菌株 TKU033 係以烏賊軟骨為唯一碳/氮源，篩選至台灣三芝土壤經鑑定為 Bacillus cereus之幾丁聚醣酶生產菌。幾丁聚醣酶之較適培養條件為，以含有1.5% 烏賊軟骨粉、0.1%K2HPO4、0.05%MgSO4．7H2O之50mL液態培養基於 37℃搖瓶(150 rpm)培養2天，所得發酵上清液經硫酸銨沉澱、DEAE-Sepharose 及Macro-Prep DEAE Cartidge離子交換樹脂等步驟，純化出一種幾丁聚醣酶。此幾丁聚醣酶之分子量經SDS-PAGE測為43 kDa ，最適反應pH、最適溫度、pH安定性及熱安定性分別為 pH 5、50℃、pH5-7及<40℃，其活性會受 Cu2+ 、Mn2+ 和EDTA所抑制。
      較適培養條件所製得B.cereus TKU033粗酵素液，幾丁聚醣酶水解水溶性幾丁聚醣，所得上清液添加於乳酸菌Lactobacillus paracasei 12193及 Lactobacillus kefri 14011的MRS液態培養基，可分別促進乳酸菌生長達141%及116% 之影響。

The chitosanase-producing strain TKU033 was isolated from soil in Taiwan, Sanzhi with squid pen powder as the sole carbon/nitrogen source and identified as Bacillus cereus. A chitosanase was purified from the culture supernatant by ammonium sulfate precipitation, DEAE-Sepharose and Macro-Prep DEAE Cartridge. The molecular mass of chitosanase determined by SDS-PAGE was approximately 43 kDa. The optimun pH , optimun temperature, pH stability, and thermal stability of chitosanase were pH 5,
50℃, pH5-7 and <40℃. The chitosanase activity was inhibited by Cu2+ , Mn2+ and EDTA.
   The supernatant of the crude enzyme from B. cereus TKU033 hydrolyzed water soluble chitosan exhibited activity of enhancing growth for L. paracasei 12193 up to 141% and L.kefri 14011 up to 116%.

目錄
頁次
中文摘要	I
英文摘要	II
目錄	III
圖目錄	VI
表目錄	VII

第一章 緒論	1
第二章 文獻回顧	2
2.1 Bacillus cereus 之簡介	2
2.2幾丁寡與幾丁聚醣	3
2.3幾丁寡醣	6
2.4 幾丁質酶與幾丁聚醣酶	7
2.5 蛋白酶	8
2.6 益生質(prebiotics)	8
2.7 幾丁類物質吸附色素	9
第三章 材料與方法	10
3.1 實驗菌株	10
3.2 實驗材料	10
3.3 實驗儀器	12
3.4 酵素生產菌株之篩選	12
3.5 幾丁聚醣酶活性測定	13
3.6 蛋白酶活性測定	13
3.7 幾丁質酶活性測定	14
3.8 較適培養條件探討	14
3.8.1 培養條件	14
3.8.2 碳/氮源濃度	14
3.9 酵素之分離純化	15
3.9.1 粗酵素液之製備	15
3.9.2 離子交換樹脂層析	15
3.10 蛋白質電泳分析	16
3.11 酵素之特性分析	16
3.11.1 酵素最適反應溫度	16
3.11.2 酵素熱安定性	16
3.11.3 酵素最適反應 pH	16
3.11.4 酵素 pH 安定性	17
3.11.5 金屬離子及化學藥品對酵素活性之影響	17
3.11.6 界面活性劑對酵素活性之影響	17
3.11.7 酵素之基質特異性	18
3.12 幾丁聚醣酶水解基質及寡醣分析	18
3.12.1 基質之水解	18
3.12.2 N-乙醯幾丁寡醣之製備	18
3.12.3 N-乙醯幾丁寡醣之組成分析	19
3.13 還原糖之測量	19
3.14 總糖量之測定 (H2SO4-Phenol 方法)	20
3.15 MALDI-TOF-MS	20
3.16 經 B. cereus TKU033粗酵素液水解基質促進乳酸菌生長	21
3.17 幾丁類物質吸附色素	21
第四章 結果與討論	22
4.1 幾丁聚醣酶與蛋白酶生產菌之篩選	22
4.1.1 菌株TKU033 之鑑定	22
4.2 酵素較適生產條件探討	22
4.2.1 培養液體基	23
4.2.2 不同碳/氮源濃度	23
4.2.3 較適培養條件探討結果	23
4.3 幾丁聚醣酶及蛋白酶之純化	25
4.3.1 粗酵素液之製備	25
4.3.2 離子交換樹脂層析	25
4.3.3 綜合結果	26
4.4 幾丁聚醣酶分子量之測定	27
4.4.1 SDS-PAGE	27
4.4.2 綜合結果	27
4.5 幾丁聚醣酶之特性分析	27
4.5.1 幾丁聚醣酶之最適反應溫度及熱安定性	27
4.5.2 幾丁聚醣酶之最適反應pH及pH安定性	28
4.5.3 金屬離子及化學藥品對幾丁聚醣酶之影響	28
4.5.4 界面活性劑對幾丁聚醣酶活性之影響	29
4.5.5 幾丁聚醣酶之基質特異性	30
4.6 水解基質之探討	30
4.6.1 還原糖與總糖含量之分析	30
4.6.2 利用 HPLC 分析幾丁寡醣組成	31
4.6.3 利用 MALDI-TOF-MS 進行幾丁寡醣組成分析	31
4.7 促進乳酸菌生長	32
4.8 幾丁類物質吸附色素	33
4.9 含硼化合物對三種不同幾丁聚醣酶活性之影響	33
第五章 結論	62
參考文獻	63

