Acknowledgements

1
Antimicrobial Activity of Kombucha Tea
Ashley Good and Navdeep Grewal Biology
Department, Skyline College, San Bruno CA
Methods 1. Commercially available SCOBYs were
purchased from Kombucha America and Happy
Herbalist. 2. According to the suppliers
directions, a 3-L beaker was filled with a
solution of water, sucrose, black tea, and white
vinegar (5 acetic acid). 3. SCOBY was added to
the beaker and incubated at 37C for 8
days. 4. The pH was recorded daily. 5. Our
kombucha and a bottled kombucha beverage
(Synergy) were tested against gram-negative
Escherichia coli (ATCC 11775) and Pseudomonas
aeruginosa (ATCC 10145), gram-positive
Staphylococcus aureus (ATCC 27659) bacteria, and
Saccharomyces cerevisiae (ATCC 9763) and
Aspergillus niger (ATCC 16404) fungi. 6. White
vinegar (5 acetic acid) or 1.0 M NaOH was added
to maintain the control pH of 2 or the test pH of
7. The negative control was black tea and sucrose
adjusted to the appropriate pH. 7. Disk
diffusion assay a. Nutrient agar plates were
aseptically inoculated with 200 µL of bacteria or
fungi grown in nutrient broth. Filter paper discs
soaked in kombucha or control black tea were
placed on the plates. b. Plates were incubated
at 37C for 24 hr. 8. Neutralized kombucha (pH
7) was tested against gram-negative
bacteria. 9. Evaporation of kombucha yielded a
gummy microbial mass with a naturally occurring
pH of 2 which was tested in a disk diffusion
assay. The pH was not adjusted. 10. Kombucha
samples were heated to 56C for 30 min. and then
used in disk diffusion assays at pH 2 and pH 7.

• Results
• The pH steadily declined during the 8-day
  fermentation (Figure 2).
• Kombucha did not inhibit the gram-positive
  bacterium (S. aureus). It did show significant
  zones of inhibition against the gram-negative
  bacteria E. coli (3 mm) and P. aeruginosa (6
  mm) (Figure 3).
• Kombucha did not inhibit fungi (Sa. cerevisiae
  and A. niger).
• The pH is a factor because inhibition of both
  gram-negative bacteria increased at a lower pH by
  an average of 80 (Figure 4).
• At a higher concentration, the evaporated
  kombucha resulted in a microbial mass that
  yielded more inhibition (19 more against E.
  coli) than the positive control (5 acetic acid)
  suggesting that the acidic pH is not the only
  factor causing inhibition (Figures 5 and 6).
• Even at an acidic pH, heating kombucha destroyed
  its ability to inhibit gram-negative bacteria.

Abstract There is a need for new antibacterial
agents due to acquired resistance to current
antibiotics. For centuries, people have prepared
teas from natural sources to treat disease. In
the 21st century, these herbal remedies are
gaining acceptance in the West and provide a
starting place to look for new antibiotics.
Kombucha is one such source it is a tea that is
fermented by a diverse consortium of
microorganisms. Our aim was to investigate the
antibacterial properties of kombucha tea against
gram-negative Escherichia coli and Pseudomonas
aeruginosa bacteria and gram-positive
Staphylococcus aureus bacteria. Teas produced
from two commercially available kombucha starter
cultures were grown in black tea and sucrose. The
pH of the kombucha decreased to 2 during the
8-day fermentation. Antimicrobial activity was
determined using a disk-diffusion assay of
kombucha tea and a control of black tea and
sucrose adjusted to a pH of 2 with 5 acetic
acid. Kombucha inhibited gram-negative bacteria
at pH 2. To test the effect of pH, we neutralized
the kombucha tea and the control using 1M NaOH.
Antibacterial activity was reduced at by an
average of 80 at pH 7 against gram-negative
bacteria. Evaporation of kombucha tea yielded a
gummy microbial mass, which increased inhibition
by 19 at pH 2 against E. coli. Heated kombucha
teas did not inhibit bacterial growth suggesting
activity is due to proteins. Kombucha may provide
compounds to develop products that inhibit
survival of E. coli in food or treat
gram-negative bacterial infections.

