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为实现藻油中ω-3多不饱和脂肪酸（ω-3 PUFAs）的简单、绿色、高效分离纯化工艺，首先利用裂殖壶菌原始菌株及定向驯化菌株ALE40为原料提取油脂，通过优势性评估及细胞通透性对比二者提油效果，并通过测定油脂指标对比3种提油方法(酸解法、超声干法、超声湿法)优劣。对超声湿法提取的粗藻油采用酯交换法进行乙酯化后，联合尿素包埋法和分子蒸馏技术富集高浓度二十碳五烯酸（EPA）和二十二碳六烯酸（DHA），对酯交换及尿素包埋工艺条件进行单因素实验优化,通过对分子蒸馏重相DHA和EPA含量测定确定分子蒸馏级数，并对纯化藻油的理化指标进行测定。结果表明：相比裂殖壶菌原始菌株，ALE40 的细胞通透性明显较好，在无需干燥的情况下，超声10 min即可达到最高油脂得率（53.2%），缩短超声时间的同时提高了破壁效率；3种提油方法中超声湿法提取的藻油理化指标较好；酯交换反应最佳条件为反应温度60 ℃、KOH 添加量3%、醇油比10∶ 1、反应时间0.5 h，尿素包埋最佳工艺条件为脲酯比0.8∶ 1、溶酯比8∶ 1、包埋温度4 ℃、包埋时间8 h；尿素包埋后的藻油经二级分子蒸馏纯化后，藻油中 EPA 和 DHA 总含量从17.40%（酯交换反应）升高到90.86%,总回收率为62.28%；得到的纯化藻油的各项理化指标均符合行标要求，且储存3个月内无沉淀，具备一定稳定性。综上，该提取分离纯化工艺全程实现试剂循环利用，且制备的藻油酸值低、无异味。To realize a simple, green and efficient separation and purification process of ω-3 polyunsaturated fatty acids (ω-3 PUFAs) from algal oil, firstly, the original strain of Schizochytrium sp. and the oriented domesticated strain ALE40 were used as raw materials to extract the algal oil, the oil extraction effects of the two strains were compared through the assessment of dominance and cell permeability, and the advantages and disadvantages of three oil extraction methods （acid digestion, ultrasonic-assisted dry extraction, and ultrasonic-assisted wet extraction）were compared through the determination of the oil indexes.After ethyl esterification by transesterification of algal oil extracted by ultrasound-assisted wet extraction, high concentrations of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) were enriched by the combination of urea complexation and molecular distillation techniques, and the conditions of the transesterification and urea complexation processes were optimized by single factor experiment, as well as the physicochemical indexes of purified oil were determined. The results showed that compared to the original strain of Schizochytrium sp. , the cell permeability of ALE40 was significantly better, and the highest oil yield of 53.2% could be achieved by ultrasound-assisted wet extraction of ALE40 for 10 min without drying, which shortened the ultrasound time and improved the wall-breaking efficiency. In the three oil extraction methods, the algal oil extracted by ultrasound-assisted wet extraction had better physicochemical indexes.The optimal transesterification reaction conditions were reaction temperature 60 ℃, KOH addition 3%, alcohol-oil ratio 10∶ 1 and reaction time 0.5 h. The optimal urea complexation process conditions were urea-ester ratio 0.8∶ 1, solvent-ester ratio 8∶ 1, crystallization temperature 4 ℃ and crystallization time 8 h. After urea complexation, the algal oil was purified by secondary molecular distillation, the total content of EPA and DHA in the algal oil increased from 1740% (transesterification reaction) to 90.86%, and the total recovery was 62.28%. The physicochemical indexes of the purified algal oil met the requirements of the industry standard, and there was no precipitation within three months of storage, with a certain degree of stability. In conclusion, the extraction, separation and purification process can realize the recyling of reagents throughout the entire procedure, and the prepared algal oil has low acid value and no peculiar odor. |
