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Please use this identifier to cite or link to this item: http://ntour.ntou.edu.tw:8080/ir/handle/987654321/15969

Title: Aeromonas salmonicida MAEF108 之 Agarase AS-IIIb 基因轉殖至乳酸菌及乳酸菌發酵含山藥藻類寡醣乳製品之探討
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
Authors: Po-Hsuan Chen
陳柏璇
Contributors: NTOU:Department of Food Science
國立臺灣海洋大學:食品科學系
Keywords: Agarase AS-IIIb 基因;乳酸菌;山藥;藻類寡醣;Lactococcus lactis 控制 nisin 基因表現系統
Agarase AS-IIIb gene;Lactic acid bacteria;Yam;Algal Oligosaccharides;Nisin controlled gene expression system
Date: 2010
Issue Date: 2011-06-30T08:26:04Z
Abstract: 本研究之目的在於探討利用食品級載體 pNZ8149 和 TA-cloning,將革蘭氏陰性菌 A. salmonicida MAEF108 之 Agarase AS-IIIb 基因轉殖至乳酸菌 Lc. lactis NZ3900 和大腸桿菌 E. coli DH5 表現系統中,做為未來以 Agarase 降解山藥所得之寡醣添加至發酵乳製品之研發基礎,並以三組乳酸菌酛生產發酵含基隆山藥和青海菜寡醣 (M. nitidum oligosaccharides, MnOS) 之乳製品及暸解儲藏期間之變化。分別對 Lc. lactis subsp. lactis BCRC 10791、BCRC 12322、與 LM2301 進行突變與篩選,共篩得 48 株乳酸菌宿主接受細胞 (lac- 和 nisr),將質體 pNZ8149 電穿孔法轉形至宿主接受細胞,初步已篩得 10 株乳酸菌轉形株。依 Agarase AS-IIIb 基因序列設計不同限制酶切位之專一性引子,成功擴增 Agarase AS-IIIb 基因序列,利用 TA-cloning 方法構築,以電穿孔法和熱休克法轉形至 E. coli DH5中,成功得到轉殖株。將 Agarase AS-IIIb 基因和載體 pNZ8149,經限制酶和 T4 polymerase 反應處理及使用乳酸菌 NZ3900 表現系統,成功得到三株 rAS-IIIb 轉殖株,命名為 L-aga I、L-aga II、和 L-aga III,並測得胞外酵素活性為 0.2-3.8 U/mL,胞內酵素活性為 0.2-0.3 U/mL。以四組酵素組合降解 2% 山藥熱萃液,分別反應 1 和 6 hr 後,A 組: MAEF108 + MA103-crude agarases、B 組: MA103 澱粉誘導粗酵素液、和 C 組: MA103 褐藻膠誘導粗酵素液皆可得到大於乳糖之寡醣分子,D 組: 市售 agarase 則可得到之醣類大小與 galactose、maltose 相似。以三組乳酸菌酛: Lb. plantarum BCRC 12250 + BCRC 10069、Ped. pentosaceus MFS + MFL、以及 Lb. bulgaricus BCRC 10696 + Strep. thermophilus BCRC 12268 發酵含基隆山藥乳製品,發酵 24 hr 後皆達到發酵終點 pH 4.6 以下,乳酸菌數 8.53-9.47 CFU/mL 和可滴定酸度 0.87%-1.23% 皆有上升的趨勢。以二組乳酸菌酛: Lb. plantarum BCRC 12250 + BCRC 10069 以及 Ped. pentosaceus MFS + MFL 發酵含基隆山藥及青海菜寡醣乳製品,發酵 12 hr 後皆達到發酵終點 pH 4.6 以下,乳酸菌數 10.19-11.56 CFU/mL 和可滴定酸度 0.64%-0.76% 皆有上升的趨勢。於發酵期間內黴菌和酵母菌及大腸桿菌群則均未被檢出。基隆山藥發酵乳製品和含基隆山藥及青海菜寡醣發酵乳製品於 4oC 下儲藏 14 天過程中之 pH 值和乳酸菌量隨時間增加持續降低,可滴定酸度則會上升,而黴菌和酵母菌及大腸桿菌群於儲藏期間均未被檢出。
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.
URI: http://ethesys.lib.ntou.edu.tw/cdrfb3/record/#G0M97320006
http://ntour.ntou.edu.tw/ir/handle/987654321/15969
Appears in Collections:[食品科學系] 博碩士論文

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