A novel enzymatic method for the measurement of lactose in lactose-free products.

BACKGROUND: In recent years there has been a surge in the number of commercially available lactose-free variants of a wide variety of products. This presents an analytical challenge for the measurement of the residual lactose content in the presence of high levels of mono-, di-, and oligosaccharides. RESULTS: In the current work, we describe the development of a novel enzymatic low-lactose determination method termed LOLAC (low lactose), which is based on an optimized glucose removal pre-treatment step followed by a sequential enzymatic assay that measures residual glucose and lactose in a single cuvette. Sensitivity was improved over existing enzymatic lactose assays through the extension of the typical glucose detection biochemical pathway to amplify the signal response. Selectivity for lactose in the presence of structurally similar oligosaccharides was provided by using a ß-galactosidase with much improved selectivity over the analytical industry standards from Aspergillus oryzae and Escherichia coli (EcLacZ), coupled with a 'creep' calculation adjustment to account for any overestimation. The resulting enzymatic method was fully characterized in terms of its linear range (2.3-113 mg per 100 g), limit of detection (LOD) (0.13 mg per 100 g), limit of quantification (LOQ) (0.44 mg per 100 g) and reproducibility (≤ 3.2% coefficient of variation (CV)). A range of commercially available lactose-free samples were analyzed with spiking experiments and excellent recoveries were obtained. Lactose quantitation in lactose-free infant formula, a particularly challenging matrix, was carried out using the LOLAC method and the results compared favorably with those obtained from a United Kingdom Accreditation Service (UKAS) accredited laboratory employing quantitative high performance anion exchange chromatography - pulsed amperometric detection (HPAEC-PAD) analysis. CONCLUSION: The LOLAC assay is the first reported enzymatic method that accurately quantitates lactose in lactose-free samples. © 2018 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Determination of Lactose Concentration in Low-Lactose and Lactose-Free Milk, Milk Products, and Products Containing Dairy Ingredients, Enzymatic Method: Single-Laboratory Validation First Action Method 2020.08.

BACKGROUND: The AOAC Stakeholder Panel on Strategic Food Analytical Methods issued a call for methods for the measurement of lactose in low-lactose and lactose-free products under Standard Method Performance Requirement (SMPR®) 2018.009. Megazyme's Lactose Assay Kit (K-LOLAC) was developed specifically to address the need for accurate enzymatic testing in lactose-free samples. OBJECTIVE: K-LOLAC was validated for measurement of lactose in low-lactose and lactose-free milk, milk products, and products containing dairy ingredients. A single-laboratory validation (SLV) of the method is reported. METHOD: K-LOLAC is an accurate and sensitive enzymatic method for the rapid measurement of lactose in low-lactose or lactose-free products. Validation analysis was performed on a sample set of 36 commercial food and beverage products and a set of 10 certified reference materials. Parameters examined during the validation included working range and linear range, selectivity, LOD, LOQ, trueness (bias), precision (repeatability and intermediate precision), robustness, and stability. RESULTS: For all samples tested within the lower range (10-100 mg/100 g or mL), recoveries varied from 93.21-114.10%. Recoveries obtained for samples in the higher range (>100 mg/100 g or mL) varied from 94.44-108.28%. All materials had repeatability relative standard deviations (RSDr and RSDir) of <9%. CONCLUSIONS: The commercial K-LOLAC assay kit, as developed by Megazyme, meets the requirements set out under SMPR 2018.009. HIGHLIGHTS: K-LOLAC is a robust, quick, and easy method for analysis of lactose in foodstuffs and beverages.

Determination of Ethanol Concentration in Kombucha Beverages: Single-Laboratory Validation of an Enzymatic Method, First Action Method 2019.08.

Kombucha is a fermented, lightly effervescent sweetened black or green tea drink. It is marketed as a functional beverage based on its proposed health benefits. Kombucha is produced by fermenting tea using a "symbiotic colony of bacteria and yeast" (SCOBY). Kombucha is marketed as a non-alcoholic beverage, however due to the production process employed, there is a high possibility that the Kombucha products will contain low levels of ethanol. Kombucha is sold in a raw and unpasteurized form and, if kept at temperatures above 4 °C, the possibility exists that it will continue to ferment, producing ethanol. This possibility of continued fermentation may lead to an increase in ethanol content from levels below 0.5%ABV at time of production to higher levels at time of consumption. Thus, there is a potential for levels rising to greater than 0.5%ABV, the threshold for certification as a non-alcoholic beverage. It is essential that Kombucha manufacturers have the capacity to accurately and quickly test for ethanol in their products. The Ethanol Assay Kit is an enzymatic test kit developed by Megazyme for the determination of ethanol in a variety of samples. The kit has been validated in a single laboratory for use with Kombucha fermented drinks, fruit juices, and low-alcohol beer samples. The commercially available Ethanol Assay Kit (Megazyme catalogue no. K-ETOH) contains all components required for the analysis. Quantification is based on the oxidation of ethanol to acetaldehyde by alcohol dehydrogenase and further oxidation of acetaldehyde by acetaldehyde dehydrogenase with conversion of NAD+ to NADH. The single laboratory validation (SLV) outlined in this document was performed on a sample set of eight different commercial Kombucha products purchased in Ireland, a set of five Cerilliant aqueous ethanol solutions, two BCR low-alcohol beer reference materials, two alcohol-free beer samples, and two fruit juice samples against SMPR 2016.001 (1). Parameters examined during the validation included Working range, Selectivity, Limit of Detection (LOD), Limit of Quantification (LOQ), Trueness (bias), Precision (reproducibility and repeatability), Robustness, and Stability. The Ethanol Assay is a robust, quick and easy method for the measurement of ethanol in Kombucha. Our data suggests this method is also reliable for similar matrices, such as low-alcohol beer and fruit juice. The assay meets all requirements set out in in AOAC SMPR 2016.001.