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Optimization of media composition for Nattokinase production by Bacillus subtilis using response surface methodology
Bioresource Technology 99 (2008) 8170–8174
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Optimization of media composition for Nattokinase production by Bacillus subtilis using response surface methodology V. Deepak 1, K. Kalishwaralal 1, S. Ramkumarpandian 1, S. Venkatesh Babu 1, S.R. Senthilkumar, G. Sangiliyandi * Department of Biotechnology, Kalasalingam University, Krishnankoil 626190, Viruthunagar District, Tamil Nadu, India
a r t i c l e
i n f o
Article history: Received 31 December 2007 Received in revised form 8 March 2008 Accepted 11 March 2008 Available online 21 April 2008 Keywords: Nattokinase Bacillus subtilis Response surface methodology Central composite rotary design Fibrinolytic activity
a b s t r a c t Response surface methodology and central composite rotary design (CCRD) was employed to optimize a fermentation medium for the production of Nattokinase by Bacillus subtilis at pH 7.5. The four variables involved in this study were Glucose, Peptone, CaCl2, and MgSO4. The statistical analysis of the results showed that, in the range studied; only peptone had a significant effect on Nattokinase production. The optimized medium containing (%) Glucose: 1, Peptone: 5.5, MgSO4: 0.2 and CaCl2: 0.5 resulted in 2-fold increased level of Nattokinase (3194.25 U/ml) production compared to initial level (1599.09 U/ml) after 10 h of fermentation. Nattokinase production was checked with fibrinolytic activity. Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction Intravenous fibrinolytic agents such as streptokinase, urokinase and tissue plasminogen activator (t-PA) have been widely used in clinical practice for thrombolytic therapy since the 1960s, 1970s and 1980s, respectively. However, the biological activity of these substances is short lived in the circulation after intravenous administration and there is a significant risk of hemorrhagic complications with a pronounced activation of fibrinolytic activity in whole blood in some circumstances (Thomas et al., 1996; Goodchild and Boylan, 1992; Hanaway et al., 1972). Nattokinase enzyme has been reported to have potent fibrinolytic activity (Sumi et al., 1987). Recently, similar fibrinolytic enzyme-producing bacteria have also been isolated from Chinese douchi (Peng et al., 2003) and Korea chungkookjang (Kim et al., 1996). Based on its food origin and relatively strong fibrinolytic activity, Nattokinase has advantages over other commercially used medicine in preventative and prolonged effects, convenient oral administration, and stability in the gastrointestinal tract (Sumi et al., 1990). Nattokinase has potent fibrinolytic activity, enhances plasminogen activators and inactivates a plasminogen activator inhibitor (Sumi et al., 1987; Urano et al., 2001). It also has fibrinolytic activity when administered orally (Sumi et al., 1990; Suzuki et al., 2003) and is widely
* Corresponding author. Tel.: +91 4563 289042; fax: +91 4563 289322. E-mail address: [email protected] (G. Sangiliyandi). 1 Contributed equally to this work. 0960-8524/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2008.03.018
available in processed and health foods containing natto (fermented soybean) extracts. Nattokinase might be an effective pharmacologic adjunct for surgery to induce posterior vitreous detachment in patients with vitreoretinal disorders (Sumi et al., 1990). Bioprocess technologies require effective problem solving methods because they involve multiple parameters to adjust and complications that inhibit application of engineering principles. Additional obstacles for bioprocess research include the lack of an accurate mathematical model equation to describe the whole process, high noise levels, interactions among variables, and complex biochemical reactions. This condition calls for a good strategy to deal with such a complicated system. Statistically designed experiments use a small set of carefully planned experiments. This method is more satisfactory and effective than other methods such as classical one-at-a-time or mathematical methods because it can study many variables simultaneously with a low number of observations, saving time and costs. The statistical experimental design provides a universal language with which people from different areas such as academia, engineering, business, and industry can communicate for setting, performing, and analyzing experiments for research (Montogomery, 2001; Myers and Montogomery, 2002). Response surface methodology (RSM) is a well-known method applied in the optimization of medium constituents and other critical variables responsible for the production of biomolecules (Ya-Hong et al., 2004). A novel fermentation medium is of critical importance because medium composition can significantly affect product yield. Culture
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medium optimization by the traditional ‘one-factor-at-a-time’ technique requires a considerable amount of work and time. An alternate strategy is a statistical approach, e.g. factorial experimental design and response surface methodology (RSM), involving a minimum number of experiments for a large number of factors, by which improvement in enzyme production has been successfully demonstrated (Ghanem et al., 2000; Bocchini et al., 2002; Senthilkumar et al., 2005). These methods have also been employed to improve the production of Nattokinase (Liu et al., 2005). The present work describes the successful optimization of a culture medium for the hyperproduction of Nattokinase by Bacillus subtilis.
