以紅糖菌發酵液開發保健食品之研究

The study of Tibicos fermentation product as pilot model of healthy drinks

凃博翔(Po-Shun Tu)

指導教授 ： 劉孝漢

義守大學/醫學院/生物科技學系/碩士(2015年)

https://doi.org/10.6343/ISU.2015.00306

摘要

紅糖菌（Tibicos）是由多種菌株附著於多醣類支架所構成的白色半透明顆粒，本實驗室進行檢測Tibicos混合菌株及Tibicos發酵液所含幾種有機酸及其他兩項成份(蛋白質、醣類)的濃度變化，以後進一步研究探討其促進身體健康的機理。
本實驗使用HPLC做Tibicos有機酸液體定量分析，化學比色法測定醣類濃度、Bradford定量法測定蛋白質濃度，本實驗運用紅糖做為主要成分來培養菌株，在不同時間0hr，24hr，48hr取出Tibicos菌株及Tibicos發酵液，分別檢測Tibicos菌株重量與Tibicos發酵液有機酸，蛋白質，醣類的檢測是在0hr，24hr，48hr培養液，量測隨時間濃度的變化。結果顯示，用紅糖液以25℃密封培養48hr，Tibicos菌株重量增加45%。
Tibicos發酵液經由HPLC結果顯示：發酵液含Lactic acid、Cl- 、Malate、Sulfate、Oxalic acid、Phosphate、Citrate等成份。另外採用革蘭式染色法(Gram staining)以確認Tibicos菌株內的組成。
菌種鑑定分為細菌及酵母菌兩部分，細菌部分:利用分子生物學的方法將菌體之16 S rDNA 以533R 與341Fgc 利用PCR 的方式將目標基因放大，膠體純化分離及送定序，定序結果比對NCBI 資料庫之16S ribosomal RNA sequences (Bacteria and Archaea)。鑑定可能菌株如下:菌株(一)Bacillus circulans、菌株(二)Bacillus eiseniae、菌株(三)Bacillus oceanisediminis、菌株(四) Bacillus atrophaeus、菌株(五) Bacillus siralis、菌株(六) Bacillus massiliosenegalensis。
酵母菌部分:採用四區畫線分離法純化單一真菌菌落以及菌種鑑定，將菌體之18 S rDNA 以FR1 與NS1 引子，利用PCR 的方式將目標基因PCR放大，膠體純化分離及定序，比對NCBI 資料庫(Nucleotide collection)。鑑定可能菌株如下:菌株(一)Sporobolomyces koalae、菌株(二)Meyerozyma guilliermondii、菌株(三)Aureobasidium pullulans。

關鍵字

紅糖菌 ； 保健飲品 ； 發酵液 ； 16S rDNA定序 ； 18S rDNA定序

並列摘要

Sugar kefir grains (Tibicos) are assembled by various strains of microbes attached polysaccharide bracket composed of white transparent particles. We measured those of the concentration changes in Tibicos strains and Tibicos broth with internal ingredients (proteins, carbohydrates, ions), and further studied their functions for promoting our health status.
Initially we used chemical colorimetric method to measure that of carbohydrate concentrations, and Bradford 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.

並列關鍵字

Tibicos ； health drink ； fermentation broth ； 18S rDNA sequence ； 16S rDNA sequence

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