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從市售納豆健康食品中篩選納豆菌並探討最適 化生產納豆激酶之培養條件

Optimization of nutritional conditions for nattokinase production by a isolated Bacillus subtilis from natto health food

研究生:吳世佑(Shih-Yu Wu) 指導教授:段國仁(Prof.Kow-Jen Duan)

大同大學 生物工程研究所 碩士論文 Thesis for Master of Science Department of Bioengineering Tatung University

中華民國九十四年八月 August 2005

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Abstract
Nattokinase, a potent fibrinolytic enzyme, was primarily isolated from a traditional fermented food in Japan, ‘‘Natto’’. Nattokinase, a serine protease, can prevent and cure cardiovascular system, for example, thrombus disease and apoplexy. Nattokinase having a strong fibrinolytic activity and produced by a Bacillus natto hokaido, which was isolated from the vegetable cheese natto. Maximum activity (63.3 U ml-1) was obtained when the bacterium was grown in a 500 ml Erlenmeyer flask containing 200 ml medium containing (g/l): molasses, 20; peptone, 20; CaCl2 •2H2O, 1; MgSO4 •7H2O, 1; K2HPO4, 2 at 37℃ for 48 h incubation period with agitation of 180 rpm. The optimum pH and temperature for activity were 37℃ and 10. Fed-batch culture was performed to maximize protease activity in a bioreactor. By using suitable feeding strategies, the protease activity and biomass production in a fed-batch process was increase by 207 ﹪and 300 ﹪over that in batch process, primarily due to the longer maintenance of cell growth and enzyme production by providing continuous and controlled supply of additional substrate and nutrients.

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中文摘要
納豆激酶(nattokinase, NK)具有很強的血纖維蛋白(fibrin)溶解 能力。它主要是利用枯草桿菌(Bacillus subtilis)的類緣菌,俗稱「納豆 菌」 。NK 是屬於一種絲胺酸蛋白酶(serine protease) ,可運用於血栓症、中風等心血管疾病的預防及治療。

本研究利用一株來自納豆食品中篩選的北海道納豆菌,以 500 ml 的三角錐瓶內含 200 ml 基礎培養基,進行以醱酵培養生產 NK 之研究。 最後以糖蜜(20 g/L) 、peptone(20 g/L) 、MgSO4(1 g/L) 、CaCl2(1 g/L) 和 K2HPO4(2 g/L)為培養基,在醱酵溫度 37℃,轉速 180 rpm,培養 47.5 小時會有最高活性(63 unit)。酵素最適 pH 值及溫度分別為 10 與 37 ℃。pH-stat 的饋料批式醱酵方式,初期以 molasses 為主要的碳源,之後 的饋料主要是增加細胞濃度為主,有如此大量菌體就可產生較多的酵 素。由於連續提供養分使的菌得以長時間生長及分泌酵素,最後的細胞 濃度比以三角錐瓶醱酵培養增加了 3 倍,而酵素的活性也比三角錐瓶醱 酵培養提高了 2.7 倍,酵素活性約為 172 unit。

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4The nattokinase of functions………………………………………….13 2..i CHINESE ABSTRACT…….…………….......….…………………………….1 Natto introduction.03 1...2 Screening the optimal nitrogen sources……………………………....………….………………....11 2.......….vi Introduction1………………………………………………01 CHAPTER 1 1...ii CONTENTS….….....………………………………….……………….7 Batch fermentation…………………………………………………..12 2....11 2.………..…………………………………………..…...1 Microorganism isolation and maintenance………………………….com .v Figures…………………………………….....05 CHAPTER2 Materials and Methods…………………………………….14 iii PDF 檔案使用 "pdfFactory Pro" 試用版本建立 www...……………………….CONTENTS ENGLISH ABSTRACT………….……………………….…….11 2...iii List List Of Of Tables…....…01 1.04 1.3 Screening the optimal nitrogen sources concentration………………12 2.5 Screening the optimal carbon sources concentration………………..4 Screening the optimal carbon sources……………………………….2 Fibrin introduction………………………………………………….pdffactory.….13 2.3 Discovery of a fibrinolytic enzyme…………………………………..6 Screening the optimal pH and growth temperature…………………..…………..

....5 Effect of pH on enzyme activity……………………………………...1 Azocasein activity………………………………………….com ......3 Effect of sodium carbonate on the protease production……………..33 CHAPTER4 Conclusions…………….37 iv PDF 檔案使用 "pdfFactory Pro" 試用版本建立 www.…….....……...1 Effect of nitrogen sources on the protease production………………19 3.…….....……….4 Effect of growing temperature on the protease production………….8 Azocaseinase activity vs Fibrin plate assay vs Fibrinolytic activity of association………………………………………………......………………......25 3..9....15 2..7 Fed-batch fermentation………………………………………………31 3....2.…..2 Fibrinolytic activity…..15 2.19 3.pdffactory.9.…………….………………………………………..6 Batch fermentation………………………………………………….29 3...2 Effect of carbon sources on the protease production………………......…………………….3 Fibrin plate assay……………………………………………….9.........21 3..27 3.23 3.9 Enzyme assay………………………………………………………....16 CHAPTER3 Results and Discussion….....8 pH stat fed-Batch fermentation………………………………………14 2..............36 REFERENCES…....……….……15 2......

