Kombucha: a tasty Symbiotic Culture of Bacteria and Yeasts

posted by Jonas Astrup Pedersen

Kombucha, also known as Kargasok Tea, Tea Fungus,
Haipao and Manchurian Mushroom, is a fermented beverage dating back several
thousand years in the East. More recently, it has become popular in the West,
specifically in ‘New Age’ circles (Battikh et al.,
2012
; Jarrell et al., 2000; Greenwalt et al., 2000). Tea fungus initially originated in China in 220
BCE during the Tsin Dynasty and prized as the ‘Divine Che’. The name ‘Kombucha’
seems associated with Doctor Kombu, who is said to have brought the ‘tea
fungus’ from Korea to Japan in 414 CE (Dufresne and Farnworth, 2000).

Increasing interest in Kombucha products is linked
to their supposed therapeutic benefits, ranging from curing cancer and AIDS to
enhancing weight loss, as well as demonstrating interesting sensory properties (Dufresne and Farnworth, 2000; Teoh et al., 2004). Although several of these claims are not proven,
Kombucha beverages exerts antimicrobial activity against Salmonella typhimurium, Staphylococcus
aureus
, Helicobacter pylori, (Greenwalt et
al.
, 1998
), Shigella
sonnei
, Salmonella enteritidis and
Escherichia coli (Greenwalt et
al.
, 1998
; Sreeramulu et al., 2001). Furthermore, Kombucha tea ingestion by mice
contributed significantly to both life elongation and weight gain inhibition (Hartmann et al.,
2000
).

The beverage is typically made with black tea,
sweetened with 5 to 15% of sucrose, and set to ferment at room temperatures for
10-12 days with a culture popularly known as a ‘tea fungus’, Medusomyces gisevii (Anken and Kappel, 1992; Jayabalan et
al.
, 2010
). Inoculation of new batches uses about 10% of
Kombucha from a previous batch. The brewing vessel is covered with a clean
cotton cloth to keep out debris while allowing aeration (Greenwalt et
al.
, 2000
). A schematic description of Kombucha production is
seen in Figure 1.

  Figure 1:  Schematic overview of producing Kombucha

Figure 1: Schematic overview of producing Kombucha

Kombucha is the expression of a symbiotic growth of
bacteria such as Acetobacter xylinum,
A. xylinoides, A. aceti, A. pasteurianus,
Bacterium gluconicum (Sreeramulu et
al.
, 2000
; Dufresne and
Farnworth, 2000
) and yeasts like Schizosaccharomyces pombe, Kloeckera
apiculata
, Saccharomycodes ludwigii,
Saccharomyces cerevisiae, Zygosaccharomyces bailii, Brettanomyces
bruxellensis
, B. lambicus, B.
custersii
and Pichia species (Dufresne and Farnworth, 2000). Though the fungus-like cellulosic matrix produced
by especially Acetobacter xylinum
might look like a fungus (Mo et al., 2008), ‘tea fungus’ is rather misleading since the ‘tea
fungus’ is in fact only a physical manifestation of the yeast and bacteria
symbiosis (Sreeramulu et
al.
, 2000
). The floating jelly-like membrane, called a zoogleal mat, is where the cell mass of
the bacteria and yeasts are attached (Jayabalan et
al.
, 2010
). The cellulose is a secondary metabolite of the
fermentation, similar in structure to a ‘mother of vinegar’ (Jayabalan et
al.
, 2010
). Within the cellulose network, investigations have
shown the Kombucha colony to be arranged in bands and layers (Anken and Kappel, 1992), see Picture 4. The composition and exact diversity of the
microbiological presence depends on the source of the Kombucha culture (Sreeramulu et
al.
, 2000
).

As yeast cells hydrolyze sucrose into glucose and
fructose, producing ethanol and carbon dioxide as metabolites, acetic acid
bacteria converts glucose into gluconic acid and fructose into acetic acid (Reiss, 1994; Loncar et al.,
2006
). The primary metabolites of ethanol and acetic
acid behave as catalyzing agents; yeast are stimulated to produce ethanol by
acetic acid, whereas ethanol stimulates the growth of acetic acid bacteria and
their production of acetic acid (Liu et al., 1996). Fructose is utilized to a lesser degree and
remains part of the fermented liquid (Greenwalt et
al.
, 1998
). The synthesis of complex B vitamins and folic
acids has also been reported during the fermentation process (Bauer-Petrovska and Petrushevska-Tozi, 2000). Additionally, the organic acids produced
throughout the fermentation and the corresponding decrease in pH value prevent
the symbiotic culture from becoming contaminated by undesirable microorganisms
not contained in the tea fungus (Greenwalt et
al.
, 1998
; Mo et al., 2008).

The properties and composition of the final product
depends on the initial substrates, to geographical and climatic conditions, as
well as the locally-specific types of wild yeast and bacteria present (Bauer-Petrovska and Petrushevska-Tozi, 2000). To obtain beneficial attributes and antimicrobial
activity against a range of pathogenic bacteria, Greenwalt et al. (1998) recommends consumption of Kombucha containing 33 g/L
total acids, 7 g/L acetic acid. Usually, the pH of a fermented Kombucha is
around 2.5, regarded by the food industry as a high-acid food since a pH of 4.0
prevents growth of most organisms linked with spoilage (Greenwalt et
al.
, 2000
).

