乳酸桿菌對口腔微生物之抗菌作用及作為健康食品之研究

The effect of Lactobacilli to oral Micro-organism and developing as a healthy food

賴威光(Wei-Kunang Lai)

指導教授 ： 錢佑

中山醫學大學/醫學院/醫學應用微生物研究所/碩士(2010年)

摘要

乳酸菌(lactic acid bacteria)具有免疫調節、擷抗、殺滅其他病原菌之功能。由於齲齒及牙周病罹患率頗高，開發出抗上述疾病之產品有其必要性。本研究之目的為建構一個鑑定乳酸菌株是否抗口腔病原菌及產品發展的平台。由文獻整理得知，乳酸菌之所以擷抗、殺滅其他病原菌之功能為其代謝產物如乳酸及細菌素(bacteriocin)等。本研究之內容為自450株乳酸菌株抽取粗細菌素、細菌素抗菌功能試驗、目標菌株高密度培養試驗、目標菌株抗菌功能確認試驗、消費性產品優化設計、產品品質安定性測試等。粗細菌素抽取係依已知之研究方法進行，細菌素抗菌功能以抑菌圈試驗進行初選，其最佳者稱為「目標菌株」。目標菌株培養試驗係以直交表(orthogonal array )實驗計劃法找出最恰當之培養條件。目標菌株培養後之抗菌功能確認試驗係將該菌株培養後經熱處理滅活，再經凍乾成粉末，以無菌液體培養基回溶成1.0×107cells/ml起三個十倍數稀釋之液體，取此液體1ml與1ml以BHI培養基調整成2.0×107cfu/ml之轉醣變形鏈球菌( Streptococcus mutans)混合培養0、6、24小時後，塗盤於固體BHI培養基培養3天，計數S. mutans之菌落數；復再取上述液體1ml與1ml以改良過之BHI培養基調整成2.0×107 cfu/ml之牙齦卟啉菌(Porphyromonas gingivalis)混合培養0、6、24 小時後，塗盤於BBAP 培養7 天，計數P. gingivalis 之菌落數。口腔病原菌落數越少代表乳酸菌株抗菌效果越佳。消費性產品優化設計係將目標菌株粉末以直交表實驗計畫找出最適口含錠處方；再調整參數使其達到目標硬度及目標崩解時間。品質安定性試驗係藉助Arrhenius 方程式，以官能品評分數、硬度及崩解時間做為指標，預測出可接受品質之窗櫥壽命(Self life)，以此方法預測之窗櫥壽命為446天。本研究以上述諸平台獲得一功能及品質良好之產品，若再經安全驗證、人體試驗確認具有抗齲齒及抗牙周病功能，有潛力作為抗口腔病原菌之健康產品。

關鍵字

乳酸桿菌 ； 口腔病原菌 ； 轉醣變形鏈球菌 ； 牙齦

並列摘要

Lactic bacteria possess functions in immunoregulation, antagonism, and destroying other pathogens. Due to the high incidence of dental caries and periodontal diseases, it is crucial to develop products that specifically against these diseases. The purpose of this study was to construct a platform for evaluating the effectiveness of lactic bacteria in countering oral pathogens, and to develop related products. We concluded from previous studies that lactic bacteria rely on metabolites such as lactic acid and bacteriocin to antagonize and destroy other pathogens. In this study, we extracted crude bacteriocin from 450 strains of lactic bacteria, and conducted the following procedures: bacteriocin antibacterial function test; target strain high-density culture test; target strain antibacterial function validation test; optimization for consumer products; and product quality and stability tests. Crude bacteriocin was extracted using standard methods. To determine the antibacterial function of bacteriocin, a zone of inhibition test was used to perform the initial selection, and the strain with the best results was designated as the “target strain”. In the target strain culture test, optimal conditions were determined by applying an orthogonal array experimental design. For the antibacterial function validation test, the bacteria was cultured, subjected to temperature attenuated, and freeze-dried to a powder form. This powder was then dissolved in a sterilized medium to a ten fold dilution, beginning from 1.0×107cells/ml. 1 ml of this fluid with 1 ml BHI medium were mixed to form a Streptococcus mutans mixture of 2.0×107cfu/ml, which was cultured for 0, 6, and 24 hours. The bacterial mixture was then plated on a solid BHI medium, cultured for 3 days, and subsequently measured for the number of S. mutans colonies by colony forming unit ( cfu). We also combined 1 ml of the powder-dissolved fluid with 1 ml modified BHI medium to form a Porphyromonas gingivalis mixture of 2.0×107cfu/ml, which was cultured for 0, 6 and 24 hours. The bacterial mixture was plated on solid a BHI medium, cultured for 7 days, and measured for the cfu of P. gingivalis. Fewer colonies indicated better antibacterial activity. We optimized the consumer products by applying an orthogonal array experimental design to determine the best prescription for oral tablets. We then modified the parameters to achieve a target hardness and disintegration time. Quality and stability were evaluated through the Arrhenius equation. Sensory evaluation scores, hardness, and integration time served as indicators to predict shelf life for the product; the shelf life was estimated to be 446 days. We believe that further safety tests and clinical trials to assess its effectiveness in preventing caries and periodontal diseases could prove the potential of this product as a viable anti-oral pathogen health product.

並列關鍵字

Lactobacillus ； oral pathogens ； Streptococcus mutans ； Porphyromonas gingivalis

參考文獻

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