圖目錄
圖 2.1 幾丁質、幾丁聚醣及纖維素之結構............................................................... 4
圖 4.1 16SrDNA 部分鹼基序列分析及API試驗結果 ........................................... 34
圖 4.2 培養體積對 B. cereus TKU033生產幾丁聚醣酶及蛋白酶之影響 ........... 35
圖 4.3 不同碳/氮源對B. cereus TKU033生產幾丁聚醣酶之影響 ....................... 36
圖 4.4不同碳/氮源對B. cereus TKU033生產蛋白酶之影響 ................................ 37
圖 4.5 B. cereus TKU033 幾丁聚醣酶之 DEAE-Sepharose CL-6B 層析圖譜 ..... 38
圖 4.6 B. cereus TKU033 幾丁聚醣酶之Macro-Prep DEAE Cartridge 層析圖譜 38
圖 4.7 B. cereus TKU033 幾丁聚醣酶之SDS-PAGE 之分子量分析 ................... 39
圖 4.8 幾丁聚醣酶之 (A) 最適反應溫度 (B) 熱安定性 ...................................... 40
圖 4.9 幾丁聚醣酶之 (A) 最適反應 pH (B) pH 安定性 ...................................... 41
圖 4.10 銅離子對幾丁聚醣酶活性影響................................................................... 42
圖 4.11 水溶性幾丁聚醣 (WSC) 中添加B. cereus TKU033粗酵素液水解不同時
間所得總醣及還原糖含量.......................................................................................... 43
圖 4.12 WSC 經B. cereus TKU033 粗酵素液水解不同時間所得幾丁寡醣HPLC 分析圖(A) standard (1-6 醣) (B) 2 hr (C) 4hr (D) 6hr ............................................... 44
圖 4.13 WSC 經B. cereus TKU033 粗酵素液水解不同時間所得幾丁寡醣HPLC 分析圖 (A) standard (1-6 醣) (B) 12 hr (C) 24hr (D) 48hr ....................................... 45
圖 4.14 水溶性幾丁聚醣經 B. cereus TKU033 粗酵素液水解所得幾丁寡醣之 MALDI-TOF-MS分析圖 ........................................................................................... 46
圖4.15 水溶性幾丁寡醣經 B. cereus TKU033 粗酵素液水解所得幾丁寡醣添加於(A) L.paracasei 12193 (B) L.kefir 14011 生長之影響 .......................................... 49

表目錄
表2.1幾丁質與幾丁聚醣之應用................................................................................. 5
表 4.1篩選菌株之蛋白酶及幾丁聚醣活性表.......................................................... 50
表4.2 B. cereus TKU033 生產酵素之較適條件 ...................................................... 51
表4.3幾丁聚醣酶./蛋白酶生產菌酵素活性之比較 ................................................ 51
表4.4微生物來源之幾丁聚醣特性比較................................................................... 52
表4.5 B. cereus TKU033 幾丁聚醣酶純化總表 ...................................................... 54
表4.6金屬離子及化學藥品對幾丁聚醣酶活性之影響........................................... 55
表4.7界面活性劑對幾丁聚醣酶活性之影響........................................................... 56
表4.8 B. cereus TKU033 幾丁聚醣酶之基質特異性 .............................................. 57
表4.9水溶性幾丁聚醣經B. cereus TKU033粗酵素液水解不同時間之MALDI-TOF-MS 幾丁寡醣組成分析 ...................................................................... 58
表4.10 水溶性幾丁聚醣經B. cereus TKU033粗酵素液水解不同時間所得幾丁寡醣添加於乳酸菌 (L.paracasei 12193和L.kefir 14011) 生長之影響 ..................... 59
表4.11發酵及未發酵烏賊軟骨粉對色素之吸附率................................................. 60
表4.12含硼化合物對幾丁聚醣酶活性之影響......................................................... 61

