探討發酵乳清蛋白之抗氧化及神經保護力對抗神經退化性疾病

Abstract

隨著現代醫學的進步，全球人口老化的問題日漸嚴重，其中老年人口的疾病預防及照顧則是我們迫切面臨到的挑戰。神經退化性疾病是一類常出現在老年人的慢性疾病，其病因主要為環境因素、發炎以及氧化壓力等，其中又以氧化壓力與神經細胞的慢性退化最為相關。目前有許多方式包含營養補充等致力於減少氧化壓力，去抵抗神經退化性疾病的病程，而乳清蛋白以及益生菌皆被證實具有抗氧化及神經保護的能力。然而目前鮮少文獻結合兩者發展機能性產品以抵抗神經退化性疾病，因此本研究將篩選乳酸菌用於發酵乳清蛋白，並以體外試驗及動物試驗驗證其神經保護能力。 在體外試驗，首先本研究篩選40株克菲爾粒的分離菌株，並且利用革蘭氏染色保留35株革蘭氏陽性菌，接續利用1,1-二苯基-2-三硝基苯肼 (DPPH) 試驗作為初篩依據，經初篩後本試驗保留15株分離菌株。接著本研究利用15株分離菌株去發酵乳清蛋白，並測定發酵乳清蛋白的抗氧化能力及發酵特性，結果顯示4株分離菌株所發酵的乳清蛋白擁有最佳的抗氧化活性，並且利用16S rRNA及rpoA基因序列搭配系統分類樹分析4株分離菌株皆屬於Lactobacillus kefiri。發酵乳清蛋白首先經過體外消化再測定其保護效應，而體外試驗將以SH-SY5Y 細胞株當作神經模式並以過氧化氫當作氧化壓力來源。結果顯示所有的消化發酵產物皆能顯著提升細胞存活率且有劑量效應 (p < 0.05)。而本研究也發現經消化發酵產物處理與不處理的細胞相比能夠顯著提升抗氧化酵素之活性以及減少活性氧化物質的產生 (p < 0.05)，此外消化發酵產物的處理能顯著抑制粒線體膜電位的減少以及截切後凋亡蛋白3的表現量 (p < 0.05)。透過體外試驗的結果本研究篩選出L. kefiri M09 (M09) 及其發酵乳清蛋白進行後續動物試驗。 動物試驗方面本研究採用D-半乳糖誘導老化小鼠模式，並且給予小鼠未發酵乳清蛋白、M09以及M09發酵乳清蛋白。在行為表現方面，各處理組對於小鼠在水迷宮的表現無顯著影響 (p > 0.05)，而在築巢試驗中給予未發酵乳清蛋白、M09以及M09發酵乳清蛋白相對老化組能夠顯著提升小鼠築巢得分 (p < 0.05)。此外，M09以及M09發酵乳清蛋白能夠顯著減少老化小鼠腦部活性氧化物質的含量 (p < 0.05)，以及減少血及腦中脂質過氧化程度 (p < 0.05)，並從切片結果中觀察到給予M09以及M09發酵乳清蛋白能夠保持腦部皮質的厚度。 總結本篇研究，利用L. kefiri發酵乳清蛋白能夠展現良好的抗氧化活性，並且能夠作為預防神經退化性疾病的潛力產品。

As the medical advances, the global ageing issue becomes severe. We are facing the challenge of the elderly care and disease prevention. Neurodegenerative disease has a higher occurrence in the elderly due to environmental changes, inflammation, and oxidative stress. Among these causes, oxidative stress has been reported to directly associate with chronic degeneration of neurons. Different approaches were used to eliminate oxidative stress including nutritional method. Recently, both whey protein and probiotics have been proven to possess antioxidative and neuroprotective effects, respectively. However, few researches combine these two ingredients together for developing novel functional product. Thus, in the present study, we aimed to select lactic acid bacteria to produce fermented whey product with a neuroprotective effect in vitro and in vivo models. In vitro, we isolated 40 bacteria from the kefir grains. After Gram staining, 35 Gram positive isolates were tested their anti-oxidative effect by DPPH (1,1-diphenyl-2-picrylhydrazyl) assay, and 15 isolates were selected. Then, we used 15 isolates to produce fermented whey protein and evaluated their antioxidative and fermentation abilities. The 4 fermented whey samples with the highest antioxidative were all fermented by Lactobacillus kefiri, which were identified by 16S rRNA and house-keeping gene (rpoA gene) combining with phylogenetic analysis. The fermented whey protein samples were then in vitro digested and determined their neuroprotective effect by SH-SY5Y cell line as neuron model and H2O2 as oxidative stress. All digested fermented whey protein significantly elevated cell viability with dose-dependent manner under oxidative stress (p < 0.05). Meanwhile, we also found that the reactive oxygen species (ROS) production significantly decreased and antioxidative enzyme activity was increased as compared with non-treated cell (p < 0.05). Besides, the levels of cleaved caspase-3 was significantly reduced (p < 0.05) and the mitochondrial membrane potential reduction was inhibited after treatment, contributing to the cell viability. L. kefiri M09 (M09) and its fermented whey protein product were selected for the following in vivo study. In vivo, D-galactose induced ageing mice were fed with unfermed whey protein, M09 and its fermented whey protein product. In the behavior test, all treatment didn’t significantly affect the performance from each other in Morris water maze (p > 0.05). However, mice fed with whey protein, M09 and M09 fermented whey protein product scored significantly higher than ageing group (p < 0.05) in nest building test. Besides, M09 and M09 fermented whey protein product could significantly reduce ROS level in brain (p < 0.05). Meanwhile, mice fed with M09 and M09 fermented whey protein product showed siginificantly lower lipid oixidative levels in blood and brain comparing with ageing group (p < 0.05). Last, supplement of M09 and M09 fermented whey protein product also exhibited protection of brain cortex thickness. In conclusion, whey protein fermented by L. kefiri demonstrated a potential ability in ameliorating oxidative stress and preventing neurodegenerative disease.