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International Baccalaureate
A study on the effect of sugar concentration on the antioxidant concentration of
Kombucha
Aim

This investigation aims to show how the sugar concentration in Kombucha affects the number of
antioxidants in this beverage at the end of the fermentation process.

Background

Kombucha tea is a sweet-and-sour beverage obtained

" from a fermentation process.
Fermentation is defined as, "the anerobic generation of ATP from glucose that generates
characteristic end products such as alcohol and lactic acid1 (Allot, 2014). This drink is a form of
sugared black tea (Camellia sinensis) with a symbiotic culture of fungi and acetic bacteria. The
creation of Kombucha in this investigation used the substrate of black tea and white
sugar and the aid of the SCOBY fermentation process (Symbiotic Culture Of Bacteria and
Yeast). The white sugar concentration that is usually added to Kombucha is ¼ of a cup.2

The SCOBY is a mixed culture of yeast and bacteria that assists the fermentation process in
Kombucha. In SCOBY there is a symbiotic relationship between bacteria and yeast because
yeast utilizes the sugars in Kombucha for the production of alcohol. Then, bacteria consumes
this alcohol and produces acetic acid, which gives the beverage the slight taste of vinegar. This
process can take from one to four weeks, depending on the SCOBY.

Kombucha tea is beneficial for health being a potential source of probiotics, containing
antioxidants, polyphenols, and killing bacteria.

1
Allot, A. (2014). Biology: for the IB diploma. Oxford: Oxford University Press
2Jayabalan, R., Malbasa, R., Loncar E., Vitas, Jasmina S., & Sathishkumar, M. (2014). A Review on
Kombucha Tea- Microbiology, Composition, Fermentation, Beneficial Effects, Toxicity, and Tea Fungus.
Comprehensive Rewiews in Food Science and Food Safety, vol. 13, no. 4, pp. 538-550.
https://doi.org/10.1111/1541-4337.12073
 Page 2 of 12

Kombucha is rich in antioxidants such as vitamin C, Bl, 82, 83, 86, and 812.3 Vitamin C is a
predominant antioxidant in Kombucha, which is fundamental for human health, and it's essential
in a human diet because it enhances iron consumption. B vitamins are essential for the body to
make sure that the body's cells are functioning properly. These vitamins also help the body
create new cells, convert food into energy, and maintain healthy cells.

Antioxidants in Kombucha are exogenous antioxidants, meaning that they come from outside
the body. They are substances that may protect against free radicals by removing them as
waste products in cells.4 These molecules are stable, which means they can donate an electron
to a free radical. This way the free radical is neutralized, and the damage is reduced. These
antioxidants are used to detain damage in cells by terminating the chain reaction. This
termination is done before fundamental molecules are harmed.

Free radicals are independent molecules produced by the human body by from metabolic
processes.. Also, free radicals can be produced by external sources such as X-rays, pollutants,
or industrial chemicals.5 These are dangerous for the human body, considering that they cause
cell damage, known as oxidative stress. Free radical reactions accumulate over time in the
human body, and they are manifested as diseases like cancer and atherosclerosis.

Research Question

To what extent does the sugar concentration on Kombucha tea affect the
concentration of antioxidants at the end of the fermentation process?

Hypothesis

After the fermentation process, different Kombucha teas with different sugar concentrations
will vary the end product of antioxidant concentrations. Consequently, there will be a
significant increase resulting in an increase in sugar concentrations will result in more
antioxidant concentration.

3 Kumar, V. & Joshi, V. (2016). Kombucha: Technology, Microbiology,Production, Composition and

Therapeutic Value. Int/. J. Food. Ferment. Technol, vo l. 6 , no . 1 , pp . 13-24 . DOI: 10.5958/2277-9396.2016.00022.2
4 Waisundara, V., Amarashinghe , H., & Weerakkody, N. (2018) Evaluation of physicochemical
properties and antioxidant activities of kombucha 'T ea Fungus" during extended periods of fermentation. Food
Science and Nutrition, vol. 6, no. 3, pp. 659-665. Doi: 10.1002/fsn3.605.
5 Phanienda, A., Jestadi D., & Periyasamy, L. (2015). Free Radicals: Properties, Sources, Targets, and Their

Implication in Various Diseases." Indian Journal of Clinical Biochemistry, vol. 30, no. 1. pp. 11-26.DOI:
10.1007/s12291-014-0446-0
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Variables

The independent variable in this investigation is the white sugar concentration, while
the dependent variable is antioxidant (Vitamin C) concentration. The controlled
variables in this investigation were all the factors that could influence the fermentation
process, such as temperature, light availability, and the amount of oxygen present.
These variables were kept constant by placing all fermenting Kombucha jars in the
same place at room temperature (25° C), and with minimum light exposure.