2. Methods 2.1. Chemicals Culture media and analytical chemicals were purchased from Hi-Media, Mumbai, India. Fibrinogen and Thrombin were purchased from Sigma chemical Co, CA, USA. 2.2. Microorganism and inoculum’s preparation B. subtilis is a gift from Dr. P. Gunasekaran, Madurai Kamaraj University (India). The strain was previously obtained from Bacillus genetic stock center, Ohio, US (strain no. 1A752, B. subtilis). The culture was maintained at 4 °C in nutrient agar slants. 2.3. Production of Nattokinase Nattokinase is produced by B. subtilis in a shake flask culture. The unoptimized fermentation medium is composed of (%): Glucose – 1.0; Peptone – 3.0; MgSO4 – 0.2 and CaCl2 – 0.5. The pH of the medium was adjusted to 7.0. Erlenmeyer flasks (250 ml) containing 45 ml of the culture medium were inoculated with 5 ml of the seed culture. The flasks were incubated at 37 °C for 10 h and the culture was centrifuged at 4000g and at 4 °C for 10 min and the clear supernatant was assayed for Nattokinase activity. For optimization studies, the composition of the culture medium was varied according to the experimental design, while the pH, temperature and time of fermentation were not varied. 2.4. Evaluation of the fibrinolytic activity The fibrinolytic activity was observed by artificial blood clot degradation. An artificial blood clot was made by spontaneous coagulation in a glass test tube using fresh human blood obtained from healthy young male volunteer with written informed consents. After 60 min, the artificial blood clot was rinsed out repeatedly using phosphate buffer (pH 7.0). The artificial blood clot was dipped in Nattokinase solution at room temperature for 60 min. For control, normal saline was used (Omura et al., 2005). 2.5. Statistical analysis The statistical software, Design-Expert, Stat-Ease, Inc., Minneapolis, USA was used for regression analysis of experimental data and to plot response surface. ANOVA was used to estimate the statistical parameters. 2.6. Optimization of medium for Nattokinase production A factorial central composite rotatory design (CCRD) for four factors with replicates at the centre point and star points was used. The variables used were Glucose, Peptone, MgSO4, and CaCl2 each at five coded levels (a, 1, 0, +1, +a) as shown in Table 2. The actual levels
Table 1 The variables and their levels for the central composite experimental design Independent variables (%)
Symbols
Glucose Peptone Magnesium sulphate Calcium chloride
A B C D
Code levels a
1
0
+1
+a
0.50 0.50 0.04 0.10
0.75 1.75 0.12 0.30
1.00 3.00 0.20 0.50
1.25 4.25 0.28 0.70
1.50 5.50 0.36 0.90
of variables for CCRD experiments were selected with reference to the composition of the initial culture medium as the centre points. The CCRD contains a total of 30 experimental trials that include 16 trials for factorial design, 8 trials for axial points (2 for each variable) and 6 trials for replications of the central points. The response value (Y) in each trial was average of duplicates (see Table 1). 2.7. Nattokinase activity Tris–HCl (50 mM, pH 7.5) of 1.3 ml and 0.4 ml of 0.72% (w/v) fibrinogen solution were taken in vials and kept in water bath (37 °C) for 5 min. Then 0.1 ml thrombin (20 U/ml) was added and kept in water bath (37 °C) for 10 min. To this clot, 0.l ml of enzyme was added. After incubation (37 °C, 60 min), 2 ml of 0.2 M trichloroacetic acid (TCA) was added. Vials were kept 20 min and centrifuged at 3000 g for 5 min. One unit enzyme activity is defined as the amount enzyme required to produce an increase in absorbance equal to 1.0 in 60 min at 275 nm (Shih-Yu Wu, 2005). 3. Results 3.1. Evaluation of the fibrinolytic effect The blood clot degradation was observed in the test tube containing Nattokinase by dissolution of the clot indicating the degraTable 2 Experimental design and results of the central composite design Run
Factor 1 glucose (%)
Factor 2 peptone (%)
Factor 3 MgSO4 (%)
Factor 3 CaCl2 (%)
Nattokinase activity (U/ml)
1 2a 3 4 5 6 7a 8 9 10 11 12a 13 14 15 16 17 18a 19 20 21 22 23 24 25 26a 27 28 29 30a
0.75 1 1.25 0.75 1.25 1.25 1 0.75 1.25 0.75 1.25 1 1.25 0.75 0.75 0.75 1.25 1 1.25 0.75 1 1.5 1 1 1 1 1 1 0.5 1
1.75 3 1.75 1.75 4.25 4.25 3 4.25 1.75 4.25 4.25 3 1.75 4.25 1.75 1.75 4.25 3 1.75 4.25 3 3 3 3 5.5 3 3 0.5 3 3
0.28 0.2 0.12 0.12 0.28 0.12 0.2 0.12 0.28 0.28 0.12 0.2 0.12 0.12 0.28 0.12 0.28 0.2 0.28 0.28 0.04 0.2 0.36 0.2 0.2 0.2 0.2 0.2 0.2 0.2
0.3 0.5 0.3 0.7 0.3 0.7 0.5 0.3 0.7 0.7 0.3 0.5 0.7 0.7 0.7 0.3 0.7 0.5 0.3 0.3 0.5 0.5 0.5 0.9 0.5 0.5 0.1 0.5 0.5 0.5
1158.56 1599.09 988.59 985.125 1949.25 2147.15 1599.09 2053.5 159.56 2670.93 2539.12 1599.09 1238.34 2743.78 815.15 912.3 2941.5 1599.09 2143.68 892.01 1239.01 1200.18 728.43 468.28 3194.25 1599.09 770.06 1390.96 364.68 1599.09
a
Center points.
