Production of goat milk protein hydrolysate enriched in ACE-inhibitory peptides by ultrafiltration

1 2 3 PRODUCTION OF GOAT MILK PROTEIN HYDROLYSATE 4 CONCENTRATED IN ACE-INHIBITORY PEPTIDES BY 5 ULTRAFILTRATION. 6 F.J. Espejo-Carpio*, R. Pérez-Gálvez, M.C. Almécija, A. Guadix and E.M. Guadix 7 Department of Chemical Engineering, University of Granada, 18071 Granada (Spain) 8 Shortened version of the title: Production of ACE-inhibitory hydrolysates 9 10 * Corresponding author. Tel.: +34 958241329; fax: +34 958 248992. E-mail address: [email protected] 2 Summary 11 A global process for the production of goat milk hydrolysates enriched in angiotensin 12 converting enzyme (ACE) inhibitory peptides was proposed. Firstly, the protein fractions 13 (caseins and whey proteins) were separated by ultrafiltration through a 0.14 m ceramic 14 membrane. The casein fraction obtained in the retentate stream of the above filtration step 15 was subsequently hydrolysed with a combination of subtilisin and trypsin. After 3 hours of 16 reaction, the hydrolysate produced presented an IC50 of 218.50 µg /mL, which represent a 17 relatively high ACE inhibitory activity. Finally, this hydrolysate was filtered through a 50 18 kDa ceramic membrane until reaching a volume reduction factor of 3. The permeate 19 produced presented an improvement of more than 30% in the ACE inhibitory activity. In 20 contrast, the retentate was concentrated in bigger and inactive peptides which originated a 21 decrease of more than 80% in its inhibitory activity. The process suggested in this work 22 was suitable to obtain a potent ACE inhibitory activity product able to be incorporated into 23 food formulas intended to control or lower blood pressure. Moreover, the liquid product 24 could be easily stabilised by spray dried if it would be necessary. 25 Keywords: Goat milk protein; enzymatic hydrolysis; ACE-inhibitory activity; membrane 26 ultrafiltration; peptide identification. 27 28 3 Most of the treatments currently available to treat hypertension are based on the inhibition 29 of the angiotensin converting enzyme (ACE). This dicarbopeptidase (EC 3.4.15.1) plays a 30 crucial role in the blood pressure rising by transforming angiotensing I into angiotensing II 31 (potent vasoconstrictor) and degrading the vasodilator bradykinin (Riordan, 2003). As a 32 consequence, a number of synthetic compounds with ACE inhibitory activity are nowadays 33 available for the treatment of hypertension. Nevertheless, these drugs present several 34 undesirable side-effects (FitzGerald et al. 2004) which could be alleviated by employing 35 ACE inhibitors from natural sources. Although, generally, these peptides present lower 36 ACE inhibitory activity than pharmaceutical drugs, they do not possess side effects even in 37 high doses (Ishida et al. 2011). Moreover, they can be easily incorporated into functional 38 food products intended to prevent or help to control hypertension. 39 Peptides with ACE inhibitory activity have been produced by enzymatic hydrolysis of 40 several substrates from animal or vegetal origin (Rui et al. 2013; Ryan et al. 2011). More 41 specifically, the ACE inhibitory activity of several peptides derived from bovine milk has 42 been extensively reported by both in vitro and in vivo assays (López-Fandiño et al. 2006). 43 In contrast, goat milk proteins have been studied in a lesser extent, but with promising 44 results as an ACE-inhibitory peptide source (Geerlings et al. 2006; Kumar et al. 2011). 45 The production of ACE inhibitory hydrolysates from goat milk would suppose a new 46 application for this kind of milk which, currently, is mostly intended to cheese making. 47 These ACE inhibitory peptides could be incorporated as ingredients in the formulation of 48 food products. Indeed, nowadays, there are currently a number of commercial products 49 which contain such peptides (Aluko, 2007). As example, BiozateTM, is a commercial whey 50 protein hydrolysate containing 3 potent antihypertensive peptides (IIAEK, IPAVF and 51 IPAVFK). 52 4 To this end, it would be desirable obtaining hydrolysates enriched in active peptides. Most 53 of the literature available is focus on isolation and identification of new inhibitory peptides. 54 These identification studies employ filtrations through polymeric membranes as a previous 55 step for selecting the most active fraction for further analysis (Pan et al., 2013; Pihlanto56 Leppälä et al, 2000). Some authors have also used polymeric membranes in order to 57 purified bioactive peptides in larger scale (Holder et al., 2013; Jiang et al., 2010). However 58 these organic membranes present numerous disadvantages when comparing with inorganic 59 membranes. Specifically, ceramic membranes present very good chemical resistance, wide 60 pH and T limits and extended operating lifetimes (Cheryan et al., 1998). Despite of these 61 favourable characteristics, ceramic membranes have been rarely evaluated for the 62 purification of ACE inhibitory peptides. Lin et al. (2011) assayed ceramic membranes in 63 the purification of corn protein hydrolysates. Nevertheless, the authors did not study 64 neither the filtration process nor the cleaning of the membrane process. From an industrial 65 point of view, both the study of the filtration mechanism as well as the implementation of a 66 cleaning stage are key factors. Especially important is to choose an adequate cleaning 67 process allowing several reutilizations of the membrane. Therefore, the study in detail of 68 both the filtration and cleaning process is desirable. 