The Effect of Temperature and Fungal:Bacterial Ratio on Kombucha Culture Fermentation

Abstract

The fermented tea beverage kombucha is a traditional drink that has been consumed for thousands of years in Asia. Over the past decades, it has gained greatly in popularity amongst consumers and researchers also in the rest of the world. Kombucha is produced through fermentation of sweetened tea by a complex community of bacteria, mainly dominated by acetic acid bacteria, and a variety of yeast. During the fermentation process, bacteria and yeast interact through forming compounds and metabolites that stimulate the growth of the other part. The aim of this project was to investigate how temperature and the fungal:bacterial ratio affects the kombucha fermentation process. First, I attempted to construct a gradient of fungal:bacterial biomass between inoculums based on filtration and sedimentation. However, as the visual evaluation as well as the PCR results implied a weak effectiveness of the method, it was decided not to continue with this approach. Instead, fermentation vessels were places in different temperature zones around 15.1 °C, 19.8 °C, 24.0 °C, 35.9 °C, and allowed to ferment for 20 days. Previous research have shown contrasting results between yeast and bacteria on optimal temperature and temperature sensitivity. Thus, given that the two groups are competing for resources, temperature conditions which favor one of the groups more would indirectly disfavor the other and an alteration of the fungal:bacterial biomass might be achieved by the temperature difference. During the 20 day fermentation time, pH and brix was measured continuously every second day and samples were saved and frozen for later analysis including qPCR and sugar content. The pH followed an expected pattern throughout the fermentation processes such as faster decrease associated with higher temperature. In contrast, the brix results were unexpected, not changing as much as expected for the three lower temperatures and increasing significantly for the highest temperature. A decrease in sucrose content and increase in glucose and fructose content could be observed from the sugar analysis.

Although few differences were significant, some tendencies could be observed when measuring the DNA amount and comparing amount of yeast and bacterial DNA between the different temperatures. Lower temperature seemed to favor the growth of both bacteria and yeast, although bacteria somewhat more. However, no evidence of a consistent effect of temperature on the fungal:bacterial ratio could be observed.

Overall, the observable effects of temperature on fungal:bacterial ratio and kombucha fermentation dynamics in this project was not as significant as was hypothesized when planning this project but further research on the subject is suggested before final conclusions are drawn.

Popular Abstract

Have you ever had the chance to try a cold glass of delicious kombucha? Perhaps you have, because over the past couple of years, this drink has become more and more popular. However, if you are not yet familiar with kombucha, it is a black tea and sugar-based drink that is fermented by a mixture of millions of yeasts and bacteria. During the production, a process called fermentation, the yeast and bacteria work together, providing each other with nutrients in order to provide a final product that is pleasantly acidic, a little sweet and slightly sparkling. This project aims to find out how the ratio of yeast and bacteria present, as well as how the temperature during production, affects the fermentation process.

One way that I investigated this was by filtrating the kombucha with the hopes of removing all the yeast, and then add specified amounts of a concentrated yeast solution into the remaining liquid. This way, a gradient of yeast to bacteria ratio, ranging from low to high, would be created. The results, both visual and molecular, did not seem promising. A tendency towards the filters either letting through or retaining both yeast and bacteria independent of the size of the pores was seen. Therefore, focus was instead placed on producing the kombucha in different temperature zones, ranging from 15 – 36 °C. Before, during and after 20 days of fermentation, the amount of DNA from yeast and bacteria respectively was analyzed in order to find out if the ratio of yeast:bacteria was affected by temperature. Also other factors that are characteristic for kombucha fermentation, such as pH and sugar as well as the taste of the final product, was tested.

The pH in all kombuchas decreased over time. This was expected as a result of an increase of organic acids which are being produced during the process. The pH decrease can be interpreted as the rate and activity of the process. The activity was higher in the higher temperatures and lower in the lower temperatures. Sugar is added to kombucha in the form of sucrose which is cleaved by the microbes into glucose and fructose and then consumed for production of for example ethanol, carbon dioxide and organic acids. Overall, the sugar analysis showed few significant results with some exceptions.

Also the results related to the yeast and bacteria DNA presence showed few changes that could be verified statistically. To sum up, no constant effect by temperature could be discovered on the fungal:bacterial ratio.

• author: Forsvall, Johanna
• supervisor: Åsa Håkansson
• organization: Food Technology and Nutrition (M.Sc.)
• course: KLTM05 20211
• year: 2021
• type: H2 - Master's Degree (Two Years)
• subject: Technology and Engineering
• keywords: Kombucha, Kombucha culture, Fermentation, Food engineering
• language: English
• id: 9057046
• date added to LUP: 2021-06-29 10:04:29
• date last changed: 2021-06-29 10:04:29