Aiba S (1994) Preparation of N-acetylchitooligosaccharides by hydrolysis of chitosan with chitinase followed by N-acetylation. Carbohydrate Research, 265:323-328.
Alves SP, Brum DM, Branco de Andrade EC, and Pereira Netto AD (2008) Determination of synthetic dyes in selected foodstuffs by high performance liquid chromatography with UV-DAD detection. Food Chemistry, 107: 489-496.
Aam BB, Heggset EB, Norberg AL, SOrlie M, Varum KM, and Eijsink VGH (2010) Production of chitooligosaccharides and their potential applications in medicine. Marines Drugs, 8:1482-1517.
Bernfeld P (1955)Amylase, α and β. Methods in Enzymology, 1:149-158.
Banik RM and Prakash M (2004) Laundry detergent compatibility of the alkaline protease from Bacillus cereus. Microbiology Research, 159: 135-140.
Bosscher D (2009) Fructan prebiotics derived from inulin in: D. Charalampopoulos, R.A. Rastall (Eds.), Prebiotics and probiotics, Science and technology, Springer, UK, pp. 164-204.
Chang WT, Chen YC, and Jao CL (2007) Antifungal activity and enhancement of plant growth by Bacillus cereus grown on shellfish chitin wastes. Bioresource Technology, 98: 1224-1230.
Chen H, Liu LJ, Zhu JJ, Xu B, and Li R (2010) Effects of soybean oligosaccharides on blood lipid, glucose levels and antioxidant enzymes activity in high fat rats. Food Chemistry, 119: 1633-1636.
Cohen-Kupiec R and Chet I (1998) The molecular biology of chitin digestion. Current Opinion in Biotechnology, 9: 270-277.
Dubois M, Gilles KA, Hamilton JK, Rebers PA, and Smith F (1956) 
Colorimetric method for  determination of sugars and related 
substances. Analytical Chemistry, 28:350-356.
EI-Shafei AM, Fouda M, Knittel D, and Schollmeyer E (2008) Antibacterial activity of cationically modified cotton fabric with carboxymethyl chitosan. Journal of Applied Polymer Science, 110: 1289-1296.
Fitches E, Wilkinson H, Bell H, Bown DP, Gatehouse JA, and Edwards 
JP (2004) Cloning expression and functional characterization of chitinase from larvae of tomato moth (Lacanobia oleracea) : a demonstration of the insecticidal activity of insect chitinase. Insect Biochemistry and Molecular Biology, 34: 1037-1050.
Franca EF, Lins RD, Freitas LC, and Straatsma TP (2008) Characterization of chitin and chitosan molecular Structure in aqueous solution. Journal of Chemical Theory and Computation, 4: 2141-2149.
Ghaouth AE, Arul J, Grenier J, and Asselin A (1992) Effect of chitosan 
and other polyions on chitin deacetylase in Rhizopus stolinifer. 
Experimental Mycology, 16: 173-177.
Gao XA, Ju WT, Jung WJ, and Park RD (2008) Purification and 
characterization of chitosanases from Bacillus cereus D-11. Carbohydrate Polymers, 72: 513-520.
Ge L, Zhang H, Chen K, Ma L, and Xu Z (2010) Effect of chitin on the antagonistic activity of Rhodotorula glutinis against Botrytis cinerea in strawberries and the possible mechanisms involved. Food Chemistry, 120: 490-495.
He H, Silo-Suh LA, Handelsman J, and Clardy J (1994) Zwittermicin A, 
an antifungal and plant protection agent from Bacillus cereus. Tetrahedron Letters, 35: 2499-2502.
Helisto P, Aktuganov G, Galimzianova N, Melentjev A, and Korpela T (2001) Lytic enzyme complex of an antagonistic Bacillus sp. X-b: isolation and purification of components. Journal of Chromatography B, 758:197-205.
Harish Prashanth KV and Tharanathan RN (2007) Chitin/chitosan: 
modifications and their unlimited application potential- an overview. Trends in Food Science & Technology, 18: 117-311.
Imoto T and Yagishita K (1971) A simple activity measurement by 
lysozyme. Agricultural and Biological Chemistry, 35: 1154-1156.
Jeon YJ, Park PJ, and Kim SK (2001) Antimicrobial effect of 
chitooligosaccharides produced by bioreactor. Carbohydrate Polymers, 44: 71-76.
Janek T, Łukaszewicz M, Rezanka T, and Krasowska A (2010) Isolation 
and characterization of two new lipopeptide biosurfactants produced by Pseudomonas fluorescens BD5 isolated from water from the Arctic Archipelago of Svalbard. Bioresource Technology, 101:6118-6123.
Kumar MNVR (2000) A review of chitin and chitosan applications. 
Reactive and Functional Polymers, 46: 1-27.


