Based On The Discovery Of Soybean Enzymatic Hydrolysate Zinc Complex Peptide And The Research On The Mechanism Of Peptide-zinc Complexation

Zinc,as an essential trace element of human bodies,participates in the metabolic regulation of carbohydrates,fats and proteins.It plays an important role in maintaining the normal physiological functions.Zinc deficiency can lead to various secondary diseases such as impaired growth,immune system defects,and neurological behavior disorders.The traditional zinc supplement is inorganic zinc such as ZnSO₄.The poor solubility of inorganic zinc in the intestine makes low absorption and side effects on the human bodies.Proteins and peptides chelate with zinc ions and to form a stable and soluble peptide-Zn complex.The peptide-Zn complex effectively improves the zinc stability in the digestive system and enhances zinc absorption and bioavailability.It provides new research idea and theoretical basis for solving the problem of zinc deficiency.In this paper,soy peptides with zinc binding capacity were prepared from soy protein through enzymatic technology.The effects on zinc binding capacity of soy peptides were investigated.A specific zinc binding peptide was isolated and identified from soy peptides.The binding mechanism and physical and chemical properties of peptide-Zn complex were explored.The details of the work would be shown as follows:Soy protein was hydrolyzed by Alcalase to prepare soy peptides.The zinc binding capacity was regarded as an indicator and the optimal condition for the preparation of soy peptides-Zn complex were investigated.The optimal condition was determined as ZnCl₂ concentration of 0.45mmol/and reaction time of 1 h at 75?and pH 5.5.The zinc binding capacity of soy peptides was26.96±1.22 mg/g under the optimal condition.The results of Ultra-violet spectroscopy and Fourier transform infrared spectra?FTIR?indicate that zinc could be binding by carboxyl,amino group and peptide bond in peptides,thus forming soy peptides-Zn complex.The results of fluorescence spectroscopy,scanning electron microscope?SEM?and zeta potential test showed that intramolecular and intermolecular folding and aggregation occurred during the formation of the chelation.Soy peptides were separated by ultrafiltration.Four fractions were obtained with molecular weights>5 kDa?F1?,1-5 kDa?F2?,0.5-1 kDa?F3?and<0.5 kDa?F4?and its zinc binding capacity was determined as 32.18±2.97 mg/g,43.83±1.25 mg/g,32.08±2.83 mg/g and8.98±0.57 mg/g,respectively.The zinc binding capacity of F2 was higher than other ultrafiltration fractions.The content of amino acids?Asp,Ser,Glu,His,Lys and Arg?with metal ion binding capacity in F2 was higher than that of other ultrafiltration fractions.F2 was separated by size exclusion chromatography to obtain three main fractions and zinc binding capacity was determined as 39.28±4.83 mg/g?F21?,54.28±4.62 mg/g?F22?and 36.69±4.23 mg/g?F23?.F22,which exhibited the highest zinc binding capacity,was separated by reversed phase-high performance liquid chromatography?RP-HPLC?to obtain three kinds of peptide with zinc binding capacity of 72.47±2.52 mg/g?F221?,39.69±2.56 mg/g?F222?and 29.05±1.95 mg/g?F223?.F221 was confirmed as zinc binding peptide with the highest zinc binding capacity.The amino acid sequence of peptide was confirmed to be Lys-Tyr-Lys-Arg-Gln-Arg-Trp?KYKRQRW?and the molecular weight was 1064.4 Da by using electrospray ionization-tandem mass spectrometry?ESI-MS/MS?.According to the results of FTIR and ¹H nuclear magnetic resonance?¹H NMR?,the zinc binding sites of peptide involved the oxygen of the carbonyl group in Trp and nitrogen of the amino group in Lys residue.The coordination with zinc ions changes the second structure of the peptide,and a part of?-sheet structure was transformed into random coil structure.The affinity for peptide and zinc ions was determined as 2.63.It was found that the thermal stability of the peptide-Zn complex is higher than that of the zinc binding peptide by using thermogravimetric-differential scanning calorimetry?TG-DSC?.It indicates that the chemical bonds of peptide-Zn complex required more heat to be destroyed.The peptide-Zn complex showed good zinc solubility under acidic conditions.Under pH 8.0,the zinc solubility of peptide-Zn complex was determined as 29.63±4.39%and higher than that of ZnSO₄?0.89±0.255%?.It demonstrates that the peptide-Zn was more stable than ZnSO₄ at various pH conditions.After simulated digestion in vitro,the zinc solubility of the peptide-Zn complex was11.74±2.96%,significantly higher than that of ZnSO₄?2.31±1.74%?,indicating that the peptide-Zn complex has the potential to improve zinc bioavailability.