OPTIMIZATION OF FLASK CULTURE MEDIUM AND CONDITIONS FOR HYALURONIC ACID PRODUCTION BY A STREPTOCOCCUS EQUISIMILIS MUTANT NC2168

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1 Brzilin Journl of Miroiology (2012): ISSN OPTIMIZATION OF FLASK CULTURE MEDIUM AND CONDITIONS FOR HYALURONIC ACID PRODUCTION BY A STREPTOCOCCUS EQUISIMILIS MUTANT NC2168 Yong-Ho Chen 1, 2 ; Jun Li 3 ; Li Liu 2 ; Hong-Zhi Liu 2 ; Qing Wng 2 * 1 Institute of Forestry n Pomology,Beijing Aemy of Agriulture n Forestry Sienes,Beijing , Chin; 2 Institute of Agro-foo Siene n Tehnology, Chinese Aemy of Agriulturl Sienes, Beijing , Chin; 3 Beijing Institute of lnspe rhiteture, Beijing , Chin. Sumitte: August 04, 2011; Approve: June 07, ABSTRACT A mutnt esignte NC2168, whih ws selete from wil-type Streptoous equisimilis CVCC55116 y ultrviolet ry omine with 60 C o -γ ry tretment n oes not proue streptolysin, ws employe to proue hyluroni i (HA). In orer to inrese the output of HA in flsk, the ulture meium n onitions for NC2168 were optimize in this stuy. The influene of ulture meium ingreients inluing ron soures, nitrogen soures n metl ions on HA proution ws evlute using ftionl ftoril esign. The mthemtil moel, whih represente the effet of eh meium omponent n their intertion on the yiel of HA, ws estlishe y the qurti rotry omintion esign n response surfe metho. The moel estimte tht, mximl yiel of HA oul e otine when the onentrtions of yest extrt, peptone, gluose, n MgSO 4 were set t 3 g/100 ml, 2 g/100 ml, 0.5 g/100 ml n 0.15 g/100 ml, respetively. Compre with the vlues otine y other runs in the experimentl esign, the optimize meium resulte in remrkle inrese in the output of HA n the mximum of the preite HA proution ws mg/l. The moel evelope ws urte n relile for preiting the proution of HA y NC2168.Cultivtion onitions were optimize y n orthogonl experimentl esign n the optiml onitions were s follows: temperture 33ºC, ph 7.8, gittion spee 200 rpm, meium volume 20 ml. Key wors: Streptoous equisimilis mutnt; hyluroni i; optimiztion; ulture meium; ulture onitions INTRODUCTION Hyluroni i (HA), liner, unrnhe polyshrie onsiste of lternting N-etyl-D-gluosmine n D- gluuroni i, exists in mny mmmlin onnetive tissues suh s joints, vitreous oies, umilil ors, rtilges, skins, n oms of fowls s onstituent; it plys n importnt role in mny iologil proesses suh s in tissue hyrtion, proteoglyn orgnistion in the extrellulr mtrix, n tissue repir (14). HA is vlule iopolymer in *Corresponing Author. Miling ress: Institute of Agro-foo Siene n Tehnology, Chinese Aemy of Agriulturl Sienes, Yunmingyun West Ro 2 #, Beijing, P. R. Chin, ; Tel/Fx: ; E-mil: swngqing@hotmil.om 1553

2 Chen, Y.H. et l. Hyluroni i proution y S. equisimilis ophthlmis, orthopeis, woun heling, tissue engineering, rug elivery, osmetis, n s vine i (2, 6, 15). In reent yers, inste of extrting HA from rooster oms n umilil or, prepring HA through miroil fermenttion is eing use more, euse the risk of rossspeies virl infetion n e voie n the isoltion proess of high purity n high moleulr weight HA n e simplifie(5). This polymer is proue ommerilly y fermenttion of the Lne fiel group C streptooi, Streptoous equi suspeies equsimilis n Streptoous equi suspeies zooepiemius whih n proue psule tht is ompose of hyluroni i polymers ientil to tht foun in mmmlin onnetive tissues (10). However, Streptoous spp. strin n proue streptolysin whih uses hemolysis. Thus, it is very importnt tht streptolysin is sent in the hyluroni i otine y ultivtion of the miroorgnism. Aoring to the reports (8, 10, 12), NTG tretment ws n effetive inue mesure to otin mutnt whih oes not proue streptolysin. However, in this stuy, mutnt esignte NC2168, whih ws selete from wil-type Streptoous equisimilis CVCC55116 y ultrviolet ry omine with 60 C o -γ ry tretment n oes not proue streptolysin, ws employe to proue HA. Beuse no generl meium works est for new strin eing teste, the optimiztion of the meium for the new mutnt is neessry (9). Menwhile, lrge numers of experiments in stirre ontrolle vessels re not prtil. However, the est meium hosen from shke flsk t n uil fountion for the lrge sle stirre tnk, thus it s essentil to improve the HA proution through the optimiztion of shke flsk meium n ultivtion onitions. As olletion of sttistil tehniques for experiment esigning, moel eveloping n ftors evluting, response surfe methoology (RSM) hs een extensively pplie in optimiztion of meium omposition, onitions of enzymti hyrolysis, fermenttion n foo mnufturing proesses (3, 11, 13, 17,). In this stuy, to inrese the yiel of HA, the optimum meium omposition ws rrie out y RSM n the optimum ultivtion onitions inluing temperture, rotte spee, ph, n meium volume were investigte through orthogonl experimentl esign. MATERIALS AND METHODS Miroorgnism n Meium The strin use in this stuy ws Streptoous equisimilis CVCC55116, from Chinese Veterinry Culture Colletion. Miroorgnisms were grown in soli ulture meium onsisting of 1 g gluose, 16 g ox-rin extrt power, 4 g oxhert extrt power (Brin Hert Infusion, BHI), 10 g yest extrt n 20 g gr. The loo gr (Joint Logistis Deprtment, Beijing Militry Are Commn) ws use for the isoltion of the mutnt without hemolysin. The selete mutnt ws ultivte in the fermenttion meium onsisting of 20 g gluose, 16 g ox-rin extrt power, 4 g ox-hert extrt power (BHI), 10 g peptone, 2 g MgSO 4 n 2 g K 2 HPO 4 per liter of istille wter. The ultivtion onitions were: temperture 37ºC, ph 7.4, n gittion spee 180 rpm. Mutgenesis Streptoous equisimilis CVCC55116 ws inute t 37 C for 24 h on the gr plte. A single olony ws inoulte in 250 ml flsk ontining 50 ml of fermenttion meium; it ws inute t 37 C for 12h. The grown ells were ollete y entrifugtion t 3,000 g for 20 min n wshe with istille wter. The ollete ells were resuspene in istille wter n performe ultrviolet ry mutgenesis in rk room. After ultrviolet ry tretment, the ells were inute t 37 C for 12 h in the fermenttion meium n plte on the loo gr plte for the seletion of nonhemolyti mutnts. The mutnt trete y ultrviolet ry ws irrite y 60 C O -γ ry with ose rte of Gy/min. The irrition oses of the two mutgenesis methos were se on irrition time n orresponing the ftlity rte. Through repete sreening, high-prouing hyluroni i mutnt esignte NC2168 ws otine. 1554