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replications of the central point and eight axial points are shown in Table 2. The responses of the CCRD design were fitted with a second-order polynomial equation
Table 3 Results of the regression analysis of the central composite rotatory design Source
Mean square
F-value
Prob > F
Model A-glucose B-peptone C-MgSO4 D-CaCl2 AB AC AD BC BD CD A2 B2 C2 D2 Lack of fit
9.985E + 005 5.242E + 005 7.197E + 006 1.502E + 005 8853.70 19443.86 1.294E + 005 6.942E + 005 87387.49 1.609E + 006 1963.49 4.266E + 005 1.753E + 006 1.518E + 005 7.516E + 005 2.516E + 005
5.16 2.71 37.19 0.78 0.046 0.10 0.67 3.59 0.45 8.32 0.010 2.20 9.06 0.78 3.88
0.0027 0.1238 <0.0001 0.3944 0.8340 0.7563 0.4283 0.0807 0.5134 0.0128 0.9213 0.1614 0.0100 0.3919 0.0704 0.0027
Nattokinase activity ¼ 1599:09 þ 147:78A þ 547:60B 79:10 þ 19:21D þ 34:86AB þ 89:93AC 208:29AD 73:90BC þ 317:15BD 11:08CD 124:72 þ 252:83B2 74:40C2 165:53D2
ð1Þ
Except for the linear term A (p < 0.05) and quadratic term A square (p < 0.05), none of the other linear, quadratic and interaction terms were statistically significant (Table 3). The overall second-order polynomial equation for Nattokinase production was where, Y is Nattokinase activity (U/ml of substrate); A is Glucose (%); B is Peptone (%); C is Magnesium sulphate (%); D is Calcium chloride. The statistical significance of the model equation was evaluated by the F-test for analysis of variance (ANOVA), which showed that the regression is statistically significant at 99% (p < 0.05) confidence level. The model F-value of 5.16 for Nattokinase production implies that the model is statistically significant. There is only a 3.03% chance that a ‘‘Model F-Value’’ this large could occur due to noise. The value of p > F less than 0.05 indicates that the model terms are also significant. The coefficient of determination (R2) was calculated to be 0.8475, indicating that the model could explain 84% of the variability. The ‘‘lack of fit test’’
dation of the fibrin net. In contrast test tube of normal saline as control, clot degradation was not observed. In order to improve the production of Nattokinase, medium optimization was performed using response surface methodology. 3.2. Response surface methodology The experimental results of Nattokinase production by a complete four-factor-two-level factorial experiment design with six
Nattokinase activity
3200
2475
1750
1025
300
4.25
1.25 3.63
1.13 3.00
B: Peptone
1.00 2.38
0.88 1.75
0.75
A: Glucose
Nattokinase activity
3200
2500
1800
1100
400
0.70
4.25 0.60
3.63 0.50
D: CaCl 2
3.00 0.40
2.38 0.30
1.75
B: Peptone
Fig. 1. Response surface plot for Nattokinase production by B. subtilis. The interaction between (a) Glucose and Peptone, (b) Peptone and MgSO4 and (c) Peptone and CaCl2.
V. Deepak et al. / Bioresource Technology 99 (2008) 8170–8174
Nattokinase activity (U/ml)
3200
B
2425
A DC
C D AB
1650
875
100
-1.000
-0.500
0.000
0.500
1.000
Deviation from Reference Point (Coded Units) Fig. 2. Perturbation graph showing the effect of each of the independent variables on Nattokinase production while keeping other variables at their respective midpoint levels. (A) Glucose, (B) Peptone, (C) MgSO4 and (D) CaCl2.
compares the residual error to the ‘‘Pure Error’’ from replicated design points. The ‘‘lack of fit F-value’’ of 5.6 implies the lack of fit is significant. There is only 0.48% chance that a ‘‘lack of fit F-value’’ this large could occur due to noise. Thus, the estimated models fit the experimental data adequately. ‘‘Adeq. Precision” measures the signal (response) to noise (deviation) ratio. A ratio greater than 4 is desirable. The ratio of 8.7 indicates an adequate signal and therefore the model is significant for the process. Three-dimensional response surfaces were plotted on the basis of the model equation, to investigate the interaction among the variables and to determine the optimum concentration of each factor for maximum Nattokinase production by B. subtilis. The effects of varying the concentration of peptone and one of the other variables are shown in Fig. 1, which demonstrates that the response surfaces for the three combinations were similar to each other. A small through in the response surface indicates an initial decrease in Nattokinase production with the initial increase of peptone (0.5– 3%) before a steep increase in Nattokinase production with a further increase in concentration (3–5.5%). From the response surface (Fig. 1) and perturbation plot (Fig. 2) it is obvious that peptone had a significant effect on Nattokinase production compared to other variables. The metal ions such as Ca2+ and Mg2+ increased and stabilized the Nattokinase activity of the enzyme; this is possible because of the activation by the metal ions. The perturbation graph clearly shows that the three components A, C, D did not have significant role in the enzyme synthesis. The component B has a major role in the production of Nattokinase also it is the limiting factor for the production of Nattokinase. The Nattokinase production was 1599 U/ml of substrate in the original medium with the four components at their central levels. The maximum Nattokinase yield is 3194.25 U/ml of substrate in optimized medium composed of (%): Glucose (1.0); Peptone (5.5); MgSO4 (0.2); and CaCl2 (0.5) which is 2-fold higher than the unoptimized medium. 4. Discussion Sumi et al. (1987) reported the presence of a strong fibrinolytic enzyme (Nattokinase) in natto (a fermented food), and Nattokinase may be an equally good protease for oral fibrinolytic therapy
