國家圖書館典藏電子全文

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1 酶 Study on the fermentation conditions for producing nattokinase and its improvement conditions of thermal stability

2 酶 3% 1% 3% 31 酶酶酶 i

3 Abstract Bacillus subtilis natto was isolated from commercial natto. The effects of carbon sources, nitrogen sources, inoculum size and cultivation temperature were investigated producing for nattokinase from Bacillus subtilis natto. The major composition and fermentation condition were soluble starch 3%, yeast extract 1%, inoculum size 3% and the cultivation temperature 31. Effect of additives on the thermal stability of nattokinase during heat treatment were studies. Trehalose, saccharose, lactose and gelatin were effective, whereas surfactant and polyethylene glycol (PEG) were ineffective. The metal ions K +, Na + and Ca + could effectively increase the thermal stability of nattokinase. ii

The major composition and fermentation condition were soluble starch 3%, yeast extract 1%, inoculum size 3% and the cultivation temperature 31.

4 ( ) iii

5 iv

6 i...ii..iii...v..ix...x (Bacillus subtilis) v

4.3........9 1.4.4....13 1.4.5....13 1.4.6......15 1.4.7.

7 1.5 酶酶酶..34 vi

.....32 2.2.7......32 2.2.8..32 2.3.33 2.3.1......33 2.3.1.1......33 2.3.1.2 酶.

8 酶酶酶酶酶酶酶酶 vii

.....40 2.3.4.3 酶...40 2.3.4.4 酶..41 2.3.4.5 酶..41 2.3.4.6 酶....42.43 3.1.....43 3.2.43 3.3.46 3.

9 酶酶酶酶酶酶酶酶酶 viii

4.4 酶..58 3.4.5 酶...61 3.4.5.1 酶.. 61 3.4.5.2 酶.

10 ix

11 酶...5 酶...7 酶...8 E. coli O 酶酶酶酶酶酶酶.60 x

coli O-157.10 酶.14.16 酶.18.26.36.....44 酶.45.47.

12 3.13 酶酶酶...65 xi

13 ~ 40% 18 ~ 20% 90% glycinin [, 1991] [Hodgson et al., 1996] [Roberts, 1995] [Adlercreutz et al., 1995] [Nagata et al., 1981 Yashiro et al., 1985] [Wolfe et al., 1981 Carroll et al., 1995] [Kritchevsky, 1995 Sirtori et al.,1995] [Wang et al, 1999] isoflavones saponin phytic acid phytosterol phenolic acid 酶 trypsin inhibitor [Anderson et al., 1995] [Wang et al, 1999] 1

, 1985] [Wolfe et al., 1981 Carroll et al., 1995] [Kritchevsky, 1995 Sirtori et al.

14 1.2 Bacillus subtilis 1872 Ferdinand Cohn Bacillus 1 ~ 5 µm 4 ~ 8 µm aerobic facultative anaerobic 20 ~ 45 ph 6 [Hawood, 1989] sporulation phase exponential phase stationary phase Bacillus [Errington et al., 1993] 65 Bacillus Food and Drug Administration FDA GRAS generally regarded as safe [Priest et al., 1977] subtilisin amylase [, 2003] Bacillus natto 1905 Shin Sawamura Bacillus natto Sawamura DNA 1946 Smith Bacillus subtilis Bergey's Manual of Systematic Bateriology 2

, 1993] 65 Bacillus Food and Drug Administration FDA GRAS generally regarded as safe [Priest et al.

15 Bacillus natto Bacillus subtilis [, 2003] 1.3 natto [Sumi et al., 1987] lecithin saponin folic acid dietary fiber B 6 E K 2 [Esaki et al., 1994] isoflavones 酶 nattokinase NK 酶 superoxide dismutase SOD 酶 angiotension converting enzyme inhibitor ACEI γ-poly-glutamic acid γ-pga 吡 dipicolinic acid [Sumi, 1987 Fukutake et al., 1996 Yamaguchi et al., 1996, 2000] [, 2004] [, 2004] 754 [, 2003]

, 1994] isoflavones 酶 nattokinase NK 酶 superoxide dismutase SOD 酶 angiotension converting enzyme

