The Study of Tibicos Fermentation Product as Pilot Model of Healthy Drinks


  • Hsiao-Han Liu 1Department of Biological Science & Technology, I-Shou University, Kaohsiung, Taiwan, ROC
  • Bo-Shun Tu 1Department of Biological Science & Technology, I-Shou University, Kaohsiung, Taiwan, ROC
  • Yew-Loom Chen Department of Hospitality Management, Tajen University, Pintung, Taiwan, ROC
  • Heng-Long Wang Department of Life Sciences, National University of Kaohsiung, Kaohsiung, Taiwan, ROC


tibicos, health drink, fermentation broth, 18S rDNA sequence, 16S rDNA sequence


Sugar kefir grains (Tibicos) are a symbiotic culture of bacteria and yeasts which were assembled by various strains of microbes attached polysaccharide bracket composed of white transparent particles. Initially, we used chemical colorimetric method to measure that of carbohydrate concentrations, and Brad ford method for that of protein concentrations. We utilized black sugar as carbohydrate source and medium to cultivate Tibicos, and collected media after 0 hr., 24 hr., and 48 hr. different time points. These collected Tibicos grains and fermentation broth were measured with the above methods.
In addition, we used Gram stain to observe the inside microbial populations of Tibicos. The results showed that the weight of cultured grains with brown sugar solution at 25 ℃ and sealed for 48 hr increased to 45% as that of original grains. The components of Tibicos fermentation broth were analyzed by HPLC for ions as following: Lactic acid, Chloride ion, Malate, Sulfate, Oxalic acid, Phosphate, Citrate, and other minor ingredients.
Furthermore, we used the Gram stain to observe the microbial composition within Tibicos grains. The more detail identification of microbial population in Tibicos was done with bacteria and yeasts. The bacteria parts used PCR amplification with the 16S rDNA primer sets (533R and 341Fgc), gel purification, fragments sequencing, and alignment with 16S ribosomal RNA sequences in NCBI database (16S rDNA of Bacteria and Archaea). The result sequences were assigned as Bacillus strain as following: (a) Bacillus circulans, (b) Bacillus eiseniae, (c) Bacillus oceanisediminis, (d) Bacillus atrophaeus, (e) Bacillus siralis, (f) Bacillus massiliosenegalensis. The Yeast parts were first isolated as single colony, purified the chromosomal DNA, and amplified by PCR method with 18 S rDNA primer set (FR1 and NS1), then gel purified, DNA sequenced, and aligned in NCBI database (Nucleotide collection). The identified yeast strains as following: (a) Sporobolomyces koalae, (b) Meyerozyma guilliermondii, (c) Aureobasidium pullulans.


T. C. Shiu, “The pilot study of tibicos as a model to develop healthy drinks,” Master thesis, Dept. of Biological Science and Technology, I-Shou University, Taiwan, 2012.

A. G. J. Stadie, M. Wenning, M. A. Ehrmann, and R. F. Vogel, “The microbial diversity of water kefir,” International Journal of Food Microbiology, vol. 151, no. 3, pp. 284-288, December 2011.

A. G. Friques, C. M. Arpini, I. C. Kalil, A. L. Gava, M. A. Leal, M. L. Porto, et al., “Chronic administration of the probiotic kefir improves the endothelial function in spontaneously hypertensive rats,” Journal of Translational Medicine, vol. 13, no. 1, p. 390, December 2015.

F. R. Maciel, G. R. Punaro, A. M. Rodrigues, C. S. Bogsan, M. M. Rogero, M. N. Oliveira, et al., “Immunomodulation and nitric oxide restoration by a probiotic and its activity in gut and peritoneal macrophages in diabetic rats,” Clin Nutr, vol. 35, no. 5, pp. 1066-1072, October 2016.

C. C. Kuo and C. M. Chao, “Gram stain for vibrio species,” The American Journal of Emergency Medicine, vol. 31, no. 5, pp. 877-878, May 2013.

R. Freitag, Encyclopedia of Separation Science, I. D. Wilson, 1st ed., Oxford: Academic Press, 2000, pp. 1201-1208.

C. Schabereiter-Gurtner, S. Maca, S. Rölleke, K. Nigl, J. Lukas, A. Hirschl, et al., “16S rDNA-based identification of bacteria from conjunctival swabs by PCR and DGGE fingerprinting,” Investigative Ophthalmology & Visual Science, vol. 42, no. 6, pp. 1164-1171, May 2001.

