Baciilus subtilis TKU007 係一株以蝦頭粉為唯一碳/氮源，篩選自台灣北部土壤之納豆激酶生產菌。TKU007生產納豆激酶之較適培養條件為-含有1%蝦頭粉、0.1% K2HPO4及0.05% MgSO4‧7H2O之100 mL 液態培養基，於 pH 7、30℃，搖瓶培養 3天。所得發酵上清液經硫酸銨沉澱、DEAE-Sepharose、Phenyl Sepharose 及Sephacryl S-100 層析分離步驟，純化出二種納豆激酶，經SDS-PAGE測得分子量分別為 30 kDa (N1)、15 kDa (N2)。於最適反應pH、最適反應溫度、pH安定性、熱安定性方面，N1分別為 pH 8、40℃、pH 6-10、<50℃。納豆激酶 N1 活性會受到Mg2+、Fe2+、Cu2+所促進，然而會受到 Zn2+、PMSF所抑制，為絲胺酸型蛋白酶。
   將B. subtilis TKU007發酵所得上清液與酪蛋白於37℃反應2小時，添加所得水解液 (最終濃度2.5 %) 至乳酸菌培養基MRS進行乳酸菌Lactobacillus paracasei TKU010之培養，於25℃、搖瓶培養3天，L. paracasei TKU010菌落數可達到5.9×1012 CFU/mL；較對照組(未添加水解物) 增加2. 3倍，添加未水解的酪蛋白反而會抑制L. paracasei  TKU010 生長。

Bacillus subtilis TKU007, cultured by shrimp head powder as the only carbon/nitrogen source, was isolated from the soil in Taiwan. For the production of nattokinase, B. subtilis TKU007 was grown in 100 mL of liquid medium in an Erlenmeyer flask (250 mL) containing 1% SHP, 0.1% K2HPO4 , 0.05%
MgSO4‧7H2O and grown in an orbital shaking incubator for 3 days at 30℃ and pH 7. After incubation, the culture broth was centrifuged (4℃ and 10,870 ×g for 15 min) , 
and the supernatant was used for further purification by DEAE-Sepharose, Phenyl Sepharose and Sephacryl S-100. The molecular mass of TKU007 nattokinases determined by SDS-PAGE was approximately 28 kDa (N1) and 15 kDa (N2),   respectively. The optimum pH, optimum temperature, pH stability, thermal stability of nattokinase N1 was pH 8, 40℃, pH 6-10、<50℃, respectively. N1 nattokinase activity was activated by Mg2+, Fe2+, Cu2+, inactivated by Zn2+, PMSF, indicating N1 nattokinase was serine protease.     
      A culture supernatant of B. subtilis TKU007 was mixed with casein and incubated for 2 hour at 37℃. The hydrolysates were added to MRS medium (final concentration 2.5 v/v ) for Lactobacillus paracasei TKU010 culture and incubated for 3 days at 25℃. The growth of L. paracasei TKU010 was 5.9×1012 CFU/mL. Which than the control group (non-add hydrolysates) increase 2.3 fold . However, the addition of unhydrolyzed casein could inhibit the growth of L. paracasei TKU010.

目錄
                                                    頁次
 
封面內頁	
授權書	
簽名頁	
誌謝	                                           Ⅰ
中文摘要	                                           II
英文摘要	                                           IV
目錄	                                           VI
圖目錄	                                           XI