Procedure

For the preparation of Kombucha, 15 jars of 1.3 liters were prepared with black tea,
different concentrations of sugar, and 3cm SCOBY. The sugar concentrations in the
preparations varied. There were five different sugar concentrations: ¼ of a cup, ½ of a
cup, ½ of a cup, ¾ of a cup, and a cup. Therefore, there were three jars for each sugar
concentration, and three trials were done for each jar. For the fermentation process,
these jars were placed for a week in a dark space at room temperature and covered
with a cheesecloth. All of the sugar concentrations are higher than the usual amount of
sugar added to Kombucha (¼ of a cup) to observe if there is an increase in antioxidant
concentration from the one present in the traditional Kombucha recipe.

To determine the antioxidant concentration, a research process was assembled to

determine an appropriate method. The most suitable process for the central objective of
this investigation was the PFRAP (potassium ferricyanide reducing power) method.6
Conserving the purpose and the available equipment in the Chemistry lab, there was an
adaptation made to this method. Instead of using Potassium ferricyanide, 5.000g of Iron
(III) nitrate reacts with 6.800g of Potassium thiocyanate causing a dark red color to the
aqueous solution. Because of the high thiocyanate concentration in the reaction. there's a

formation of [Fe(SCN)6]•3-.

To test the process, a 20.00ml sample of Kombucha tea was

• measured with a 10ml
pipette and was transferred into a 125 Erlenmeyer flask with the pipette. Adding
•

distilled water to the Erlenmeyer flask was not necessary because when [Fe(SCN)6]3
reacted with the Kombucha tea the change in color occurred rapidly. The titration
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technique was done to determine theconcentration of vitamin C. A 50ml burette

• used
in the titration was filled with the [Fe(SCN)6]3- solution. The first trial consisted of
0.0200g of vitamin C titrated with [Fe(SCN)6]3- to see the change in color to an
orange color. Then to find out the antioxidant concentration in Kombucha, the process
was repeated by filling out a 20ml pipette of the Kombucha tea with ¼ of sugar and
transferring it to the Erlenmeyer flask and performing three different trials for the three
different jars of the same sugar concentration. Afterward, the same process was
repeated nine times for each of the Kombuchas with different sugar concentrations: ½,
½, ¾, and 1 cup of sugar. This added up to a total of 45 trials for different sugar
concentrations. The antioxidant concentration was measured when the 20.00ml of
Kombucha tea changed color from brown to a dark gray color.

In this Internal Assessment, an ethical consideration

• is the waste of Kombucha, since
some were not drinkable because of the high amounts of sugar in it. It would be
unethical to discard the sweet Kombucha because it is a waste of food items. Another
ethical consideration is the precautions, such as washing hands and use of goggles
and aprons at all times. These safety measures have to be taken because
[Fe(SCN)6]3- can cause irritation in skin because it is a strong oxidizer. Also,
Potassium thiocyanate is moderately toxic by ingestion.

6
Pisoschi, A. & Negulescu, G. 2011. Methods for Total Antioxidant Activity Determination : A Review. ts1ocnem1srry &
Analytical Biochemistry, vol. no. 1, pp. 1-10. DOI: 10.4172/2161-1009.1000106.

7
Marzzacco, C. (1998). The Effects of Salts and Nonelectrolytes on the Solubility of Potassium
 Page 5 of 12

Research Methodology

To select the procedure, there was a research process to select the method to measure
antioxidant concentration. A scientific article that explained different methods to measure
antioxidants was used as a secondary source, and it provided information on the PFRAP
(potassium ferricyanide reducing power) method. Later on, this method was adapted to the
chemical compounds available in the Chemistry lab. Furthermore, there was more literature
investigation for background descriptive research. When testing the relationship between
the independent and dependent variable, the methods of data collection in this investigation
included quantitative and qualitative data in the titration process. The titration technique was
utilized because it was a process repeatedly done in Chemistry class, and it was interesting
to explore how it can be applied to Biology. The measurement of the volume of

[Fe(SCN)6]. 3- used is part of the quantitative data, while observing the change in color in
Kombucha was part of the qualitative data of the investigation.

