different solvent extraction methods including chloroform/methanol (1:1, v/v), hexane/isopropanol (3:2, v/v), dichloromethane/methanol (1:1, v/v), dichloromethane/ethanol (1:1, v/v), dichloromethane/acetone (1:1, v/v) and diethyl ether were tested to identify the most efficient solvent extraction method. Furthermore, various cellular pre-treatment methods, including autoclaving, ultrasonication, microwaves, and osmotic shock were examined to identify the most effective cell disruption method. The result showed that a chloroform/methanol solvent mixture gave the highest efficiency to extract lipids from R. opacus PD630. Also, ultrasonication, microwave and osmotic shock cellular pre-treatment method have higher efficiency for cell wall disruption than the autoclave method. Analysis of lipid class by thin layer chromatography (TLC) showed that lipids from R. opacus PD630 were mainly composed of triacylglycerols (TAG). Saturated fatty acids (SFA) contribute a large portion (51.50 ± 0.49%) of total fatty acids. A high proportion of fatty acids with an odd number of carbon atoms (32.08 ± 0.39%) were detected. Lipids from R. opacus PD630 contained a high level of unsaponifiable matter (USM). The lipid was high in red and yellow colour parameters. Lipids from R. opacus PD630 was oxidatively stable and had good quality based on moisture and volatile content, peroxide value (PV), p-anisidine value (p-AnV), TOTOX value, free fatty acid (FFA), acid value (AV), conjugated diene and triene values, and polar compounds content. Positional distribution of fatty acids in lipids of R. opacus PD630 were determined via pancreatic lipase treatment followed by thin layer chromatography and gas chromatography analysis. In TAG of R. opacus PD630, the SFA (70.56 ± 1.95%) prefer sn-2 position against sn-1,3 position; C16:0 (39.35 ± 0.99%) was the dominant SFA at sn-2 position. On the other hand, unsaturated fatty acids were predominantly found at sn-1,3 position. A large proportion of C16:1, C17:1 and C18:1 were found at sn-1,3 position which contribute 88.61 ± 1.76% monounsaturated fatty acids (MUFA). Melting and crystallization characteristics of lipids from R. opacus PD630 were investigated using Differential Scanning Calorimetry (DSC) from -40 °C to 60 °C under a nitrogen atmosphere. The melting and crystallization of lipid from R. opacus PD630 occurred over a large temperature range from -7.45 °C to 32.37 °C and 17.62 °C to -19.93 °C respectively, due to its high degree of saturation. Oxidative stability of lipids from R. opacus PD630 was studied using DSC and Thermogravimetric Analysis (TGA) at 100 °C, 110 °C and 150 °C (TGA only) under air atmosphere. Results showed that lipids of R. opacus PD630 were very stable against oxidation at 100 °C and 110 °C isothermal conditions. At the temperature of 150 °C, the lipid started to decompose at 126.3 minutes but without an oxygen uptake peak. Analysis of thermal stability of lipids from R. opacus PD630 was carried out using TGA. Lipids from R. opacus PD630 decomposed in four stages during non-isothermal heating, which were decomposition of USM, MUFA and SFA, followed by decomposition of MUFA and SFA degradation products. Antioxidants present in the lipid including α- and γ-tocopherols, chlorophyll, and total phenolics and flavonoids may protect the lipid against oxidation. There were no α- and γ-tocopherols or chlorophyll detected in the lipids of R. opacus PD630. On the other hand, small amounts of phenolic acids and flavonoids were found in the lipids which may enhance oxidative stability of lipid. The antioxidant activity was determined by using 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2’-azino-bis(3- ethylbenzothiazoline-6-sulphonic acid), known as ABTS. The antioxidation effects of bioactive compound of lipid were determined by DPPH and ABTS+ free radical scavenging tests, the IC50 value was 675.2 μg/mL for DPPH and 510.0 μg/mL for ABTS. These results indicated that lipid from R. opacus PD630 was a good quality oil, and very stable against oxidation and thermal decomposition.