16 [, 2003] 酶酶 Nattkinase [Sumi et al., 1987] 1.1-A 酶 U urokinase 酶 P plasmin N nattokinase 1.1-B fibrin 酶 C [Sumi et al., 1987] 酶酶酶 serine proteinase 8.6 SDS-PAGE 27,728 Da 275 肽 [Takashi et al., 1992 Fujita et al., 1993 Urano et al., 2001] ph 6~12 60 [Sumi et al., 1987] 1990 酶 [Sumi et al., 1990] 酶酶 4 酶 250mg / 4

1-C [Sumi et al., 1987] 酶酶酶 serine proteinase 8.6 SDS-PAGE 27,728 Da 275 肽 [Takashi et al.

17 1.1 酶 (A) (B) U 酶 P 酶 N (C) [Sumi et al., 1987] 5

18 酶 A B 1996 酶 12 酶 fibrin degradation products FDP 酶酶 2 ~ 8 酶 [Sumi et al., 1996] 酶 tissue plasminogen activator t-pa 酶 t-pa Arima Genkai [, 2003] 酶 lysozyme 酶 Fleming lyso zyme 6

19 A B 1.2 酶 (A) (B) 5 [Sumi et al., 1990] 7

20 Prourokinase 酶 Urokinase 酶 B Plasminogen 酶 Plasmin 酶 Blood clot / Fibrin / Fibrin decompose products A Nattokinase 酶 C tissue plasminogen activator t-pa 酶 1.3 酶酶 (A) (B) 酶酶 (C) 酶 [, 2001] 8

21 酶 Bacillus Micrococus [Fleming, 1922] 酶酶酶酶 [, 2003] endospores 吡 dipicolinic acid DPA [Gould et al., 1969] 1983 Escherichia coli Salmonella 1996 E. coli O-157 E. coli O-111 O E. coli O cell/ml 10 6 cell/ml 1.4 E. coli O-157 E. coli O cell/ml E. coli O-157 E. coli O-157 [Sumi et al., 1997] [, 2004] 12 9

22 12 10 E. coli (O-157) E. coli (O-157) + Bucillus subtilis ( log/ml ) ( days ) 1.4 E. coli O-157 E. coli O-157 E. coli O-157 E. coli O cell/ml [Sumi et al., 1997] 10

23 1.1 [, 2001] mm

24 9 3 genistein daidzein [Meksem et al., 2001 Eisen et al., 2003] genistin daidzin Enterococci spp. Lactobacillus spp. Bacteroides spp. Fusobacterium spp. Bifidobacterium spp. -glucosidase genistein daidzein [Hawksworth et al., 1971 Eisen et al., 2003 Hendrich, 2002 Xu et al., 1995] [Birt et al., 2001 Dixon et al., 2002 Ishimi et al., 2002] genistein [Uckun et al., 1995] genistein biochanin A biochanin A [Yanagihara et al., 1993] estrogen phytoestrogen [Meksem et al., 2001] 17 -estradiol 4 -OH 7-OH 11.5 Å estrogen receptor sex hormone binding globulin SHBG [Kurzer et al., 1997 Dixon et al., 2002] [Hsieh et al., 1998] 12

25 1968 Ehrlich 2 ~ 3 [Kameda et al., 1968] mmhg 1998 [Maruyama et al., 1998] 1.5 I angiotensin I 酶 angiotensin-conterving enzyme ACE II angiotensin II angiotensin-conterving enzyme inhibitors ACEIs 酶 I II [Okamoto et al., 1995] [Vaughan, 2001] Osteo-porosis 13

26 SBP (mmhg) DBP (mmhg) 1.5 酶 [Maruyama et al.,1998] 14

27 Osteocalcin K 2 K 2 [Yamaguchi et al., 2000] 6 65 K 2 K 2 K Nishimura 酶 [Nishimura et al., 1994] ~ 70% 酶 50% 90% 50 ~ 60% 酶 10% [Kiers et al., 2000] 15

