Preparation,characterization Of Peptide-calcium Chelate From Antler Bone And The Study On Its Chelation Mode

As the most abundant mineral element in the body,calcium plays a very important role in maintaining bone structure and physiological functions.Calcium deficiency is associated with the development of many diseases,such as osteoporosis,arterial hypertension,and kidney stones.Therefore,the study of calcium fortification drugs and foods that can be effectively absorbed has attracted a lot of attention from scholars.In recent years,peptide-calcium chelates have been shown to be more eadily absorbed and utilized by the intestinal tract due to their unique chelation system and transport mechanism,thus improving calcium bioavailability.As the only renewable organ in mammals,antler has a very rapid growth rate of 1.7cm/day,but its roots are underutilized due to severe ossification,and studies have shown that antler bone are rich in inorganic elements,amino acids and proteins;therefore,the antler bone are considered to be a good source of calcium chelating peptides.In this study,neutral protease,alkaline protease,trypsin,flavorourzyme and papain were selected to prepare calcium chelating peptides from the antler bone,and the most suitable proteases were screened for calcium chelation rate and degree of hydrolysis.The bone calcium was extracted by acid solubilization method,and malic acid,citric acid and mixed acid(malic acid and citric acid 1:1)were selected to prepare bone calcium,and the most suitable acid was screened by bone calcium content as an indicator.The peptide enzymatic digestion process and bone calcium extraction process were further optimized by single-factor test.The results showed that the best enzymatic hydrolysis conditions for peptide were 7000U/g,10%substrate concentration,p H8.5,and 50?for 5h;the best extraction conditions for bone calcium were malic acid,10%acid,50?,and 1h extraction time.Caco-2 cells were used to detect the effect of different concentrations of peptide on cell viability,and to evaluate the effect of peptide on calcium transport.The results showed that the cell viability was 1.29±0.02 at 1000?g/m L.Compared with the blank control group(Ca Cl₂),the calcium transfer volume of bone calcium was nearly twice that of Ca Cl₂,indicating that the antler bone could be a new source.Compared with the bone calcium group,the addition of polypeptide could significantly promote the transport of bone calcium(P<0.05).In order to further isolate the specific functional peptides in the calcium chelating peptide and analyze the peptide-calcium binding mechanism,the peptides were separated and purified by hydroxyapatite chromatography(HAC),gel filtration chromatography and reversed-phase high performance liquid chromatography(RP-HPLC),and the peptide sequences were identified by LC-MS/MS,and the chelation mechanism was further analyzed by Fourier transform infrared spectroscopy(FTIR),UV-vis spectroscopy(UV-vis),X-ray diffraction(XRD)and molecular dynamics simulation.The results showed that the peptide sequences obtained were TKLGTQLQL,LETVILGLLKT and KMVFLMDLLK,and their calcium chelating capacities were87.68±2.86%,80.72±0.93%and 67.97±0.98%,respectively.The FTIR spectra,UV-vis spectra and X-ray diffraction results showed that the peptide and calcium binding sites might involve the carboxy oxygen and amino nitrogen of the side chains of the peptides.The molecular dynamics simulations revealed that the calcium ions preferred to form complexes with the negatively charged carboxylic acid groups of leucine or threonine.In conclusion,this study established the peptide enzymatic hydrolysis process and bone calcium extraction process of antler bone,verified the efficiency of peptides to promote calcium transport.The functionsl peptides with specific calcium affinity by chromatographic methods,among which TKLGTQLQL and LETVILGLLKT with calcium chelating rate of 87.68±2.86%and 80.72±0.93%,respectively.The peptide-calcium chelation mechanism were determined by characterization methods.This research promoted the progress of peptide-calcium chelation research,and also proved that antler bone could be a very promising source of peptide-calcium chelate,providing a theoretical basis for its application in functional food.This study also provided a new idea for improving the utilization value of antler bone.