乳酸菌發酵豆粕之健康促進功效及活性成分

黃豆 (Glycine max) 是一含有豐富蛋白質、油脂及活性成分之食品原料，其萃油後之副產物－豆粕，更可作動物飼料之蛋白質來源。但是，動物卻無法有效利用豆粕的高營養價值，原因是其中含有多種抗營養因子 (Anti-nutritional factors, ANFs)，使其產生營養利用率低、過敏型腹瀉及免疫力低下等健康問題。目前在實務上，大多透過乳酸菌發酵豆粕 (Lacto-fermented soybean meal, LFSM)，混入飼料中進行餵食，發現能有效改善上述狀況，但詳細機制不明。本研究旨在透過不同的乳酸菌發酵菌株及條件，期望能釐清 LFSM 可能具有之健康促進功效，並找出主要活性成分，可作為商業上控制製程之指標。以四株特性迥異之乳酸菌 (Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus salivarius, Lactobacillus helveticus ) 進行豆粕發酵，並以 in vitro 細胞模式 (小鼠巨噬細胞、大鼠小腸上皮細胞、大鼠嗜鹼性球)，探討營養利用率低、過敏型腹瀉及影響免疫功能三項健康問題，評估 LFSM 改善健康之功效，亦同步分析發酵產物中可能活性成分，並以統計分析比較其相關性。結果顯示，乳酸菌發酵豆粕之效果皆與未發酵豆粕有顯著差異，且依不同菌株而有分別。在 LPS 活化 RAW264.7 模式中，發酵樣品能顯著抑制 LPS 誘導產生之一氧化氮，以 L. helveticus 效果最佳，在高濃度下能達 67-77% 之抑制率；在未活化 RAW264.7 模式中，發酵樣品有促進一氧化氮分泌之效果，以 L. plantarum 效果最佳，在高濃度下能促進約 8-13 µM之一氧化氮分泌；在 LPS 誘導 IEC-6 傷害模式中及 IgE 致敏 RBL-2H3 模式中，觀察到發酵樣品能恢復 TEER 值及產生較低程度之釋粒反應。綜上所述，乳酸菌發酵豆粕在 in vitro 模式中顯示有免疫調節、增加腸道屏障完整性及減緩過敏之健康促進功效。本研究同時分析乳酸菌發酵對胜肽、乳酸、胞外多醣含量及抗營養因子的影響。各菌株均能有效降解主要的過敏原 (β-conglycinin、glycinin)、植酸及寡醣 (棉子醣、水蘇醣)。綜合實驗結果，配合多元線性迴歸統計分析，以及文獻資料，可知，任何單一生理活性其實有多個關鍵成分共同調控。由於發酵豆粕在免疫調節 (刺激一氧化氮分泌、抑制 LPS 誘導之高量一氧化氮) 上之功效顯著，且統計模型顯示胜肽是一關鍵成分因子，為了釐清乳酸菌發酵豆粕產生之胜肽對於免疫調節功效之影響，則選擇在免疫調節上效果較好的 L. helveticus 及 L. plantarum 配合蛋白水解酵素 (protamex 及bromelain) 之添加進行發酵，以提升胜肽產量，期望提升其生理活性。結果顯示，雖然酵素水解提高了胜肽含量，但是在免疫調節生理活性的表現上仍不及單純菌株發酵組。推測其原因為，對免疫有調節活性之胜肽具有結構特異性，特定胺基酸組成及大小之胜肽都會影響生理活性。添加蛋白水解酵素可能影響了胜肽的結構，也影響到乳酸菌本身的酵素系統對蛋白質及胜肽的分解效果，上述結果同時也確定了胜肽是影響 LFSM 活性的重要因子。

關鍵字

豆粕；乳酸菌發酵；活性成分；抗營養因子；健康促進功效；胜?

並列摘要

Soybeans are rich in proteins, lipids, variety of bioactive compounds and beneficial for human health. Soybean meal (SBM) is the by-product of soybean after oil extraction and can be used as a protein source in animal feed. However, effective use of high nutritional soybean meal is still limited due to its anti-nutritional factors (ANFs), which lead to less nutrient absorption, allergic diarrhea and immunity problems. In feed industry, these problems are often solved by partial replacement of SBM with lacto-fermented soybean meal (LFSM). However, its health promoting mechanism is still not clear. Therefore, the aims of this study were to investigate the possible health-promoting effects of LFSM and identify the major bioactive components which are responsible for the health benefits. Four lactic acid bacteria, including L. acidophilus, L. plantarum, L. salivarius, L. helveticus were used for fermentation. Three health problems, including nutrients uptake, allergic diarrhea and immune function were simulated using three in vitro cell models (murine macrophages, rat intestinal epithelial cells, rat basophils). Possible bioactive components of the fermentation product were analyzed simultaneously. Multivariate linear regression analysis was used to compare their relevance. The results showed that LFSM could significantly inhibit nitric oxide production on LPS-activated RAW 264.7, and L. helveticus-LFSM had the highest (67~77%) inhibition rate at high concentration. On the other hand, LFSM also promoted the secretion of nitric oxide of RAW 264.7. L. plantarum-LFSM had the best effect, which induced 8~13 μM of nitric oxide secretion at high concentration. The LFSM also recovered the TEER value and alleviated dagranulation reaction in the LPS-induced IEC-6 cells and IgE sensitized RBL-2H3 cells, respectively. Thus we could conclude that LSFM has health promoting effects including immune modulation, enhancing the integrity of the epithelial barrier, and alleviation of degranulation in vitro model. In this study, the content of peptides, organic acids, extracellular polysaccharides and ANFs were also analyzed. All of the four lactic acid bacteria could effectively degrade the main allergens (β-conglycinin and glycinin), phytic acid and oligosaccharides (raffinose and stachyose). Immune modulation effect (stimulating nitric oxide secretion and inhibiting LPS-induced high amount of nitric oxide) is the most prominent bioactivity among the activities investigated. Based on our results with multivariate linear regression statistical analysis and information from literatures, we could conclude that there are multiple bioactive components to regulate a physiological activity. The peptide content is the most significant factor correlated with immune modulation activity. In order to clarify the effect of the peptide in LFSM on immune modulation, L. helveticus and L. plantarum were used for fermentation and proteolytic enzymes (protamex and bromelain) were also added during fermentation to increase the yield of the peptides. The results showed that the longer the hydrolysis time, the higher the peptides content in the fermented products. However, the immune activity of the fermentation/enzyme hydrolyzed products was still lower than that of LFSM without protease hydrolysis. We suspect that the peptide has structural specificity for immunomodulatory activity, and the specific amino acids composition and size of the peptides would influence their bioactivity. Addition of protease may have altered the structure of the peptides, in turn, their bioactivities. Further, exogeneous enzyme may also affect the enzyme system of lactic acid bacteria acting on the protein and the peptide. Nevertheless, these results confirmed that the peptide is one of the major factors affecting the bioactivity.