Study On Preparation Of Egg White Peptides Binding With Calcium And Their Effect On Promoting Calcium Absorption In Vivo

In order to obtain a kind of calcium supplement agent with high bioavailability, and increase the added value of egg white protein, enzymatic modification, enzyme screening and orthogonal test were carried out. The emzymolysis conditions of preparing egg white peptides (EWP) and egg white peptides binding with Ca2+ (EWP-Ca) were optimized. Egg white peptides were fractioned by ultrafiltration technology, and primary structures of the peptides with a high affinity to Ca were identified by HPLC-MS/MS. The ligand compounds of peptides and calcium were confirmed by infrared spectroscopy (IR), circular dichroism spectroscopy (CD), transmission electron microscopy (TEM) and amino acid analysis. In vivo effects of EWP-Ca on promoting Ca absorption were further examined in rats of calcium deficiency. Finally, egg shell was used as a natural source of calcium in the preparation of EWP-Ca. The main results were shown as follows:1. Study on preparation of egg white peptide with enhancing calcium absorptionThe optimum emzymolysis condition was as follow:material to water ratio was 6%, the [E]/[S] of Alcalase was 1:100, pH8.0,3h; then the [E]/[S] of Neutrase was 1:25, pH7.0,3h. Then the degree of hydrolysis (DH) of EWP was 8.66%, and soluble calcium binded by EWP was 21.15mg/L. The hydrolysis method of egg white protein using two kinds of enzymes was better than that using single enzyme.2. Study on preparation of EWP-Ca(1) The optimal conditions for preparation of ligand compounds was as follow: the ratio of EWP and CaCl2 was 2.5:1 (w/w), pH 9.5, substrate concentration was 4%, temperature was 55℃, reaction time was 20 minutes. The calcium content of product was 8.75%.(2) After ultrafiltration treatment, the binding calcium capacity of EWP was negatively correlated with their molecular mass. The fraction with Mm<1 kDa showed the strongest binding calcium capacity, and the calcium mass percent enhanced three times higher than that of the original hydrolytes, up to 23.95%.(3) The optimal conditions for preparation of calcium lactate by eggshell was as follow:the amount of eggshell power was 5g, lactic acid was 6 mL, the ratio of liquid and solid (w/w) was 15:1, temperature was 30℃, reaction time was 3.2h. Under these conditions, the purity of calcium lactate was up to 96.94%, the yield was 99.35%. 3. Study on the effect of EWP-Ca on promoting the calcium absorption in vivoCompared with the calcium deficiency model group, high dose of EWP-Ca (the dose of Ca is 106.4 mg/g-bw) could significantly increase the femur diameter, dry weight, calcium content and bone mineral density (P<0.01). Morever, compared with the CaCO3 group with the same calcium content, femur diameter (P<0.01) and bone calcium content were significantly increased (P<0.05). It shows EWP-Ca could make bone stronger and the calcium supplement effect was better than CaCO3 with the same calcium content.4. Study on the structure of EWP and EWP-Ca(1) The differences between EWP and EWP-Ca were exhibited by infrared spectroscopy and circular dichroism spectroscopy. Infrared spectra showed that both amino groups and carboxyl groups of EWP were involved in the coordination with Ca2+, and the egg white peptides formed a kind of bridging bidentate complex with calcium. The differences between them in circular dichroism spectroscopy showed the chain conformation and chain-chain interactions of EWP have changed obviously and we infered that EWP binded with calcium.(2) The microstructure of EWP and EWP-Ca were observed by TEM. It showed that salt bridge can be formed between two or more carboxyl by Ca2+, becoming aggregate, when concentration of Ca2+ was high enough.(3) The result of amino acid analysis showed that total content of acidic amino acid and amide amino acid in EWP-Ca was higher than that in EWP. Using HPLC-MS combined with online searching in tandem mass spectrometry database, the primary structures of six kinds of small peptides were revealed as follow:QNQ, QAF, QMM, MVLV, NVLQPS and KLKPI. The content of amino acid amide (Q and N) was high in six kinds of peptides, and the free amino groups in side chain of amide compounds may coordinate with Ca2+. The terminal group atoms in tripeptide and tetrapeptide can coordinate with Ca2+, while both the terminal group atoms and the side chain atoms of Q or K in pentapeptide and hexapeptide can coordinate with Ca2+. All these six kinds of short peptides could promote calcium absorption.