Enzymatic Determination of L-Malic Acid in Selected Foods and Beverages: AOAC Official Method 2025.03 First Action

BACKGROUND: L-malic acid is one of the most important acids present in fruits. Its determination is important in the manufacture of beer, fruit and vegetable products. In the wine industry, its level is monitored during malolactic fermentation. It is also added to a variety of products as food preservative and flavor enhancer. OBJECTIVE: To validate the performance of the Enzytec™ Liquid L-Malic acid test kit for the determination of L-Malic Acid in foods and beverages such as fruit juice (concentrates and products), vegetable juice, wine, beer, sauces, and carbonated soft drinks. METHODS: The kit contains two ready-to-use components only which makes handling easy and suitable for automation. L-malic acid is oxidized to oxaloacetate by L-malate dehydrogenase and NAD+. The NADH produced is equivalent to the amount of L-malate converted and is measured at 340 nm. RESULTS: The Limit of Detection (LOD) was determined to be 8 mg/L for 100 µL test volume and the measurement range is 15 to 500 mg/L. Trueness was checked by analyzing a reference wine sample resulting in a recovery between 96 and 99%. Recovery was checked by spiking and resulted in mean values from 97 to 105% for red wine, beer, soft drink, carrot juice, apple juice, acerola juice concentrate, BBQ sauce, and quince jelly. Relative intermediate precision is between 1.5 and 3.6% for carrot juice, BBQ sauce, NIST SRM 3282, and a reference wine. D-tartaric acid and meso-tartaric acid interfere and cause a creep during the reaction. Sulfite interferes at concentrations higher than 0.025 g/L but can be masked by glyoxal. For automation, three applications with different test volumes were validated. Linearity is given from 3.5 to 2500 mg/L. CONCLUSIONS: The method was approved as AOAC Official Method of Analysis℠. HIGHLIGHTS: Real time and in-use stability are at least 24 months.