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because of its confirmed safety for long-term intake, stability and the strong fibrinolytic activity. The fermented soybeans produced using bacteria, however, may have a preventive effect on not only thrombosis but also cancer. Statistical experimental design has not been widely used in the biological sciences even though it has been commonly employed in many other areas such as industrial, chemical, engineering, agricultural, medical, and food sciences. The primary reason for this is that most biological research has not been involved in many manufacturing processes. However, since genetic engineering, biomaterials, and bioprocess technologies like biodegradation and bioremediation have emerged, more people are getting interested in experimental designs to improve their biological processes and productions by shortening time and increasing efficiencies (Kwang-Min and David, 2005). The fermentation media for Nattokinase production was optimized using statistical method. According to the previous report using the medium components peptone, calcium chloride and yeast extract resulted in maximum Nattokinase activity of 1300 U/ml (Liu et al., 2005). In case of our report using the medium components Glucose, Peptone, Calcium chloride and Magnesium sulphate resulted in maximum Nattokinase activity of 3194.25 U/ml. In addition to establishing optimal fermentation medium compositions, the present methodology also makes it possible to predict the yield if the composition of the medium is altered in some way, by using the quadratic equation (Eq. (1)). Central composite experimental design maximizes the amount of information that can be obtained, while limiting the numbers of individual experiments required. Thus small and less time consuming experimental designs could generally suffice for the optimization of many fermentation processes. Therefore, with the increase in yield and productivity and simultaneous cost reduction, the industrial Nattokinase production by B. subtilis can be regarded as possible and economically attractive. References Bocchini, D.A., Alves-Prado, H.F., Baida, L.C., Roberto, I.C., Gomes, E., Da Silva, R., 2002. Optimization of xylanase production by Bacillus circulans D1 in submerged fermentation using response surface methodology. Proc. Biochem. 38, 727–731. Goodchild, C.S., Boylan, M.K., 1992. Reversal of streptokinase-induced bleeding with aprotinin for emergency cardiac surgery. Anaesthesia 47, 226–228. Ghanem, N.B., Yusef, H.H., Mahrouse, H.K., 2000. Production of Aspergillus terreus xylanase in solid state cultures: application of the Plackett–Burman experimental design to evaluate nutritional requirements. Biores. Technol. 73, 113–121. Hanaway, J., Torack, R., Fletcher, A.P., Landau, W.M., 1972. Intracranial bleeding associated with urokinase therapy for acute ischemic hemispherical stroke. Stroke 7, 143–146. 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, 2482–2488. Kwang-Min, L., David, F.G., 2005. Formulation and process modeling of biopolymer (polyhydroxyalkanoates: PHAs) production from industrial wastes by novel crossed experimental design. Process Biochem. 40, 229–246. Liu, J., Xing, J., Chang, T., Ma, Z., Liu, H., 2005. Optimization of nutritional condition for Nattokinase production by Bacillus natto NLSSE using statistical experimental methods. Process. Biochem. 40, 2757–2762. Montogomery, D.C., 2001. Design and Analysis of Experiments, fifth ed. John Wiley and Sons, NY. Myers, R.H., Montogomery, D.C., 2002. Response Surface Methodology: Process and Product Optimization Using Designed Experiments, second ed. John Wiley and Sons, NY. Omura, K., Hitosugi, M., Zhu, X., Ikeda, M., Maeda, H., Tokududome, S., 2005. A newly derived protein from Bacillus subtilis natto with both antithrombotic and fibrinolytic effects. J. Pharmacol. Sci. 99, 247–251. 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. Senthilkumar, S.R., Ashokkumar, B., Chandra Raj, K., Gunasekaran, P., 2005. Optimization of medium composition for alkali-stable xylanase production by Aspergillus fischeri Fxn 1 in solid-state fermentation using central composite rotary design. Biores. Technol. 96, 1380–1386.
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Sumi, H., Hamada, H., Tsushima, H., 1987. A novel fibrinolytic enzyme (Nattokinase) in the vegetable cheese Natto; a typical and popular soybean food in the Japanese diet. Experientia 43, 1110–1111. Sumi, H., Hamada, H., Nakanishi, K., Hiratani, H., 1990. Enhancement of the fibrinolytic activity in plasma by oral administration of Nattokinase. Acta. Haematol. 84, 139–143. Suzuki, Y., Kondo, K., Matsumoto, Y., Zhao, B.Q., Otsuguro, K., Maeda, T., Tsukamoto, Y., Urano, T., Umemura, K., 2003. Dietary supplementation of fermented soybean, natto, suppresses intimal thickening and modulates the lysis of mural thrombi after endothelial injury in rat femoral artery. Life Sci. 73, 1289– 1298. Thomas, G.R., Thibodeaux, H., Errett, C.J., Badillo, J.M., Wu, D.T., Refino, C.J., Keyt, B.A., Bennett, W.F., 1996. Limiting systemic plasminogenolysis reduces the
bleeding potential for tissue-type plasminogen activators but not for streptokinase. Thromb. Haemost. 75, 915–920. Urano, T., Ihara, H., Umemura, K., 2001. The profibrinolytic enzyme subtilisin NAT purified from Bacillus subtilis cleaves and inactivates plasminogen activator inhibitor type 1. J. Biol. Chem. 276, 24690–24696. Wu, S.Y., 2005. Optimization of nutritional conditions for Nattokinase production by a isolated Bacillus subtilis from natto health food. A Master of Science Thesis, Tatung University. Ya-Hong, X., Jian-Zhong, L., Hai-Yan, S., Liang-Nian, J., 2004. Enhanced production of extracellular ribonuclease from Aspergillus niger by optimization of culture conditions using response surface methodology. Biochem. Eng. J. 21, 27–32.
