IOSR Journal of Engneerng (IOSRJEN) ISSN (e): 2250-3021, ISSN (p): 2278-8719 Vol. 08, Issue 3 (March. 2018), V2 PP 29-36 www.osrjen.org Producton of Mlk Clottng Enzyme n Submerged Fermentaton wth Streptococcus Lacts by Usng Whey Medum A. S. Santhaln Shellomth 1 and B. Preetha 2 1,2 Department of Chemcal Engneerng, Annamala Unversty, Annamala Nagar 608 002, Chdambaram, Taml Nadu, Inda Correspondng Author: A. S. Santhaln Shellomth Abstract: In ths study, utlzaton of whey for the producton of mlk clottng enzyme by Streptococcus lacts was carred out under submerged fermentaton. Effects of dfferent medum components for the producton of mlk clottng enzyme were determned under statonary and shakng condtons. Hghest mlk clottng actvty was observed n the whey medum contanng casen under shakng condtons. Central composte desgn experments were carred out to examne the mutual nteracton between the varables and to determne the optmal values that brngs out hgh yeld mlk clottng enzyme. Maxmum mlk clottng actvty of 0.4975 unts/mg was obtaned at optmum process condtons namely ntal substrate concentraton 29 g/l, ntal ph 6.2, temperature 39.9ºC and bomass concentraton 1.6 g/l. Keywords: Mlk clottng enzyme, Whey, Response Surface Methodology, Mlk clottng actvty, Proteolytc actvty. ----------------------------------------------------------------------------------------------------------------------------- ---------- Date of Submsson: 26-02-2018 Date of acceptance: 15-03-2018 ----------------------------------------------------------------------------------------------------------------------------- ---------- I. INTRODUCTION Mlk clottng enzyme s known as Rennet whch s composed of rennn and pepsn. Rennn wth hgh mlk clottng actvty and low proteolytc actvty s the potent enzyme source whch s commercally acceptable for cheese makng n the food processng Industres. The calf rennet used for cheese makng s the oldest method and t s extracted from the fourth stomach of young calves. Due to the legal problems aganst the anmal sacrfce for the research purpose leads to the new search for alternate rennet producton from plant and mcrobal sources.the present study deals wth the Producton of Mlk clottng enzyme by Streptococcus lacts usng whey water as a medum for the submerged fermentaton. The Streptococcus lacts s the bacteral culture whch s wdely used n dary ndustres. Whey contans hghly nutrtous consttuents and the most valuable components are the whey protens whch are desgnated superor to most of the other protens such as egg, beef, casen and soya protens n nutrtve value [1]. Gupta et al. [2] reported that whey protens are generally regarded as safe for food applcatons [3,4] because of ther good nutrtonal and functonal propertes. Whey protens are next to egg proten n terms of nutrtve value. Whey Lactose s the mlk sugar whch s the prmary mlk consttuent n whey whch contrbutes around 80-90% of whey solds. The major mneral components n lqud whey are the mneral catons lke sodum, potassum, calcum and magnesum as well as anons lke chlorne, ctrate and phosphate. [5] Whey Protens exhbt excellent functonal propertes such as solublty, foamng, emulsfyng, gellng and water bndng etc. apart from ther nutrtonal and therapeutc value [6,7]. Because of the presence of large part of organc consttuents, the bologcal oxygen demand (BOD) of whey s very hgh (40,000-50,000 mg/kg)leads to a major burden of dsposal as waste through the regular channel. In recent tmes, the envronmental regulatons have become severe across the world necesstatng the treatment of whey pror to dsposal through sewage system. To eradcate the problems nvolved wth the whey dsposal, efforts are beng made towards product dversfcaton usng whey components wthout much change n the exstng nfrastructure. Ths wll be qute feasble to reduce the polluton. In the present study, dfferent medum namely basal medum, casen and sucrose 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 Streptococcus lacts. The am of ths study was to fnd out the optmum process condtons for the selected operatng varables namely ntal substrate concentraton, ntal 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 29 P a g e
