益生菌應用於食品發酵已有多年歷史。近年來許多研究指出，藉由益生菌(probiotics)的攝取可平衡腸道菌相及有益人體健康。本項研究的目的是以Lactobacillus paracasei TKU010所生產之蛋白酶純化和定性及對酸和膽汁耐受性之探討。由嬰兒吐奶篩選到之菌株Lactobacillus paracasei TKU010這株能以烏賊軟骨粉末作為生產蛋白酶之主要碳氮源之益生菌。TKU010生產蛋白酶之較適培養條件為: 由含有1%烏賊軟骨粉末、0.1%K2HPO4、0.05%MgSO4．7H2O之100mL液體培養基(pH9)於25℃振盪培養3天後，可得較佳蛋白酶活性。所得發酵液經硫酸銨沉澱、DEAE-Sepharose離子交換層析及Sephacryl S-100膠體層析的分離步驟，利用SDS-PAGE所分析出來的分子量約為49kDa。TKU010 蛋白酶之最適反應溫度、最適反應pH、熱安定性及pH安定性分別為50℃、pH10、＜60℃和pH5-10;活性會受到EDTA的抑制，屬於金屬型的蛋白質酶;且純化後的酵素在0.5(mM)SDS, 0.5%(v/v)Tween 40, 0.5%(v/v)Triton X-100界面活性劑存在下殘餘活性分別為73% ,54% ,及102%。有機溶劑對酵素活性及安定性影響方面，在(20% ,v/v)甲苯、乙醚、與丙酮等有機溶劑下，蛋白質酶仍有50%以上的殘餘活性，而在乙酸乙酯和甲醇有機溶劑下，蛋白質酶只剩約10%的殘餘活性;將酵素與(25% ,v/v)有機溶劑於4℃與25℃下，待反應10天後，皆仍然保有75%以上的殘餘活性。
此外, TKU010於酸的耐受性上, 當磷酸鹽緩衝液pH為2.5時 可達到10 CFU/ml。耐膽鹽性試驗方面，在含有0.3%牛膽鹽之MRS broth 中培養24 小時菌數的存活率達90％以上。經過耐酸性和耐膽鹽性測試, TKU010於模擬消化過程測試中也可達到80%以上。於植物生長方面，TKU010之發酵上清液對萵苣及芥蘭的生長明顯有幫助。

Probiotics have been used in various fermented foods for many years. Resent reports indicated that digest probiotics are microbial food supplements, which beneficially affect the balance of intestinal microflora and host’s healthy. The purpose of this study was to investigate the purification and characteristics of a protease from Lactobacillus paracasei TKU010 and its acid and bile resistant ability discussion . The protease-producing strain, Lactobacillus paracasei TKU010, was isolated from infant Spits milk . The optimized culture medium was composed of 1% squid pen powder(SPP), 0.1%K2HPO4,0.05%MgSO4．7H2O at pH9. The strain was incubated in 100mL of above liquid medium and kept shaking at 25℃ for 3 days. The TKU010 protease was purified from the culture supernatant by ammonium sulfate precipitation, DEAE-Sepharose column chromatography and Sephacryl S-100 gel chromatography. The molecular mass of TKU010 protease determined by SDS-PAGE was approximately 49,000Da.The optimum temperature, optimum pH , pH stability and thermal stability of TKU010 protease was 50℃, pH 10, pH5-10 and＜60℃ . The protease was characterized as a metalloprotease because it was inactivated by EDTA. Additionally,the pure protease retained 73% ,54% ,and 102% of its original activity in the presence of 0.5(mM)SDS, 0.5%Tween 40 and 0.5%Triton X-100, respectively.
    In the presence of organic solvent such as toluene, ethyl ether and acetone, the protease retained more than 50% of its activity. In contrast, in the presence of organic solvent such as ethyl acetate and methanol, it retained only 10% of its activity. The TKU010 protease retain over 75 % of its activity by pre-incubation in the organic solvent at 4℃ and 25℃ for 10 days.  
    Additionally, TKU010 showed tolerance properties in this test on pH 2.5 phosphate buffer, it could achieve 10 CFU/ml . In the bile resistance test, it viability can achieve higher than 90% survival population in MRS plus broth (include 0.3% bull bile salt) after being incubated for 24hrs.According to the acid and bile resistance test, TKU010 take the imitating digestive tract test and it was higher than 80% survival population.

