牦牛乳酪蛋白磷酸肽纯化工艺优化及其促钙吸收作用

Optimization of the Purification Process of Yak Casein Phosphopeptide and Its Calcium Absorption-promoting Effect

摘要:

为了探究牦牛乳酪蛋白磷酸肽（Casein Phosphopeptide, CPP）产品性质和持钙能力，该试验以牦牛乳酪蛋白为原料，采用胰蛋白酶酶解，静态吸附试验确定洗脱剂，同时分析上样量、上样流速、洗脱液浓度对CPP纯化工艺的影响；测定纯化后CPP的基本成分、功能性质、体外抑制磷酸钙和亚铁离子沉淀能力；建立低钙小鼠模型，以体质量、脏器指数、血清生化、股骨指数、骨密度和骨钙为指标，探究CPP在小鼠体内促钙吸收能力。结果表明：以0.2 mol/L稀盐酸为洗脱液，上样流速3 mL/min，上样量100 mL条件下获得的CPP纯化效果最优，其氮磷摩尔比为6.8，磷回收率为95.5%，谷氨酸含量18.55%，丝氨酸含量6.01%；体外促钙实验表明纯化后CPP能有效阻止磷酸钙、亚铁离子沉淀；动物实验结果表明：经28 d喂养，所有小鼠内脏指数均在合理范围内，纯化后CPP无慢性毒性，中高剂量组血钙、血磷含量显著高于模型组（P<0.05），碱性磷酸酶（Alkaline Phosphatase, ALP）值极显著低于模型组（P<0.01），高剂量组股骨指数显著高于碳酸钙组（P<0.05），CPP中、高剂量组骨钙、骨密度显著高于模型组（P<0.05）。综上，该实验可为牦牛乳功能性食品开发提供理论依据，为后续牦牛乳生物活性肽的相关研究提供参考。

Abstract:

To investigate the properties and calcium-holding capacity of yak casein phosphopeptide (CPP), this experiment was carried out using yak casein as the raw material, with trypsin being used for enzymatic hydrolysis. Static adsorption test was performed to determine the eluent, and the effects of sample amount, sample flow rate and eluent concentration on the purification process of CPP. The basic composition, functional properties, and in vitro inhibitory capacity against calcium phosphate and ferrous ion precipitation of purified CPP were examined. A low-calcium mouse model was established, and the ability of CPP to promote calcium absorption in mice was investigated using body weight, organ index, serum biochemical parameters, femur index, bone density and bone calcium as the indicators. The experimental results showed that the best purification effect on the CPP was obtained under the following conditions: 0.2 mol/L dilute hydrochloric acid as the eluent, 3 mL/min as the loading flow rate, and 100 mL as the loading volume, leading to a nitrogen to phosphorus molar ratio of 6.8, a phosphorus recovery of 95.5%, a glutamic acid content of 18.55% and a serine content of 6.01%. In vitro calcium promotion experiments showed that the purified CPP could effectively prevent the precipitation of calcium phosphate and ferrous ions. The results of animal experiments showed that after 28 d of feeding, the organ indices of all mice were within a reasonable range. After purification, CPP showed no chronic toxicity. The medium- and highdose groups had significantly higher blood calcium and phosphorus levels than those of the model group (P<0.05), and had extremely significantly lower alkaline phosphatase (ALP) values than those in the model group (P<0.01). The femoral bone index in the high-dose group was significantly higher than that in the calcium carbonate group (P<0.05). The bone calcium and bone density of the medium- and high-dose CPP groups were significantly higher than those of the model group (P<0.05). In conclusion, this experiment can provide a theoretical basis for the development of functional yak milk foods and a reference for the subsequent research on yak milk’s bioactive peptides.