臺灣博碩士論文加值系統

去乙醯度95%之幾丁聚醣（DD95）在55℃，pH 5.2以纖維素酶水解10小時所得之中性水溶液具有最大抗菌活性，分離其中小分子幾丁聚醣（low-molecular-weight chitosan, LMWC）及幾丁寡醣。本研究旨在探討DD95、LMWC及幾丁寡醣對產氣莢膜梭菌（Clostridium perfringens）孢子萌發及菌體生長的影響，其次探討幾丁聚醣在豬肉香腸加工上應用之可行性。DD95對兩株產氣莢膜梭菌CCRC13019及CCRC10648菌體最低致死濃度（minimal lethal concentration, MLC）均為250 ppm，但CCRC13019較快死亡。1000 ppm LMWC可分別降低CCRC13019及CCRC10648分別約為1.5 Log CFU / mL及0.55 Log CFU / mL；幾丁寡醣則對此兩株菌幾乎無抗菌活性。250 ppm DD95對此兩菌株孢子幾乎無抗菌活性。同株菌其營養體菌體對DD95之敏感性與菌體表面之陰電荷與陽電荷比值成正相關，以對數生長期末期之菌體最敏感，其次為對數生長期中期，平穩期末期最不敏感。250 ppm DD95可使CCRC13019之D80及D100從40.98及4.64分鐘降低至39.21及3.26分鐘，其可使CCRC10648之D80及D100則從41.15及6.46分鐘降低至39.52及3.78分鐘。添加3000 ppm及6000 ppm DD95在豬肉香腸中抗菌活性明顯大於120 ppm亞硝酸鹽。在25℃儲藏48小時後，40 ~ 120 ppm亞硝酸鹽並不影響豬肉香腸中總生菌數，但可降低其內人為添加之產氣莢膜梭菌CCRC13019菌數約1.24 ~ 1.55 Log CFU / g。添加3000 ppm DD95與0 ~ 120 ppm亞硝酸鹽者，可使總生菌數及產氣莢膜梭菌降低約0.5 ~ 0.9 Log CFU / g及3.09 ~ 3.39 Log CFU / mL。不論是於25℃或4℃儲藏，亞硝酸鹽或（和）DD95之添加並不影響豬肉香腸色澤之L值及b值。但添加DD95可延緩a值之下降，且添加40 ppm亞硝酸鹽與3000 ppm DD95者，促使a值增加程度與120 ppm亞硝酸鹽之效果相近。亞硝酸鹽或（和）DD95之添加對豬肉香腸之剪切力及硬度均無顯著影響。DD95與亞硝酸鹽同時添加更可延緩揮發性鹽基態氮（volatile basic nitrogen；VBN）之生成，有助於延長肉品之保存期限。

The hydrolysate of chitosan with 95% degree of deacetylation ( DD95 ) by cellulase digestion at 55℃, pH 5.2 for 10 h was shown to have the strongest activity against Escherichia coli. A low - molecular - weight chitosan ( LMWC ) and chitooligosaccharides were separated from the this hydrolysate. The effects of DD95, LMWC and chitooligosaccharides on the spore germination and cell proliferation of Clostridium perfringens CCRC10648 and CCRC13019 were investigated. Then, the applicability of DD95 into pork sausage was evaluated . The minimal lethal concentration of DD95 for both strains was 250 ppm. However, the strain CCRC 13019 was more sensitive to chitosan than the strain CCRC 10648. After reaction at 37℃ for 16 h, LMWC at 1000 ppm decreased the counts of CCRC10648 and CCRC13019 by 0.5 Log CFU / mL and 1.5 Log CFU / mL, respectively; while the chitooligosaccharides had little activity against the 2 strains tested. DD95 at 250 ppm had little activity to effectively inhibit the spore germination of Clostridium perfringens 2 strains. There was a good positive correlation between the relative negative charge ratio of 2 strains tested, and the susceptibility of cells to DD95, with cells in late - log phase being the highest, followed by in mid - log phase and in late - stationary phase being the least. DD95 could decrease the heat resistance of the spores of both CCRC10648 and CCRC13019. The D80 and D100 for the strain CCRC 13019 changed from 40.98 and 4.64 min for the control to 39.21 and 3.26 min, respectively, in the presence of 250 of ppm DD95, respectively; while the D80 and D100 for CCRC10648 from 41.15 and 6.46 min for the control to 39.52 and 3.78 min, respectively, in the presence of 250 of ppm DD95. In pork sausage DD95 showed much stronger antibacterial activity than nitrite did. After storage at 25℃for 48 h, the total aerobic counts of pork sausage were not significantly different between the control and the nitrite added ( 40 ~ 120 ppm ) groups; while the counts for C. perfringens were decreased by 1.24 ~ 1.55 Log CFU / g for the groups with nitrite added. At the same condition, the addition of 3000 ppm of DD95 could decrease the total aerobic counts by 0.5 ~ 0.9 Log CFU / g, and the counts of C. perfringens by 3.09 ~ 3.39 Log CFU / mL. The addition of nitrite and ( or ) DD95 didn’t significantly change the L value and b value in pork sausage; while the addition of DD95 could significantly retard the decreasing rate of a value in pork sausage. DD95 could replace pent of nitrite added to sausage to retain similar “ a “ value for color enhancing effect. The addition of nitrite and ( or ) DD95 didn’t significantly change the shear force and hardness of pork sausage. The increasing of the volatile basic nitrogen of pork sausage was significantly retarded by the addition of nitrite and DD95.