目錄

頁次
封面內頁
簽名頁
授權書
中文摘要………………………………………………………....….i
英文摘要……………………………...……………………..…..iii
誌謝…………………………………………………………………...vi
目錄……………………………………………………………......vii
圖目錄………………………………………………………………..xii
表目錄……………………………………………………………….xiv

第一章 緒論...............................................1
第二章 文獻回顧……………………………………….............2
       2.1烏賊軟骨之微生物再利用………………………………..2
           2.1.1烏賊軟骨所含主要成分………………………….2
       2.2 蛋白酶簡介……………...…………..……………….3
           2.2.1蛋白酶分類……………………………………..3
2.2.2乳酸菌蛋白酶之分類………………………….........5
2.2.3 乳酸菌之分類與型態……………………………....5
2.2.4乳酸菌對人體之益處……………….……………....6

第三章 材料與方法…………………………………………………..9
       3.1實驗菌株…………………………………………………9
       3.2實驗材料……………………………………………..…..9
       3.3實驗儀器………………………………………………..10
       3.4 蛋白酶生產菌株之篩選……………………………….11
       3.5 蛋白酶活性之測定…………………………………….12
       3.6 蛋白酶之較適培養條件探討…………………….....12
            3.6.1 碳源之選擇……………………………….……12
            3.6.2 培養基之酸鹼值………………………….……13
            3.6.3 培養溫度……………………….………………13
3.6.4 培養基之通氣量……………………….……...13
3.6.5 較適培養時間……………………….………...13
3.7 酵素之純化分離………………….……………………..14
3.7.1 粗酵素液之製備…………………….………...14
3.7.2 離子交換樹脂層析法….…...…. .....…...…..…14
3.7.3 膠體過濾層析法……………….……………...14
3.8蛋白質電泳分析………………….…………………….....15
3.9蛋白質定量分析………………….……………………….15
3.10 酵素生化特性分析…………….………………………..15
3.10.1 酵素最適反應溫度…………………………....15
3.10.2 酵素熱安定……………….…………...……....15
3.10.3 酵素最適反應pH………………………..……..16
3.10.4 酵素pH安定性……………….……………......16
3.10.5 金屬離子與抑制劑對酵素活性之影響………...16
3.10.6 界面活性劑對酵素活性之影響 ......................17
3.10.7 蛋白酶之基質特異性................................ 17
3.11有機溶劑對酵素活性之影響............................18
3.11.1酵素抗菌測試......................................18
3.11.2 酵素抗黴菌測試...................................18
3.12利用TKU010發酵液進行萵苣及芥蘭生長測試..............19
3.12.1萵苣及芥蘭之預培養................................19
3.12.2 促進萵苣及芥蘭生長試驗………………………19
3.13 乳酸菌對酸液、膽鹽的耐受性試驗…………………………20
3.13.1  耐鹽試驗………………………………………..20
3.13.2  耐酸試驗…………………………..……………20
3.13.3  耐膽鹽試驗…………………………..…………20
3.13.4  模擬腸道消化過程試驗……………..…………21
3.13.5  pH值與可滴定酸測定………………...……….22
3.13.6  貯藏試驗……..……………………………..…..23
3.13.7  溶菌酶試驗……………………………………..23
第四章 結果與討論………………………………………………...24
     4.1 蛋白酶生產菌之篩選……………………….….……...24
     4.2 菌株特性……………………………….…….………..24  
4.3 碳氮源的選擇…………………………….………………..28 
      4.4 蛋白酶較適生產條件探討………………………….…..29
      4.5 TKU010生產蛋白酶之時間變化………………………….30
4.6 蛋白酶之純化分離..……………………………………….33
4.6.1 粗酵素液製備…………………………….…….33
4.6.2 離子交換樹脂層析……………………………..33
4.6.3 膠體過濾層析…………………………………..33
4.6.4 酵素之分子量………………….……………….34
4.7 蛋白酶之生化特性……………………………………...…..35
4.7.1 蛋白酶最適反應溫度及熱安定性…...………….35
4.7.2 蛋白酶最適反應pH 及 pH安定性…….………35
4.7.3 各種化學藥品對蛋白酶之影響………………....36
4.7.4 各種界面活性劑對蛋白酶之影響……………....37
4.7.5 蛋白酶之基質特異性…………………………....37
4.7.6 有機溶劑對蛋白酶活性及安定性之影響……....38
4.7.7 TKU010發酵上清液對E.coli之抑制作用………..38
4.7.8 TKU010發酵上清液對Aspergillus fumigatus及Fusarium solani之抑制作用……..………………………….39
4.7.9 TKU010發酵上清液對萵苣及芥蘭生長之影響…. ...39
4.8 不同環境對乳酸菌生長之影響…….………….…..…….....40
4.8.1氯化鈉對乳酸菌生長之影響……..…………. …..40
4.8.2酸對乳酸菌生長之影響…………………….........40
4.8.3膽鹽對乳酸菌生長之影響…………. ……….......60
4.8.4模擬腸道試驗………. ……. …… ……………....61
4.8.5溶菌酶對乳酸菌之影響………………………......61
4.8.6乳酸菌之貯藏試驗……………………………… 62
第五章 結論與未來展望……………………………………….....68
參考文獻……………………………………………………………..69
附錄…………………………………………………………………..77



