| Summary: | 碩士 === 臺北醫學大學 === 醫學科學研究所 === 96 === Ｂacillus licheniformis, one of the genus of Bacillus, was recognized as a probiotic in human. Probiotics could interact with intestinal cells to develop the barrier effect by presenting cell surface molecules or releasing metabolites. Our study focused on the bioactive metabolites produced byＢacillus licheniformis, by performing the isolation, purification, and structure analysis of the metabolic molecules in order to realize the roles and the mechanism of the strain existing in human intestines.
The experimental outline was divided as following: 1. the optimal condition for fermentation, 2. the screening model for fermentation by antimicrobial activity and scavenging activity of free radicals, 3. the isolation and purification of bioactive metabolites, 4. the characterization and identification of bioactive metabolites.
In this experiment, Bacillus licheniformis No.TMI-H1221 was selected as the target strain in executing liquid shaking and static fermentation, respectively. The optimal condition for fermentation screened by the antibacterial activity and free radical scavenging ability was: 1. Medium: M1 containing yeast extract, peptone, glucose as the main nutrient source, 2. Fermentation model: temperature 37℃, liquid shaking fermentation with the reciprocal 75 rpm in three days, 3. Fermentation vessel: 1.5L/5L(v/v) in erlenmeyer flask.
The recovery procedure of the active metabolites was described as following: after centrifuging the cultural broth, the fermentation supernatant was obtained. The acid-base fractionation to the supernatant was processed successfully with solvent extraction by ethyl acetate. The acidic fraction, being confirmed to be the bioactive fraction, was further burnished by performing column liquid chromatography (CLC, silica gel 60F developed by chloroform: methanol: acetate = 100:0:1) followed by thin layer chromatography (TLC, silica gel F254 developed by benzene: methanol= 90:10). One of the pure compounds, named BL-A-01, was obtained through the process of gel scratch and solvent elution. The compound was identified to be phenylacetic acid, which was also known as one of the antimicrobial metabolites of Ｂacillus licheniformis. According to the isolation procedure described above, the separation condition of chloroform: methanol: acetate = 95:5:1 for CLC in combination with chloroform: methanol: acetic acid = 80:20:1 for TLC was undertaken. By recovering the band existing on Rf =0.6 of the TLC, the bioactive compound, named BL-A-02, was obtained successfully. The result of HPLC analysis indicated that BL-A-02 was a new component different from phenylacetic acid. The purity of the compound was further confirmed by gas chromatography. In addition, a colorless needle crystal was also obtained from the neutral/weakly-acidic fraction. The purity of this compound could be accelerated by re-crystallization with the aid of warm ethyl acetate, thus named it as BL-N-01.
The biological and physico-chemical characteristics of the three metabolites were: BL-A-01 was active against Staphylococcus aureus(SA), methicillin resistant Staphylococcus aureus(MRSA), and Candida albicans(CA). The UV spectrum of BL-A-01 showed a maximal absorption at 280 nm. BL-A-02 was active against SA, but not MRSA and CA. The UV spectrum of BL-A-02 showed a maximal absorption at 236 and 278nm. BL-N-01 was inactive to all the test organisms being used in our experiment. The melting point of BL-N-01 was 198~200℃ and the maximal of UV spectrum at 280 nm. Based on the understanding of the biological functions and the molecular structures of the bioactive metabolites derived from Ｂacillus licheniformis, our study may provide a prospective clue in dealing with the functions of Bacillus licheniformis in immune-regulation and disease prevention of the intestinal tract as well as the development of related drugs.
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