目錄
圖表目錄................................................VI
中文摘要................................................XI
英文摘要.................... ................................XIII
壹、前言................................................1
貳、文獻整理 ...........................................3
2.1.幾丁質與幾丁聚醣......................................................3
2.1.1.幾丁質量 .........................................3
2.1.1.1. α型為斜方晶系（rhombic）......................3
2.1.1.2. β型為單晶斜體（monochinic）...................3
2.1.1.3. γ型...........................................4
2.2. 幾丁聚醣...........................................5
2.2.1. 製備幾丁聚醣.....................................6
2.2.1.1. 化學法.........................................6
2.2.1.2. 微生物法.......................................6
2.3. 幾丁質及幾丁聚醣之應用.............................6
2.4. N-乙醯基幾丁寡醣與幾丁寡醣.........................8
2.4.1. 製備N -乙醯基幾丁寡醣與幾丁寡醣..................8
2.4.1.1. 化學法.........................................8
2.4.1.2. 酵素法.........................................9
2.4.2. N-乙醯基幾丁寡醣與幾丁寡醣之生理活性.............9
2.4.2.1. N-乙醯基幾丁寡醣與幾丁寡醣之抗菌活性..........9
2.4.2.2. N-乙醯基幾丁寡醣與幾丁寡醣之免疫及抗癌活性....10
2.5. 幾丁聚醣之抗菌活性.................................10
2.5.1. 對細菌之抗菌活性.................................11
2.5.2. 對酵母菌之抗菌活性...............................11
2.5.3. 對黴菌之抗菌活性.................................12
2.5.4. 抗菌作用機制.....................................13
2.5.4.1. 真菌方面.......................................13
2.5.4.2. 細菌方面.......................................13
2.6. 產氣莢膜梭菌（Clostridium perfringens).............14
2.6.1. 簡介.............................................14
2.6.2. 菌種特性.........................................14
2.6.3. 產氣莢膜梭菌所導致之疾病.........................16
2.6.3.1. 氣性壞疽.......................................16
2.6.3.2. 食物中毒.......................................16
2.6.3.3. 急性壞疽性腸炎.................................17
2.6.4 產氣莢膜梭菌之致病因子...........................17
2.7. 幾丁聚醣在加工肉品上之應用........................18
2.7.1. 加工肉品中添加幾丁聚醣對菌相變化之影響..........18
2.7.2. 對加工肉品風味品質之影響.........................19
2.7.2.1. 脂質氧化方面...................................19
2.7.2.2. pH值及保水性方面...............................20
2.7.2.3. 在色澤方面.....................................20
參、材料設備與實驗方法..................................22
3.1. 實驗流程.......................................... 22
3.2. 食品原料...........................................23
3.3. 菌株 ...............................................23
3.4. 培養基.............................................23
3.5. 材料及化學藥品.....................................24
3.6. 器材及設備.........................................24
3.7.實驗方法............................................26
3.7.1. 幾丁聚醣之製備...................................26
3.7.1.1. 製備去乙醯度95 %之幾丁聚醣.....................26
3.7.1.2. 幾丁聚醣去乙醯之測定...........................26
3.7.2. 幾丁聚醣抗菌測試.................................27
3.7.2.1. Stock chitosan製備.............................27
