Validation of Enzytec™ Liquid D-Lactic acid for Enzymatic Determination of D-Lactic acid in Selected Foods and Beverages: Official Method 2024.06 First Action.

Abstract

Background: D- and L-lactic acid are produced naturally by lactic acid bacteria and are found in fermented milk products, pickled vegetables, and cured meats. D-lactic acid is formed by some microorganisms only, e.g., Lactobacillus lactis, and Leuconostoc cremoris. D-Lactic acid is not formed or only in traces by "higher organisms", e.g., by animals. Therefore the presence of D-lactate may serve as an indicator for microbial contamination or spoilage, assuming that fermentation techniques have not been used.

Objective: To validate the performance of the Enzytec™ Liquid D-Lactic acid for the determination of D-lactic acid in food and beverages such as milk and (fermented) milk products, fermented vegetable products, wines, beer, fruit and vegetable juices, egg and egg powder.

Methods: The kit contains two ready-to-use components which makes handling easy and suitable for automation. D-lactic acids react in the presence of NAD and D-lactate dehydrogenase to pyruvate and NADH. The NADH formed is equivalent to the amount of D-lactic acid converted.

Results: Ascorbic acid, 3-hydroxybutyric acid, and sulfite interfere at concentrations higher than 0.2, 0.2, and 0.05 g/L, respectively. Oxaloacetic acid, pyruvic acid and D-fructose do not interfere at or below concentrations of 0.2, 1, and 10 g/L, respectively. The calculated LOD when using a test volume of 100 µL is 5.4 mg/L and the LOQ is 15 mg/L. The practical upper measurement range is 600 mg/L. Relative intermediate precision was between 3.5 and 5.7% for pineapple juice, sauerkraut juice, wine and liquid egg. A reference material (wine) showed recoveries of 108%. For automation, three applications with different test volumes were validated. Linearity is given from 0.75 up to 3125 mg/L.

Conclusions: The method is robust and accurate and was approved as AOAC Official Method of Analysis℠.

Highlights: The ready-to-use components of the test kit have a shelf life of at least 24 months.