3 Chen, Y.H. et l. Hyluroni i proution y S. equisimilis Anlytil Methos CTAB Turiimetri metho ws pplie to etermine the HA ontent (1). Ultrviolet n visile spetrophotometer (Rili, Chin) ws use here. Experiment esign Bse on the single ftor experiments, the ron n nitrogen soure n inorgni slt were etermine, n the response surfe methoology (RSM) ws employe to optimize the meium for the mutnt NC2168.Then, 23 runs were rrie out using ftoril entrl omposite esign to stuy four inepenent vriles eh t five levels. The four inepenent vriles X 1 (yest extrt), X 2 (peptone), X 3 (surose), n X 4 (MgSO 4 ) t five levels n the experimentl esign were given in Tle 1. A mthemtil moel, esriing the reltionships etween the proess inies (the output of HA) n the meium omponent ontents in qurti eqution, ws evelope. The ury n generl ility of the ove polynomil moel oul e evlute y the oeffiient of etermintion R 2. As for the optimiztion of the ulture onitions, the experiment ws rrnge se on the 3 levels of orthogonl experimentl esign with 4 ftors n 9 runs s shown in Tle 3. Four inepenent vriles representing temperture, rotte spee, initil ph n meium volume were employe respetively. In eh se, the output of HA ws monitore n ll the experiments were rrie out in triplite for the verge lultion. Sttistil nlysis Sttistil evlutions were performe using F-test. A vlue of p < 0.05 ws onsiere signifint. The qurti moel ws otine y stnr multinomil regression metho for four ftors using the softwre Sttisti, version 6.0. The extremum ws lso quire y ifferentil oeffiient lultion n nonil nlysis whih ws use to preit the shpe of the urve generte y the moel. Vrine nlysis on the orthogonl experiment results ws performe y SAS, version8.0. RESULTS AND DISCUSSION Optimiztion of ulture meium Effet of nitrogen soures on the output of HA: The effet of orgni n inorgni nitrogen soures on the yiel of HA is shown in Figure 1. It inites tht the output of HA se on inorgni soure (NH 4 ) 2 SO 4 ws the lowest while those se on most of orgni nitrogen soures were superior. The min reson might e tht orgni soures were more esily tken up, trnsporte n sore while inorgni nitrogen oul restrin the metolism of the strin to some extent. It is esy to see tht BHI ws the est orgni nitrogen soure to ultivte the mutnt NC2168 n the highest output of HA se on BHI touhe mg/l, even with the meium ontining 1 g/100 ml BHI, the output ws higher thn tht se on other nitrogen soures. However, the ost of BHI is severl times higher thn tht of other nitrogen soures so tht it is essentil to fin out sustitute to BHI. A omposite meium ontining multiple growth ftors might meet the higher emn of Streptoous on nutrition. As shown in Figure 1, when the onentrtions of yest extrt n peptone were 3 g/100 ml n 2 g/100 ml, the output of HA got mg/l n mg/l, respetively, whih were the two etter nitrogen soures esies BHI. The omposite nitrogen soure onsisting of yest extrt n peptone might e promising nitrogen soure whih oul provie more neessry growth ftors. Bse on the previous experiments, 3 g/100 ml yest extrt n 2 g/100 ml peptone were fixe respetively, n e 0.5, 1.0, 1.5, 2.0 n 2.5 g/100 ml peptone n yest extrt into the meium to onstitute the omposite nitrogen soures. The results in Figure 1 shows tht mg/l yiel of HA ws otine using 3 g/100 ml yest extrt n 1.5 g/100 ml peptone, whih ws inrese y 23.97% ompre with mg/l, using yest extrt only. 1555

