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| Optimization of extraction and separation conditions of milk-clotting protease from Moringa oleifera seeds |
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| DOI： |
| KeyWord:Moringa oleifera seeds milk-clotting protease ammonium sulfate fractional precipitation ion exchange chromatography milk coagulating activity SDS-PAGE |
| FundProject:国家自然科学基金地区项目（31560431）；云南省高校食品加工与安全控制重点实验室开放基金（YJK〔2014〕16KF03）；云南省现代农业奶牛产业技术体系资助项目（2019KJTX0014） |
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| Abstract: |
| To provide theoretical basis for the development and utilization of milk-clotting protease from Moringa oleifera seeds, the milk-clotting protease were extracted from defatted Moringa oleifera seed powder by salt method, and separated by ammonium sulfate (AS) fractional precipitation and ion exchange chromatography. The salt extraction conditions of milk-clotting protease from Moringa oleifera seeds were determined by single factor experiment and orthogonal experiment, and the ion exchange chromatography separation conditions were optimized by single factor experiment. The molecular weight and purity of the milk-clotting protease from Moringa oleifera seeds were analyzed using SDS-PAGE and RP-HPLC. The results showed that the optimal extraction conditions of salt method were obtained as follows: solid-liquid ratio 1∶ 10, extraction temperature 30 ℃, pH 7.15, sodium chloride concentration 0.3 mol/L and extraction time 0.5 h. Under 20%-40% AS saturation, the precipitated protein had the best milk coagulating activity and the total percentage of extracted protein reached 61.71%. The optimal separation conditions of ion exchange chromatography were obtained as follows: injection mass concentration 30 mg/mL, mobile phase pH 5.15, flow rate 1.68 mL/min, and mobile phase consisted of 0.05 mol/L sodium acetate and 0.05 mol/L sodium chloride. Under these conditions, the milk coagulating specific activity value of the milk-clotting from Moringa oleifera seeds reached 24 SU/mg.The molecular weight of milk-clotting protease was mainly distributed in the range of 35-45 kDa, and the purity was over 90%. This method can effectively extract and separate milk-clotting protease from Moringa oleifera seeds, and maintain its milk coagulating activity well. |
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