Y. T. Hoshino and S. Morimoto, “Comparison of 18S rDNA primers for estimating fungal diversity in agricultural soils using polymerase chain reaction-denaturing gradient gel electrophoresis,” Soil Science and Plant Nutrition, vol. 54, no. 5, pp. 701-710, October 2008.

L. S. Magwaza and U. L. Opara, “Analytical methods for determination of sugars and sweetness of horticultural products-A review,” Scientia Horticulturae, vol. 184, pp. 179-192, March 2015.

T. Zor and Z. Selinger, “Linearization of the Bradford protein assay increases its sensitivity: theoretical and experimental studies,” Analytical biochemistry, vol. 236, no. 2, pp. 302-308, May 1996.

A. C. M. de Sena Aquino, M. S. Azevedo, D. H. B. Ribeiro, A. C. O. Costa, and E. R. Amante, “Validation of HPLC and CE methods for determination of organic acids in sour cassava starch wastewater,” Food Chemistry, vol. 172, pp. 725-730, April 2015.

C. Puerari, K. T. Magalhães, and R. F. Schwan, “New cocoa pulp-based kefir beverages: Microbiological, chemical composition and sensory analysis,” Food Research International, vol. 48, no. 2, pp. 634-640, October 2012.

A. M. O. Leite, M. A. L. Miguel, R. S. Peixoto, P. Ruas-Madiedo, V. M. F. Paschoalin, B. Mayo, et al., “Probiotic potential of selected lactic acid bacteria strains isolated from Brazilian kefir grains,” Journal of Dairy Science, vol. 98, no. 6, pp. 3622-3632, June 2015.

R. Barczynska, K. Jochym, K. Slizewska, J. Kapusniak, and Z. Libudzisz, “The effect of citric acid-modified enzyme-resistant dextrin on growth and metabolism of selected strains of probiotic and other intestinal bacteria,” Journal of Functional Foods, vol. 2, no. 2, pp. 126-133, April 2010.

B. van de Wier, J. M. Balk, G. R. M. M. Haenen, D. Giamouridis, J. A. Bakker, B. C. Bast, et al., “Elevated citrate levels in non-alcoholic fatty liver disease: The potential of citrate to promote radical production,” FEBS Letters, vol. 587, no. 15, pp. 2461-2466, August 2013.

B. Sharif Kashani, P. Tahmaseb Pour, M. Malekmohammad, N. Behzadnia, F. Sheybani-Afshar, M. Fakhri, et al., “Oral l-citrulline malate in patients with idiopathic pulmonary arterial hypertension and Eisenmenger Syndrome: A clinical trial,” Journal of Cardiology, vol. 64, no. 3, pp. 231-235, September 2014.

A. Di Francesco, L. Ugolini, L. Lazzeri, and M. Mari, “Production of volatile organic compounds by Aureobasidium pullulans as a potential mechanism of action against postharvest fruit pathogens,” Biological Control, vol. 81, pp. 8-14, February 2015.

D. Yogesh and P. M. Halami, “A fibrin degrading serine metallo protease of Bacillus circulans with α-chain specificity,” Food Bioscience, vol. 11, pp. 72-78, September 2015.

H. J. Kim, Y. J. Lee, H. Jeong, D. W. Lee, and S. J. Lee, “Insights into diversity and specificity of heavy metal resistance and efflux systems in Bacillus oceanisediminis 2691,” New Biotechnology, vol. 31, Supplement, p. S154, July 2014.

R. Coda, C. G. Rizzello, R. Di Cagno, A. Trani, G. Cardinali, and M. Gobbetti, “Antifungal activity of Meyerozyma guilliermondii: Identification of active compounds synthesized during dough fermentation and their effect on long-term storage of wheat bread,” Food Microbiology, vol. 33, no. 2, pp. 243-251, October 2013.

P. Aryuman, S. Lertsiri, W. Visessanguan, N. Niamsiri, A. Bhumiratana, and A. Assavanig, “Glutaminase-producing Meyerozyma (Pichia) guilliermondii isolated from Thai soy sauce fermentation,” International Journal of Food Microbiology, vol. 192, pp. 7-12, January 2015.




How to Cite

H.-H. Liu, B.-S. Tu, Y.-L. Chen, and H.-L. Wang, “The Study of Tibicos Fermentation Product as Pilot Model of Healthy Drinks”, Proc. eng. technol. innov., vol. 8, pp. 23–31, Apr. 2018.