副乾酪乳酸桿菌 NTU 101 及胚芽乳酸桿菌 NTU 102 益生特性探討及其基因表現系統之建立

Abstract

乳酸菌廣泛應用於食品發酵及保存上，被認為是安全級之菌種，某些菌株經實驗證實為可促進人體及動物腸道健康之益生菌。部分乳酸菌基因序列已於2004年解序完成，對於乳酸菌遺傳工程之研究具有實質的幫助，使乳酸菌之開發與應用有更多元化的發展。近年來的研究中，除了乳酸菌本身生理代謝機制之探討外，表現系統之建立亦漸受矚目，高效之表現系統可使菌株大量表現重組異源蛋白質，以利運用於食品工業或醫藥用途。本研究評估 Lactobacillus paracasei subsp. paracasei NTU 101 與 Lactobacillus plantarum NTU 102 之益生特性及其建立乳酸菌基因工程表現系統，以利異源基因之表現，首先以兩株由台灣本土分離之乳酸菌株 (L. paracasei subsp. paracasei NTU 101 及 L. plantarum NTU 102) 及十株乳酸菌株以體外試驗評估抑制癌細胞及提高抗氧化之能力。研究結果顯示 L. acidophilus BCRC 14079 熱致死細胞 (heat-killed cell, HKC) 具有最高之總抗氧化之活性(82.3%)。清除1,1-diphenyl-2-picryl-hydrazyl (DPPH) 自由基活性部份，除了L. salivarius subsp. salivarius BCRC 14759 及 Bifidobacterium breve BCRC 11846 之 HKC 無顯著活性外，其餘乳酸菌樣品約呈現 14.8-32.9% 的清除活性。還原力 (reducing activity) 部份，L. acidophilus BCRC 14079之細胞質區分 (cytoplasmic fraction, CF) 相較於B. adolescentis BCRC 14606之 HKC (全部樣品中活性最低) 有明顯的增加 (約 6 倍)。細胞存活率部份，不同的乳酸菌株對於抑制癌細胞生長皆有不同的影響。利用本土分離乳酸菌株 L. paracasei subsp. paracasei NTU 101 及 L. plantarum NTU 102 處理乳癌細胞株 (MDA-MB-231)，並以流式細胞儀分析該細胞週期，結果顯示可有效抑制乳癌細胞停滯於G0/G1期間。上述結果顯示本土乳酸菌株具有強的抗氧化能力及抑制癌細胞生長能力。近年來食品微生物學研究中，許多研究著重於益生菌之免疫調節作用，當中如乳酸菌所生產之胞外多醣能刺激巨噬細胞產生相關細胞激素。本研究萃取 L. paracasei subsp. paracasei NTU 101及 L. plantarum NTU 102 之胞外多醣 (101EP 及 102EP)，進行抗氧化及免疫調節特性探討。結果顯示 101EP 及 102EP 可刺激巨噬細胞 RAW264.7 誘發細胞激素生成 (IL-6、TNF-α 及 IL-1β)。抗氧化層次經體外試驗證實，10 mg/mL 之 101EP 與 102EP 具有高的抗氧化能力 (DPPH scavenging activity、chelating effect on ferrous ions、inhibition of linoleic acid peroxidation 及 reduction power)。乳酸菌基因工程表現系統部份，本研究以L. paracasei subsp. paracasei NTU 101 及 L. plantarum NTU 102 為遺傳工程操作宿主，進行最適之轉形條件及建構表現載體中表現元件的選殖。研究結果顯示，L. paracasei subsp. paracasei NTU 101 及 L. plantarum NTU 102 在弱化細胞壁程度及抗生素之耐受試驗中，2% glycine可有效弱化細胞壁的結構，而10 μg/mL 之 chloramphenicol可有效抑制宿主生長；電轉形效率部份，以固定電容 (25 μF) 與電阻 (200 ohms) 搭配不同電壓條件，將 pNZ8020 電轉形入本土乳酸菌中，研究顯示，L. paracasei subsp. paracasei NTU 101 (12.5 kV/cm) 及 L. plantarum NTU 102 (10 kV/cm) 於不同的電壓條件下，分別可得到 4.6x104 transformant/μg plasmid DNA 及 1.5x104 transformant/μg plasmid DNA 之最適電轉形效率。質體穩定度 L. plantarum NTU 102/pNZ8020 轉形株約 96% 以上的質體皆可穩定存在於宿主中。乳酸菌表現元件選殖，目前已成功選殖相關表現元件包括啟動子、訊息胜肽、報導基因等，此外，目前已成功構築並挑選正確之乳酸菌表現載體 pNZP23G，未來將持續構築表現功能性基因相關之表現質體，並於乳酸菌表現系統中表現。