表目錄	XIII

 
第一章 緒論                                               1
第二章 文獻回顧                                         3                                                        
      2.1 蝦、蟹殼與烏賊軟骨廢棄物的組成                3 
        2.2  納豆激酶                                     4
          2.2.1納豆激酶的發現                           4
             2.2.2 納豆激酶分解血栓的能力驗證              6
             2.2.3納豆激酶溶解血栓的機制                   6
       2.3 血栓溶解劑                                     7
        2.4益生菌                                         8
      2.5 益生質                                        11
  2.6 活性胜肽                                      13 
第三章 材料與方法                                        15
       3.1 實驗菌株                                      15
       3.2 實驗材料                                      15
       3.3 實驗儀器                                      16
       3.4 納豆激酶之活性測定                            17
       3.5 蛋白酶之活性測定                              17
       3.6 納豆激酶較適生產條件探討                      18
        3.6.1 碳/氮源種類之影響                       18
            3.6.2 碳/氮源濃度之影響                       18
            3.6.3 培養基酸鹼值之影響                      19
            3.6.4 培養體積對酵素產量之影響                19
            3.6.5 培養溫度之影響                          19
            3.6.6 較適培養時間                            20
            3.6.7 較適培養條件                            20
      3.7 酵素之純化分離                                20
           3.7.1 粗酵素液之製備                           20
           3.7.2 離子交換樹脂層析法                       20
           3.7.3 疏水性層析法                             21
           3.7.4 膠體過濾層析                             21
           3.7.5 蛋白質電泳分析                           22
    3.8 酵素生化特性分析                                22
          3.8.1 酵素最適反應溫度                          22
          3.8.2 酵素熱安定性                              22
 3.8.3 最適反應 pH                              23
          3.8.4 酵素 pH 安定性                           23
     3.8.5 金屬離子與抑制劑對酵素活性之影響          24
          3.8.6 界面活性劑對酵素活性的影響                24
          3.8.7 納豆激酶之基質特異性                      25
   3.9 發酵所剩烏賊軟骨粉末 (SPP)之去蛋白處理           25
    3.10 還原醣之測定                                    25
   3.11 胜肽促進乳酸菌生長                              26
        3.11.1 水解時間之探討                          26
           3.11.2 水解液添加濃度之探討                    27
           3.11.3 不同酵素之探討                          27
           3.11.4 活菌計數法                              28
第四章 結果與討論                  29       
4.1酵素較適生產條件探討                             29
            4.1.1碳/氮源之選擇　                          29
            4.1.2培養之pH值探討                         30
            4.1.3 培養之通氣量探討                        31
            4.1.4培養之溫度探討                           31
           4.1.5 綜合結果                                31
        4.2納豆激酶之分離純化                            32
4.2.1 粗酵素液製備                            32
            4.2.2離子交換樹脂層析                         32
            4.2.3疏水性層析                               33
            4.2.4膠體過濾層析                             33
            4.2.5 綜合結果                                34
       4.3蛋白質電泳分析                                34
4.3.1分子量測定                               34
            4.3.2 胺基酸序列鑑定                          34
            4.3.3 綜合結果                                35
        4.4納豆激酶之生化特性分析                        50
            4.4.1納豆激酶之最適反應溫度及熱安定性         50
            4.4.2納豆激酶之最適反應 pH及pH安定性       50
            4.4.3金屬離子對納豆激酶之影響                 50
            4.4.4納豆激酶對於基質之特異性                 51
            4.4.5各種界面活性劑對納豆激酶之影響           52
       4.5 發酵所剩烏賊軟骨粉末 (SPP)之去蛋白效果        52
       4.6促進乳酸菌生長                                59
            4.6.1水解時間之探討                           60
            4.6.2 水解液添加濃度之探討                    60
            4.6.3不同酵素之探討                           61
            4.6.4 活菌計數法                              61
            
第五章 結論與未來展望                                    69
參考文獻                         70