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Optimization of media composition for Nattokinase production by Bacillus subtilis using response surface methodology V. Deepak 1, K. Kalishwaralal 1, S. Ramkumarpandian 1, S. Venkatesh Babu 1, S.R. Senthilkumar, G. Sangiliyandi * Department of Biotechnology, Kalasalingam University, Krishnankoil 626190, Viruthunagar District, Tamil Nadu, India
a r t i c l e
i n f o
Article history: Received 31 December 2007 Received in revised form 8 March 2008 Accepted 11 March 2008 Available online 21 April 2008 Keywords: Nattokinase Bacillus subtilis Response surface methodology Central composite rotary design Fibrinolytic activity
a b s t r a c t Response surface methodology and central composite rotary design (CCRD) was employed to optimize a fermentation medium for the production of Nattokinase by Bacillus subtilis at pH 7.5. The four variables involved in this study were Glucose, Peptone, CaCl2, and MgSO4. The statistical analysis of the results showed that, in the range studied; only peptone had a significant effect on Nattokinase production. The optimized medium containing (%) Glucose: 1, Peptone: 5.5, MgSO4: 0.2 and CaCl2: 0.5 resulted in 2-fold increased level of Nattokinase (3194.25 U/ml) production compared to initial level (1599.09 U/ml) after 10 h of fermentation. Nattokinase production was checked with fibrinolytic activity. Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction Intravenous fibrinolytic agents such as streptokinase, urokinase and tissue plasminogen activator (t-PA) have been widely used in clinical practice for thrombolytic therapy since the 1960s, 1970s and 1980s, respectively. However, the biological activity of these substances is short lived in the circulation after intravenous administration and there is a significant risk of hemorrhagic complications with a pronounced activation of fibrinolytic activity in whole blood in some circumstances (Thomas et al., 1996; Goodchild and Boylan, 1992; Hanaway et al., 1972). Nattokinase enzyme has been reported to have potent fibrinolytic activity (Sumi et al., 1987). Recently, similar fibrinolytic enzyme-producing bacteria have also been isolated from Chinese douchi (Peng et al., 2003) and Korea chungkookjang (Kim et al., 1996). Based on its food origin and relatively strong fibrinolytic activity, Nattokinase has advantages over other commercially used medicine in preventative and prolonged effects, convenient oral administration, and stability in the gastrointestinal tract (Sumi et al., 1990). Nattokinase has potent fibrinolytic activity, enhances plasminogen activators and inactivates a plasminogen activator inhibitor (Sumi et al., 1987; Urano et al., 2001). It also has fibrinolytic activity when administered orally (Sumi et al., 1990; Suzuki et al., 2003) and is widely
* Corresponding author. Tel.: +91 4563 289042; fax: +91 4563 289322. E-mail address: [email protected] (G. Sangiliyandi). 1 Contributed equally to this work. 0960-8524/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2008.03.018
available in processed and health foods containing natto (fermented soybean) extracts. Nattokinase might be an effective pharmacologic adjunct for surgery to induce posterior vitreous detachment in patients with vitreoretinal disorders (Sumi et al., 1990). Bioprocess technologies require effective problem solving methods because they involve multiple parameters to adjust and complications that inhibit application of engineering principles. Additional obstacles for bioprocess research include the lack of an accurate mathematical model equation to describe the whole process, high noise levels, interactions among variables, and complex biochemical reactions. This condition calls for a good strategy to deal with such a complicated system. Statistically designed experiments use a small set of carefully planned experiments. This method is more satisfactory and effective than other methods such as classical one-at-a-time or mathematical methods because it can study many variables simultaneously with a low number of observations, saving time and costs. The statistical experimental design provides a universal language with which people from different areas such as academia, engineering, business, and industry can communicate for setting, performing, and analyzing experiments for research (Montogomery, 2001; Myers and Montogomery, 2002). Response surface methodology (RSM) is a well-known method applied in the optimization of medium constituents and other critical variables responsible for the production of biomolecules (Ya-Hong et al., 2004). A novel fermentation medium is of critical importance because medium composition can significantly affect product yield. Culture
8171
V. Deepak et al. / Bioresource Technology 99 (2008) 8170–8174
medium optimization by the traditional ‘one-factor-at-a-time’ technique requires a considerable amount of work and time. An alternate strategy is a statistical approach, e.g. factorial experimental design and response surface methodology (RSM), involving a minimum number of experiments for a large number of factors, by which improvement in enzyme production has been successfully demonstrated (Ghanem et al., 2000; Bocchini et al., 2002; Senthilkumar et al., 2005). These methods have also been employed to improve the production of Nattokinase (Liu et al., 2005). The present work describes the successful optimization of a culture medium for the hyperproduction of Nattokinase by Bacillus subtilis.
