Process Optimization for the Production of Bio-functional Whey Protein Hydrolysates: Adopting Response Surface Methodology

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1 Int J Pept Res Ther (2013) 19: DOI /s x Process Optimization for the Production of Bio-functional Whey Protein Hydrolysates: Adopting Response Surface Methodology Laxmana Naik Bimlesh Mann Rajesh Bajaj R. B. Sangwan Rajan Sharma Accepted: 27 December 2012 / Published online: 12 January 2013 Ó Springer Science+Business Media New York 2013 Abstract Whey is a protein complex derived from milk, exhibit highest protein quality rating among other proteins, being touted as a functional food with number of health benefits. In the present investigation, whey proteins hydrolysates produced using trypsin enzyme to augment antioxidant activity and to assess angiotensin converting enzyme (ACE) inhibition activity. Hydrolysis parameters were standardized applying response surface methodology. The response antioxidant activity in terms of Trolox equivalent antioxidant capacity (TEAC) values was determined by radical scavenging assay method. Optimum conditions for maximum antioxidant activity were standardized at 88 C of preheating, 7.3 ph, 0.05 enzymes to substrate ratio and hydrolysis was carried up to 8 h at 36.5 C. Resulting peptide fractions obtained at 11.8 % of degree of hydrolysis displayed antioxidant capacity with TEAC values of 1.37 ± The designed model found to be significant with R 2 value of for antioxidant activity and lack of fit test-as non significant, indicating that the optimized conditions were best suited. The hydrolysate further investigated for antihypertensive activity. The outcome indicate that to affect decrease in ACE inhibition activity 4, lg of native whey protein is required when compared to lg of hydrolysates. These results indicate hydrolysate produced under these conditions could be an effective nutraceutical. Keywords Whey protein Trypsin Antioxidant activity Antihypertensive activity Response surface method L. Naik (&) B. Mann R. Bajaj R. B. Sangwan R. Sharma Department of Dairy Chemistry, National Dairy Research Institute, Karnal , Haryana, India laxmandcnaik@gmail.com Introduction Milk proteins are considered as power house of bioactive peptides, they are known to carry wide range of biological and technofunctional peptides. In bovine milk there are two major groups of proteins. On acidification to its isoelectric ph (4.7) at 30 C a portion of proteins from milk precipitates out of solution are called as casein and the remaining protein fraction which is soluble under these conditions referred as whey protein or serum protein or non-casein nitrogen (Fox and McSweeney 1998). Of the total protein inventory casein accounts 80 % and the remaining fraction is whey protein, which accounts 20 %. Whey protein comprises 50 % b-lactoglobulin, 20 % a-lactalbumin, 15 % glycomacropeptide (in renneted whey; depends on methods of preparation), and 15 % minor protein/peptide components (e.g., immunoglobulins, lactoferrin, lactoperoxidase, serum albumin, lysozyme, and growth factors). Whey is a major co-product of cheese industries. Historically, whey has primarily been considered as a waste stream and nuisance by dairy industry. But in the late 20th century, regulations prevented disposal of untreated whey. From thereafter, recognition of the nutritional value of whey components accelerated, additionally in the early 21st century developments in modern science and technology advanced whey-processing regimes and unraveled the secrets of whey proteins by establishing a sound basis for their biofunctional and technofunctional values. Smithers (2008), in his review highlighted the milestones on these whey proteins as how they transformed from gutter-to-gold. Industrially whey proteins can be prepared by ultrafiltration or diafiltration of whey (to remove lactose and salts), followed by spray drying and these products referred to as whey protein concentrates (WPC), in which whey protein content varies from 30 to 80 %.

