Transcript Water Kefir Microbial Composition and Rapidly Growing Yeast

Water Kefir Microbiological Composition and Rapidly Growing Yeast
Wei Ma, Ron Peck
BI451 Research Project, Biology Dept., Colby College, Waterville, Maine
CONCLUSIONS
INTRODUCTION
• Kefir could be used as a highly
selective cancer chemotherapy
• Must determine microbial composition
in order to determine safety for
consumption and to reproduce
uniform anti-cancer complex
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Fig1: Stock kefir culture using brown sugar,
sucanat, supplemented with dried apricots
Stock Kefir
Culture
METHODS
•
Culture-Dependent Method:
•
•
•
Stock water kefir culture were grown/streaked on trypic
soy, MRS, and YM liquid and agar media. Same culturing
techniques were performed with the addition of
oxytetracycline HCL and ampicillin to select for yeast
species
Colonies were examined via Gram staining techniques
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Fig. 5 steak plate from stock kefir culture,
notice the two distinct morphotypes
Fig.2 agarose gel containing metagenomic
DNA for E. coli transformation
Culture Independent
Method
Gel excision
Culture-Independent Method:
•
Stock water kefir culture were extracted for DNA using
GenScript Plasmid Miniprep Kit with modification;
vortexing were performed after the addition of lysis
buffer to extract genomic DNA. DNA samples were
sequenced using improved Sanger sequencing method
RESULTS
• Culture Dependent Method:
• Culture-dependent method
revealed two distinct
morphotypes: clear and white.
The white morphotype was
successfully transferred and
cultured in liquid media,
whereas the clear morphotype
did not grow in any liquid media.
The clear morphotype was
purified for DNA identification.
• Culture Independent Method:
• We found many different species
of Lactobacillus, including L.
satsumensis, L. paracasei, L.
ghanesis, L. rennaqif7, L.
JCM7745, and L. hilgardii using
bacterial primers 33 and 34.
Transform into
competent E.
coli
Culture Dependent
Method
Metagenomic Analysis
Two Distinct
Morphotypes
Species Identification
Microscopy
Fig. 6 selective media
containing ampicillin and
oxytetracycline to isolate
yeast and other antibiotic
resistant bacteria
• We were able to identify
various Lactobacillus strains in
our stock culture. We did not
identify any potentially
harmful Bacteria.
• Ampicillin and oxytetracycline
media revealed potentially
useful strains of yeast. These
yeasts grow extremely fast,
causing high turbidity in
culture media within 24hrs.
Further study is required to
determine whether or not
these strains of yeast are
medically useful.
Culture in super
ampicillin plate
by streaking
Colony picking
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Plasmid
purification
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Fig. 7-11, rapidly growing fungi
from super antibiotic
concentrates, unidentified,
under 40x magnification
Sequencing
Fig.3 sample 16sRNA DNA sequences from
metagenomic analysis showing base pair
scaled by confidence
Bacterial Composition Percentage
L. hilgardii
L. JCM 7745
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L. ghanensis
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THANKS
Thanks to Prof. Ron Peck, Rachel Bolender, Shayla William, and Dan
Saunderand for their helpful guidance. Special thanks to Prof. Frank Fekete
and Lindsey Colby for the kefir samples. Colby’s the best!
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40%
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Fig.12 chart depicting the relative Lactobacillus Bacterial
composition identified in 19 samples of community-sampling
metagenomic analysis
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