2. Methods 2.1. Chemicals Culture media and analytical chemicals were purchased from Hi-Media, Mumbai, India. Fibrinogen and Thrombin were purchased from Sigma chemical Co, CA, USA. 2.2. Microorganism and inoculum’s preparation B. subtilis is a gift from Dr. P. Gunasekaran, Madurai Kamaraj University (India). The strain was previously obtained from Bacillus genetic stock center, Ohio, US (strain no. 1A752, B. subtilis). The culture was maintained at 4 °C in nutrient agar slants. 2.3. Production of Nattokinase Nattokinase is produced by B. subtilis in a shake flask culture. The unoptimized fermentation medium is composed of (%): Glucose – 1.0; Peptone – 3.0; MgSO4 – 0.2 and CaCl2 – 0.5. The pH of the medium was adjusted to 7.0. Erlenmeyer flasks (250 ml) containing 45 ml of the culture medium were inoculated with 5 ml of the seed culture. The flasks were incubated at 37 °C for 10 h and the culture was centrifuged at 4000g and at 4 °C for 10 min and the clear supernatant was assayed for Nattokinase activity. For optimization studies, the composition of the culture medium was varied according to the experimental design, while the pH, temperature and time of fermentation were not varied. 2.4. Evaluation of the fibrinolytic activity The fibrinolytic activity was observed by artificial blood clot degradation. An artificial blood clot was made by spontaneous coagulation in a glass test tube using fresh human blood obtained from healthy young male volunteer with written informed consents. After 60 min, the artificial blood clot was rinsed out repeatedly using phosphate buffer (pH 7.0). The artificial blood clot was dipped in Nattokinase solution at room temperature for 60 min. For control, normal saline was used (Omura et al., 2005). 2.5. Statistical analysis The statistical software, Design-Expert, Stat-Ease, Inc., Minneapolis, USA was used for regression analysis of experimental data and to plot response surface. ANOVA was used to estimate the statistical parameters. 2.6. Optimization of medium for Nattokinase production A factorial central composite rotatory design (CCRD) for four factors with replicates at the centre point and star points was used. The variables used were Glucose, Peptone, MgSO4, and CaCl2 each at five coded levels (a, 1, 0, +1, +a) as shown in Table 2. The actual levels
Table 1 The variables and their levels for the central composite experimental design Independent variables (%)
Symbols
Glucose Peptone Magnesium sulphate Calcium chloride
A B C D
Code levels a
1
0
+1
+a
0.50 0.50 0.04 0.10
0.75 1.75 0.12 0.30
1.00 3.00 0.20 0.50
1.25 4.25 0.28 0.70
1.50 5.50 0.36 0.90
of variables for CCRD experiments were selected with reference to the composition of the initial culture medium as the centre points. The CCRD contains a total of 30 experimental trials that include 16 trials for factorial design, 8 trials for axial points (2 for each variable) and 6 trials for replications of the central points. The response value (Y) in each trial was average of duplicates (see Table 1). 2.7. Nattokinase activity Tris–HCl (50 mM, pH 7.5) of 1.3 ml and 0.4 ml of 0.72% (w/v) fibrinogen solution were taken in vials and kept in water bath (37 °C) for 5 min. Then 0.1 ml thrombin (20 U/ml) was added and kept in water bath (37 °C) for 10 min. To this clot, 0.l ml of enzyme was added. After incubation (37 °C, 60 min), 2 ml of 0.2 M trichloroacetic acid (TCA) was added. Vials were kept 20 min and centrifuged at 3000 g for 5 min. One unit enzyme activity is defined as the amount enzyme required to produce an increase in absorbance equal to 1.0 in 60 min at 275 nm (Shih-Yu Wu, 2005). 3. Results 3.1. Evaluation of the fibrinolytic effect The blood clot degradation was observed in the test tube containing Nattokinase by dissolution of the clot indicating the degraTable 2 Experimental design and results of the central composite design Run
Factor 1 glucose (%)
Factor 2 peptone (%)
Factor 3 MgSO4 (%)
Factor 3 CaCl2 (%)
Nattokinase activity (U/ml)
1 2a 3 4 5 6 7a 8 9 10 11 12a 13 14 15 16 17 18a 19 20 21 22 23 24 25 26a 27 28 29 30a
0.75 1 1.25 0.75 1.25 1.25 1 0.75 1.25 0.75 1.25 1 1.25 0.75 0.75 0.75 1.25 1 1.25 0.75 1 1.5 1 1 1 1 1 1 0.5 1
1.75 3 1.75 1.75 4.25 4.25 3 4.25 1.75 4.25 4.25 3 1.75 4.25 1.75 1.75 4.25 3 1.75 4.25 3 3 3 3 5.5 3 3 0.5 3 3
0.28 0.2 0.12 0.12 0.28 0.12 0.2 0.12 0.28 0.28 0.12 0.2 0.12 0.12 0.28 0.12 0.28 0.2 0.28 0.28 0.04 0.2 0.36 0.2 0.2 0.2 0.2 0.2 0.2 0.2
0.3 0.5 0.3 0.7 0.3 0.7 0.5 0.3 0.7 0.7 0.3 0.5 0.7 0.7 0.7 0.3 0.7 0.5 0.3 0.3 0.5 0.5 0.5 0.9 0.5 0.5 0.1 0.5 0.5 0.5
1158.56 1599.09 988.59 985.125 1949.25 2147.15 1599.09 2053.5 159.56 2670.93 2539.12 1599.09 1238.34 2743.78 815.15 912.3 2941.5 1599.09 2143.68 892.01 1239.01 1200.18 728.43 468.28 3194.25 1599.09 770.06 1390.96 364.68 1599.09
a
Center points.
