During the fermentation process of kombucha, with the help of bacteria and yeasts cultures, the sugared tea turns into a refreshing drink with an acidic, fruity aroma. How does the choice of microorganisms influence the aroma? What matters most is their mix.

Kombucha – a complex soft drink

Kombucha, a fermented tea beverage of traditional origin, has been enjoying growing popularity for several years. Its growing demand and thus the increasing need to devise industrial processes for kombucha and other acidic fermented beverages is primarily due to the health-promoting properties attributed to such products. Studies on acidic fermented beverages, especially kombucha, mostly focus on the involved microorganisms and the associated primary ingredients of the final product, acetic acid and ethanol. Although kombucha is classified as a non-alcoholic beverage (containing less than 0.5% ABV), ethanol formation plays a major role during fermentation. In an initial step, yeasts break down the sucrose into glucose and fructose. These are then converted by the yeasts into ethanol. The alcohol is subsequently converted to acetic acid in the aerobic region of the fermentation broth. In addition, the glucose is also processed by the contained acetic acid bacteria into cellulose fibers, which float on the surface in aggregated form. The microorganisms involved in fermentation are associated with each other in this structure, which is often referred to as a tea fungus. The consortium of yeasts and bacteria, which is undefined in type, species and amounts, and their distribution, especially in the traditional manufacturing process, is generally referred to as SCOBY (symbiotic culture of bacteria and yeasts). For industrial processes, however, an undefined and thus also failure-prone consortium of microorganisms is undesirable from a quality assurance perspective. Recent studies have therefore focused on using pure cultures as starter cultures to produce kombucha. But for consumers to accept a product, a beverage’s organoleptic impression, i.e. its odor, taste and appearance, is decisive. This also depends on the consortium of microorganisms used, which is why studies on the influence of the applied yeasts and bacteria on the aroma of kombucha are essential.

More than just sweet and sour

The consortia used for fermentation are not only complex in their composition but also extremely diverse regarding origin, age and fermentation parameters, resulting in a variety of products that are highly diverse in sensory terms. Previous sensory studies mostly focused on the primary taste sensations of sweet and sour because the balance of these two attributes is critical to the fundamental character of the beverage [1]. However, upon closer examination, it quickly becomes apparent that additional descriptors are necessary to address the diversity of kombucha products. In the course of the research at the Versuchs- und Lehranstalt für Brauerei in Berlin (VLB), therefore, various market samples were first tasted and, on that basis, an evaluation scheme for sensory attributes developed that would be as broad as possible. In addition to the classic gustatory parameters such as sweet, sour and bitter, it was also important to list tactility parameters such as mouthfeel and fizziness, as well as olfactory parameters such as fruitiness. Visual parameters such as color and turbidity were not considered in the developed scheme.

In addition to a comprehensive scheme with meaningful descriptors, the taster panel is also critical to the dependability of such studies. In order to reliably and objectively evaluate the properties of a product, the range to be expected from commercially available products must be established for each descriptor. Furthermore, there must be specific training for individual aromas and off-flavors so that they can be reliably detected even in a complex matrix. Regular tastings and panel comparisons, like those that also take place at the VLB for acidic fermented beverages, ensure the quality of the sensory evaluations and allow different kombucha products to be compared. In addition to objective criteria, subjective parameters such as preference can optionally also be recorded. They can be of particular interest in product development.

Aroma formation in co-culture

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Fig. 1 Microbiology determines the odor and taste of kombucha. Points awarded for preference of odor and taste of the various kobucha drinks (1 = do not like at all, 5 = like very much). H1: B. bruxellensis; H2: B. anomalus; H3: P. membranifaciens; A1: A. lovaniensis; A2: A. tropicalis; G1: G. liquefaciens; G2: K. hansenii

To investigate the aroma profile, the microorganisms in traditional kombucha beverages were first isolated and identified. Subsequently, organisms frequently described in connection with kombucha were selected for combinational experiments. The selection included three yeasts (Brettanomyces bruxellensis (H1), B. anomalus (H2), Pichia membranifaciens (H3)), and four bacteria (Acetobacter lovaniensis (A1), A. tropicalis (A2), Gluconacetobacter liquefaciens G1, Komagataeibacter hansenii G2)). Black tea spiked with 60 g/L of sucrose served as the beverage base. Tasting took place after a fermentation period of 14 days at 26 °C. In addition to the objective descriptors, preferences with respect to both smell and taste were also recorded.

