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Chemical and biological changes accompanying kombucha fermentation and

sensory evaluation on teas fermented with different concentration of sugars.
1.Introduction:
Kombucha originated from the Manchurian and is made by fermenting tea with cultures of
bacteria and yeasts [1,2]. The most commonly used bacteria is Acetobacter xylinum, while
yeasts usually belong to Saccharomyces family [2]. Fermentation in Kombucha involves the
production of ethanol by yeasts during respiration and oxidation of ethanol by bacteria to
produce acetic acid [3,4]. Generally, fermentation occurs for 7 to 12 days at 22˚C to 30˚C with
30 to 100 g/L of sugar [1,5].
Kombucha has been reported to have anti-inflammatory and antioxidant effects [2]. However,
the antioxidative effect varies between different Kombucha teas depending on the symbiotic
cultures used [1]. Nonetheless, extended fermentation can result to the build-up of organic acids
harmful to the human body [1]. Moreover, one should be cautious when brewing kombucha
tea at home due to potential foodborne illness [1].
This experiment aims to record different chemical and biological aspects accompanying stages
of fermentation, further consolidating the knowledge of the fermentation process. In addition,
it also aims to assess sensory properties of tea with different concentration of sugars.
The hypothesis is that the amount of sugar and pH would decrease during fermentation.
Meanwhile, SCOBY weight would increase due to favorable conditions. Concentration of
vitamin C would remain constant throughout.
2.Method:
Sugar and 1.5 L of hot water were dissolved in a glass jar. The amount of sugar added were as
following: group A 5g/L, group B 1g/L, group C 5g/L and group D 10g/L. Next, two tea bags
were added into the mixture and let cool for half an hour. Then ¼ cup of starter tea was added
with 25g of SCOBY for fermentation. The jar was covered tightly with chux and left at room
temperature, away from sunlight for 21 days. After 14 days of fermentation, ¼ cup of lemon
juice was added and the fermentation process continued.
The concentration of sugar, weight of SCOBY, vitamin C and pH level were recorded before
and at every two weeks after fermentation. Sugar concentration was recorded using a
refractometer. Vitamin C concentration was measured using test strips. Weight of SCOBY was
measured using digital scale. pH level was recorded with digital pH meter. Sensory analysis
was recorded using a 9-point Hedonic test at 28 days and 42 days after fermentation.
3.Results and discussion:
3.1 SCOBY weight

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3.500

3.000

2.500
Fold change (g)

2.000

1.500

1.000

0.500

0.000
5g 10 g 50 g 100 g
Amount of sugar (g/L)

Figure 1: Average fold change in weight of SCOBY after fermentation in grams in four different
sugar concentrations 5g, 10g, 50g and 100g.

The total weight of SCOBY across four groups increased significantly after 14 days of
fermentation. The differences observed across four groups can be explained by varied sugar
concentrations available for yeasts and bacteria’s growth. The sharp increase in total count of
SCOBY correlates with the results from other studies obtained in kombucha fermented with
100 g sucrose. For instance, Chen and Liu observed increasing bacteria and yeast counts as
incubation time lengthens, with maximum values recorded from 6 to 14 days of fermentation
[6]. Teoh et al. also reported a peak in the number of yeast and bacteria species from day 6 to
8 of fermentation [7].
3.2 pH levels

5
Group A
pH

4
Group B
3
Group C
2 Group D
1

0
Day 0 Day 14 Day 28 Day 42
Day of fermentation

Figure 2: Mean pH of four experimental groups at day 0, day 14, day 28 and day 42

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Mean pH decreased sharply from day 0 to day 14 across all four groups. The drastic drop in
pH is due to formation of gluconic acid and acetic acid produced from glucose and ethanol by
bacteria [8]. From day 14 to day 28, the pH level slightly decreased and finally remained
relatively constant at day 42. This correlates with studies done by Malbaša et al and Loncar et
al, both reported a significant drop of pH after several days of fermentation and a very slow
decrease onwards [2,9]. However, the pattern was different for group A, where the pH
increased slightly at day 42. Similar results obtained by Jayabalan et al also showed a slight
increase in pH towards the end of fermentation [10]. The observed change could be due to the
buffering reaction between weak organic acids and mineral composition of tea [2]. Moreover,
different sucrose concentration used can produce slight changes in pH across the four groups
[9], as visible in figure 2.