multvarate equatons smultaneously. The response surfaces are the graphcal representaton to explan the ndvdual and cumulatve effect of the test varable response surfaces and to fnd out the nteracton between the test varables [8,9]. II. MATERIALS AND METHODS 2.1 Mcroorgansm. The bacteral culture Streptococcus lacts(ncim 2114) was obtaned from NCL Pune, Inda.. Ths culture was mantaned by sub culturng perodcally at 30 o C for 24 hours and stored at 4 o C. 2.2 Growth and Producton medum The mcroorgansm was grown aerobcally n MRS meda contanng followng composton n 1000 ml dstlled water: protease peptone, 10g; yeast extract, 5g; Beef extract, 10g; dextrose, 20g; tween 80, 1.0g; ammonum ctrate, 2.0g;sodum acetate, 5.0g; Magnesum sulphate, 0.1g; Manganese sulphate, 0.05g; Dpotassum phosphate, 2.0g. The ph of the medum was adjusted to 6.5 usng dlute hydrochlorc acd, ncubated at 30 o C for 24 hours and stored at 4 o C. The producton was carred out by usng known volume of 1 day noculum n the above medum usng whey at 30 C both statc and shaker at 120 rpm for 3 days. Fermentaton medum of above composton namely plan whey medum, Sucrose and Casen along wth the basal medum n whey and plan basal medum were used for ths study. Samples were taken from the soluton at regular tme ntervals for the analyss of mlk clottng actvty, proteolytc actvty, bomass concentraton and proten content. All experments were carred out n duplcates. 2.3 Expermental Desgn and Statstcal Analyss The factors affectng the producton of mlk clottng enzyme from whey by Streptococcus lacts 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 submerged 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 0.5, 1.0,1.5, 2.0,and2.5 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 1 1 1 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. 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 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. 30 P a g e
2.4 Preparaton of whey The mlk whey was provded by Ponlat Dary products Ltd., Pondcherry, Inda. The whey was fltered by Whatmann No. 1 flter paper to remove the suspended partcles. 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 10000 rpm n the coolng centrfuge. Then the supernatant was used for the further analyss. 2.6 Analyss of crude enzyme 2.6.1 Estmaton of Mlk clottng actvty: Mlk clottng actvty (MCA) was determned by the method explaned by Arma, et al [10] and Balls,,et al [11] 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 and the mlk clottng actvty was calculated. MCU / mg T M mnutes xw g (3) 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) 2.6.2 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. The tyrosne standard was set up (0.2mg/ml) n the range of 0.1-0.5ml, 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 [12,13]. Unts / ml enzyme μ mole tyrosne equvalents released 11 1 10 2 (4) 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 determnaton(ml). 2.6.3 Determnaton of Proten Proten was estmated by Lowry method [14] by usng BSA as a standard. The optcal densty was measured for 660 nm. 2.6.4 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 DISCUSSION 3.1 Effect of dfferent medum components on the producton of mlk clottng enzyme The effect of dfferent medum components on the Producton of mlk clottng enzyme was carred out by supplemented wth scurose and casen along wth the basal medum of whey, plan basal medun and plan whey medum under agtated and statonary condton.fg 1 shows the mlk clottng actvty and proteolytc actvty levels obtaned n a whey. The plan whey medum, plan basal medum, casen and sucrose along wth the basal medum were denoted as S1,S2,S3 and S4 respectvely. Maxmum mlk clottng enzyme concentraton 0.498 unts/mg was obtaned for S.lacts under shakng condton. Fg 1. clearly ndcates the addton of 31 P a g e