20 Table 3.22 Table 3.35 v PDF 檔案使用 "pdfFactory Pro" 試用版本建立 www...pdffactory..6 Azocaseinase activity vs Fibrin plate assay vs Fibrinolytic activity of association……………………………………………………………….4 Effect of growing temperature on the protease production…….2 Effect of carbon sources on the protease production…………….com .LIST OF TABLES Table 3.1 Effect of nitrogen sources on the protease production………….3 Effect of sodium carbonate on the protease production…………24 Table 3.26 Table 3.5 Optimum pH of the protease activity……………………………28 Table3.

10 Fig2.4 The fibrin and nattokinase of mechanism………………………….1 Batch fermentation………………………………………………30 Fig.3.pdffactory.3 Azocaseinase activity vs Fibrin plate assay vs Fibrinolytic activity of association………………………………………………………………….3.8 Fig1.1 Fibrin plate…………………………………………………………8 Fig1.LIST OF FIGURES Fig1.3.34 vi PDF 檔案使用 "pdfFactory Pro" 試用版本建立 www.2 Threads…………………………………………………………….3 Nattokinase of 3D structure and its active site︰Asp32、His64 、 Asn155及Ser221 ……………………………………………………………9 Fig1.32 Fig.com .1 Fibrin plate…………………………………………………………18 Fig.2 Fed-batch fermentation……………………………………………..

” or meat of the field. is the third most popular type of fermented soybean in the Japanese diet. natto has slightly less protein (16. the natto produces more calories. but this soybean is different﹗What makes it different is simple. Japan has the highest average longevity in the world.2grams). fiber.CHAPTER 1 INTRODUTION 1. and vitamin B2. calcium. This nickname appears well deserved. protein. Its high protein and economical price in terms of protein per gram has earned it the sobriquet “hata-ke no niko.1 Natto introduction Say It Ain’t Soy﹗Yes. the boiled soybeans metamorphose to an ancient medicinal food called “natto” pronounced “nah’-toe”.pdffactory. Natto may just be the “perfect food” producing 18 valuable amino acids and an enzyme nattokinase that may challenge the pharmaceutical industry’s best “blood-clot busters”.com . After hours of fermentation. 1 PDF 檔案使用 "pdfFactory Pro" 試用版本建立 www.5grams to 21. as in comparison with an equivalent amount of beef. which has recently attracted attention throughout the world. Natto. When compared with ordinary soybeans. which may partly be attributed to a high consumption of natto. but contains more carbohydrates and fiber. potassium.

and is also higher in calcium. the boiled soybeans were packed hastily into the rice straw bags without cooling or drying. One day. If the army was on a rapid deployment.pdffactory.D. Initially. To his astonishment. The rice straw just happened to contain a harmless and naturally occurring microorganism. Typically. Minamoto demonostrated tremendous courage and dipped his finger into the seemingly “rotten goo”. dried in the sun and packed immediately in rice straw bags for transport with the army. Bacillus subtilis that fermented the soybeans and produced natto with its characteristic sticky texture. the soybeans were presumed to have spoiled until Yoshiie Minamoto observed that his horses were “picky eaters” and demonstrated a preference for the “spoiled” soybeans or natto. it has nearly double the calcium and far more vitamin E to boot. ion and vitamin B2. Plus.). the first person to originate traditional Japanese natto was the famous warrior Yoshiie Minamoto during the Heian era of Japanese history (794-1192 A. the fermented soybeans were not only 2 PDF 檔案使用 "pdfFactory Pro" 試用版本建立 www. boiled soybeans were cooled down.com . The horse was an extremely important to the Japanese samurai warrior of the period and great care was given to provide suitable provisions for the horses when armies were on the move. phosphorous. According to legend.

and dissolve fibrin [3.5-8].edible but had a distinct Umami flavor. The most distinctive features of natto are the adhesive surrounding the soybeans and the strong flavor. especially people from other countries. however. Natto. Thrombolytic enzymes ( enzymes that break down blood clots ) are normally generated in the endothelial cells of 3 PDF 檔案使用 "pdfFactory Pro" 試用版本建立 www. Furthermore. heart and vascular diseases. it had recently been found that natto can help to prevent the viral infection O-157 [4.19]. 1. which has recently attracted attention throughout the world. high blood pressure [17-18].pdffactory. osteoporosis [9-14]. The sticky material has been show to consist of poly-g-glutamic acid (D and L) and polysaccharides and the strong “cheese-like” flavor is due to the presence of pyrazine. Mimamoto was responsible for introducing natto to northwestern Japan where he ruled. dysentery. these are the main factors which give natto the outstanding properties. This is essential to stop excess blood loss. To this day natto is especially popular in that region of Japan and a folk remedy for fatigue. atherosclerosis [15-16].com . These features sometimes make it hard for some people.2 Fibrin introduction Fibrin is a protein that forms in the blood after trauma or injury. beriberi. is a familiar part of the Japanese diet [1-3]. to accept natto.

such as in the arteries. making blood more prone to coagulation. as well as other conditions. M. production of these enzymes begins to decline. veins and lymphatic system.1). Sumi took the natto that he was eating for lunch and dropped a small portion into the artificial thrombus (fibrin) plate(fig. As the body ages. 1.2).3 Discovery of a fibrinolytic enzyme Dr. Since endothelial cells exist throughout the body. exhibit a strong fibrinolytic activity. Sumi found that the sticky part of natto. virtually anywhere in the body [5-8]. The natto gradually dissolved the thrombus and had completely resolved it in 18 hours﹗Dr. He was searching for a natural agent that could successfully dissolved thrombus associated with cardiac and cerebral infarction ( blood clots associated with heart attacks and stroke ). had long researched thrombolytic enzymes.com . Hiroyuki Sumi. One day in 1980 Dr.the blood vessels. commonly called “threads” (fig. poor production of thrombolytic enzymes can lead to the development of blood clots and the conditions caused by them. He named the corresponding fibrinolytic enzyme 4 PDF 檔案使用 "pdfFactory Pro" 試用版本建立 www. a researcher of the Japan Ministry of Education and majoring in the physiological chemistry at the blood laboratory of the University of Chicago. This mechanism can lead to cardiac or cerebral infraction.D.pdffactory.