Equipment
• 5.004g of Iron (Ill) nitrate
• 6.800g of Potassium thiocyanate
• 0.0208g of Vitamin C
• 20ml of Kombucha tea
• Volumetric flask of 1000ml (±30) ml
• Burette (±0.03) ml
• Pipette of 20ml (±0.030) ml
• Graduated cylinder of 50ml (±0.05) ml
• Erlenmeyer flask of 125ml (±0.05) ml
• Balance (±0.001) g

Bitartrate: An Introductory Chemistry Discovery Experiment. ACS Publications, vol. 75, no. 12. DOI:
https://doi.org/10.1021/ed075p1628
 Page 6 of 12

Figure I: Kombucha tea color Figure II:

dark red color

Method Background

Iron (III) Nitrate is a chemical compound with the formula Fe(NO3)3, and since it tends to
become liquid it is common for it to be found in the form of Fe(NO3)3.9H20 in which it forms
violet crystals. This crystalline chemical compound is an iron source for uses compatible
with nitrates. Nitrate compounds such as are soluble and water and serve as oxidizing
agents. 8

Potassium thiocyanate is a chemical compound with the formula of KSCN. An important property
of this compound is its low melting point. This compound is an important salt in thiocyanate anion,
the conjugate base of thiocyanic acid.

Iron (III) reacts with thiocyanate to give red color in aqueous solution (Refer to Figure II).

8
Schmidt, H., Bok, F.. Asztalos, A., & Voigt, W. (2012). New iron(III) nitrate hydrates: Fe(NO 3) 3 · x H 2 0 with x =
4, 5 and 6. Crystal Structure Communications, vol. 68, no. 6, pp. 29-33. DOI: 10.1107/S0108270112015855
 Page 7 of 12

Assumptions

In this Internal Assessment, it is assumed that the antioxidants in Kombucha are all Vitamin
C because the method only assesses the method of vitamin C. It is also assumed that all
vitamins in Kombucha are proportional: if there is an increase of the concentration of
Vitamin C with an increase of sugar concentration, it can be assumed that there will also be
an increase of Vitamins B1, 8 6, and B12. It is assured that only Vitamin C is taken into
consideration in the present investigation because in the first trial of the titrations was done
with Vitamin C as the indicator. This assumption is based on an investigation made by the
Institute of Food Science & Technology where they quantified the concentrations of soluble
vitamins in Kombucha by using a thin layer chromatography and eventually compare the
values with their chromatographic UV spectra. The results of this investigation were: vitamin
B1 0.74 mg/ml-1, vitamin B6 0.52 mg/ml -1, vitamin 812 0.84 mg/ml -1, and vitamin C 1.51
mg/ml-1 (Bauer-Petrovska and Petrusevska-Tozi, 2001). Hence. the antioxidant with greater
concentration in Kombucha is vitamin C.

Table I. Amount of each type of antioxidant in Kombucha according to the Institute of Food
Science & Technology 9

mg/ml-1
Vitamin B1 74
Vitamin B6 52
Vitamin B12 84
Vitamin C 152

9 Bauer-Petrovska B, Petrushevska-Tozi L. "Mineral and water soluble vitamin content in the Kombucha drink."
International Journal of Food Science & Technology, vol. 35, no. 2, 2000, pp. 201-205.DO:I
10.1046/j.1365-2621.2000.00342.x
 Page 8 of 12

Data
Table II. The effect of sugar concentration on antioxidant concentration

Volume used of [Fe(SCN)6]3-, (±0.03) ml

1/4 cup of sugar 1/3 cup of sugar 1/2 cup of sugar 3/4 cup of sugar 1 cup of sugar
Trial 1 0.98 1.17 1.30 1.30 1.59
Trial 2 0.97 1.18 1.20 1.29 1.54
Jar 1 Tria13 0.96 1.19 1.25 1.28 1.50
Trial I 1.08 1.13 1.30 1.40 1.48
Trial 2 1.06 1.17 1.35 1.30 1.25
Jar2 Trial 3 1.07 1.15 1.25 1.36 1.30
Trial 1 1.01 1.19 1.27 1.40 1.30
Trial 2 0.99 1.15 1.24 1.20 1.20
Jar3 Trial 3 1.00 1.17 1.25 1.30 1.25
Average 1.01 1.17 1.27 1 .31 1.38
Standard Deviation 0.04 0.02 0.04 0.06 0. 14

