Sirindhorn International Institute of Technology Thammasat University GROWTHS, ANTIBACTERIAL AND...

Sirindhorn International Institute of TechnologyThammasat University

GROWTHS, ANTIBACTERIAL AND ANTIFUNGAL ACTIVITIES OF KEFIR GROWN

IN DIFFERENT CULTURE MEDIASathika Jariyatamkitti, Somrutai Kitchalao, Pattarawan Wuttisan,

Rachnarin Nitisoravut*, Issara Poljungreedand and Siwarutt Boonyarattanakalin


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OUTLINES

• Background• Statement of Problem•Objectives•Methodology• Results and Discussion• Conclusion


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WHAT IS KEFIR?

Kefir Fermented drink Natural prebiotic: rich in

enzymes and beneficial live bacteria or microbial

Benefits Nutrition Additive product Therapeutic effects http://ecofriendlyfreckles.blogspot.com/2010/01/eco-friendly-freckles-is-now-

selling.html


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WHAT IS KEFIR GRAINS?

Diameter: 3 to 20 mm

Grain matrix

Polysaccharide

Protein (dry weight)

Cellular debris and unknown components

Microorganisms: A complex symbiotic relationship

Mostly lactic acid bacteria and yeasts

http://en.wikipedia.org/wiki/File:Kefirpilze.jpg


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WHAT ARE KEFIRANS?

Exopolysaccharides (EPS)Mainly produced from bacteriaProvide…

Specific rheology and texture of fermented milk products

Biological Properties- Antimicrobial and

antifungal- Antitumor

o Fernando Lopitz-Otsoa, A. R., Natalia Elguezabal and Javier Garaizar, Rev Lbaroam Micol, (2006) 23, 67.

o Hsi-Chia Chen, S.-Y. W., Ming-Ju Chen, Food Microbiology, (2008) 25, 492.

- Anti-inflammatory- Antimutagenic and

antioxidant


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STATEMENT OF PROBLEM

Can milk kefir grown in media other than cow milk Would kefir grown in other media possess health benefits Alternative culturing liquid… Tropical country Plenty of coconut trees Relatively Cheap

Growth of kefir grains grown in three different culture mediaMicroorganism profiles of kefir and kefir grainsCompare antibacterial and antifungal activities of milk, coconut milk and coconut water kefir

Coconut milkCoconut water


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OBJECTIVES

To see the effects of coconut milk and coconut water, as a culturing medium for kefir, on kefir culture growth

To classify microorganism profiles in kefir and kefir grains

To examine antibacterial and antifungal activities of kefir products


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METHODOLOGY


ISOLATION AND CULTIVATION OF LACTIC ACID BACTERIA, ACETIC ACID BACTERIA AND YEASTS

Suspend grains in phosphate buffer and homogenize using mortar

Perform serial dilutions for enumerations

Isolate bacteria using MRS agar, DECA agar, and YM agar and incubate

Count and observe the colonies


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ANTIBACTERIAL AND ANTIFUNGAL TESTING

Inoculum Preparation

Inoculation of Test Plates

Interpreting Results

Application of Disc to Inoculated Plates


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INOCULUM PREPARATION

Select isolated colonies and transfer them into a broth tube

Incubate the broth culture

Compare the turbidity with 0.5 McFarland Standard

http://www.bio-world.com/productinfo/3_43_287_688/10171/Potato-Dextrose-Broth.html


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INOCULATION OF TEST PLATES

Streak the swab over the entire agar surface

http://www.ehow.com/info_8681698_purpose-streaking-agar-plate.htmlhttp://www.flickr.com/photos/medmicro/page10/


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APPLICATION OF DISC TO INOCULATED PLATES

Place on inoculated plates

Incubate plates

http://butterflyandbiotech.blogspot.com/2012/06/pepper-antimicrobial-effect.html

Soak ø5 mm no.1 filter paper by fermented solutions and positive

control solutions


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INOCULATION OF TEST PLATES

Measure the diameter of clear zones

Sliding calipers

A ruler

http://ftp.cdc.gov/pub/infectious_diseases/artesting/images_photos/disk%20diffusion/


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RESULTS AND DISCUSSION


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RESULTS AND DISCUSSIONCoconut milk and coconut water can be used as an alternative culturing medium for kefir culture