8]. Sumi presented the results of his research in Japan for the first time at the Japan Agricultural Chemistry Society. but it 5 PDF 檔案使用 "pdfFactory Pro" 試用版本建立 www. wt and pI were about 27. There are many traditional foods for the prevention and treatment of thrombosis (e.728 Da and 8.pdffactory. Japanese water dropwort) but most of these foods inhibit platelet aggregation similar to aspirin.4 The nattokinase of functions Nattokinase was a single strain protein composed of 275 pieces of amino acids ( fig3). and the mol. Sumi commented that nattokinase showed “a potency matched by no other enzyme”.6± 0.. Dr. Only nattokinase acts only on the fibrinolytic system to dissolve within the blood vessels. The enzyme had a strong fibrinolytic activity at pH 6〜12. Dr.5.g. Sumi conducted research on about 200 kinds of food from all over world and he found that natto had the highest fibrinolyic activity among all those foods. Later he wrote a similar article for the International Thrombolytic Association where the audience began to believe that the dietary intake of natto was the major contributor to the longevity of Japanese people [3. Dr.com . azuki beans. 1.6. Korean ginseng.“nattokinase”. In 1986.3.

com . The nattokinase was easily extracted with saline [22]. More importantly. prolonged effects. It is a naturally occurring.Nattokinase may actually be superior to conventional clot-dissolving drugs such as recombinant tissue plasminogen activators (rt-PA).000 yen [3. including urokinase (endogenous) (fig4). Moreover. it also enhances the body’s production of both plasmin and other clot-dissolving agents.20-22]. the efficiency of a fibrinolytic injection lasts only 4 〜 20 minutes.pdffactory. but a packet of natto (about 50 grams) has the same efficacy as an amount of urokinase costing 200. The properties of nattokinase closely resemble those properties of plasmin so it dissolves fibrin directly. The average activity was calculated at about 40 CU(plasmin units)/g wet weight. whereas nattokinase maintains it activity for 2〜12 hours. and can be used preventatively. urokinase is often utilized as a fibrinolytic enzyme. In hospitals at present. 6 PDF 檔案使用 "pdfFactory Pro" 試用版本建立 www. cost effectiveness. Nattokinase is a particularly potent treatment because it enhances the body’s natural ability to fight blood clots in several different ways and has many benefits including convenience of oral administration. urokinase. food dietary supplement that has demonstrated stability in the gastrointestinal tract. confirmed efficacy.fibrinolytic activity slowed down when temperature was over 60℃.

Nattokinase.and strepkinase. may help prevent the conditions leading to blood clots with an oral daily dose of as little as 2.com .pdffactory.5-8.22]. 7 PDF 檔案使用 "pdfFactory Pro" 試用版本建立 www. however. which are only effect therapeutically when taken intravenously within 12 hours of a stroke or heart attack.000 fibrin units (FU) or 50 grams of natto [3.

2 Threads 8 PDF 檔案使用 "pdfFactory Pro" 試用版本建立 www. U. nattokinase extract. urokinase and P.com .Fig1.pdffactory. Fig1. plasmin were applied to a fibrin plate.1 Fibrin plate 10ml of N.

com .pdffactory..2005 9 PDF 檔案使用 "pdfFactory Pro" 試用版本建立 www.3 Nattokinase of 3D structure and its active site︰Asp32、His64 、 Asn155及Ser221 Zhenga et al.Fig1.

2003 10 PDF 檔案使用 "pdfFactory Pro" 試用版本建立 www.com .Fig1.pdffactory.4 The fibrin and nattokinase of mechanism A: direct dissolve fibrin B: active pro-urokinase to urokinase C: increase the amount t-PA 蘇.

CHAPTER 2 Material and Methods 2. 0. The natto was diluted in sterile water. 2. 0. A clear zone of milk hydrolysis gave an indication of protease producing organisms. and 100 ml milk. Depending upon the zone clearance. cells were removed by centrifugation. 2. CaCl2.2H2O. soybean meal. The diluted samples were plated onto milk agar plates containing( ﹪): beef extract. 2. supplemented with 4﹪of molasses and the 11 PDF 檔案使用 "pdfFactory Pro" 試用版本建立 www.3. After 48 hours of fermentation.2. YE. The isolates were maintained on milk agar plate and stored at 4℃. K2HPO4.5. (NH4)2SO4. In the investigation of nitrogen sources. and the supernatant were used for nattokinase activity assay.3.5. 0. 0.1.2 Screening the optimal nitrogen sources Five kinds of nitrogen sources were investigated at 37℃with shaking at 180 rpm for 48 hours. the microorganisms was selected for further experimental work. Nitrogen sources and corresponding concentration were( ﹪): peptone. peptone. 2.pdffactory. 2. 0. 0.com .1. casamino acid. Plates were incubated at 37℃for 24 h. corn steep liquor. growth was carried out in the minimal synthetic medium containing( ﹪): MgSO4.7H2O. 0.1 Microorganism isolation and maintenance Protease-producing isolates were isolated from the natto food (Hokaido)and screened using a milk agar plate [23].

