Parameters influencing water kefir grain growth dynamics by image analysis

Water kefir is a fermented beverage that is produced by adding water kefir grains to a sucrose solution with dried fruits. The approximately 1-10 mm large grains consist of a bacterial exopolysaccharide matrix and are mainly colonised by lactic acid bacteria, acetic acid bacteria and yeasts. The production depends on the successful cultivation and propagation of the existing grains: During fermentation cycles, the mass of the grains increases, but growth dynamics between the start and end points of fermentation and the parameters that can influence this process have not yet been analysed. Monitoring growth is potentially relevant for the industrial production of water kefir, as no defined starter cultures are available. Our aim was therefore to establish a method for the continuous documentation of grain growth and thus enable a kinetic description of the involved processes.
We developed a documentation system that takes high quality pictures, which are then further processed with Fiji image-analysis software. Kefir grains were selected by size and incubated in standardized water kefir medium at room temperature for 72 h. The number of and increase in white pixels in the pictures against a black background represent a measure of the two-dimensional grain size and growth, respectively.
The developed method enables the visualization of grain growth curves as a function of white pixels over fermentation time. These are comparable to classic bacterial growth curves, as they exhibit lag, exponential and stationary phases. The influence of the number of starter grains and their size could be evaluated based on curve progression and total growth. It was found that an increasing number of grains and larger grain sizes have a negative effect on the overall growth rate. However, the exponential and stationary growth phases tend to be reached faster as the number of grains increases. In ongoing experiments, the influence of the sugar content and the type of water in the medium is being investigated.