3.7.2.2. 幾丁聚醣對產氣莢膜梭菌之抗菌試驗...............27
3.7.2.2.1. 菌株保存及活化...............................27
3.7.2.2.2. 孢子製備.....................................27
3.7.2.2.3. 對菌體之抗菌測試.............................28
3.8 幾丁聚醣對孢子之抗菌測試............................28
3.9. 纖維素酶水解幾丁聚醣...............................28
3.9.1. 以纖維素酶水解幾丁聚醣...........................28
3.9.2. 還原醣量的分析...................................29
3.9.2.1. Somogyi reagent I 製備.........................29
3.9.2.2. Somogyi reagent II製備........................29
3.9.2.3. Nelson reagent製備.............................29
3.9.3. 幾丁聚醣水解液抗菌活性之測試.....................30
3.9.4. 幾丁聚醣水解產物之分離...........................30
3.9.5. 幾丁聚醣水解產物對產氣莢膜梭菌之抗菌測試.........30
3.9.5.1. Stock LMWC製備.................................30
3.9.5.2. 抗菌測試.......................................31
3.9.6. 產氣莢膜桿菌表面電荷與對幾丁聚醣敏感關係之探討...31
3.9.6.1. 生長曲線測定..................................31
3.9.6.2. 不同菌齡菌株之收集............................31
3.9.6.3. 對不同菌齡之抗菌測試..........................32
3.9.6.4. 表面電荷之測定................................32
3.9.7. 幾丁聚醣對產氣莢膜梭菌孢子耐熱性之影響測試.......33
3.9.7.1. D value測定...................................33
3.9.7.2. 幾丁聚醣對孢子D value之影響....................33
3.10. 豬肉香腸中幾丁聚醣對產氣莢膜梭菌之抗菌測試........34
3.10.1. 豬肉香腸中幾丁聚醣對產氣莢膜梭菌生長之影響......34
3.10.1.1. 豬肉香腸之製備................................34
3.10.1.2. 儲藏試驗.......................................34
3.10.1.2.1. 總生菌數檢測（Total plate count, TPC).......34
3.10.1.2.2. 產氣莢膜梭菌菌數檢測........................34
3.10.2. 豬肉香腸中幾丁聚醣與亞硝酸鹽對產氣莢膜梭菌生長之影響 ...............................................35
3.10.2.1. 豬肉香腸之製備................................35
3.10.2.2. 儲存試驗 ......................................35
3.10.2.2.1. 總生菌數檢測（Total plate count, TPC).......35
3.10.2.2.2. 產氣莢膜梭菌菌數檢測........................36
3.10.2.2.3. 色澤測定....................................36
3.10.2.2.4. 物性測定....................................36
3.10.2.2.5. pH值測定....................................36
3.10.2.2.6. 揮發性鹽基態氮含量分析......................36
3.11. 統計分析..........................................37
肆、結果與討論..........................................38
一、幾丁聚醣（DD95）及幾丁寡醣對產氣莢膜梭菌的抗菌活性 ...............................................38
1.1. DD95對產氣莢膜梭菌營養體之抗菌效果................38
1.2. DD95水解液之抗菌活性探討..........................38
1.3. 低分子量幾丁聚醣及幾丁寡醣對產氣莢膜梭菌之抗菌效果 ...............................................39
1.4. 產氣莢膜梭菌對DD95敏感性與菌體表面電荷之關係......40
1.5. DD95對產氣莢膜梭菌孢子之抗菌效果..................42
1.6. DD95對產氣莢膜梭菌孢子耐熱性之影響................43
二、DD95於豬肉香腸之應用................................44
2.1 DD95對豬肉香腸中總生菌數之影響.....................44
2.2 DD95對豬肉香腸中產氣莢膜梭菌生長之影響.............46
2.3 DD95對豬肉香腸pH值之影響...........................48
2.4 DD95對豬肉香腸色澤之影響...........................48
2.5 DD95對豬肉香腸質地之影響...........................51
2.6 DD95對豬肉香腸揮發性鹽基態氮之影響.................52
伍、結論................................................55
陸、參考文獻............................................57

王綺芬。1989。蟹殼幾丁質產品理化性質測定及製備方法之研究。國
