| Phosvitin phosphopeptides (Abbr. PPP) is a mixture that contains many kinds of short peptides, which is extracted from phosvitin. Most of the peptides contain conglomerate phosphoserine sequence (Serp-Serp-Serp-Glu-Glu) that is correlated with functional group of PPP. The biological function of PPP mainly indicated that, it can promote the absorption of calcium, iron and zinc absorptions and so on.The influence of concentration of substrate, concentration of alkaline and time on dephosphorylation was investigated, respectively. The optimized parameters for dephosphorylation were that: concentration of substrate 10g/L, Concentration of NaOH 0.1mol/L, time 20min.And then the influence of concentration of alkaLine on hydrolization was researched, the optimized parameters for hydrolization were that: concentration of substrate 1%, quantity of enzyme 5%, pH 8.0, time 4h. The uniform design was employed to optimize the step-by-step hydrolization in the study of trypsinase and dispase process and the equation was as follows:Y=-0.401-0.390×X1+1.37×X2 +8.04×10-3×X4+0.139X12 + 4.02×10-4×X32.Through an analysis of the equation, the optimized conditions for the hydrolization were that: quantity of enzyme 1.5%, pH 8.0, and temperature 40℃, time 120min. The equation for the step-by-step hydrolization in the study of trypsinase and dispase process was as follows:Y=15.12+0.46×X1+1.18X2+1.65X3 +0.99X1X2-0.14X1X3+0.1X2X3-1.37X12-0.62X22-1.32X32.And the optimized conditions were that: quantity of enzyme 5%, pH 9.0, and temperature 40℃, time 90min. Response surface design was used to optimize the coordinative hydrolization of trypsinase and alcalase and the equation was obtained:Y=15.12+0.46×X1+1.18X2+1.65X3+0.99X1X2- 0.14X1X3+0.1X2X3-1.37X12-0.62X22-1.32X32.And the optimized parameters for the process were that: temperature 51.13℃, time 159.66min, pH 8.54, and the DH reached 16.64%.The GB method was used to analyze the common nutritional ingredients in the materials and the samples. The atomic absorption spectrophotometry (AAS) was used to determine the Ca contents in the samples. And the results indicated that, the Ca contents in PPP1 was 8.446%, PPP2 10.35%, PPP3 11.375%, respectively. Besides, the verification test of AAS was conducted, and the results showed that, the RSD in the analysis of the precision, reproducibility, recovery rate was all less than 1.2%, which showed that the AAS was reliable to analyze the Ca contents in the samples.The solubility and stability of PPP was studied in this paper. Firstly, the influence of the NaCl, EDTA, and citric acid on the solubility was compared, and the results indicated that the PPP had the best solubility in EDTA, approaching to 90%. Besides, the pH value had some impact on the solubility of PPP, when the pH value was 6.0 the solubility was maximized. In the research of PPP stability, when the temperature was 100℃and time 30min, there was less change of the absorbance that also indicated that the thermo-stability of PPP was good. On the other hand, when the Ca2+ existed in the PPP samples solution the absorbance was stable. Through a comparison with the crude protein, the emulsibility of the PPP decreased by 1.72 times.Based on the pre-column derivation, the HPLC was employed to analyze the ingredients of amino acids of PPP. On the one hand, the equation of linear regression and the coefficient correlation were calculated, and the coefficient correlation varied from 0.9961877 to 0.9999369, the linear ranged from 0.0005μmol/mL to 0.003μmol/mL. On the other hand, the verification test was conducted and the results indicated that, the RSD ranged from 0.373% to 1.986%, and the RSD of reproducibility from 0.316% to 1.817%, the recovery rate from 0.41% to 1.93%, above of which showed that the HPLC was reliable to analyze amino acids of PPP.In vitro research of PPP referring to maintaining Ca levels, different PPP products prepared by different enzyme system were analyzed. The results indicated that the PPP3 was the best to prevent the precipitation of calcium acid phosphate, which was in accordance with the reported references that maintaining Ca levels was better when the ratio of N/P was lower. Besides, when there was different concentration of PPP, the precipitation of calcium acid phosphate varied to some extent, meanwhile, the higher the concentration of the PPP, the better the precipitation of calcium acid phosphate was prevented. In vivo, calcium apparent absorptivity, serum calcium and phosphorus, serum alkaline phosphatase, tartrate-resistant acid phosphatase in growing period rats fed phosvitin phosphopeptides(PPP) diets was evaluated. Thus absorption ability by bounding and promoting calcium of phosvitin phosphopeptides was investigated. The apparent absorptivity in the rats fed with PPP were significantly higher (P<0.05) than that of the control rats, the level of strum calcium and strum phosphorus were no statistically significant at all groups (P>0.05). The level of ALP of experiment groups were significant lower then blank group(P<0.05),The amount of StrACP of experiment groups were significant lower then blank group(P<0.05),the diameter, length,weight and bone density of femur in rats of experiment groups were significant higher then blank group(P<0.05).The results indicate that,PPP can heighten significantly at promoting bone calcium absorption, stimulating to produce osteoblast, increasing activity of osteoblast, decreasing the amount of osteoclast, restraining activity of osteoclast, promoting development and healthy of skeleton. Three different PPP were adopted to this study,and N/P mole ratio of PPP3 was lowest,and the results illustrated that, this simples had the best role at promoting calcium absorption.In addition, among three dosages of PPP3, the high dose of PPP3 cheleted calcium was the best, and exhibited a dose-dependent pattern. |
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