casen(s3) gves the hgher mlk clottng actvty than the other components of medum(s1,s2,s4) Dutt et al [15] reported that the ncreased MCA was found n the casen contanng medum.fg2. Shows the bomass concentraton under statc and shakng condtons. Maxmum bomass concentraton of 15.2 g/l was obtaned n the presence of casen, followed by 11.5 g/l n sucrose, 11.0g/l n basal medum,whle plan whey had the least yeld of 8.9 g/l under shakng condtons. Fg 1.Effects dfferent medum components on mlk clottng enzyme producton by Streotococcus lacts Fg 2.Effects dfferent medum components on bomass concentraton by Streptococcus lacrs 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=0.495-0.000A+0.008B-0.003C+0.006D-0.026A 2-0.020B 2-0.020C 2-0.030D 2-0.014AB+0.016AC- 0.005AD+0.001BC+0.021BD+0.014CD... (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). 32 P a g e
Table 1 Central composte desgn for the producton of mlk clottng enzyme by Streptococcus lacts Independent Varable Range and Level -2-1 0 +1 +2 Intal Substrate Concentraton (whey medum) (%v/v)) (A) 10 20 30 40 50 Intal ph (B) 5.0 5.5 6.0 6.5 7.0 Temperature( o C) (C) 30 35 40 45 50 Bomass Concentraton (g/l) (D) 0.5 1.0 1.5 2.0 2.5 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 1-1 1-1 1 0.450 0.458 2 1-1 -1 1 0.325 0.355 3 0 0 0-2 0.334 0.362 4 1-1 1-1 0.422 0.421 5 0 0 0 2 0.365 0.386 6-1 -1-1 -1 0.399 0.420 7 0-2 0 0 0.401 0.395 8-1 -1 1 1 0.366 0.358 9 2 0 0 0 0.388 0.389 10 0 0 0 0 0.499 0.495 11 0 0 0 0 0.489 0.495 12 1 1-1 -1 0.354 0.369 13 0 0 0 0 0.490 0.495 14 0 2 0 0 0.374 0.429 15 1 1-1 1 0.422 0.382 16 0 0 0 0 0.495 0.495 17-1 1 1-1 0.378 0.355 18 1-1 -1-1 0.450 0.425 19-1 1 1 1 0.480 0.449 20 0 0 0 0 0.496 0.496 21 1 1 1-1 0.385 0.368 22 0 0-2 0 0.399 0.418 23 1 1 1 1 0.455 0.441 24-2 0 0 0 0.343 0.392 25 0 0 2 0 0.375 0.405 26 0 0 0 0 0.497 0.495 27-1 1-1 -1 0.480 0.442 28-1 -1 1-1 0.364 0.348 29-1 -1-1 1 0.411 0.372 30 0 0 0 0 0.499 0.495 31 1-1 1 1 0.407 0.409 Table 3 Sgnfcance of regresson coeffcents for the producton of mlk clottng enzyme usng Mntab 16 software Model Term Parameter estmate ( Coeffcents) Constant 0.495 37.982 0.000 A -0.000-0.107 0.0916 B 0.008 1.22 0.240 C -0.003-0.485 0.634 D 0.006 0.864 0.400 33 P a g e
A *A -0.026-4.068 0.001 B * B -0.020-3.215 0.005 C * C -0.020-3.234 0.005 D * D -0.030-0.468 0.000 A * B -0.014-1.711 0.106 A * C 0.016 1.958 0.068 A * D -0.005-0.638 0.532 B * C 0.001 0.131 0.898 B * D 0.021 2.436 0.027 C * D 0.014 1.697 0.109 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 *A B * B C * C D * D A *C B * D Table 4 Analyss of Varance (ANOVA) for the selected quadratc model for the Producton of mlk clottng enzyme Sources of Sum of Degrees of Mean varaton squares Freedom square F P Regresson 0.076 14 0.005 4.5 0.002 Lnear 0.002 4 0.000 0.62 0.655 Square 0.054 4 0.013 11.38 0.000 Interacton 0.019 6 0.003 2.67 0.055 Resdual error 0.019 16 0.001 Total 0.095 30 Square The student t dstrbuton and correspondng p values, along wth the parameter estmate were gven n Table 3. The squared effects of all the parameters A * A,B * B,C * C,D * D were found to be sgnfcant and the Interactve effect of the parameters A*C and B*D were also found to be 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 4. ANOVA of the regresson model was sgnfcant and t was evdent from the calculated F value (4.5) 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. Fg 3 to 6 shows the response surface plots aganst each of the ndependent varables whle keepng the other varables at ther 0 levels. The smallest surface 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 and the optmum range of process varable are found by the response surfaces of the contour plots. 34 P a g e
