Kinetic Study of Enzymatic Hydrolysis of Lactose in Whey

Transcription

1 International Journal of Cheical Engineering Research. ISSN Volue 9, Nuber 2 (2017), pp Research India Publications Kinetic Study of Enzyatic Hydrolysis of Lactose in Whey Dr. Sangita Bhattacharjee 1 and Dwaipayan Sarker 2 1,2 Departent of Cheical Engineering, Heritage Institute of Technology, Kolkata, West Bengal, India. Abstract Dairy industry is one of the largest industries that generate significant quantity of waste strea of which casein/cheese whey is the ost abundant. Major constituent of whey is lactose and it contributes very high value of BOD and COD. Effort has been taken to convert the lactose, a disaccharide to ferentable sugar through enzyatic hydrolysis using coercially available enzye β-galactosidase. The bioethanol derived fro hydrolyzed wheylactose could thus be a cost effective and environent friendly solution for treating this highly polluting waste strea. In this work, the enzyatic hydrolysis of lactose in whey was studied at roo teperature of 32 0 C. The lactose concentration was estiated using DNSA (Dinitro salicylic acid) ethod. The concentration of the hydrolyzed product, glucose was easured using GOD-POD test. On the basis of the data analyzed, Michaelis-Menten kinetic odel has been represented. The paraeters were estiated using lineweaver-burk plot. The axiu rate of hydrolysis was found to be 4.38 L/ol/in. The catalytic efficiency of the enzye for the reaction has also been deterined and reported. Keywords: Lactose, whey, enzyatic hydrolysis, kinetic study, Lineweaver- Burk plot 1. INTRODUCTION: Whey, the liquid reaining after separation of casein/cheese and fat during ilk coagulation, is the principal by-product of dairy industry. Every year ore than 3.2 illion tonnes of lactose, dissolved in whey, is accrued by the cheese production worldwide. Alost half of this aount is used for huan and anial nutrition. The

2 224 Dr. Sangita Bhattacharjee and Dwaipayan Sarker rest is waste and it is very difficult to dispose off the rest as it would cause severe environental pollution. Therefore, there is a need for investigation about further utilization possibilities of lactose fro whey. One of these applications with a high technological and dietetic interest is the enzyatic hydrolysis of lactose, whose econoic iportance has been increasing ever since the 1960s 1.In this process, the disaccharide lactose is converted into two siple onsaccharides, glucose and galactose. Fig.1 Conversion of lactose to glucose and galactose Also, lactose present in ilk or other dairy products cannot be digested by a large section of the huan population creating various lactose intolerance syptos. Therefore hydrolysis of lactose helps people to consue food products derived fro ilk/whey who suffer fro lactose intolerance. Hydrolyzed whey can be effectively converted to bio-ethanol with the help of the ost popular icroorganis involving ferentation, Saccharoyces cerevisiae; the bioethanol thus fored could be blended with gasoline for use in otor vehicle. Two ethods can be applied for lactose hydrolysis in whey and other dairy products: enzyatic hydrolysis and acidic hydrolysis. Enzyatic hydrolysis is preferable than acidic hydrolysis as the forer process allows ilder conditions of ph and teperature, and does not cause bad flavours, odors and colours. Furtherore, acidic ethod can cause protein denaturation which can be present in lactose solution and yield of undesirable by-products that could inhibit the hydrolysis 2,3. In general, there are several technologies for enzyatic hydrolysis of lactose 4. The easiest way is the discontinuous batch-process. In this context, the present work has been undertaken with an objective to study the enzyatic hydrolysis of lactose solution in free enzye ode. Attept would be taken to find suitable kinetic odel to represent the enzyatic hydrolysis reaction.

3 Kinetic Study of Enzyatic Hydrolysis of Lactose in Whey METHODOLOGY In the laboratory, casein whey was prepared following iso-electric precipitation of casein protein of ilk. The casein whey thus obtained has a ph of 4.8 and it was straw coloured. The lactose content of the whey thus fored was easured using DNSA (Dinitro salicylic acid) ethod 5 by easuring the absorbance of the solution at 540n with the help of the calibration curve. Since lactose is a reducing sugar, it responds to the test wherein Fig.2: DNSA reaction 3,5-dinitrosalicylic acid (DNS) is reduced to 3-aino,5-nitrosalicylic acid under alkaline conditions. The lactose standard curve (Fig.3) was used to deterine the concentration of lactose in whey. Fig 3: Lactose standard curve To study the kinetics of lactose hydrolysis, 5 different lactose solutions (10, 20, 30,40, 50g/L i.e. 29 M M) were prepared separately on a buffer of 0.01 M K2HPO4, M KCL and M MgCl2.6H2O at ph 6.75 adjusted with citric acid. 1 L of the lactose saple was incubated with 1 L of the enzye solution (0.143 ole /L) prepared on the sae buffer for 10 inutes at roo teperature of 32 C, and after

