液化澱粉芽孢桿菌Bacillus amyloliquefaciens JN68之培養及其抗真菌脂胜肽iturin A之純化及特性分析

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液化澱粉芽孢桿菌Bacillus amyloliquefaciens JN68之培養及其抗真菌脂胜肽iturin A之純化及特性分析

Culture conditions of Bacillus amyloliquefaciens JN68 and purification and characterization of an antifungal lipopeptide - iturin A

黃立彬 , Masters　　Advisor：陳錦樹　　

繁體中文

液化澱粉芽孢桿菌；天然抑菌劑；酸沉澱法；固相萃取法； Iturin A ； Bacillus amyloliquefaciens ； natural antibacterial agent ； acid precipitation ； solid phase extraction ； iturin A

Abstract │ Reference (111) │ Altmetrics

Abstract 〈TOP〉

液化澱粉芽孢桿菌（Bacillus amyloliquefaciens）能產生多種抑生物質（如iturin A, fengycin及surfactins），抑制多種細菌、真菌的生長，具作爲天然抑菌劑之潛力。本研究探討培養基組成及培養條件對B. amyloliquefaciens JN68抑菌能力之影響，再分別以酸沉澱法(acid precipitation)、固相萃取法(solid phase extraction)及以上兩種方法純化組合iturin A，進而建立較適純化條件，探討其最後對多種食品中常見病原菌之抑生能力。
  實驗結果顯示，調整培養基成份為氮源12.5 g/l tryptone、添加1 mmol L-1 FeSO4·7H2O及4 mmol L-1 MgCl2·6H2O、培養條件為溫度30℃、起始pH 6.7-6.8、接種量1%、振盪速率180 rpm、培養48小時，其培養上清液對Aspergillus niger抑菌能力最好，抑菌圈直徑為17.05 mm。
  B. amyloliquefaciens JN68培養上清液抑菌成分之耐受性方面，於不同pH（pH 2.0-10.0）環境下一小時之抑菌能力並無明顯差異；於-80℃ ~ 60℃對其抑菌能力並無影響。但於100℃下一小時後，抑菌活性下降30%，而在121℃下一小時則完全失去活性。不同水解酵素對B. amyloliquefaciens JN68培養上清液之抑菌能力並無任何影響。
酵素活性試驗方面，B. amyloliquefaciens JN68培養液之中性蛋白酶活性於第一天即達最高峰，為160 U/ml；其澱粉酶在一天後快速上升，然後緩慢上升並於第五天活性達到最高峰，約1.33 U/ml。
  Iturin A之分離與純化方面，取50 ml B. amyloliquefaciens JN68培養上清液分別利用酸沉澱及固相萃取進行iturin A之純化， iturin A產量分別為0.50 mg及1.05 mg。經固相萃取純化後之iturin A對測試之病原菌及真菌都有明顯抑制作用，其中真菌Actinomucor taiwanensis BCRC31159之抑菌圈直徑最大，為22.57 mm。而經固相萃取純化之iturin A對Staphylococcus aureus BCRC12154及Salmonella enterica BCRC10747等細菌有較好之抑菌效果，其抑菌圈直徑介於22.82-23.79 mm之間。
  調整iturin A為10 μg/ml對S. aureus BCRC12154及Sal. enterica BCRC10747抗菌指數為50%，對Escherichia coli BCRC11634及Listeria monocytogenes BCRC15330抑菌分別為13.2%及25%。濃度為7 μg/ml之iturin A對Act. taiwanensis BCRC31159具有較佳之抗菌指數，為68.9%，其次為Asp. niger BCRC32073，為48.3%，而Aspergillus oryzae BCRC32279最差，為21.7%。

Parallel Abstract 〈TOP〉

Bacillus amyloliquefaciens, that has the potential to be a natural antibacterial agent, has the ability to produce various antimicrobial substances that can effectively inhibit the growth of bacteria and fungi. The aim of the study was to produce, purify and characterize the antimicrobial substance, iturin A, from a B. amyloliquefaciens JN68 isolate. 
  The optimal medium composition and culture conditions for B. amyloliquefaciens JN68 were determined with shaking flask (capacity 100 ml/250 ml). The results showed that 12.5 g/l tryptone as nitrogen source, 1 mmol L-1 FeSO4·7H2O and 4 mmol L-1 MgCl2·6H2O as additional metal ions, incubation temperature at 30℃, initial pH at 6.7-6.8, inoculum size was 1% (v/v), and shaking rate at 180 rpm for 48 hours, the cell-free supernatant gave the best antimicrobial activity against Aspergillus niger, with diameter of inhibition zone being 17.05 mm. 
  The antimicrobial activity of cell-free supernatant retained over a pH range of 2.0-10.0. The antimicrobial activity (diameter of inhibition zone > 15.78 mm) was stable ranging from -80 to 60℃, but residual activity decreased at 100℃. All activity was lost after incubation at 121℃. No obvious variation tendency was observed after hydrolytic enzyme degradation.
  The neutral protease activity of B. amyloliquefaciens JN68 was 160 U/ml incubated after 1 day, while amylase activities about 1.16-1.33 U/ml in the culture broth were obtained between 2-5 days under optimal conditions.
  The cell-free supernatant (50ml) from B. amyloliquefaciens JN68 was purified by acid precipitation and C18 solid phase extraction, respectively. The amount of crude extracted iturin A obtained from acid precipitation method and C18 solid phase extraction were about 0.50 mg and 1.05 mg respectively. Crude iturin A extract obtained from C18 solid phase extraction showed the best antifungal activity against Actinomucor taiwanensis BCRC31159, which diameter of inhibition zone was 22.57 mm; and showed better antibacterial activity on Staphylococcus aureus BCRC12154 and Salmonella enterica BCRC10747, which radius of inhibition zone ranged from 22.82 to 23.79 mm. 
  Concentration of crude iturin A extract at 10 μg/ml showed 50% antibacterial index to S. aureus BCRC12154 and Sal. enterica BCRC10747, while showed that only 13.2% and 25% to Escherichia coli BCRC11634 and Listeria monocytogenes BCRC15330, respectively. Concentration of crude iturin A extract at 7 μg/ml showed the largest antibacterial index, about 68.9%, on Act. taiwanensis BCRC31159. However, the antibacterial index to Asp. niger BCRC32073 and Aspergillus oryzae BCRC32279 decreased to 48.3% and 21.7%, respectively.

Reference ( 111 ) 〈TOP〉

邱安隆。2007。植物病害生物製劑研發與應用。花蓮區農業專訊。48 : 21-24。
連結：
林秀芬，陳滄海，劉顯達。2011。枯草桿菌Bacillus subtilis BS-99-H之iturin A 抗菌物質對蓮霧果腐病原菌Pestalotiopsis eugeniae 之生物活性。植病會刊。19：225-233。
連結：
郭建志，陳俊位，廖君達，陳葦琳，蔡宜?。2014。液化澱粉芽孢桿菌在作物病害防治的開發與應用。農業生物資材產業發展研討會專刊。121：69-86。
連結：
農藥資訊服務網: 行政院農業委員會動植物防疫檢疫局
連結：
Alcaraz L.D., Moreno-Hagelsieb G., Eguiarte L.E., Souza V., Herrera-Estrella L., Olmedo G. 2010. Understanding the evolutionary relationships and major traits of Bacillus through comparative genomics. Biomedical Central Genomics, 11: 332.
連結：

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