HOl NGHI KHOA HQC C O N G NGH? SINH HOC TOAN QUOC 2013
OPTIMIZATION OF GROWING CONDITIONS FOR THE PRODUCTION OF FIBRINOLYTIC ENZYMES BY BACILLUS SUBTILIS USING RESPONSE SURFACE METHODOLOGY
Dinh Bui Quynh Anh. Do Ngoc Anh Huy and Pham Van Hung' International UnhrersHy, Vietnam National Univ&sity, Ho Chf Mlr^ City
SUMMARY
The fomiation of fibrin clots in blood vessels is one of the soious reasons affecting to the transposition of blood in human body Fibrinolytic enzymes are i^eats dissolving fibrin clots, therefore, preventing human from vascular diseases such as stroke, coronary artery. Fibrinolytic enzymes can be extracted hcsn food or non - fijod sources and irucroorganisms are considered aa potential sources for producing fibrinolytic enzymes. In this study. Bacillus subtilis was used to produce fibrinolytic enzymes by liquid fennemaiion and (be growing conditions of this bacterium w o e optimized using die Response Surface Mediodology to obtam the hi^Kst filninolytic enzyme activity. The optimum combination of selected &ctors was investigated b y die Box-Behnken design.
Four &ctors, shrimp shell p o w d a ^ S P ) and NaCI as main nutritional and mineral sources as well as tenqierature and duration time at three levels were used. M a result, die e n ^ m e activity reached to 8.15 FU/ml at the optimum condition with 1.36% SSP. 1.18%
NaCi, 36°C and 51-hour fennentaiioa. Thus, the fil^inolytic enzyme could be produced widi die high activity by SanZ/ur «u!>ri/u at the t^timum condition in this study and diese enzymes can be applied as thrombolytic agents in pharmaceuiical bdustries.
Key yards. Bacillus subtilis. Box Behnken design, fibrinolytic enzyme. Response Surface Methodology INTRODUCTION
Despite the considerable innovations of thrombdyttc therapies have been applied in heart disease treatments, there is still a high mortality rate in the world caused by vascular diseases such as stroke, coronary artery. As reported by the World Health Organization (WHO) in 21300. heart diseases are responsible for 29% of total mortality rate in the world.
CanJIovastxilar diseases, especially stroke and heart diseases, account for 17 million people death annual and predicted thai the number mil be increased to 23.3 million in 2030 (Mathers et aL, 2006). The accumulation of fibrin clots in blood vessels is a main cause of stroke and many serious cardiovascular diseases. Many thrombolytic agents such as urokinase, streptokinase, genetically tissue-type (riasminogen activators (t-PA) have been applied ir\, thrombolytic treatments but they seem not to be effective because of many undesirable side efTects such as gastrointestinal bleeding, resistarice to repercussion or. allergk: reactions (Blann et al., 2002). Therefore, thrbmbolytic agents derived from food sources are expected to become safe dierapies for patients. Fibrinolytic enzymes can be widely found not only in nature but also in a variety of foods such as fennented red bean (Chang et al.. 2012), fnjiting bodies of Korean Cordyceps m/Waris (Choi efa/.. 2011), chive (Chung ©(a/.,2010). Many kinds of bacteria presented In fennented food products are able to secrete fibrinolytic enzymes and Badllus sp. is conadered as a potential source for fibnnolytic enzyme production (Peng e( a/.. 2005).