Kim HB and An CS (2002) Differential expression patterns of an acidic 
chitinase and a basic chitinase in the root nodule of Elaeagnus 
umbellate. Molecular Plant-Microbe Interactions: MPMI, 15: 209-215.
Kurita K (2006) Chitin and Chitosan: Functional biopolymers from marine crustaceans. Marine Biotechnology, 8: 203- 226.
Khoushab F and Yamabhai M (2010) Chitin research revisited. Marine Drugs, 8:1988-2012.
Kong CS, Kim JA, Ahn B, Byun HG, and Kim SK (2010) Carboxymethylations of chitosan and chitin inhibit MMP expression and ROS scavenging in human fibrosarcoma cells. Process Biochemistry, 45 :179-186.
Laemmli UK (1970) Cleavage of structural proteins during the assembly 
of the head of bacteriophage T4. Nature, 227:680-685.
Langlands SJ, Hopkins MJ, Coleman N, and Cummings JH (2004) Prebiotic carbohydrates modify the mucosa associated microflora of the human large bowel. Gut, 53:1610-1616.
Lee YS, Yoo JS, Chung SY, Lee YC, Cho YS, and Choi YL (2006) Cloning, purification, and characterization of chitosanase from Bacillus sp. DAU101. Applied Microbiology and Biotechnology, 73: 113-121.
Liu BL, Kao PM, Tzeng YM, and Feng KC (2003) Production of 
chitinase from Verticillium lecanii F091 using submerged fermentation. 
Enzyme and Microbial Technology, 33: 410-415.
Li TC, Sun XJ, Bi Y, Ge YH, and Wang Y (2009) Antifungal activity of 
chitosan on Fusarium sulphureum in relation to dry rot of potato tuber. Agricultural Sciences in China, 8: 597-604.
Liang TW, Chen YJ, Yen YH, and Wang SL (2007) The antitumor 
activity of the hydrolysates of chitinous materials hydrolyzed by crude enzyme from Bacillus amyloliquefaciens V656. Process Biochemistry, 42: 527-534.
Liang TW, Wu YY, Huang TY, Wang CY, Yen YH, Liu CP, Chen YC, 
and Wang SL (2010a) Conversion of squid pen by a novel strain Lactobacillus paracasei subsp. paracasei TKU010, and its application in antimicrobial and antioxidants activity. Joural of General and Applied Microbiology, 56:481-489.




Liang TW, Kuo YH, Wu PC, Wang CL, Dzung NA, and Wang SL 
(2010b) Purification and characterization of a chitosanase and a protease by conversion of shrimp shell wastes fermented by Serratia marcescens subsp. sakuensis TKU019. Journal of the Chinese Chemical Society, 57:857-863.
Liang TW, Hsieh JL, and Wang SL (2012) Production and purification 
of a protease, a chitosanase, and chitin oligosaccharides by Bacillus cereus TKU022 fermentation.Carbohydrate Research, 362:38-46.
Miller GL (1959) Use of dinitrosalicylic acid reagent for determination 
of reducing sugar. Analytical Chemistry, 31:426-428.
Milner JL, Silo-Suh L, Lee JC, He H, Clardy J, and Handelsman J (1996) Production of kanosamine by Bacillus cereus UW85. Applied and Environmental Microbiology, 62: 3061-3065.
Macfarlane GT, Cummings JH (2006) Review article: Prebiotics in the gastrointestinal tract. Alimentary Pharmacology and Therapeutics, 24: 701-714.
Ma C, Ni X, Chi Z, Ma L, and Gao L (2007) Purification and 
characterization of a 36-kDa chitinase from Bacillus thuringiensis 
subsp. colmeri, anf its biocontrol potential. Enzyme and Microbial Technology, 45: 252-256.
McCann D, Barrett A, Cooper A, Crumpler D, Dalen L, Grimshaw K, Kitchin E, and Stevenson J (2007) Food additives and hyperactive behaviour in 3-year-old and 8/9-year-old children in the community: a randomised, double-blinded, placebo-controlled trial. The Lancet, 370: 1560-1567.
Nagahama H, Kashiki T, New N, Jayakumar R, Furuike T, and Tamura H (2008) Preparation of biodegradable chitin/gelatin membranes with GlcNAc for tissue engineering applications. Carbohydrate Polymers, 73: 456-463.
Neri DFM, Balcao VM, Costa RS, Rocha ICAP, Ferreira EMEC, Torres 
DPM, Rodrigues LRM, Jr LBC, and Teixeira JA (2009) Galacto-oligosaccharides production during lactose hydrolysis by free Aspergillus oryzae β-galactosidase and immobilized on magnetic polysiloxane-polyvinyl alcohol. Food Chemistry, 115: 92-99.
Pool-Zobel BL (2005) Inulin-type fructans and reduction in colon cancer risk: review of experimental and human data. British Journal of Nutrition, 93:73-90.