4 Chen, Y.H. et l. Hyluroni i proution y S. equisimilis The output of HA (mg/l) BHI YE PEP BE (NH 4 ) 2 SO 4 The output of HA ( mg/l) % YE 2% PEP e Nitrogen soures (g/100ml) Composite nitrogen soures (g/100ml) Figure 1. Effet of nitrogen soures on the output of HA BHI rin hert infusion, YE yest extrt, PEP peptone, BE eef extrt. The -e in Figure1-3 represente the signifintly ifferene (p<0.05) etween the ifferent onentrtion of the sme meium omponent. Effet of ron soures on the output of HA: In this stuy, gluose, frutose, gltose, surose, mltose, xylose were use s the ron soures respetively to enhne the yiel of HA. Their influenes t ifferent onentrtions re presente in Figure 2. It shows tht the output of HA oul e ffete gretly y the kin n quntity of ron soures. As shown in Figure 2, the highest output of HA ws se on 0.5 g/100 ml gluose s the ron soure. Other ron soures inluing mltose t 1.5 g/100 ml, frutose t 0.5 g/100 ml n surose t 0.5 g/100 ml oul otin higher output of HA. However, the gltose n xylose oul not e utilize well n the orresponing output of HA ws reltively low. We speulte tht oth UDP gluuroni i n N-etmie uriine iphosphogluose re the synthesize preursors of HA n thlli. Mltose n surose oul e esily eompoune into gluose y the thlli n frutose oul e esily trnsforme into gluose so tht they oul e utilize etter. The output of HA (mg/l) Gluose Mltose Surose Frutose Gltose Xylose e e Cron soures Figure 2. Effet of ron soures on the output of HA. The numers liste in the X xis represente 0.1, 0.5, 1.0, 2.0, 3.0, 4.0 g/100 ml ron soures respetively. 1556

5 Chen, Y.H. et l. Hyluroni i proution y S. equisimilis Effet of metl ions on the output of HA: As for ell, mny metl ions re inispensle for tive enters of n enzyme. During the proess synthesizing HA, the HA synthetse is key enzyme, whih tlyzes the preursor of HA to synthesize HA (16). In this stuy, the effets of Mg 2+ n K 1+ on the output of HA were explore onsiering their possile roles in the HA synthetse. As shown in Figure 3, the highest output of HA ws hieve up to mg/l t MgSO 4 onentrtion of 0.15 g/100 ml n the ifferene of output se on the ifferent onentrtion of MgSO 4 ws pronoune (p<0.05). However, s for KH 2 PO 4, the output of HA se on the ifferent onentrtion ws lmost the sme, thus the effet of KH 2 PO 4 onentrtion ws negligile in the ssigne rnge while Mg 2+ ws selete to e performe in the following experiment. 220 MgSO 4 KH 2 PO The output of HA (mg/l) Figure 3. Effet of metl ions on the output of HA Metl ions (mg/100 ml) Orthogonl rottle entrl omposite esign In the previous experiments, the suitility of vrious nitrogen soures, ron soures, n metlli ions for the superior proution of HA ws evlute. After the influene ftors were etermine, response surfe metho ws use to optimize the ulture meium for enhning the output of HA through the qurti rotry omintion esign (4, 7). A set of 23 experiments were require with four vriles, n eh vrile eing t five levels (α = 1.682). The inepenent n epenent vriles were nlyze to get regression eqution, whih ws n empiril reltionship etween the HA output n the test vrile in oe units. The regression eqution otine for the output of HA is s follows: Y= X X X X X X X X X 1 X X 1 X X 1 X X 2 X X 2 X X 3 X 4 (1) Y represents the response, whih is epenent vrile, the output of HA (mg/l), n X 1, X2, X 3, n X 4 represent vlues of the inepenent vriles, yest extrt, peptone, gluose n MgSO 4, respetively. Eh of the oserve vlue ws ompre with the preite vlue whih ws lulte from the moel, s epite in Tle 1. The sttistil signifine of the qurti moel eqution ws heke y nlysis of vrine (ANOVA), s shown in Tle 2. The multiple oeffiient of orreltion R= inites lose greement etween experimentl n preite vlue of the HA output. The totl etermintion oeffiient, R 2 = implies tht 90.95% of the vriility in the response oul e expline y the moel. 1557