Each observation was done in triplicae. 2.2. and the supernatant were used for nattokinase activity assay. supplemented with 4 ﹪of molasses and peptone to be investigated. and the supernatant were used for nattokinase activity assay. In the investigation of carbon sources. 0. 2. 12 PDF 檔案使用 "pdfFactory Pro" 試用版本建立 www. 0.0. 0.1.6. K2HPO4.1. maltose. 3. supplemented with 2 ﹪of peptone and the carbon sources to be investigated. CaCl2.2H2O.4 Screening the optimal carbon sources Five kinds of carbon sources were investigated at 37℃with shaking at 180 rpm for 48 hours. After 48 hours of fermentation.1.nitrogen sources to be investigated. cells were removed by centrifugation. growth was carried out in the minimal synthetic medium containing( ﹪): MgSO4.7H2O. sucrose.6. 3. 0.2. cells were removed by centrifugation. 2. 1.0. 0. CaCl2. 2H2O.0. Each observation was done in triplicate. K2HPO4. In the investigation of peptone concentration.1.pdffactory. growth was carried out in the minimal synthetic medium containing( ﹪): MgSO4.7H2O. Carbon sources were lactose. After 48 hours of fermentation.com . and molasses and the concentration of every carbon sources was ( ﹪). Peptone concentration were( ﹪): 0.3 Screening the optimal nitrogen sources concentration Five kinds of peptone concentration were investigated at 37℃with shaking at 180 rpm for 48 hours. glucose. 0.

and the supernatant were used for nattokinase activity assay. 2. 0.2. In the investigation of molasses concentration. 2. 37.5. 4. 2. After 48 hours of fermentation. Sodium carbonate concentration were ( ﹪): 0. MgSO4.7H2O.6 Screening the optimal pH and growth temperature Five kinds of sodium carbonate concentration and five kinds of growth temperature were investigated at 37℃with shaking at 180 rpm. 0.0.com .5. 13 PDF 檔案使用 "pdfFactory Pro" 試用版本建立 www.pdffactory. 40. 4. 0. 35. 1. 0.1. 3. peptone. 2. growth was carried out in the minimal synthetic medium containing( ﹪): MgSO4 7H2O.5 Screening the optimal carbon sources concentration Nine kinds of molasses concentration were investigated at 37℃with shaking at 180 rpm for 48 hours.2. 2.75. 1. K2HPO4. cells were removed by centrifugation. cells were removed by centrifugation. 0.25. 5. and the supernatant were used for nattokinase activity assay.0. supplemented with 2 ﹪of peptone and molasses to be investigated.Each observation was done in triplicate. 0.5.5.1. Each observation was done in triplicate.0.5. K2HPO4. After 48 hours of fermentation. 0.0 and growth temperature were ( ℃ ): 30.0. . 0. CaCl2.2H2O. 45. CaCl2. 2H2O.1.1. Molasses concentration were ( ﹪): 0. Cell grown in the medium composed of ( ﹪): molasses. 0. 3. 2.

The fermentor’s medium is the same seed medium.8 pH stat fed-Batch fermentation The grown cells from a milk agar plate were added to seed medium composed of ( ﹪): molasses. CaCl2.2H2O. 0.pdffactory.1. The cultivation was carried out for 48 hours at 37℃with agitation at 400 rpm aeration at 4 l min-1 1 l. 14 PDF 檔案使用 "pdfFactory Pro" 試用版本建立 www. The batch fermentation medium was composed of ( ﹪): molasses. 0.7 Batch fermentation The grown cells from a milk agar plate were added to seed medium composed of ( ﹪): molasses. Fermentation was performed in a laboratory 5 l fermentor with a working volume of 1 l. peptone. The subsequent cultivation lasted for 12 at 37℃hours and 180 rpm in an orbital shaker to obtain seed culture with a OD600 value of 11. 0. K2HPO4. 0. K2HPO4. 2.2 → 6.2.com . K2HPO4.25. 2.1. YE.2. and pH 6.2. 0. peptone. CaCl2. 2H2O. MgSO4.7H2O. Then seed culture was added to fermentor.8). and pH 6.2.1. 2.1. 0. 0. 2.5.625.3〜6. MgSO4.7H2O. CaCl2. 2H2O. 2. YE.5. The subsequent cultivation lasted for 12 at 37℃hours and 180 rpm in an orbital shaker to obtain seed culture with a OD600 value of 11. 2. 0. 5. When pH value risen to set point (pH 6. we fed the glucose-based medium composed of ( ﹪): glucose.1. 0.1. MgSO4.7H2O. 2. 11.3〜6.