69 The aim of this paper was to study a three-stage integral process to produce hydrolysates 70 exhibiting ACE-inhibitory activity from goat milk protein. Firstly, since not commercial 71 goat caseins or whey proteins are available, a membrane filtration process was proposed to 72 isolate caseins and whey protein fractions. Subsequently, the hydrolysis of the protein 73 fractions obtained were studied employing three enzyme treatments with the objective of 74 maximize the ACE inhibitory activity. Finally, different ultrafiltration processes with 75 ceramic membranes were assayed for improving the ACE inhibitory activity of the 76 hydrolysates. The fouling mechanisms of the filtration stages as well as the cleaning 77 procedures were studied. 78 5 79 Materials and methods 80 Production of protein fractions 81 Commercial UHT goat milk was centrifuged at 4800 g and 4ºC for 30 min in order to 82 remove fat. Protein fractions, caseins and whey proteins, were obtained after concentration 83 with a 0.14 µm ultrafiltration tubular ceramic membrane (TAMI, Nyons, France), 1.20 m 84 length, 3 channels and 0.045 m2 of filtration area. 85 Firstly, the membrane was hydrated with demineralised water at 50ºC for 1 hour. At this 86 point, the permeability of the unfouled membrane was determined, reported as the slope of 87 the water flux (demineralised water at 50ºC) against transmembrane pressure. 88 Subsequently 6 L of the skimmed milk were concentrated at 50ºC to a volume reduction 89 factor (VRF) of 3, attaining 2 L of retentate (enriched in caseins). The operation conditions 90 were 1 bar of transmembrane pressure and cross-flow velocity 3.3 m/s, with recycle of the 91 retentate stream while permeate was continuously removed from the ultrafiltration rig. 92 In order to model the dynamic behavior of the permeate flux, the experimental data were 93 fitted to a several blocking models according to the expressions for crossflow filtration 94 proposed by Field (1995). The better model fitting the experimental data was the complete 95 pore blocking model (1). This model assumes that the flux decline is provoked by the 96 deposition of particles which seal the membranes pores and decrease the effective filtration 97 area. The flux can be expressed mathematically as follows: 98 J = J*+(J0-J*)·exp(-k1·t) (1) 99 6 where J is the flux of permeate, J0 is the initial value of flux of permeate, J* is a critical 100 value of flux, below which there is no fouling phenomena and k1 is a constant related to the 101 deposition of particles onto the pores. 102 Finally, a cleaning procedure was conducted on the fouled membrane to restore the initial 103 water flux. Two consecutive cleaning stages were proposed. Firstly, a mechanical cleaning 104 consisting in an initial rinse with demineralised water at room temperature (1 bar, 3.3 m/s). 105 Secondly, a chemical cleaning stage employing alkaline (sodium hydroxide 0.5 N plus 2 106 g/L SDS as surfactant agent). Sodium hydroxide is effective to hydrolyse and remove 107 protein deposits on the membrane surface. The cleaning solution was pumped at total 108 recycle mode for 30 min at 50ºC, 1 bar of transmembrane pressure and cross-flow velocity 109 of 3.3 m/s. Finally, both permeate and retentate ports were rinsed with de-mineralised 110 water until neutrality. The hydraulic resistance of the membrane was determined before 111 and after the cleaning procedure to determine its effectiveness. 112 The efficiency of the cleaning protocol was assessed by evaluating the decrease of the 113 hydraulic resistance of the fouled membrane throughout the cleaning steps, until attaining 114 its intrinsic value prior to the ultrafiltration. To this end, the total hydraulic resistances (RT) 115 after each cleaning stage were determined as the inverse of the slope of the water flux (Jw) 116 against the transmembrane pressure (TMP): 117 RT= JW / TMP (2) 118 Assuming that the total resistance provided by the membrane is the contribution of that of 119 the membrane material (i.e. membrane intrinsic resistance RM) and that provided by the 120 fouling deposits (RF), the latter could be calculated by: 121 RF = RT - RM (3) 122 7 Finally, a cleaning efficiency index was defined for the cleaning treatment, which was 123 calculated by means of the equation (4): 124 E= (R0 – RClean)/(R0-RM) · 100 (4) 125 where RM is the intrinsic membrane resistance (i.e. that provided by the unfouled 126 membrane), R0 is the hydraulic resistance after the concentration and RClean is that 127 determined after performing the cleaning procedure. The cleaning efficiency index 128 represents the percentage removal of the fouling resistance after the cleaning procedure. 