28 Before After 1.6 [Nishimura et al., 1994] 16

29 1.4.8 ethanol acetaldehyde [Sumi et al., 1995] Yokota 酶 1.7 LDL [Yokota et al., 1996] [Iwai et al., 2002] Nagata 4800 [Nagata et al., 1998] 38% [Meeker et al., 1941] lipoxygenase cyclooxygenase peroxidase heme [Patel et al., 2001] [Ames, 1990 Giugliano et al., 1995 Jialal et al., 1996 Spector et al., 1995] 17

30 15 Before After Total cholesterol (mg/ml) 酶 [Yokota et al., 1996] Time (week) 18

31 [Ishikawa, 1992] [Ebine, 1990 Ohshita, 1990 Takahashi, 1990] 1979 [Pratt et al., 1979] DNA [Birt et al., 2001] 肽肽 L-Arg L-Arg [Wallner et al., 2001] 1.5 酶 (1) [, 1982 Minihane et al., 1986] 酶酶酶酶 [, 1999, 2001, 2001 Liu et al., 2004 Liu et al., 2005] (2) 腖 [, 1982 Minihane et al., 1986 Cohen, 1994] 酶 19

32 酶腖酶 [, 1999, 2001, 2001 Liu et al., 2004 Liu et al., 2005] (3) 酶酶 ph [, 1982 Stanbury et al., 1984 Egorov, 1985 Ward, 1989 Cohen, 1994] 酶 [, 1999, 2001, 2001 Liu et al., 2004 Liu et al., 2005] (4) 20 ~ 40 [Minihane et al., 1986 Ward, 1989] [, 1982 Minihane et al., 1986 Cohen, 1994] 酶 30 ~ 37 [,

33 , 2001, 2001 Liu et al., 2004 Liu et al., 2005] (5) ph ph 酶 ph [, 1982 Minihane et al., 1986] ph ph 4.0 ~ 8.5 [Cohen, 1994] 酶 ph 7.0 ~ 7.2 ph 7.0 [, 1999, 2001, 2001 Liu et al., 2004 Liu et al., 2005] (6) Hinton s [Minihane et al., 1986 Cohen, 1994] [Stanbury et al., 1984] [, 1982 Ward, 1989] Hinton s 100 ~ 150 rpm 300 ~ 400 rpm 0.6 ~ 0.8 vvm [, 1999, 2001, 2001] (7) [Stanbury et al., 1984] 21

34 [Egorov, 1985] 酶 [Cohen, 1994] 酶 36 ~ 48 [, 1999 Liu et al., 2004 Liu et al., 2005] X-ray denaturation [, 1991] (1) [, 1991] 35 ~ ~ 80 active site [Kleinsmith et al., 1995] (2) [Garret et al., 1995] 22

35 (3) 3000 [Dressler et al., 1991] (4) ph ph ph ph 3 ~ 9 ph [, 1991 Dressler et al., 1991 Garret et al., 1995] (5) [, 1991 Dressler et al., 1991 Garret et al., 1995] Porter 酶 urokinase UK 酶 ph [Porter et al., 1993] substrate 酶 coenzyme [, 1991] 酶酶 23

36 proteins CP 酶 20% [Porter et al., 1993] 酶 [Vrkljan et al., 1994] gelatin glycerol hydroxyethyl starch HS 吡 polyvinylpyrrolidone PVP polyethylene glycols PEG 4000 [Vrkljan et al., 1994] 酶 porteases 肽酶 60% 酶酶 [Germano et al., 2003] Ca 2+ Mg 2+ Mn 2+ 酶 [Paliwal, 1994 Adinarayana et al., 2003] Hg 2+ 酶 [Rahman et al., 1994 Shimogaki et al., 1991] 酶酶酶 [Ramos et al., 1997] coat 1.7 酶酶酶酶 24

37 酶酶酶酶酶 25

38 酶 26

39 WA CE FD OR NA FA FA alcohol agarose dextrin fructose galactose glucose lactose corn starch potato starch soluble starch maltose saccharose 腖 casein peptone malt extract beef extract Ultra biotechnology E.U. Panreac E.U. Panreac HiMedia HiMedia HiMedia 27

