Study On The Preparation And Osteogenic Activity Of Calcium-binding Peptides From Black Bean

Calcium deficiency has become a global nutritional problem that plagues the human health.Calcium supplement is of great significance for maintaining the balance of bone metabolism,preventing or reducing bone metabolism diseases such as osteoporosis.Food-derived calcium binding peptide is a new type of calcium supplement with high efficiency and low toxicity.It has the potential to positively regulate bone metabolism and enhance the activity of osteoblasts.However,there are few studies on the effect of plant-derived calcium binding peptides on osteogenic activity,and the structure and action mechanism of active components are still unclear.In this paper,with the help of enzymatic hydrolysis technology and fingerprint,the structure of calcium binding peptide derived from black bean protein was identified and characterized,and the mechanism of calcium binding was analyzed.Then the MC3T3-E1 cell model was used to explore its osteogenic activity and action mechanism.The paper aimed to provide scientific basis for the development of plant-based calcium supplement.The main results were as follows:Firstly,a series of black bean protein hydrolysates were prepared using enzymatic hydrolysis technology,and their calcium binding activity and structural characteristics were evaluated.The results showed that the calcium binding activity of the hydrolysates prepared by the four proteases was ficin>alkaline>bromelain>trypsin.The optimal capacity of ficin-treated black bean protein hydrolysates(BPHs)reached 77.54±1.61?g/mg,where the optimal hydrolysis conditions of ficin were temperature of 70?,p H value of 6.2,enzyme concentration of 1.61%(w/v)and time of 3 h.The structural changes of BPHs were obvious after the chelation of calcium.The content of aspartic acid and glutamic acid in the complex(BPHs-Ca)were significantly higher than that of BPHs(p<0.05).Compared with BPHs,the ultraviolet absorption value and fluorescence intensity of BPHs-Ca were significantly reduced,and the infrared absorption peaks were shifted.It is speculated that the binding mainly occurring on carboxyl oxygen and amino nitrogen atoms.In addition,the peptide surface appeared irregularly rough after the formation of calcium complex,which was due to the adhesion of calcium ions to the surfaceSecondly,the high performance liquid chromatography(HPLC)fingerprint of BPHs was constructed to identify and characterize the structure and activity of calcium binding peptides.The results showed that 28 common characteristic peaks were obtained in the spectrum.Among them,characteristic peak 2 and 4 had the strongest calcium binding activity,which were91.02±4.37?g/mg and 98.84±7.04?g/mg,respectively.After the analysis of liquid chromatography-mass spectrometry(LC-MS/MS),there were 31 and 67 peptides identified from characteristic peak 2 and 4 with a molecular weight ranging from 307.1 to 3301.5 Da.Eight small peptides without digestive protease action sites in the structure were selected,including TVVP,LTP,DIV,ITDI,NIDI,TISSENI,IEI,and CVE.The calcium binding capacity ranged from 110.56±4.10?g/mg to 161.52±3.64?g/mg.The molecular docking technology was used to explore the binding mechanism of black bean peptides and calcium ions.It was found that the above 8 peptides could form stable complexes with calcium ions.Among them,the binding mode of NIDI with calcium ion was via the?mode,and the binding modes of the other seven peptides was via the unidentate mode.The main binding sites of peptides were the carboxyl oxygen atoms of the terminal amino acid of the peptide chains.Finally,the effects of black bean calcium-binding peptides on the proliferation,differentiation and mineralization of MC3T3-E1 cells were studied,and their action mechanism was explored.The results showed that BPHs and CVE could significantly promote the proliferation,differentiation and mineralization of MC3T3-E1 cells.At the stage of proliferation,the promotion effects of BPHs and CVE on MC3T3-E1 cells presented a time and concentration-dependent manner,and the cell proliferation rate was 162.56±20.03%and147.99±12.70%,respectively.At the differentiation stage,BPHs and CVE significantly increased the activity of cellular alkaline phosphatase(ALP)(p<0.05).After 7 days,there was no significant difference with the change of CVE concentrations(0.1?100?M).The growth rate of ALP activity reached the highest value(460.14±8.20%)when treated with 200?g/m L BPHs.At the mineralization stage,BPHs and CVE increased the mineralization level of extracellular matrix(p<0.05),and the former showed an obvious concentration-dependence manner.The combination of CVE and 4 m M Ca Cl₂ showed a synergistic effect on the mineralization of MC3T3-E1 cells,and the superior effects occurred when they were added together.In addition,BPHs and CVE significantly increased the expression levels of BMPs,Smads,and transcription factors including Runx2 and Osterix(p<0.05),and up-regulated the expression of differentiation-related genes such as ALP and OC.It is speculated that BPHs and CVE might promote the osteogenic activity of MC3T3-E1 through the BMP/Smad signaling pathway.