圖目錄

圖2.1幾丁質、幾丁聚醣之製備  	5
圖2.2納豆激酶在血栓溶解系統之直接與間接作用	9
圖4.1 SHP濃度對B. subtilis TKU007納豆激酶之影響	36
圖4.2不同碳/氮源對B. subtilis TKU007納豆激酶之影響	37
圖4.3 pH對B. subtilis TKU007納豆激酶之影響	38
圖4.4通氣量對B. subtilis TKU007納豆激酶之影響	39
圖4.5溫度對B. subtilis TKU007納豆激酶之影響	40
圖4.6 B. subtilis TKU007 於1% SHP (w/v)生產納豆激酶、蛋白酶、幾丁聚醣之生長曲線圖	42
圖4.7 B. subtilis TKU007所生產酵素之純化分離流程圖	43
圖4.8 B. subtilis TKU007之納豆激酶DEAE-Sepharose CL-6B層析圖譜	44
圖4.9 Phenyl Sepharose之納豆激酶層析圖譜	45
圖4.10 Sephacryl S-100之納豆激酶層析圖譜	46
圖4.11 B. subtilis TKU007納豆激酶SDS–PAGE 之分子量分析 48
圖4.12溫度對B. subtilis TKU007納豆激酶活性之影響  	53
圖4.13 pH值對B.subtilisTKU007納豆激酶活性之影響	54
圖4.14 B. subtilis TKU007發酵 1% 烏賊軟骨粉末( w/v )之去蛋白應用	 63
圖4.15 B.subtilis TKU007 於1%烏賊軟骨粉末( w/v )生產納豆激
酶、蛋白酶及幾丁聚醣之生長曲線圖	64
圖4.16水解時間對於L. paracasei TKU010生長之影響	65
圖4.17水解液添加濃度對於 L. paracasei TKU010生長之影響	66
圖4.18不同水解液對於 L. paracasei TKU010生長之影響	67

表目錄

表2.1臨床上常見的血栓溶解藥劑	10
表2.2益生菌臨床功效   	12
表2.3日本各種胜肽原料的來源與機能	14
表4.1 B. subtilis TKU007生產酵素之較適條件之比較	41
表 4.2 B. subtilis TKU007 納豆激酶純化總表	47
表 4.3 B. subtilis TKU007納豆激酶胜肽質譜鑑定結果	49
表4.4金屬離子對 B. subtilis TKU007納豆激酶活性之影響	55
表4.5 B. subtilis TKU007納豆激酶對於基質之特異性	56
表4.6 界面活性劑對B. subtilis TKU007納豆激酶、蛋白酶活
性之影響	57
表 4.7 Bacillus sp.生產納豆激酶特性比較	58
表4.8 添加2.5% 酪蛋白及酪蛋白水解物對於 L. paracasei 
TKU010 菌落數之變化	68

潘子明 ( 2001) 機能性發酵製品。第 227 頁。藝軒圖書出版社。台北。台灣
日本「食品與開發，2004年7月」

Bielecka, M. E., Majkowska, A., 2002 .Selection of probiotics and prebiotics for sysnbiotics and confirmation of their in vivo effectiveness. Food Res Int , 35, 125-31.

Bhatia, S.J., Kochar, N., Abraham, P., Nair, N.G., Mehta, A.P., 1989. Lactobacillus acidophilus inhibits growth of Campylobacter pylori in vitro. J Clin Microbiol, 27, 2328-30.

Borchers, A.T., Selmi, C., Meyers, F.J., Keen, C.L., Gershwin, M.E., 2009. Probiotics and immunity. J Gastroenterol, 44, 26-46.

Brown, I.L.,Wang , D.L ., 1998. High amylose maize starch as a verstaile prebiotic for use with probiotic bacteria. Food Australia, 50, 603-10 .

Chang, C.T., Fan, M.H., Kuo, F.C., Sung, H.Y., 2000. Potent fibrinolytic enzyme from a mutant of Bacillus subtilis IMR-NK1. J Agric Food Chem, 48, 3210-6.

Chiang, C. L., Chang, C.T., Sung, H.Y., 2003. Purification and properties of chitosanase from a mutant of Bacillus subtilis IMR-NK1.Eznyme   Microb Techol,32, 260-7.

Collado, M. C., Isolauri, E., Salminen, S., Sanz, Y., 2009. The impact of probiotic on gut health. Curr Drug Metab, 10, 68-78.

Cummings, J.H., Macfarlane, G.T., 1991. The control and consequences of bacterial fermentation in the human colon. J Appl Bacteriol, 70,443-59.

