利用體外模式探討發酵豆粕衍生肽對腸黏膜免疫調節作用

An in vitro study on gut mucosal immunomodulation by peptides derived from fermented soybean meal

紀明慧(Ming-Hui Chi)

指導教授 ： 蔣丙煌

國立臺灣大學/生物資源暨農學院/食品科技研究所/碩士(2014年)

https://doi.org/10.6342/NTU.2014.01521

摘要

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肽具有免疫調節活性。因此，本研究的目的為評估發酵豆粕衍生肽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酛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，產生生物活性肽。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肽在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，不同菌酛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。接著將不同菌酛發酵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肽，結果顯示B. subtilis natto發酵水萃物確實有隨著發酵時間增加，分子量較小之胜肽有增加的趨勢。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肽。

關鍵字

黏膜免疫 ； 大豆粕 ； 小麥 ； 固態發酵 ； 胜肽 ； 小腸上皮細胞

並列摘要

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.

並列關鍵字

Mucosal immunity ； soybean meal ； wheat ； solid-state fermentation ； peptides ； intestinal epithelial cell

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