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Please use this identifier to cite or link to this item: http://ntour.ntou.edu.tw:8080/ir/handle/987654321/48977

Title: 不同分子量幾丁聚醣抗菌與抗致突變活性及其作用機制探討
Antibacterial and antimutagenic activity and mechanism of chitosan with various molecular weights
Authors: Chang, Shun-Hsien
張順憲
Contributors: NTOU:Department of Food Science
國立臺灣海洋大學:食品科學系
Keywords: 幾丁聚醣;分子量;超過濾;抗菌活性;抗致突變活性
chitosan;molecular weight;ultra-filtration;antibacterial activity;antimutagenic activity
Date: 2016
Issue Date: 2018-08-22T06:40:00Z
Abstract: 本研究以纖維素酶於 55oC水解去乙醯度 95%幾丁聚醣 (Degree of deacetylation, DD 95),將水解液經不同分子量範圍超過濾膜處理,經離心及甲醇區分沈澱法得到不同分子量幾丁聚醣。分析不同分子量大小幾丁聚醣於不同pH下之溶解度與電荷度變化,以探討對其抗菌活性影響,並同時以Salmonella typhimurium TA98、TA100 為模型,分析其對直接型致突變劑 4-nitroquinoline-N-oxide (4-NQO)、間接型致突變劑benzo[a] pyrene (B[a]P) 之抗致突變活性變化。 DD 95 於水解 1、3、6、9、18、24 小時所得水解液分離而得幾丁聚醣平均分子量分別為 300、156、72、29.2、7.1、3.3、2.2 kDa。7種幾丁聚醣樣品於酸性環境下 (pH 5.0、6.0) 及溫度下 (4、15、37、45oC) 對測試菌株Staphylococcus aureus BCRC 10780 及Escherichia coli BCRC 10675抗菌活性皆隨分子量增加而增強,並以 300 kDa幾丁聚醣活性最強,且呈溫度效應 (37、45oC最佳)。pH 7.0 時,300、156、72 kDa幾丁聚醣失去活性;反觀低於 30 kDa組別 (29.2、7.1、3.3、2.2 kDa) 幾丁聚醣仍具抗菌活性。此現象經分析其於不同pH中之溶解度即界面電位量得知各幾丁聚醣樣品於酸性環境 (pH 5.0、6.0) 下呈溶解性及正電荷;300、156、72 kDa幾丁聚醣於中性環境時失去溶解度及呈負電荷。藉由此現象結果與其抗菌活性呈明顯相關聯性,故推測其為主要抗菌活性因子。 不同分子量幾丁聚醣抗致突變活性方面,7 種幾丁聚醣經毒性、致突變能力測試確認樣品本身對測試菌株Salmonella typhimurium TA98、TA100無毒性及致突變能力後。將其以濃度 1、2.5、5.0 mg/plate下與測試菌株分別對 4-NQO、B[a]P之抗致突變活性。結果顯示各幾丁聚醣樣品之抗致突變活性隨其分子量減少而增強,以分子量低於 30 kDa幾丁聚醣組別 (29.2、7.1、3.3、2.2 kDa) 活性較佳,各濃度下對 4-NQO、B[a]P均超過 50%以上抑制率,其中以 2.2 kDa幾丁聚醣活性最佳。將 7 種幾丁聚醣樣品先分別與 4-NQO、B[a]P、S9 mix、B[a]P metabolites預培養 0、20、40 分鐘後,再與測試菌株共培養 20 分鐘以進一步探討其去致突變活性能力 (desmutagenicity) 。結果顯示各樣品先與致突變劑共培養後之抗致突變活性明顯增加,並以低分子量幾丁聚醣組別明顯較佳,29.2、7.1、3.3、2.2 kDa對 4-NQO、B[a]P metabolites組別均可超過80%以上抑制率,幾丁聚醣具去致突變活性,可抑制其致突變能力,其中並以 29.2、7.1、3.3、2.2 kDa幾丁聚醣組別明顯較強。
Chitosan samples with molecular weights (MWs) of 300, 156, 72.1, 29.2, 7.1, 3.3, and 2.2 kDa were prepared by cellulase degradation of chitosan (300 kDa) for 0–24 h, followed by ultrafiltration separation techniques. We examined the combined effects of chitosan MW, reaction temperature, and pH on the bacterial growth of Escherichia coli and Staphylococcus aureus, as well as the effects of pH on the water solubility and zeta potential (ZP) of chitosans. The antibacterial activity of chitosan increased as the pH value decreased and as the temperature increased, irrespective of the chitosan MW and the bacteria tested. The pH value affected the correlation between the antibacterial activity and chitosan MW. In acidic pH conditions (pH 5.0 and 6.0), the chitosan activity increased with increasing MW, irrespective of the temperature and bacteria tested. However, at neutral pH (7.0), little activity was observed for chitosans with MW > 29.2 kDa, for example, 72.1, 156, and 300 kDa, and the activities of the smaller chitosans (MW = 29.2, 7.7, 3.3, and 2.2 kDa) increased as the MW decreased. The pH value of the reaction mixture obviously affected the water solubility and ZP of chitosans. At pH 5.0 and 6.0, all the tested chitosans exhibited good water solubility and ZP decreased with the MW, whereas the solubility and ZP of the chitosans decreased with increasing MW at pH 7.0. Particularly, low solubility and negative ZP values were determined for chitosans with MWs of 300, 156, and 72.1 kDa, which may explain the loss of their antibacterial activity at pH 7.0. 7 chitosan samples exhibit anitmutagenic activities against the direct [4-nitroquinoline N-oxide (4-NQO)] and indirect-mutagen [benzo[a]pyrene (B[a]P)] with Salmonella typhimurium TA 98 and TA 100 in presence of simulated human liver enzyme mix (S9 mix), which were higher than did against 4-NQO, with former being 16.12-64.67% and 29.01-71.60% and later being 31.03-75.65% and 21.88-82.58%, respectively. Moreover, desmutagenicities obtained by chitosan samples preincubated with tested mutagen, then with treated strains. The desmutagenic activities of the chitosans below 30 kDa (80.4-82.8, 77.9-85.3% and 68.4-83.3, 78.3-87.1%) against 4-NQO and B[a]P with S. typhimurium TA 98 and TA 100 were significantly higher than did those of higher MW ones (20.2-46.3, 36.6-57.8% and 27.4-32.5, 28.2-39.6%). The desmutagenic activities of 5 mg/plate smaller chitosans (< 30 kDa) were greater than 50%, among which 2.2 kDa chitosan being highest. The desmutagenic effect of this chitosan against 4-NQO and B[a]P for TA 98 and TA 100 were 82.8, 85.3, 83.3, and 87.1%, respectively.
URI: http://ethesys.lib.ntou.edu.tw/cgi-bin/gs32/gsweb.cgi?o=dstdcdr&s=G0D98320002.id
http://ntour.ntou.edu.tw:8080/ir/handle/987654321/48977
Appears in Collections:[食品科學系] 博碩士論文

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