| Abstract: | 益生菌應用於食品發酵已有多年歷史。近年來許多研究指出，藉由益生菌(probiotics)的攝取可平衡腸道菌相及有益人體健康。本項研究的目的是以Lactobacillus paracasei TKU010所生產之蛋白酶純化和定性及對酸和膽汁耐受性之探討。由嬰兒吐奶篩選到之菌株Lactobacillus paracasei TKU010這株能以烏賊軟骨粉末作為生產蛋白酶之主要碳氮源之益生菌。TKU010生產蛋白酶之較適培養條件為: 由含有1%烏賊軟骨粉末、0.1%K2HPO4、0.05%MgSO4．7H2O之100mL液體培養基(pH9)於25℃振盪培養3天後，可得較佳蛋白酶活性。所得發酵液經硫酸銨沉澱、DEAE-Sepharose離子交換層析及Sephacryl S-100膠體層析的分離步驟，利用SDS-PAGE所分析出來的分子量約為49kDa。TKU010 蛋白酶之最適反應溫度、最適反應pH、熱安定性及pH安定性分別為50℃、pH10、<60℃和pH5-10;活性會受到EDTA的抑制，屬於金屬型的蛋白質酶;且純化後的酵素在0.5(mM)SDS, 0.5%(v/v)Tween 40, 0.5%(v/v)Triton X-100界面活性劑存在下殘餘活性分別為73% ,54% ,及102%。有機溶劑對酵素活性及安定性影響方面，在(20% ,v/v)甲苯、乙醚、與丙酮等有機溶劑下，蛋白質酶仍有50%以上的殘餘活性，而在乙酸乙酯和甲醇有機溶劑下，蛋白質酶只剩約10%的殘餘活性;將酵素與(25% ,v/v)有機溶劑於4℃與25℃下，待反應10天後，皆仍然保有75%以上的殘餘活性。
此外, TKU010於酸的耐受性上, 當磷酸鹽緩衝液pH為2.5時 可達到10 CFU/ml。耐膽鹽性試驗方面，在含有0.3%牛膽鹽之MRS broth 中培養24 小時菌數的存活率達90％以上。經過耐酸性和耐膽鹽性測試, TKU010於模擬消化過程測試中也可達到80%以上。於植物生長方面，TKU010之發酵上清液對萵苣及芥蘭的生長明顯有幫助。
Probiotics have been used in various fermented foods for many years. Resent reports indicated that digest probiotics are microbial food supplements, which beneficially affect the balance of intestinal microflora and host’s healthy. The purpose of this study was to investigate the purification and characteristics of a protease from Lactobacillus paracasei TKU010 and its acid and bile resistant ability discussion . The protease-producing strain, Lactobacillus paracasei TKU010, was isolated from infant Spits milk . The optimized culture medium was composed of 1% squid pen powder(SPP), 0.1%K2HPO4,0.05%MgSO4．7H2O at pH9. The strain was incubated in 100mL of above liquid medium and kept shaking at 25℃ for 3 days. The TKU010 protease was purified from the culture supernatant by ammonium sulfate precipitation, DEAE-Sepharose column chromatography and Sephacryl S-100 gel chromatography. The molecular mass of TKU010 protease determined by SDS-PAGE was approximately 49,000Da.The optimum temperature, optimum pH , pH stability and thermal stability of TKU010 protease was 50℃, pH 10, pH5-10 and<60℃ . The protease was characterized as a metalloprotease because it was inactivated by EDTA. Additionally,the pure protease retained 73% ,54% ,and 102% of its original activity in the presence of 0.5(mM)SDS, 0.5%Tween 40 and 0.5%Triton X-100, respectively.
In the presence of organic solvent such as toluene, ethyl ether and acetone, the protease retained more than 50% of its activity. In contrast, in the presence of organic solvent such as ethyl acetate and methanol, it retained only 10% of its activity. The TKU010 protease retain over 75 % of its activity by pre-incubation in the organic solvent at 4℃ and 25℃ for 10 days.
Additionally, TKU010 showed tolerance properties in this test on pH 2.5 phosphate buffer, it could achieve 10 CFU/ml . In the bile resistance test, it viability can achieve higher than 90% survival population in MRS plus broth (include 0.3% bull bile salt) after being incubated for 24hrs.According to the acid and bile resistance test, TKU010 take the imitating digestive tract test and it was higher than 80% survival population. |
