[目的]通过凝乳酶酶解牛乳得到乳源糖巨肽（CGMP）,利用CGMP对三氯乙酸的耐受差异,实现CGMP的分离与纯化。利用β-酪蛋白在低温低酸条件下容易游离的特性,从凝乳酶酶解后的酪蛋白中分离出β-酪蛋白。[方法]鲜牛乳经过脱脂后,加入适量凝乳酶在35 ℃条件下充分酶解,酶解后样品离心后得到上清液和沉淀。在上清液中加入三氯乙酸实现CGMP的除杂和沉淀,得到固体CGMP采用高效液相色谱仪测定其纯度;沉淀经过碱性复溶降低温度后,在全程低温条件下调节pH值到3.5,低温离心取上层清液,升温至35 ℃,再次常温离心后得到β-酪蛋白,并用高效液相色谱仪测定其纯度。[结果]当TCA浓度达到10%时,溶液中CGMP全部析出。收集分离纯化后的CGMP固体,经高效液相色谱法检测,CGMP纯度为80%~90%。在低温低酸性条件下,分离得到纯度较高的β-酪蛋白,纯度为85%~90%。
[Objective]The milk-derived glycomacropeptide（CGMP） was obtained by enzymatic hydrolysis of cow's milk using rennet.Due to the tolerance difference of CGMP to trichloroacetic acid,the separation and purification of CGMP was realized. The β-casein was separated from the casein hydrolyzed by rennet by the characteristic that β-casein was easily freed under low temperature and low acid conditions.[Method]The defatted fresh milk was fully enzymatically hydrolyzed by adding an appropriate amount of rennet at 35 ℃,and centrifuged to obtain the supernatant and precipitate. Trichloroacetic acid was added to the supernatant to remove the impurities of CGMP,and the purity of the obtained precipitated CGMP was measured by high performance liquid chromatography.The precipitation was redissolved in alkaline and the temperature was lowered.Then the pH value was adjusted to 3.5 under the condition of low temperature throughout the whole process.The supernatant was collected by centrifugation at low temperature.After that,the temperature was raised to 35 ℃ to gain β-casein with centrifugation at room temperature.Its purity was determined by high performance liquid chromatography.[Result]3% trichloroacetic acid could better remove impurities after CGMP enzymatic hydrolysis.CGMP was completely precipitated by 10% trichloroacetic acid.The purity of the final CGMP product was 80% to 90% and the β-casein with treatment of low temperature and low acid had a purity of 85% to 90%.
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