40 腖 soya peptone yeast extract yeast malt agar YM Agar 腖 meat peptone polipeptone gelatin HM LM corn steep liquor 酶 thrombin 酶 fibrinogen sorbose trehalose dioctyl sulfosuccinate, AOT 99% mannitol sodium chloride, AR sodium tetraborate, ACS magnesium sulfate heptahydra potassium chloride, ACS Polysorbate 80, Tween 80 Polysorbate 20, Tween 20 HiMedia HiMedia HiMedia Pronadisa Pronadisa Sigma Sigma Sigma Sigma Sigma Sigma Riedel-de Haen Riedel-de Haen Riedel-de Haen Riedel-de Haen J.T.Baker J.T.Baker J.T.Baker 28

41 glycerol trichloroacetic acid, ACS potassium dihydrogen phosphate disodium hydrogen phosphate calcium chloride dihydrate, ACS citric acid monohydrate, ACS eisen chloride tetrahydrat J.T.Baker Merck Merck Merck Merck Merck Merck manganese sulfate pentahydrate, iron sulfate heptahydrate, copper sulfate pentahydrate, cobalt sulfate heptahydrate, calcium sulfate, calcium acetate, sodium sulfate UV-1500 RC-5C Plus Z200A S305R ph meter MP220 HL-341 B403L 8210R-MT Vortex-2 Genie Hitachi Sorvall Hermle Firstek Scientific Metter Toledo Huxley Haake Bransonic Bohemia ( ) AG285 Metter Toledo 29

42 LVDV-E TE-2000U LR L MDL-8C Brookfield Nikon Yamato Marubishi beef extract powder 0.3 % 腖 meat peptone 0.5 % NaCl 0.5 % Agar 1.5 % 2. ph petri dish mm ph % w/v 2 酶 10 unit/ml % w/v 酶 parafilm 4 30

43 mm 150 mm ph g/mol g/mol 1 ph 酶 20 unit/ml 1.5 酶 74 unit/ml % w/v M g/mol beef extract 0.3 % 腖 meat peptone 0.5 % NaCl 0.5 % 2. ph glucose 1 % yeast extract 1 % KH 2 PO % MgSO 4 7H 2 O 0.05 % 2. ph

44 % 腖 soya peptone 3 % 2. ph soluble starch 3 % 14 3 % 2. ph soluble starch 3 % yeast extract 1 % 2. ph

45 酶酶 Fibrin-plate method clear zone area cm 2 酶 [, 1998, 2003] Fibrin-digestion method [Sumi al., 1993, 2003] ~ / 酶 M

46 ,000 g 5 酶酶 µl cm 酶酶 thrombin 酶 fibrinogen fibrin - O.D. 275 nm tyrosine 毎 0.01 fibrinolytic unit FU Japan Bio Science Laboratory Co., Ltd Assay Method of Nattokinase 酶 1. Ac (1) 酶 37 5 (2) 0.1 酶 (3) 酶 20 (4) 12,000 g 5 - O.D. 275 nm 2. Ar (1) 酶 37 5 (2) 0.1 酶

47 (3) 0.1 酶 (4) 2 20 (5) 12,000 g 5 - O.D. 275 nm 3. (Ar Ac) Nattokinas e Activity ( FU/g ) = D Ac 275 Ar 酶 D D g FU/ml rpm 3. 3% v/v rpm 4. - O.D. 600 nm 12,000 g

48 0.25 Y ( Cell density ) = X( O.D.600 nm ) r 2 = Cell concentration ( mg/ml ) O.D. 600 nm

49 酶 rpm 3. 5 L rpm 0.4 vvm 4. 12,000 g 5 - O.D. 600 nm cell concentration 酶腖 soya peptone glucose fructose galactose maltose malt extract saccharose lactose soluble starch potato starch corn starch dextrin 13 酶 rpm 37

50 3. 3% v/v rpm 4. 酶腖 soya peptone 腖 meat peptone 腖 casein peptone polipeptone beef extract powder yeast extract malt extract corn steep liquor 14 酶 rpm 3. 3% v/v rpm 4. 酶 % w/v % w/v 酶 % w/v rpm 3. 3% v/v 38

51 rpm 4. 酶酶 rpm % v/v ph rpm 4. 酶酶 rpm 3. 3% v/v ph rpm 4. 酶酶酶酶 % 酶 39

52 1. 5 酶酶 3. % 酶 saccharose sorbose lactose mannitol trehalose 酶酶 mm 2. 酶 500 mm 酶 5. % 酶 AOT dioctyl sulfosuccinate [Goto et al. 2000] Tween 20 Tween 80 AOT 酶酶 % w/v 2. 酶 0.01 %