6 Chen, Y.H. et l. Hyluroni i proution y S. equisimilis Tle 1. Experimentl esign mtrix of RSM n the responses of the epenent vriles Design mtrix (g/100 ml) Output of HA( mg/l) Run Yest extrt Peptone Gluose MgSO 4 7H 2 O Experiment vlue Fitte vlue 1 1( 3.83 ) 1( 2.60 ) 1( 0.74 ) 1( 0.23 ) ( 3.83 ) -1( 1.41 ) 1( 0.74 ) -1( 0.07 ) ( 2.17 ) 1( 2.60 ) 1( 0.74 ) -1( 0.07 ) ( 2.17 ) -1( 1.41 ) 1( 0.74 ) 1( 0.23 ) ( 3.83 ) 1( 2.60 ) -1( 0.26 ) -1( 0.07 ) ( 3.83 ) -1( 1.41 ) -1( 0.26 ) 1( 0.23 ) ( 2.17 ) 1( 2.60 ) -1( 0.26 ) 1( 0.23 ) ( 2.17 ) -1( 1.41 ) -1( 0.26 ) -1( 0.07 ) ( 1.60 ) 0( 2.00 ) 0( 0.50 ) 0( 0.15 ) ( 4.40 ) 0( 2.00 ) 0( 0.50 ) 0( 0.15 ) ( 3.00 ) - 0( 0.50 ) 0( 0.15) 1.68( 1.00 ) ( 3.00 ) 1.68( 3.00 ) 0( 0.50 ) 0( 0.15 ) ( 3.00 ) 0( 2.00 ) - 0( 0.15 ) 1.68( 0.10 ) ( 3.00 ) 0( 2.00 ) 1.68( 0.90 ) 0( 0.15 ) ( 3.00 ) 0( 2.00 ) 0( 0.50 ) -1.68( 0.01 ) ( 3.00 ) 0( 2.00 ) 0( 0.50) 1.68( 0.29 ) ( 3.00 ) 0( 2.00 ) 0( 0.50 ) 0( 0.15 ) ( 3.00 ) 0( 2.00 ) 0( 0.50 ) 0( 0.15 ) ( 3.00 ) 0( 2.00 ) 0( 0.50 ) 0( 0.15 ) ( 3.00 ) 0( 2.00 ) 0( 0.50 ) 0( 0.15 ) ( 3.00 ) 0( 2.00 ) 0( 0.50 ) 0( 0.15 ) ( 3.00 ) 0( 2.00 ) 0( 0.50 ) 0( 0.15 ) ( 3.00 ) 0( 2.00 ) 0( 0.50 ) 0( 0.15 ) The numers in rkets were the tul vlues of the vriles. Tle 2. Vrine nlysis for qurti moel n prmeter estimtes Vriles SS DF MS F p-vlue X X X X X 1 X X 2 X X 3 X X 4 X X 1 X X 1 X X 1 X X 2 X X 2 X X 3 X X 2 X Moel F2= Resiul Lk of fit F1= Pure error R= ; R 2 = ; R 2 (Aj) = ; SS, sum of squres; DF, egrees of freeom; MS, men squres 1558

7 Chen, Y.H. et l. Hyluroni i proution y S. equisimilis The signifine of eh oeffiient ws heke using the vrine rtio test (F-test) n p- vlue in Tle 2. On the one hn, the F-test result shows tht F 0.05 (2, 6) = 5.14<F 1 = 7.581<F 0.01 (2, 6) = 10.92, whih implies tht the Lk of Fit ws signifint wherein α= 0.05 level ut not signifint wherein α= 0.01 level. Bse on the nlysis of the ulture meium omposition, it mnifests tht the existene of the Lk of Fit ftors were minly ue to the high orer intertion. On the other hn, F 2 = 5.746> F 0.01 (14, 8) = 5.56 inites tht the regression eqution ws signifint t α= 0.01 level. The p-vlue less thn 0.05 inites tht the moel terms were signifint. The orresponing vriles will e more signifint if the solute F-vlue eomes lrger n the p- vlue eomes smller. In this se, it n e seen tht the vriles with the remrkle effets on the level of HA proution were the qurti term of gluose (X 3 X 3 ), peptone (X 2 X 2 ), MgSO 4 (X 4 X 4 ), yest extrt (X 1 X 1 ).The p-vlue greter thn 0.10 inite tht the moel terms were not signifint. The mximum of the moel ws otine from the eqution when the onentrtion of yest extrt, peptone, gluose n MgSO 4 were t 3.00 g/100 ml, 2.00 g/100 ml, 0.5 g/100 ml n 0.15 g/100 ml, respetively. The mximum of the preite HA proution ws mg/l. The triplite experiments were lso rrie out to verify the ury of the result for HA proution in similr wy s tht of proution moel. Uner the lulte optiml ulture onition, the men vlue of HA proution ws ±3.50 mg/l, whih ws in greement with the preite vlue well. The verge reltive error etween tul vlue n preite vlue ws 4.95%. Therefore, the moel evelope ws onsiere to e urte