Water Kefir Microbiological Composition and Rapidly Growing Yeast

Wei Ma, Ron Peck

BI451 Research Project, Biology Dept., Colby College, Waterville, Maine
CONCLUSIONS
INTRODUCTION
• We were able to identify
• Kefir could be used as a highly
selective cancer chemotherapy various Lactobacillus strains in
• Must determine microbial composition our stock culture. We did not
in order to determine safety for WM, 2014
identify any potentially
Fig1: Stock kefir culture using brown sugar,
consumption and to reproduce sucanat, supplemented with dried apricots harmful Bacteria.
uniform anti-cancer complex
Stock Kefir • Ampicillin and oxytetracycline
Culture media revealed potentially
METHODS
• Culture-Dependent Method: WM, 2014 useful strains of yeast. These
• Stock water kefir culture were grown/streaked on trypic WM, 2014
Fig. 5 steak plate from stock kefir culture,
soy, MRS, and YM liquid and agar media. Same culturing Fig.2 agarose gel containing metagenomic notice the two distinct morphotypes yeasts grow extremely fast,
techniques were performed with the addition of DNA for E. coli transformation
oxytetracycline HCL and ampicillin to select for yeast Culture Independent Culture Dependent
causing high turbidity in
species Method Method
• Colonies were examined via Gram staining techniques
Gel excision culture media within 24hrs.
Fig. 6 selective media
• Culture-Independent Method:
Metagenomic Analysis
Two Distinct containing ampicillin and Further study is required to
• Stock water kefir culture were extracted for DNA using Morphotypes oxytetracycline to isolate
GenScript Plasmid Miniprep Kit with modification; Transform into yeast and other antibiotic determine whether or not
vortexing were performed after the addition of lysis competent E. resistant bacteria
buffer to extract genomic DNA. DNA samples were coli Species Identification Microscopy these strains of yeast are
sequenced using improved Sanger sequencing method
medically useful.
Culture in super
RESULTS ampicillin plate
by streaking
• Culture Dependent Method:
• Culture-dependent method
Colony picking
revealed two distinct WM, 2014

morphotypes: clear and white.

The white morphotype was Plasmid
Fig. 7-11, rapidly growing fungi
from super antibiotic
purification
successfully transferred and concentrates, unidentified,
7 WM, 2014 8 WM, 2014
under 40x magnification
cultured in liquid media,
whereas the clear morphotype
Sequencing
did not grow in any liquid media. Fig.3 sample 16sRNA DNA sequences from
metagenomic analysis showing base pair
The clear morphotype was scaled by confidence
purified for DNA identification.
Bacterial Composition Percentage
• Culture Independent Method:
• We found many different species L. hilgardii 

of Lactobacillus, including L. L. JCM 7745 

9 WM, 2014 10 WM, 2014
satsumensis, L. paracasei, L. REFERENCES
L. ghanensis 1. Beshkova DM, Simova ED, Simov ZI, Frengova GI, Spasov ZN. 2002. Pure cultures for making kefir. Food Microbiology 19:537-544.

ghanesis, L. rennaqif7, L. 2. Botelho PS, Maciel MIS, Bueno LA, Marques MDFF, Marques DN, Sarmento Silva TM. 2014. Characterisation of a new exopolysaccharide obtained from of
fermented kefir grains in soymilk. Carbohydrate Polymers 107:1-6.
3. Cevikbas A, Yemni E, Ezzedenn FW, Yardimici T, Cevikbas U, Stohs SJ. 1994. Antitumoural antibacterial and antifungal activities of kefir and kefir grain.
Phytotherapy Research 8:78-82.

JCM7745, and L. hilgardii using L. rennanqilf7 4.

