本實驗的目的在初步去證實幾丁聚醣抑制細菌生長的機轉為抑制細菌胞外 蛋白質酵素的活性，當蛋白質做為唯一氮源時，細菌無法利用蛋白質酵素去水 解大分子蛋白質變成小分子蛋白質，作為其營養的來源，而達到抑制細菌生長 的效果。以石蕊反應（ litmus reaction ）分析了革蘭氏陽性菌與革蘭氏陰性菌
，結果 Bacillus subtilis 、 Staphylococcus aureus 以及 Methicillin resistant Staphyl ococcus aureus （ MRSA ）具有水解 gelatin 的酵素。再將明膠（ gelatin ）平板 培養基加入各種濃度的幾丁聚醣去了解細菌的生長情況，只有 Bacillus subtilis
、 Staphylococcus aureus 以及 MRSA 的控制組可生長，也可被幾丁聚醣抑制生 長。其次，幾丁聚醣對 Bacillus subtilis 的最小抑菌濃度（ MIC ）是在 0.2 mg/
ml ，對 Staphylococcus aureus 為 0.2 mg/ml ，而對 MRSA 為 0.06 mg/ml 。再者
，以 mineral medium 培養菌種，證實了幾丁聚醣抑菌的途徑並非抑制利用氮源 為營養的途徑，顯示抑菌的途徑為抑制利用小分子蛋白質為營養的途徑。根據 幾丁聚醣抑制了 chymotrypsin 的活性，推測幾丁聚醣抑制蛋白質酵素，可能藉 由相互的結合，而影響到蛋白質酵素活性。由受質凝膠電泳（ zymography ） 也說明了此一論點。最後，以 Bacillus subtilis 的菌液，投與幾丁聚醣以 zymog raphy 分析 Bacillus subtilis 胞外蛋白質酵素活性的變化。幾丁聚醣可抑制住 Ba cillus subtilis 胞外蛋白質酵素的活性。本研究證實了幾丁聚醣抑制細菌生長的 機轉為抑制蛋白質酵素的活性，亦可說明幾丁質或幾丁聚醣做為食品保存劑時
，食物系統中，不得存在氨基酸或銨鹽。
幾丁質與幾丁聚醣對革蘭氏陽性菌抑菌機轉
The purpose of the present study was to elucidate the binding ability of chitin and chito san to proteases produced by gram-positive bacteria that resulted in growth inhibition of bacteria with protein as a sole nitrogen source. The hypothesis proposed that bacteria w ould be unable to obtain amino acids through hydrolyzing protein in presence of chitin a nd chitosan and the growth would be rendered if small molecule nitrogen sourced were deprived. Litmus gelatin medium was used to detect gelatinase production of bacteria a nd it was found that Bacillus subtilis, Staphylococcus aureus and a methicillin resistant S. aureus (MRSA) were positive in the test. Minimal inhibitory concentrations (MIC) of chitosan in gelatin medium to the bacteria were 0.06, 0.2 and 0.2 to MRSA, B. subtilis a nd S aureus, respectively. In minimal medium, all the bacteria including gram positive a nd gram negative grew normally, in presence or absence of chitosan. It is evident that b acteria were able to proliferate by small inorganic nitrogen molecule (NH4+) and chitin or chitosan were not effective to inhibit the growth of bacteria. Spectrophotometry were performed to detect the binding of chitin and chitosan to proteases from various sources . The inhibition of the proteases revealed that chitin and chitosan might effect on those proteases of metallo-type. Zymographic gel analysis indicated that proteases from B. su btilis, S. sureus and MRSA were inhibited by chitosan with a dose response reaction. Th e result might be useful to explain the bacterial static effect on skin wound when chitin or chitosan dressing was applied. It is also meaningful that chitin and chitosan might be a good food preservative when amino acid or ammonium salts were absent in the food s ystem.
Anti-Gram(+)-bacterial Mechanism of Chitin and C hitosan
