Production of Milk Clotting Enzyme by Penicillium camemberti using Whey medium

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1 IOSR Journal of Botechnology and Bochemstry (IOSR-JBB) ISSN: , Volume 4, Issue 1 (Jan. - Feb 2018), PP Producton of Mlk Clottng Enzyme by Pencllum camembert usng Whey medum A. S. Santhaln Shellomth, * and B. Preetha** Department of Chemcal Engneerng, Annamala Unversty, Annamala Nagar , Chdambaram, Taml Nadu, Inda Abstract: Mlk clottng enzyme s commercally known as Rennet whch s composed of Rennn and Pepsn. It plays a vtal role n Cheese makng wth good flavor and fne texture. The use of cheaper substrates nstead of synthetc medum such as glucose, sucrose, etc wll result lower cost of the fnal product. Approxmately half of the cheese whey produced worldwde s dscarded wthout treatment. The utlzaton of the whey to valuable bo products s the best way to avod the dary waste polluton. Pencllum camembert, a fungal culture was used for the producton of mlk clottng enzyme by usng whey as a substrate n ths study. Pencllum camembert has the ablty to produce a hgh mlk clottng enzyme n submerged fermentaton. Keywords: Mlk clottng enzyme, Rennn, Proteolytc actvty. Response surface methodology Date of Submsson: Date of acceptance: I. Introducton The development of food technology leads to ncrease the applcatons of food grade enzymes. Enzymes for food applcatons are obtaned from anmal, plant and mcrobal sources and the hydrolases play vtal role n the food ndustres. Mlk clottng enzyme s bochemcally known as Aspartc protease, an extracellular hydrolase enzyme. Bovne Chymosn n the form of Calf Rennet has been used for cheese makng, datng back to approxmately by 6000 BC (srmayegn, et al., 2011) Tradtonal cheese producton by usng Bovne rennet s the oldest method. Natural calf rennet s extracted from the fourth stomach (abomasum) of mlk fed young calves.the lmted avalablty of anmal rennet leads to a search for alternate rennet substtutes for cheese makng ndustry. Many plants have coagulatng propertes for example, extract of fg juce, Papaya & Pne apple to coagulate mlk.mcrobal rennet are produced from the mcroorgansms both fung and bactera. Some molds such as Rhzomucormehe are able to produce mlk clottng enzyme Mcro organsms, EndothaParastca Bacllus reseus, mucorpuslus, Cryptococcus albdus and Mucormehe are known to produce mlk clottng enzymes whch substtute calf rennet (Fox, 1991; and Baley et al., 1988).Most of the plant rennets have prooved not sutable for cheese makng because of the btter taste. But the mcrobal rennets are more promsng because ts cheaper producton,greater bochemcal dversty and the genetc modfcaton s easer.(tanboly etal.2013) Whey s the by-product of cheese and curd manufacturng, once t was consdered as a waste product. The dscovery of whey as a functonal food wth nutrtonal applcatons elevated whey to a co-product n the manufacturng of cheese (Walzem.et al 2002). Mlk contans two sources of proten, the casens and whey. Durng processng, the casens are the protens responsble for makng curds, whle whey remans n an aqueous envronment. Whey s a major source of lactose, good source of valuable proten and mnerals and water soluble vtamns (Bande, 2011).. The bologcal components of whey, ncludng lactoferrn, beta lactoglobuln, alphalactalbumn, glycomacropeptde, and mmunoglobulns, have mmune-enhancng propertes. In addton, whey act as an antoxdant, anthypertensve, anttumor, hypolpdemc, antvral, antbacteral, and chelatng agent. A number of clncal trals have successfully been performed usng whey n the treatment of cancer, HIV, hepatts B, cardovascular dsease, osteopoross, and as an antmcrobal agent. ( Ker Marshall, ND, MS.,2004). Dary ndustres