可降低膽固醇能力的乳酸菌之篩選及其特性研究

Isolation and Characterization of Lactic Acid Bacteria Strain with Cholesterol Reducing Ability

卡斯帝佑(Brandon Rafael de Jesus Castillo Corea)

指導教授 ： 鄭達智

國立屏東科技大學/國際學院/熱帶農業暨國際合作系/碩士(2022年)

https://doi.org/10.6346/NPUST202200157

摘要

心血管疾病是因營養失衡而導致體内膽固醇水平升高所引起，若不治療可能會致命，現已是全球常見死因之一。治療方式包含飲食控制及藥物治療，例如限制食物的種類，而藥物會抑制患者體內與膽固醇合成和附帶效應相關的幾種酶。益生菌是這些藥物治療的更健康替代品。 眾所周知，益生菌可作為人類食用的補充食品調節膽固醇水平，也可作為功能性動物飼料生產具有較低膽固醇水平的畜產品。本研究的重點是從豆腐和泡菜中分離且評估具有降低膽固醇特性的乳酸菌株，採用基因篩選技術，透過快速分離DNA技術在分離菌株中鑑定BSH2基因。採用酶試驗法取出BSH2陽性菌株的DNA，並鑑定出BSH1、BSH2、BSH3和BSH4基因。酶活性試驗中，包括膽鹽耐受性 (0.3% w/v)、BSH 平板瓊脂試驗、與 (0.3% w/v) 的牛磺膽酸鈉去结合膽鹽與 (0.3% w/v) 進行膽固醇同化 牛膽汁和 100 μg/mL 膽固醇。 還通過 RT-qPCR評估膽鹽脅迫牛磺膽酸鈉 (0.3% w/v) 下的 BSH 基因表達。益生菌測試包括自動聚集性、疏水性、溶血反應及抗生素抗藥性，並使用 16S rRNA序列鑑定菌株。在每個測試中使用三種細菌菌株作為對照菌株，分別為嗜酸乳桿菌 (C1)、乾酪乳桿菌(C2)和植物乳桿菌(P1)。泡菜中的菌株分離並純化得到90株以及豆腐中得到196株。在To1、To2、To3、To4和To5細菌分離物中鑑定出BSH2基因，且BSH基因鑑定顯示菌株之間具差異性。菌株間膽汁之耐受性試驗無顯著差異（78 %～89 %），但疏水性（31.87 %～49.88 %）、自動聚集性試驗（29.31 %～55.31 %）有顯著差異。所有菌株均皆無溶血反應，對氨苄青黴素和青黴素具抗生素敏感性，紅黴素、氯黴素、鏈黴素以及萬古黴素具抗藥性和中度抗藥性。To1、To2、To3和To4菌株被鑑定為植物乳桿菌，To5被鑑定為Weisella cibaria。釋放的膽酸具顯著差異，其中P1的早期解離最高，為345.6 µMol/mL；To1、To2和To4 與 C2（233～160 µMol/mL）相比具有相似的結果。在這些條件下的基因表達表現為To1 和P1的BSH1和BSH4顯著上調，而C2的BSH1被下調。膽固醇同化試驗顯示To1和To4可使膽固醇分別降低62%和61 %，其次是To2和P1菌株，分別降低52 % 和59 %。本研究得出結論為To1和To4是可以從豆腐中分離出的安全菌株，這些菌株在牛磺膽酸鹽中含有BSH活性，可作為具降低膽固醇的益生菌，並補充到動物飼料中。

關鍵字

膽鹽 ； 膽固醇同化 ； 基因篩選 ； 植物乳桿菌 ； 益生菌

並列摘要

Cardiovascular disease is a global health concern caused by a nutritional imbalance leading to high cholesterol levels in the body. If not treated, cardiovascular diseases can be fatal. Dietary intervention and drug therapy are medical prescriptions. Dietary will restrict the selection of food; drugs will inhibit several enzymes related to cholesterol synthesis and collateral effects in patients. Probiotics are healthier alternatives to these medical treatments. Probiotics are known to regulate cholesterol levels as supplementary food for human consumption and also as a functional animal feed to produce livestock products with lower cholesterol levels. This study focused on isolating and evaluating lactic acid strain from tofu and kimchi with cholesterol-reducing properties. A gene screening technique was used to identify the BSH2 gene by fast isolation DNA technique in the isolated strains. DNA from BSH2-positive strains was extracted by using an enzymatic method and BSH1, BSH2, BSH3, and BSH4 genes were identified. The enzyme activity tests were linked to the tolerance of bile salt 0.3 % (w/v), BSH plate agar test, deconjugation of bile salt supplemented with sodium taurocholate 0.3 % (w/v), and cholesterol assimilation supplemented with 0.3 % (w/v) of ox bile and supplemented with 100 μg/mL of cholesterol. The BSH gene expression under bile salt stress sodium taurocholate 0.3 % (w/v) was also evaluated by RT-qPCR. A probiotic test that included: autoaggregation, hydrophobicity, hemolytic reaction, and antibiotics resistance was done. Strains were identified using a 16S rRNA sequence. Three bacterial strains were used in each test L. acidophilus (C1), L. casei (C2), and L. plantarum (P1) were used as control strains. The isolation and purification of bacterial strains resulted in 90 strains from kimchi and 196 strains from tofu. BSH2 genes were identified in To1, To2, To3, To4, and To5 bacterial isolates. The BSH genes identification showed divergence among strains. There was no significant difference in the bile tolerance test from 78% to 89%, however, there was significant differences in the hydrophobicity from 31.87% to 49.88% and autoaggregation from 29.31% to 55.31% test among strains. All strains were non-hemolytic, showed antibiotic susceptibility to ampicillin and penicillin, intermediate resistance to erythromycin and showed resistance to chloramphenicol, streptomycin, and vancomycin. Strains (To1, To2, To3, To4) were identified as Lactobacillus plantarum and To5 was identified as Weisella cibaria. There was a significant difference in the cholic acid liberated, where P1 presented the highest deconjugation of 345.6 µMol/mL; To1, To2, and To4 had similar results compared with C2 (233 to 160 µMol/mL). The gene expression under these conditions presented a significant up-regulation of BSH1 and BSH4 for To1 and P1; BSH1 for C2 was down-regulated. Cholesterol assimilation assay showed that To1 and To4 can reduce cholesterol by 62 and 61 %, followed by To2 and P1 strains with 52 and 59 %. This study concludes that To1 and To4 are safe bacterial strains that can be isolated from tofu, these strains contain BSH activity in taurocholate bile salt and can be used as a potential probiotic with cholesterol-reducing ability and be supplemented in animal feed.

並列關鍵字

bile salt ； cholesterol assimilation ； gene screening ； Lactobacillus plantarum ； probiotic

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