基因轉殖乳酸球菌NZ9000/pNZPNK之致敏性及副乾酪乳酸桿菌 NTU 101 與胚芽乳酸桿菌 NTU 102 益生功能之評估

Abstract

利用本研究室已建立的乳酸菌表現 nattokinase 之基因轉殖微生物為試驗樣品，為了解基因轉殖微生物與非基因轉殖微生物在因應日後上市前安全評估項目之結果，進行致敏性安全評估實驗，並確認此檢測方法之可行性。基因轉殖乳酸菌之致敏安全性試驗主要結果為: (1)經由腹腔注射方式進行小鼠致敏實驗結果顯示，並不會造成小鼠產生如正控制組 ovalbumin (OVA) 誘導 IgE 及 IgG1 大量產生的致敏現象 (p<0.05)，且會透過IgG2a的分泌使免疫反應朝向 Th1 的耐受性反應路徑進行。(2) 在口服餵食致敏試驗中，於第 2 週開始 OVA 組之特異性 IgG1 及 IgE 濃度均顯著高於其他處理組(p<0.01)，餵食基因轉殖或非基因轉殖乳酸菌之處理組，於第2週之血清中其IgG2a含量明顯高於OVA及phosphate buffered saline (PBS) 餵食組 (p<0.01)，但餵食4週後則無明顯差異。小鼠脾臟細胞分泌細胞激素顯示，Th2 細胞分泌的IL-4及IL-10含量，非基因轉殖或基因轉殖乳酸菌在自然發生 (spontaneous)、裂殖原 (mitogen) 及OVA誘發處理下，較控制組為低 (p<0.01)。綜合上述結果可知，二株乳酸菌經腹腔注射或餵食方式處理BALB/c小鼠均不會引發過敏現象，顯示所開發的基因轉殖乳酸菌應屬於無致敏性之安全菌株。 利用體外細胞模式評估本土性乳酸菌Lactobacillus paracasei subsp. paracasei NTU 101及 L. plantarum NTU 102 之細胞水解區分物對巨噬細胞RAW264.7 增生及細胞激素分泌之影響，結果顯示水解物之沉澱區分及熱致死全菌會抑制巨噬細胞增生及型態改變為類樹突狀細胞，誘導 NO生成、環氧化酶活性表現及促發炎細胞激素分泌。細胞水解之可溶性區分能促進巨噬細胞增生，減少TNF-α, IL-6 及 IL-1β 之分泌，提高其IL-12/IL-10比值，使其免疫反應朝向Th1-type路徑進行。口服餵食熱致死乳酸菌可作為誘導免疫反應活化T淋巴細胞，並刺激相關細胞激素之分泌。 本土性乳酸菌應用於減低過敏模式之結果顯示，口服餵食乳酸菌株能減低由OVA誘發專一性IgE抗體之形成，達到調節Th1/Th2 免疫反應之平衡狀態與淋巴細胞增生之作用，能有效減低未致敏動物於餵食後，再接觸過敏原時所可能產生之過敏現象；但在已有過敏症狀之動物模式中，其過敏相關之抗體及細胞激素含量遠高於未致敏組，故減敏效果較不顯著。 以L. paracasei subsp. paracasei NTU 101及 L. plantarum NTU 102 乳酸菌株豆漿牛奶發酵乳、未發酵豆漿牛奶及福善美藥物為餵食樣品，利用卵巢切除小鼠動物模式，評估乳酸菌發酵乳改善骨質疏鬆症狀之能力。結果顯示，發酵豆漿牛奶含高量之非糖苷鍵結型大豆異黃酮、多醣、胜肽類、維生素D及可溶性鈣質等有利骨質生成之成分。由微電腦斷層掃描分析及電顯結果可知，小鼠透過卵巢切除後明顯產生骨小樑數目及體積減少的現象，若以L. paracasei subsp. paracasei NTU 101發酵豆漿牛奶餵食後，能有效增加骨體積(BV/TV)及骨小樑數目(Tb.N.)，其增加倍數為卵巢切除對照組的1.48及1.74倍，可知其對停經後雌激素缺乏所造成之骨質疏鬆症狀有明顯的緩解與預防的功效。

By introducing aprN into Lactococcus lactis NZ9000, the genetically modified L. lactis NZ9000/pNZPNK successfully expressed the nattokinase. The safety assessment of this novel strain was based on allergenicity of murine model serologic identity. Subjecting to the genetically modified (GM) strain and host were injected by intraperitoneal to BALB/c mice. The productions of IgG1 and IgE by L. lactis NZ9000/pNZPNK or L. lactis NZ9000 were significantly lower than that of ovalbumin (OVA) group. Feeding with OVA resulted in significantly higher production of IgG1 and IgE as compared to that of L. lactis NZ9000/pNZPNK or L. lactis NZ9000. Further, serum IgG2a level increased dose-dependently at week 2 and induced immune reaction toward Th1 pathway. Secretion of cytokines IL-4 and IL-10 fed with lactococci was significantly lower than OVA group. L. lactis NZ9000/pNZPNK did not increase the proliferation of type 2 helper T cells in spleen or induced allergenicity in BALB/c mice. Based on the results, the new GM lactic acid bacterium is regarded as safe to use. To elucidate the immunomodulation effects of dead lactobacilli, whole cells and gastrointestinal enzymatic hydrolysates of supernatants and precipitates from L. paracasei subsp. paracasei NTU 101 and L. plantarum NTU 102 on RAW264.7 macrophages and splenocytes were investigated. Increased NO, COX-2 expression, IL-10 and IL-12 were observed in high-dose precipitates and whole cells of both strains after 24-h stimulation. All of the hydrolysates and whole cells from both strains induced lower pro-inflammatory cytokines (IL-1