立臺灣大學食品科技研究所碩士論文。台北。台灣。
王有忠。1998。食品添加物。p.177-191。華香園出版社。台北。台灣。
方紹威。1990。幾丁直及幾丁聚醣在廢水處理、生化、食品和醫藥上之研究發展現況。藥物食品檢驗局調查研究報告，8：20-30。
江晃榮。1998。生體高分子（幾丁質、膠原蛋白）在食品上的應用。原料應用，150（6）：9-25。
宋宜真。2003。不同去乙醯度與濃度幾丁聚醣對法蘭克福香腸保存性之影響。東海大學畜產學研究所碩士論文。台中，台灣。
吳仁永。1999。以酵素生產抑菌性幾丁寡醣及其在牛乳保鮮上的應用，
國立臺灣海洋大學食品科學研究所碩士論文。基隆。台灣。
吳照男。2003。不同乙醯化程度幾丁聚醣珠粒固定脂肪酶之探討。國立臺灣海洋大學食品科學研究所碩士論文。基隆，台灣。
阮進惠，林翰良，羅淑珍。1997。幾丁聚醣水解物之連續式生產及其
抑菌作用。中國農業化學會誌，35: 596-611。
莊榮輝，宋賢一，蘇仲卿。1988。以蝦殼幾丁質固定鏈黴蛋白酶。中國農業化學會誌，26（3）：330-337。
張展榮。1996。剪力、超音波或兩種合併作用對幾丁聚醣物化性質之影響及其在水溶性幾丁聚醣製備上的應用。國立台灣海洋大學水產食品科學研究所碩士論文。基隆。台灣。
陸德源。2001。醫學微生物，第五版。p.732-766。人民衛生出版社。中國。
黃怡雯。2001a。以纖維素水解幾丁聚醣所得之水溶性產物對腸內細
菌組成之影響。國立台灣海洋大學食品科學研究所碩士論文。基隆。台灣。
黃珊萍。2001b。不同去乙醯度幾丁聚醣對腸內細菌生長之影響。國立台灣海洋大學食品科學系碩士學位論文。基隆。台灣。
彭仁信。2002。醋酸酐乙醯化反應之幾丁聚醣特性及幾丁聚醣酵素水
解產物對微生物生長之影響。國立台灣海洋大學食品科學系碩士學位論文。基隆。台灣。
詹淑玲。2002。由纖維素水解幾丁聚醣而得之小分子量幾丁聚醣與
幾丁寡醣對腸道菌群生長之影響。國立台灣海洋大學食品科學研究所碩士論文。基隆。台灣。
趙仁佑。2000。幾丁聚醣應用於中式減脂香腸之研究。靜宜大學食品營養研究所碩士論文。台中，台灣。
蔡政家。1996。以化學法由蝦殼製備幾丁質及幾丁質及幾丁聚醣之處
理條件與反應動力之探討。國立台灣海洋大學水產食品科學研究所碩士論文。基隆。台灣。
蔡孟佐。2003。幾丁聚醣及其水解產物對仙人掌桿菌的作用及於米飯保存上的應用。國立臺灣海洋大學食品科學研究所碩士論文。基隆，台灣。
蔡鳳城。1997。水產食品中水生產氣單胞菌分離株產毒之影響因子及其細胞毒素的分離純化與特性鑑定。國立台灣海洋大學水產食品科學研究所碩士論文。基隆。台灣。
蘇文慧。1998。幾丁聚醣之抑菌作用及其在食品保存上的應用。國立
台灣海洋大學食品科學系碩士學位論文。基隆。台灣。

Adam, D. 2002. Maximun shields: the assembly and function of the bacterial spore coat. Trends Microbiol. 10: 251-254.
Ahn, H. J., Kim, J. H., Jo, C., Lee, C. H., and Byun, M. W. 2002. Reduction of carcinogenic N-nitrosamines and residual nitrite in model system sausage by irradiation. J. Food Sci. 67(4):1370-1373.
Aiba, S. 1992. Studies on chitosan: 4. Lysozymic hydrolysis of partially
N-acetylated chitosans. Int. J. Biol. Macromol. 14: 225-228.
Aiba, S. 1993. Sudies on chitosan: 6. Relationship between N-acetyl group distribution pattern and chitinase digestibility of partially N-acetylated chitosans. Int. J. Biol. Macromol. 15: 241-245.
Aiba, S. 1994. Preparation of N-acetyl-chitooligosaccharides by hydrolysis of chitosan with chitinase follow by N-acetylation. Carbohydr. Res. 265:323-328.
Alderton, G., Ito, K. A., and Chen, J. K. 1976. Chemical manipulation of the heat resistance of Clostridium botulnium spores. Appl. Environ. Microbiol. 31:492-498.
Alderton, G., and Snell, N. 1969. Bacterial spores:Chemical sensitization to heat. Science. 168:1212-1213.
Allan, G. G., and Peyron, M. 1995. Molecular weight manipulation of
chitosan. I: Kinetics of depolymerization by nitrous acid. Carbohydr. Res. 277: 257-272.
Arneth, W. 2001. Chemistry of curing meat flavour. Fleischwirtschaft. 81(3):85-87.
Beaman, T. C. and Gerhardt, P. 1986. Heat resistance of bacterial spores
correlated with protoplast dehydration, mineralization, and thermal adaptation. Appl. Environ. Microbiol. 52: 1242-1246.

Beaman, T. C., Greenamyre, J. T., Corner, T. R., Pankratz, H. S., and Gerhardt, P. 1982. Bacterial spore heat resistance correlated with water content, wet density, and protoplast / sporoplast volume ratio. J. Bacteriol. 150:870-877.
