Study On Variation Law In The Storage And Processing Of Astaxanthin Compounds In Litopenaeus Vannamei

Astaxanthin, one of natural carotenoids, is widely present in shrimp, crab, algae and other marine plants and animals, which has various physiological activities, such as anti-tumor, anti-inflammatory, immune regulation, reducing oxidative damage, etc. In this paper, we established three methods to analyze astaxanthin and astaxanthin ester molecular species, astaxanthin optical isomers, and oxidation products of astaxanthin and astaxanthin ester in aquatic products. A deep insight was obtain on the changes of composition, molecular species and pattern of astaxanthin and astaxanthin esters in Litopenaeus vannamei during storage and processing. This paper provided a theoretical foundation and scientific basis for the utilization and quality evaluation of shrimps and the follow-up study of astaxanthin compounds. The content and results of this study are as follows:1. A C30-HPLC-DAD-(APCI)-MS/MS method was established for analyzing astaxanthin and astaxanthin esters molecular species in aquatic products. A YMC C30 column (4.6mm×250mm,5μm) is employed for separation. And a gradient elution by using methyl tert-butyl ether and methanol as mobile phase is performed to achieve a good separation of astaxanthin compounds in different sample extracts. The astaxanthin and astaxanthin ester molecular species in Litopenaeus vannamei, Haematococcus pluvialis, Euphausia superba, Portunus trituberculatus and Salmon salar were analyzed by this method. The results showed that astaxanthin monoester is the main form of astaxanthin in Haematococcus pluvialis and Litopenaeus vannamei, and accounts for 57.05% and 58.65% in total astaxanthin compounds, respectively. While in Euphausia superba, astaxanthin diester accounts for 68.20% in total astaxanthin compounds. Free astaxanthin accounts for 68.53% in the total astaxanthin compounds of Portunus trituberculatus. And only all-trans astaxanthin exists in salmon.2. A high performance liquid chromatography method for chiral separation and analysis of astaxanthin stereoisomers in several biological organisms was developed. The separation was performed on a Chiralpak IC column (4.6mmx250mm,5μm) by using methyl tert-butyl ether (MTBE)-acetonitrile as the mobile phase at a flow rate of 1.0 mL/min. The detection wavelength of diode array detector (DAD) was 476nm. Haematococcus pluvialis, Phaffia yeast, Penaeus vannamei, Penacus orientalis, Japanese tiger prawn, salmon and crab were selected as research subjects. The linear range of astaxanthin stereoisomers calibration curve was 0.1～50mg/L with a regression correlation coefficient of 0.9999. The average recovery rate for astaxantin stereoisomers was 86.2%～98.3%. These results demonstrate that the proposed method is feasible, rapid, simple and accurate.3. A high-performance liquid chromatographic-atmospheric pressure chemical ionization mass spectrometry (HPLC-(APCI)-MS/MS) method for analyzing autoxidation products of astaxanthin and astaxanthin esters was developed. A YMC C30 column (4.6mmx250mm,5μm) is employed for separation, and a gradient elution by using methyl tert-butyl ether and methanol as mobile phase is performed to separate 17 kinds autoxidation of astaxanthin and astaxanthin DHA esters. The product ion mode and precursor ion mode of mass spectra were used to identify the autoxidation products and resolve their fragmentation patterns, in which the fragmentation of apo-astaxanthinals or apoastaxanthinone is dehydration as the main feature, and for the po-astaxanthinal or apo-astaxanthinone docosahexaenoic keto acid esters is based on the loss of the fatty acid ester as the main feature. In astaxanthin oxidation products, apo-12’-astaxanthinal and apo-14’-astaxanthinal were the main products, and their relative contents were 43.9% and 19.2%. For astaxanthin docosahexaenoic acid (Asta-C22:6) monoester, apo-13- astaxanthinone docosahexaenoic acid ester and apo-14’-astaxanthinal accounted for 18.0% and 17.5% in total products, respectively. In astaxanthin docosahexaenoic acid (Asta-C22:6/C22:6) diesters, apo-13- astaxanthinone docosahexaenoic acid ester is the most abundant, accounting for 42.3% of total products.4. The effect of ice and frozen storage on astaxanthin compounds in Litopenaeus vannamei were investigated by the established methods. The content of total astaxanthin, staxanthin ester and free astaxanthin decreased slowly in the early storage, and then fell fast. The content total astaxanthin and TVB-N showed a significant negative correlation. During 7 days of ice storage and 12 weeks of frozen storage, all-trans-astaxanthin decreased by 52.35% and 49.85% respectively, while the content of 13-cis-astaxanthin increased first and then decreased, which was accompanied by the generation of 9-cis-astaxanthin. In Litopenaeus vannamei, astaxanthin octadecane acid monoester(Asta-C 18:1) and Asta-C22:6 lost 35.15% and 33.78% during 7 days of ice storage respectively, and lost lost 7.96% and 6.33% during 4 weeks of frozen storage respectively. Asta-C22:6/C22:6 and astaxanthin docosahexaenoic acid/ octadecenoic acid diester (Asta-C22:6/C18:1) lost 20.13% and 33.27% during 7 days of ice storage, and lost 6.50% and 7.13% during 4 weeks of frozen storage. The main oxidation product of astaxanthin and astaxanthin esters will increase along with the storage time, and showed a significant positive correlation with the phenol oxidase and lipoxygenase activity.5. The effect of thermal processing on astaxanthin in Litopenaeus vannamei were investigated. A high-performance liquid chromatographic-atmospheric pressure chemical ionization mass spectrometry (HPLC-(APCI)-MS/MS) method was used to identify and quantify all-trans- and cis-isomers of astaxanthin, and molecule species of astaxanthin esters in fresh and thermal processed shrimps. Total astaxanthin losses ranged from 7.99% to 52.01% in first 3 min under three thermal processing. All-trans-astaxanthin was most affected, with a reduction from 32.81 to 8.72μg/100 g, while 13-cis-astxanthin had a rise (from 2.38 to 4.58 μg/100 g). Esterified astaxanthin was shown to hydrolyze and degrade, furthermore astaxanthin diesters had a better thermostability compare to astaxanthin monoesters. Astaxanthin monoesters with eicosapntemacnioc acid (EPA, C20:5) and docosahexaenoic acid (DHA, C22:6), had a lower thermal stability than those with saturated fatty acids, however, it was the opposite of astaxanthin diesters.