To valdate the optmal parameters, confrmatory experments were carred out by lab scale producton n the Bofermentor. The observed results were compared wth the predcted results. The process condtons for the maxmum producton of mlk clottng enzyme by Streptococcus lacts under optmzed condtons were gven n Table 5. Mlk Clottng Actvty 0.491unts/mg(MCA), Proteolytc Actvty 0.387unts/mg (PA), the rato MCA/PA1.26 and proten content 0.297mg/ml were found under optmum condtons. These values agree wth the values from the response surface analyss (MCA=0.4975unts/mg) confrmng that the RSM usng statstcal desgn s the effectve tool to optmze the process parameters and to study the 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 suggests ths s a sutable enzyme source for Mlk clottng n cheese Industres. Good coagulaton was observed after17 mn under the optmzed condtons. 35 P a g e
Table 5 Optmum values of varables obtaned from regresson equatons for the producton of mlk clottng enzyme by Streptococcus lacts Parameter Optmum value for mlk clottng enzyme producton Intal Substrate Concentraton (whey medum) (%v/v) 29 Intal ph 6.2 Temperature( o C) 39.9 Bomass Concentraton (g/l) 1.6 Mlk Clottng Actvty (unts/mg) 0.4975 IV. CONCLUSION The fermentatve producton of mlk clottng enzyme by Streptococcus lacts has been studed usng whey as a substrate.the results reported that the whey basal medum contanng casen under shakng condtons enhanced the mlk clottng actvty of 0.491 unts/mg wth low proteolytc actvty 0.387unts/mg. Statstcal expermental desgn s a valuable tool for studyng the nfluence of process parameters on mlk clottng actvty. The results suggested that the whey medum s the hgh nutrent substrate for the producton of mlk clottng enzyme by the bacteral culture Streptococcus lacts.the whey could be used as a cheap and good source for the producton of mlk clottng enzyme.. ACKNOWLEDGEMENT The authors acknowledge the fnancal support receved from the Unversty Grant commsson, New Delh, Inda, (Grant No:F1-17.1/2015-2016/RGNF-2015-2017-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] Poonam, M. (2007) Incorporaton of concentrated whey n the producton of bun and pzza. M. Tech. Thess submtted to NDRI Deemed Unversty, Karnal. [2] Gupta, V. K., and Reuter, H. (1993). Frmness and meltng qualty of processed cheese foods wth added whey proten concentrates. Le Lat, 73(4), 381-388. [3] Hugunn, A.G. (1987). Applcaton of UF whey proten: Developng new market n trends n whey utlsaton. Int. Dary Fed. Brussels, Belgum, 135. [4] Morr, C.V., and Ha, E.Y.W. (1991). Off-flavors of whey proten concentrates: A lterature revew. Internatonal Dary Journal, 1(1), 1-11. [5] IDF (2003). Bulletn of the Internatonal Dary Federaton. WDS Forum, 384: 20. [6] Matthews, M.E. (1984). Whey proten recovery processes and products. Journal of Dary Scence, 67(11), 2680-2692. [7] Patel, M.T., and Klara, A. (1990). Studes on whey proten concentrates. 2. Foamng and emulsfyng propertes and ther relatonshps wth physcochemcal propertes. Journal of Dary Scence, 73(10), 2731-2740. [8] Khur, A. I and Cornell J.A. (1982) Response Surfaces: Desgn and Analyss. Marcel Dekker, New York. [9] Montgomery, D. C. (1981),Desgn and Analyss of experments.3rd edn, Wley, New York, 13.Anson, M.L., J.Gen. Physol,1938,22: p. 79-89. [10] 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.35,221-228. [11] Balls A.K and Hoover, S.R., J.Bol, (1937) Chem.121, 737. [12] Anson, M.L.,J.Gen.Phsol,(1938),22:p.79-89. [13] Chwen-Jen Sheh, Lan-Anh Phan Th, Ing-Lung Shn, (2009), Mlk clottng Enzyme Produced by culture of Bacllus natto,. Bochemcal engneerng journal, 43: 85-91. [14] Lowry, O.H. et al (1951). Proten Measurement wth Foln Phenol Reagent.J.Bol.Chem.193, p.265-275. [15] Dutt., et al (2008). Role of casen on nducton and enhancement of producton of a bacteral mlk clottng protease from an ndgenously solated Bacllus subtls, Letters n Appled Mcrobology, 46(5), p.513 518. A. S. Santhaln Shellomth "Producton of Mlk Clottng Enzyme n Submerged Fermentaton wth Streptococcus Lactc by Usng Whey Medum. IOSR Journal of Engneerng (IOSRJEN), vol. 8, no. 3, 2018, pp. 29-36. 36 P a g e