Elmer, G. W., Surawicz, C.M., McFarland, L.V., 1996.
Biotherapeutic agents. A neglected modality for the treatment and prevention of selected intestinal and vaginal infections. J Am Med Assoc, 275, 870-6.

Fabian, E., Majchrzak, D., Dieminger, B., Meyer, E., 2008. Influence of probiotic and conventional yoghurt on the ctatus of vitamins B1, B2 and B6  in young healthy women . Ann Nutr Metab, 52, 29-36.

Fotiadis, C.I., Stoidis, C.N., Spyropoulos, B.G., Zografos, E.D., 2008. Role of probiotics, prebiotics and synbiotics in chemoprevention for colorectal cancer. World J Gastroenterol, 14, 6453-7.

Fujita, M., Nomura, K., Hong, K., Ito, Y., Asada, A., Nishimuro, S., 1993. Purification and characterization of a strong fibrinolytic enzyme (nattokinase) in the vegetable cheese natto, a popular soybeanermented food in Japan. Biochem Biophys Res Commun, 197, 1340-7.

Gagne, N., Simpson, B.K., 1993. Use of proteolytic enzymes to acilitate recoveryof chitin from shrimp wastes. Food Biotechnol, 7, 253-63.

Gibson, G. R., Roberfroid, M. B., 1995. Dietary modulation of the human    colonic microbiota: Introducing the concept of prebiotics. J Nutr 125, 1401-12.

Gibson, G. R., 1999. Dietary modulation of the human gut microflora using the prebiotics oligofructose and inulin. J Nutr, 129 ,1438-41.

Goldhaber, S.Z., Bounameaux, H., 2001. Thrombolytic therapy in pulmonary embolism. Semin Vasc Med, 1, 213-20.

Guan, Z., Hederstedt, L., Li, J., Su, X. D., 2001. Preparation and crystallization of a Bacillus subtilis arsenate reductase. Acta Crystallogr D Biol Crystallogr, 57, 1718-21.

Hart, A.L., Stagg, A.J., Kamm, M.A, 2003. Use of probiotics in the treatment of inflammatory bowel disease. J Clin Gastroenterol, 36 , 111-9.

Hyronimus, B., Le Marrec, C., Sassi, A.H., Deschamps, A., 2000. Acid and bile tolerance of spore-forming lactic acid bacteria. Int J FoodMicrobiol, 61, 193-7.

Hua, Y., Jiang, B., Mine, Y., Mu, W., 2008. Purification and characterization of a novel fibrinolytic enzyme from Bacillus sp. nov. SK006 isolated from an Asian traditional fermented shrimp paste. J Agric Food Chem, 56, 1451-7.

Isolauri, E., Juntunen, M., Rautanen, T., Sillanaukee, P., Koivula, T., 1991. A human Lactobacillus strain (Lactobacillus casei sp strain GG) promotes recovery from acute diarrhea in children. Pediatrics, 88, 90-7.

Imoto, T.,Yagishita, K, A.,1971. A simple activity measurement by lysozyme . Agric Biol Chem, 35,1154-6.

Jeong, Y.K., Park, J.U., Baek, H., Park, S.H., Kong, I.S., 2001. Purification and biochemical characterization of the a fibrinolytic enzyme from Bacillus subtilis BK-17. World J Microbiol Biotechnol , 17,89-92.

Jeong,Y.K., Kim, J.H., Gal, S.W., Kim, J.E.,Park, S.S.,2004.Molecular cloning and characterization of the geneencoding a fibrinolytic enzyme from Bacillus subtilis Strain A1. World J. Microbiol. Biotechnol , 20,711-717.

Kim, W., Choi, K., Kim, Y., Park, H., Choi, J., Lee, Y., Oh, H., Kwon, I., Lee, S., 1996. Purification and characterization of a fibrinolytic enzyme produced from Bacillus sp. strain CK 11-4 screened from Chungkook-Jang. Appl Environ Microbiol, 62, 2482-8.

