- 學位論文
篩選具抗氧化能力之益生菌及探討其延緩老化之研究
Selecting Probiotics with Anti-Oxidative Ability and Investigating Their Anti-Aging Effects
摘要
老化是一種逐漸失去身體性能及繁殖能力的現象，並且隨著年齡增加，死亡率逐漸提高；而眾多引起老化之因素中，最廣泛者即為活性氧物質之傷害。隨著人口老化，延緩老化之機能性產品逐漸受重視。已有研究發現老化、慢性疾病及腸道菌相改變之關連性，並且目前益生菌之相關研究中，亦發現特定益生菌株具有抗氧化壓力之效果，並可減少體內活性氧群之累積，使其成為值得探討之對象。本實驗室先前之研究亦發現，攝取Lactobacillus paracasei subsp. paracasei BCRC 12188、Lactobacillus plantarum BCRC 12251及Streptococcus thermophilus BCRC 13869三株益生菌之混合液Pm-1後，可減少D-galactose誘導老化之小鼠體內丙二醛(malondialdehyde, MDA)濃度，顯示減少小鼠體內脂質過氧化程度。因此，本實驗便希望可藉其抗氧化特性，進一步探討益生菌是否能為我們帶來延緩老化之機能性。 實驗先以28株益生菌進行體外抗氧化能力之測試，從中發現Lb. kefiranofaciens HL1這株菌較其他菌株有顯著較高的清除1,1-二苯基-2-三硝基苯肼(1,1-diphenyl-2-picrylhydrazyl, DPPH)自由基及螯合鐵離子能力，並且可顯著抑制亞麻油酸過氧化，因此便以此菌株(108 CFU/daily and 109 CFU/daily)搭配先前研究之Pm-1混菌(108 CFU/daily)餵予以D-galactose誘導老化之小鼠，並對小鼠進行Morris水迷宮試驗之行為觀察及腦部海馬迴cleaved caspase-3表現量，以評估小鼠學習及記憶能力與海馬迴受損程度。實驗結果發現，攝取挑選之益生菌可顯著增強小鼠長期、短期及空間上的學習與記憶能力，並且餵予益生菌可顯著減少小鼠海馬迴中的cleaved caspase-3的表現量(P < 0.05)，顯示特定益生菌之攝取可以減緩老化造成的行為退化現象。再來，進一步透過小鼠體內抗氧化酵素、MDA濃度及菌相之分析，探討益生菌延緩老化可能機制之實驗結果中，發現餵予老化小鼠Lb. kefiranofaciens HL1及Pm-1，可降低小鼠肝臟及腦部MDA濃度，且其中Pm-1組還可顯著減少血漿中MDA濃度(P < 0.05)；另外，所有益生菌組皆表現較高的血液超氧歧化酶(superoxide dismutase, SOD)及過氧化氫酶(catalase)活性，並且其中高劑量HL1組腦部SOD活性及肝臟catalase活性顯著高於負對照組(P < 0.05)。而在腸道微生物分析的結果可以發現益生菌組有較高乳酸桿菌屬(Lactobacillus)，較低腸桿菌科(Enterobacteriaceae)比例之現象。綜合以上結果得到，利用體外抗氧化之測定可作為先前篩選菌株的方式，並且由結果推測益生菌帶來之延緩老化現象，可能是透過增強體內抗氧化的機制。 藉由實驗之結果，期望對於益生菌延緩老化之路徑能有進一步了解，並可應用於開發具延緩老化機能性之益生菌產品。
並列摘要
The primary manifestation of aging is an overall decline in the capacity of various organs to maintain homeostasis. Recent studies have shown that the generation of reactive oxygen species (ROS) and the corresponding response to oxidative stress are key factors in aging. As the elderly population increases, leads to the importance of health care and health food. Earlier studies have found certain probiotic strains with antioxidant effect. Our previous study also found that administrated Pm-1, the mixture of Lactobacillus paracasei subsp. paracasei BCRC 12188, Lactobacillus plantarum BCRC 12251 and Streptococcus thermophilus BCRC 13869, could decrease concentration of malondialdehyde (MDA) in D-galactose induced aging mice, which reflects decreased production of free radicals by lipid peroxidation. However, few studies are focused on the anti-aging role of probiotic strains. In the present study, we screened and identified the potential strain, Lb. kefiranofaciens HL1, with antioxidant properties by measuring the inhibition of linoleic acid peroxidation, chelation ability for Fe2+ and 1, 1-diphenyl-2-picrylhydrazyl scavenging activity. The further in-vivo study was conducted using D-galactose-induced aging mice with daily administrating the selected strains, Lb. kefiranofaciens HL1 (108 CFU/daily and 109 CFU/daily) and Pm-1 (108 CFU/daily). In comparison of D-galactose-treated mice, all three probiotic groups significantly reduced the D-galactose-induced learning and memory impairment by performing shorter latency to platform and longer target quadrant search time (P < 0.05) in Morris water maze test at week 10. The result for immunohistochemistry also showed that D-galactose induced the activation of caspase-3 was reversed by treatment of probiotics in the hippocampus (P < 0.05). To study the possible anti-aging mechanism, lipid peroxidation induced by ROS were indirectly estimated by measuring MDA. The activities of anti-oxidative enzymes, including catalase (CAT) and superoxide dismutase (SOD), were also measured. Results indicated that lower MDA level were observed in the probiotic treating groups when compared with D-galactose-treated control in both liver and brain tissue (P < 0.05). For SOD, higher enzyme activities were also found in all three probiotic groups in plasma (P < 0.05). In addition, higher CAT activities were shown in high dosage HL1 group compared with D-galactose-treated group in plasma and liver tissues. Finally, the alteration of microbiota was analyzed during experimental period and after sacrificed. For the cecum microbiota analysis, all probiotic groups exhibited higher ratio of Lactobacillus and lower ratio of Enterobacteriaceae compared with D-galactose-treated group. These results suggest that both Lb. kefiranofaciens HL1 and Pm-1 may exhibit some anti-aging properties by strengthening the resistance to oxidative stress and modulating the composition of gut microbiota. The study may extend usage for probiotics.