Mousy off-flavor describes N-heterocycles compounds related to spoilage in the brewing industry. It has also been identified in sour beers through sensory analysis. Therefore, preventing spoilage N-heterocycles development is essential to preserve end-products and obviate economic losses. To this day, no methods or protocols have been reported to identifying mousy off-flavor compounds in a beer matrix. The main objective of this work was to develop a standardized quantification method for 2-acetyl-3,4,5,6-tetrahydropyridine (ATHP) in beer matrix, by Liquid Chromatography Mass Spectrometry with Electrospray Ionization (LC-MS-ESI). Extraction of ATHP in the samples was performed using QuEChERS (quick, easy, cheap, effective, rugged, and safe) technique. Over a dozen different potentially mousy cask-aged sour beers including other spontaneously fermented beverages were provided, based on sensory analysis, to determine the variation in ATHP levels. Results indicated ATHP was found in all the samples, ranging from 1.64 ± 0.06 to 57.96 ± 2.15 µg L-1. Herein, we described our detection method of mousy-off flavor compounds which enables future research to mitigate the occurrence of such defects in fermented beverages matrix.•ATHP content in samples varied from 1.64 ± 0.06 to 57.96 ± 2.15 µg L-1.•The recovery range of ATHP using LC-MS-ESI varied from 71% to 97%.•Basified QuEChERS salting-out procedure is applicable for ATHP extraction from beer and other fermented beverages matrices.

Beer has over 600 flavor compounds and creates a positive tasting experience with acceptable sensory properties, which are essential for the best consumer experience. Spontaneous and mixed-culture fermentation beers, generally classified as sour beers, are gaining popularity compared to typical lager or ale styles, which have dominated in the USA for the last few decades. Unique and acceptable flavor compounds characterize sour beers, but some unfavorable aspects appear in conjunction. One such unfavorable flavor is called "mousy". This description is usually labeled as an unpleasant odor, identifying spoilage of fermented food and beverages. It is related as having the odor of mouse urine, cereal, corn tortilla chips, or freshly baked sour bread. The main compounds responsible for it are N-heterocyclic compounds: 2-acetyltetrahydropyridine, 2-acetyl-1-pyrroline, and 2-ethyltetrahydropyridine. The most common beverages associated with mousy off-flavor are identified in wines, sour beers, other grain-based beverages, and kombucha, which may contain heterofermentative lactic acid bacteria, acetic acid bacteria, and/or yeast/fungus cultures. In particular, the fungal species Brettanomyces bruxellensis are associated with mousy-off flavor occurrence in fermented beverages matrices. However, many factors for N-heterocycle formation are not well-understood. Currently, the research and development of mixed-cultured beer and non/low alcohol beverages (NABLAB) has increased to obtain the highest quality, sensory, functionality, and most notably safety standards, and also to meet consumers' demand for a balanced sourness in these beverages. This paper introduces mousy off-flavor expression in beers and beverages, which occurs in spontaneous or mixed-culture fermentations, with a focus on sour beers due to common inconsistency aspects in fermentation. We discuss and suggest possible pathways of mousy off-flavor development in the beer matrix, which also apply to other fermented beverages, including non/low alcohol drinks, e.g., kombucha and low/nonalcohol beers. Some precautions and modifications may prevent the occurrence of these off-flavor compounds in the beverage matrix: improving raw material quality, adjusting brewing processes, and using specific strains of yeast and bacteria that are less likely to produce the off-flavor. Conceivably, it is clear that spontaneous and mixed culture fermentation is gaining popularity in industrial, craft, and home brewing. The review discusses important elements to identify and understand metabolic pathways, following the prevention of spoilage targeted to off-flavor compounds development in beers and NABLABs.