圖 目 錄

頁次
圖 4.1 L. paracasei TKU010 之顯微照片……………………….25
圖 4.2 L. paracasei TKU010 之16S rDNA部份序列…………...26
圖 4.3 L. paracasei TKU010 之API鑑定系統分析……………...27
圖 4.4不同碳/氮源對於TKU010生產蛋白酶活性之影響…........31
圖 4.5 L. paracasei TKU010培養於烏賊軟骨培養基生產蛋白酶之生長曲線圖………………………………………………………....32
圖 4.6  L. paracasei TKU010所生產蛋白酶之純化分離流程圖…..41
圖 4.7 TKU010蛋白酶之DEAE-Sepharose CL-6B層析圖譜........42
圖 4.8   TKU010蛋白酶之Sephacryl S-100層析圖譜…………...43
圖 4.9 TKU010蛋白酶之SDS-PAGE……………………...…….....45
圖 4.10蛋白酶之最適反應溫度及熱安定性………………………..46
圖 4.11蛋白酶之最適反應pH及pH安定性………………..…......47
圖 4.12 有機溶劑對蛋白酶活性之影響………………….......…53
圖 4.13有機溶劑對蛋白酶安定性之影響……………………......54
圖 4.14 L. paracasei TKU010發酵上清液對E.coli之抑制作用…55
圖 4.15 L. paracasei TKU010發酵上清液對Aspergillus fumigatus及Fusarium solani之抑制作用…....……...…………56
圖 4.7.1 L.paracasei TKU010發酵上清液對萵苣生長之影響…..58
圖 4.7.2 L.paracasei TKU010發酵上清液對芥蘭生長之影響……59
圖 4.16 L. paracasei TKU010培養在不同鹽濃度下之生長變化…64
圖 4.17 L. paracasei TKU010培養在不同膽鹽濃度下之生長變化65



表 目 錄

頁次
表2.1 乳酸桿菌及乳酸球菌之蛋白酶分類…………………………..7表2.2 乳酸菌對人體之益處…………………………….………...…8
表4.1  L. paracasei TKU010生產蛋白酶之較適條件…………….31
表4.2  L. paracasei TKU010蛋白酶之純化總表 …………………44
表4.3 各種化學藥品對蛋白酶之影響……………………………….48
表4.4 各種乳酸菌所生產蛋白酶之特性比較……………..……….49
表4.5 各種界面活性劑對蛋白酶之影響………..………………….51
表4.6 蛋白酶之基質特異性…..…………………...............52
表4.7  L. paracasei TKU010發酵液對萵苣及芥蘭生長之影響….57
表4.8  L. paracasei TKU010培養在不同pH下之生長變化….....63
表4.9  L. paracasei TKU010培養在不同膽鹽濃度下之生長變化
(以存活率表示) …………...……...........................66
表4.10 L. paracasei TKU010之模擬腸道試驗.................66
表4.11 L. paracasei TKU010之耐溶菌酶試驗.................67
表4.12 L. paracasei TKU010之貯藏試驗.....................67