Experiments

Herbs or wood and boiling water (1 L each) were added to separate containers, closed with a lid and left to infuse at room temperature with different infusing times for optimal flavour intensity. These included dried yarrow flowers (1% w/v, 10 min.), juniper wood (5% w/v, 1 hour), dried chamomile (1% w/v, 10 min.), dried lemon verbena (1% w/v, 10 min.), dried woodruff (1% w/v, 10 min.) and dried cèpes (5% w/v, 12 hour). Dried kelp (2.3% w/v, 1 hour) was sealed in a vacuum bag and treated sous vide at 60 °C. To all solutions were added 50 g of sucrose.

  Picture 1-3:   herbs, cepes and wood extraction (top); straining tea (bottom left); inoculated yarrow flower tea (bottom right).

Picture 1-3:  herbs, cepes and wood extraction (top); straining tea (bottom left); inoculated yarrow flower tea (bottom right).

The Kombucha mothers were carefully cut into
approximately equal sizes and added to the teas. Additionally, 100 mL (10% v/v)
of the liquid medium (tea kvass) were also added. Containers were covered with
a cloth and set into a closed cabinet at an ambient temperature of
approximately 21 ± 2 °C.

  Table 1:  pH of the different infusions at initial stage and after 12 days of fermentation

Table 1: pH of the different infusions at initial stage and after 12 days of fermentation

Based on sensory evaluation among employees at
Nordic Food Lab (NFL) during the period of fermentation and at day 12, lemon
verbena (Aloysia triphylla) was
selected as a basis for further elaboration. The absence of data for cepe and
carrot is due to loss of samples to mold, most likely because of too high
initial pH.

To produce sufficient quantity of the lemon verbena
Kombucha for further investigation, a batch of 17 L of tea was brewed:
dried lemon verbena (1% w/v, 10 min.) was steeped in boiling water, to which
was added 50 g of sucrose per liter. The batch was inoculated with tea kvass
(0.95 mL) from the previous batch. The container was put into an incubator-box
to control fermentation temperatures, which measured in the range of 27 ± 2 °C.

  Picture 4:  the cellulose network, zoogleal mat (Kombucha mother) produced by  Acetobacter xylinum.

Picture 4: the cellulose network, zoogleal mat (Kombucha mother) produced by Acetobacter xylinum.

Lemon verbena Kombucha

The herb mentioned as lemon verbena (Aloysia triphylla (L’Hérit.) Britt. Syn.
Lippia citriodora) belongs to the
family of Verbenáeae (in Danish: jernurt-familien) (Vogel et al., 1999). It
is well known for the pleasant odour of its leaves, comparable to that of a
lemon. Responsible for its aromatic properties are essential oils found in
concentrations of 0.4% (Montes et al., 1973) to
1.2% (Vogel et al., 1999). Its
pronounced lemon-like odour is due to the chemical compound citral found in
concentration of lemon verbena oils between 11% and 54% (Montes et al., 1973; Vogel et al., 1999).

Lemon verbena, also called cedrón in its countries of origin, is a shrub native to Peru, Chile
and Argentina where it is cultivated for domestic consumption as an herb tea (Vogel et al., 1999). It
was brought to Europe during the 18th century and grown as potted
plants due to its high sensitivity to cold (Vogel et al., 1999).
Despite its origin in South America, lemon verbena has caught the attention of
chefs and is now found in many Nordic kitchens and dishes.

In cooking circles, an often-heard misunderstanding
of its name is the simple use of (in Danish) jernurt. Jernurt refers merely to the family Verbenáeae, which
contains some 25-34 genera and 500-1200 species comprising a great variety of
small trees, lianas, shrubs and herbs (Yuan et al., 2010).

In an attempt to speed up the fermentation process,
aeration was tested on four brews. An aquarium pump (AM-TOP model CR10) was
connected to the containers during the entire fermentation process. Different
kinds of sugar, either ‘alone’ or in combination, were tested as different energy
sources for the yeast and bacteria. This was done with the purpose of observing and detect possible variation in sensory qualities and pH of end products. Teas were
prepared as in the preliminary trials: herb extraction (1% w/v, 10 min.), added
sugar (5% w/v) and finally inoculation with tea kvass (10% v/v) at 21 ± 2 °C,
see Table 2 and Table 3.
Where fructose is stated, Danish honey from Søborg was used.

  Table 2:  pH of different infusions at initial stage and after 7 days of fermentation

Table 2: pH of different infusions at initial stage and after 7 days of fermentation

  Table 3:  pH of different infusions at initial stage and after 7 days of fermentation

Table 3: pH of different infusions at initial stage and after 7 days of fermentation

Results

With the aerated batches, enhancing the
fermentation speed did not seem to succeed in favouring the acetic acid
bacteria. Of the four batches, none were as balanced, as complex in flavor, or
as refreshing as Kombucha can be. Although Kombucha is acidic, this tartness
was not satisfying when complementary aroma was absent or expressed in a flat manner.

Testing different sugars revealed great difference
in end product. Some Kombuchas turned out almost vinegar-like and not
appropriate for a soft drink, though these could have interesting applications
for culinary exploration and use. By far the most aromatic and interesting
Kombucha developed in this experiment, contained sucrose as the sugar
substrate. It expressed herby notes as well as an interesting ginger-like
association, well balanced, pleasantly acidic and complex.

Alongside these small-batch trials, a continuous
batch has served as staff Kombucha. Sweetened lemon verbena tea has been added, throughout the project, to a large vessel used during the process, while Kombucha
has been tapped and enjoyed. The Kombucha turned out very tasty in
expression: fizzy, refreshing, and especially delicious when served ice-cold. This
encourages further investigation into continuous fermentations with e.g. higher
levels of inoculum to amount of sweetened tea, as well as second fermentations in terms of added juice or other flavourfull sugar containing liquids, is a path believed worth investigating. KOMBOOOUCHA!

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