Lactic acid bacteria (LAB) are widely used in food fermentation, preservation and are generally recognized as safe. Some strains of LAB are “probiotic” that have several beneficial effects on the health of the digestive tract of human and animals. In 2004, part of the LAB genome had been successfully decoded, that provided practical assistance for genetic engineering of LAB, and the applications of LAB are leaded to diversification. In recent studies, in addition to better know the metabolic capacities of LAB, people pay more attention to establish the efficient expression system, for large scale production of heterologous recombination proteins and hope to apply to food industry and medical therapy. In this thesis, we evaluate the probiotic properties of L. paracasei subsp. paracasei NTU 101 and L. plantarum NTU 102 and established the LAB gene expression system. Firstly, we used two local Lactobacillus strains (L. paracasei subsp. paracasei NTU 101 and L. plantarum NTU 102) and other 10 LAB strains from the Biosource Collection and Research Center (BCRC) to evaluate the health promoting capability including inhibitory effects on cancer cell lines and antioxidant activities in vitro. The heat-killed cells (HKC) of L. acidophilus BCRC14079 have the highest total antioxidative activity (82.3%). 1,1-diphenyl-2-picryl-hydrazyl (DPPH) radical scavenging effects were in the range of 14.8-32.9%, except for the HKC of L. salivarius subsp. salivarius BCRC 14759 and Bifidobacterium breve BCRC 11846. Twelve strains showed varying degrees of reducing activity. The reducing activity of cytoplasmic fraction (CF) of L. acidophilus BCRC 14079 was found to be significantly more pronounced (six-fold) than that of the HKC of B. adolescentis BCRC 14606, which was the lowest of all. In the cell viability, the inhibitory effects on cancer cells were very different which depended on various LAB strains. Flow cytometry study on human breast adenocarcinoma cells (MDA-MB-231) treated with local lactobacilli strains (L. paracasei subsp. paracasei NTU 101 and L. plantarum NTU 102) isolated in Taiwan showed the highest percentage of arrested at G0/G1 phase in the cell cycle. These results show that local lactobacilli strains have strong antioxidative and anticancer activities. Recently, immunomodulation by probiotic microorganism is a topic of increasing interest in food microbiology. Some probiotics such as LAB also can produce exopolysaccharide which stimulated and produced the cytokine in macrophage. The aim of this work is to study the effects of the exopolysaccharide from L. paracasei subsp. paracasei NTU 101 (101EP) and L. plantarum NTU 102 (102EP) on antioxidant ability and immunomodulation properties in vitro. The result shows 101EP and 102EP are able to induce cytokine production (including IL-6, TNF-α, and IL-1β) in RAW264.7. In the antioxidant status, 10 mg/mL of 101EP and 102EP suggest high antioxidant ability (DPPH scavenging activity, chelating effect on ferrous ions, inhibition of linoleic acid peroxidation and reduction power) in vitro. In LAB genetic engineering expression system, we had used L. paracasei subsp. paracasei NTU 101 and L. plantarum NTU 102 as a host for genetic engineering to discuss the optimal conditions for the transformation of LAB by electroporation and cloning the expression signal for LAB expression vector. Our result shows that two percentage of glycine can retard the growth rate of L. paracasei subsp. paracasei NTU 101 and L. plantarum NTU 102 and 10 μg/mL of chloramphenicol suppresses their growth completely. Using the fixed capacitance (25 μF), fixed resistance (200 ohms), and different voltages, the optimal electro-transformation efficiency of L. paracasei subsp. paracasei NTU 101 (voltage, 12.5 kV/cm) and L. plantarum NTU 102 (voltage, 10 kV/cm) were 4.6x104 and 1.5x104 transformant/μg plasmid DNA, respectively. In the test of plasmid stability, 96% of the L. plantarum NTU 102/pNZ8020 transformants are stable after 96 hr in the non-antibiotic culture. Cloning related expression signals for LAB, had successfully screened such as promoter, signal peptide and reporter gene. Besides, we have successfully constructed a LAB-expression plasmid (pNZP23G). Functional plasmids will be constructed and express in the LAB expression systems in the future.