2 232 Int J Pept Res Ther (2013) 19: Whey proteins contain all the essential amino acids and have the highest protein quality rating among other proteins. Beside nutritional aspects whey proteins exhibit specific physiological properties, demonstrate a range of health and immune-enhancing properties. In addition to this whey protein hydrolysates have the ability to act as antioxidant, antihypertensive, antitumor, antiviral, antibacterial, hypolipidemic, antithrombotic and chelating agents. A number of clinical trials have successfully been performed using whey proteins and their hydrolysates for the treatment of cancer, HIV, hepatitis-b, cardiovascular disease and osteoporosis. As a result of these above findings whey proteins are viewed as value added ingredient in infant formulas, specialized enteral and clinical protein supplements, sports nutrition products, products specific to weight management and mood control and many more functional foods (Korhonen et al. 1998; Xu 1998; Clare and Swaisgood 2000; Pihlanto- Leppala 2001; Bajaj and Sangwan 2002; Meisel and FitzGerald 2003; Korhonen and Pihlanto 2003; Amiot et al. 2004; Marshall 2004; Meisel 2004; Richard et al. 2004; Geoffrey 2007; Korhonen 2009; Madureira et al. 2009; Sarmadia and Ismail 2010; Mine et al. 2010; Bhat and Bhat 2011). Whey proteins offer multifunctional characteristics (Hou et al. 2003); techno-functionally acts by the ability to modify the texture, stabilizing emulsions, foams and gels, to impart viscosity, water-holding capability and also as fat mimetic. Hydrolysis of WPC by enzymatic treatment to improve specific biological activities and as potential ingredients in functional or health-promoting foods have been studied (Peng et al. 2009; Contreras et al. 2011; Tavares et al. 2011a; Ryan et al. 2012). Oxidative metabolism is essential for survival of cells, but it generates free radicals and other reactive oxygen species as a side effect, which can cause oxidative damage. Whey proteins are shown to good source of natural antioxidants as well as angiotensin-converting enzyme (ACE) inhibitory activity. ACE has been implicated in hypertension. In many tissues and biological fluids ACE occurs and it plays an important physiological role in up-regulation of blood pressure (Lopez-Fandino et al. 2006). Conventional antihypertensive drugs thus act essentially as ACE inhibitors; however, they cause several adverse secondary effects, so safer alternatives have been sought from whey proteins. Attempts were made for the production of biofunctional whey protein hydrolysates using starter culture, digestive enzymes and alternatively combinations thereof. As the hydrolysis process depends on many parameters viz: pretreatment, time and temperature of hydrolysis, ph, substrate concentration and so on. Many research investigation attempted to carry out hydrolysis only on in and around of optimum parameters. Hence, in the present study we have developed a model and optimized the different Table 1 Process parameters and the levels of screening applied to optimize whey protein hydrolysis for maximum antioxidant activity Process parameters -1 0?1 ph E/S Temperature Table 2 Experimental design for the optimization of antioxidant activity in central composite design showing antioxidant activity and degree of hydrolysis of WPH prepared under different hydrolysis conditions Code ph E/S Temp ( C) TEAC DH (%) TEAC Trolox equivalent antioxidant capacity (lm of Trolox/mg of protein), DH degree of hydrolysis hydrolysis parameters to produce maximum antioxidant activity from WPC using trypsin enzyme and further to study antihypertensive activity. Response surface methodology (RSM) was employed for design and optimization of the hydrolysis condition. This section of research article deals with optimization of hydrolysis parameters for bioactivity, other aspects of works are being published elsewhere. Materials and Methods Materials WPC sample was procured from Mahan Proteins Ltd.; Commercial trypsin enzyme (39 crystallized, lyophilized powder with an activity min BEAE (N a-benzoyl-l-arginine ethyl ester) U/mg) was purchased from SRL Pvt. Ltd. 2,2 0 -azinobis (3 ethyl beizothiazoline)-6-sulfonic acid (ABTS), Trolox (6-hydroxy 2,5,7,8-tetramethyl chroman-2-carbocyclic acid),

