臺灣博碩士論文加值系統

感染性疾病(Infectious diseases)主要是由致病微生物所引起的，在人類和動物體中，此疾病主要發生在黏膜表面，特別是在腸道中。黏膜組織是體內免疫系統與外界各種抗原接觸作用的主要部位，可防止病原微生物入侵生物體。在人體中，小腸是最大的淋巴器官，含有許多淋巴球。近年來已有文獻顯示，小腸上皮細胞(Intestinal epithelial cells；IEC)在調節腸道平衡和免疫反應上扮演著關鍵角色。大豆粕(soybean meal；SBM)俗稱豆粉，為大豆經過溶劑萃取油脂後所留下之副產物。大豆粕因含有高量蛋白質，所以在飼料工業中已被廣泛的作為蛋白的來源。有研究指出，來自大豆蛋白的生物活性&;#32957;具有免疫調節活性。因此，本研究的目的為評估發酵豆粕衍生&;#32957;對於腸黏膜免疫的調節作用。將豆粕與小麥依比例混合做為基質，之後利用三種不同的菌&;#37211;在不同時間點下進行發酵，分別為Aspergillus oryzae BCRC 30428、Rhizopus oryzae BCRC 31150、Bacillus subtilis BCRC 14715。這些菌株常用在發酵東方傳統製品上，而在本實驗中則用以分解豆粕中的蛋白質，產生生物活性&;#32957;。IEC-6細胞株為來自大鼠的正常小腸上皮細胞，本研究用它作為體外細胞模式，以評估發酵豆粕衍生&;#32957;在LPS的誘導下是否可以降低細胞間的滲透性和細胞激素IL-6的分泌，以及增加IL-10的分泌。同時也評估在單獨給予發酵豆粕水萃物時是否可以增加IL-6和IL-10的分泌量，以間接證實對於IgA生成量的關係。首先測定IEC-6細胞株電阻，知其電阻值約為80.82 ± 2.75 Ω‧cm2，與文獻中所顯示之大鼠完整小腸組織(50-100 Ω ‧ cm2)電阻相似，因此，可確定IEC-6細胞株可做為細胞單層膜模式，用以評估上皮細胞的障壁功能。實驗結果顯示，不同菌&;#37211;在不同發酵時間下所得之發酵產物，皆顯示隨著發酵時間增加，48、60、72 h之豆粕水萃物對於大鼠小腸上皮細胞均有促進增生的效果。接著將不同菌&;#37211;發酵48、60、72 h之豆粕水萃物，進一步做細胞通透性的測定，結果顯示，利用B. subtilis natto發酵之豆粕水萃物，可維持上皮細胞之屏障完整性，對於黏膜並無損傷作用。因此，進一步的以Lipopolysaccharides（LPS）作為誘導劑，觀察B. subtilis natto發酵之豆粕水萃物對於上皮細胞是否有保護效果。結果可知，B. subtilis natto在不同發酵時間下之豆粕水萃物對於LPS所誘導降低的細胞跨膜電阻值(Transepithelial resistance; TER)皆有顯著增加的情形，可有效改善細胞通透性減緩黏膜損傷。而在IL-6細胞激素分泌量的實驗中，A. oryzae和B. subtilis natto的發酵水萃物，隨著發酵時間的增加，其對於LPS所誘導增加的IL-6分泌量有顯著性降低之作用，顯示這些發酵水萃物可有效降低發炎激素的產生。同時B. subtilis natto的發酵水萃物，在與LPS共培養下，也可有效提升IL-10的分泌量。綜合以上結果，可知B. subtilis natto的發酵水萃物，確實具有較好的生理活性，因此，接著利用分子篩層析法分離B. subtilis natto發酵水萃物中之不同大小片段之胜&;#32957;，結果顯示B. subtilis natto發酵水萃物確實有隨著發酵時間增加，分子量較小之胜&;#32957;有增加的趨勢。因此，之後會進一步的將分離出之區分物，進行生物活性評估，以找出具有黏膜免疫作用的生物活性&;#32957;。

