Studies on Cloning and Expression of MAEF108 Agarase AS-IIIb Gene from Aeromonas salmonicida to Lactic Acid Bacteria and Lactic Acid Bacteria Fermented with Yam and Algal Oligosaccharides Dairy Product
| Autor: | Po-Hsuan Chen, 陳柏璇 |
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| Rok vydání: | 2010 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 98 The object of this study was using TA-cloning to clone gram negative Agarase gene AS-IIIb into lactic acid bacteria (LAB) with food grade vector pNZ8149 of Lactococcus lactis express system. Another way utilized LAB to ferment Keelung yam and Monostroma nitidum oligosaccharides (MnOS) cultured milk and examined the effect of their storage period at 4oC. 48 lactic acid bacteria mutant were found from Lc. lactis subsp. lactis BCRC 10791, BCRC 12322, and LM2301. 10 LAB survived after the protoplast electroporated to lactis acid host cell. Specific primers include restricted enzyme were designed to clone the Agerase AS-IIIb gene into E.coli DH5 by TA-cloning,used heat shock and electroporated to obtained AS-IIIb clone. By using lactic acid bacteria expression system and restriction enzyme SpeI, SacI with T4 polymerase mothed, three rAS-IIIb clones L-aga I, L-aga II, and L-aga III were contructed; both have extra- and intra-cellular agarase activity, but the extracellular agarase activity is too low to be detected. Hot water extact yam solution degraded by used four group, A: MAEF108 + MA103-crude agarases, B: MA103-crude amylase, C: MA103-crude alginate lyase, and D: commeicial agarase. group A, B, C to obtained oligosaccharides that molecular weight larger than lactose after reacting 1 and 6 hr, commeicial agarase group to obtained saccharides similar to galactose and maltose. In the fermentation experiment, yam cultured milk was fermented by Lb. plantarum BCRC 12250 + BCRC 10069, Ped. pentosaceus MFS + MFL, and Lb. bulgaricus BCRC 10696 + Strep. thermophilus BCRC 12268, respectively. The pH could reach 4.6 in 24 hr, LAB counts were 8.53-9.47 CFU/mL and titratable acidity rised 0.87%-1.23%. The pH of yam and MnOS cultured milk by Lb. plantarum BCRC 10069 + 12250 group and Ped. pentosaceus MFL + MFS group could reach 4.6 in 12 hr, LAB counts were 10.19-11.52 log CFU/mL, titratable acidity rised 0.64-0.76%. Both mold/yeast count and coliforms count have not been detected during the fermentation period. In the storage period at 4oC experiment of yam cultured milk and yam with MnOS cultured milk, the lactic acid bacteria and pH value decreased and titratable acidity increased after 14 days. Both mold/yeast count and coliforms count have not been detected during the storage period at 4oC. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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