| Summary: | 碩士 === 淡江大學 === 生命科學研究所碩士班 === 95 === 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.
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