基因改造乳酸菌 Lactococcus lactis NZ9000/pNZPNK 安全性評估

Abstract

本研究為評估基因改造乳酸菌之安全性，使用基因改造乳酸菌 Lactococcus lactis NZ9000/pNZPNK，以三種實驗進行分析：一、抗藥性基因檢測：以基因改造乳酸菌所攜帶之質體及非基改乳酸菌染色體為模板進行聚合酶鏈反應 (polymerase chain reaction, PCR)，於質體增幅所得抗藥性基因序列其大小正確，且經定序、比對後顯示為原本之抗藥性基因；非基改乳酸菌染色體則無抗藥性基因之存在。二、將非基因改造乳酸菌及基因改造乳酸菌破菌後分為可溶性蛋白質及細胞碎片兩部分，於基因改造乳酸菌之細胞碎片中以 SDS-PAGE 觀察到納豆激酶 (nattokinase)，顯示乳酸鏈球菌素 (nisin) 可成功誘導基改乳酸菌胞內生產納豆激酶；對所得可溶性蛋白質及細胞碎片進行胃蛋白酶水解，以 SDS-PAGE 分析發現所有蛋白質皆無法抵抗胃蛋白酶之水解；以具納豆激酶專一性之抗體對水解前後片段進行西方墨點法分析，於未水解片段中觀察到納豆激酶但已水解者則否，顯示所表現納豆激酶被降解。三、參考衛生署公告之腸道菌相改善評估辦法，於實驗中檢測大鼠腸道中產氣莢膜梭菌 (Clostridium perfringens)、雙歧桿菌 (Bifodobacterium) 兩株指標菌；其中 C. perfringens 若減少或不變而 Bifodobacterium 增加，則可認定受試物質有改善腸道菌相之功能。此外，本研究檢測腸道中常見之菌叢包括大腸桿菌群 (coliform bacteria) 及乳酸桿菌 (Lactobacillus)，觀察乳酸菌與重要腸道菌之互動，並檢測所餵食之基因改造乳酸菌以評估其存活力。實驗結果顯示，於所有組別中 C. perfringens 皆無法被偵測，Bifidobacterium 在餵食乳酸菌的數個組別，於四週時有顯著的增加 (p＜0.05)，六週時則未出現此現象。大腸桿菌群於四週時各組間並無顯著差異，六週時餵食乳酸菌組則普遍較控制組低 (p＜0.05)；乳酸桿菌於四週時沒有差異，六週時餵食乳酸菌之組別明顯增加 (p＜0.05)。以上菌相變化顯示餵食基因改造乳酸菌或非基因改造乳酸菌，對腸道菌相並沒有不良的影響。於腸道菌相評估實驗後收集相關檢體進行血清生化學及血液學檢查，結果顯示肝腎功能指標、血糖、血脂、電解質及血球組成，各組沒有顯著差異。細菌位移試驗對肝臟、脾臟、腎臟及血液進行檢測，於所有樣本中都未觀察到轉移現象。總結實驗結果，餵食基因改造乳酸菌 L. lactis NZ9000/pNZPNK 對大鼠之腸道菌相、體重、血清生化值及血液組成並無不良影響，於大鼠模型中能安全使用。未來若有類似的基因改造微生物，本實驗架構可為其安全性評估之參考。

The genetically modified microorganism (GMM) is derived from the techniques of modern biotechnology which is purposed to use in food and pharmaceutical industry. However, safety assessment for the potential risk of GMM must be considered. We introduced detection of antibiotic resistance genes, pepsin digestion assay and evaluation of intestinal microflora to assess the safety of GMM. The samples applied in this study were genetically modified lactic acid bacteria Lactococcus lactis NZ9000/pNZPNK and the host strain L. lactis NZ9000. Polymerase chain reaction (PCR) analysis of GM strain resulted in correct sequence of antibiotic resistance gene cat, indicating that antibiotic resistance gene remained unchanged after genetic engineering. By applying GM strain and host strain to pepsin digestion assay, the protein fractions of both soluble protein fractions and cell debris were degraded completely and rapidly. The produced nattokinase was undetectable after digestion with western blotting analysis. To assess the interaction between GM strain and intestinal microflora, Wistar rats received GM strain and host strain. Several important intestinal bacteria were counted. A significant increase in the count of Bifidobacterium in rat feces was observed after fed both GM and host strains for 4 weeks, but the count of Bifidobacterium had showed no significant change after fed GM and host strains for 6 weeks and the count of Clostridium perfringens in rat feces had no difference in all groups. These results suggest that Lactococcus lactis NZ9000/pNZPNK has low allergenicity and likely to be safe for human consumption.