5.
College C. 2013. Food Microbiology: The Making of Yogurt, Pickles and Kefir Water (Tibicos) BI 248L Laboratory Five.
Diosma G, Romanin DE, Rey-Burusco MF, Londero A, Garrote GL. 2014. Yeasts from kefir grains: Isolation, identification, and probiotic characterization.
World Journal of Microbiology and Biotechnology 30:43-53.
6. Ghoneum M, Gimzewski J. 2014. Apoptotic effect of a novel kefir product, PFT, on multidrug-resistant myeloid leukemia cells via a hole-piercing mechanism.

bacterial primers 33 and 34. L. paracasei subsp. paracasei

International Journal of Oncology 44:830-837.
7. Hosseini H, Hippe B, Denner E, Kollegger E, Haslberger A. 2012. Isolation, identification and monitoring of contaminant bacteria in Iranian Kefir type drink
by 16S rDNA sequencing. Food Control 25:784-788.
8. Liu JR, Wang SY, Lin YY, Lin CW. 2002. Antitumor activity of milk kefir and soy milk kefir in tumor-bearing mice. Nutrition and Cancer 44:182-187.
9. Maeda H, Zhu X, Suzuki S, Suzuki K, Kitamura S. 2004. Structural characterization and biological activities of an exopolysaccharide kefiran produced by
Lactobacillus kefiranofaciens WT-2B T. Journal of Agricultural and Food Chemistry 52:5533-5538.
10. Marsh AJ, O'Sullivan O, Hill C, Ross RP, Cotter PD. 2013. Sequence-based analysis of the microbial composition of water kefir from multiple sources. FEMS

L. satsumensis Microbiology Letters 348:79-85.

11. Miguel MGCP, Cardoso PG, Magalhães KT, Schwan RF. 2011. Profile of microbial communities present in tibico (sugary kefir) grains from different Brazilian

THANKS
States. World Journal of Microbiology and Biotechnology 27:1875-1884.
12. Nalbantoglu U, Cakar A, Dogan H, Abaci N, Ustek D, Sayood K, Can H. 2014. Metagenomic analysis of the microbial community in kefir grains. Food
Microbiology 41:42-51.
0% 10% 20% 30% 40% 13. Punaro GR, Maciel FR, Rodrigues AM, Rogero MM, Bogsan CSB, Oliveira MN, Ihara SSM, Araujo SRR, Sanches TRC, Andrade LC, Higa EMS. 2014. Kefir
administration reduced progression of renal injury in STZ-diabetic rats by lowering oxidative stress. Nitric Oxide - Biology and Chemistry 37:53-60.

Thanks to Prof. Ron Peck, Rachel Bolender, Shayla William, and Dan 14. Rodrigues KL, Gaudino Caputo LR, Tavares Carvalho JC, Evangelista J, Schneedorf JM. 2005. Antimicrobial and healing activity of kefir and kefiran extract.
International Journal of Antimicrobial Agents 25:404-408.

Saunderand for their helpful guidance. Special thanks to Prof. Frank Fig.12 chart depicting the relative Lactobacillus Bacterial 15. Sefidgar SAA, Gharekhani S, Ghasempour M. 2014. Identification of yeasts and bacteria isolated from Iranian Kefir drink. Jundishapur Journal of
Microbiology 7.

Fekete and Lindsey Colby for the kefir samples. Colby’s the best! composition identified in 19 samples of community-sampling 16. Simova E, Beshkova D, Angelov A, Hristozova T, Frengova G, Spasov Z. 2002. Lactic acid bacteria and yeasts in kefir grains and kefir made from them.
Journal of Industrial Microbiology and Biotechnology 28:1-6.

metagenomic analysis 17. Simova E, Simov Z, Beshkova D, Frengova G, Dimitrov Z, Spasov Z. 2006. Amino acid profiles of lactic acid bacteria, isolated from kefir grains and kefir
starter made from them. International Journal of Food Microbiology 107:112-123.
18. Wei Sheng Hong HCC, Yen Po Chen, Ming Ju Chen. 2009. Effects of kefir supernatant and lactic acid bacteria isolated from kefir grain on cytokine production
11 WM, 2014 by macrophage. International Dairy Journal:244-251.