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replications of the central point and eight axial points are shown in Table 2. The responses of the CCRD design were fitted with a second-order polynomial equation
Table 3 Results of the regression analysis of the central composite rotatory design Source
Mean square
F-value
Prob > F
Model A-glucose B-peptone C-MgSO4 D-CaCl2 AB AC AD BC BD CD A2 B2 C2 D2 Lack of fit
9.985E + 005 5.242E + 005 7.197E + 006 1.502E + 005 8853.70 19443.86 1.294E + 005 6.942E + 005 87387.49 1.609E + 006 1963.49 4.266E + 005 1.753E + 006 1.518E + 005 7.516E + 005 2.516E + 005
5.16 2.71 37.19 0.78 0.046 0.10 0.67 3.59 0.45 8.32 0.010 2.20 9.06 0.78 3.88
0.0027 0.1238 <0.0001 0.3944 0.8340 0.7563 0.4283 0.0807 0.5134 0.0128 0.9213 0.1614 0.0100 0.3919 0.0704 0.0027
Nattokinase activity ¼ 1599:09 þ 147:78A þ 547:60B 79:10 þ 19:21D þ 34:86AB þ 89:93AC 208:29AD 73:90BC þ 317:15BD 11:08CD 124:72 þ 252:83B2 74:40C2 165:53D2
ð1Þ
Except for the linear term A (p < 0.05) and quadratic term A square (p < 0.05), none of the other linear, quadratic and interaction terms were statistically significant (Table 3). The overall second-order polynomial equation for Nattokinase production was where, Y is Nattokinase activity (U/ml of substrate); A is Glucose (%); B is Peptone (%); C is Magnesium sulphate (%); D is Calcium chloride. The statistical significance of the model equation was evaluated by the F-test for analysis of variance (ANOVA), which showed that the regression is statistically significant at 99% (p < 0.05) confidence level. The model F-value of 5.16 for Nattokinase production implies that the model is statistically significant. There is only a 3.03% chance that a ‘‘Model F-Value’’ this large could occur due to noise. The value of p > F less than 0.05 indicates that the model terms are also significant. The coefficient of determination (R2) was calculated to be 0.8475, indicating that the model could explain 84% of the variability. The ‘‘lack of fit test’’
dation of the fibrin net. In contrast test tube of normal saline as control, clot degradation was not observed. In order to improve the production of Nattokinase, medium optimization was performed using response surface methodology. 3.2. Response surface methodology The experimental results of Nattokinase production by a complete four-factor-two-level factorial experiment design with six
Nattokinase activity
3200
2475
1750
1025
300
4.25
1.25 3.63
1.13 3.00
B: Peptone
1.00 2.38
0.88 1.75
0.75
A: Glucose
Nattokinase activity
3200
2500
1800
1100
400
0.70
4.25 0.60
3.63 0.50
D: CaCl 2
3.00 0.40
2.38 0.30
1.75
B: Peptone
Fig. 1. Response surface plot for Nattokinase production by B. subtilis. The interaction between (a) Glucose and Peptone, (b) Peptone and MgSO4 and (c) Peptone and CaCl2.
V. Deepak et al. / Bioresource Technology 99 (2008) 8170–8174
Nattokinase activity (U/ml)
3200
B
2425
A DC
C D AB
1650
875
100
-1.000
-0.500
0.000
0.500
1.000
Deviation from Reference Point (Coded Units) Fig. 2. Perturbation graph showing the effect of each of the independent variables on Nattokinase production while keeping other variables at their respective midpoint levels. (A) Glucose, (B) Peptone, (C) MgSO4 and (D) CaCl2.