Graph I: The effect of sugar concentration on the volume used of [Fe(SCN)6]3-•

2
Volume used of [Fe(SCN)6]3- (±0.03)mL

1,5

0,5

0
1/4 cup of sugar 1/3 cup of sugar 1/2 cup of sugar 3/4 cup of sugar 1 cup of sugar
 Page 9 of 12

Evaluation and Conclusion

The Potassium Ferricyanide Reducing Power method resulted effectively at the time of
exploring the relationship between sugar concentration and antioxidant concentration since the
method displayed a trend in the results. The results obtained in the titration of Kombucha tea
with [Fe(SCN)6]3- demonstrated in table II, reflect that the concentration of antioxidants
increased as the sugar concentration increased in each trial. When in the Kombucha tea a
greater concentration of sugar was added, more volume of [Fe(SCN)6]3- was necessary to each
endpoint of the reaction, which was the color change. Therefore, there were more antioxidants
in solutions with more sugar. Graph I evidenced a positive correlation between antioxidant
concentration and sugar concentration. Also, there is a clear increasing trend line in the results.

To answer my research question, "To what extent does the sugar concentration on Kombucha
tea affect the concentration of antioxidants at the end of the fermentation process? " It appears
that sugar concentration is a factor that significantly affects antioxidant concentration. However,
in the results for ¾ cups of sugar and 1 cup of sugar, there is an insignificant change because
the Standard Deviation of the results overlaps. It can be concluded that the hypothesis in this
internal assessment was accurate in the aspect that there is a positive correlation between
sugar and antioxidants: the increase of the independent variable causes an increase in the
dependent variable. Nevertheless, the hypothesis was inaccurate with the last two results
because the volume increase between the two results was 0.07, reflecting an insignificant
increase. Connected to scientific context, sugar does have an effect on antioxidants because, in
the fermentation process, any change in the composition of the medium will have an effect on
the nutritional quality beverage.10

10
Villarreal Soto, S., Beaufort, S., Bouajila, J., Souchard, J., & Taillandier, P. (2018). Understanding
Kombucha Tea Fermentation: A Review. Journal of Food Science, vol. 83, no. 3, pp.580-588. Bauer-Petrovska, B. &
Petrushevska-Toz,i L. (2000). Mineral and water soluble vitamin content in the Kombucha drink. International Journal
of Food Science & Technology, vol. 35, no. 2, pp. 201-205.DOI: 10.1046/j.1365-2621.2000.00342.x
Evaluation

A weakness is that there was not a measurement of the six types of antioxidants in
Kombucha, therefore it had to be assumed that only Vitamin C was present in the sample.
Hence, there is a reduction in the accuracy of the results. In addition, another weakness is
the adaptation that had to be done of the PFRAP (potassium ferricyanide reducing power)
method replacing Potassium ferricyanide with Potassium thiocyanate. Even though both
chemical compounds work similarly,it could affect the accuracy of the results.

Possible improvements

A shortcoming of this investigation is the use of the unit cups, since this unit is not part of
the International System of Units. Therefore, a possible improvement is using grams
instead, to abide by the SI units. Another improvement for this internal assessment is
the possibility to repeat the trials by making
· the [Fe(SCN)6]3- solution less concentrated,
this way more volume will be necessary to change the Kombucha's color. By diluting the
titre by adding water, there will be a greater volume of the [Fe(SCN)6]3- necessary to
change the indicator's color. Therefore, it will be easier to read the volumes from the
burette, and the investigation would contain more detailed results. Additionally, the
investigation could be repeated by implementing more trials for each of the sugar
concentrations to obtain more accurate averages in the antioxidant concentrations in
my response. Furthermore, another aspect of changing for the better in the internal
assessment is reassuring that all of the different jars of Kombucha ferment at the same
time since the titrations were done during different days. This is a fundamental possible
improvement because the time of fermentation could have an effect on the end
nutritional value of the Kombucha, therefore could create inaccuracies in the results.
 Bibliography