王三郎 編著。(2005)。應用微生物學:第56~57頁。高立圖書出版社。 
   台北，台灣。
Adler-Nissen J (1986) Enzymatic hydrolysis of food proteins. pp.
  19-20. Elselier Applied Science Publ. London and New York.
Akuzawa R, Yagi N, Kimura M, Okitani A (1994) Purification and 
   characterization of a serine proteinase from Lactococcus lactis spp
   lactis IAM 1198. Anim Sci Techno1. 65:22-32.
Akuzawa R, Okitani A (1995) Purification and characterization of a  
   cysteine proteinase with low activation energy from Lactococcus lactis  
   spp. lactis IAM 1198. J Dairy Sci. 78:2609-2616.
Akuzawa R, Tottori A, Tsukahara K, Okitani A (1997) Purification and  
   characterization of a cysteine proteinase from Lactococcus lactis ssp. 
   lactis IAM 1198 . Intl Dairy J. 7:429-434.
Axelsson L (1998) Lactic acid bacteria: classification and physiology.
   In: Salminen S, von Wright A (eds) Lactic acid bacteria. Microbiology  
   and functional aspects. Marcel Dekker. New York :1–72.
Blow D M, Birktoft J J, Hartley B S (1969) Role of buried acid group in  
   the mechanism of action of chymotrypsin. Nature. 221:337-340.
Bockelmann W, Seyler T H, Heller K J (1996) Purification and 
   Characterization of an Endopeptidase from Lactobacillus delbrueckii 
   subsp. bulgaricus B14. Int Dairy Journal. 6:1167-1180.
Brashears M M, Jaroni D, Trimble J (2003) Isolation, selection, 
   and characterization of lactic acid bacteria for a competitive exclusion
   product to reduce shedding of Escherichia coli O157:H7 in cattle. 
   J Food Prot. 66:355–363.
Chou L S, Weimer B (1999) Isolation and characterization of acid and 
   bile-tolerant isolates from strains of Lactobacillus acidophilus. J Dairy 
   Sci. 82:23–31.
Coconnier M H, Lievin V, Lorrot M, Servin A L (2000)    
   Antagonistic activity of Lactobacillus acidophilus LB against
   intracellular Salmonella enterica serovar typhimurium infecting
   human enterocyte-like Caco-2/TC-7 cells. Appl Environ Microbiol.
   66:1152–1157.
Enan G, el Essawy A A, Uyttendaele M, Debevere J (1996)
 Antibacterial activity of Lactobacillus plantarum UG1 isolated
   from dry sausage: characterization, production and bactericidal
 action of plantaricin UG1. Int J Food Microbiol. 30:189–215.
Fuller R, Brooker B E (1974) Lactobacilli which attach to the crop 
   epithelium of the fowl. Am J Clin Nutr. 27:1305–1312.
Gilliland S, Staley T, Bush L (1984) Importance of bile tolerance of 
   Lactobacillus acidophilus used as dietary adjunct. J of Dairy Sci. 
   67:3045-3051.
Gilliland S E, Walker D K (1990) Factors to consider when selecting a 
   dietary adjunct to produce a hypocholesteroleric effect in humans.    J Dairy Sci. 73:905–911.
Gupta M N (1992) Enzyme function in organic solvents. Eur J Biochem. 
   203:25-32.	
Hartmann R , Meisel H (2007) Food-derived peptides with biological  
   activity:from research to food applications. Current Opinion in  
   Biotechnology.18:163–169.
H´ebert E M, Raya R R,de Giori GS (1999) Characterisation of a  
   cell-envelope proteinase from Lactobacillus helveticus. Biotechnol Lett.   21:831–834.        
Hyronimus B, Le Marrec C, Hadj Sassi A, Deschamps A (2000) 
   Acid and bile tolerance of spore-forming lactic acid bacteria. Intl J  
   Food Microbio. 61:193–197.
Jones B L, Fontanini D, Jarvinen M, Pekkarinen A (1997) Simplified   
   endoproteinase assays using gelatin or azogelatin. Anal Biochem.  
