Sequential Extraction Of Antarctic Krill Oils And Evaluation Of Their Function

Antarctic krill oil refers to the lipid extracted from Euphausia superba.Recently,it has attracted great attention from academic and industrial circles due to its unique composition.However,the absent standards,complicated composition and diverse processing technologies easily result in krill oil products with unstable qualities,further limiting the in-depth studies on their functionalities and hampering the development of krill oil industry.Therefore,the relationships among the processing technologies,chemical composition and functional properties of krill oil were explored in the present study.Based on the evaluation of the effects of different extraction solvents on the lipid yield and oil quality,a sequential extraction method was developed for obtaining three grades of krill oils with significantly different compositions from whole krill meal.Then,the antioxidant and antiinflammatory properties of the three grades of krill oils were evaluated with the aim to explore the relationships between chemical compositions and functionalities and scientifically guide the processing of krill oil.Firstly,seven types of extraction solvents commonly used in oil industry were used to extract krill oil from whole krill meal.The effects of different solvents on the lipid yield and oil quality were investigated.The results indicated that alcohols solvents(ethanol and isopropanol)gave higher lipid yields and high phospholipids contents,but lower levels of the minor components.Alkanes solvents(isohexane,n-hexane and subcritical butane)and ethyl acetate resulted in lower lipid yields and phospholipids contents and moderate levels of the minor components compared with alcohols solvents.Even though acetone resulted in the lowest lipid yield and PL content,the highest levels of sterols,astaxanthin and higher vitamins were detected in the extracted oil.Phosphatidylcholine and phosphatidylethanolamine were the only two types of phospholipids detected in all extracted krill oil samples,with relative proportions of 87.93%~95.16%and 4.84%~12.07%.The solvents had no significant effects on the relative proportions of the two types of phospholipids.Besides,the eicosapentaennoic acid(EPA)and docosahexaenoic acid(DHA)in krill oil were mainly bound to phospholipids,and their contents positively correlated with the content of phospholipids.In general,the extraction solvents showed significant effects on the lipid yield and phospholipids,EPA,DHA and minor components contents of krill oil products.Then,a novel three-step sequential extraction was developed.In the first step,acetone was adopted to extract lipid from whole krill meal.The first grade krill oil(KO1)was obtained by extraction at 5 ~oC for 15 min with 1:2 ratio of krill meal to solvent.KO1 contained 2.39 g/100g of phospholipids,519.00 mg/kg of astaxanthin,29.65 mg/100 g of tocopherols,33.54 mg/100g of vitamin A,28.13 mg/g of cholesterol,and 11.52%of EPA and DHA;In the second step,n-hexane was selected to extract lipid from the krill meal partly defatted in the last step.The second grade krill oil(KO2)was obtained by extraction at 30 ~oC for 15 min with 1:2 ratio of krill meal to solvent.KO2 contained 35.02 g/100 g of phospholipids,30.03 mg/kg of astaxanthin,11.57 mg/100 g of tocopherols,11.84 mg/100 g vitamin A,18.69 mg/g of cholesterol,and 26.17%of EPA and DHA;In the last step,ethanol was selected to extract lipid from the krill meal partly defatted in the last two steps.The third grade krill oil(KO3)was obtained by extraction at 30 ~oC for 15 min with 1:3 ratio of krill meal to solvent.KO3 contained62.79 g/100 g of phospholipids,9.50 mg/kg of astaxanthin,3.73 mg/100 g of tocopherols,2.62mg/100 g of vitamin A,9.01 mg/g,and 41.39%of EPA and DHA.The three grades of krill oils had significant differences in phospholipids contents and minor components levels.This not only enriched the product varieties of krill oil,but also provided raw materials for the following study on the relationship between the composition and functional properties of krill oil.Next,the antioxidant abilities of the three grades of krill oils were evaluated by chemical methods of scavenging free radicals,cellular antioxidant activity(CAA)model,oxidative induction time determination and Schaal oven test with the aim to explore the relationships between the chemical compositions and antioxidant abilities of krill oils.Among the four chemical methods,FRAP and DPPH methods were not applicable in evaluating the antioxidant abilities of krill oils.Although the determination values by ORAC and ABTS methods were different,they showed similar trends:KO2>KO1>KO3,which were also in accordance with the results of CAA and oxidative induction time.In addition,it was found that peroxide value and thiobarbituric acid-reactive substances value were not applicable for assessment of krill oil oxidation in the Schaal test.The changes of phospholipids,pyrroles and free amino acids indicated non-enzymatic browning was involved in the oxidation process,but the degrees of non-enzymatic browning differed among the three grades of krill oils due to their different compositions.KO1 contained the highest content of tocopherols and astaxanthin,both of which decreased steadily during the storage period of 3 months,showing a protective effect on KO1.However,it generated the least content of pyrroles which are antioxidant Maillard products.Although KO3 produced the highest content of pyrroles,its acid value was still significantly increased and phospholipids were rapidly degraded due to the lack of tocopherols and astaxanthin.KO2 exhibited the highest antioxidant capacity during storage period.The protection of tocopherols and astaxanthin,moderate production of pyrroles,and synergistic antioxidant effects of phospholipids and tocopherols in the system were responsible for the superior oxidative stability of KO2.Finally,the antiinflammatory properties of the three grades of krill oils were evaluated based on the lipopolysaccharide(LPS)-stimulated RAW264.7 macrophage inflammation model.The effects of the three grades of krill oils with different concentrations on cell viability,nitric oxide(NO)generation,production of tumor necrosis factor(TNF-α),interleukin(IL)-1β,IL-6,and the relative mRNA expression of TNF-α,IL-1β,IL-6,induced nitric oxide synthase(iNOS),cyclooxygenase 2(COX-2)were investigated.Both the maximum tolerant dosages of RAW264.7 cells to KO1 and KO2 were 200μg/mL,and that to KO3 was 100μg/mL.At the concentrations of 25,50 and 100μg/mL,all the three grades of krill oils inhibited the production of NO,the secretion of TNF-α,IL-1βand IL-6,and their relative mRNA expression in LPS-induced inflammatory cells,as well as iNOS and COX-2 which are related to inflammation.At the medium and high concentrations(50 and 100μg/mL),KO3 showed significantly higher antiinflammatory activity than KO2 and KO1,but at the lowest concentration,the order of antiinflammatory activities was KO3≈KO2>KO1.In general,krill oil with high phospholipids or EPA and DHA contents had high antiinflammatory property.In conclusion,the chemical composition of krill oil is influenced by the extraction method,which further affects its antioxidant stability and antiinflammatory activity.Future research should focus on the influence of typical processing process on the composition,structure and function of krill oil and their related mechanisms,which would be helpful to precisely regulate and efficiently produce krill oil products with target functionalities.