53 酶 5. % 酶 polyethylene glycols PEG PEG [Clenland et al., 1992 Vrkljan et al., 1994] Daltons PEG 酶酶 % PEG 酶 1. PEG 10% w/v 2.PEG 酶 PEG 5% 酶 5. % 酶 MgSO 4 ZnSO 4 (NH 4 ) 2 SO 4 KCl 2 SO 4 NaCl 2 SO 4 CaCl 2 Ca(CH 3 COO) 2 MnCl 2 MnSO 4 CuSO 4 CoSO 4 CaSO 4 FeSO 4 FeCl 2 酶酶 41

54 % 酶 mm 10 mm 2. 酶 100 mm 5 mm 酶 5. % 酶 gelatin glycerol HM LM 酶酶 % 酶 1. 10% w/v 2. 酶 5% 酶 5. % 42

55 a 酶酶 [Sumi et al., 1987] WA CE 1.33 cm 2 酶 FA-1 FA FA-2 FA WA CE 3.1 b WA CE ± ± FU/g CE lag phase 4 18 exponential phase 18 43

56 (a) Clean zone area ( cm 2 ) Time ( hr ) Nattokinase activity ( FU/g ) (b) 0 WA Strain CE 3.1 (a) 6 ( )WA ( )CE ( )FD ( )OR ( )NA ( )FA-1 ( )FA-2 (b) WA CE 44

57 5 6 Cell concentration ( mg/ml ) Nattokinase activity ( FU/ml ) Incubation time ( hr ) 3.2 酶 ( ) 酶 ph rpm 0.4 vvm 22 45

58 stationary phase 3.2 酶酶 ± FU/ml 酶 Primary metabolites 腖 soya peptone 酶 3.3 soluble starch 酶 ± FU/ml lactose dextrin potato starch ± ± ± FU/ml fructose 酶 ± FU/ml soluble starch 酶酶 3.4 yeast extract 酶 ± FU/ml corn steep liquor malt extract ± ± ± ±0.150 FU/ml 腖 soya peptone 46

59 5 Nattokinase activity ( FU/ml ) Carbon source (glucose) 2. (fructose) 3. (galactose) 4. (maltose) 5. extract) 6. (saccharose) 7. (soluble starch) 8. (potato starch) 9. (corn starch) 10. (lactose) 11. (dextrin) ph rpm 72 47

60 10 9 Nattokinase activity ( FU/ml ) Nitrogen source 腖 (soya peptone) 2. 腖 (meat peptone) 3. 腖 (casein peptone) 4.polipeptone 5. (beef extract) 6. (yeast extract) 7. (malt extract) 8. (corn steep liquor) ph rpm 72 48

61 酶 ± FU/ml yeast extract 酶酶 % w/v % w/v ph 酶 3.5 3% ± mg/ml ± FU/ml 2% ± cp ph 7.0 3% 3.6 1% 酶 ± FU/ml 3% ± cp 2% ± mg/ml ph 7.0 3% 1% γ-poly-glutamic acid γ-pga [Yamaguchi et al., 1996, 2003] % ph 酶

62 ph Cell concentration ( mg/ml ) Nattokinase activity ( FU/ml ) Viscosity ( cp ) Soluble starch concentration ( % ) 3.5 ( ) ( ) ph ( ) 酶 ph rpm 48 50

63 ph Cell concentration ( mg/ml ) Nattokinase activity ( FU/ml ) Viscosity ( cp ) Yeast extract concentration ( % ) 3.6 ( ) ( ) ph ( ) 酶 ph rpm 48 51

64 ph Cell concentration ( mg/ml ) Nattokinase activity ( FU/ml ) Viscosity ( cp ) Inoculum size ( % ) 3.7 ( ) ( ) ph ( ) 酶 ph rpm 48 52