Kim, H. K., Kim, G.T., Kim, D.K. 1997. Purification and Characterization of a Novel Fibrinolytic Enzyme from Bacillus sp. KA-38 originated from Fermented Fish. J.Ferment Bioeng, 84,307-12.

Kimura, K., McCartney, A. L., McConnel, M.A., Tannock, G.W., 1997. Analysis of fecal population of bifidobacteria and lactobacilli and  investigation of the immunological responses of their human hosts to predominant strains. Appl Environ Microbiol, 63, 3393-8.

Kim, S.H., Choi, N.S., 2000. Purifircation and characterization of subtilisin DJ-4 secredted by Bacillus sp.strain DJ-4 screednted from Doen-Jang .Biosci Biotechnol biochem, 64,1722-5.

Knorr, D., 1984. Functional properties of chitin and chitosan. J Food Sci, 47, 593-5.

Ko, J.H., Yan, J.P., Zhu, L., Qi, Y.P., 2004. Identification of two novel fibrinolytic enzymes from Bacillus subtilis QK02. Comp Biochem Physiol C Toxicol Pharmacol, 137, 65-74.

Krasakoopt, W., Bhandari, B., Deeth, H., 2003. Evaluation of encapsulation techniques of probiotics for yogurt. Int Dairy J ,13, 3-13.

Laemmli, U, K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680-5.

Lee, S.K., Bae, D.H., Kwnon, T.J., Lee, S.B., Lee, H. H., 2001. Purification and characterization of fibrinolytic enzyme from Bacillus sp. KDO-13 isolated from soybean paste. J Microbiol Biotechnol, 11,845-852.

Lee, S.Y., Kim, J. S., Kim, J.E., Shen, M.H., 2005. Purification and characterization of fibrinolytic enzyme from cultured mycelia of Armillaria mella. Protein Expr Purif, 43, 10-17.

Liu, X.L., Du, L. X., Zheng, X.Q., 2005. Purification and characterization of a novel fibrinolytic enzyme from Rhizopus chinensis 12. Appl Microbiol Biotechnol, 67, 209-14.

Meisel, H., Bockelmann, W., 1999. Bioactive peptides encrypted in milk proteins: proteolytic activation and thropho-functional properties. Antonie Van Leeuwenhoek, 76, 207-15.

McDonagh, D., Fitzgerald, R, J., 1998. Production of caseinophosphpertides (CPPs) from sodium caseinate using a range of commercial protease preparations. Int Dairy J, 8, 39-45.

Mukhametova, L. I., Aisina, R. B., Lomakina, G., Varfolomeev, S. D., 2002. Characterization of urokinase type plasminogen activator modified by phenylglyoxal. Bioorg Khim, 28, 308-14.

Ohtakara, A., Izume, M. 1987. Preparation of D-Glucosanmine oligosaccharide by enzymatic hydrolysis of chitosan. J Agric Food Chem, 51, 1189-91.

Park, Y.S., Lee, J.Y., Kim, Y.S., Shin, D.H., 2002 .Isolation and characterization of lactic acid bacteria from feces of newborn baby and from dongchimi. J Agric Food Chem, 50, 2531-6.

Paik, H.D., Lee, S.K., Heo, S., Kim, S.Y., Lee, H., 2004. Purification and characterization of the fibrinolytic enzyme produced by Bacillus subtilis KCK-7 from
Chungkookjang. J Microbiol Biotechnol,14, 829-35.

Peng, Y., Huang, Q., Zhang, R.H., Zhang, Y.Z., 2003. Purification and characterization of a fibrinolytic enzyme produced by Bacillus amyloliquefaciens DC-4 screened from douchi, a traditional Chinese soybean food. Comp Biochem Physiol B Biochem Mol Biol, 134, 45-52.

Scholz-Ahrens, K. E., Ade, P., Marten, B., Weber, P., Timm, W., Acil, Y., Gluer, C. C., Schrezenmeir, J., 2007. Prebiotics, probiotics, and synbiotics affect mineral absorption, bone mineral content, and bone structure. J Nut r, 137, 83S-46S.