Growth data for kefir grains grown in three different culture media (50 mL) at 25°C

4.7g/100g Sugar level


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Species of bacteria and yeasts in kefir and kefir grains can be found

Selective plates GECA MRS YMShape of microorganisms

Bacillus Coccus Yeast Bacillus Coccus Yeast Bacillus Coccus Yeast

Milk kefir + + - + + - + + -

Milk kefir grain + + + + - - + - +

Coconut milk kefir + - + + - - + - +Coconut milk kefir grain + - + - - - + - +

Coconut water kefir + - + + - - + - +Coconut water kefir grain + - + + - - + - +

*+ indicates the presence of the microbial where – indicates the absence of the microbials

Table 1*: Microscopic examination of kefir and kefir grains in three different culturing media


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Species of bacteria and yeasts in kefir grains can be found

Selective plates GECA MRS YMShape of microorganisms

Bacillus Coccus Yeast Bacillus Coccus Yeast Bacillus Coccus Yeast

Milk kefir + + - + + - + + -

Milk kefir grain + + + + - - + - +

Coconut milk kefir + - + + - - + - +Coconut milk kefir grain + - + - - + - +

Coconut water kefir + - + + - - + - +Coconut water kefir grain + - + + - - + - +

*+ indicates the presence of the microbial where – indicates the absence of the microbials

Table 1*: Microscopic examination of kefir and kefir grains in three different culturing media


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RESULTS AND DISCUSSIONKefir grown in coconut milk and coconut water have antibacterial and antifungal activities

Strains

Antibiotic Agents Kefir Products

Flucozole Ampicillin Chloram-phenicol Penicillin Milk Coconut

milk Coconut water

E. Coli (-) 0.0 15.5 25.0 0.0 8.9 11.5 11.0S. Typhi (-) 0.0 27.0 35.5 8.5 11.5 15.5 11.0S. Aureus (+) 8.0 29.5 25.5 33.5 12.5 12.0 10.0B. Subtilis (+) 10.5 51.5 34.5 53.5 11.5 13.0 14.5C. albican 18.5 0.0 20.5 0.0 11.0 0.0 0.0

** The results represent the mean of clear zone diameters (mm). The non fermented media (milk, coconut milk and coconut water) show no inhibition zone

Table 2**: Antibacterial and antifungal activities using disk diffusion method of 24 h kefir fermentation in various culturing media


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Strains



milk Coconut water





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Strains



milk Coconut water





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CONCLUSION

Coconut milk and coconut water can be used as the alternative to cow milk

Bacillus and Yeasts All kefir and grainsCoccus Milk kefir and grains

Antibacterial activities- Coconut milk kefir- Coconut water kefirAntifungal activities- Only milk kefir


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REFERENCESSemih Otes , a. O. C., Pakistan Journal of Nutrition, (2003) 2, 54.Fernando Lopitz-Otsoa, A. R., Natalia Elguezabal and Javier Garaizar, Rev Lbaroam Micol, (2006) 23, 67.Hsi-Chia Chen, S.-Y. W., Ming-Ju Chen, Food Microbiology, (2008) 25, 492.Zhou Jianzhonga, L. X., Jiang Hanhub, Dong Mingshengb, Food Microbiology, (2009) 26, 770.Chujian Chen, McGill University Montreal, (2005), p 222.Prof.Dr.Lalitha Kameswaran, Manual on Antimicrobial Susceptibility Testing, In (2004).http://www.neogen.com/Acumedia/pdf/ProdInfo/7101_PI.pdf (Retrieved September 26,2012)Kamila Leite Rodrigues, L. R. G. C., Jose Carlos Tavares Carvalho, Joao Evangelista and Jose Mauricil Schneedorf, International Journal of Antimicrobial Agents, (2005) 25, 404.Michel Andre Wattiaux, Milk Composition and Nutritional Value, In Dairy Essentials - Lactation and Milking.Muhammad Majeed, P. D., Lakshmi Prakash, Ph.D., CococinTM: The Nourishment FactorTM Freeze-Dried Coconut Water Solids, In (2007).http://www.fineli.fi/food.php?foodid=29014&lan g=en (Retrieved September 15, 2012)


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QUESTIONS?

Sirindhorn International Institute of Technology Thammasat University GROWTHS, ANTIBACTERIAL AND...

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