Preparation And Characteristics Of Iron Chelated With Bone-meat Polypeptide From Rana Chensinensis

Iron plays an important role in normal substance metabolism and composition.At the same time,hemoglobin,myoglobin and other substances can not be lack of iron participation.It is also an important part of many enzymes in the body.How to improve the absorption and utilization of metal ions in human body has become the focus of scholars' research.Rana chensinensis is a famous medicinal herb with the reputation of "nourishing soft gold" in Northeast China,which is rich in nutrients.However,the development of bone and meat polypeptide products of Rana chensinensis has not been fully developed.In this paper,Rana chensinensis bone and meat polypeptide liquid was prepared by enzymatic hydrolysis.The polypeptide liquid was chelated with inorganic iron source to obtain the chelated iron of Rana chensinensis bone and meat polypeptide,a powerful iron supplement.The optimum protease for the hydrolysis of frog bone and flesh was determined by single factor,and the chelating process of frog bone and flesh polypeptide solution with ferrous chloride was further optimized by orthogonal test.The physical and chemical properties of iron chelated by frog bone and flesh polypeptide were studied by quantitative and qualitative analysis techniques.The digestion of iron chelated by frog bone and flesh polypeptide was investigated by simulating gastrointestinal environment in vitro,and the chelating process of iron chelated by frog bone and flesh The effect of bone-meat polypeptide chelated iron on Caco-2 cells was studied.The specific test results are as follows:(1)The effects of four enzymes on bone and meat hydrolysis of Rana chensinensis were investigated by single factor and control variable method.Papain was chosen as the best enzymes and the optimum pH was 7.0.L9(34)orthogonal experiment was designed to study the effects of hydrolysis time,temperature,material-liquid ratio and enzyme dosage on the hydrolysis degree of frog bone and meat,and to select the optimum conditions for enzymatic hydrolysis.When the ratio of material to liquid is 1:5,the enzyme dosage is 3000 U/g,the hydrolysis time is 120 min at 60?,the hydrolysis degree of frog bone and meat is the highest,and the content of amino nitrogen is 1.963 mg/ml.(2)Chelating the polypeptide solution of frog bone and flesh with ferrous chloride to determine the chelating rate.Orthogonal experiments were designed to study the effects of chelating time,temperature,pH value of reaction system,dosage of compound antioxidant and molar ratio of peptide to metal ion on the chelating rate.When the molar ratio of bone-meat polypeptide to iron ion is 2:0.5,pH is 6.0,the amount of compound antioxidant is 1%,and the temperature of water bath at 60? is 45 min,the chelating effect of bone-meat polypeptide of Rana chensinensis is the best.The chelating rate was 93.0312%.(3)Polypeptide chelated iron of Rana chensinensis was purified by organic solvent precipitation method.The effects of the kinds of organic solvents,the amount of organic solvents used and the purifying time were investigated with the purifying rate as the index.When isopropanol was used as the solvent,the volume ratio of chelated iron solution with frog bone and meat polypeptide was 1:7,and the reaction time was 120 minutes,the purity rate reached 88.9237%.(4)To study the physical and chemical properties of iron chelated by bone and flesh polypeptide of Rana chensinensis.BCA method showed that protein content increased after chelation of bone and meat polypeptide of Rana chensinensis.GPC method showed that the molecular weight of bone and meat polypeptide mainly concentrated in 2-60 kDa,while after chelation,the molecular weight mainly distributed in 2-400 kDa,and the molecular weight distribution became wider.GPC method showed that the total content of free amino acids decreased after chelation,especially those with negative charge,mainly due to the binding of iron ions to polypeptide.Multiple functional sites combine to form proteins with larger molecular weight,and negative charge amino acids play a major role.Infrared spectroscopy showed that-COOH and amide III were involved in chelating reaction to form new substances.In the ultraviolet spectrum,the characteristic absorption peaks of iron chelated by bone and flesh polypeptide of Rana chensinensis were purple shift compared with that before chelating,and the absorption intensity was higher than that before chelating,which proved that the structure of bone and flesh polypeptide of Rana chensinensis changed after chelating.(5)The digestion process of stomach and intestinal juice was simulated by bone-meat polypeptide chelated iron of Rana chensinensis,which proved that the chelated iron of bone-meat polypeptide of Rana chensinensis was stable under the action of pepsin and trypsin.The results of cytotoxicity test with Caco-2 and protein content test with BCA showed that a certain concentration of iron chelated with frog bone-meat polypeptide could significantly promote cell growth,promoting the growth of cell protein content,laying a theoretical foundation for further study of the effect of bone and flesh polypeptide chelated iron on the cells of Rana chensinensis.