   263:214-220.
Johnson T R, Case C L (1995) Laboratory Experiments in  
   Microbiology,fourth ed. The Benjamin/CummingsPublis hing Co. 
   Inc.,Mealopark, CA.
Jung W J, Kuk J H, Kim K Y, Park R D (2005) Demineralization of red 
   crab shell waste by lactic acid fermentation. Appl Microbiol Biotechnol.  67: 851–854.
Jung W J, Jo G H ,Kuk J H, Kim K Y, Park R D (2006) Extraction of 
   chitin from red crab shell waste by cofermentation with Lactobacillus 
   paracasei subsp. tolerans KCTC-3074 and Serratia marcescens FS-3.  
  Appl Microbiol Biotechnol. 71: 234–237.
Jung W J, Jo G H, Kuk J H, Kim Y J, Oh K T, Park R D (2007) Production 
   of chitin from red crab shell waste by successive fermentation with 
   Lactobacillus paracasei KCTC-3074 and Serratia marcescens FS-3. 
   Carbohydr Polym. 68:746–750.
Kaizu M, Sasaki M, Nakajima H, Suzuki Y (1993) Effect of antioxidative  
   lactic acid bacteria on rats fed a diet deficient in vitamin E.  
   J Dairy Sci. 76:2493–2499.
Karadzic I, Masui A, Fujiwara N (2004) Purification and characterization 
   of a protease from Pseudomonas aeruginosa grown in cutting oil.
   J Biosci Bioeng. 3:145-152.
Laemmli U K (1970) Cleavage of structural during assembly of
 the head of bacteriophage T4. Nature. 227:680-685.
Lin W H, Hwang C F,Chen L W, Tsen H Y (2006) Viable counts, 
   characteristic evaluation for commercial lactic acid bacteria products. 
   Food Microbiol. 23: 74–81.
Maria de Fa¨tima Silva Lopes , Ana Lu¨ cia Leita , J.J. Figueiredo Marques   
   Manuel Jose¨ Teixeira Carrondo , Maria Teresa Barreto Crespo (1999)  
   Processing of extracellular lipase of Lactobacillus plantarum:  
   involvement of a metalloprotease. FEMS Microbio Lett. 00:483-487.
Magboul A A A,. Fox P F , McSweeney Paul L H (1997) Purification and  
   Characterization of a Proteinase from Lactobacillus plantarum 
   DPC2739. Int Dairy Journal. 7:693-700.
Manzi P, Marconi S, Pizzoferrato L (2007) New functional milk-based   
   products in the Italian market. Food Chem. 104:808–813.
Michida H, Tamalampudi S, Pandiella S S,Webb C, Fukuda H, Kondo A 
   (2006) Effect of cereal extracts and cereal fiber on viability of 
   Lactobacillus plantarum under gastrointestinal tract conditions. Biochem Eng J. 28:73–78.
Mohamed S A, Fahmy A S, Mohamed T M, Hamdy S M (2005) Proteases   
   in egg, miracidium and adult of Fasciola gigantica.
 Characterization of serine and cysteine proteases from adult. Comp
 Biochem Physiol B Biochem Mol Biol. 142:192-200.
Muset G, Monnet V ,Gripon J C (1989) Intracellular proteinase
 of Lactococcus lactis subsp lactis NCDO 763. J Dairy Res. 56:
 765-778.
Nowak A, Libudzisz Z (2006) Influence of phenol, p-cresol and indole on  
   growth and survival of intestinal lactic acid bacteria. 
 Ecol/environ microbial Anaerobe. 12:80–84.
Ohmiya K , Sato Y (1975) Purification and properties of intracellular 
   proteinase from Streptococcus cremoris. Appl Microbiol. 30: 738-745.
Okereke A, Montville T J (1991) Bacteriocin inhibition of
   Clostridium botulinum spores by lactic acid bacteria. J of
   Food Protect. 54:349–353.
Pereira C I, Barreto Crespo M T, San Rom˜ao MV (2001)         
 Evidence for proteolytic activity and biogenic amines production in  
 Lactobacillus curvatus and L. homohiochii. Intl J Food Microbiol. 
 68:211–216.

