Isolation,Structure Characterization And Thermal Processing Characteristics Of Black Sesame Melanin

Black sesame is a traditional edible and medical crop in china.Except for protein,lipids,carbohydrates,vitamins,minerals,melanin is also abound in black sesame seeds.Black sesame showed potent biological activity,such as anti-oxidation,scavenging free radicals,anti-tumor,liver protection,and prevention of neurological diseases in previous studies.Melanin is a heterogeneous polyphenol-like polymer with a very complex structure,which makes its structure difficult to identify.At present,the chemical structure of black sesame melanin（BSM）has not been elucidated clearly.Thermal processing is a common way of food processing,and black sesame seeds are generally processed or eaten after being fried or roasted.Moreover,as an active substance,the thermal processing characteristics of BSM have not been clarified so far.Therefore,BSM was extracted from black sesame seed coats by alkali extraction and acid precipitation,and then the crude melanin was isolated.Followed the structure of purified melanin was elucidated by multiple spectroscopic methods.The in vitro antioxidant activity of obtained melanin was also evaluated.The main content and results were as follows:（1）Chemical Structure Elucidation of BSMThe crude melanin was purified by the methods of alkali extraction and acid precipitation as well as acid hydrolysis.And the structure of melanin was studied by extensive spectroscopic methods including ultraviolet-visible absorption spectroscopy（UV-Vis）,fourier transform infrared spectroscopy（FT-IR）,elemental analysis（EA）,chemical degradation,pyrolysis-gas chromatography mass spectrometry（Py-GC-MS）,gas chromatography mass spectrometry（GC-MS）,nuclear magnetic resonance（NMR）,X-ray photoelectron spectroscopy（XPS）,electron paramagnetic resonance（EPR）,X-ray diffraction（XRD）.The results showed that the content of nitrogen of BSM was lower than eumelanin and it had a characteristic melanin color reaction.BSM had the typical characteristics of ultraviolet and infrared spectral absorption of natural melanin,and there were functional groups such as benzene ring,alkyl,carboxyl,hydroxyl,amino,carbonyl in the structure of BSM.BSM also demonstrared the X-ray diffraction characteristics of amorphous compounds and paremegnetic resonance characterictics of natural melains.The pyrolysis products of BSM contain several eumelanin characteristic degradation fragments.And the result of solid nuclear magnetic resonance of BSM was close to that of dopa melanin.Above results indicated that BSM might be a polymer formed by polymerization of phenolic substance and dopa unit.Four in vitro antioxidant activity tests including DPPH,ABTS,FRAP and ORAC showed that BSM had excellent antioxidant activity.The IC₅₀values of DPPH and ABTS free radical scavenging of BSM were 18.98μg/m L and 21.00μg/m L,respectively,which were higher than that of dopa melanin(The IC₅₀values were 30.19μg/m L,33.00μg/m L,respectively,p<0.05).The FRAP and ORAC antioxidant capacity values of BSM were 3.03 mmol Fe E/g DW and 6049.02μmol TE/g DW,respectively,which were also higher than that of dopa melanin（The antioxidant capacity values were 2.72mmol Fe E/g DW and 3711.03μmol TE/g DW,respectively,p<0.05）.（2）Isolation,Structure Characterization and Antioxidant Activity of BSMCrude extract of BSM was isolated by HW-40C size exclusion column and the yields,color value of obtained fractions were determined.The structure of each fraction was studied by extensive spectroscopic methods including UV-Vis,EA,FT-TR,¹H-NMR,¹³C-NMR,XPS,EPR and XRD.Four methods including DPPH and ABTS radical scavenging assay,FRAP and ORAC assay were used to evaluate the in vitro antioxidant activity of each fraction of BSM.The results showed that two fractions,black Fr1 and brown Fr2 with the yields 60%and 24%were isolated from BSM extract.The purity of Fr1 was higher than Fr2 and Fr1was the main fraction of melanin.The molecular weights of two fractions were 38800 Da and 6000 Da,respectively.Elemental analysis showed that Fr1 was eumelanin and Fr2 might be allomelanin.Functional groups such as carbonyl,hydroxyl,amino,aromatic ring and nitrogen heterocycle existed in both fractions,and the aromaticity of Fr2 was higher than that of Fr1.Both fractions had strong paramagnetic resonance characteristics and there was a planar stacked structure in Fr1.The IC₅₀values of DPPH free radical scavenging of Fr1 and Fr2 were 83.00 and54.00μg/m L and the IC₅₀values of ABTS free radical scavenging were53.00 and 30.00μg/m L,respectively.The FRAP antioxidant capacity values were 1.05 and 1.62 mmol Fe E/g DW and the ORAC antioxidant capacity values were 3141.80 and 4143.76μmol TE/g DW,respectively.Above results showed that Fr2 possessed better DPPH,ABTS,FRAP and ORAC antioxidant capacity than Fr1（p<0.05）.（3）Effect of Thermal Processing on the Structure and Antioxidant Activity of BSMTwo methods,roasted and fried were used to thermally process black sesame seeds.Melanin was extracted from black sesame seeds by the methods of alkali extraction and acid precipitation.Structural characterization methods including UV-Vis,EA,FT-IR,NMR,XPS,EPR,XRD and four in vitro antioxidant activity methods including DPPH,ABTS,FRAP,ORAC were used to evaluate the effect of thermal processing on the structure and antioxidant activity of BSM.The results showed that the proportion of unsaturated structural units and heteroatoms in the structure of BSM were reduced,while functional group types,free radical properties and spatial characteristics（interlayer distance）were not significantly affected after thermal processing.Moreover,the effect of being fried on unsaturated structural units and element composition in the structure of BSM was significantly higher than that of being roasted.The DPPH,ABTS free radical scavenging capacity,FRAP and ORAC antioxidant capacity of BSM was significantly reduced after thermal processing.The DPPH and ABTS free radical scavenging capacity of BSM decreased by 31.91%（p<0.05）and10.71%and FRAP and ORAC antioxidant capacity decreased by 4.23%and 12.76%（p<0.05）after being roasted,respectively.The DPPH and ABTS free radical scavenging capacity of BSM after being fried were40.43%（p<0.05）and 25.00%（p<0.05）lower than that of the BSM untreated,and the antioxidant capacity of FRAP and ORAC were 23.94%（p<0.05）and 16.00%（p<0.05）lower than unteated,respectively.The antioxidant activity of BSM was affected more significantly after being fried than that of being roasted.