The growth of bacteria is affected by not only intrinsic but also extrinsic conditions. They can be nubients, temperature, GUlHvation time, pfl levels, moisture, light, radiation, osmotic pressure, hydrostatic pressure and humidity. Therefore, it is very critKal Io find out important factors among fliese factors because the optimizaBon process requires much time and expense for doing experiments. Opdnuzahon can be camed out using conventional method conducted lay changing one Independent variable at a time while others are fixed. This method is quite simple but may consume time and the results are not confidem. The solution for optimizaton problem is Response Surface Methodology (RSM). a collection of statsBcal and'mitftemalical techniques for contributing experiments, designing model, analyzing input data from e>q»nrnents, evaluating effects of variables in onJer to find out the optimum response. This method requires -doing several eiqwnments. using the results of empirical study to contribute the relationship between variables and response and then figure out the best combinadon of variables for obtaining the maximizing or minimizing response depending on S ! ^ ^ desire. Because RSM covers all interaction of different variables al a time, therefore it is considered as a
^ v t S n ^ » ? ! ^ ^ * ^ ^ . ^ ''^'"ot^:! * ™ ^"'^ resources and die results are reliable and confident in comparison with S ^ a t T h S ^^f- ^fJ^^ ^ ^ ™ ^ ^"^'^^^ ^"'' "'^'"' ''^*^"''^' ^ ^ ^ " d ^°' investigating the optimal condition
^ ^ ™ t ^ f ^ 1 L " ^ ^ " * " ^ " ' ^°°^- '^'^'""^ «' ^'- 2010)' f>'e aim of this study was to establish die optimum Z S v ^ r ™ ^ " ^ r ^ ' ^ - ^ "^ temperature; shrimp shell powder, sodium chloride, duradon bme for maximizing fibnndybc enzyme producbon using Response Surface Methodology based on Box Behnken design. " « ' " " ^ n s MATERIALS AND METHODS
Materials
^oStHo^SfHnn!"'*' dtoinogen. Ihmmbir,, scxSum cWonde, yeast extract peptone. KjHPO,. 3H;0,
«19!.o,,7H!0. CaCt2H,0, D-glucose were purchased from Sigma Chimical Company (SingapTre)-
1 2 f f ? ! S . , ^ ' L ' ! ^ " ^ " " ^ P " " ! ^ ' sl^™? shell was collected from marf<et. washed with lap water and then dried at ..Oi I,, nner JU mjn. It was ground into powder form and stored al desrccalore with knobbed lid to minimize the moisture.
HOI NGH! KHOA HQC C O N G NGH? SINH HQC T O A N Q U 6 C 2013
Fig. 1. Morphology of Bacillus subtilis
Fibrinolytic activity evaluation
Fibrinolytic enzyme activity was evaluated according to Fibrin degradation assay provided by Japan Bio Science Laboratory Co.. Ltd. (JBSL) with slight modifications. Rrst of all. 0.4 n i l of fibrinogen and 0.1 mL of 245 mM phosphate buffer (pH 7.0) were loaded to a lest tube and incubated at 37° C. After 5 min. 0.1 mL of thnambin solution was added and incubated at 37* C for 10 min to fonn fibrin d o t After adding 0.1 mL enzyme solution, incubating at 37° C and sUm'ng after 20 min. 40 min. 60 mm, 2.0 mL of 0.2 M trichloroacetic a d d (TCA) was added and mixed well to stop enzyme reaction. The reaction mixture was then centrifuged at 6,500"g for 5 min and removed pellet. Then, 1 m L of supematant containing fibrinolytic enzyme was measured the absorbance at 275 nm. For control of each sample, all steps were done except for the enzyme addition step, in which the e n z ^ e solution was added after temiinating the reaction by TCA. In this assay, 1 unit (Fibnn degradation unit, FU) of enzyme activity Is defined as a 0.01-per-minute increase in. absorbance at 275 nm of the reaction solution.
Enzyme activity was calculated based on this fonmulation Rbrinolytic enzyme acUvity (FU/mL) = ( O D , - ODc)/(0.01 x 60 x 0.1) Where: OD,: Optical density value of sample; ODc: Optical density value of control S o x Behnken design
Four factors affecting to grov/th of bacteria, temperature (Xi). shrimp shell powder (Xz), sodium chloride (X3), duration time (X4), were chosen as independent variables and fibrinolytic activity (Y) wa^ dependent response. Three levels of each factor were shown in table 1. A modeling involved 25 experiments was set up by Box Behnken design according to Design Expert software 7.0.0 trial version (Sfat Ease, USA).
