| Summary: | 博士 === 國立嘉義大學 === 食品科學系研究所 === 103 === Spirulina, also known as blue-green algae, is a uni-filamentous cyanobacterium. It is classified under the plant kingdom, blue-green algae phylum, blue-green algae class, hormogon order, Oscillatoriales family, Spirulina genus or Arthira genus. The species mainly used for edible purpose are Spirulina maxima and Spirulina platensis. Research has revealed that spirulina contains approximately 6% of fatty acid, in which contains rich amount of γ-linolenic acid (GLA) with physiological activity. Many researchers have proposed that an appropriate intake of GLA can alleviate rheumatoid arthritis, pre-menstrual syndrome, cardiovascular diseases, and cancers.
The aim of the current study was to investigate the optimal conditions of fractionation and anti-inflammatory performance of GLA in Spirulina platensis using a continuous supercritical CO2 fractionation process. The content of fatty acids components in the fractions was measured to determine the optimal fractionation conditions. The anti-inflammatory performance was determined by the ability to inhibit lipopolysaccharide (LPS)-induced inflammatory responses in RAW 264.7 macrophages under different concentration of extracts and fractions that contained 100 or 200 μM GLA.
In this study, a continuous supercritical CO2 fractionation process was employed using a Box-Behnken factorial design of a three-factor by three-level 13 experimental responses based on the response surface methodology (RSM). Under a fixed mass ratio of CO2, that is by changing the pressure (10, 20, 30 MPa), temperature (40, 50, 60°C), and the flow velocity of samples (1, 2, 3 mL/min), the responses were determined using multiple regression analyses to investigate the optimal fractionation conditions of GLA and other fatty acids. The results revealed that:
(1) γ-Linolenic acid (C18:3): Content of purified GLA in fractionation could be enhanced with an increase in sample flow velocity, temperature, and pressure. When the condition was 30 MPa, 60°C with a sample flow velocity of 3 mL/min, the yield of GLA was predicted to reach 165 μg/g dry weight, followed by 147 μg/g dry weight at 50°C and 124 μg/g dry weight at 40°C. The results revealed that the increase in temperature and pressure of CO2 fluid significantly enhanced GLA content after fractionation.
(2) Other fatty acids: For myristic acid (C14:0), the combination of a high sample flow velocity, a high density, and a high viscosity could lead to the highest fraction content, with the optimal fractionation condition at 60°C, 30 MPa, and a sample flow velocity of 3 mL/min. In addition, an increase in the fractionation pressure and temperature in combination with a low flow velocity, the optimal fractionation condition of palmitic acid (C16:0), palmitoleic acid (C16:1), stearic acid (C18:0), oleic acid (C18:1), and linoleic acid (C18:2) was found to be at 30 MPa, 60°C, and a sample flow velocity at 1 mL/min. This fractionation condition can increase the content of these fatty acids.
(3) Anti-inflammatory performance: Based on the MTT assay, there were no significant difference in the survival rate of RAW 264.7 macrophages between culture of spirulina extract (50, 100, 200 μg/mL), sample fluid containing GLA after supercritical fractionation (50, 100, 200 μg/mL), or cultured with 0.1 μg/mL LPS for 24 hours. The results of LPS-induced RAW 264.7 macrophages inflammatory response suggested that the sample fluid containing GLA after fractionation could significantly inhibit the generation of nitric oxide (NO), tumor necrosis factor (TNF-α), prostaglandin E2 (PGE2), cyclooxygenase-2 (COX-2), and interleukin-1β (IL-1β). In addition, the anti-inflammatory performances of fractionation fluid were better than those of the extract.
In conclusion, findings from this research indicated that a continuous supercritical CO2 fractionation process could effectively improve the issue of insufficient GLA concentration with traditional spirulina extraction methods. In addition, the GLA content and the inhibitory capacity of the inflammatory factors were significantly associated.
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