The majority of infectious diseases of animals and humans occur at mucosal surfaces, especially in the intestinal tract. The mucosa is the principal site for the immune system’s interaction with the outside environment, it can protect our body from pathogenic organisms. Intestine is the largest lymphoid organ in human body. Recent researches showed that intestinal epithelial cells (IEC) may play key role in maintaining the homeostasis and modulating the immune response. Soybean meal is the byproduct of soybean oil production. It has been used extensively as an important source of dietary protein in the feed industry because of their high protein content. It has been reported that peptides derived from soy protein have immunomodulating activity. Thus, the aim of this research was to evaluate the effects of peptides derived from fermented soybean meal on mucosal immune response. Mixture of soybean meal and wheat was fermented with different microorganisms, including Aspergillus oryzae BCRC 30428, Rhizopus oryzae BCRC 31150, Bacillus subtilis BCRC 14715. These microbes are commonly used as starters in the fermentation of many traditional, oriental food products. The IEC-6 cell line from the rat normal small intestine cell was used as the cell model to evaluate whether peptides from fermented soy products could increase IL-6 and IL-10 cytokines secretion, inhibit LPS induced inflammatory cytokines and decrease intracellular permeability. First, we determined the transepithelial resistance (TER) value of IEC-6 cell line, and found its TER value is approximately 80.82 ± 2.75 Ω ‧ cm2, which is similar to the complete rat intestinal tissue (50-100 Ω ‧ cm2). Therefore, the IEC-6 cell can be used as a cell monolayer to assess the epithelial barrier function. Results showed that disregard the kind of starter used, the water extract of fermented soybean meal could increase proliferation of the rat intestinal epithelial cells, and this effect would be enhanced when fermentation time was increased. For the permeability of IEC-6 cells, our results showed that only the water extract of B. subtilis natto fermented soybean meal could maintain the integrity of the epithelial barrier. Therefore, we further treated the cells with LPS to increase of permeability in IEC-6 cells, and in the meantime the cell culture were also given water extract of B. subtilis natto fermented soybean meal. We found the water extract of the fermented product could significantly recover the level of transepithelial resistance. This result demonstrated that the water extract of B. subtilis natto fermented soybean meal had protective effect on rat intestinal epithelial cells and can decrease mucosal injury. In addition, the water extracts of soybean meal fermented by B. subtilis natto could inhibit the LPS induced inflammation in intestinal epithelial cells, and its bioactivity increased with increase of fermentation time. And it could also up-regulate the level of IL-10. We further used the size exclusion chromatograph to separate the peptides based on their molecular weights, and then identified the peptides which has the bioactivity of mucosal immunomodulation.

目錄
口試委員會審定書 I
中文摘要 II
Abstract IV
目錄 VI
圖目錄 IX
表目錄 X

第一章、文獻回顧 1
第一節、小腸組織之組成與功能 1
(一)小腸上皮細胞之屏障功能 1
(二)小腸細胞間之結合作用 3
第二節、腸黏膜的防禦機制 5
(一)黏膜屏障 6
(二)免疫防禦 7
(三)腸黏膜免疫系統的組成 8
第三節、大豆及其副產物大豆粕 11
(一)大豆之特性 11
(二)大豆粕之特性 11
(三)發酵大豆粕的特性 12
(四)固態發酵之特性 13
第四節、發酵菌&;#37211; 14
(一) Aspergillus oryzae 14
(二) Rhizopus oryzae 14
(三) Bacillus subtilis 15
第五節、機能性蛋白質與生物活性&;#32957; 16
(一)機能性蛋白質與生物活性&;#32957; 16
(二)蛋白質與胜&;#32957;在腸道之吸收 17
第六節、以IEC-6細胞株作為體外模式評估腸黏膜免疫反應 19

第二章、實驗目的與設計 20
第一節、實驗目的 20
第二節、實驗設計 21
(一)發酵豆粕的發酵製程 21
(二)發酵豆粕活性&;#32957;之基本成分分析與生物活性評估 22