Box Behnken design Is a three-level second-order design-used in Response Surface Methodology (Box and Behnken, 1960). The Box-Behnken design used in this study was established for four independent factors with three levels of each lector and Uie relationship betv/een independent variables and dependent response was fitted by second-order model as shown in equation (1)
Where Y is the response, po, Pi, pa, Pu are Uie intercept, linear coefficient of Xi, quadratic coefficient of Xi, and two-
y=p^i:i3,x^i:p,.x>T.i:p,x.Xj
(1)factor interaction between Xt and )^. respectively. The estimated coefficient and p-value ft^om F test were used t o evaluate the significance of model. The coeffident of determination, denoted R , indicated how well date points fit Uie line or curve. The relationships among variables and response in Response Surface Methodology were expressed in three- dimensional surface.
Table 1. The coded level of factors for Box Behnken design
Temperature (°C) Shrimp shell powder (%) Sodium chloride {%) Duration time (tir^)
Coded - X, XJ Xl X,
-1 30 0.5 0.5 24
Range and level 0 35 1 1 43
1 40 1.5 1.5 72
OptirJilzation of growing condition using Response Sur^ee Methodology
According to method o f Prafuiia et BL (2010). bacterium was cultured into an Erlenmeyer flask containing 25 mL of liquid medium consisted of D-gluoose, yeast extract. KjHP04. SHjO. MgS04.7H20 and pH of medium at 7.0 and then incubated a l 37° C in orbitel shaker at 180 rpm for 24 h. For the optimization process, 1.25 mL of inoculums was taken and dropped Into the Erienmeyer fiask containing 25 mL u n c ^ m i z e d medium composed of D-glucose, peptone, yeast- extract, KiHP04.3H20, MgS04.7HiO, CaCl2.2H20. Temperature, SSP and NaCI concentrations, duration time were continuously varied ftom 30° C to 40° C, 0,5% to 1.5%, 0.5% to 1.5%. and 24 hrs to 72 hrs, respectively, and pH of
H O I N G H ! K H O A H Q C C O N G N G H E S I N H H Q C T O A N Q U O C 2 0 1 3
medium was adjusted at 7.0. After appropriate incubaton temperature and fennentation t m e at 180 rpm, samples were c e n t r i f u g e d a l 1 0 . 0 0 0 r p m f i 3 r 3 0 m i n to get supematant Centrifugestepwas conducted at 4 ° C .
Statistical analysis
Analysis of variance was used to analyze data. F test was carried out to compare treatment means at p < 0.05 a n d p- value from F test was compared with 0.05. T h e higher F value and lower p-value (p-value < 0.05), ttie more significant model was. Based on p value and estimated coeffident, the significance of variables to response was evaluated.
Design Expert 7.0.0 trial version (Stat Ease. USA) was used to design matrix of relationsfiip among variables and response. The optimum treatment was contributed b y solving regress equation and analyzing response surface contour ptots. The predk^ed maximize/minimize response was supplied together wiUi the optimum conditkNi based o n the input response f ^ m empirical eiqieriments.
RESULTS AND DISCUSSION
Fibrinolytic enzyme p r o d u c t i o n f r o m Bacillus subtilis under o p t i m i z a t i o n c o n d i t i o n s u s i n g R e s p o n s e S u r f a c e Methodology based o n Box Behnken d e s i g n
Optimization conditions induding temperature, shrimp shell powder (SSP), sodium chloride and duration time were chosen as four important variables affecting te the fibrinolytic enzyme secreted by Bacillus subtilis. Table 2 shows ttie matrix displaying relationship among response and four variables.
Table 2. Box Batinkon design matrix with variables and empirical response
Run,
1 2 3
Temperature CO
X, -1 1 0
Variables SSP
X, 0 1 0
NaQ
X l 0 0 1
Duraton time (hours)
x*
-1 0 1
Response Rbnnolytic activity
(FU/mL) Y 1.00 4.58 0 59
5.70 5 52 0 70 0.35 3.67 0.53 2.67 2.87 0 23 0 53
The data were analyzed by multiple regression analysis and the regression coefficients for equation were determined as bellowed equ^ation:
^ ° y ° * l Q i ° ? ' i ' J v " ' ^ , ° d ' ' ? " * " ^ ^ ^ ^ 1 . 2 6 X , X , - 0 . 0 5 8 X , X , * 0.067X,X. * 2.87X,X, + 0 . 9 7 X , X , - 1.13X, ^ - J . a J X ) - ^ . 2 5 X 2 - 2 . 3 7 X i - 3 . 7 1 X ,
l I i T r i ' , r t ^ i ^ ™ * * ^ " . ^ - " " f ? ' " ' V ^ ^ " ' variaMes of temperature, SSP, NaCI, duration time, respectively and Y was preorcted response offibnnolytc activity.
