Study On Lipofuscin-like Pigments Formation Mechanism In Postharvest Walnut (Carya Cathayensis Sarg.)

Lipofuscin-like pigments(LFLP)also called age pigment,which are mainly the end products of lipid oxidation and non-enzymatic glycation.Accumulation of LFLP in cells may affect cell normally function and protmote cell senescence.In addition to their physiological role in plant aging biology,LFLP also play an important role in food quality.Increase of LFLP accumulation could not only affect the appearance and color but can also cause loss of nutritional value.Moreover,LFLP of food sources will induce a variety of chronic diseases,if ingested by human.The LFLP was selected as the subjects,its accumulation in postharvest walnut during storage was studied.Formation mechanism and product characteristics of LFLP was also explored through model systems in this study.The results of this study will offer some references for LFLP accumulation in real systems.And the results were of great significance in LFLP further research and application.The mainly results were as follows:1.The effects of different factors on LFLP accumulation of the postharvest walnut during storage were studied.(1)The present study focused on the effect of different initial moisture contents(4%,6%,12%,16%)on postharvest biochemical changes and accumulation of LFLP during walnut storage.The results showed that,water content is an important factor affecting physiological,biochemical metabolism and accumulation of LFLP:The accumulation of LFLP in walnut with high initial moisture(12%and 16%)were through non-enzymatic glycosylation pathway,while through lipid peroxidation pathway in walnut with low initial moisture(4%and 6%).The LFLP accumulation of walnuts with low moisture(4%-6%)was found to be positively correlated with lipoxidation products.Lipid oxidation process of walnut with high initial water content(12-16%)were not obvious,while the soluble sugar obviously decreased and the content of reducing sugar increased.The LFLP accumulation of walnuts with high initial moisture(12%-16%)was found to be closely related to the process of nonenzymatic glycosylation and be positively correlated with HMF.In addition,the accumulation of LFLP increased,antioxidant substances and antioxidant capacity was heading downwards during storage of all walnuts.(2)Effect of three kinds of packaging(package filled with nitrogen,vacuum packaging and filled with oxygen packaging)on the LFLP accumulation and quality deterioration of walnut under different storage temperature(10℃,25℃and 40℃)have also been studied in this paper.The results showed that,the storage temperature and packaging are also important factors affecting lipid oxidation,so they are also the main reasons for the accumulation of LFLP:package filled with nitrogen and vacuum packaging were good ways to inhibit lipid oxidation,antioxidants content decreases and LFLP accumulation under low temperature storage.The quality deterioration and LFLP accumulation of walnut were significantly promoted under high temperature and high oxygen environment.And the results of variance analysis showed that,the effect of temperature on lipid oxidation and LFLP accumulation was much higher than that of packaging.The phenomenon of protein body fusion with increase of the degree of walnut oxidation was found in the experiment of cell ultrastructure.Loss of genomic DNA integrity was found on highly oxidized walnut(storage temperature was 40℃,filled with oxygen packaging),which was also comfirmed by the AFLP experiment.(3)Active oxygen(O2·-、H2O2、OH)and metal ions were selected for treatment of walnut oil and powder.Effect of active oxygen and metal ions on walnut oil oxidation and LFLP accumulation were discussed in this paper.The results showed that,active oxygen and metal ions could promote lipid oxidation and LFLP accumulation:Active oxygen can promote the formation of primary products of lipid oxidation,while not significantly for secondary oxidation products.But the metal ions could greatly promote both the primary and secondary of lipid oxidation products.Lipid oxidation products is an important precursor of LFLP.The results of GC-MS showed that the volatile products from walnut oil oxidation were mainly straight chain aldehyde compounds,including mono-unsaturated aldehydes and poly-unsaturated aldehydes.Moreover,active oxygen and metal ions could promote the LFLP accumulation of walnut powder.2.The reaction between oxidation oil or reactive carbonyl and protein were carried out to simulate LFLP formation.The purpose was to further explore the formation mechanism of LFLP and its characteristics.Model system can provide some theoretical basis and reference data for the real system,though it is some way behind the two system.(1)Oxidation linoleic acid-protein(BSA)reactions was used for the model experimental of LFLP formation.The results showed that,the protein side chain modified,carbonyl value increased and the typical of LFLP fluorescence emerged along with the oil oxidation process.Moreover,the browning degree of products deepen with the oxidation time.The maximum wavelength of intrinsic fluorescence blue shift,thioflavin T fluorescence value increase,congo red absorption and CD spectra results showed that,the conformational of oxidation protein changed during LFLP formation.In addition,reducing SDS-PAGE analysis showed that the molecular weight of LFLP from oxidation oil-protein interaction were increased,and the LFLP products exhibited resistance to protease enzymatic properties and certain degree of cytotoxicity.(2)The product properties of LFLP coming from MDA,heptenal,heptadienal-protein(BSA)interaction were explored in this paper.The result showed that,reactive carbonyl species from lipid oxidation also play an important role in LFLP formation.Incubation of protein(BSA)with all these three aldehydes resulted in modification of Lys and Arg residues,conformational changes,and formation of colored,fluorescent,lipofuscin-like pigments(exhibiting strong lipofuscin-like fluorescence of MDA-protein,but much weaker fluorescence for the other two aldehyde-protein molecules).Moreover,the generated LFLP complexes exhibited ultraviolet-visible(UV-Vis)absorption,i.e.,the LFLP derived from MDA exhibited increased UV-Vis absorbance at 290 and 400 nm,while the LFLP derived from the other two aldehyd exhibited increased UV-Vis absorbance at 280 and 300-350 nm,respectively,and cross-linking,similar to the characteristics of Maillard reaction products.The formation of aggregates were detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis,and MDA plays a more significant role in formation of the LFLP aggregates.