3.3 Vitamin C concentration:

140
Concentration of vitamin C (mg/L)

120

100

80 Group A

60 Group B
Group C
40
Group D

20

0
Day 0 Day 14 Day 28 Day 42
Day of fermentation

Figure 3: Mean vitamin C concentration of four groups at day 0, day 14, day 28 and day 42 of
fermentation.

The patterns for vitamin C contradicted with the hypothesis, as the results showed that vitamin
C concentration increased greatly during fermentation. This is because yeast and bacteria
produce vitamin C along with other metabolites during fermentation [8]. Both studies done by
Bauer-Petrovska and Malbaša et al found an increase in vitamin C concentration with the
highest amount observed at day 10 of fermentation [2,11]. However, this does not correlate
with the results obtained, which shows that the maximum concentration of vitamin C is at day
28 of fermentation. This could be due to failure to follow the procedure outlined, as some
groups had initial vitamin C values when it was only added during day 14. Moreover, errors
can also result from incorrect reading of test strips.
3.4 Sugar concentration

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Sugar concentration (%Brix)

Group A
Group B
Group C
Group D

Day 0 Day 14 Day 28 Day 42

Day (s)

Figure 4: Mean sugar concentration of four groups at day 0, day 14, day 28 and day 42 of fermentation.

Overall, sugar concentration decreased for all the groups as shown, however the amount of
unmetabolized sugar in group C and D remained relatively large compared to group A and B.
This is because a large amount of sugar remained unfermented during the process [2]. Malbaša
et al reported a 19.28% residual of sugar in fermented kombucha after 21 days [2]. Additionally,
Chen and Liu also found a very slow decrease of sugar concentration after 30 days of
fermentation, which further supports the recorded results [6].
3.5 Sensory properties

Sourness

Sweetness

Taste and flavour

Day 42
Odor Day 28

Color

Appearance

0 1 2 3 4 5 6 7 8 9

Figure 5: Comparison of sensory characteristics of sample A between day 28 and day 42

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Sourness

Sweetness

Taste and flavour

Day 42
Odor Day 28

Color

Appearance

0 1 2 3 4 5 6 7 8 9

Figure 6: Comparison of sensory characteristics of sample B between day 28 and day 42

Sourness

Sweetness

Taste and flavour

Day 42
Odor Day 28

Color

Appearance

0 1 2 3 4 5 6 7 8 9

Figure 7: Comparison of sensory characteristics of sample C between day 28 and day 42

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Sourness

Sweetness

Taste and flavour

Day 42
Odor Day 28

Color

Appearance

0 1 2 3 4 5 6 7 8 9

Figure 8: Comparison of sensory characteristics of sample D between day 28 and day 42

Personally, the fermented kombucha tasted more fruity and sweeter after 28 days of
fermentation. Prolonged fermentation, specifically 42 days, resulted in a sourer and more
vinegar-like flavor, which was not desirable. In addition, kombucha teas with higher
concentration of sugar like those made by bench B, C and D are more preferred due to their
sweeter taste. The color and odor in all the kombucha samples were relatively similar at day
28 and day 42 for most of the groups, therefore not contributing as much to overall acceptability
of the products.
4.Conclusion:
In conclusion, the obtained results mostly reflect the outlined hypothesis and correlate with
other studies, as pH and sugar concentration decreased while SCOBY weight increased. The
results for vitamin C was contradicted with the hypothesis, however the validity of the results
remains unclear as there were errors in the preparation of kombucha. More reliable and accurate
data would be needed for proper evaluation of such results.

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