Repeated backslopped culture usage and prolonged beverage storage alter kombucha microbial composition and physco-chemical properties

• Kombucha has gained popularity due to its purpoted health benefits. • Production, however, is unstandardized posing a challenge to beverage integrity. • Changes in microbial composition and chemical consitutents in finished stored beverages necessitate the need for standardization to preseve the beverage intergity Kombucha, a low-alcohol fermented beverage, is believed to possess health benefits associated with its microbial consortium. However, it is unknown whether repeated culture usage results in changes to the microbial and physicochemical properties of the beverage over time. Twelve kombucha cultures were brewed for 10 repeated cycles. Sampling from each culture system was collected at a pH of 3 and subsequently stored at 4°C for microbial and physicochemical analyses at every 2 week increments for 12 weeks. Microbial assay consisted of enumeration of aerobic plate count (APC), lactic acid bacteria (LAB), acetic acid bacteria (AAB) and yeast; while physicochemical analysis consisted of simultaneous detection of major organic acids, sugars, and alcohol using high performance liquid chromatography (HPLC). The microbial population and physicochemical profile of kombucha varied with repeated brewing cycles and storage. Repeated culture usage resulted in an increase in LAB (from 5.3 to 6.7 Log CFU/mL) and AAB (from 5.8 to 6.5 Log CFU/mL) in the fermentate. After 12 weeks of refrigerated storage, these populations decreased by 0.7-1.4 Log CFU/mL. Ethanol concentration and degree of sucrose hydrolysis increased with storage. These data revealed that the microbial composition and physicochemical properties of kombucha change during repeated brewing cycles and storage, suggesting the need for standardization to ensure reproducibility and preservation of beverage integrity.