Structural Identification And Preliminary Study On Antioxidant Activity Of Soy Sauce Melanoidins

Soy sauce is a fermented condiment produced and consumed in large quantities in China.At present,the impact of fermented food on health has been widely concerned.Melanoidins are a class of bioactive substances produced in the final stage of Maillard reaction during soy sauce fermentation.It has anti-oxidation,antibacterial,anti-blood pressure,anti-inflammatory,anti-cancer and similar effects to dietary fiber,and greatly affects the color and flavor of soy sauce.Melanoidins have broad application prospects.At present,the research on melanoidins mainly focused on the model system and coffee melanoidins.There are few studies on melanoidins in soy sauce,whose composition and structure are still unclear,and the quantitative studies on the contribution of melanoidins to the color and antioxidant activity of soy sauce have not yet been reported.Therefore,this paper used macroporous adsorption resin combined with ultrafiltration to separate melanoidins from raw soy sauce.The content,composition,color and structure of melanoidins in soy sauce were characterized,the antioxidant activity and mechanism of melanoidins in soy sauce were discussed and the contribution of melanoidins to the color and antioxidant activity of soy sauce were quantified,which provided data support and preliminary theoretical basis for the in-depth study of melanoidins in soy sauce.The main research results are as follows:(1)DA201-E macroporous adsorption resin was used to purify and enrich the melanoidins of soy sauce.The content of melanoidins was about 2.44 g /100 m L soy sauce.Five groups were obtained by further ultrafiltration: 1-3 k Da,3-10 k Da,10-30 k Da,30-50 k Da and >50 k Da.The content of >50 k Da was the highest,accounting for 36.9% of the total content of melanoidins in soy sauce.The basic components,content of free amino acids and color of melanoidins in soy sauce were analyzed by ultraviolet-visible spectrophotometer,HPLC and colorimeter.The results showed that the contents of protein(10.52%)and total sugar(2.83%)in >50 k Da group was the highest,and the content of phenols(0.88%)in 10-30 k Da group was the highest.The content of free amino acids in 1-3 k Da group was the highest,and the content of melanoidins in all groups was more than 83%.The color results showed that 10-30 k Da group had the largest browning degree and the deepest color,while the melanoidins of the group >50 k Da had the largest contribution to the color of soy sauce(25.72%),and the contribution value of all the melanoidins groups to the color of soy sauce was 64.42%.(2)Scanning electron microscope(SEM),laser particle size analyzer,UV-visible absorption spectrum,fluorescence spectrum,fourier transform infrared spectroscopy(FT-IR),nuclear magnetic resonance spectroscopy(NMR),differential scanning calorimeter(DSC)were used to identify the microscopic morphology,structural characteristics and thermodynamic properties of melanoidins with different molecular weights.The findings indicated that the molecular weight positively correlated with the structural fragments,particle size,and Zeta potential of melanoidins in soy sauce.The UV-visible absorption spectrum characteristics showed that the extractive was in line with the typical characteristics of melanoidins,and it was inferred that the melanoidins with small molecular weight,especially the 1-3 k Da group,were mainly intermediate products of Maillard reaction,while the melanoidins in soy sauce were mainly medium and high molecular weight polymers larger than 10 k Da.Fluorescence spectra showed that the fluorescence intensity of melanoidins decreased with increasing molecular weight.The results of FT-IR and NMR analysis showed the presence of protein skeleton and other small molecules such as carbohydrates and phenols in soy sauce melanoidins.The results of DSC analysis showed that the structure of melanoidins in the group larger than 10 k Da was more complex and stable,and the peak thermal cracking temperature of 10-30 k Da group was the highest,the heat required for thermal cracking was also higher,and the structure was more compact.(3)The antioxidant activity in vitro of soy sauce melanoidins was evaluated by metal ion chelating activity,ferric reducing antioxidant power,DPPH radical scavenging activity,ABTS radical scavenging activity,oxygen radical absorbing capacity,inhibition of lipid peroxidation induced by iron and xanthine oxidase(XOD)inhibition activity.The results showed that 1-3 k Da group had the lowest antioxidant activity,while 10-30 k Da group showed the highest antioxidant activity at the same concentration.And the melanoidins of the group larger than 50 k Da contributed the most to the antioxidant activity of the model solution and soy sauce,which were27.82-34.64% and 11.20-25.61%,respectively.Based on the seven antioxidant activity assays,the contribution of all melanoidins groups to the antioxidant activity of soy sauce ranged from 33.98 to 76.35%.In addition,soy sauce melanoidins can inhibit the occurrence of oxidation reactions by chelating metal ions,scavenging free radicals and inhibiting XOD activity at source.