牛奶营养丰富、口感香醇，是老少皆宜的营养食品。可是，一些人食用牛奶后会出现腹痛、腹胀甚至腹泻等不良症状，这是因为这部分人体内的乳糖酶含量较少或者乳糖酶缺失而导致的乳糖不耐受症。研究和生产质优价廉的低乳糖含量的牛奶，对于解决这一类人群的牛奶饮用问题具有十分重要的意义。 本研究在对3个厂家生产的乳糖酶进行筛选，选择出综合性能最优的乳糖酶基础上，对乳糖酶用不同的载体、方法进行固定化，对固定化后的乳糖酶的活性及重复次数、使用效果进行研究。选取最佳固定乳糖酶方案进行生产试验，研究最佳生产工艺参数，固定化乳糖酶的清洗、消毒方法，重复利用率等，验证其工业化生产的可行性。通过固定化乳糖酶的水解，得到水解率≥70 %的低乳糖牛奶，并通过固定化乳糖酶的重复利用达到节约成本的目的。主要结果如下： （1）通过市场调研，初选出科汉森（丹麦）、哈尔滨美华生物科技、山西恩泽生物科技公司生产的β-半乳糖苷酶。对酶活性测定、乳糖水解效率、最适宜pH值范围测定，发现：科汉森（丹麦）公司生产的游离β-半乳糖苷酶酶活性 5005 u/g，最适宜的pH值范围为 6.0~7.0，37℃±2℃、2.0 h~2.5 h条件下，在牛奶中的降低乳糖含量的水解率≥98 %。与其他2家生产的乳糖酶相比，该公司生产的乳糖酶具有酶活性高，pH范围适宜，乳糖水解率高，价格适中等显著优势。 （2）用食品级的海藻酸钠作固定化载体，对遴选出的乳糖酶进行固定化试验。研究发现：用4 %海藻酸钠溶液，将其与酶液在室温下混匀静置0.5 h 后，加入到1 % 的CaCl2固定化液中，固定1 h后，制得固定化酶。此种工艺制得的固定化酶最适水解温度为37℃±2℃，pH值6.0～7.0的范围内最佳，且活力稳定，机械强度表现良好。 （3）本研究对同一奶样使用四种检测方法开展对比试验。用高效液相法、碘量法、葡萄糖试剂盒法、Foss成分分析仪法分别进行牛奶中乳糖水解效果的测定，计算乳糖水解率。从检测准确度、快捷度、检测成本等方面对检测过程和结果进行综合对比，发现：Foss成分分析仪法测定1个样品的平均时间为10 min，2次独立测定数据的绝对值差为0.06 %。与其它3种检测方法相比，Foss成分分析仪法测定低乳糖牛奶的乳糖水解率最为方便、快捷，准确度高，稳定性好；不足之处为仪器和耗材费用高。 （4）将固定化后的乳糖酶加入牛奶中进行降低乳糖含量的工业化生产。水解温度为37℃±2℃，水解时间为2.0 h～2.5 h的生产条件下，牛奶中的乳糖含量降低比率≥70 %。对固定化的乳糖酶用浓度为3.0 %～5.0 %的H2O2溶液进行冲洗，再置于O3浓度为10~20 ppm的消毒室内消毒60 min后重复利用，可重复使用5次。

Milk is characterized by rich nutrients, mellow taste, which is suitable for people at different ages. However, after drinking milk, many persons have abdominal pain, abdominal distension and diarrhea and other symptoms. It is probably due to the low content of lactose in human body or lactose intolerance attributed to lactase deficiency. Therefore, it is necessary to study the production of low-lactose milk with high quality and low price. In this study, optimal lactase was selected from three different factories. In the meantime, the resulting lactase was immobilized by different carrier agents using various methods. The activity and using time of immobilized lactase were evaluated. Furthermore, the process parameters to produce immobilized lactase was optimized. The cleaning, sterilization and repeating using rate were investigated. Through immobilized lactase hydrolysis, low-lactase milk with the hydrolysis rate higher than 70% was obtained. In addition, the cost can be reduced through using immobilized lactase for many times. The main results are shown as follows: (1)Through market survey and primary screening investigation, beta galactosidase from Chuke Hansen (Denmark), Harbin Meihua biotechnology, Shanxi Enzebiotechnology company were selected. After determination of enzyme activity, lactose hydrolysis efficiency and optimum pH range, it was found that a free beta glycosidase produced by Hansen (Denmark) had the glycosidase activity of 5005 U / g and its optimal pH range was 6.0-7.0. After hydrolysis at 37℃±2℃ for 2-2.5 h, more than 98% of lactose in milk was hydrolyzed. Compared to the lactase produced by other two companies, lactase produced by Hansen was characterized by high enzyme activity, wide pH range, high hydrolysis rate and relatively low price. (2)Using food-grade sodium alginate as the immobilization carrier, the immobilization of selected lactase was carried out. 4% sodium alginate water solution was mixed with enzyme at room temperature and the mixture was incubated for 0.5 h. After that, 1% calcium chloride solution was added. The mixture was incubated for another 1h to obtain the immobilized enzyme. The optimal temperature of the resulting immobilized enzyme was 37℃±2℃ and the optimal pH was 6.0-7.0. The enzyme activity was high and the mechanical properties were also satisfactory. (3)Four detecting methods were compared using the same milk sample. Specifically, HPLC, iodine value method, glucose reagent box method and Foss component analyzer were used. The hydrolysis effectiveness of lactase was used as the detecting parameter. In terms of detecting accuracy, efficacy and cost, the time needed for the Foss component analyzer was 10 min and the absolute difference value between two samples was 0.06%. Analysis using Foss component analyzer for detection of hydrolysis rate had several advantages, such as fast, convenient, accurate and stable. However, the detection cost was high. (4)The immobilized lactase was added to milk to reduce lactose in an industrial scale. More than 70% of lactose was hydrolyzed if the temperature was 37℃±2℃ and hydrolysis time was 2.0-2.5h. Immobilized lactase was first cleaned using 3.0-5.0% H2O2. Next, it was sterilized by ozone at 10-20 ppm for 60 min. In this case, the immobilized lactase can be used for 5 times.