dspose strong waste water characterzed by hgh BOD and COD representng ther hgh organc content (Demrelet al., 2005). Worldwde producton of whey s about 145 mllon tons, wth 60% recovered by several methods and 40% dscarded drectly nto rvers (Cela Mara et al., 2001).) The utlzaton of whey for the producton of valuable commercal Products s the best soluton for the envronmental problem caused by the dsposal of whey (Morr.et al., 1993). In the present study, dfferent medum namely basal medum, casen and lactose along wth basal medum n whey medum and plan whey were studed under statonary and shakng condtons for the producton of mlk clottng enzyme by Pencllum camembert. The am of ths study was to fnd out the optmum process condtons for the selected operatng varables namely ntal substrate concentraton, ntal DOI: / Page

2 ph, temperature and bomass concentraton for the maxmum producton of mlk clottng enzyme usng whey as substrate by Response surface methodology. Response surface methodology (RSM) s an emprcal statstcal method utlzed for multple regresson analyss of quanttatve data obtaned from statstcally desgned experments by solvng the multvarate equatons smultaneously. The response surfaces are the graphcal representaton of the quadratc equatons to explan the ndvdual and cumulatve effect of the test varable response surfaces and to fnd out the nteracton between the test varables.( Khur , Montgomery.1932). II. Materals and methods 2.1 Mcroorgansm. The fungal culture Pencllum camembert (MTCC 418) was obtaned from MTCC Chandgarh, Inda. Ths culture was mantaned by sub culturng perodcally at 30 o C for 5 days and stored at 4 o C. 2.2 Growth and Producton medum The composton of the growth and fermentaton medum were mentoned below(g/l)czapek concentrate 10 ml; K2HPO4 1.0; Yeast extract 5.0;Sucrose 30.0; Dstlled water 1000ml.Composton of czapek concentrate (g/100ml) are NaNo3 30.0;Kcl 5.0; Mgso4.7H2O 5.0; FeSo4.7H2O 0.1; The 250 ml Erlenmeyer Flask Contaned 100ml of the medum and autoclaved for 15 mn at 121k gf whch were then noculated and ncubated at 30 C for 5 days. The producton was carred out by usng known volume of 5 days noculums n the above medum usng whey at 30 C both statc and shaker at 120 rpm for 5 days. Fermentaton medum of above composton namely plan basal medum, Lactose and Casen along wth the basal medum n whey medum and plan whey were used for ths study. All experments were carred out n duplcate and repeated at least twce. Samples were taken from the soluton at regular tme ntervals for the analyss of mlk clottng actvty, proteolytc actvty, bomass concentraton and proten content. 2.3 Expermental Desgn and Statstcal Analyss The factors affectng the producton of mlk clottng enzyme from whey by Pencllum camembert was studed usng Central Composte Desgn (CCD) experments. The ntal substrate concentraton (A) g/l, ntal ph (B), temperature (C) C and bomass concentraton (D) g/l were chosen as the ndependent varables as shown n Table 1. Mlk clottng actvty (Y) was chosen as the dependent output varable. An orthogonal 2 4 full factoral central composte desgn wth eght star ponts ( =2) and seven replcaton at the centre pont, all n duplcates, resultng n a total of 31 experments were used to optmze the chosen key varables for the producton of Mlk clottng enzyme n a batch reactor. The experments wth varous ntal substrate concentratons (whey medum) namely 10,,20,30.40 and 50(%v/v), dfferent ntal ph values of 5.0, 5.5, 6.0, 6.5 and 7.0, dfferent temperatures of 30, 35, 40, 45 and 50 o C and fve dfferent bomass concentratons of 3.0, 6.0, 9.0, 12.0 and 15.0 g/l were employed and vared smultaneously to cover the combnatons of varables n the desgn. The range and the levels of the