Cassens, R. G. 1995. Use of sodium nitrite in cured meats today. Food Technol. 49(7):72-80, 115.
Chen, R. H., Chang, J. R., and Shyur, J. S. 1997. Effect of ultrasonic conditions and storage in acidic solution on changes in molecular weitht and polydisperfity of treated chitosan. Carbohydr. Res. 299:287-294.
Chui, V. W. D., Mok, K. W., Ng, C. Y., Luong, B. P., and Ma, K. K. 1996. Remove and recovery copper（II）,chromium（III）,and nickel（II）from solutions using crude shrimp chitin packed in small columns. Environ. Int. 22:463-468.
Chung,Y. C., Wang,H. L., Chen,Y. M., and Li, S. L. 2003. Effect of abiotic factors on the antibacterial activity of chitosan against waterborne pathogens. Bioresource Technol. 88:179–184.
Darmadji, P., and Izumimoto, M. 1994. Effect of chitosan in meat
preservation. Meat Sci. 38:243-254.
Domard, A., and Cartier, N. 1989. Glucosamine oligomers: 1. Preparation
and characterization. Int. J. Biol. Macromol. 11: 297-302.
Feng, J., Zhao, L., and Yu, Q. 2004. Receptor-mediated stimulatory effect of oligochitosan in macrophages. Biochem. Biophys. Res. Commun. 317: 414-420.
Ge, S. J., Bai, H., and Zhang, L. X. 1996. Trypsin immobilization on shrimp chitin with formaldehyde and its application to continous hydrolysis of casein. Biotech. Appl. Biochem. 24:1-5.
Ghaouth, E., Arul, A., Asselin, J., and Benhamou, N. 1992a. Antifungal activity of chitosan on post-harvest pathogens: Induction of morphological and cytological alterations in Rhizopus stolonifer. Myco. Res. 96: 769-779.
Ghaouth, E., A Arul., J., Grenier, J., and Asselin, A. 1992b. Antifungal
activity of chitosan on two pothogens of strawberry fruits. Phytopathology 82: 398-402.
Granum, P. E. 1990. Clostridium perfringens toxins involved in food poisoning. Int. J. Food Microbiol.10(2):101-11.
Gray, J. I., and Pearson, A. M. 1984. Cured meat flavor. Adv. Food Res. 29:1-86
Grener, A. B. G., and Ruiter. A. 2001. Prevention of Clostridium outgrowth in heated and hermetically sealed meat products by nitrite - a review. Eur. Food Res. Technol. 213:165-169.
Hadwiger, L. A., Kendra, D. F., Frislensky, B. W., and Wagoner, W. 1986. Chitosan both activates genes in plants and inhibits RNA synthesis in fungi. In: Chitin in Nature and Technology. pp. 209-214. Muzzarelli, R. A. A., Jeuniaux, C. and Goody. C. W. (Eds). Plenum Press. New York. U.S.A.
Hasegawa, M., Isogai, A., and Onabe, F. 1993. Preparation of
low-molecular-weight chitosan using phosphoric acid. Carbohydr. Polym. 20: 279-283.
Hirano, S. 1989. Production and application of chitin and chitosan in Janpan. In: Proceeding of the forth international con ference on chitin and chitosan. pp. 37-43. Skjak-Braek, G., Anthonsen, T., and Sandford, P. (Eds) Elsevier Applied Sci. Publishers, London.


Hirano, S., and Nagno, N. 1989. Effects of chitosan, pectic acid, lysozyme and chitinase on the growth of several phytopathogens. Agric. Biol. Chem. 43: 33-37.
Hirano, S. 1997. Application of chitin and chitosan in the ecological and environmental fields. In: Application of chitin and chitosan. pp. 31-54. Goosen, M. F. A. (Eds.) Technomic Publishers. Tottori.
Huang, Y. C., Su, L. H., Wu, T. L., Liu, C. E., Young, T. G., Chen, P. Y., Hseuh, P. R., and Lin, T. Y. 2004. Molecular epidemiology of clinical isolates of methicillin-resistant Staphylococcus aureus in Taiwan. J. Clinical Microbiol. 42(1):307-310.
Ingram, M.. 1977. Problems in the use of nitrite. Fleischwirtschaft 57(2):
211-217.
Ispolatovskaya, M. V. 1973. Structure and function of Clostridium perfringens toxin. Vestnik Akademii Meditsinskikh Nauk SSSR. 11:83-9.
Jha, D. F., Iyenganr, L., and Prabhakara Rao, A. V. S. 1988. Removal of cadmium using chitosan. J. Env. Eng. 11:962-974.
