Expert Review Panel for Kombucha Tea

4. Based on the supporting information, is the method written clearly and concisely? If no, please specify the needed revisions.

The Following clarification about method will help

1.8 Sample Preparation: c. Samples diluted 1:200 with distilled water. The authors may state how the dilution was performed (and recommended ), any minimum volume of Kombucha need to be pipetted to get good precision data?. d. Analyze the samples as per the Megazyme Ethanol Assay Kit (K-ETOH) (We were not provided with a copy of the instructions). Is the method in the kit instructions different that listed here. “-------, lengthen the incubation time to 10 minutes to ensure completion of the reaction”. Does the K-ETOH kit instruction mention a shorter incubation time ? Is it expected for the analysts to watch every sample for about 15 minutes to confirm that reaction has stopped in 10 minutes? The authors may provide some guidelines. 1.9 Manual Assay procedure Was the assay performed directly in curettes or certain size of tubes? How were solutions mixed in the container of the reaction before incubations? – whether hand shaking or vortex or some other way? How did you manage the incubation time? – whether fixed 10 minutes or checked it in every case? 2.1 Selectivity, accuracy and interference: “If the conversion of ethanol has been completed within time specified in the assay (approx. 10 minutes), it can be generally concluded that no interference has occurred”. Authors may provide data or justify this statement specific to this method and matrix in scope. “However, this can be further checked by adding ethanol (about 5 µg in 0.1 mL) to the cuvette on completion of reaction”. Please clarify this further and whether authors had an opportunity to perform the mentioned experiment to establish selectivity of the assay. It may be important to establish selectivity of the method. It will help to know that some of the components which can be in Kombucha like, glucose, fructose, sucrose, gluconic acid, acetic acid, lactic acid and citric acid , caffeine etc. don’t interfere in the ethanol assay by this method. Stability of the Kit: The authors claim the shelf-life of the most of the components of the kit is >1 year at 4 °C. The authors may provide data to support this claim. It's an easy, simple and very promising method for the matrix. Establishing Selectivity and accuracy of the method will help further validate the method for the targeted application. The method is quite promising but some additional information as specified earlier about the method selectivity and accuracy is required for further recommendation of the method for First Action. Simple assay

5. Based on the supporting information, what are the pros/strengths of the method? 6. Based on the supporting information, what are the cons/weaknesses of the method?

7. Any general comments about the method?

Do you recommend this method be adopted as a First Action and published in the Official Methods of Analysis of AOAC INTERNATIONAL? Please specify rationale.