Poulsen PHB, Moller J, and Magid J (2008) Determination of a relationship between chitinase activity and microbial diversity in chitin amended compost. Bioresource Technology, 99: 4355-4359.
Pacheco N, Garnika-Gonzalez M, Ramirez JY, Flores-Albino B, Gimeno M, Barzana E, and Shirai K (2009) Effect of temperature on chitin and astaxanthin recoveries from shrimp waste using lactic acid bacteria. Bioresource Technology, 100: 2849-2854.
Park JK, Chung MJ, Choi HN, and Park YI (2011) Effects of the molecular weight and the degree of deacetylation of chitosan oligosaccharides on antitumor activity. Molecular Science, 12: 266-277.
Robinson-Lora MA and Brennan RA (2009) The use of crab-shell chitin for biological denitrification: Batch and column tests. Bioresource Technology, 100: 534-541.
Steer T, Carpenter H, Tuohy K, and Gibson GR (2000) Perspectives on the role of the human gut microbiota and its modulation by pro- and prebiotics. Nutrition Research Reviews, 13: 229-254.
Suetsuna K (2000) Antioxidant peptides from the protease digest of prawn (Penaeus japonicus) muscle. Marine Biotechnology, 2:5-10.
Schallmey M, Singh A, and Ward OP (2004) Developments in the use of 
Bacillus species for industrial production. Canadian Joural of Microbiology, 50 : 1-17.
Su CX, Wang DM, Yao LM, and Yu ZL (2006) Purification, characterization, and gene cloning of a chitosanase from Bacillus species strain S65. Journal of Agricultural and Food Chemistry, 54; 4208-4214.
Shikha, Sharan A, and Darmwal NS (2007) Improved production of 
alkanine protease from a mutant of a alkalophilic Bacillus pantotheneticus using molasses as a substrate. Biosource Technology, 98: 881-885.
Todd EW (1949) Quantitative studies on the total plasmin and the trypsin inhibitor of human blood serum. Journal of Experimental Medicine, 39: 295-308.
Trimukhe KD and Varma AJ (2008) Complexation of heavy metals by crosslinked chitin and its deacetylated derivatives. Carbohydrate Polymers, 71: 66-73.
Tuohy KM (2009) Commentary on prebiotics, immune function, infection and inflammation: A review of the evidence. British Journal of Nutrition, 101: 631-632.
Varshosaz J (2007) The promise of chitosan microspheres in drug 
delivery systems. Expert opinion on drug delivery. 4: 263-273.
Waldeck J, Daum G, Bisping B, and Meinhardt F (2006) Isolation and molecular characterization of chitinase-deficient Bacillus licheniformis strains capable of deproteinization of shrimp shell waste to obtain highly viscous chitin. Applied and Environmental Microbiology, 72: 7879-7885.
Wang SL and Chio SH (1998) Deproteinization of shrimp and crab shell with the protease of Pseudomonas aeruginosa K-187. Enzyme and Microbial Technology, 22: 629-633.
Wang SL and Yeh PY (2006) Production of a surfactant- and solvent-stable alkaliphilic protease by bioconversion of shrimp wastes fermented by Bacillus subtilis TKU007. Process Biochemistry, 41:1545-1552.
Wang SL and Yeh PY (2008) Purification and characterization of a chitosanase from a nattokinase producing strain Bacillus subtilis TKU007. Process Biochemistry, 43: 132-138.
Wang SL, Yen YH, Tzeng GC, and Chienyan H (2005) Production of antifungal materials by bioconversion of shellfish chitin wastes fermented by Pseudomonas fluorescens K-188. Enzyme and Microbial Technology, 36: 49-56.
Wang SL, Kao TY, Wang CL, Yen YH,  Chern MK,  Chen YH (2006) A solvent stable metalloprotease produced by Bacillus sp. TKU004 and its application in the deproteinization of squid pen for β-chitin preparation. Enzyme and Microbial Technology, 39: 724-731.
Wang, S. L., Lin, H. T., Liang, T. W., Chen, Y. J., Yen, Y. H., & Guo, S. P. (2008a). Reclamation of chitinous materials by bromelain for the preparation of antitumor and antifungal materials. Bioresource Technology, 99: 4386-4393
Wang, S. L., Chen, S. J., & Wang, C. L. (2008b). Purification and characterization of chitinases and chitosanases from a new species strain Pseudomonas sp. TKU015 using shrimp shells as a substrate. Carbohydrate Research, 343: 1171-1179.
Wang SL, Peng JH, Liang TW, and Liu KC (2008c) Purification and characterization of a chitosanase from Serratia marcescens TKU011. Carbohydrate Research, 343:1316-1323.


