Determination of the lactose content of fluid milk by spectrophotometric enzymatic analysis using weight additions and path length adjustment: collaborative study.

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.

Effectiveness of temperature modification in decreasing the bias in milk fat test results between the Babcock and Ether extraction methods.

Both the Babcock (AOAC Method 989.04, revised Final Action 2000) and modified Mojonnier ether extraction (AOAC Method 989.05) methods are used in the dairy industry to determine the fat content of milk. Prior to revision in 1997, the Babcock method gave consistently higher fat test results than did the ether extraction. In 1997, a modification of the Babcock method was introduced to bring the results of the Babcock test into closer agreement with the ether extraction. The Babcock method was modified by lowering the temperatures used at various points in the method from about 57.5 to 48 degrees C to increase the density of the material in the Babcock column. A collaborative study of the modification indicated it was successful in bringing the Babcock and ether extraction results into agreement but suggested that performance of the modified method was not as good as that of the unmodified method. In the present study, substantial evidence is presented to validate the success of the Babcock modification in bringing test results into agreement with ether extraction, and to document that temperature modification does not adversely affect method performance. Data were evaluated from an on-going proficiency testing program where 8-15 laboratories tested 7 milk samples in blind duplicate once every 2 months. Laboratories used the unmodified method from 1995 through 1996 and the modified method from 1998 through 1999. Compared with ether extraction, test results from the unmodified Babcock test were consistently higher by an average of 0.022% fat. For the modified Babcock test, average test results were -0.003% fat lower than with ether extraction and not significantly different from zero. AOAC method performance statistics (within- and between-laboratory precision) were equivalent for both the unmodified (Sr = 0.027, SR = 0.041, RSDr = 0.73%, RSDR= 1.08%) and modified (Sr = 0.023, SR = 0.038, RSDr = 0.60%, RSDR = 1.02%) Babcock methods. Modification of the Babcock method was successful in bringing test results into agreement with those of ether extraction.

Determination of the total nitrogen content of hard, semihard, and processed cheese by the Kjeldahl method: collaborative study.

The objective of this collaborative study was to determine interlaboratory performance statistics for a modified and optimized version of AOAC Method 920.123 for the determination of the total nitrogen content of hard, semihard, and processed cheese by Kjeldahl analysis. Details included addressing the issues of material homogeneity, test portion size (1 g), quantitative transfer (weighing on to filter paper), ensuring system suitability (nitrogen recoveries), and using AOAC Method 991.20 as the basis for nitrogen analysis. Fifteen laboratories tested 18 pairs of blind duplicate cheese materials with a crude protein content between 18 and 36%. Materials represented hard, semihard, and processed commercial cheeses with a wide range of composition. Statistical performance parameters expressed as crude protein (nitrogen x 6.38), g/100 g, with invalid and outlier data removed were mean = 26.461, repeatability standard deviation (Sr) 0.111, reproducibility standard deviation (S(R)) = 0.153, repeatability relative standard deviation (RSDr) = 0.42%, reproducibility relative standard deviation (RSDR) = 0.58%, repeatability (r) = 0.312, and reproducibility (R) = 0.428. The interlaboratory study results were acceptable and comparable to those for the milk Kjeldahl nitrogen method on a relative nitrogen basis. The Study Directors recommend that this modified method for the determination of total nitrogen in hard, semihard, and processed cheese by Kjeldahl analysis be adopted First Action as an improved method to replace Method 920.123.