| Astaxanthin is widely present in nature,especially in shells of shrimps and crabs,trout,algae and fungi,and mainly exists in esterified form in most organisms.Due to the complex structure of ester astaxanthin,many structural standards are difficult to obtain,so it is impossible to directly measure ester astaxanthin.To take full advantage of the astaxanthin resources in aquatic products,this research systematically analyzed the composition and content of astaxanthin in different parts of six typical aquatic products,and established methods for accurately quantifying astaxanthin in different forms.Finally,by establishing a D-gal-induced liver injury model in aging mice,representative sources of astaxanthin were selected for intervention,and the protective effects of different sources of astaxanthin on the liver of D-galactose-induced aging mice were explored.Provide basic data for the evaluation and rational utilization of astaxanthin resources from different sources.The main contents of this study are as follows:1.A screening database of astaxanthin and its derivatives based on high performance liquid chromatography-high resolution mass spectrometry was established and used to accurately verify the composition of astaxanthin and its derivatives in different parts of typical aquatic products.(1)Explore the primary and secondary mass spectra of different forms of astaxanthin and analyze the structure of secondary mass spectrometry fragments,infer the fragmentation law of the mass spectrum,and screen mass spectrometry fragmentation markers that can identify astaxanthin compounds.(2)Combine astaxanthin with one or two of the 40 fatty acids,and calculate the theoretical molecular weight of the corresponding MS fragments based on the 23 screened out mass spectrometry cleavage markers that can identify astaxanthin compounds.A database containing 860 astaxanthin ester molecules,molecular formulas,molecular weights,and3440 mass spectrometry fragments has a theoretical molecular weight database.(3)Taking Haematococcus pluvialis,Procambarus clarkia,Antarctic krill,Penaeus vannamei,Rainbow trout and Eriocheir sinensis as the research objects,the data was collected in Full MS/dd-MS2 mode and analysised by self-built mass spectrometry database.The results show that the composition and content of astaxanthin and astaxanthin esters in different aquatic products are different,and there are also differences between different parts of the same aquatic product.19 species of astaxanthin esters were screened out from Haematococcus pluvialis,which mainly combined C16:0,C18:1,C18:2 and C18:3.42 species of astaxanthin esters were identified in the shell of Procambarus clarkia,there are 16 and 18 kinds of shrimp head and meat respectively,which are mainly combined with saturated fatty acids such as C12:0,C13:0,and C16:0.Antarctic krill heads(30 types of esters)are more abundant than shrimp shells(11 types)and shrimp meat(9 types),mainly C16:0,C18:1,and C20:5.Compared with the first two species of shrimp,there are fewer astaxanthin esters in Penaeus vannamei,8 species in shrimp heads,16 species in shrimp shells,and only free state and no esterified state in shrimp meat.There are only free forms in Rainbow trout muscles,and only 2 monoesters are screened out in fish skin.The composition of the astaxanthin esters in the head breastplate(20 species)of the Eriocheir sinensis is similar to that in the body(19 species),and 4 monoesters have been identified in the gonads.2.The hydrolysis conditions of the enzymatic hydrolysis method were optimized,and the effects of enzymatic hydrolysis and saponification methods on the composition and quantitative analysis of esterified astaxanthin were investigated and used in the accurate quantitative detection of different forms of astaxanthin in typical aquatic products.(1)First,taking the astaxanthin mono-and di-esters isolated and prepared from Antarctic krill as the research object,the best process of enzymatic hydrolysis is optimized through single factor and orthogonal experiments,which can maximize the conversion of ester-state astaxanthin into Free state and apply it to actual samples.The results showed that when the substrate concentration of the monoester was 0.5 ?g/m L,the enzyme concentration of the reaction system was 1.14 U/m L,the reaction temperature was 25°C,and the reaction time was 75 minutes,the yield of free astaxanthin was(94.56±1.24)%.When the substrate concentration of the diester was 1.0 ?g/m L,the enzyme concentration of the reaction system was 0.92 U/m L,the reaction temperature was 20°C,and the reaction time was 75 minutes,the free astaxanthin yield was(98.28±0.84)%.The optimized monoester and diester optimal processes were applied to Haematococcus pluvialis and Antarctic krill,and the free astaxanthin yields were as high as(98.56±0.74%)and(95.24±1.93%),compared with other enzymatic hydrolysis methods,the free astaxanthin yield is higher.