Jeon, Y. J., and Kim, S. K. 2000. Production of chitooligosaccharides using an ultrafiltration membrane reactor and their antibacterial activity. Carbohydr. Polym. 41(2): 133-141.
Joen, Y. J., Park, P. J., and Kim, S. K., 2000. Preparation of chitin and chitosan oligomers and teir application in physiological functional foods. Food Rev. Int. 16(2):159-176.
Justin, N., Eaton, J., Naylor, C., Howells, A., Moss, D., Titball, R., and Basak, A. 1999 . Structure function studies of Clostridium perfringens alpha-toxin; A gas gangrene causing protein. J. Chem. Society Pakistan . 21(3):239-247.

Kendra, D. F., and Hadwiger, L. A. 1984. Characterization of the smallest
chitosan oligomer that is maximally antifungal to Fusarium solani andelicits pisatin formation in Pisum sativum. Exp. Mycol. 8: 276-281.
Kim, Y. K., and Sakano, Y. 1996. Analyses of reducing sugars on a thin-layer chromatographic plate with modified Somogyi and Nelson reagents, and with copper bicinchoninate. Biosci. Biotechnol. Biochem. 60(4):594-7.
Knorr, D. 1984. Use of chitinous polymers in food. Food Technol. 45:
114-122.
Knorr, D., Wampler, J. P., and Teutonico, R. A. 1985. Formation of pyrazine by chitin pyrolysis. J. Food Sci. 50:1762-1763.
Kobayashi, M., Watanabe, T., Suzuki S., and Suzuki M. 1990. Effect of
N-acetyl-chitohexaose against Candida albicans infection of
tumor-bearing mice. Microbiol. Immunol. 34: 413-426.
Kono, M., Matsuy, T., Shimizu, C., and Koga, D. 1990. Purifications and
some properties of chitinase from the liver of a prawn, Penaeus japonicus. Agric. Biol. Chem. 54: 2145-2147.
Labbe, R. G., and Duncan, C. L. 1974. Sporulation and enterotoxin production by Clostridium perfringens type A under condition of pH and temperature. Can. J. Microbiol. 20:1493-1501.
Lang, E. R., Kienzle-Sterzr, C. A., Rodriiquez-Sanchez, D., and Rha, C. K. 1982. Rheological behavior of a typical randon coil polyelectrolyte: Chitosan. In: Chitin and Chitosan. pp. 34-38. Hirano, S., and Tokura, S. (Eds.) The Japanese Society, Tokyo. Japan..


Lepri, L and Desideri, P. G. 1978. Separation and identification of water-soluble food dyes by ion-exchange and soap thin-layer chromatography. J. Chromatog. 161:279-286.
Leuba, J. L., and Stossel, P. 1986. Chitosan and other polyamines: antifungal activity and interaction with biological membranes. In: Chitin in Nature and Technology, pp. 215-221, Muzzarelli, R., Jeuniarx, C., and Gooday, G. (Eds), Plenum Press, N. Y.
Mahoney, A. W., Hendricks, D. G., Gillett, T. A. Buck, D. R., and Miller, C. G. 1979. Effect of sodium nitrite on the bioavailability of meat iron for the anemic rat. J. Nutr. 109(12):2182-9.
McClane, B.A. 2000. The action, genetics, and synthesis of Clostridium perfringens enterotoxin. In: Microbial Food Diseases: Mechanisms of Pathogenesis. pp. 247-271. Cary, J. W., Linz, J. E., and Bhatnagar, D (Eds.), Landcaster, Pennsylvania: Technomic Publishing Company, Inc.
Minke, R. and Blackwell, J. 1978. The structure of α-chitin. J. Mol. Biol.120: 167-181.
Miwa, N., Masuda, T., Kwamura, A., Terai, K., and Akiyama., M. 2002. Survial and growth of enterotoxin-positive and enterotoxin -negative Clostridium perfringens in laboratory media. Int. J. Food Microbiol. 72:233-238.
Mitani, T., Moriyama, A., and Ishii, H. 1992. Heavy metal uptake by swollen chitosan beads. Biosci. Biotech. Biochem. 56:985-986.
Muhammed, S. I., Morrison, S. M.,and Boyd, W. L. 1975. Nutritional requirements for growth and sporulation of Clostridium perfringens. J. Appl. Bacteriol. 38(3):245-53.


Muraki E., Yaku F., and Kojima H. 1993. Preparation and crystallization of D-glucosamine oligosaccharides with DP 6-8. Carbohydr. Res. 239:227-237.
Murao, S., Kawada, T., Itoh, H., Oyama, H., and Shin, T. 1992. Purification and characterization of a novel type of chitinase from Vibrio alginolyticus TK-22. Biosci. Biotechol. Biochem. 56: 368-369.
