| In recent years,polyunsaturated fatty acids(PUFAs)have been proved to be essential substances to treat some chronic diseases,so the development of PUFAs has a broad market prospects and many researchers have turned their research focus on the PUFAs production of microalgae.Since PUFAs have been widely applied in pharmaceuticals and food industries,the safety of microalgae oil extraction technology is requested.Therefore,economic and green oil extraction methods to obtain microalgae-derived PUFAs should be developed.In addition,utilizing microalgae biomass for the co-production of bioactive compounds is an important research direction at present.If other high value-added biomass of microalgae can be rationally utilized during the process of microalgae oil extraction to balance the high cost of microalgae oil extraction,it will greatly promote the industrial development of microalgae oil.In this study,a green and environmentally-friendly technology to comprehensively exploit Chlorella biomass was established by integrating with enzymatic hydrolysis process and n-hexane-ethanol two-phase extraction technology,which could obtain high value CGF(Chlorella growth factor)and microalgae oil rich in PUFAs.This novel integrated process provided a new idea for the comprehensive development of microalgae resources.Also,the anti-inflammatory function of the synthetic process products was evaluated.Meanwhile,the nutritional composition and anti-inflammatory function of CGF obtained by enzymatic hydrolysis process and commercial CGF were compared.1.The hydrolysis effect of four food grade hydrolases(cellulase,hemicellulase,pectinase and papain)on the cell wall of Chlorella was compared.It was concluded that pectinase had the best hydrolysis effect on the cell wall of Chlorella.The optimum process parameters were as follows: enzyme amount of 200 U/g for Chlorella powder,0.75% normal saline as solvent,solid-liquid ratio of 1:20(W/V(g/m L)),p H= of 5,shaked by 150 rpm in 50? water bath,reaction time of 4h.Under the optimal conditions,the hydrolysis rate of Chlorella was 17.52% that was 1.49 times higher than that in the no-enzyme group;the oil extraction rate of Chlorella was 20.65% in 45 mins,which was32.97% higher than that in the no-enzyme group.In addition,the supernatant of Chlorella enzymolysis can be collected and treated to obtain CGF.2.A green,safe and efficient two-phase extraction method mediated with n-hexane and ethanol was used to extract the oil from Chlorella after enzymatic hydrolysis.And the lipid composition of the obtained oil was analyzed.The optimal extraction parameters were as follows: solid-liquid ratio of 1:50(g/m L),solvent volume ratio(n-hexane/ethanol)of 1:2,volume ratio of water with organic solution of 0.25 and temperature of 25?.Under the optimal conditions,the total oil extraction rate of Chlorella was 26.83%,which was 104.93% of that of chloroform-methanol extraction method.The extraction rate of n-hexane phase is 17.79%,and the proportion of neutral lipid is 83.20%.The oil extraction rate of ethanol phase was 9.04%,the ratio of glycolipids and phospholipids was 53.65% and 40.37%,respectively.The results showed that unsaturated fatty acids were the main fatty acids in n-hexane phase microalgae oil,and saturated fatty acids were the highest in ethanol phase microalgae oil.After oil extraction,microalgae residue still contained polysaccharide,protein and other nutrients,which could be used for the secondary development of microalgae resources,improving economic benefits to balance the process cost.3.The mouse macrophage RAW 264.7 induced by LPS(lipopolysaccharide)was applied to evaluate the anti-inflammatory activity of microalgae oil obtained by the two-phase extraction method.Both n-hexane microalgae oil and ethanol-phase microalgae oil obtained by the two-phase extraction method had anti-inflammatory effect on mouse macrophages RAW264.7,and ethanol-phase microalgae oil had better anti-inflammatory effect than n-hexane microalgae oil.Both of them could decrease the secretion of TNF-? and IL-6,but did not decrease the secretion of IL-1? in mouse macrophages.4.The nutritional composition and anti-inflammatory function of commercialized CGF and enzymatic hydrolyzed CGF were compared.The results showed that,compared with enzymatic hydrolyzed CGF,the contents of polysaccharide and protein in commercialized CGF were higher,while the contents of fatty acids in enzymatic hydrolyzed CGF were higher.Both enzymatic hydrolyzed CGF and commercial CGF had anti-inflammatory effects on mouse macrophages RAW264.7.Enzymatic hydrolyzed CGF could reduce the secretion of inflammatory cytokines TNF-? and IL-6 in mouse macrophages RAW264.7,while commercial CGF could only reduce the secretion of IL-6.Both of them had no effect on the secretion of IL-1?.In this paper,a process for the comprehensive utilization of Chlorella that combining mild enzymatic hydrolysis and green and efficient double liquid extraction method was applied to obtain CGF and microalgae oil of different compositions.The process conditions were optimized and the anti-inflammatory function of the extracted products was evaluated.Meanwhile,the nutritional composition and anti-inflammatory function of CGF obtained by enzymatic hydrolysis process and commercial CGF were compared. |
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