2 ^ r l ^ T ? h ! ^ msulls wtiich was tested by the F-test for analysis o f variance (ANOVA) using Design Expert S S a o n ^ e l S ^ Z 7 ! " Z ' : ^ ^ " ^ " ^ " « " " " ! " = « • " ' ^ o a f f l ^ n t R l THe d o s e ! o f R ' value to 1 wes. t h e more S S e * S i m S i S T , ' • J ^ f ^ " ^ » a s - ' " " ^ atudy, the R v a l u e was found to be 0.82 indicated that
" n d S r w T r ^ a 2 r . n 5 ^ n ; « °°"'?^ T^'' ™ ^ ™ ™ * "^ "^ I " ' " " * ' ™g'assion modal shovred F-value
„ H , ^ S ^ ? « i : . . 1 i ^ ° ^ ^ ! L ™ ^ ' * * ™ ' ' - ™ « = » " " " " a ' s indicated Ihat the model w a s significant (p < 0.05). T h e b e S ^ I a S S S 1Z " " ^ ^ ^ a ^ P O n J l a S ^ P - v a l u e Indicated how signilicanl o f each factors and Interaction between factors wem. In th.s case. X j , X , X , , X , ' , X.= were found to be significart model l e m s .
l ^ l ^ l ^ ' J ' " ' * " " " " " ' » " « ' ° " S suPPliad by software and the targeted conditions canied out in this study. The 2 J r » ^ ' ' * ° ™ " ^ ' » ™lnl>"<ed based on ec,uatlon (1), and the response was also predicted by Design expert software. The conditon consisted o f 1.36% of SSP (X,), 1 18% of NaCI (X,), 36.21'C ( X , ) and 50.99 hrs ( X . ) v i e , .
B '
HQI NGH! KHOA HQC C O N G N G H f SINH HQC TOAN QUOC 2013
corfespdnded with 9:3T1=U/mt-ofpredicted response.-ln-pratical-experimentjbe-optimuin.condition was conducted with slight modification for suitable setting. The optimum conditions were targeted as tempearture of 36°C and duration time of 51 hrs whereas SSP and NaCI concentrations were maintained at 1.36% and 1.18%, respectively. Under targeted conditions, the fibrinolytic activity obtained after doing practical experiment was 8.15 FU/mL. The practical obtained data was not significantiy difi'erent to predicted value (9.31 FU/mL). Therefore, ttie targeted conditions can be applied in pnxJudng fibrinolytic enzyme in large scale. , . . ,. .
Table 3. Analysis of variance (ANOVA) for the experimental results of response surface quadratic model Source
Model X, X J X l X.
X1iXa X,Xt X.X, XjXi X2X4 X3X4 X,^
X J ' Xa' Residual
x/
R' Table 4. Tlie optimum
Response df 14
10
Coeffident Estimate 8 55 1.02 1.73 -0.14 0.63 1.26 -0.050 0.067 2.87 0.97 -1.13 -3.93 -2.25 -2.37 -3 71
Fit>rviolytic activity F-value
3,25 3.65 10.50 0.072 1J8 '- 184 3 963E-0,03 5.176E-0 03 9 59 1.10 149 12.69 4.18 4,61 11.29 0 82
Prob>F 0.0335 0.0851 0.0089- 0.7938 0.2679 0.2043 0.9510 0.9441 0.0113*
0.3196 0.2496 0.0052*
0.0682 0.0573 0.0072*
condition for fibrinolytfc enzyme production tiy using Response Surface Method
Temperature CC) Shrimp shell powder (%)
Sodiui 1 chionde (%}
Duration bme (hrs) Fibnnolybc activity (FU/mL)
Opti num values (in a range)
36.21 1.36 50.99 9.31
Optimum values (targeted)
36 1.36
51 8.15
Several previous studies on fibrinolytic e n z ^ e production have been done wiUi different fibrinolytic e n ^ m e s from our study. Wang ef al. (2011) reported that the fibrinolyti'c enzyme produced by Bacillus subtilis TKU007 was 6.7 FU/mL.
whereas the fibrinolytic enzyme activity was only 2.3 FU/mL using Pseudomonas sp. TKU015 (Wang ef al. 2009). These results indicate that Uie production of firbinolytic enzyme depends on growing condition and microbial strains.
