| Nannochloropsis sp.is a type of marine microalgae rich in Eicosapentaenoic acid(EPA).Due to its easy cultivation,short growth cycle,and high oil production,it is considered to be one of the effective sources of EPA to replace deep-sea fish oil in the future.The EPA in Nannochloropsis sp.is mostly concentrated in polar lipids.Therefore,when extracting lipids containing EPA from Nannochloropsis sp.,the extraction of membrane polar lipids should also be considered.Secondly,the cell wall of Nannochloropsis sp.is a double-layer dense structure,which is three times stronger than the cell wall of the plant and hinders the extraction of intracellular lipids.Therefore,before extracting lipids in Nannochloropsis sp.,it is necessary to break the wall of Nannochloropsis sp.In response to the above problems,this article uses Nannochloropsis sp.as a raw material to study the EPA lipid extraction method of Nannochloropsis sp.and the process of enzymatic wall-breaking.Combining with the analysis of the composition and structure of Nannochloropsis sp.before and after the enzymatic wall-breaking,it is hoped to clarify the mechanism of enzymatic wall-breaking to promote the lipid extraction rate of Nannochloropsis sp.The specific research content and results are as follows:(1)Study on the extraction process of EPA lipid from Nannochloropsis sp.First,compare the effects of different solvent systems on the extraction of EPA lipids from Nannochloropsis sp.and taking EPA lipid extraction rate and EPA content as evaluation indicators,it was found that ethanol is more suitable for extracting polar lipids from Nannochloropsis sp.,and the obtained extract contains higher EPA content.Secondly,using EPA lipid extraction rate and EPA content as indicators,combined with single-factor experiment and orthogonal experiment,the ethanol extraction process of Nannochloropsis sp.EPA lipid was optimized.The optimized process conditions obtained are:ethanol concentration 80%(v/v),material-to-liquid ratio 1:17.5,extraction temperature 65°C,and extraction time 2 h.Under these process conditions,the extraction yield of EPA lipid from Nannochloropsis sp.is 15.87%±0.21%,EPA content is 14.49±0.14 g/100g.Finally,after the acute toxicity test of the EPA lipid from Nannochloropsis sp.extracted by ethanol method,there were no death or poisoning symptoms.It proves that the EPA lipid of Nannochloropsis sp.obtained by ethanol extraction has food safety and can be used in the food industry in the future.(2)The influence of enzymatic wall-breaking on the extraction of EPA lipid from Nannochloropsis sp.Using the lipid extraction rate as an indicator,compare the wall-breaking effects of different single enzymes on Nannochloropsis sp.,select the best enzyme as the base enzyme for compounding with other enzymes.Finally,the better compound combination is laccase+cellulase.Further based on the single factor test,PB(Plackett-Burman)test and BB(Box-Behnken)test were used to optimize the enzymatic breakage of Nannochloropsis sp.The optimized process conditions obtained are enzyme dosage 21 mg/g,laccase:cellulase=1:2.5(laccase enzyme activity in the system:7.2×10-2 U/m L,cellulase enzyme activity:75 U/m L),buffer solution p H=5.0,enzymatic temperature 45.0?,enzymatic time 6 h.Under these conditions,the extraction rate of EPA lipid of Nannochloropsis sp.26.39%was 66.29%higher than that without enzyme treatment(15.87%).The amount of EPA in lipids 20.67 g/100g was 42.63%higher than that without enzymatic wall-breaking(14.49 g/100g).(3)Study on the mechanism of enzymatic wall-breaking to improve the extraction yield of EPA lipids from Nannochloropsis sp.The compound enzyme enzymatic wall-breaking can more effectively improve the lipid extraction rate of Nannochloropsis sp.than a single enzyme.It is speculated that the synergistic effect of cellulase and laccase effectively promotes the extraction of neutral lipids and polar lipids from Nannochloropsis sp.Through the analysis of the lipidomics of the obtained EPA,it was found that it contained 17 types of lipids.The content of EPA lipids in the non-enzymatic wall-breaking group was 108.45 mg/g,and in the enzymatic wall-breaking group was 166.21 mg/g which increased by 53.26%compared with the untreated group,indicating that enzymatically breaking the walls of Nannochloropsis sp.can effectively increase the EPA content in the EPA lipid of Nannochloropsis sp.Furthermore,the analysis of the lipid composition obtained showed that the content of lysoacylglycerol trimethylhomoserine(LDGTS)in the lipid extracted after enzymatic wall-breaking was significantly increased.In this paper,the ethanol method is used to extract EPA lipids,the EPA content in the obtained lipids is significantly increased by enzymatic wall-breaking and has preliminarily explained the mechanism of enzymatic wall breaking to promote EPA lipid extraction.It lays the foundation for the production and application of EPA lipid nutrition products from Nannochloropsis sp. |
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