4 226 Dr. Sangita Bhattacharjee and Dwaipayan Sarker that 1 L was extracted. The reaction was stopped by ixing with 1 L of 0.1N trichloroacetic acid 6. Afterwards the glucose concentration was easured by the GOD-Perid ethod 7 in each case by easuring the absorbance of Quinoneiine dye solution at 505 n in a UV-Visible Spectrophotoeter (Thero, Genesis). 3. RESULTS AND DISCUSSION The casein whey was straw coloured and was found to have a ph of 4.8. The lactose content was estiated using GOD-Perid ethod and has a concentration of 3.2%. Michaelis-Menten equation was used to odel the enzyatic hydrolysis of lactose solution dp Vax S v (1) dt K S dp where v is reaction rate, is rate of product foration, Vax represents axiu dt rate achieved by the syste at axiu (saturating) substrate condition, Michaelis constant K is the substrate concentration at which the reaction rate is half of Vax. K is a reflection of the affinity of enzye for its substrate and is characteristic for a particular enzye-substrate syste. The saller the value of the enzye binds the substrate 8. K, the ore strongly The paraeters Vax and K were estiated using Lineweaver-Burk plot 9. Fro Fig.4 the values of Vax and K have been estiated and those are found to be 4.38 ol/l/in and M respectively. The sall value of enzye-substrate binding is sufficiently strong. K signifies that Fig. 4: Line-weaver Burk plot at 32 0 C

5 Kinetic Study of Enzyatic Hydrolysis of Lactose in Whey 227 The catalytic efficiency of the reaction was calculated by the following Equation: Vax k cat (2) [ E 0 ] where V ax is axiu reaction velocity, [ E0] is total enzye concentration and kcat is catalytic efficiency. The catalytic efficiency was found to be in CONCLUSION In this study, the lactose content of casein whey, a dairy effluent has been estiated to be 3.2%. For proper utilization of whey, enzyatic hydrolysis was carried out. Product glucose concentration was found. The kinetic study of enzyatic hydrolysis reaction reveals that Michaelis Menten equation has been followed. The paraeters of Vax and K have been found to be 4.38 ol/l/in and M respectively. Catalytic efficiency was also reported. REFERENCES [1] S. Novalin, W. Neuhaus, K.D. Kulbe, 2005, A New Innovative Process to Produce Lactose reduced ski ilk, Journal of Biotechnology, 119, [2] Deirhan, E., Apar, D.K., Özbek, B., A odelling study on hydrolysis of whey lactose and stability of β-galactosidase. Korean J. Che. Eng. 27, [3] S ener, N., Apar, D.K., O zbek, B., A odelling study on ilk lactose hydrolysis and b-galactosidase stability under sonication. Process Bioche. 41, [4] Pivarnik, L.F., Senecal, A.G., Rand, A.G., Hydrolytic and transgalactosylic activities of coercial _-galactosidase (lactase) in food processing. In: Kinsella, J.E., Taylor, D.L. (Eds.),Advances in Food and Nutrition: Research, vol. 3. Acadeic, NY, pp [5] Miller GL (1959) Use of dinitrosalicylic acid reagent for deterination of reducing sugar. Anal. Che. 31 p [6] Jurado E, Caacho F. Luzón G, Vicaria, JM (2002) A new kinetic odel proposed for enzyatic hydrolysis of lactose by a β-galactosidase fro Kluyveroyces fragilis. Enzye and Microbial Technology 31 p [7] Werner W, Rey HG, Wielinger H (1970) Properties of a new chroogen for the estiation of glucose in blood sugar according to the GOD/POD ethod. Anal. Che., 252 p [8] Dalziel, K. (1962), Bioche. J., 83, 28, P.

6 228 Dr. Sangita Bhattacharjee and Dwaipayan Sarker [9] Lineweaver H, Burk D (1934) The Deterination of Enzye Dissociation Constants. Journ of the A. Che. Soc. 56 (3): p