Preparation And Physicochemical Properties Of Enzymatic Hydrolysates And Ferrous Complexes Of Antarctic Krill

Antarctic krill is abundant in high quality protein with reasonable amino acid composition,which meets the ideal protein model recommended by FAO/WHO.At present,the exploitation and utilization of Antarctic krill in China are still in the primary stage,and the utilization of protein is not enough,resulting in a waste of resources.The mineral element iron plays an important physiological role in promoting human growth,development and metabolism.Iron deficiency can cause many diseases,which can be improved by metal chelate of having potential value and has become one of the hot spots in the world.In this paper,the optimal enzymatic hydrolysis process for the preparation of enzymatic hydrolysates of Antarctic krill?AKH?was determined,and the physicochemical properties and antioxidant activities of AKH were analyzed.The Antarctic krill enzymatic hydrolysates-ferrous chelate?AKH-Fe?was prepared and its structure was analyzed.The effect of AKH on iron uptake was evaluated by Caco-2 cell model.The specific contents and conclusions are as follows:The water content,crude protein content,crude fat content,ash content and total sugar content of Antarctic krill were?75.61±0.27?%,?15.09±1.49?%,?4.50±0.35?%,?3.47±0.13?%and?0.24±0.01?%,respectively.Ferrous chelating rate as the main evaluation index,the degree of hydrolysis as auxiliary evaluation index,selected from the five kinds of protease Alcalase 2.4 L was the suitable enzyme.hydrolysis process were optimized using one-factor-at-a-time method and response surface methodology?RSM?.The optimal hydrolysis conditions were obtained as follows:the enzyme solution temperature 57.59?,enzyme/substrate ratio 5.5%,pH 8.79,enzymolysis time 300 min.The actual iron-chelating rate of the enzymatic hydrolysates prepared under the optimized condition was?77.77±0.72?%,DH was?23.22±0.16?%,which was in good agreement with the predicted value.The relative molecular weight of AKH was amount of 180¹000 Da and accounted for 89.28%.Amino acid composition analysis showed that specific amino acid contents were relatively high in AKH such as Lys,Glu,Asp,Arg,etc.In vitro free radical scavenging experiments showed that AKH had good scavenging ability against DPPH free radicals,hydroxyl free radicals and superoxide anions,and showed a good dose-effect relationship,with IC₅₀ of 1.92 mg/mL,2.09 mg/mL and 15.48 mg/mL,respectively.When AKH concentration was 10 mg/mL,DPPH radical scavenging rate was up to 87.52%,hydroxyl radical scavenging rate was 90.89%,superoxide ion scavenging rate was 40.05%,and the reduction force was relatively weak,only 0.3391.Functional analysis shows that AKH has good solubility.When the pH value is 6,the solubility reaches the maximum value?98.06±1.14?%.The oil absorption was peaked at 60?for?317.12±0.79?%.AKH has good foaming property in the pH range of 4¹2.When the pH value was 8,the foaming property was the lowest,and the foam stability first decreased and then increased with the increase of pH value,and then gradually leveled off.In the range of pH 2¹0,AKH's emulsifying property first increased and then decreased,and reached the maximum value?25.35±2.10?%when the pH value was 6,while the emulsifying stability was the lowest.Antarctic krill enzymatic hydrolysates prepared before was used as raw materials,ferrous chelating rate and chelate yield were acted as the evaluation index,one-factor-at-a-time method and orthogonal experiment were used to optimize the experimental design,the optimal chelate condition were obtained as follows:peptide mass ratio 3:1,iron chelation temperature 40?,chelation time 40 min,pH value 5.5,peptide concentration 4%,ethanol volume ratio 6.Under these conditions,the ferrous chelation rate and chelate yield of AKH-Fe were?77.25±2.11?%and?41.03±0.68?%,respectively.Na₂S method indicated that there was no free polypeptide in the chelate and the sample purity was high.Ultraviolet scanning showed that compared with AKH and FeCl₂·4H₂O,the atomic spatial structure of AKH-Fe was changed,and the valence electron transition was different,so that the position of characteristic absorption peak was changed.Fourier transform infrared?FTIR?spectroscopy suggested that after Fe²⁺chelated with AKH,the blue shift of amido A,red shift of amido I,and the disappearance of the absorption peak of ammonium salt indicate that the amino and carboxyl groups of AKH were involved in the chelation reaction.Scanning electron microscopy?SEM?showed that the microstructure of AKH changed dramatically after chelated with Fe²⁺,turning from loose and sparse flake structure into granular aggregates.It is confirmed that the AKH-Fe was a new substance different from AKH.The Caco-2 cell model was established by in vitro culture,and the integrity of the cell model was evaluated by transmembrane resistance and alkaline phosphatase activity.Experimental results showed that the cells were grown in 15 days later,the resistance value is higher than 400?·cm²,resistance has been stable since.After 21 days of culture,the AKP activity on AP side was 9.64 times that on BL side,and caco-2 cells were completely differentiated.The results of Scanning electron microscopy?SEM?and Transmission electron microscope?TEM?suggested that the cells had differentiated into distinct microvilli with formed dense and neat brush edges.The results indicated that caco-2 cell model was successfully constructed.Cytotoxicity experiments showed that AKH had good biological safety,because it had no obvious toxic and side effects on caco-2 cells.Iron bioavailability experiments showed that AKH had a good effect on promoting iron absorption.