Study On Enzymatic Synthesis And Physiological Function Of Human Milk Structural Lipids From Nannochloropsis-derived Oil

The production of human milk structural lipids is one of the important research directions in the market of infant milk products.The human milk structural lipids（HMSLs）rich inω-3 polyunsaturated fatty acids（PUFAs）have universally been paid attention by nutritionists,due to its special biological functions such as preventing the loss of lipids and mineral elements,anti-tumor,anti-inflammation,anti-bacteria,and providing enough energy to maintain the normal growth and development of early infants,Thus,exploiting HMSLs rich inω-3 PUFAs is also one of the hot research directions in the field of lipids.Considering the structural composition of human milk structural lipids,vegetable oils or animal fats were usually selected as a raw materials to produce the human milk structural lipids.However,the yield and fatty acid composition of vegetable oils are greatly influenced by environmental factors.As an animal fats,the fatty acid composition of lard is greatly affected by the given oil source in feed and the breed of pig,resulting in the instability of the sn-2 palmitic acid content in lard.As for polyunsaturated fatty acids,deep-sea fish or fish oils are usually the main resource for human to takeω-3polyunsaturated fatty acids.However,in recent years,due to the continuous deterioration of the global ecological environment,the global marine fishery resources are gradually exhausting,which makes the raw material rich inω-3 polyunsaturated fatty acids increasingly used up.All of these factors have limited the development and application of human milk structural lipids rich inω-3 polyunsaturated fatty acids.Microalgae have been found to be a potential resource to replace the animal fats and vegetable oils for the production of human milk structural lipids rich inω-3 polyunsaturated fatty acids,due to its fast growth rate,high oil yield and easy cultivation.This project used a microalgal strain named by Nannochloropsis oculata（N.oculata）rich in palmitic acid in sn-2position of its triacylglycerides as a experimental microalga to explore the effects of different iron concentrations and salinity on the growth and lipid accumulation of Nannochloropsis oculata.Microalgal oil of Nannochloropsis was extracted by enzyme-assisted three phase partitioning（EATPP）,and the microalgal oil was separated and purified by silica gel column to obtain the triacylglycerides of Nannochloropsis.Whereafter,using microalgal oil of Schizochytrium limacinum rich in DHA as a material,the triacylglycerides of Nannochloropsis was jointly catalyzed by Lipozyme RM IM in an anhydrous system to prepare the human milk structural lipids rich in DHA.Finally,the safety and anti-inflammatory effect of the human milk structural lipids rich in DHA were also investigated.1.Using Nannochloropsis oculata as a experimental microalga,the effects of different combination of iron concentrations and salinity on the growth and lipid accumulation of Nannochloropsis oculata were explored in a photobioreactor.The experimental results demonstrated that:（1）The optimal iron concentrations and salinity for the growth of Nannochloropsis oculata were 250 mg/L and 2.5%,respectively.（2）The optimal iron concentrations and salinity for the lipid accumulation of Nannochloropsis oculata were 0mg/L and 5.5%,respectively.（3）Compared with the iron deficiency,high salinity stress had a greater inhibitory effect on the growth of Nannochloropsis oculata.（4）There were no significant difference between the effect of iron deficiency and high salinity on the oil accumulation of Nannochloropsis oculata,but it could significantly increase the oil accumulation of Nannochloropsis oculata when iron deficiency and high salinity stress were combined.2.Using wet Nannochloropsis sp.biomass as raw material,the enzyme-assisted three phase partitioning（EA-TPP）was used to extract microalgal oil of Nannochloropsis,and the different parameters on the lipid extraction efficiency of enzyme-assisted three phase partitioning were been optimized.The results showed that:（1）In the pretreatment process of microalgal biomass by four hydrolytic enzymes for disrupting the cell wall of Nannochloropsis,compared with hemicellulase,pectinase and papain,the treatment process by cellulase enzymatic hydrolysis obtained the the highest TFAs extraction efficiency of Nannochloropsis.