225.4 ml Na3BO3(50 mM) and 0.2 Fibrinolytic activity(FU) 1. 0. Glasses were kept 10 min and centrifuged at 12000×g for 2 N NaOH. 2. Fermentation was performed in a laboratory 5 l fermentor with a working volume of 2 l. 0.4 ﹪(w/v) azocasein solution in tris-NaOH buffer ( 0.9.05. the activity was always expressed by this method.9 Enzyme assay 2.111.CaCl2.2H2O. Then 0. Na2HPO4.pdffactory. The cultivation was carried out for 51 hour at 37℃with agitation at 400 rpm aeration at 4 l min-1 1 l.226. 2. To this 10 ul enzyme was added.com .l ml of diluted enzyme 15 PDF 檔案使用 "pdfFactory Pro" 試用版本建立 www. MgSO4.7H2O. NH4Cl. To this 0.9. 0.1 Azocasein activity 340 ml of 0. 0. pH 10 ) was taken in a glass tube and kept in water bath (37℃) for 5 minutes. 10 min ).4 ml 0. unless said especially. After incubation ( 37℃.1 ml thrombin (20 U/ml) in Na3BO3 buffer (pH 8.0 in 30 min [23]. 50 mM ) was added and kept in water bath (37℃) for 10 minutes.1 M. Absorbance was measured at 420nm and appropriate blanks included. One unit enzyme activity was defined as the amount enzyme required to produce an increase in absorbance equal to 1.72 ﹪(w/v)fibrinogen solution were taken in a glass tube and kept in water bath (37℃) for 5 minutes. In results and discussion. 650 ul of 10 ﹪(w/v) trichloroacetic acid (TCA) was added.

/0. Let plate set at room temperature for 10 min.com .3 Fibrin plate assay Fibrinogen solution: 80 mg Human fibrinogen 7. 60 min ).06 ml H2O Thrombin (1000U/ml) solution : 20.pdffactory.62 g NaBarbital、500 ml 0.8 mg thrombin 1 ml Saline-Barbital gelatin buffer ( 5.1 2.12 g NaCl )、 1350 ml H2O) ,pH=7. Incubated plate at 37 16 PDF 檔案使用 "pdfFactory Pro" 試用版本建立 www.75。 1. 2 ml of 0.031 g NaBarbital 200 ml 、 、 0. Absorbance was measured at 275nm (Ar) and appropriate blanks (Ac) included. One unit enzyme activity was defined as the amount enzyme required to produce an increase in absorbance equal to 1.85 g NaCl 1. Sake gently after every well contains both solution.4 ml Fibrin plate buffer (20.98 g CaCl2.2H2O、2.1 M HCl、100 ml Salt Solution(4.9. Glasses were kept 20 min and centrifuged at 15000× g for 5 min.01×60×0.75。 Place 300 ul of fibrinogen solution and 10 ul thrombin solution into each well of a 24 well plate.2 M trichloroacetic acid (TCA) was added.0 in 60 min [3]. Nk (FU/ml)=(Ar-Ac)×diluted NO. After incubation ( 37℃.1 N HCl、25 g gelatine、725 ml H2O ),pH=7.78 g MgSO4.7H2O、190.was added.

After 10 min pour off TCA. Fibrinolytic activity was measured by the area cleared in the bottom of the well. The uncleared fibrin will remain milkly white.25]. Place 2 ul of diluted enzyme into each well.1).0 cm2[24. Put about 700 ul of 10 ﹪(w/v) trichloroacetic acid (TCA) into each well.℃ for 3 h. and the cleared area will be totally clear (fig2.com . 17 PDF 檔案使用 "pdfFactory Pro" 試用版本建立 www.pdffactory. Incubated at 37℃ overnight. One unit enzyme activity was defined as the amount enzyme required to produce an increase in area equal to 1.

and cleared area was active site.pdffactory.1 Fibrin plate The uncleared area was fibrin. 18 PDF 檔案使用 "pdfFactory Pro" 試用版本建立 www.Fig2.com .

1.3 ﹪(w/v) YE ( 19.com . 0.pdffactory.CHAPTER 3 RESULTS and DISCUSSION 3.6 U/ml).5 ﹪(w/v) (NH4)2SO4 (12. 0. cells produced protease when grown in a basal medium supplemented with 2 ﹪ (w/v) casamino acid ( 19. 19 PDF 檔案使用 "pdfFactory Pro" 試用版本建立 www.1 Effect of nitrogen sources on the protease production The effects of nitrogen sources were showed in table 3. In addition. peptone exhibited a prominent effect on the production activity and higher protease yield was achieved using a basal medium supplying 2 ﹪ (w/v) peptone (25. Among the different nitrogen sources test.8 U/ml).7 U/ml).7 U/ml).7 U/ml). 2 ﹪(w/v) soybean meal ( 7. 2 ﹪(w/v) corn steep liquor ( 12 U/ml).

0.7 23 .0 3 .3 18 .0 3 .1. 20 PDF 檔案使用 "pdfFactory Pro" 試用版本建立 www.7 Cells grown in the basal medium containing ( ﹪) molasses.0 2 .5 0 .2 and supplemented with each nitrogen sources at 37℃hours and 180 rpm in an orbital shaker. 4. 0.pdffactory.8 19 .Table 3.0 7 .6 12 .0 0 . MgSO4. 7H2O.com .6 1 .0 P ep to n e 2 . K2HPO4.5 19 .1 Effect of nitrogen sources on the protease production n itro gen so u rces co ncentration ﹪ en zym e activityU /m l 0 .0 2 .7 12 .4 24 .1 25 .4 casam in o acid co rn steep liq u or so yb eab m eal (N H 4 ) 2 S O 4 YE 2 .1. 0. CaCl2.2H2O.3 12 .