第三章、材料與方法 23
第一節、實驗材料 23
(一)發酵菌種 23
(二)實驗原料 23
(三)細胞株來源 24
(四)藥品試劑 24
第二節、儀器設備 26
第三節、實驗方法 27
(一) 發酵豆粕之製備 27
(二) 發酵豆粕水萃液之製備 29
(三) 發酵豆粕成分分析 29
(四) 樣品配置 32
(五) 細胞培養 32
(六) 細胞存活率分析( MTT assay ) 34
(七) 以細胞模式探討發酵豆粕衍生&;#32957;免疫調節功能 35

第四章、結果與討論 40
第一節、發酵豆粕水萃液之成分分析 40
(一) 發酵豆粕水萃液之水溶性蛋白含量 40
(二) 發酵豆粕水萃液之胜&;#32957;含量 43
(三) 發酵豆粕水萃液之pH值變化 45
第二節、不同菌&;#37211;發酵豆粕水萃液對大鼠腸上皮細胞IEC-6之存活率影響 46
(一) 不同蛋白質濃度的發酵豆粕水萃液處理IEC-6細胞之MTT結果 46
第三節、不同菌&;#37211;發酵豆粕水萃物對大鼠腸上皮細胞IEC-6之細胞緊密型結合
作用( Tight junction )之影響 51
(一)以大鼠腸上皮細胞IEC-6作為評估黏膜上皮細胞屏障功能之模式 51
(二)不同菌&;#37211;發酵豆粕水萃物對於IEC-6細胞通透性之影響 53
(三) B. subtilis natto發酵豆粕水萃物抑制LPS所誘導IEC-6之損傷 57
第四節、以細胞模式探討不同菌&;#37211;發酵豆粕水萃物之免疫調節功能 60
(一)發酵豆粕水萃物對於LPS所誘導產生之發炎激素IL-6之抑制能力 60
(二)發酵豆粕水萃物對於LPS所誘導降低抗發炎激素IL-10之上調能力 65
第五節、利用分子篩層析法分離豆粕水萃物中之活性胜&;#32957; 70
(一)發酵豆粕水萃液之分子量大小 70

第五章、結論 72

第六章、參考文獻 73








圖目錄
圖1-1、調節上皮細胞通透作用的兩個主要途徑 2
圖1-2、小腸細胞間結合作用及上皮細胞接合蛋白之型態 4
圖1-3、黏膜免疫的途徑和各部位的作用功能 5
圖1-4、腸道黏膜免疫的機制 9
圖1-5、腸道黏膜免疫系統 10
圖3-1、OPA法反應式 31
圖3-2、細胞跨膜電阻(TER)測定裝置 36
圖4-1、不同發酵菌&;#37211;對於大豆粕中蛋白質含量的影響 42
圖4-2、不同發酵菌&;#37211;對於大豆粕中胜&;#32957;含量的影響 44
圖4-3、不同發酵菌&;#37211;在發酵大豆粕期間pH值之變化 45
圖4-4、不同蛋白濃度的發酵豆粕水萃液處理大鼠腸上皮細胞IEC-6達24小時之存活率結果 48
圖4-5、在不同時間下IEC-6單層膜之電阻值變化 52
圖4-6、不同菌&;#37211;在不同發酵時間下之豆粕水萃物對於IEC-6細胞通透性影響 54
圖4-7、B. subtilis natto發酵之豆粕水萃物抑制LPS所誘導改變IEC-6細胞之通透性 59
圖4-8、不同菌&;#37211;在不同發酵時間下之豆粕水萃物抑制LPS所誘導產生的發炎細胞激素 62
圖4-9、不同菌&;#37211;發酵豆粕水萃物在不同發酵時間下上調LPS所誘導減少IL-10
之能力 67
圖4-10、納豆菌發酵豆粕水萃物在不同發酵時間下胜&;#32957;之分子量分佈 71








表目錄
表3-1、定量蛋白之檢量線配製表 30

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