expermental varables nvestgated n ths study were gven n Table 1. The chosen ndependent varables used n ths experment were coded accordng to Eq. (1): x x o (1) Where x s the coded value of the th varable, s the uncoded value of the th test varable and o s the uncoded value of the th test varable at the centre pont The behavour of the system s explaned by the followng second- degree polynomal Eq. (2): k k k 1 k 2 Y o j j j 2 (2) Where Y s the predcted response, o s the offset term, s the coeffcent of lnear effect, s the coeffcent of squared effect and j s the coeffcent of nteracton effect. Ths regresson model can be used to estmate the ellptcal contours of a constant surface. A statstcal desgn package, Mntab 16 was used for regresson analyss of the data obtaned and to estmate the coeffcents of the second-degree polynomal equaton. The equatons were valdated by the statstcal tests called the analyss of varance (ANOVA), to determne the sgnfcance of each term n the equaton and to estmate the goodness of ft n each varable. Response surfaces were drawn to determne the ndvdual and nteractve effects of test varables on mlk clottng actvty. DOI: / Page

3 2.4 Preparaton of whey The fresh mlk whey was provded by Ponlat Dary products Ltd., Pondcherry, Inda. To remove the suspended partcles contaned n raw whey, fltraton step was performed by Whatmann No. 1 flter paper. The clarfed whey was used as a substrate for mlk clottng enzyme producton. 2.5 Preparaton of the Crude enzyme The fermented medum was fltered to separate the bomass from the culture fltrate usng whatman no 40 flter paper. The fltrate was centrfuged at 4 o C for 10 mn at rpm n the coolng centrfuge. Then the supernatant was used for the enzyme assays. 2.6 Analyss of crude enzyme Estmaton of Mlk clottng actvty: Mlk clottng actvty (MCA) was determned by the method explaned by Arma., etal(1964) and Balls.,etal(1937) usng 0.1 (w/v) of rennn std and the substrate s 10g of skmmed mlk powder n 0.01 mol. calcum chlorde. The reacton mxture contans 5 ml of skm mlk and 1ml of enzyme. It was kept at 37 C for MCA. The curd formaton was observed by manually rotatng the test tube from tme to tme. The end pont s the sem lquefed flm appears on the sde of the test tube above the mlk. The clottng tme was noted. M MCU / mg (3) T mnutes xw g Where M s the mlk factor, T s the clottng tme of sample (mn) and Ws the grams of enzyme added to the substrate n 2.0 ml alquot (g wt. x 2) Estmaton of Proteolytc actvty Proteolytc actvty was determned by the Unversal Protease actvty assay by usng casen as a substrate. The reacton mxture contanng 5 ml of 0.65% pre ncubated casen soluton (37 o C/10mn) and 1ml of enzyme was ncubated for 10 mn at 37 o C. And 5 ml of TCA was added to stop the reacton and ncubated at 37 o C for 30 mn. Durng the tyrosne standard was set up (0.2mg/ml) n the range of ml, made up to 2ml wth dstlled water. Then the test solutons are centrfuged at4 o C at 10000rpm for 10 mn and the 2ml of alquots are used for PA. To all the tubes (ncludng standard), 5 ml of sodum carbonate, 1ml of Foln s phenol s added and ncubated at 37 o C for 30 mn. Then the optcal densty was measured at 660 nm by usng uv- Bospectrophotometer.