(2)The content change of astaxanthin ester during the saponification process was explored.When the saponification time is 0-18 h,the loss of free astaxanthin(6.21%-19.89%)is higher than that of enzymatic hydrolysis(4.84%-8.9%);The free astaxanthin yield of monoester reached the maximum(89.54%)at 14 h of saponification;the highest free astaxanthin yield of diester at 16 h was 89.76%,which was significantly lower than that of enzymatic hydrolysis.when the monoesters and diesters were saponified for 2 hours,which indicates that the conversion efficiency of monoesters was higher than that of diesters.(3)Comparative analysis of the changes of geometric isomers of astaxanthin during saponification and enzymatic hydrolysis.The results show that free astaxanthin was easier to isomerize compared with monoester and diester astaxanthin.And the sequence of conversion of free astaxanthin into isomers was13-cis,9-cis.In addition,compared with enzymatic hydrolysis,the saponification method produces a higher proportion of isomers.(4)The main molecular species of astaxanthin esters in the monoesters and diesters during the saponification process were analyzed and it was found that when astaxanthin was combined with one fatty acid,the stability of the astaxanthin ester combined with saturated fatty acids > monounsaturated fatty acids > polyunsaturated fatty acid.When two fatty acids are combined,the astaxanthin diester combined with saturated fatty acid is the most stable.(5)Enzymatic hydrolysis and saponification methods are used to determine the content of astaxanthin in typical aquatic products.The results show that the results obtained by the two methods are significantly different.The enzymatic hydrolysis method is not suitable for samples with higher lipid content,and is more suitable for Saponification method.The two methods have their own advantages and disadvantages,and the appropriate method can be selected according to the sample matrix and the actual situation of the experiment.3.To study the effects of different sources of astaxanthin on D-gal-induced liver oxidative damage and related genes in aging mice.Sixty mice were randomly separate into six groups compriseing Blank control group(CON),D-galactose group(D-gal),Antarctic krill group(ANT),Haematococcus pluvialis group(HAE),Phaffia rhodozyma(PHA),chemically synthesized astaxanthin group(SYN).Body weight and liver index of mice were recorded.Malondialdehyde(MDA)content and the activities of superoxide dismutase(SOD),glutathione peroxidase(GSH-px),catalase(CAT)and total antioxidant capacity(T-AOC)were measured by biochemical methods.The relative m RNA expression levels of glutathione S-transferase(GSTA1),heme oxygenase-1(HO-1),quinone oxidoreductase 1(NQO1),Bax,Bcl-2 and Caspase3 were measured by fluorescence quantitative PCR.HE staining method for pathological analysis of liver tissue.The results showed that:(1)Comparing with other groups,the body weight of the mice in the D-gal group was slightly lower,but there was no significant difference(P>0.05).Comparing with the D-gal group,the group of ANT,HAE,PHA,SYN could significantly increse liver coefficients(P<0.05).The liver coefficients of HAE group was significantly higher than the other astaxanthin groups(P<0.05),and the ANT group was higher than PHA and SYN group,but no significant difference(P>0.05).(2)Comparing with the D-gal group,the activities of CAT,GSH-PX,T-AOC and SOD in the liver tissues of mice in the 4 astaxanthin group were significantly increased(P<0.05),while the MDA content was significant Decrease(P<0.05).The antioxidant enzyme activity of astaxanthin from different sources was higher in HAE and ANT groups than in PHA and ANT groups,and MDA content was lower in HAE and ANT groups than in PHA and ANT groups.Comparing with the D-gal group,the relative expression of the four astaxanthin groups of the antioxidant-related genes HO-1,GSTA1,NQO1 and Bcl-2were significantly increased(P<0.05),while Bax and Caspase3 were significantly reduced(P<0.05).(4)The mice in the D-gal group had severe liver damage,enlarged sinusoids,and disordered arrangement of liver cells.The 4 astaxanthin groups all protected liver damage to varying degrees.In conclusion,astaxanthin can protect mouse liver cell morphology,increase liver antioxidant level,regulate the expression of antioxidant enzymes and apoptosis-related enzyme m RNA in liver tissue,thereby alleviating liver oxidative stress caused by D-gal and reducing liver damage.In addition,the protective effects of HAE group and ANT group on liver oxidative damage induced by D-gal in aging mice were stronger than that of PHA group SYN group. |
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