1. Allot, A. (2014). Biology: for the IB diploma. Oxford: Oxford University Press

2. Fu, C., Yan, F., Cao, Z., Xie, F., & Lin, J. (2014). Antioxidant activities of kombucha prepared from
three different substrates and changes in content of probiotics during storage. Food Science and
Technology(Campinas),vol. 34, no.1, pp.123-126. Doi: 10.1590/S0101-20612014005000012

3. Jayabalan, R., Malbasa, R., Loncar E., Vitas, Jasmina S., & Sathishkumar, M. (2014). A Review on
Kombucha Tea- Microbiology, Composition, Fermentation, Beneficial Effects, Toxicity, and
Tea Fungus. Comprehensive Rewiews in Food Science and Food Safety, vol. 13, no. 4,
pp. 538-550.https://doi.org/10.1111/1541-4337.12073

4. Kumar, V. & Joshi, V. (2016). Kombucha: Technology, Microbiology, Production, Composition and
Therapeutic Value. Intl. J. Food. Ferment Technol, vol. 6, no. 1, pp. 13-24. DOI:
10.5958/2277-9396.2016.00022.2

5. Marzzacco, C. (1998). The Effects of Salts and Nonelectrolytes on the Solubility of Potassium
Bitartrate: An Introductory Chemistry Discovery Experiment. ACS Publicaitons, vol. 75, no. 12. DOI:
bttps'[/doi org/10 1021/ed075p1628

6. May , A., Narayanan , S., Alcock, J., Varsani, A., Maley, C., & Aktipis, A. (2019). Kombucha: a novel
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7. Muhialdin, B., Meor H., Anis S., Sapawi, C., & Wen, Y. (2019). Effects of sugar sources and
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Research Journal, vol. 26, no. 2, pp. 481-487.

8. Pisoschi, A. & Negulescu, G. 2011. Methods for Total Antioxidant Activity Determination: A Review.
Biochemistry & Analytical Biochemistry, vol. no. 1, pp. 1-10. DOI: 10.4172/2161-1009.1000106.

9. Phanienda, A., Jestadi D., & Periyasamy, L. (2015). Free Radicals: Properties, Sources, Targets, and
Their Implication in Various Diseases. " Indian Journal of Clinical Biochemistry, vol. 30, no. 1.
pp.
11-26.DOI: 10.1007/s12291-014-0446-0

10. Schmidt, H., Bok, F., Asztalos, A., & Voigt, W. (2012). New iron (III) nitrate hydrates: Fe(NO3)3·x
H 2 0 with x = 4, 5 and 6. Crystal Structure Communication,svol. 68, no. 6, pp. 29-33. DOI:
10.1107/S0108270112015855

11. Waisundara, V., Amarashinghe, H., & Weerakkody, N. (2018) Evaluation of physicochemical
properties and antioxidant activities of kombucha "Tea Fungus" during extended periods of
fermentation. Food Science and Nutrition, vol. 6, no. 3, pp. 659-665. Doi: 10.1002/fsn3.605.

12. Velicansk,i A., Cvetkovic, D., Markov, S., Tumbas Saponjac, V., & Vulic, J. (2014). Antioxidant and
Antibacterial Activity of the Beverage Obtained by Fermentation of Sweetened Lemon
Balm (Melissa officinalis L.) Tea with Symbiotic Consortium of Bacteria and Yeasts. Food
Technology and Biotechnology, vol. 52, no. 4, pp. 420-4 29. Doi: 10.17113/ftb.52.04.14.3611.

13. Villarreal Soto, S., Beaufort, S., Bouajila, J., Souchard, J., & Taillandier, P. (2018). Understanding
Kombucha Tea Fermentation: A Review. Journal of Food Science, vol. 83, no. 3, pp.580-588.
Bauer-Petrovska, B. & Petrushevska-Tozi, L. (2000). Mineral and water soluble vitamin content
inthe Kombucha drink. International Journal of Food Science & Technology, vol. 35, no.
2, pp. 201-205.DOI: 10.1046/j.1365-2621.2000.0034

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