(Anson,1937.,Chwen-jensheh etal.2009) Unts / ml enzyme μmole tyrosne equvalents released Where 11 s the total volume of assay(ml), 10 s the tme of assay as per the unt defnton (mn), 1s the volume of enzyme used(ml) and 2s the volume used n colormetrc detrmnaton(ml) Determnaton of Proten Proten was estmated by Lowry method (1951) by usng BSA as a standard. The optcal densty was measured for 660 nm Estmaton of Bomass concentraton Samples from the producton medum were fltered through whatmann no.40 flter paper to separate the bomass. The settled bomass was collected and dred and expressng the dry weght as grams per ltre of growth medum. III. Results and Dscusson 3.1 Effect of dfferent medum components on the producton of mlk clottng enzyme The effect of addton of lactose and casen along wth the basal medum of whey, plan basal medun and plan whey medum under agtated and statonary condton for the producton of mlk clottng enzyme was evaluated wth remarkable observaton. Fg 1 shows the mlk clottng actvty and proteolytc actvty levels obtaned n a whey. The plan whey medum,plan basal medum,casen and lactose along wth the basal medum were denoted as M1, M2,M3 and M4 respectvely. Maxmum mlk clottng enzyme concentraton 0.50 unts/mg was obtaned for P. camembert under statonary condton. Fg 2 clearly ndcates the addton of casen gves the hgher mlk clottng actvty than the lactose, plan basal medum and plan whey. Casen wth other substrates s an effectve enhancer of mlk coagulaton (slva et al.,2014). Fg 2 shows the bomass concentraton under statc and shakng condtons. Maxmum bomass concentraton of 35.8 g/l was obtaned n the presence of casen, followed by 30.5 g/l n lactose, 28.5g/l n basal medum,whle plan whey had the least yeld of 13.2 g/l under statc condtons. DOI: / Page (4)

4 Enzyme Actvty (unts/mg) Bomass Concentraton (g/l) Producton of Mlk Clottng Enzyme by Pencllum camembert usng Whey medum 0.6 Mlk clottng actvty 0.5 Proteolytc actvty M1 M2 M3 M4 Medum Fg 1 Effects of dfferent medum components on mlk clottng enzyme producton by Pencllum camembert M1 M2 M3 M4 Medum Statonary Shakng Fg 2 Effect of dfferent medum components on bomass concentraton by Pencllum camembert 3.2 Central composte desgn and optmzaton usng response surface methodology for the producton of mlk clottng enzyme The coded values of the ndependent varables along wth observed responses n each case were gven n Table 2. By applyng multple regresson analyss, a predctve quadratc model was ftted wth expermental results, and the equaton for the producton of mlk clottng enzyme was n the form of the followng equaton: Y= A+0.017B-0.020C+0.024D-0.035A B C D AB+0.010AC-0.011AD+0.016BC-0.030BD+0.036CD... (5) where Y s the mlk clottng actvty (unts/mg), A s the ntal substrate concentraton(whey medum) (%v/v), B s the ntal ph, C s the temperature ( o C) and D s the bomass concentraton (g/l).the mlk clottng observaton durng enzyme analyss for all 31 expermental run were gven n Table 3.It was found that the mlk clottng actvty manly depends on clottng tme and enzyme concentraton. Good coagulaton was observed after15mn when the ph was mantaned at 6.0 and mlk clottng was also observed at hgh temperature level wthn 20mn.Good clottng wth fne curd wthn 15 mn. was observed at the p H range of 6.0 at 40 o. Table 1 Central composte desgn for the producton of mlk clottng enzyme by Pencllum camembert. Independent Varable Range and Level Intal Substrate Concentraton (whey medum) (%v/v)) (A) Intal ph (B) Temperature( o C) (C) Bomass Concentraton (g/l) (D) Table 2 Full factoral central composte desgn matrx of orthogonal values along wth observed responses for the producton of mlk clottng enzyme Run order Independent Varable Mlk Clottng Actvty (unts/mg) Orthogonal Value Expermental Predcted A B C D DOI: / Page

5 Table 3 Central composte desgn matrx of orthogonal values along wth respectve observed mlk clottng observaton DOI: / Page