A selection of the results is presented below. They illustrate the distinctiveness of aroma formation in co-culture. Although preference is a highly subjective criterion, the results indicate that harmony between odor and taste matters. Most of the tasted samples diverged significantly for aroma in smell and taste (Fig. 1), which tended to lead to a much lower appreciation of these products, since the expectations unwittingly developed during smelling were not fulfilled by the taste. In the case of fermented beverages, nutrient deficiencies can also lead to the formation of sulfur-containing, aroma-active substances with odors strongly perceived as off-flavors.

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Fig. 2: Aroma profile of kombucha produced with defined co-cultures. Assessment results of tasting in a spider chart for combinations of (A) two and (B) four different microorganisms. H1: B. bruxellensis; H2: B. anomalus; H3: P. membranifaciens; A1: A. lovaniensis; A2: A. tropicalis; G1: G. liquefaciens; G2: K. hansenii. 1 = not pronounced, 5 = strongly pronounced

Although traditional kombucha production uses undefined mixed cultures of usually more than four different organisms, using only one yeast and one bacterium led to an equally complex aroma that was rated as quite pleasant. This is of particular interest in the industrial sector because having to use fewer pure culture microorganisms means lower production costs. Looking closer at the aroma profiles of the two-organism approaches (see Fig. 2), it becomes clear that bacterial acid formation directly depends on the yeast, while yeast aromas are masked by the acid. Although the acetic acid concentration for H1+G2 and H2+G2 were both about 7 g/L, the H2+G2 kombucha drink was perceived as less acidic, and its yeast aromas as more prominent than in the other drinks.

Where four organisms were used, the microbial metabolic performance sometimes stagnated, which led to the original tea characteristics being retained. To better understand these phenomena, a focus of current research at FIBW is to characterize microbial interactions and potential nutrient limitation during complex beverage fermentations.

Outlook

Since every organism forms a different aroma in pure culture than in co-culture, limited predictive statements can be made on aroma formation as a consequence of interactions with other microorganisms, even if sufficient data is available. For kombucha, there are two forms of co-culture: while there is competition between individual yeasts and between individual bacteria, a more or less pronounced symbiosis exists between yeasts and bacteria. Today, it is assumed that volatile substances serve to communicate between microorganisms, which is why the type and quantity of the various aroma components depend strongly on the mix of microorganisms [2]. At the same time, other parameters such as the raw materials used (and thus the nutrient source and its availability) and technical fermentation parameters also play a role in aroma formation.

From a technological perspective, the production of kombucha is an exciting process whose control can be achieved through different strategies. From a scientific perspective, kombucha offers a large set of parameters for a better understanding of microbial metabolites in co-culture. Current research projects at VLB focus on building industrially useful knowledge around the metabolic processes of yeasts and bacteria in co-culture, bridging the gap between the two fields.

Acknowledgement

The authors would like to thank Katarzyna Woźniak for her energetic and valuable work that formed the basis of the data presented here, and Marie Ludszuweit for the kind provision of microscopic images of co-cultures in kombucha.

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Category: Food Biotechnology | Kombucha

Literature:
[1] Tran T, Grandvalet C, Verdier F, Martin A et al. Microbiological and technological parameters impacting the chemical composition and sensory quality of kombucha. Compr Rev Food Sci Food Saf. 2020;19:2050-2070. DOI:10.1111/1541-4337.12574
[2] Bader J, Brigham CJ, Stahl U, Popović MK. 3 - Fermented Beverages Produced by Mixed Cultures, Pure Cultures, and Defined Cocultures. In Fermented Beverages, Volume 5: The Science of Beverages. Eds. Grumezescu, AM, Holban, AM. Woodhead Publishing. 2019;67-101. DOI:10.1016/B978-0-12-815271-3.00003-8