3 Int J Pept Res Ther (2013) 19: hippuryl histidine lysine (HLL) and angiotensin-1-converting enzyme (ACE) from rabbit lung were procured from Sigma-Aldrich. Production of Hydrolysate RSM was employed in the design of experimental conditions. In this investigation preheating and time of hydrolysis were standardized during preliminary studies. Afterwards enzyme to substrate (E/S) ratio, ph and temperature of ± at 734 nm. After addition of 3 ml of diluted ABTS working solution (A734 nm = ± 0.020) to 10 ll of hydrolysate, the absorbance reading was taken at 30 C exactly 5 s after initial mixing and up to 10 min. Results Trolox equivalent antioxidant capacity were expressed as lm of Trolox equivalence/mg of the protein. The % inhibition of absorbance at 734 nm was calculated and plotted as a function of the concentration of antioxidants and Trolox for the standard reference data using the following equation. % Inhibition ¼ 734 nm control 734 nm sample 734 nm control 100 hydrolysis were selected as process variables (Table 1) for optimization using central composite design (CCD). Fresh reconstitute was prepared from the WPC to 5 % protein concentration in distilled water. As per the design conditions (Table 2), respective ph was adjusted, E/S maintained and hydrolysis was carried out at their specified temperature. Immediately after achieving the optimum hydrolysis enzyme activity was terminated by heating to 90 C for 10 min. Hydrolysate obtained was separated using ultra filtration system (Amicon, Ultra Free CL) through 5,000 molecular weight cut-off membranes using positive pressure. Permeate and retentate obtained was separately collected and stored at -20 C. Bioassay Protein content in the hydrolysate was estimated according to the method of Lowry et al. (1951). Degree of hydrolysis (DH) was assessed by the alkali titration curve using the method of Adler-Nissen (1986). Tricine sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) technique was used and the gel image was scanned under Spectronics Corporation ImageAide (Version: ) for the study of molecular weight distribution. Radical scavenging activity for antioxidant activity of samples was determined by the ABTS radical cation decolorization assay with some modification Pellegrini et al. (1999) Hernandez-Ledesma et al. 2005). ABTS? was generated by oxidation of ABTS with potassium persulphate. A stock solution of ABTS was prepared by dissolving it in deionized water and potassium persulphate added to a concentration of 140 mm. The reaction mixture was left to stand at room temperature overnight (12 16 h) in the dark before use for the study. The ABTS? stock solution was diluted with phosphate buffer (ph 7.4) to an absorbance of Whey protein hydrolysate showing the maximum antioxidant activity was assessed for ACE inhibitory activity by the method of Cushman and Cheung (1971), with some modification given by Hernandez-Ledesma et al. (2003). In this assay HHL was dissolved in 0.1 M sodium borate buffer (ph 8.3) containing 0.3 M NaCl. Then 190 ll of 5 mm HHL solution was mixed with 35 ll of whey protein hydrolysate fraction (10 mg/ml, ph 8.3) and then preincubated for 3 min at 37 C. Reaction was initiated by addition of 20 ll of reaction mixture and contents were incubated for 30 min at 37 C. Then reaction was terminated by addition of 250 ll of 1 N HCl. The hippuric acid liberated by the ACE was extracted with 1.7 ml ethyl acetate, which is then dissolved by addition of 1 ml of distilled water, ethyl acetate was removed by evaporation and liberated HHL catalyzed by ACE was measured spectrophotometrically at an optical density of 228 nm. The extent of inhibition was calculated as follows: ðb AÞ= ðb CÞ100 where A = AbsorbanceinthepresenceofACEandACE inhibitory component,b = Absorbance without ACE inhibitory component, and C = Absorbance without ACE. Inhibition was expressed as the concentration of component that inhibits 50 % of ACE activity (IC 50 ), and 1 unit of ACE inhibitory activity was expressed as the potency showing 50 % ACE inhibition under these conditions. Results and Discussions The values of the processing factors were selected to cover a wide range of conditions as shown in Table 2. In this experimental design, quadratic model was used to screen the parameters influencing the hydrolysis condition.

