Paenibacillus sp. TKU036 發酵烏賊軟骨所生產抗氧化物之分離與鑑定

Purification and identification of antioxidants from Paenibacillus sp. TKU036 fermented with squid pen as the sole carbon/nitrogen source

李欣庭(Hsin-Ting Li)

指導教授 ： 王三郎

共同指導教授 ： 郭曜豪(Yao-Haur Kuo)

淡江大學/理學院/化學學系碩士班/碩士(2016年)

https://doi.org/10.6846/TKU.2016.00021

摘要

抗氧化物能夠直接地清除活性氧化物質，基本上區分兩類，天然抗氧化物及合成抗氧化物。合成的抗氧化物如butylated hydroxyanisole（BHA）、butylatedhydroxytoluene（BHT），因便宜而常添加於食品中作抗氧化劑，防止食品脂質過氧化而腐壞，但由於 BHA 及 BHT 這類合成抗氧化物長期服用下有致癌風險，因此遭許多國家限制使用，並致力於從天然資源獲取抗氧化物來添加於食品中。
實驗使用菌株Paenibacillus sp. TKU036以烏賊軟骨粉（SPP）做碳/氮源，於 37 ℃ 培養 3 天有最好 DPPH 自由基清除率，將此離心之上清液經冷凍乾燥後由乙醇在 60 ℃ 下萃取，使用矽膠管住層析分得 14 個分液，在第 4 個分液有最佳的 DPPH 自由基清除活性，接著將此分液進一步以 HPLC 分離純化後能得到色胺酸及尿黑酸。比較 TKU036 發酵不同碳/氮源之上清液，及發酵前後 SPP 培養基上清液的 HPLC 指紋圖譜，發現 TKU036 只有以烏賊軟骨作碳/氮源發酵才能有效生產色胺酸及尿黑酸，而此 2 化合物原本是不存在於未發酵之培養基。

關鍵字

色胺酸 ； 尿黑酸 ； 抗氧化 ； 烏賊軟骨

並列摘要

Antioxidants can scavenge reactive oxygen species. Synthetic antioxidants, such as butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) may be added to food products to retard oxidation reactions. Though synthetic antioxidants are effective and inexpensive compared to natural ones, however, their applications are limited because many are suspected to be carcinogenic.
Paenibacillus sp. TKU036 using squid pen powder (SPP) as carbon/nitrogen source culturing at 37℃ 3 days shows the highest DPPH radical scavenging, this supernatant extracted by ethanol at 60℃, then obtained 14 fraction by silicone chromatography. The 4th fraction exhibited the highest DPPH radical scavenging activity, then the 4th fraction was further purified by HPLC and got tryptophan and homogentisic acid. Comparing the HPLC fingerprint of different carbon/nitrogen source fermented by TKU036, and the result shows that only TKU036 using squid pen as carbon/nitrogen source can effectively produce tryptophan and homogentisic acid, and these 2 compounds are not originally present in the unfermented SPP.

並列關鍵字

Paenibacillus sp. ； squid pen ； antioxidant ； tryptophan ； homogentisic acid

參考文獻

Ash C., Priest F.G., Collins M.D. (1993). Molecular identification of rRNA group 3 bacilli (Ash, Farrow, Wallbanks and Collins) using a PCR probe test. Proposal for the creation of a new genus Paenibacillus. Antonie Van Leeuwenhoek, 64, 253-260.

Bongaerts J., Kramer M., Muller U., Raeven L., Wubbolts M. (2001). Metabolic engineering for microbial production of aromatic amino acids and derived compounds. Metabolic Engineering, 3, 289-300.

Braconi D., Millucci L., Bernardini G., Santucci A. (2015). Oxidative stress and mechanisms of ochronosis in alkaptonuria. Free Radical Biology and Medicine, 88, 70-80.

Branen A. L. (1975). Toxicology and biochemistry of butylated hydroxyl anisole and butylated hydroxy toluene. Journal of the American Oil Chemists' Society, 52, 59-63.

Cabras P., Angioni A., Tuberoso C., Floris I., Reniero F., Guillou C., Ghelli S. (1999). Homogentisic acid:  a phenolic acid as a marker of strawberry-tree (Arbutus unedo) honey. Journal of Agricultural and Food Chemistry, 47, 4064-4067.