Powell JE, Witthuhn R C, Todorov S D, Dicks L M T (2007)
 Characterization of bacteriocin ST8KF produced by a kefir isolate   
 Lactobacillus plantarum ST8KF. Intl Dairy J. 17:190–198.
Rawlings N D, Barrett A J (1993) Evolutionary families of peptidases.  
   Biochem J. 290:205–218.
Saavedra J (2000) Probiotics and infectious diarrhea. Ameri J   
   Gastroenterol. 95:16-18.
Siezen R J (1999) Multi-domain, cell-envelope proteinases of lactic acid 
   bacteria. Antonie van Leeuwenhoek. 76:139-155.
Stefanitsi D, Sakellaris G , Garel JR (1995) The presence of two  
   proteinases associated with the cell wall of Lactobacillus bulgaricus.  
   FEMS Microbiol Lett. 128:53-58.
Stepaniak L, Gobbetti M, Fox PF (1996) Partial purification and  
   characterization of intracellular proteinases from Lactococcus lactis   
   subsp lactis MG 1363. Lait. 76:489-499.
Sushil K, Neeru S, Sharma M, Saharan R, Randhir S (2005)      
   Extracellular acid protease from Rhizopus oryzae：purification and   
 characterization. Process Biochem. 40:1701-1705.
Todorov S D, Dicks L M T (2006) Screening for bacteriocin-producing 
   lactic acid bacteria from boza, a traditional cereal beverage from 
   Bulgaria. Comparison of the bacteriocins. Process Biochem. 41:11–19.
Todorov S, Onno B, Sorokine O, Chobert J M, Ivanova I, Dousset X  
   (1999) Detection and characterization of a novel antibacterial substance  
   produced by Lactobacillus plantarum ST 31 isolated from sourdough. Intl J Food Microbiol. 48:167–177.
Todd E W (1949) Quantitative studies on the total plasmin and
   trypsin inhibitor of human blood serum. J Exp Med. 39:295-308.
Vitini E, Alvarez S, Medina M, Medici M, de Budeguer M V, Perdigon G 
   (2000) Gut mucosal immunostimulation by lactic acid bacteria. Biocell. 
   24:223–232.
Wang S L, Yieh T C, Shih I L (1999) Production of antifungal compound by Pseudomonas aeruginosa K-187 using shrimp and crab shell powder as a carbon source. Enzyme Microb Technol. 25:142- 148. 
Wang S L, Yieh T C, Shih I L (1999) Purification and characterization of a new antifungal compound produced by Pseudomonas aeruginosa K-187 in a shrimp and crab shell powder medium. Enzyme Microbl Technol. 25:439-446. 
Wang S L, Yeh P Y (2006) Production of a surfactant and solventstable  
    alkaliphilic protease by bioconversion of shrimp shell
 wastes fermented by Bacillus subtilis TKU007. Process Biochem.
 41:1545-1552.
Wang S L, Kao T Y, Wang C L, Yen Y H, Chern M K, Chen Y H (2006)  
    A solvent stable metalloprotease produced by Bacillus sp. TKU004  
    and its application in the deproteinization of squid pen for β-chitin 
    preparation. Enzyme Microb Technol. 39:724-731.
Xanthopoulos V, Tzanetaki E L , Tzanetakis N (2000) Characterization of  
    Lactobacillus isolates frominfant faeces as dietary adjuncts. Food Microbiology. 17: 205-215.
Yoon K Y, Woodams E E, Hang Y D (2006) Production of probiotic 
   cabbage juice by lactic acid bacteria. Biores Technol. 97:1427–1430.
Yoon K Y, Woodams E E, Hang Y D (2005) Fermentation of beet juice by 
   beneficial lactic acid bacteria. Lebensm.-Wiss. u.-Technol. 38:73–75.
Yoo J J, Lee Y S, Song C Y, Kim B S (2004) Purification and 
   characterization of a 43-kilodalton extracellular serine proteinase from 
   Cryptococcus neoformans. J Clin Microbiol. 42:722-726.
Zhu W M, Liu W, Wu D Q (2000) Isolation and characterization of a new  
   bacteriocin from Lactobacillus gasseri KT7. J Appl Microbiol.  
   88:877–886.