Purification and application of anticarious substances produced by Lactobacillus paracasei subsp. paracasei NTU 101 and Lactobacillus plantarum NTU 102

乳酸菌 (lactic acid bacteria, LAB) 廣泛存在自然界，泛指一群能發酵碳水化合物產生多量乳酸的細菌。乳酸菌製作的發酵食品及發酵飲品食用歷史悠久，原本是為食品保存，後來漸漸發展成地區特有的傳統發酵食品。乳酸菌的代謝產物包括有機酸 (乳酸與醋酸)、二乙醯、過氧化氫、細菌素與低分子量的化合物等，這些具有抗菌能力的代謝產物是延長發酵食品保存時間的關鍵。這些乳酸菌代謝產物中，最具抑菌活性的是由細菌產生具有抑菌或殺菌活性的胜肽或小分子化合物。在目前的研究中，學者對其抑菌能力的廣效性及作為天然食品添加物延長食品保存期限最感興趣。本研究室先前的研究結果顯示，分離的乳酸菌株 Lactobacillus paracasei subsp. paracasei NTU 101 與Lactobacillus plantarum NTU 102 具有廣效的抑菌範圍。本研究評估此二株乳酸菌上清液純化所得抗菌活性物質之抗菌範圍及活性，並以核磁共振光譜儀分析其結構。以誘導齲齒之動物模式測試抗菌物質是否具抗齲能力，結果顯示 L. paracasei subsp. paracasei NTU 101 及 L. plantarum NTU 102 上清液所含抗菌物質可以抑制大鼠口腔中變形鏈球菌菌數增長並降低齲齒指數，與未塗抹抗菌物質的組別有顯著差異 (p < 0.05)，顯示其良好抗齲能力，可做為開發口腔保健產品之開發素材。為提升其應用的可能性，另以反應曲面法探討最適替代培養基，以豆漿與糖蜜為替代培養基可大幅降低生產成本，如要應用於大規模商業生產，仍須提升產量以降低耗能與生產成本。Lactic acid bacteria (LAB) are widely distributed in nature such as in dairy, fish as well as vegetables and grains. LAB fermented foods are less susceptible to microbial spoilage and prolong shelf life in food preservation. Previous studies showed that metabolites produced from LAB including organic acids (lactic acid, acetic acid), diacetyl, hydrogen peroxide, bacteriocins and some low molecular weight compounds have antimicrobial abilities. Among these antimicrobial metabolites, peptides or the low molecular weight compounds with bacteriostatic and bactericidal action, attracted interest of researchers and food producers for the potential to be biopreservative additives. In this study, we investigated the antimicrobial activity of supernatant of Lactobacillus paracasei subsp. paracasei NTU 101 and Lactobacillus plantarum NTU 102 and demonstrated the broad inhibitory spectrum. The antimicrobial substances were purified, characterized and identified by nuclear magnetic resonance (NMR) techniques. In animal experiments, we assessed Streptococcus mutans counts and caries scores to evaluate the decay process during a 68-day period. These agents significantly reduced oral cariogenic bacteria and caries scores (p < 0.05). The results revealed that the bacteriological factors involved in the caries process can be suppressed. These results suggested that the antimicrobial substances produced by L. paracasei subsp. paracasei NTU 101 and L. plantarum NTU 102 have protective functions against dental caries development. The production of antimicrobial substances was optimized by the Box-Behnken experimental design. An empirical model was developed through response surface methodology to describe the relationship between variables (molasses, soy-milk, and incubation temperature). The results revealed that an alternative medium could be used for large-scale commercial production of the antimicrobial substances, with potential prospects for application