Murata, T., and Usui, T. 1997. Preapration of oligosaccharides units library and its utilization. Biosci. Biotech. Biochem. 61: 1059-1066.
Muzzarelli, R. A. A. 1977. Chitin. Pergamon, Oxford, UK.
Nakashio, S., and Gerhardt, P. 1985. Protoplast dehydration correlated with heat resistance of bacterial spores. J. Bacteriol. 162:571-578.
Orozco, A. S., Zhou, X., and Filler, S. G. 2000. Mechanisms of the proinflammatory response of endothelial cells to Candida albicans infection. Infection and Immunity. 68(3):1134-1141.
Park, G. B., Lee, H. G., Kim, J. S., Kim, Y. J., Park, T. S., Shin, T. S., and Lee, J. I. 1994. Effect of sodium nitrite levels and curing temperatures on preservation and production of antihygienic chemicals of cured pork. Han'guk Ch'uksan Hakhoechi. 36(3): 330-339.
Pantaleone, D., M., Yaipani, M., and Scollar, M. 1992. Unusual susceptibility of chitosan to enzymic hydrolysis. Carbohydr. Res. 237: 235.
Papineau, A. M., Hoover, D. G., Knorr, D., and Farkas, D. F. 1991.
Antimicrobial effect of water-soluble chitosans with high hydrostatic pressure. J. Food Biotechnol. 5: 45-57.
Rane, K. D., and Hoover, D. G. 1993. Production of chitosan by fungi. Food Biotech. 7: 11-13.
Reddy, D., Lancaster, J. R., and Cornforth, D. P. 1983. Nitrite inhibition of Clostridium botulinum: Electron spin resonance detection of iron-nitric oxide complexes. Science 221:769-770.
Roberts, G. A. F. 1992. Chitin chemistry. The MacMillan Press Ltd, London.
Roberts, G. A, F. 1995. Structure-property relationships in chitin and chitosan. In: Chitin and Chitosan-The Versatile Environmentally Friendly moger material. pp. 95-108. Zakaria, M. D., Muda, W. M. W., and Abdullah, M. P. (Eds.). Penerbit Universiti Kebangaan. Malaysia Bangi.
Rolfe, R. D., Hentges, D. J., Campbell, B. J., and Barrett, J. T. 1978. Factors related to the oxygen tolerance of anaerobic bacteria. Appl. Environ. Microbiol. 36(2):306-13.
Roy, R. J., Busta, F. F., and Thompson, D. R. 1981. Thermal inactivation of Clostridium perfringens after growth at several constant and linearly rising temperatures. J. Food Sci. 46:1586-1591.
Sannan, T., Kurita, K., and Iwakura, Y. 1976. Studies on chitin 2: Effect of deacetylation on solubility. Makromol. Chem. 177:3589-3600.
Sanford, P. A., and Hutchings, G. P. 1987. Industrial polysaccharides. In: Genetic Engineering, Structure/Property Relation and Application. pp. 363-375. Yalpani, M. (Ed) Elsevier. Amsterdam.
Sashia, H., and Shigemasa, Y. 1999. Chemical modification of chitin and chitosan 2: preparation and water soluble property of N-acylated or N-alkylated partially deacetylated chitins. Carbohydr. Poly. 39:127-138.
Shahidi, F., Arachchi, J. K. V., and Jeon, Y. J. 1999. Food applications of deacetylated chitin and chitosans. Food Sci. Technol. 10:37-51.
Simpson, B. K., Gagne, N., Ashie, I. N. A., and Noroozu E. 1997. Utilization of chitosan for preservation of raw shrimp (Pandalus borealis). Food Biotechnol. 11: 25-44.
Soto-Peralta, N. V., Muller, H., and Knorr, D. 1989. Effect of chitosan treatment on the clarity and color of apple juice. J. Food Sci. 54:495-496.
Strong, D. H., Duncan, C. L., and Peran, G. 1971.Clostridium perfringens type A food poisoning. II. Response of the rabbit ileum as an indication of enteropathogenicity of straims of Clostridium perfringens in human beings. Infect. Immun. 3:171-178.
Suzuki, K., Okawa, Y., Suzuki, S., and Suzuki, M. 1985. Enchancing effects of N-acetyl-chitooligosaccharides on the active oxygenation and microbiocial activities of peritoneal exudates cell in mice. Chem. Pharm. Bull. 33:886-892.
Suzuki, K., Midami, T., Okawa, Y., Tokoro, A., Suzuki, S., and Suzuki, M. 1986a.Antitumer effects of hexa-N-acetyl-chitooligosaccharides chitohexise. Carbohydr. Res. 151:403-408.