6 Table 4 Sgnfcance of regresson coeffcents for the producton of mlk clottng enzyme usng Mntab 16 software Model Term Parameter estmate ( Coeffcents) Constant A B C D A *A B * B C * C D * D A * B A * C A * D B * C B * D C * D A, B, C, D = Lnear effects A 2, B 2, C 2, D 2 = Squared effects AB, AC, AD, BC, BD, CD = Interacton effects A B C D A *A B * B C * C D * D Table 5Analyss of Varance (ANOVA) for the selected quadratc model for the Producton of mlk clottng enzyme Sources of Degrees of Sum of squares varaton Freedom Mean square F P Regresson Lnear Square Interacton Resdual error Total Lnear Square Interacton The student t dstrbuton and correspondng p values, along wth the parameter estmate were gven n Table 4.The lnear effect of all the parameters were found to be sgnfcant. The squared effects of all the parameters A * A,B * B,C * C,D * D were also found to be sgnfcant and the coeffcent of the effect of temperature(p = )was found to be hghly sgnfcant. The statstcal sgnfcance of each term n the quadratc model was valdated by the statstcal tests called the Analyss-of-varance (ANOVA) and the results were gven n Table 5. ANOVA of the regresson model was sgnfcant and t was evdent from the calculated F value (7.99) and a very low probablty. The coeffcent for the squared effect was hghly sgnfcant (p=0.0001) when compared wth the lnear and nteractve effects. Response surface contour plots descrbe the relatonshp between the response and expermental levels of each varable and These plots explan the type of nteracton between test varables and help to obtan the optmum condtons..fg3 to 5 shows the response surface plots aganst each of the ndependent varables whle keepng the other varables at ther 0 levels. The surface confned n the smallest curve of the response surface dagram ndcated the maxmum product yeld. The ellptcal nature of the contour ndcates that ths nteracton s sgnfcant on the response. The response surfaces can also fnd the optmum range of process varables. DOI: / Page

7 Fg.3 Response surface contour plot showng nteractve effect of ntal substrate concentraton and ntal ph on the producton of mlk clottng enzyme Fg.4 Response surface contour plot showng nteractve effect of ntal ph and temperature on the producton of mlk clottng enzyme Fg.5 Response surface contour plot showng nteractve effect of temperature and ntal substrate concentraton on the produc ton of mlk clottng enzyme To valdate the optmal parameters, confrmatory experments were carred out by lab scale producton. The observed results were compared wth the predcted results.the process condtons for the maxmum producton of mlk clottng enzyme by Pencllum camembert under optmzed condtons were gven n Table 6. Mlk Clottng Actvty 0.585unts/mg(MCA), Proteolytc Actvty0.395unts/mg (PA), the rato MCA/PA1.44 and proten content 0.356mg/ml were found under optmum condtons. These values agree wth the values from the response surface analyss (MCA=0.5933unts/mg) confrmng that the RSM usng statstcal desgn s the effectve tool can be used to optmze the process parameters and to study the mportance of ndvdual, cumulatve and nteractve effects of the test varables n mlk clottng enzyme producton. The confrmatory experments showed the hgh mlk clottng actvty and low proteolytc actvty whch s essental for the perfect mlk coagulaton.it was reported that the producton of cheese s necessary to use rennn wth strong mlk clottng actvty and the least proteolytc acton to mnmse dssoluton of the curd. (Hashem.,1999) Table 6 Optmum values of varables obtaned from regresson equatons for the producton of mlk clottng enzyme by Pencllum camembert Parameter Optmum value for mlk clottng enzyme producton Intal Substrate Concentraton (whey medum)(%v/v) 33 Intal ph 6.0 Temperature( o C) 40 Bomass Concentraton (g/l) 10.3 Mlk Clottng Actvty (unts/mg) IV.Concluson The producton of mlk clottng enzyme by Pencllum camembert has been studed n submerged fermentaton usng whey as a substrate.the whey basal medum wth casen shows the hgh mlk clottng actvty and t s an effectve substrate for the producton of mlk clottng enzyme by Pencllum camembert. DOI: / Page