4 234 Int J Pept Res Ther (2013) 19: Table 3 Other statistics for central composite design used for optimization Table 4 Regression coefficient and their p values for central composite design Parameters Values Factor Coefficient estimate Prob [ F SD Mean R Adj R Pred R Adeq precision Residual Lack of fit (p value [0.6348) not significant Pure error F value (p value \0.0001) significant Intercept ph E/S ratio Temp ph \ E/S \ Temp ph 9 E/S ph 9 Temp E/S 9 Temp Main variables determining the antioxidant activity of the whey protein during hydrolysis are temperature, ph, E/S and the reaction time. First three factors determine the reaction rate and may influence the specificity of the enzyme mixture. The reaction time only determines the final extent of hydrolysis. According to several preliminary screening studies preheat treatment and times of hydrolysis were kept constant at 88 C and 8 h respectively. Then effect of ph, E/S and temperature of hydrolysis were investigated using second order model which was derived from CCD and following regression equation was obtained after the analysis of variance. Y ¼ 22:88019 þ 0:7826A 0:7435B þ 0:000707C þ 1:831A 2 þ 1:354B 2 0:1804C 2 þ 0:4909A B 1:0366A C 0:0179B C where Y is the response (antioxidant activity) and A, B and C are coded values of the test variables ph, E/S and temperature of hydrolysis respectively. The R 2 value calculated for antioxidant activity was , this implies that the sample variation of % could be attributed to the independent variables and the model s F-value of (Table 3) and p value \0.0001, which is less than 0.01, implies that the model is significant. There is only 0.01 % chance that this large Model F value could occur due to noise. The lack of fit F-value of implies that lack of fit is not significant relative to the pure error. There is a chance that a lack of fit F-value this large could occur due to noise at p value [ Non significant lack of fit is desirable for the model to fit. Therefore, the model is suitable to predict antioxidant activity of samples. Interactive effects between the hydrolysis parameters also influence the hydrolysate composition. In this case, antioxidant (TEAC) values of whey protein hydrolysates were mainly affected by the linear effect of ph and E/S ratio individually and interactions of ph 9 E/S ratio and Design-Expert Plot X = A: ph Y = B: E/S Ratio Actual Factor C: Temp = B: E/S Ratio 0.04 ph 9 temperature of hydrolysis (Table 4; p values , , and 0.003, respectively). The regression coefficients and the response surfaces were used to study the effects of various parameters on antioxidant activity. The coefficients for E/S ratio have negative sign (Table 4), so an increase in E/S ratio reduces the antioxidant activity. The effect of E/S is more important as it is reflected by the higher absolute regression coefficient. For all the factors ph, temperature of hydrolysis and E/S ratio of the quadratic effects was highly significant, which means that the optimal values exist within these experimental conditions. The effect and the interaction of ph, temperature and E/S ratio are illustrated in the response surface plot by keeping one variable constant. The optimum antioxidant activity was observed around ph and E/S at (Fig. 1). In case of Fig. 2; it was found that the antioxidant activity increased with the increase in temperature of hydrolysis. Similarly, Fig. 3 shows that at low temperature, TEAC value decreased with an increase in E/S ratio but TEAC value increased with an increase in A: ph Fig. 1 Response surface 3-D plot for antioxidant activity of whey protein hydrolysate on ph versus E/S 7.50

5 Int J Pept Res Ther (2013) 19: Design-Expert Plot Response X = A: Y = C: Actual Factor B: E/S Ratio C: Temp A: ph Fig. 2 Response surface 3-D plot for antioxidant activity of whey protein hydrolysate on ph and hydrolysis temp Design-Expert Plot X = A: E/ /S Ratio Y = C: Temp Actual Factor B: ph: Table 6 Antioxidant activity of WPC, WPH, permeate and degree of hydrolysis of WPH Sample Protein (mg/ml) TEAC DH (%) Native WPC ± 0.07 WPH ± Permeate ± TEAC Trolox equivalent antioxidant capacity (lm of Trolox/mg of protein), DH degree of hydrolysis Table 7 ACE inhibition activities of WPC, WPH and it s permeate Sample Concentration (mg/ml) ACE % inhibition IC 50 (lg/ml) Native ± , WPC WPH ± Permeate 64.1 ± C: Temp A: E / S Ratio Fig. 3 Response surface on 3-D plot for antioxidant activity of whey protein hydrolysate on hydrolysis temp and E/S Table 5 Optimized model predicted solution and antioxidant activity values Sl ph E/S Temp ( C) TEAC Desirability TEAC Trolox equivalent antioxidant capacity (lm of Trolox/mg of protein) temperature of hydrolysis, this may be due to exposure of reactive groups from