菌株TKU035係以烏賊軟骨粉為唯一碳/氮源，篩選自台灣北部土壤之一株胞外多醣及生物界面活性劑生產菌，經鑑定為Paenibacillus sp.。TKU035生產胞外多醣之較適培養條件為含有2%烏賊軟骨粉、0.1% K2HPO4及0.05% MgSO4．7H2O之100 mL液態培養基(pH 4)於37℃下震盪培養4天(150 rpm)。 TKU035發酵所得上清液(4.03 g/L)經脫色、沉澱並利用Sevag reagent去蛋白，可得水溶性粗胞外多醣，再將粗胞外多醣以陰離子交換樹脂進行純化，所得純化之胞外多醣藉由HPLC分析得知其分子量約為5.07×104 Da。將胞外多醣(20 mg)利用4M鹽酸(10 mL)在90℃下水解5天，對水透析並收集含有胞外多醣分解成寡醣類物質之外液，利用核磁共振(NMR)光譜及基質輔助雷射脫附游離飛行時間質譜儀(MALDI-TOF)分析其寡醣組成。 此外，培養第5天所得發酵上清液有較高的DPPH清除能力(75%)及較佳的總酚含量、還原力。微生物抑制測試顯示，TKU035之發酵上清液對黴菌(Fusarium oxysporum)和細菌(E. coli、Pseudomonas aeruginosa K187)皆有抑制效果。 In this study, an exopolysaccaride(EPS) and biosurfactant-producing strain was isolated from the soil in the northern Taiwan by using squid pen powder (SPP) as the sole carbon/nitrogen source and identified as Paenibacillus sp.. The optimal condition for EPS production was in 100 mL medium containing 2% squid pen powder(SPP), 0.1% K2HPO4 and 0.05% MgSO4‧7H2O(pH 4) incubation at 37oC for 4 days(150 rpm). The water-soluble crude EPS was obtained from the culture supernatant (4.03 g/L) by decoloring, precipitating and deproteinization with Sevag reagent. The crude EPS obtained was then purified subsequently by DEAE-Sepharose. The purified EPS showed molecular weight of 5.07 × 104 Da by HPLC. The EPS was hydrolyzed by 4M HCl at 90oC for 5 days, and the oligosaccharides obtained were collected by dialyzing against water. The oligosaccharide components were analyzed by using nuclear magnetic resonance (NMR) and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF). Additionally, DPPH free radical scavenging ability, total phenolic contents and reducing activity were all found highest at the 5th day-culture supernatant. TKU035 culture supernatant also showed antimicrobial activities against fungus of Fusarium oxysporum and bacteria of E. coli and Pseudomonas aeruginosa K187.
