複合菌與酵素輔助豆粕發酵以提升製程效率與產物功能性

豆粕是萃取黃豆油脂後剩餘的副產物，它具有高的粗蛋白含量、平衡的胺基酸組成並具有多項生物活性成分，如異黃酮、多醣、生物活性胜肽等，因此常作為動物飼料或製成功能性食品。然而豆粕內含有多種抗營養因子與過敏性蛋白質，可能會引起消化吸收不良和過敏反應，為了減少抗營養因子和蛋白質過敏原，飼料工業常採用單一菌株培養系統進行豆粕發酵，但是單一菌株發酵可能不是最有效的方法。因此，本研究旨在開發一種更有效的加工製程，在適當的條件下生產具有促進健康特性的發酵豆粕，本研究使用Streptococcus thermophilus搭配其他乳酸菌作為複合菌發酵系統發酵豆粕，同時添加鳳梨酵素與乳酸鈣，期望提升發酵產物的營養價值，如促進活性物質的含量、促進抗營養因子的降解、改善動物體消化利用率、強化動物體免疫力等。實驗結果證明，複合菌發酵確實可以提升發酵產物的乳酸含量，搭配鳳梨酵素使用，可以產生更多小分子蛋白質及胜肽，而添加乳酸鈣確實可以增強鳳梨酵素水解豆粕蛋白的活性，進而獲得較多的胜肽，具有增進免疫調節功效之潛力。利用S. thermophilus搭配Ligilactobacillus salivarius發酵的豆粕，在未活化RAW264.7模式中，發酵產物能刺激巨噬細胞分泌適量的NO，在0.5 mg/mL之低劑量下即可促進NO含量至17.76 ± 1.45 μM，足以誘發足夠的免疫反應；在LPS誘導RAW264.7發炎模式中，發酵產物能顯著抑制活化的巨噬細胞分泌過量的NO，在劑量為0.25 mg/mL時可達到最佳的NO抑制效果，使NO含量降低至19.89 ± 1.98 μM；在LPS誘導Caco-2損傷模式中，保護效果最為優異，TEER數值僅下降至71.54 ± 4.01 %；在Caco-2與RAW264.7的共培養模型中，觀察到發酵產物能夠促進IL-6與TNF-α 的分泌。綜上所述，酵素輔助複合菌發酵豆粕在in vitro實驗中證實具有強化免疫、抗發炎及保護腸道屏障完整性之健康促進功效。本研究亦分析發酵產物中的活性成分含量 (如可溶性固形物、胜肽、乳酸) 及抗營養因子含量 (如寡糖、胰蛋白酶抑制劑及過敏性蛋白質)，各發酵組別豆粕的活性成分含量均顯著提升並有效地降解抗營養因子。利用S. thermophilus搭配L. salivarius進行豆粕發酵對於過敏性蛋白的降解較為優異並且可以產生較多乳酸；而S. thermophilus搭配Lactobacillus helveticus進行豆粕發酵能夠產生較多的小分子蛋白質及胜肽。綜合上述實驗結果，本研究所製備出的發酵豆粕具有作為安全的腸黏膜免疫調節之功能性食品的潛力。

關鍵字

豆粕；抗營養因子；過敏性蛋白質； Streptococcus thermophilus ；複合菌發酵系統；鳳梨酵素；乳酸鈣

並列摘要

Soybean meal (SBM) is a by-product remaining after soybean oil is extracted. It has high crude protein content, balanced amino acid composition and multiple bioactive compounds, such as isoflavones, polysaccharides and bioactive peptides. Therefore, it is often used as animal feed or made into functional food. However, SBM also contains anti-nutritional factors and certain allergic proteins, which may cause low digestion, low absorption, and allergic reactions. To reduce the anti-nutritional factors and protein allergens, pure culture lactic acid bacterium fermentation is often used in feed industry. However, pure culture fermentation is probably not the most efficient way to do the process. In this study, we investigated the possible benefit of co-culture fermentation of SBM using Streptococcus thermophilus with other lactic acid bacteria. Besides, addition of bromelain at the appropriate time point during fermentation, with addition of calcium ion to enhance enzyme activity, was also studied for the production of peptides. Overall speaking, this study aimed to develop a more efficient processing protocol with proper conditions to produce a fermented soybean meal (FSBM) with better health promoting properties. The experimental results showed that the co-culture fermentation indeed could produce lactic acid more effectively in a shortened fermentation time. Addition of calcium ion to enhance bromelain activity could obtain more peptides in the FSBM. The FSBM prepared by S. thermophilus and Ligilactobacillus salivarius, in the unactivated RAW264.7 model, could stimulate macrophages to secrete an appropriate amount of NO. At a low dose of 0.5 mg/mL, the NO content was increased to 17.76 ± 1.45 μM, enough to induce sufficient immune response. In the LPS-induced RAW264.7 inflammation model, the fermentation product significantly inhibited the activated macrophages to secrete excessive NO, and the best NO inhibitory effect was achieved at a dose of 0.25 mg/mL, reducing the NO content to 19.89 ± 1.98 μM. In the LPS-induced Caco-2 injury model, the protective effect of FSBM was significantly better than the unfermented SBM, the TEER value only droped to 71.54 ± 4.01%. In the co-culture model of Caco-2 and RAW264.7, it was observed that FSBM could promote the secretion of IL-6 and TNF-α. In summary, the enzyme-assisted co-culture fermented soybean meal was proved to have health-promoting effects, including enhancement of immunity, anti-inflammatory, and protecting the integrity of the intestinal barrier in in vitro experiments. This study also analyzed the content of active compounds such as soluble solids, peptides, lactic acid, and anti-nutritional factors such as oligosaccharides, trypsin inhibitors, and allergic proteins in the fermented products. Co-culture fermentation significantly increased the bioactive compounds in FSBM and effectively degraded the anti-nutritional factors. The FSBM prepared by S. thermophilus and L. salivarius had excellent effect on degrading allergic proteins and produced more lactic acid; while the FSBM prepared by S. thermophilus and Lactobacillus helveticus produced more small molecule proteins and peptides. Based on the above experimental results, the FSBM prepared in this research has the potential as a safe functional food for intestinal mucosal immune regulation.