CONCLUSION
Four factors affecting to the growth of Bacillus subtilis (temperature, shrimp shell powder, sodium chloride a n d duration time) were selected as independent variables to find out &ie dependent response (fibrinolytic activity) using Response Surfece MeUiodology. Under the optimum cohditions viriUi 1.36% of SSP, 1.18% of NaCI, Se'C and 51 hour cultivation.
the fibrinolytic activity w a s achieved to 8.15 FU/mL. With Uie ability of fibrinolytic enzyme secrection, Bacillus subBlis isolated from fermented shrimp paste can be used to produce thrombolytic agents in industrial and phamaceuUcal fields.
Acknowledgement!
This research is funded try Vietnam National University in Ho Chi Mink City (VNU-HCM) under grant number C2013-28-04.
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T 6 | LfU H 6 A OieU KIEN SiNH TRl/d-NG CClA VI KHUAN BACILLUS SUBTILIS TRONG SAN XUAT FIBRINOLYTIC ENZYMES-SO" DUNG PHI/ONG PHAP T 6 | UU
H 6 A BE MAT OAPO'NG
Dinh Bill Quynh Anh, 0& Ngpc Anh Huy, P h ? m Van Hiding' Tnrdng D9i hgc Qudc ti, Dei hgc Qudc Gia Tp. H6 Chl Mlnh
T6M TAT
Sv hlnh thAnh h u y ^ khoi t^ong m^cfa win IA m^t trong nhOng nguyin nhan n ^ n n irgog Anh hucmg d£a kha oiiiig v$a chuyen mau trong CO dli ngir6i. Fitmnolytic enzyme U tac nhin cd khA ning lAm taa huyet khdi gi£ip n g i n ngite c i c bfnh v€ tim tn^ch n h u dot quy, d^ng m^ch vAnh. Fibrinolytic enzyme cd th£ dugc chiJi xuit ti^ cAc nguyen lifu Ihirc pham hogc tCt c i c nguon ty: nhi&i khAc trong d6 vi smh vgt IA ngucln chinh d£ SAD xuil fibrinolytic enzyme. Trong nghiSn c i ^ nAy, Bacillus subtilis d u g c su dung ii san xuit Sbiinolytic enzyme ttAng quy trinh len men ctiim v i ^hi k i ^ song ciia vi khuin du^c toi uu h6a bing phuong p b i p toi u u hoa be m$t dAp img nhim ihu du^c h o ^ tinh enzyme cao nhit. Sv ket hop t6i uu giiia cAc y ^ t6 chon tpc d u ^ k h i o s i t t)6i thiit k i cOa Box Beteikcn. B6n yiu t i g i m b$[ v6 tdm (SSP) vA NaCI du^c xem nhu ngu^n dinh dudng vA khoing chit thiit y l u ciing v d i nhift d$ vA thbi gian b i ^ d i i d 3 cip do d l duTc sir dyng. K i t q u i l i bo^t tinh enzyme d^t S.15 FU/mL d dieu k i | a t6i ini gom 1,36%
SSP, 1,18% NsCl, 36° C v i 51 giir len men. VI thi, co t h i sAn xuil fibrinolytic enzyme ho;il tinh cao tir Bacillus subtilis d di£u k i ^ t i i uu trong nghiin ciru n i y v i enzynM t ^ la cA d i i dugc si> dyng nhu c i c tac nhln lAm tan huyit kh6i trong Snh vvrc y dugc.
Td khia • Bacillus subtilis. Box Behnken design, fibrinolytic enzyme. Response Surface Methodology 'Author for correspondence: DT 08-37244270 {exL 3814). E ma il:-pvhung@hcmiu .edu.vn