2 U/ml).6 U/ml). galactose (23. Of all the carbon sources that were investigated.5 ﹪(w/v) lactose (17. glucose (22.6 U/ml). cells produced protease when grown in a basal medium supplemented with 2.8 U/ml).3. sucrose (25. fructose (24.9 U/ml). maltose (28.2. 2 ﹪(w/v) molasses was the most promosing and the corresponding nattokinase activity was 63. In addition. 21 PDF 檔案使用 "pdfFactory Pro" 試用版本建立 www.com .3 U/ml.2 Effect of carbon sources on the protease production The effects of nitrogen sources were showed in table 3.pdffactory.2 U/ml).

0 2. 2.5 2.8 28.1.5 2. 0.4 33.5 5.2 25.6 22.6 43.9 Cells grown in the basal medium containing ( ﹪) peptone.7 30. MgSO4.7H2O.3 45.0 2.5 2.3 37. 0. K2HPO4.2 and supplemented with each carbon sources at 37℃hours and 180 rpm in an orbital shaker. 0.0 4.1.2 24.9 63.5 2.5 2.2 Effect of carbon sources on the protease production carbon sources concentration ﹪ enzyme activity U/ml molasses lactose sucrose glucose maltose galactose fructose 1. 22 PDF 檔案使用 "pdfFactory Pro" 試用版本建立 www.4 25.0 3.Table 3.5 2.5 37.8 17. CaCl2.2H2O.5 4.pdffactory.com .6 23.5 3.

The high concentration inhibit the nattokinase activity and the corresponding activity were 3.25 to 1 ﹪ (w/v).3 Effect of sodium carbonate on the protease production To investigate the effect of sodium carbonate on the protease production.52 ml-1 (table 3.3). 23 PDF 檔案使用 "pdfFactory Pro" 試用版本建立 www. An optimal protease production (63. sodium carbonate solution was sterilized separately and then added to a basal medium containing ( ﹪) peptone. 2 and molasses.pdffactory.3.com .17 U ml-1 and 2. 2 at a concentration ranging from 0.3 U/ml) was observed at a concentration of 0 ﹪(w/v) sodium carbonate.

pdffactory.2 and sodium carbonate at 37℃and 180 rpm in an orbital shaker. 2.1. 0. 2.5 0.3 Effect of sodium carbonate on the protease production Na2CO3 concentration(%) 0 0.7 50.52 Cells grown in the basal medium containing ( ﹪) peptone.1.75 1 Enzyme activity U/ml 63. 24 PDF 檔案使用 "pdfFactory Pro" 試用版本建立 www.17 2. MgSO4.7H2O. 0.3 60. K2HPO4. molasses.1 3. CaCl2.2H2O.25 0.com . 0.Table 3.

4 Effect of growing temperature on the protease production Growth temperature was another critical parameter that needs to be controlled. 25 PDF 檔案使用 "pdfFactory Pro" 試用版本建立 www.pdffactory.3 U/ml) was achieved when the cells were cultivated using optimized medium at 37℃with shaking at 180 rpm (table3.3. The optimum growth temperature for protease production was fond to be 37℃ and the highest yield (63.com .4).

4 Effect of growing temperature on the protease production temperature enzyme activity U/ml 30 35 37 40 45 34.0 Cells grown in the basal medium containing ( ﹪) peptone.0 55.2 and incubated with each temperature at pH 6. CaCl2.2H2O.Table 3. 0. molasses.0 63. 0.pdffactory. 2.com . K2HPO4.5 and 180 rpm in an orbital shaker.1.1.1 58. 2.0 35. MgSO4.7H2O. 26 PDF 檔案使用 "pdfFactory Pro" 試用版本建立 www. 0.

3.5 Effect of pH on enzyme activity The optimal pH for protease was determined to be around 10. and enzyme activity decreased rapidly below 7 (table3. 27 PDF 檔案使用 "pdfFactory Pro" 試用版本建立 www.com .pdffactory.5).

com .1 100.6 99.5 Optimum pH of the protease activity pH 7 8 9 10 11 enzyme activity U/ml 87.5 Activity of protease was measured at different pH value and at 37℃.0 112.6 108.pdffactory.Table 3. 28 PDF 檔案使用 "pdfFactory Pro" 試用版本建立 www.

However.6 Batch fermentation The results of a batch protease production by Bacillus natto hokaido was presented in Fig. pH started to rise again till it reach 9.pdffactory. maximum protease activity of 65.3. 29 PDF 檔案使用 "pdfFactory Pro" 試用版本建立 www.0 in 6 h then.3 U ml-1 was secreted at 30 h during the decelerating growth phase and subsequent stationary phase signifying non-growth associated production phase. The medium pH declined from 6. protease production increased with in cell mass and seemed to be growth associated.21 at 24 h. This might be due to a requirement of a critical cell mass level for starting protease synthesis. 3.5 to 6. Maximum cell growth was obtain at 24 h. A rise in culture pH due to production of free amino during protease fermentation.com .1. Following the initial 6 h. This decline of medium pH might be due to the result of acid production from molasses utilization during growth phase and once growth rate reduced. pH started to rise. Cell growth started immediately after inoculation of the bioreactor where as the secretion of protease could be observed after 6 h.

00 3.00 0.00 5. 0.00 50. CaCl2.2H2O. molasses.00 2.00 4.00 pH Cells grown in the basal medium containing ( ﹪) peptone.1 Batch fermentation 10. K2HPO4.1. 0.00 6.00 10. ◆OD600 ▲activity ■pH 30 PDF 檔案使用 "pdfFactory Pro" 試用版本建立 ÿÿ www.00 0 5 10 15 20 25 30 35 40 45 time (h) 7.00 0.pdffactory.2 and incubated for 48 hours at 37℃with agitation at 400 rpm aeration at 4 l min-1 1 l. 2.00 OD600 40.00 1.00 60.00 70.com .1. 2. 0.Fig.00 9.00 8. MgSO4.7H2O.3.00 30.00 20.