Suzuki, K., Tokoro, A., Okawa, Y., Suzuki, S., and Suzuki, M. 1986 b. Effects of N-acetyl-chitooligosaccharides on activation of phagocytes. Microbiol. Immunol. 30:777-785.
Titball, R. W., Naylor, C. E., and Basak, A. K. 1999. The Clostridium perfringens α-toxin. Anaerobe 5:61-64.
Tompkin, B. L., Christiansen, L. N.,and Shaparis, A. B. 1978. Enchancing nitrite inhibition of Clostridium botulinum with isoascorbate in perishable canned cured meat. Appl. Environ. Microbiol. 35:59-61.



Tokoro, A., Kobayishi, M., Tatewaki, N., Susuki, K., Okawa, Y., Mikami, T., Susuki, S., and Susuki, M. 1989. Protective effect of N-acetyl-chitooligosaccharides on Listeria monocytogens infection in mice. Microbiol. Immunol. 33:357-367.
Tsai, G. J., and Su, W. H. 1999. Antibacterial activity of shrimp chitosan against Escherichia coli. J. Food Prot. 62:239-243.
Tsai, G. J., Su W. H., Chen H. C., and Pan C. L. 2002. Antimicrobial
activity of shrimp chitin and chitosan from different treatments and application to fish preservation. Fish. Sci. 68: 170-177.
Tsai, G. J., Wu, Z. Y., and Su, W. H. 2000. Antibacterial activity of a chitooligosaccharide mixture prepared by cellulase digestion of shrimp chitosan and its application to milk preservation. J. Food Prot. 63:747-752.
Tsai, G. J., Zhang, S. L., and Shien, P. L. 2004. Antimicrobial activity of low-molecular-weight chitosan obtained from cellulose digestion of chitosan. J. Food Prot. 67:396-398.
Tsen, H. Y., Hu, H. H., Lin, J. S., Huang, C. H., and Wang, T. K. 2000. Analysis of the Salmonella typhimurium isolates from food-poisoning cases by molecular subtyping methods. Food Microbiol. 17(2):143-152.
Usui, T., Hayashi, Y., Nanjo, F., Sakai, K., and Ishido, Y. 1987.
Transglycosylation reaction of a chitinase purified from Nocardia orientalis. Biochim. Biophys. Acta. 923: 302-309.
Usui, T., Matsui, H., and Isobe, K. 1990. Enzyme synthesis of useful
chito-oligosaccharides utilizing transglycosylation by chitinolytic enzymes in a buffer containing ammonium. Carbohydr. Res. 203:65-77.

Uchida, Y., Izum, M., and Ohtakara, A. 1989. Preparation of chitosan oligomers with purified chitosanase and its application. pp. 373-382. Skjak-Break, G., Anthonsen, T., and Sandford (Eds), Chitin and chitosan. Elsevier, London.
Wang, G. H. 1992. Inhibition and inactivation of five speceies of foodborne pathogens by chitosan. J. Food Prot. 55(11): 916-919.
Wang, W., Bo, S., Li, S., and W. Qin. 1991. Determinartion of the
Mark-Houwink equation for chitosans with different degrees of
deacetylation. Int. J. Biol. Macromol. 13: 281-286.
Weiss, K. F., and Strong, D. H. 1967. Some properties of heat-resistant and heat-sensitive strains of Clostridium perfringens. I. Heat resistance and toxigenicity. J. Bacteriol. 93:21-26.
Williams O. B. and Robertson W. J. 1954. Studies on heat resistance. VI.
Effect of temperature of incubation at which formed on heat
resistance of aerobic thermophilic spores. J. Bacteriol. 67: 377-378.
Wirth, F. 1985. Curing today. Schweizer Archiv fuer Tierheilkunde. 127(2):109-27.
Wong, H. C., Liu, S. H., Ku, L. W., Lee, I. Y., Wang, T. K., Lee, Y. S., Lee, C. L., Kuo, L. P., Shih, D. Y. C. 2000. Characterization of Vibrio parahaemolyticus isolates obtained from foodborne illness outbreaks during 1992 through 1995 in Taiwan. J. Food Prot. 63(7):900-906.
Xie, W., Xu, P., Wang, W., and Liu, Q. 2002. Preparation of water-soluble chitosan derivatives and their antibacterial activity. J. Appl. Polymer Sci. 85:1357-1361


Young, D. H., and Kauss, H. 1983. Relaease of calcium from suspension -cultured Glycine max and Phaseolus vulgaris cells by chitosan, other polycations, and polyamines in relation to effects on membrane permeability. Plant Physiol. 73: 698-702.