Shimamura, S. F., Ishibashi, H., Miyakawa, T., Yaeshima, T., Araya, M.T., 1992. Relationship between oxygen sensitivity and oxygen metabolism of Bifidobacterium species. J. Dairy Sci, 75, 3296-306.

Steer, T. H., Carpenter, K., Gibson, G. R., 2000.
Perspectives on the role of the human gut microbiota and its modulation by proand prebiotics. Nutr Res. Rev, 13, 229-54.

Sumi, H., Hamada, H., Tsushima, H., Mihara, H., Muraki, H., 1987. A novel fibrinolytic enzyme (nattokinase) in the vegetable cheese Natto; a typical and popular soybean food in the Japanese diet. Experientia, 43, 1110-1.

Sumi, H., Hamada, H., Nakanishi, K., Hiratani, H., 1990. Enhancement of the fibrinolytic activity in plasma by oral administration of nattokinase. Acta Haematol, 84, 139-43.

Todd, E.W., 1949. Quantitative studies on the total plasmin and the trypsin inhibitor of human blood serum; methods for the titration of total plasmin and of trypsin inhibitor. J Exp Med, 89, 295-308.

Tough, J., 2005. Thrombolytic therapy in acute myocardial infarction. Nurs Stand, 19, 55-64.

Wang, S.L., Chiou, S.H., Chang, W.T., 1997. Production of chitinase from shellfish waste by Pseudomonas aeruginosa K-187. Proc Natl Sci Counc ROC (B), 21, 71-8.

Wang, C.T., Ji, B.P., Li, B., Nout, R., Li, P.L., Ji, H., Chen, L.F., 2006. Purification and characterization of a fibrinolytic enzyme of Bacillus subtilis DC33, isolated from Chinese traditional Douchi. J Ind Microbiol Biotechnol, 33, 750-8.

Wang, S.L., Yeh, P.Y., 2006. Production of a surfactant and solvent- stable alkaliphilic protease by bioconversion of shrimp shell wastes fermented by Bacillus subtilis TKU007. Process Biochem, 41, 1545-52. 

Wang, S. H., Zhang, C., Yang, Y.L.,2007.Screening of a high fibrinolytic enzyme producing strain and characterization of the fibrinolytic enzyme produced from Bacillus subtilis LD-8547. World J Microbiol Biotechnol , 24, 475-82.

Wang, S.L., Yeh, P.Y., 2008. Purification and characterization of a chitosanase from a nattokinase producing strain Bacillus subtilis TKU007. Process Biochem, 43 , 132–8.

Wang, S.L., Huang, T.Y., Wang, C.Y., Liang, T.W., Yen, Y.H., Sakata, Y., 2008. Bioconversion of squid pen by Lactobacillus paracasei subsp paracasei TKU010 for the production of proteases and lettuce growth enhancing biofertilizers. Bioreours Technol, 99, 5436-43.

Wang, S.L., Chen, S.J., Wang, C.L., 2008. Purification and characterization of chitinases and chitosanases from a new species strain Pseudomonas sp. TKU015 using shrimp shells as a substrate. Carbohydr Res , 343, 1171-9.

Xiao, J.Z., Kondo, S., Takahashi, N., Miyaji, K., Oshida, K., Hiramatsu, A., Iwatsuki, K., Kokubo, S., Hosono, A., 2003. Effects of milk products fermented by Bifidobacterium longum on blood lipids in rats and healthy adult male volunteers. J Dairy Sci, 86, 2452-61.

Yang, S., Huang, H., Zhang, R., Huang, X., Li, S., Yuan, Z., 2001. Expression and purification of extracellular penicillin G acylase in Bacillus subtilis. Protein Expr Purif, 21, 60-4.

Ziemer, C.J., Gibson, G.R., 1998. An overview of probiotics. Prebiotics and synbiotics in the functional food concept: perspectives and future strategies. Int Dairy J, 8473-9.