益生菌寡醣發酵液抑制大腸癌細胞增生及其對人類結腸細胞株Int-407抗細胞毒性研究

Abstract

益生質大多為非消化性寡醣，可通過胃酸及消化道而不被完全分解，到達大腸被腸道微生物利用。許多文獻指出，益生菌在利用非消化性寡醣發酵後所產生之二次代謝物和有機酸具有降低大腸癌細胞活性的功效。本研究欲評估四種寡醣 (包括菊醣、果寡醣、異麥芽寡醣和木寡醣) 對於乳酸桿菌和雙叉桿菌 (Lactobacillus casei 01、Bifidobacterium lactis Bb-12 和 Lactobacillus plantarum) 之助生性，並利用去除菌體之發酵液進行細胞試驗，觀察寡醣發酵液對於人類大腸癌細胞HT-29是否有抑制生長之效果，以及利用 4-nitroquiniline-1-oxide (4NQO) 誘導人類腸道細胞株 Int-407產生細胞和基因毒性，評估寡醣發酵液降低細胞和基因毒性的效果。最後分析寡醣發酵液之有機酸的組成，探討其組成和抑癌效果及抗基因毒性的關聯性。 從生長曲線和 pH 值變化發現，三珠菌株在添加不同寡醣之培養基中均可生長，除了菊醣添加之培養基的 pH 值變化並不顯著外，其它三種寡醣添加之培養基的pH值會隨著時間增加而降低。利用去除菌體後之寡醣發酵液與大腸癌細胞株 HT-29 共同培養 24 小時，除了 L. plantarum 之異麥芽寡醣發酵液無法抑制 HT-29 細胞株生長外，其它發酵液均有抑制 HT-29 細胞生長的效果 (p < 0.05)，又以木寡醣的發酵液抑制癌細胞增生效果最好。而在抑制 4NQO 誘導之細胞毒性的實驗中，4NQO 組的細胞致死率約在 54% ，在添加寡醣發酵液共同培養後，多數寡醣發酵液均可顯著性提升細胞株的存活率 (p < 0.05)。在彗星電泳的顯影發現，添加寡醣發酵液的組別其 DNA 拖尾程度顯著性小於僅添加 4NQO 之細胞的 DNA 拖尾，平均又以B. lactis Bb-12之果寡醣發酵液效果較好。分析有機酸之含量發現，平均而言木寡醣發酵液的總短鏈脂肪酸濃度較高，推論可能與其抑制癌細胞能力有關；果寡醣發酵液中之丁酸濃度平均較其它寡醣發酵液高 (p < 0.05)，推論可能與其抗細胞毒性與抗基因毒性的能力有關。

Prebiotics are usually non-digestible oligosaccharides to human. They are resistant to gastric acid and digestive enzymes so that they could reach into the large intestines and were fermented by the intestinal microbes. Recently, some research has focused on the functions of the metabolites and short chain fatty acids that they might suppress the proliferation of colon adenocarcinoma cells. The aim of this study was to evaluate the anticytotoxic and antigentoxic effects of fermented supernatant by Lactobacillus casei 01, Bifidobaterium lactis Bb-12 and Lactobacillus plantarum in inulin, fructooligosacchardie (FOS), isomaltooligosaccharide (IMO) and xylooligosaccharide (XOS) contained medium. The growth curves showed that three strains of probiotics could grow in the oligosaccharides-contained medium and the pH values were decreasing during the fermentation. The results showed that the fermented supernatant could suppress the proliferation of HT-29 (p < 0.05) except L. plantarum-IMO group. In addition, the XOS-fermented supernatant showed the best suppression. The viability of 4NQO group was 54% and almost of cell viability in the test groups increased by treating with the fermented supernatant (p < 0.05), and the FOS-fermented supernatant showed the best ability in the promoting the cell viability after 4NQO induced cell damages. Furthermore, we observed that the DNA tails reduced in the test groups significantly by comet assay (p < 0.05). From the analysis of organic acids, the XOS-fermented supernatant contained higher concentration of total SCFAs that might contribute to the decreasing viabilities on HT-29. FOS-fermented supernatant contained the highest butyrate concentration which might contribute to the anticytotoxicity and antigenotoxicity.