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標題: | 藉由體外試驗評估國產羊乳對腸道抗發炎之效果 Evaluation of Taiwan goat milk on its intestinal anti-inflammatory effect in vitro |
作者: | Tsu-Ting Teng 滕祖廷 |
指導教授: | 陳明汝(Ming-Ju Chen) |
關鍵字: | 台灣羊乳,乳蛋白質,殺菌條件,體外消化,腸道抗發炎, Taiwan goat milk,milk protein,pasteurized conditions,in vitro digestion,intestinal anti-inflammatory, |
出版年 : | 2018 |
學位: | 碩士 |
摘要: | 發炎性腸道疾病 (Inflammatory bowel disease, IBD) 是一種結腸和小腸的慢性發炎。在已開發國家,IBD具有高發生率及高盛行率的特性,而目前的治療方針對於IBD病患的治療效果尚不夠理想;因此,受限於現有的藥物治療所造成的副作用或其成效不彰,有效的食品輔助療法則顯得十分重要。過去的研究顯示,羊乳具有預防腸道發炎的潛力。因此,本研究的目的旨在評估不同來源與殺菌條件之台灣國產羊乳在腸道抗發炎的效果,並進一步分析羊乳中何種蛋白質成分對腸道發炎具有最佳之生物活性。
我們自台灣本土市售羊乳取得三種不同品牌來源,並分別進行不同的熱殺菌條件,分為四個組別:品牌A的高溫短時間殺菌法 (high-temperature short-time, HTST) (A-H)、品牌B的HTST (B-H) 和低溫長時間殺菌法 (low-temperature long-time, LTLT) (B-L),以及品牌C的超高溫瞬間殺菌法 (ultra-high temperature, UHT) (C-U)。首先,以人類腸道上皮結腸癌細胞株Caco-2於葡聚糖硫酸鈉 (dextran sodium sulfate, DSS) 有無的試驗條件下,評估不同乳源及殺菌條件之羊乳的抗發炎活性。在細胞存活率的試驗結果中,所有處理組對Caco-2細胞皆不具有細胞毒性,且不論在預處理的共培養後或DSS所造成的細胞傷害下,各組皆具有促進Caco-2細胞存活率並抵抗DSS對細胞傷害的效果。接著,測量極化的腸道上皮細胞Caco-2單層膜的跨膜上皮電阻 (transepithelial electrical resistance, TEER) 值,觀察各處理組對腸道上皮屏障功能之效果。結果顯示,在預處理的48小時共培養後,羊乳A-H和C-U能使TEER值提升,而各處理組在DSS造成Caco-2單層膜受損時,皆能有效減緩DSS引起之TEER值的降低。亦即羊乳具有加強腸道上皮細胞完整性及其屏障功能的能力,進而預防或抵抗腸道上皮細胞通透性的增加。 此外,CCL20為一種腸道上皮修復的趨化因子,我們發現處理組A-H能有效促進Caco-2細胞生成CCL20,並在DSS造成Caco-2細胞受損時,維持CCL20的生成量。此外,我們也發現羊乳A-H能促使Caco-2細胞生成促發炎激素IL-8,其為嗜中性顆粒球的趨化因子;而在DSS傷害下,各處理組皆具有維持Caco-2細胞分泌IL-8之特性。因此,取自不同乳源及殺菌條件的各處理組中,以羊乳A-H在腸道抗發炎上具有最佳之效果,是以該組進行後續的試驗。 為了進一步探討羊乳中有效的抗發炎成分之所在,我們將羊乳蛋白質分離為酪蛋白與乳清蛋白,並發酵羊乳之kefir,再以模擬消化液進行水解。在細胞存活率的試驗中,消化後的全乳 (dW) 和消化後的酪蛋白 (dC) 最具細胞保護效果。而所有處理組皆可在DSS的細胞傷害下維持TEER值。另外,我們發現dC在DSS造成的細胞傷害下,仍具有促進Caco-2細胞生成CCL20和IL-8的能力。因此,我們進一步以dC分析其潛在之機能性成分。 利用十二烷基硫酸鈉聚丙烯醯胺凝膠電泳 (SDS-PAGE) 分析羊乳A-H消化前後之全乳與酪蛋白之蛋白質分子量與其圖譜差異。結果顯示,羊乳蛋白質在消化後被水解成較小之片段,使消化後許多蛋白質條帶消失,而消化後的一些胜肽片段在未消化的組別中並不存在。 綜上所述,本研究體外試驗之成果證明台灣國產羊乳具有維持腸道細胞恆定功能與抗腸道發炎之潛力,未來需進一步以動物實驗證實體外試驗之結果。 Inflammatory bowel disease (IBD) is a chronic inflammatory condition of the colon and small intestine. With high incidence and prevalence of IBD in developed countries, lots of patients with IBD still do not show an appropriate response to existing therapies; therefore, instead of pharmacological therapy with side effects and high rate of treatment failure, the importance of effective dietary supplementation can’t be overemphasized. Several studies revealed that goat milk had potential preventive effects on intestinal inflammatory injury. Therefore, the aim of this study is to evaluate the intestinal anti-inflammatory effect of local goat milk from various origins with different pasteurized conditions, and further explore which protein fractions in goat milk has the best biological effect on inflammatory damages. Three brands of goat milk products with different thermal processes were used and divided into four groups, brand A with high-temperature short-time (HTST) (A-H), brand B with HTST (B-H) and low-temperature long-time (LTLT) (B-L), and brand C with ultra-high temperature (C-U). The effect of goat milk samples from different brands with specific thermal processes on their anti-inflammatory effect were evaluated using Caco-2 cells with or without dextran sodium sulfate (DSS). MTT assay results indicated that all the treatments showed no cytotoxicity to Caco-2 cells, and were effective in protecting cells against DSS-induced damage. We further measured the transepithelial electrical resistance (TEER) value to evaluate the effect of treatments on intestinal epithelial monolayer. Treatment of A-H and C-U enhanced TEER value after 48 hours co-culture. Further, pre-treatment of all groups could protect Caco-2 intestinal epithelial monolayer against DSS-induced loss of barrier function. In addition, we measured the level of CCL20, an intestinal epithelial restitution and migration chemokine. CCL20 was upregulated by the groups A-H with or without DSS treatment. IL-8, a neutrophil-attracting chemokine, was upregulated by A-H without DSS treatment; on the other hand, all treatments could upregulate the level of IL-8. Based on the above results, we selected A-H group for the following study. To further investigate which components of goat milk involving in the anti-inflammatory effect, we separated goat milk into casein and whey protein, and further hydrolyzed by in vitro digestion. Meanwhile, the goat milk kefir was also used for comparison. MTT assay results indicated that all the treatments showed no cytotoxicity to Caco-2 cells. Under DSS-induced damage condition, digested whole goat milk (dW) and digested casein (dC) were more effective in protecting cells. TEER values were restored by all treatments after treating DSS. Additionally, we found that digested casein could upregulate CCL20 and IL-8 levels with DDS damage. Thus, we selected digested casein to investigate the potential functional compounds. We further analyzed the protein profile of undigested and digested goat milk and goat milk casein by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) to estimate their molecular weight and characterize those proteins. Goat milk proteins were hydrolyzed to smaller fragments after in vitro digestion with disappearance of several proteins bands. The findings of this study prove the potential intestinal anti-inflammatory activity of Taiwan goat milk in sustaining intestinal homeostasis in various aspects. Further animal study is necessary to confirm the in vitro results. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/70480 |
DOI: | 10.6342/NTU201803128 |
全文授權: | 有償授權 |
顯示於系所單位: | 動物科學技術學系 |
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