Paenibacillus mucilaginosus TKU032生產生物界面活性劑與胞外多醣之條件與特性分析

Production and characterization of biosurfactant and extracellular polysaccharide from Paenibacillus mucilaginosus TKU032

曾詩純(Shih-Chun Tseng)

指導教授 ： 王三郎

淡江大學/理學院/化學學系碩士班/碩士(2013年)

https://doi.org/10.6846/TKU.2013.00668

摘要

本研究初始是以篩選生物界面活性劑生產菌為主，然而於篩菌過程中，意外發現P. mucilaginosus TKU032這株細菌發酵烏賊軟骨所得上清液，除了生產界面活性劑之外，置於室溫中會逐漸形成膠狀物質，而且顏色會漸漸加深，初步實驗發現此膠狀物為一多醣類物質，故將研究方向分為P. mucilaginosus TKU032這株細菌所產生生物界面活性劑及胞外多醣之研究。
以烏賊軟骨粉( SPP)、蝦頭殼粉( SHP)作為碳/氮源，依不同比例 (0.5%-2%)之添加濃度，於25℃、30℃、37℃培養0-5天，發現以2%烏賊軟骨、37℃培養3天之發酵上清液能得到最低的表面張力，相同條件培養四天可得到最多之胞外多醣，至第五天觀察發酵上清液顏色變黑導致不易測量，因此及選用上述條件作為較佳培養條件培養P. mucilaginosus TKU032。

TKU032發酵所得上清液，其表面張力約為36.3 mN/m經過NaOH調整p H值至12後，置放於4℃、24小時，待其沉澱並以離心方法去除沉澱物，將其上清液冷凍乾燥，再以甲醇溶出可溶物質，減壓濃縮至乾可得棕褐色油狀物，即為純化後之生物界面活性劑(1 g/50 mL)。
TKU032發酵所得上清液經過加熱(121℃、20min)脫色後可得粗胞外多醣( 14.8 g/L)，再將粗胞外多醣利用Sevag reagent 去蛋白，得到較純的多醣物質;使用酵素水解與另外取用三種不同酸水解胞外多醣，也可得到水解後結構較小之醣類，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)分析水解後之寡糖結構。
此外以烏賊軟骨作為發酵碳/氮源，TKU032在培養第1天所的上清液有較高的DPPH清除能力(80%)及較佳的總酚含量、還原力。
表單編號 ：ATRX-Q03-001-FM0030-01

關鍵字

Paenibacillus mucilaginosus ； 胞外多醣 ； 生物界面活性劑 ； 烏賊軟骨粉

並列摘要

This study is based on an initial option of screening of biosurfactant-producing bacteria,after we choose thebacteria which produce biosurfactant,we found the strain of P. mucilaginosus TKU032 have some gell-like substance in the supernatant,and the color of supernatant will become deeper gradually.Preliminary experiments found that this gum is a polysaccharide.This study will be into two part:P. mucilaginosus TKU032 producing bio-surfactants and study of extracellular polysaccharides.
With squid pen powder (SPP), head of the prawn shell powder (SHP) as a carbon / nitrogen source, according to different proportion (0.5% -2%) of the added concentration, at 25 ℃, 30 ℃, 37 ℃ cultured 0-5 days found to be 2% squid cartilage, 37 ℃ fermentation supernatant of cultured for 3 days to get the lowest surface tension.
The same conditions can be obtained up to four days of training extracellular polysaccharide, to observe the fifth day of fermentation supernatant color black lead difficult to measure, and therefore a better choice of culture conditions as the above conditions are cultured P. mucilaginosus TKU032.
TKU032 fermentation resulting supernatant, the surface tension of about 36.3 mN / m after NaOH to adjust pH values through 12, placed at 4 ℃, 24 hours, wait until the precipitate and the precipitate removed by centrifugation, the supernatant lyophilized and then eluted with methanol-soluble substance was concentrated under reduced pressure to dryness to obtain tan oil which is purified of the bio-surfactant.
TKU032 fermentation supernatant obtained after heating (121 ℃, 20min) after bleaching crude extracellular polysaccharide (14.8g / L), then the use of crude extracellular polysaccharide Sevag reagent to protein, enzymatic hydrolysis, dialysis, it can be oligosaccharide structure into smaller substances; another access three different acid hydrolysis extracellular polysaccharide can be obtained in the structure of the carbohydrate is small, the use of nuclear magnetic resonance (NMR) spectra and matrix-assisted laser desorption ionization time of flight mass Instrument (MALDI-TOF) analysis after hydrolysis of oligosaccharide structures.
In addition to fermentation squid pen as carbon / nitrogen source, in the first day of TKU032, the culture supernatant have higher DPPH scavenging ability (80%), and preferably the total phenolic content, reducing power.

表單編號 ：ATRX-Q03-001-FM0030-01

並列關鍵字

Paenibacillus mucilaginosus ； EPS ； Biosurfactant

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