• Discussion Conclusion
• The antibacterial activity of kombucha is not due
  solely to pH. Kombucha does inhibit growth of
  gram-negative bacteria, independent of pH.
• The antibacterial action appears to be due to a
  heat-sensitive protein.
• The active chemical may be a bacteriocin produced
  by one of the microbes in the kombucha
  consortium.
• Currently we are attempting to isolate the
  chemical that is causing inhibition.
• We are culturing the kombucha microbes to
  determine whether one or more is responsible for
  the antimicrobial activity.

Figure 3. Zones of inhibition. Kombucha at pH 2
inhibits gram-negative bacteria.
Hypothesis Kombucha will exhibit antimicrobial
activity, most significantly in an acidic
environment.

• Background
• Kombucha is a fermented beverage made by a
  Symbiotic Culture Of Bacteria and Yeast (SCOBY,
  Figure 1) (5).
• The SCOBY consortium of bacteria and yeasts is
  traditionally grown in brewed tea and sucrose
  (7).
• A wide variety of microbes have been isolated
  from kombucha SCOBYs (3).
• Due to the diversity among kombucha SCOBYs, there
  is no single species universally associated with
  kombucha fermentation nor is it possible to
  define an exact microbial composition for the
  drink.
• During incubation, yeast ferment sucrose and
  produce ethanol. After 1 to 2 days, acetic acid
  bacteria use the ethanol and produce acetic acid
  (9)
• High sugar and acetic acid tolerances have been
  confirmed among the predominant species
  identified (9).
• Every batch of kombucha is different and over 50
  different compounds have been identified from
  kombucha with 11 being the most common (6).
• Hailed as an immortal health elixir in the
  orient, kombucha dates back to the Tsing Dynasty
  in ancient China around 250 B.C.E. (5). It is now
  popular with home brewers. Claims that it
  promotes immunity and is staphylocidal are
  unsubstantiated (2).
• Kombucha SCOBYs are grown and shared among people
  for starting their own cultures. Consumption of
  home-produced kombucha has been the suspected
  cause of some illnesses and one possible related
  death (2, 8).
• Conventional antibiotics are becoming less
  effective. Perhaps kombucha will provide a new
  source (4).
• Our objective was to determine whether kombucha
  cultures produce an antimicrobial.

• Literature Cited
• Benefits of Kombucha. ltwww.geocities.com/ladyfan
  gs.geo/benefits.htmlgt.
• Centers for Disease Control and Prevention. 1995.
  Unexplained severe illness possibly associated
  with consumption of kombucha tea. Morbidity and
  Mortality Weekly Report 44(48)892-893, 899-900.
• Greenwalt, C. J. 2000. Kombucha, the fermented
  tea Microbiology, composition, and claimed
  health effects. Journal of Food Protection
  73(7)976-981.
• Kapil, A. 2005. The challenge of antibiotic
  resistance Need to contemplate. Indian Journal
  of Medical Research 12183-91.
• Kasper, E. Kombucha Mushroom Tea.
  ltwww.happyherbalist.com/ kombucha.htmgt (18 May
  2009).
• Roussin, M. R. 1996. Analyses of Kombucha
  Ferments. Fruita, CO Michael R. Roussin.
  ltwww.kombucha-research.comgt
• Sreeramulu, G., Y. Zhu, and W. Knol. 2000.
  Kombucha fermentation and its antimicrobial
  activity. Journal of Agricultural and Food
  Chemistry 48(6)2589-2594.
• SungHee Kole, A. 2009. A Case of Kombucha Tea
  Toxicity. Journal of Intensive Care Medicine
  24(3)205-207.
• Teoh, L. A., G. Heard and J. Cox. 2004. Yeast
  ecology of kombucha fermentation. International
  Journal of Food Microbiology 95119-126.

Figure 4. Effect of pH. Inhibition of
gram-negative bacteria (E. coli and P.
aeruginosa) increased with decreased pH.
Figure 1. Daughter SCOBY after fermentation. Size
bar 1 cm

Figure 2. Change in pH during 8-day fermentation.
Average pH for three different SCOBY starter
cultures.

Figure 5. Disk diffusion assay against P.
aeruginosa. a. 5 acetic acid b. Kombucha (pH
2) c. Concentrated microbial mass Size
bar 1 cm
Acknowledgements Dr. Christine Case, Biology
Professor, Skyline College Patricia Carter,
Biology Technician, Skyline College Stephen
Fredricks, MESA Director, Skyline College Tiffany
Reardon, Assistant Director, California
MESA Funded by NIH/SFSU Bridges to Baccalaureate