8 The results reported that the whey basal medum contanng casen under statc condtons enhanced the mlk clottng actvty of unts/mg wth low proteolytc actvty 0.404unts/mg. Statstcal expermental desgn s an effectve tool for studyng the nfluence of process parameters on mlk clottng actvty. The results recommended that the whey medum s the hgh nutrent substrate for the producton of mlk clottng enzyme by the fungal culture Pencllum camemebert.it could be concluded that the whey could be used as a cheap source for the producton of mlk clottng enzyme as a medum for mcrobal growth.. Acknowledgement The authors acknowledge the fnancal support receved from the Unversty Grant commsson, New Delh, Inda, (Grant No:F1-17.1/ /RGNF SC-TAM-4979 (SAIII Webste)) A.S. Santhaln Shellomth wshes to thank Ponlat Dary Products Ltd, Pondcherry, Inda. for provdng the whey to carry out the present study. References [1]. Amal M. Hashem (1999) Optmzaton of mlk-clottng enzyme productvty by Pencllum oxalcum. Boresource Technology [2]. Anson, M.L.,J.Gen.Phsol,(1938),22:p [3]. Arma, K., et al.,(1964). Cheese makng by usng the mlk clottng enzyme of Mucorpusllus. I Rennet propertes of the enzyme Jap.J.Zootech.Sc.35, [4]. Baley, J.E., and D.F.Olls, (1986) Bochemcal Engneerng Fundamentals. McGraw-Hll, New York, [5]. Balls A.K and Hoover, S.R., J.Bol, (1937) Chem.121, 737. [6]. Bande,S.V.(2011)Incorporaton of concentrated Lactose hydrolyzed whey n the soup stcks. M.Tech thess submtted to NDRI deemed Unversty, Karnal. [7]. Cela Mara. Galvao, AsteraF.Souza(2001). Controlled Hydrolyss of cheese whey Protens Usng Trypsn and αchymotrypsn.appled Bochemstry and Botechnology Vol [8]. Chwen-Jen Sheh, Lan-Anh Phan Th, Ing-Lung Shn, (2009),Mlk clottng Enzyme Produced by culture of Bacllus natto,.bochemcal engneerng journal, 43: [9]. De lma C.J.B., Marana Cortez,(2008).World Appled scences Journal 4(4): [10]. Dmerel, B.,O.Yengun and T.T.Onay(2005),Anaerobc treatment of dary waste water:a revew,process Bochemstry,40: [11]. Fox, Paul McSweeney, Tmothy M. Cogan, Tmothy P. Gunee (2004). "Cheese: Major cheese groups". Academc Press: 2. Retreved [12]. Ker Marshall, ND, MS.,(2004)Therapeutc Applcatons of Whey Proten.Alternatve Medcne Revew Volume 9, Number [13]. Khur, A. I and Cornell J.A. (1982) Response Surfaces: Desgn and Analyss. Marcel Dekker, New York. [14]. Lowry, O.H. et al (1951). Proten Measurement wth Foln Phenol Reagent.J.Bol.Chem.193, [15]. Montgomery, D. C. (1981),Desgn and Analyss of experments.3rd edn, Wley, New York, 13.Anson, M.L., J.Gen. Physol,1938,22: p , [16]. Morr,C.V.and Ha,E.Y.W(1993)Cr.rev.FoodSc.Nutr.33, [17]. Slva,B.L.,The Producton and Characterzaton of a Mlk-clottng Protease Produced n Submerged Fermentaton by the Thermophlc Fungus Thermomucorndcae-seudatcae N31,ApplBochemBotechnol 172,2014 [18]. Srmayegn,et al.,(2010) Aspartc protenases from Mucor spp. Appled mcrobol Botechnol.89: [19]. ) EL-Sayed El-Tanboly,Mahmoud El-Hof,Youssef Bahr Youssef Bahr Yousuff,Wased El-Desok,Azza Ismal.(2013).Utlzaton of salt whey from Egyptan Ras (Cephalotyre Cheese whey n Mcrobal Mlk Clottng enzymes Producton.Acta Sc. Pol.,Technol.Alment.12(1) [20]. Walzem RL, Dllard CJ, German JB. (2002) Whey components: mllenna of evoluton create functonaltes for mammalan nutrton: what we know and what we may be overlookng. Crt Rev Food Sc Nutr.42: IOSR Journal of Botechnology and Bochemstry (IOSR-JBB) s UGC approved Journal wth Sl. No. 4033, Journal no A. S. Santhaln Shellomth "Producton of Mlk Clottng Enzyme by Pencllum camembert usng Whey medum." IOSR Journal of Botechnology and Bochemstry (IOSR-JBB) 4.1 (2018): DOI: / Page