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Determination of crude fat, EPA and DHA content of marine microalgae
In nature, organisms capable of synthesizing EPA and DHA are only part of marine microalgae and a small amount of bacteria. Among them, marine microalgae are the primary primary productivity in the marine ecosystem, and provide the polyunsaturated fatty acid directly or indirectly through the food chain for advanced animals. EPA and DHA in fish are obtained by eating marine microalgae. Therefore, it is of great significance to study the content of EPA and DHA in marine microalgae. This article makes an analysis of the EPA and DHA content in eight kinds of marine microalgae, and provides the basis for the development and utilization of marine microalgae.
1 Materials and methods
1. 1 Materials
The experimental microalgae were Phaeodactylumtricornuru, 2038; Skeletonema costa 2tum, No. 2; 2042; Nitzschia closterium, 2034; No. 4 Isochrysis (Chrysophyceae isochrysis galba2na), 3011; No. 5 Pavlova viridis, 3012; Chlorella miutissima, 1014; No. 7 Nannochloropsis oculata, 1001; No. 8 purple ball Porphy2ridium cruentum, 8001,; Miller's seawater enriched with fP2 in culture medium; The reagents used in the experiment are all analytically pure; 721 visible spectrophotometer; centrifuge; electrothermothermic water bath; sp6800a gas chromatograph ; SZF-06B crude fat analyzer, crude fiber analyzer.
1. 2 methods
1. 2. 1 Microalgae cultivation method Natural illumination, average light intensity 6000LX, light to dark ratio 16:8, stationary culture, shaking twice a day at regular intervals. Temperature 23 ± 2 °C.
1. 2. 2 harvest logarithmic growth phase of the final harvest, determination of OD680, 4000rPmin centrifugation, the algae vacuum dried and then spare.
1. 2. 3 Sample Preparation Centrifuged algal biomass is extracted by the Bligh-Dy2er method [2], distilled under reduced pressure and quickly weighed and transferred to a 25 mL ground tube. After drying the solvent with N2, add 5 mL of 2mol PL. Potassium hydroxide 2 methanol solution was sealed after filling with N2. It was placed in a 75 °C water bath for 40 min. After cooling, n-hexane (two times) was added to extract the fatty acid methyl ester. The extract was transferred to a 5 mL conical centrifuge tube, dried with N2, and dissolved in n-hexane to provide chromatographic analysis [3–5].
1.2.4 Gas Chromatographic Analysis Gas chromatographic analysis conditions: The column was a stainless steel packed column (2m × 3mm), the support was a rosazabor, filled with 16% of DEGS. The carrier gas is N2, the flow rate is 30mLPmin, no shunt, and the temperature is 190 °C. Hydrogen flame detector with a temperature of 250 °C. The attenuation is 4 and the sensitivity is 4. Injection volume is 2μL. The determination of fatty acids was performed with reference to a standard sample and quantified using an external standard method.
2 Results and Discussion
2.1 Crude fat content
The experimental microalgae harvest density, biomass, and crude fat content are shown in Table 1. Chlorophyta harvest density is highest, in which chlorella harvest OD6801. 372, biomass 491. 2mgPL, crude fat is 26% of the dry weight; and the lowest harvest density of Chrysophyta biomass is 61. 8mgPL, but crude fat The highest content was 57% of the dry weight; the harvesting densities of three microalgae of diatoms were 0. 592, 0. 710, 0. 724, of which the No. 3 algae had the largest biomass of 509. 4mg PL, and the crude fat accounted for 26% of the dry weight; Rhodochloa erythraea individual is larger, its biomass is 475. 6mgPL, crude fat accounts for only 5% of dry weight.
2. 2 Determination of the EPA and DHA contents of eight marine microalgae As shown in Table 2, the EPA content of diatoms in the eight microalgae is the highest, of which, No. 3 algae, EPA content is 16mgPL, accounting for 26% of the dry weight, accounting for coarse The fat is 3.3%, almost free of DHA, which facilitates the extraction and purification of EPA and can be used as an algae strain for the industrial production of EPA. The EPA content of No. 1 algae was 7.1 mgPL, accounting for 12% of the dry weight; the DHA content was almost negligible. The content of DHA in No. 4 alga of Chrysophyta was the highest, which was 1. 34mgPL, accounting for 9.1% of the dry weight, accounting for 5. 2% of the crude fat. The EPA in the No.7 Chlorophyta was 7.3% of the dry weight of 7. 7mgPL accounted for 1.7% of the crude fat, while No. 6 algae contained almost no EPA and DHA. The EPA production of P. vulgaris of Rhodophyta was 2. 3mgPL, accounting for 9. 2%, accounting for 0.5% of crude fat, and did not contain DHA.