8) was obtained at 19. followed by a decelerating growth phase and subsequently.5 h. 3. fed-batch culture produced enhanced cell growth and protease activity. 1 L of medium was used for initial batch culture feeding ( at a constant rate of 80 ml h-1) was started after 4 h of fermenter inoculation using a pump. Maximum cell growth (OD600 99.5 h and maximum protease activity (172 U ml-1) was secreted at 16. 31 PDF 檔案使用 "pdfFactory Pro" 試用版本建立 www.pdffactory. the culture entered stationary phase.3. As compared with batch culture.7 Fed-batch fermentation The results of a fed-batch protease production by Bacillus natto hokaido was presented in Fig.2.com . Cell growth was observed for a short period (3-5 h).

com activity[uint] OD600nm pH .2 Fed-batch fermentation 10 120 180 160 9 100 140 8 80 120 100 7 60 80 40 60 40 6 5 20 OD600nm protease pH 20 0 0 3 6 9 12 15 18 21 24 27 30 33 36 39 42 45 48 51 4 0 Tim [h] e Cells grown in the basal medium and glucose-based medium incubated for 51 hours at 37℃with agitation at 400 rpm aeration at 4 l min-1 1 l. 32 PDF 檔案使用 "pdfFactory Pro" 試用版本建立 www.pdffactory.3.Fig.

3.com .6).pdffactory. 33 PDF 檔案使用 "pdfFactory Pro" 試用版本建立 www.3.table3.8 Azocaseinase activity vs Fibrin plate assay vs Fibrinolytic activity of association The azocasein activity is more bigger. and the ability is stronger to dissolve fibrin(fig3.

9881 2 250 200 150 100 50 0 100 150 200 250 300 350 400 Azocasein activity unit/ml □-pH=7.pdffactory.◇-pH=8 —azocaseinase activity vs fibrin plate assay --azocaseinase activity vs fibrinolytic activity 34 PDF 檔案使用 "pdfFactory Pro" 試用版本建立 www.3 Azocaseinase activity vs Fibrin plate assay vs Fibrinolytic activity of association 65000 60000 55000 50000 45000 40000 35000 30000 25000 20000 15000 10000 5000 0 0 50 450 R = 0.9297 350 300 R = 0.3.com Fibrinolytic activity FU/ml Fibrin plate assay cm /ml 2 400 2 .Fig.75.

3 61.com .Table3.5 46.9 54.5 357.5 43.5 69.6 Azocaseinase activity vs Fibrin plate assay vs Fibrinolytic activity of association azocaseinase activity U/ml 13.2 fibrinolytic activity Fu/ml 20.6 51.5 62.3 93.0 107.9 fibrin plate assay cm2/ml 0 11873 14130 9813 9813 9813 16583 19233 14130 19233 25120 56520 35 PDF 檔案使用 "pdfFactory Pro" 試用版本建立 www.8 91.6 91.1 72.8 102.2 95.1 416.pdffactory.5 59.7 52.3 88.1 82.6 139.9 62.

3.CHAPTER 4 COCLUSIONS 1.com . 2. MgSO4.7H2O.1. CaCl2.2H2O.pdffactory. Nattokinase is a particularly potent treatment because it enhances the body’s natural ability to fight blood clots in several different ways and has many benefits including convenience of oral administration.2. 36 PDF 檔案使用 "pdfFactory Pro" 試用版本建立 www. K2HPO4. Bacillus natto hokaido was grown in a 500 ml Erlenmeyer flask containing ( ﹪) peptone. confirmed efficacy. By using suitable feeding strategies.1.8) in a fed-batch process was increased by 207 ﹪ and 300 ﹪over that in batch process. cost effectiveness. prolonged effects. and can be used preventatively. 0. 2. A high level of extra cellular protease activity (63 U ml-1) was obtained at 37℃ for 48 hours and 180 rpm in an orbital shaker. 0. pH stat fed-batch operation was carried out in maximum biomass production . molasses. 2. the protease activity (172 U ml-1) and biomass production (OD600 99. 0. The optimum temperature and pH for activity were 37℃ and 10.

Hamada. Nomura. [8]Sumi H. Takahashi . H. Mihara and H. H. Ito.生物 3 資源生物技 術第四卷第一期,37-45。 [2]陸兆新. [6]Sumi. N. H.H. In vitro and in vivo fibrinolytic properties of nattokinase. H. K. Nishimuro. 1993. Nakanishi. Experientia. 43:1110-1111.2001。值得發展的保健食品-納豆,中國食品技術網。6:59。 [3]蘇遠志, 2003。 納豆代謝產物的開發與應用 。Bioindustry 14(2): , 45-58。 [4]鍾清萍. Kawamura . Acta Haematol. Nakajima.pdffactory.197(3):1340-1347.a typical and popular soybean in the Japanese diet. H.. K. 84:139-143. K. Sugimura and 37 PDF 檔案使用 "pdfFactory Pro" 試用版本建立 www.. [7]Fujita. [5]Sumi.89:1267.. Enhancement of the fibrinolytic Activity in Plasma by Oral Administration of Natto Kinase. M. M. T. [9]Fukutake. Hong. 1990. Asada and S. Ishida . Mihara. M. 1992. Purification and Characterization of a strong fibrinolytic enzyme (NATTOKINASE) in the vegetable cheese natto. A novel fibrinolytic enzyme in the vegetable cheese Natto .余世望. A. A popular soybean fermented food in Japan.com . Thromb Haemost. K.Muraki. Biochemical and Biophysical research communication. . H. Hamada. T. 1987.REFERENCES [1]施英隆、范宜琮著,2002。納豆~神奇之保健食品.梁生媛,2001。納豆菌的排菌作用及其培養基的優化。 食品工業科技。5:20-22. Hajime Hiratani.

