Lynch Joanna M, Barbano David M, Fleming J Richard
Department of Food Science, Northeast Dairy Foods Research Center, Cornell University, Ithaca, NY 14853, USA.
J AOAC Int. 2007 Jan-Feb;90(1):196-216.
The objective of this collaborative study was to determine the method performance characteristics of a spectrophotometric enzymatic assay for measuring the lactose content of fluid milk. The principle behind the method is similar to that of AOAC Method 984.15 but with significant modifications and added quality control. Additionally, lactose concentration is expressed on a weight/weight (wt/wt) rather than a weight/volume (wt/vol) basis. The principle of the method is the hydrolysis of lactose to D-glucose and D-galactose by beta-galactosidase, followed by the oxidation of beta-D-galactose by nicotinamide adenine dinucleotide (NAD+) in the presence of beta-galactose dehydrogenase. The reaction is catalyzed by the addition of aldose-l-epimerase, which accelerates the mutarotation of alphha-D-galactose to beta-D-galactose. The amount of reduced nicotinamide adenine dinucleotide (NADH) formed is measured at 340 nm and is proportional to the amount of lactose present. Important aspects of the assay include preparing the assay solution by weight (rather than volume), mixing the contents of the spectrophotometric cuvette without losing solution, inclusion of aldose-l-epimerase, specifying spectrophotometer characteristics, and accounting for the optical path length of the spectrophotometric cuvettes. In the collaborative study, 11 laboratories tested one lactose standard and 8 pairs of blind replicate raw, processed, and formulated milks with an anhydrous lactose content between 3.0-7.2%. Statistical performance, in units of g/100 g anhydrous lactose, for the milk materials within the applicability of the method was as follows: mean = 4.4040, Sr = 0.0130, SR = 0.0250, RSDr = 0.29%, RSDR = 0.57%, r = 0.0364, and R = 0.0700. Standard and marginal recoveries were 98.66 and 99.53%, respectively. Method performance represented a significant improvement over what would be achieved if path length was not accounted for or the assay was done volumetrically. The Study Directors recommend that the method for determination of the lactose content of fluid milk by the spectrophotometric enzymatic method using weight additions and path length adjustment be adopted Official First Action.
这项合作研究的目的是确定一种用于测量液态奶乳糖含量的分光光度酶法的方法性能特征。该方法背后的原理与美国分析化学家协会（AOAC）方法984.15类似，但有重大修改并增加了质量控制。此外，乳糖浓度以重量/重量（wt/wt）而非重量/体积（wt/vol）为基础表示。该方法的原理是通过β-半乳糖苷酶将乳糖水解为D-葡萄糖和D-半乳糖，然后在β-半乳糖脱氢酶存在下，烟酰胺腺嘌呤二核苷酸（NAD +）将β-D-半乳糖氧化。通过添加醛糖-1-表异构酶催化该反应，醛糖-1-表异构酶可加速α-D-半乳糖向β-D-半乳糖的变旋。在340nm处测量形成的还原型烟酰胺腺嘌呤二核苷酸（NADH）的量，其与存在的乳糖量成比例。该测定的重要方面包括通过重量（而非体积）制备测定溶液，在不损失溶液的情况下混合分光光度比色皿中的内容物，加入醛糖-1-表异构酶，规定分光光度计特性，以及考虑分光光度比色皿的光程长度。在合作研究中，11个实验室测试了一种乳糖标准品以及8对盲样重复的生乳、加工乳和配方乳，其无水乳糖含量在3.0 - 7.2%之间。在该方法适用范围内，奶样的统计性能（以g/100g无水乳糖为单位）如下：均值 = 4.4040，Sr = 0.0130，SR = 0.0250，RSDr = 0.29%，RSDR = 0.57%，r = 0.0364，R = 0.0700。标准回收率和边际回收率分别为98.66%和99.53%。与不考虑光程长度或采用体积法进行测定相比，该方法的性能有显著提高。研究负责人建议采用通过重量添加和光程长度调整的分光光度酶法测定液态奶乳糖含量的方法作为官方首次行动方法。
