豆豉中產孢益生菌之篩選及其抗發炎活性探討

Abstract

益生菌被定義為在一定數量下對宿主具有功效之活的微生物，近年在無論是在醫藥或健康食品方面都被廣泛的研究，其相關產品之種類、功能與市場規模也逐年增長，顯示益生菌產業非常具有發展潛力。 本研究主要目的在於從醃漬或發酵之園產加工食品中分離篩選出具有抗發炎功效之潛力產孢益生菌株，分別從市售之鹽漬蘿蔔、豆豉、豆腐乳及醃薑中以95℃高溫熱水浴篩選出具有產孢能力之菌株共28株。為篩選出能符合益生菌篩選條件之菌株，後續以抑制受誘導之巨噬細胞生成一氧化氮與細胞存活性試驗 (MTS assay) ，作為抗發炎功效之初步篩選指標；同時模擬人體胃、腸道之環境，以pH 2.5酸性等張溶液及膽鹽溶液測試菌株能否對其具有一定程度之耐受性。初步篩選後，共5株菌符合篩選標準且均為從豆豉中分離之菌株，認定為潛力益生菌株，並以此5株菌進行第二部分實驗。 對潛力菌株之生長條件進行分析，5株菌最適生長條件均為pH 9、37℃，以不同有機溶劑分析菌株細胞表面性質，D1在乙酸乙酯與氯仿中的溶解率分別達到54.66和63.21%，為分離菌株中最高者。在細胞聚合性方面，D1與D7之細胞聚合率均高於30%，D7在靜置3小時後聚合率達到35.7%，為所有菌株中細胞聚合率最高者。將菌株之16S rDNA與美國國家生物技術資訊中心資料庫進行比對後發現均為Bacillus屬之微生物，也確認所有潛力菌株均為產孢菌。同時也分析了潛力菌株對於抗生素之抗性，發現潛力菌株普遍對四環黴素具有較高之抗性，推測可能該屬之菌本身具有抑制四環黴素之生理機制。 潛力菌株在不同起始濃度下具有不同的抗發炎效果，故以不同起始濃度之潛力菌株與受到誘導之小鼠癌化巨噬細胞株Raw 264.7進行共培養，並同時與商業菌株Bacillus coagulans GBI-30, 6086進行比較，在抑制一氧化氮生成方面，D1與D7於起始菌與細胞數比值為10之下一氧化氮生成量較未受脂多醣 (Lipopolysaccharide, LPS) 誘導之組別更低，對一氧化氮合成酶(inducible nitric oxide synthase, iNOS) 亦有相同抑制效果，推測菌株之抑制機制可能與調控iNOS之基因表現有關。最後，測定潛力菌株與受誘導細胞共培養後之前發炎細胞激素腫瘤壞死因子-α (Tumor necrosis factor-α, TNF-α) 、介白素1β (Interleukin-1β, IL-1β) 與介白素6 (Interleukin-6, IL-6) 之生成量，發現D9a能抑制99%以上之TNF-α與IL-6生成，D4菌株則能抑制85%以上之IL-1β生成，相較於商業菌株BC30之生成量，抑制率明顯高出許多。 從豆豉中分離，並經過篩選選定之5株潛力菌株，具有可作為益生菌之特性，且能抑制免疫細胞在受到外來抗原誘導之後一氧化氮及前發炎細胞激素之生成，認為具有抗發炎潛力，日後可繼續開發成益生菌產品，而其確切之抗發炎機制也可再深入探討研究。

According to the definition of probiotics in WHO (2002), “Live micro-organisms which, when administered in adequate amounts, confer a health benefit on the host.” Many researches have studied probiotics for medicine or health supplements in recent years. The products of probiotics have many types and functions; the global market of probiotics are growing too. It shows probiotics have great potential in the future. This research aims to isolate potential spore-forming probiotic strains with anti-inflammatory activity from fermented or pickled horticultural foods. 28 spore-forming strains have been isolated from douchi, pickled radish, sufu and pickled ginger by 95℃ water bath. Then tested the ability of inhibiting LPS-induced macrophage Raw 264.7 producing NO and cell viability by MTS assay to preliminary indicate of strain’s anti-inflammatory activity. Also we tested tolerance to pH 2.5 buffered solution and 0.3% bile salt medium represent whether strain survive through the gastrointestinal tract. After preliminary screening, we chose 5 strains as potential strain and proceeded the second part experiment. pH 9 and 37℃ are the most moderate condition of all potential strains. We use different organic solvents to investigate cell hydrophobicity. Strain D1 had highest solubility 54.66 and 63.21% in ethyl acetate and chloroform respectively. Cell aggregation have positive correlation with the ability of adhesion to intestinal surface. Strain D1 and D7 has the best aggregation property over 30% after 3h. Comparing strain 16S rDNA with NCBI, and confirm all of our strains are Bacillus species. All the potential strains have highly resistance to tetracycline which may cause by expressing anti-tetracycline gene in Bacillus. Potential strains have anti-inflammatory activity with different initial concentration. Strain D1 and D7 with initial concentration 10 have the best NO inhibiting activity. The production even lower than the control. Potential strains have the same inhibition activity to nitric oxide synthase mRNA expression. Finally, potential strains can also inhibit pro-inflammatory cytokine generation. Strain D9a can inhibit over 99% Tumor necrosis factor-α (TNF-α) and Interleukin-6 (IL-6) generation with initial concentration 10, and all the potential Strain can inhibit over 80% Interleukin-1β (IL-1β) generation. Compared with the commercial strain BC30, our strains have better anti-inflammatory activity. In conclusion, our 5 strains isolated from douchi have great potential to be probiotics with anti-inflammatory activity. The research can continue to develop the strains into commercial products or further investigate the mechanism of its anti-inflammatory activity.