大米肽中压离子交换色谱分离及其免疫活性评价

Isolation and identification of immunoactive peptides from rice protein hydrolysates with medium-pressure ion exchange chromatography

为进一步提高经过DA201-C型大孔吸附树脂脱盐、40%乙醇梯度洗脱的大米免疫活性肽RPHs-A3组分的纯度和免疫活性,采用中压离子交换色谱对其进行进一步分离纯化,并对分离得到组分的免疫活性进行了评价。结果表明,采用WorkBeads 40Q强阴离子交换树脂、2.6cm×40cm色谱柱对RPHs-A3组分进行分离的最佳条件为:上样浓度30mg/mL,上样量5 mL,起始缓冲液A为pH 9.0的0.01mol/L Tris-HCl,洗脱缓冲液B为含1 mol/L NaCl的pH 9.0、0.01 mol/L Tris-HCl,上样、洗脱流速分别为1,10mL/min。共收集到5个组分,其中RPHs-A3-B3组分在MTT试验和小鼠巨噬细胞脂多糖(LPS)炎症模型中皆表现出较高免疫活性,对RAW264.7细胞具有显著增殖作用,其SI值为1.315;且对炎症模型中细胞内NO释放量具有显著抑制作用(P<0.05),抑制率为8.28%。采用中压离子交换色谱能有效地对RPHs-A3组分进行分离纯化,使SI值从1.273提高到1.315,且对细胞内NO释放量具有显著抑制作用。

Medium-pressure ion exchange chromatography was used for further fractionation of rice immunoactive peptides obtained from Trypsin hydrolyzation and gradient ethanol elution with DA201-C macroporous adsorption resin（40% ethanol fraction）.The results showed that the optimal parameters for fractionation of RPHs-A3 fractions by WorkBeads 40 Qon a 2.6 cm×40 cm columns were as follows：Sample：5 mL,30 mg/mL 40% ethanol fraction;Equilibration buffer A：pH 9.0,0.01 mol/L Tris-HCl;Elution Buffer B：pH 9.0,0.01 mol/L Tris-HCl containing 1 mol/L NaCl;Sample flow rate：1 mL/min;Elution flow rate：10 mL/min.Five fractions were obtained after NaCl gradient elution.The results of MTT assay and LPS-stimulated RAW264.7 macrophages inflammation model showed that fraction RPHs-A3-B3 had the highest immune activity.RPHs-A3-B3 produced a significant effect on the proliferation of RAW264.7 macrophages with a stimulation index value of 1.315.Meanwhile,RPHs-A3-B3 significantly decreased the production of NO in LPS-stimulated RAW264.7 macrophages（P〈0.05）.The inhibition rate was 8.28%.The research indicated that medium-pressure ion exchange chromatography can effectively fractionate rice immunoactive peptides.The SI value increased from 1.273 to 1.315.And RPHs-A3 had a significant inhibitory effect on intracellular NO release.