Food and Chemical Toxicology. Koshihara. Role of K Vitamins in the regulation of tissue calcification.K. Taguchi. Y. Joumal of Bone and Mineral Metabolism. H. K. 2001. M.. 1. H. M. S. Tadano & Y. 1996.19:201-206. [11]Yamaguchi . N. Yamazaki & S. [10]Yamaguchi . Int. Taguchi. Osteoporosis International. A. Makabayashi. K. Ontachi . 38 PDF 檔案使用 "pdfFactory Pro" 試用版本建立 www. Quantification of Genistein and Genistin in Soybeans and Soybean Products . A. Nakao.com . Gao.T.Even in Those with Suspected Vitamin K. 1996. Gao. [13]Vermeer. Joumal of Bone and Mineral Metabolism.25-dihydroxyvitamin D3 Promotes Vitamin K2 Metabolism in Human Osteoblasts. Tsukamoto.Y.pdffactory. 2000. [12]Miyaka. The Effect of Antioxigant containing Fraction from Fermented Soybean Food on Atherosclerosis Development in Cholesterol -Fed Rabbits.18:71-76. Hattori. Lebensm.. M. Joumal of Bone and Mineral Metabolism. Sano.12:680-687. 17:23-29. Myou .. Vitamin K and Adminstration to Elderly Patient with Osteoporsis Induces No Hemostatic Activation . Katanabe . Hasegawa and K. [14]Asakura. Ohishi. C.. Saito. Effect of vitamin K2 (menaquinone-7) in fermented soybean (Natto) on bone loss in ovariectomized rats. 1999. T. Igarashi and Y. and L. 2001. H. Y. Kikuchi. Hoshi.. Y. Tsukamoto. 12:996-1000. H. Braam. Igarashi and Y. Mizutani.. 2001. [15]Yokota. K. Prolonged intake of fermented soybean (Natto) diets containing vitamin K2 (menaquinone-7) prevents bone loss in ovariectomized rats. T. K. M.. 34:457-461.

[16]Yokota. Z. Tsai. and H. Nishimuro. [19]Sumi. Sumi. 1995. [18]Okamoto.watanabe. Z. [21]Nakamura. Kawamura & F. T. 14:47. 2: 1-3. Liu. Biosci. Ichishima. A. Bioindustry. a novel fibrinolytic enzyme from Bacillus natto A novel nucleophilic catalytic mechanism for nattokinase.. 1995. [22]Fujita. Biochem. Basic and Clinical Aspects of Japanese Traditional Food Natto.natto plant foods for human nutrition 4:39-47.1992. [17]Maruyama. K.C. Z. H. 2002... M.L. Y.Wiss. Y. 9:157. 23 :373–380. Hong. 2005. Construction of a 3D model of nattokinase. Nucleotide sequence of the subtilisn NAT gene..G. Hanagata. Hasegawa and K. M. Ito. A. S. Yangida.U. Fibrinolysis . Journal of Molecular Graphics and Modelling. Antioxidative Functions of Natto.Food Chem. 1998. 1997.. Effect of natto diet on blood pressure. G. H.K. T. T. 29:751 -755.164. H.com . Anti-hypertensive Substance in fermented soybean .. Characterization of Nattokinase-degraded Products from Human Fibrinogen or Cross-linked Fibrin. Antibacterial activity of bacillus natto growth inhibition against Escherichia coli O-157. 56 (11) :1869.. Biotech. Ohishi .L. K.pdffactory. Zou.Y. Zuo. E. aprN of Bacillus Subtilis (natto).Yamagata.Agric . 39 PDF 檔案使用 "pdfFactory Pro" 試用版本建立 www. A kind of Fermented Soybean: Effect on LDL Oxidation and Lipid metabolism in Cholesterol –Fed Rats J.F. Hattori. Liu.50:3597-3601. Y. [20]Zheng..Technol.

I. J.pdffactory. C.Biotec.H.84(4):307-312. Samama. S. SSR1..36:781-785. Park.. Raven Press.T. Veldhuyzen-Stolk.K. H. Choi. Kim. Kim. in Davidson. J. [24]Kim.C. Jeong.. P. KA38 originated from fermented fish. Y. R.[23]Singh. W.K. D.C. [25]Kluft. 1997.1976. New York.S Kong. of fermentation and bioengineering. Sobti. Serine alkaline protease from a newly isolated Bacillus sp. Purification and characterization of a Novel fibrinolytic enzyme from Bacillus sp. G. Screening of fibrinolytic activity in plasma euglobulin fractions on the fibrin plate. Progress in chemical fibrinolysis and thrombolysis.com . Batra. Brakman. 2001.K. 2 :57-65 40 PDF 檔案使用 "pdfFactory Pro" 試用版本建立 www. Desnoyers. E. N.