Wang SL, Chen TR, Liang TW, and Wu PC (2009a) Conversion and degradation of selfish wastes Bacillus cereus TKU018 fermentation for the production of chitosanases and bioactive materials. Biochemical Engineering Journal, 48: 111-117.
Wang SL, Chao CH, Liang TW, and Chen CC (2009b) Purification and characterization of protease and chitosanase from Bacillus cereus TKU006 and conversion of marine wastes by these enzymes. Marine Biotechnology, 11: 334-344.
Wang SL, Lin CL, Liang TW, Liu KC, and Kuo YH (2009c) Conversion of squid pen by Serratia ureilytia for the production of enzymes and antioxidants. Bioresource Technology, 100:316-323.
Wang SL, Liou JY, Liang TW, and Liu KC (2009d) Conversion of squid pen by using Serratia sp. TKU020 fermentation for the production of enzymes, antioxidants, and N-acetyl chitooligosaccharides. Process Biochemistry, 44:854-861.
Wang SL, Wu PC, and Liang TW (2009e) Utilization of squid pen for the efficient production of chitosanase and antioxidants through prolonged autoclave treatment. Carbohydrate Research, 344:979-984.
Wu Y, Wang Y, Luo G, and Dai Y (2009) In situ preparation of magnetic Fe3O4-chitosan nanopraticles for lipase immobilization by cross-linking and oxidation in aqueous solution. Bioresource Technology, 100: 3459-3464.
Wang SL, Li JY, Liang TW, Hsieh JL, and Tseng WN (2010a) Conversion of shrimp shell by using Serratia sp. TKU017 fermentation for the production of enzymes and antioxidants. Joural of Microbiology and Biotechnology, 20: 117-126.
Wang SL, Chang TJ, and Liang TW (2010b) Conversion and degradation of shellfish wastes by Serratia sp. TKU016 fermentation for the production of enzymes and bioactive materials. Biodegradation, 21: 321-333.
Wang SL, Liang TW, and Yen YH (2011a) Bioconversion of chitin-containing wastes for the production of enzymes and bioactive materials. Carbohydrate Polymers, 84: 732-742.
Wang SL, Tseng WN, and Liang TW (2011b) Biodegradation of 
shellfish wastes and production of chitosanases by a squid 
pen-assimilating bacterium, Acinetobacter calcoaceticus TKU024. Biodegradation, 22: 939-948.

Wan Ngah WS, Ariff NFM, Hanaflash MAKM (2010) Preparation, 
characterization, and environmental application of crosslinked chitosan-coated bentonite for tartrazine adsorption from aqueous solution. Water, Air, and Soil Pollution, 206: 225-236.
Xu Q, Chao YL, and Wan QB (2009) Health benefit application of functional oligosaccharides. Carbonhydrate Polymers, 77: 435-441.
Yen MT, Yang JH, and Mau JL (2009) Physicochemical characterization of chitin and chitosan from crab shells. Carbohydrate Polymers, 75:  15-21.
Ziemer CJ and Gibson GR (1998) An overview of probiotics, prebiotics and synbiotics in the functional food concept: Perspectives and future stratedies. International Dairy Journal, 8: 473-479.