（2）The optimal condition for extracting microalgal oil of Nannochloropsis by EA-TPP were as follows:20%ammonium sulphate,6-7 p H,1:2slurry/tert-butanol ratio and 70℃for 2 h incubation time and two extraction cycles.Under the optimal condition,the TFAs extraction efficiency of Nannochloropsis extracted by EA-TPP reached 90.40%.（3）Under the optimal condition,the TFAs extraction process of Nannochloropsis extracted by EA-TPP was further evaluated in a 20L reactor.The result showed that:under the optimal condition,the TFAs extraction efficiency achieved 88.70%in a 20 L reactor.The extracted microalgal oil of Nannochloropsis was separated and purified by silica gel column to produce triacylglycerides,providing a raw material for the production of human milk structural lipids rich inω-3 polyunsaturated fatty acids.3.Using triacylglycerides（TAGs）of Nannochloropsis and free fatty acids（FFAs）rich in DHA from the microalgal oil of Schizochytrium limacinum as substrates,the production of human milk structural lipids rich in DHA were catalyzed by Lipozyme RM IM,so as to improve the content ofω-3 polyunsaturated fatty acids and nutritional value of human milk structural lipids.The results showed that:（1）The optimal reaction conditions for the production of human milk structural lipids catalyzed by Lipozyme RM IM were optimized as follows:substrate molar ratio（TAG/FFAs）was 1:4,lipase loading was 7%,reaction temperature was 40℃and reaction time was 24 h.Under the optimal conditions,the incorporation rate of DHA in triacylglycerides（TAGs）of Nannochloropsis was 29.91%.（2）As for the reusability of immobilized lipase Lipozyme RM IM,it was found that the catalyst could be reused 22 times without significantly affecting the incorporation rate of DHA.（3）The melting temperature of SL₁structure lipid ranged from-57.09℃to 35.08℃,and the crystallization temperature ranged from-60.00℃to 13.52℃.The melting temperature of SL₂structure lipid ranged from-37.08℃to 31.67℃,and the crystallization temperature ranged from-42.50℃to 14.33℃.The melting temperature of SL₃structural lipids ranged from-59.92℃to 34.32℃,and the crystallization temperature ranged from-60.00℃to 20.36℃.4.The safety of human milk structural lipids rich in DHA was evaluated,using small intestinal recess epithelial cells IEC-6 of SD rats and 15-day-old SD rats as an evaluation model in vitro and in vivo,respectively.The results were listed as follows:（1）In the cytotoxic activities tests,human milk structural lipids rich in DHA did not significantly reduce the cell viability of small intestinal recess epithelial cells IEC-6 of SD rats.（2）During the 28-day acute toxicity study,the human milk structural lipids rich in DHA did not produce any meaningfully significant changes in body appearance,physiological biochemistry,hematology,histopathology and other parameters on SD rats.（3）Human milk structural lipids rich in DHA were regarded as a safe oil source.5.Mouse macrophage RAW 264.7 was used as an inflammatory cell model to investigate the anti-inflammatory effects of triacylglycerides（TAGs）of Nannochloropsis（Oil 1）and human milk structural lipids rich in DHA（Oil 2）,respectively.The results showed that:（1）Both Oil 1 and Oil 2 had no cytotoxicity on mouse macrophage RAW264.7 in the range of tested concentration.（2）After being pretreated by Oil 1 and Oil 2,all of the mice macrophage RAW 264.7 showed anti-inflammatory effect caused by lipopolysaccharides（LPS）stimulation.But there presented a stronger anti-inflammatory effect in mice macrophage RAW 264.7 after being pretreated by the human milk structure lipids rich in DHA.（3）Human milk structure fats rich in DHA could inhibit the phosphorylation process of p65 protein in the NF-κB inflammatory signaling pathway,and presented a dose-dependent manner.（4）Both Oil 1 and Oil 2 pretreatment could induce Ca²⁺influx in mouse macrophages RAW 264.7,but there were not significant difference between both of them.（5）Pretreatment with human milk structural lipids rich in DHA could reduce calmodulin（Ca M）expression in mouse macrophages RAW 264.7,and showed a dose-dependent manner.