compares the residual error to the ‘‘Pure Error’’ from replicated design points. The ‘‘lack of fit F-value’’ of 5.6 implies the lack of fit is significant. There is only 0.48% chance that a ‘‘lack of fit F-value’’ this large could occur due to noise. Thus, the estimated models fit the experimental data adequately. ‘‘Adeq. Precision” measures the signal (response) to noise (deviation) ratio. A ratio greater than 4 is desirable. The ratio of 8.7 indicates an adequate signal and therefore the model is significant for the process. Three-dimensional response surfaces were plotted on the basis of the model equation, to investigate the interaction among the variables and to determine the optimum concentration of each factor for maximum Nattokinase production by B. subtilis. The effects of varying the concentration of peptone and one of the other variables are shown in Fig. 1, which demonstrates that the response surfaces for the three combinations were similar to each other. A small through in the response surface indicates an initial decrease in Nattokinase production with the initial increase of peptone (0.5– 3%) before a steep increase in Nattokinase production with a further increase in concentration (3–5.5%). From the response surface (Fig. 1) and perturbation plot (Fig. 2) it is obvious that peptone had a significant effect on Nattokinase production compared to other variables. The metal ions such as Ca2+ and Mg2+ increased and stabilized the Nattokinase activity of the enzyme; this is possible because of the activation by the metal ions. The perturbation graph clearly shows that the three components A, C, D did not have significant role in the enzyme synthesis. The component B has a major role in the production of Nattokinase also it is the limiting factor for the production of Nattokinase. The Nattokinase production was 1599 U/ml of substrate in the original medium with the four components at their central levels. The maximum Nattokinase yield is 3194.25 U/ml of substrate in optimized medium composed of (%): Glucose (1.0); Peptone (5.5); MgSO4 (0.2); and CaCl2 (0.5) which is 2-fold higher than the unoptimized medium. 4. Discussion Sumi et al. (1987) reported the presence of a strong fibrinolytic enzyme (Nattokinase) in natto (a fermented food), and Nattokinase may be an equally good protease for oral fibrinolytic therapy
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because of its confirmed safety for long-term intake, stability and the strong fibrinolytic activity. The fermented soybeans produced using bacteria, however, may have a preventive effect on not only thrombosis but also cancer. Statistical experimental design has not been widely used in the biological sciences even though it has been commonly employed in many other areas such as industrial, chemical, engineering, agricultural, medical, and food sciences. The primary reason for this is that most biological research has not been involved in many manufacturing processes. However, since genetic engineering, biomaterials, and bioprocess technologies like biodegradation and bioremediation have emerged, more people are getting interested in experimental designs to improve their biological processes and productions by shortening time and increasing efficiencies (Kwang-Min and David, 2005). The fermentation media for Nattokinase production was optimized using statistical method. According to the previous report using the medium components peptone, calcium chloride and yeast extract resulted in maximum Nattokinase activity of 1300 U/ml (Liu et al., 2005). In case of our report using the medium components Glucose, Peptone, Calcium chloride and Magnesium sulphate resulted in maximum Nattokinase activity of 3194.25 U/ml. In addition to establishing optimal fermentation medium compositions, the present methodology also makes it possible to predict the yield if the composition of the medium is altered in some way, by using the quadratic equation (Eq. (1)). Central composite experimental design maximizes the amount of information that can be obtained, while limiting the numbers of individual experiments required. Thus small and less time consuming experimental designs could generally suffice for the optimization of many fermentation processes. Therefore, with the increase in yield and productivity and simultaneous cost reduction, the industrial Nattokinase production by B. subtilis can be regarded as possible and economically attractive. References Bocchini, D.A., Alves-Prado, H.F., Baida, L.C., Roberto, I.C., Gomes, E., Da Silva, R., 2002. Optimization of xylanase production by Bacillus circulans D1 in submerged fermentation using response surface methodology. Proc. Biochem. 38, 727–731. Goodchild, C.S., Boylan, M.K., 1992. Reversal of streptokinase-induced bleeding with aprotinin for emergency cardiac surgery. Anaesthesia 47, 226–228. Ghanem, N.B., Yusef, H.H., Mahrouse, H.K., 2000. Production of Aspergillus terreus xylanase in solid state cultures: application of the Plackett–Burman experimental design to evaluate nutritional requirements. Biores. Technol. 73, 113–121. Hanaway, J., Torack, R., Fletcher, A.P., Landau, W.M., 1972. Intracranial bleeding associated with urokinase therapy for acute ischemic hemispherical stroke. Stroke 7, 143–146. 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, 2482–2488. Kwang-Min, L., David, F.G., 2005. Formulation and process modeling of biopolymer (polyhydroxyalkanoates: PHAs) production from industrial wastes by novel crossed experimental design. Process Biochem. 40, 229–246. Liu, J., Xing, J., Chang, T., Ma, Z., Liu, H., 2005. Optimization of nutritional condition for Nattokinase production by Bacillus natto NLSSE using statistical experimental methods. Process. Biochem. 40, 2757–2762. Montogomery, D.C., 2001. Design and Analysis of Experiments, fifth ed. John Wiley and Sons, NY. Myers, R.H., Montogomery, D.C., 2002. Response Surface Methodology: Process and Product Optimization Using Designed Experiments, second ed. John Wiley and Sons, NY. Omura, K., Hitosugi, M., Zhu, X., Ikeda, M., Maeda, H., Tokududome, S., 2005. A newly derived protein from Bacillus subtilis natto with both antithrombotic and fibrinolytic effects. J. Pharmacol. Sci. 99, 247–251. 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. Senthilkumar, S.R., Ashokkumar, B., Chandra Raj, K., Gunasekaran, P., 2005. Optimization of medium composition for alkali-stable xylanase production by Aspergillus fischeri Fxn 1 in solid-state fermentation using central composite rotary design. Biores. Technol. 96, 1380–1386.
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bleeding potential for tissue-type plasminogen activators but not for streptokinase. Thromb. Haemost. 75, 915–920. Urano, T., Ihara, H., Umemura, K., 2001. The profibrinolytic enzyme subtilisin NAT purified from Bacillus subtilis cleaves and inactivates plasminogen activator inhibitor type 1. J. Biol. Chem. 276, 24690–24696. Wu, S.Y., 2005. Optimization of nutritional conditions for Nattokinase production by a isolated Bacillus subtilis from natto health food. A Master of Science Thesis, Tatung University. Ya-Hong, X., Jian-Zhong, L., Hai-Yan, S., Liang-Nian, J., 2004. Enhanced production of extracellular ribonuclease from Aspergillus niger by optimization of culture conditions using response surface methodology. Biochem. Eng. J. 21, 27–32.