milk proteins (Tables 5 and 6). Bulk samples were prepared under above optimized conditions and analyzed for antioxidant activity which exhibited an increase in antioxidant value (TEAC) of 1.37 ± 0.12 with 11.8 % DH. The results obtained under these conditions are comparable with the findings of Fig. 4 Tricine SDS-PAGE pattern traced under Spectronics Corporation ImageAide document (fluorescence mode): Track 1-Native WPC, Track 2-marker protein, Track 3 and 4 WPC hydrolysates prepared at optimum conditions (increasing protein concentration 25 and 65 ll) Pihlanto-Leppala (2001) and Tavares et al. (2011b). ACE inhibition activity of WPC, WPH and WPH permeate are shown in Table 7, WPH is showing maximum ACE inhibition activity with IC 50 values of lg/ml with 76.1 % inhibition and these results are close to the findings of van der Ven et al. (2002), with IC 50 values of 202 lg/ml at 11.8 % DH. In this investigation 3.79 % inhibition with IC 50 values of 4, lg/ml for WPC and 64.1 % inhibition for WPH permeate was observed. The results indicate that to affect decrease in a unit ACE inhibition activity 4, lg of WPC is required when compared to

6 236 Int J Pept Res Ther (2013) 19: Table 8 Molecular weight distribution and profile height of Tricine SDS-PAGE scanned under Spectronics Corporation ImageAide document (Fluorescence mode): Track 1-Native WPC, Track 2-Marker protein, Track 3 and 4 WPC hydrolysates prepared at optimum conditions (increasing protein concentration 25 and 65 ll) Band number Track 4 Track 3 Track 2 Track 1 Mol. weight Height Mol. weight Height Mol. weight Height Mol. weight Height 1 100, , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , lg of WPH. Mullally et al. (1997), have also reported that hydrolysis of WPC with trypsin generally results in good ACE-inhibiting activity. Increase in antioxidant activity and ACE inhibitory bioactivities are attributed to hydrolysis condition; probably due to change in molecular properties of proteins, like decreased molecular weight, increased charge, exposure of hydrophobic groups and disclosure of reactive amino acid side-chains all these contribute to augmentation in bioactivity. The resultant hydrolysate was subjected to ultra filtration system permeate and retentate obtained was collected separately and subjected for antioxidant and ACE activity. Majority of low molecular weight peptides (LMW) contributing for higher ACE and antioxidant activity from permeate fraction suggesting that the activating effect is produced by LMW peptides those are released to the medium. It is clear from the electrophorogram data (Fig. 4; Table 8) that the WPC band corresponding to 66 kda is completely broken down and a numbers of LMW peptide bands were obtained below 8 kda (Track 3 and 4) on WPH. It also indicating that degradation of few whey proteins bands and appearance of excessive numbers of LMW peptide bands in hydrolysate which may be due to association or aggregation of LMW components occurred during hydrolysis. Conclusion Functionality of whey proteins depends on bioactive peptides encrypted within its native structure. Trypsin, a proteolytic digestive enzyme used to hydrolyze the whey proteins, as it offers an aesthetic advantage being naturally produced in our body and also offers great specificity without undesirable side reactions. The present work reveals that the antioxidant activity of the whey protein hydrolysates could be controlled by regulation of different hydrolysis parameters. Therefore, the effect of these conditions was systematically optimized using RSM. The novelty of the process lies in the step of producing whey protein hydrolysates under these optimized conditions. These conditions offers specific environment for enzymatic reaction to attain optimum degree of hydrolysis which resulted in maximum antioxidant activity and excellent antihypertensive properties. The results entail that hydrolyzed whey protein produced under these conditions could be an interesting protein source with enhanced bioactivity and finds application as biofunctional ingredients in various therapeutic functional food formulations. Acknowledgments Author Laxmana Naik, N. gratefully acknowledges financial assistance in the form of junior research fellowship (JRF) during the course of the investigation from Indian Council of Agricultural Research, New Delhi, India. References Adler-Nissen J (1986) A review of food protein hydrolysis-specific areas. In: Enzymic hydrolysis of food proteins. Elsevier, New York, pp Amiot J, Germain L, Turgeon S, Lemay M, Ory-Salam C, Franc ois FA, Auger A (2004) Peptides from milk protein hydrolysates to improve the growth of human keratinocytes in culture. Int Dairy J 14:

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