利用兩階段式固態發酵豆粉開發具有免疫調節活性之產品

Abstract

自然殺手細胞為負責對抗病毒感染及腫瘤細胞的第一道防線，在免疫系統中扮演著關鍵的角色。近年來以飲食調節免疫功能為研究的一大趨勢，由天然來源分離出的醣蛋白被證實具有免疫調節的潛力。黃豆富含高量蛋白質且乳酸菌發酵黃豆產品已被證實具有免疫調節活性。然而，目前之發酵產品多以液態發酵方式生產為主。本研究則以黃豆粉為基質，以兩階段式固態發酵方式，先以 Saccharomyces cerevisiae 預發酵後，再接種 Lactobacillus acidophilus、Streptococcus thermophilus及Bifidobacterium infantis發酵，期望透過微生物發酵使基質產生微生物轉化作用，生成具有免疫調節活性之醣蛋白。 首先探討不同水分含量黃豆粉中微生物生長情形，確定固態發酵之可行性。之後以 NK-92 MI 細胞為免疫調節活性測試平臺，探討不同水分含量、微生物預發酵時間及乳酸菌發酵時間等因素，對於細胞存活率之影響。實驗結果發現，以 60% 水分含量，黃豆粉經酵母菌預發酵一天後，再由乳酸菌發酵一天及三天之組別，對免疫功效提升之效果最佳，且具有此效果之生物活性成分可能為蛋白質。 本實驗進一步以陰離子交換層析分離純化粗蛋白萃取液，發現經過發酵後蛋白質及醣組成有改變之情形，將區分物以 NK-92 MI 細胞測試其免疫調節活性，結果顯示，黃豆粉經酵母菌預發酵一天後，再由乳酸菌發酵一天及三天之組別，能顯著促進 NK-92 MI 細胞增生，並增加其毒殺 K-562 細胞之活性。

Natural killer cells play a vital role in immune system, which is the first line defence against viral infection and in preventing the development of cancers. To modulate immune function by diets has been a topic of interest to researchers in recent years. Glycoproteins isolated from natural sources have been proven to be an immunomodulatory potentiators. Soybean contains high quality protein, moreover, it has been reported that lactic acid bacteria fermented soy-based products possesses immunomodulatory activity. However, the soy-based products fermented by lactic acid bacteria were conducted majorly by submerged fermentation. In this study, we fermented soymeal by using two stage solid state fermentation. The soymeal inouculated Lactobacillus acidophilus, Streptococcus thermophilus and Bifidobacterium infantis after Saccharomyces cerevisiae pre-fermentation. We expected that the fermentation process could produce immunomodulatory glycoprotein due to bio-conversion of microorganism. The effect of soymeal with various moisture contents on microorganism growth was examined to evaluate feasibility of solid-state fermentation. In addition, the effect of various moisture contents, Saccharomyces cereviseae pre-fermentation time and lactic acid bacteria fermentation time on NK-92 MI cell viability were also be investigated. It was found that soymeal fermented by lactic acid bacteria for one and three days after yeast pre-fermentation for one day could promote NK-92 MI proliferation and cytotoxic activity of NK-92 MI cells against K562 cell. Furthermore, the bioactive compound is likely to be protein. The crude protein extract was fracrionated by anion-exchange chromatography. The elution profile of fermented sample was different from raw material. It was found that the fraction 4 of soymeal fermented by lactic acid bacteria for one and three days after yeast pre-fermentation for one day could promote NK-92 MI cells proliferation, and enhanced the cytotoxic activity of NK-92 MI cells against K562 cells.