Aquatic Producs Preservation And Transportation Investigation Based On Metabolomics

This research achieved the application of metabolomics in the investigation of aquatic products preservation and transportation. For the targeted metabolomics, this research applied it on the parameter optimization of the typical marine bivalve, scallops of the semi-anhydrous living-preservation. For the untargeted metabolomics, this research applied it on the discriminate of recovery insufficient of anaesthesia transportation with MS-222. This research extended the application of metabolomics to the traditional research area of aquatic products preservation and transportation. Additionally, the aspects of aquatic products preservation and transportation were renewed by this research too.This research was mainly composed from the following 5 parts:1. The energy conservation substances of typical marine bivalve in the preservationObject: The survival of scallops was investigated. The commercial acceptable duration of preservation was tested. The reference of the experiment design of the following targeted metabolomics was also offered. The physiology alterations especially the energy conservation substances was investigated.Method: A time series experiment of scallops preserved in semi-anhydrous was conducted. The LT10 and LT50 value was tested. Some indexes including glycogen level, lactate level and protein level were tested.Result: The survival curve was plotted. The LT10 value was 3 days. The LT50 value was 6 days. During the preservation the glycogen level was not altered. The lactate level was temporarily elevated. The protein level was also unchanged.Conclusion: Scallops performed good survival ability in the preservation. The maintenance of glycogen level might be caused by elevation of glycogeneogenesis. The temporarily elevation of lactate level indicated the energy metabolism pattern switching. The information of these experiments were limited.2. The investigation of metabolism features of typical marine bivalve in living preservation based on targeted metabolomicsObject: In purpose of discover the physiological detail of scallops in preservation, the targeted metabolomics research was conducted. The biomarkers were screened out.Method: semi-anhydrous preservation with a duration of 3 days, the LT10, was applied on the scallops. MCF derivation and GC-MS were involved in the chemical analysis. MVA methods including PCA and PLS-DA were applied on the dataset. The outliers were eliminated by PCA. The candidate biomarker screening was according to the VIP value of PLS-DA and the p value of t-test. Based on the biomarkers pathway elucidation was conducted.Result: with the data filtration PLS-DA model was established. Carboxyl acids related to respiration(malic, fumaric and succinic acids), fatty acids(C18:0, C16:0, and C22:6) and amino acids(phenylalanine, glutamic acid, aspartic acid, isoleucine, glycine, pyroglutamic acid, proline, leucine and 2-aminoadipic acid) were identified as biomarkers.Conclusion: Our results demonstrated that scallops performed an elevated anaerobiosis and depressed aerobiosis, which were not caused by oxygen insufficiency. The switching of energy metabolism patterns and disorder of the osmotic regulation system were also observed, suggesting that oxygen supply is less important for optimizing semi-anhydrous living-preservation.3. Metabolism features of plasma of typical aquatic vertebrate in anaesthetized transportationObject: the residual of MS-222 after recover in the plasma of Chinese Carps was tested. Some plasma biochemical indexes were investigated as also.Method: The carps were divided into three groups, two were simulated of anesthetized transportation with or without enough withdrawal time and one were transported without anesthesia. The plasma biochemical indexes including TP, ALB, AST, ALT, ALP and CREA were tested by auto analyzer.Result: MS-222 in all plasma samples were under LOD. The TP level of recovery insufficient group and the group transported without anesthesia was upregulated. The CREA level of recovery insufficient group was upregulated to the other two groups.Conclusion: The elimination rate of MS-222 was too fast for detection. This index is not capable for discriminant of recovery insufficiency. The regulation of CREA level indicated the reversible renal disfunction.4. Investigation of metabolism pattern of typical aquatic vertebrate in anaesthetized transportation based on untargeted metabolomicsObject: The model to discriminate withdrawal insufficiency of carps after anesthetized transportation was established.Method: GC-MS, Hilic-MS and RP-MS were applied jointly in chemical analysis. Support vector machine based machine learning was performed after feature selecting with Random forest and visualized with Self organizing map. Biomarkers screened by feature ranking were used to elucidate metabolism pathway alteration.Result: There are 110, 589, 1272 and 1971 annotated and combined features obtained by GC-MS, Hilic-MS, RP-MS and in total, respectively. The value were tuned to 0.1574901 and 0.003100393 as C and γ, respectively. The discriminating model was validated by testing samples. The accuracy of present method on the testing samples reached 100%. RF based feature selection and SOM based visualization were introduced to eliminate the disadvantage of SVM.Conclusion: Further pathway elucidating illustrated that the biological principle of the discriminating was represented by the relative long term impact of different treatment on metabolism pathway of central carbon, tyrosine, phenylalanine and tryptophan. Biomarkers related to inflammation and injury were also screened out. This phenomenon indicates anesthetized transportation with MS-222 caused gill injury leading to disturbed gas exchange. The hypoxia and hypercapnia induced by the injury were put claim on the impact in post-revive recovery.5. Label-free proteomics investigation of typical aquatic vertebrate in anaesthetized transportationObject: The alteration on proteome of anesthesia transportation of the carps were investigated. The conclusion of metabolomics research was confirmed.Method: The samples were inter-group pooled and FASP was applied on. Peptide analyst was performed on nano-HPLC-MS. Label-free protein quantification was conducted with Maxquant software.Result: 12251 peptides and 1353 protein groups were detected. Most of the peptides were with length of 7 to 11. The average length was 10.9. After the quantification and 1.8 fold change screening the altered protein quantities between 6 hours recovery group and 5 days recovery group were 22 and 29 respectively, the quantity between the latter two groups was 31.Conclusion: According to the pathway elucidation, different anaesthetized transportation with recovery had alterations on the energy metabolism pathway. The biomarkers related to hypercapnia including citrate synthase and fumarate hydratase were identified. And the alteration biomarker related to hypoxia of fumarate hydratase was revealed as well. The results of proteomics was in good agreement with metabolomics.