n relile for preiting the proution of HA y NC2168. The 3-D response surfe n the 2-D ontour plots were the grphil representtions of the regression eqution (13) n showe the effet of eh inepenent vrile n lso their mutul effets on the proution of HA. The response surfe urves n lso ontour plots for vritions in HA proution were epite in Figure 4. The response surfe representing HA yiel ws funtion of the onentrtions of the two meium omponents with other ingreients eing t n optiml level. By using the response surfe plot, the intertion etween two vriles n their optimum levels n e esily unerstoo n lote. The HA output ws influene y ll the four inepenent vriles. The mximum point ws hieve when gluose, peptone, yest extrt n MgSO 4 onentrtions were t their zero levels. Figure 4. Response surfe n ontour plot of eh inepenent vrile effet n lso their mutul effets on the proution of HA 1559

8 Chen, Y.H. et l. Hyluroni i proution y S. equisimilis Optimiztion of ulture onition The effet of four ftors: temperture, rotte spee, originl ph n meium volume on the output of HA uner the orthogonl experimentl esign re presente in Tle 3. The intuitionisti nlysis shows tht the mximum HA proution ws mg/l, whih ws got y the fifth run. The optimum ulture onition ws: initil ph=7.8; meium volume=20 ml; temperture=33ºc; rotte spee=150 rpm. The rnge nlysis mnifests tht the ftors orer for the effet ws: initil ph> meium volume > temperture > rotte spee. The vrine nlysis (Tle 4) showe tht the effets of ph n meium volume were extremely signifint (p<0.01) n the effet of temperture ws signifint (p<0.05). The result ws in greement with the rnge nlysis. The vlite experiment ws run t the optimum onition mentione ove. HA output touhe mg/l, whih ws lso in greement with the orthogonl experiment result. Tle 3. Orthogonl esign n responsive vlue Run Temperture Rotte spee ph vlue Meium volume Output of HA (ºC) (rpm) (ml) ( mg/l ) 1 1( 31 ) 1( 100) 1( 7.0 ) 1( 20 ) ( 31 ) 2( 150) 2( 7.4 ) 2( 30 ) ( 31 ) 3( 200) 3( 7.8 ) 3( 40 ) ( 33 ) 1( 100) 2( 7.4 ) 3( 40 ) ( 33 ) 2( 150) 3( 7.8 ) 1( 20 ) ( 33 ) 3( 200) 1( 7.0 ) 2( 30 ) ( 35 ) 1( 100) 3( 7.8 ) 2( 30 ) ( 35 ) 2( 150) 1( 7.0 ) 3( 40 ) ( 35 ) 3( 200) 2( 7.4 ) 1( 20 ) R Tle 4. Vrine nlysis on the orthogonl experiment results Soure Degr. of f F p-vlue Signifine lok Temperture * Rotte spee ph <.0001 ** Meium volume <.0001 ** CONCLUSION As to the new mutnt Streptoous equisimilis NC2168 without prouing streptomyin, of whih the optimum ulture meium for mximum HA proution ws rrie out with orthogonl rottle entrl omposite esign n RSM. The moel evelope ws onsierly urte n relile for preiting the proution of HA y NC2168 n mximum of mg/l HA ws otine using the optiml ulture meium s follows: yest extrt=3 g/100 ml, peptone=2 g/100 ml, gluose=0.5 g/100 ml, MgSO 4 =0.15 g/100 ml. The optimize ulture onition ws: temperture 32, rotte spee 200 rpm, initil ph 7.8, n meium volume 20 ml. The mximum HA proution ws mg/l uner the optimum onition. It is evient tht the output of HA ws ffete signifintly y ulture meium n onitions optimiztion. The NC2168 showe % inrese in HA proution uner the optimum ulture meium n onitions. This esign prove to e ruil in hieving inrese in the proution of HA. ACKNOWLEDGEMENTS This work ws supporte y Key Projets in the Ntionl 1560

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