65 3% ± mg/ml ± FU/ml 3% 7.38 ± cp ph 7.0 3% [Puri et al., 2005] % ph 酶酶 ± FU/ml ± cp ± mg/ml ph 31 酶酶酶 3.4 酶 50 酶 60 酶 70 酶 [Sumi et al., 1987] 酶吡 53

66 ph Cell concentration ( mg/ml ) Nattokinase activity ( FU/ml ) Viscosity ( cp ) Culture temperature ( o C ) 3.8 ( ) ( ) ph ( ) 酶 ph rpm 48 54

67 [Vrkljan etal., 1994] 酶酶酶 [Ramos et al., 1997] Ca 2+ Mg 2+ Mn 2+ 酶 [Paliwal, 1994] 酶 60 酶酶酶酶 60 酶 [Sumi et al., 1987] 60 酶 hr 100% ± % ± % ± % 酶酶酶酶 hr 100% mm

68 Relative activity ( % ) Heat treatment time ( hr ) 3.9 酶 56

69 o C ; 5 hr 60 o C ; 5 hr Relative activity ( % ) Sugar ( 500 mm ) 3.10 酶 1.Control 2. (saccharose) 3. (sorbose) 4. (lactose) 5. (mannitol) 6. (trehalose) 57

70 9.625 ± % ± % ± % ± % 340 Daltons Daltons Daltons 酶酶酶 AOT 0.01% AOT ± % 0.01% 酶 PEG 酶 PEG 酶 PEG ± % ± % PEG 58

71 o C ; 5 hr 60 o C ; 5 hr Relative activity ( % ) Surfactant ( 0.01 % ; w/v ) 3.11 酶 1.Control 2.Tween 20 3.Tween 80 4.AOT 59

72 o C ; 5 hr 60 o C ; 5 hr Relative activity ( % ) PEG ( 5 % ; w/v ) 3.12 酶 1.Control 2.PEG PEG PEG

73 PEG Daltons 5% PEG 酶酶酶酶 % ± % ± % 酶 ± % ± % ± % 酶 ± % ±2.007 % ± % 61

74 o C ; 5 hr 60 o C ; 5 hr Relative activity ( % ) Metal ions ( 100 mm ) 3.13 酶 1.Control 2. (MgSO 4 ) 3. (ZnSO 4 ) 4. ((NH 4 ) 2 SO 4 ) 5. (KCl) 6. 2 SO 4 ) 7. (NaCl) 8. 2 SO 4 ) 9. (CaCl 2 ) 10. (Ca(CH 3 COO) 2 ) 62

75 酶酶酶 ± % ± % ± % 5 mm 酶 ± % ± % 酶酶酶酶 HM ± % HM 63

76 Relative activity ( % ) o C ; 5 hr 60 o C ; 5 hr Metal ions ( 5 mm ) 3.14 酶 1.Control 2. (MnCl 2 ) 3. (MnSO 4 ) 4. (CuSO 4 ) 5. (CoSO 4 ) 6. (CaSO 4 ) 7. (FeSO 4 ) 8. (FeCl 2 ) 64

77 o C ; 5 hr 60 o C ; 5 hr Relative activity ( % ) Other additives ( 5 % ; w/v ) 3.15 酶 1.Control 2. (gelatin) 3. (glycerol) 4. (HM) 5. (LM) 65

78 100% ± % 66

79 4.1 酶 1. Fibrin-plate method Fibrin-digestion method CE 2. soluble starch yeast extract 3% 1% 3% 酶 AOT HM 50% PEG

80 68

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9 04 Vol. No. Serial No.6 China Brewing Research Report sanning electron microscope SEM g ml = 4 4h UV-800PC 0000r/min 5min SLY-B 0μL - LHS-50SC 7 6h.

9 04 Vol. No. Serial No.6 China Brewing Research Report sanning electron microscope SEM g ml = 4 4h UV-800PC 0000r/min 5min SLY-B 0μL - LHS-50SC 7 6h. 04 6 9 DNAout [].. 5g/L g/l nattokinase NK g/l K HPO 4 0.5g/L KH PO 4 0.g/L [-4] MgSO 4 ph7.0~7. LB 0g/L 5g/L [5-7] 0g/L NaCl 5g/L ph7.0 urokinase UK streptokinase SK tissue-type plasminogenactivator t-pa