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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. |