129 The closer is RClean to the intrinsic resistance of the membrane material, the larger the 130 efficiency index is, approaching to 100%. 131 SDS-Electrophoresis 132 After filtration, the proteins fractions obtained were analysed by SDS-Page in order to 133 certify the separation between caseins and whey proteins. SDS-polyacrylamide gel 134 electrophoresis (SDS-PAGE) was performed using the method described by Laemmli 135 (1970). The protein concentration of each sample was diluted with cracking buffer 4x 136 (0.0625 M Tris HCl pH 6.8, 2% SDS, 0.71 M b-mercaptoetanol, 0.025% bromophenol 137 blue, 10% glycerol) until obtain a 1x concentration cracking buffer. The mixed samples 138 were heated in a water bath at 95 °C for 5 min. The protein sample (∼20 uL) was applied 139 to the gel and run at a constant current of 200 V per gel. Upon the completion of 140 electrophoresis, the gel was stained in a staining solution which consisted of 2.5 g/l 141 Coomassie Brilliant Blue R-250 in 50% methanol and 9% acetic acid on an orbital shaker. 142 After 2 h of staining, the gel was destained in a quick destaining solution, which consisted 143 of 40% methanol and 10% acetic acid, for 3−4 h to remove background stain. A broad 144 range of molecular mass standard proteins of 116.0, 66.2, 45.0, 35.0, 25.0, 18.4 and 14.4 145 kDa (Thermo Scientific) were used to calculate molecular weight. Milk and retentate 146 8 samples were previously diluted to 1:10 and 1:50 respectively, due to their high protein 147 concentration. 148 Enzymatic hydrolysis 149 The proteins fractions, caseins and whey proteins, obtained in the ultrafiltration stage 150 through 0.14 µm were employed as substrates in the hydrolysis processes. The enzymes 151 assayed were subtilisin (EC 3.4.21.62) and trypsin (EC 3.4.21.4). Both are serine 152 endoproteases purchased from Novozymes (Denmark). Subtilisin is an endoprotease of 153 broad specifity, while trypsin only cleaves peptide bonds involving Arg or Lys residues 154 (Adler-Nissen, 1986). Both subtilisin (Geerlings et al. 2006; Jiang et al. 2007) and trypsin 155 (Pan et al. 2012; Pintado & Malcata, 2000) have been reported to release ACE inhibitory 156 peptides. 157 Both protein fractions were subjected to three different treatments: using subtilisin (S), 158 trypsin (T) and the mixture of both enzymes (ST). These experiments were carried out in a 159 stirred tank reactor of 0.2 L at 50ºC and pH 8, since these operational conditions are 160 adequate for both enzymes (Adler Nissen, 1986; Mota et al. 2006). The enzyme 161 concentration in the reaction was adjusted to 5 and 0.5 g/L for retentate and permeate, 162 respectively. The extent of the hydrolysis was followed by the pH-stat method (Adler163 Nissen, 1986) throughout the reaction time (3h). The evolution of ACE inhibitory activity 164 was determined by taking 100 µL samples at 0, 5, 10, 20, 30, 40, 50, 60, 75, 90, 105, 120, 165 150 and 180 min. The samples were deactivated by heating at 100 ºC for 15 min and 166 subsequently frozen it at -20 ºC until analysis. 167 The hydrolysate selected for the filtration studies was obtained as explained above. In this 168 case, a 2 L stirrer reactor was employed, in order to produce enough volume for each 169 filtration. After 3 h, the reaction was stopped by thermal deactivation. 170 9 Concentration of active peptides 171 The hydrolysate displaying the highest ACE inhibitory activity was then subjected to a 172 two-step ultrafiltration, with the aim to produce a concentrate in active peptides. According 173 to the molecular weight cut-offs commercially available, ceramic membranes of 50, 15, 3 174 and 1 kDa from Tami (Nyons, France) were assayed, all with a length of 0.25 m. The 175 filtration area was 0.0094 m2 and the hydraulic diameter was 3.6 mm for the highest cut-off 176 membranes (50 and 15 kDa). In the case of the smaller ones (3 and 1 kDa) the filtration 177 area was 0.0042 m2 and the hydraulic diameter was 6 mm. 178 A total of four strategies (Table 1) consisting in two consecutive filtrations were studied. 179 Each strategy comprised a first step, where the membranes with higher MWCO (50 or 15 180 kDa) were employed to remove the larger peptides from the hydrolysate. The permeate 181 obtained from the first stage was further treated by a second filtration process through 3 or 182 1 kDa membrane in order to concentrate the active peptides in the retentate. 183 The filtrations experiments were carried out following the same procedure employed in the 184 production of protein fraction. Firstly the membrane was hydrated and its permeability was 185 determined with ultrapure water. Then, the selected hydrolysate was ultrafiltrated at batch 186 concentration mode. The operation conditions were 50 ºC, 1 bar and a cross-flow velocity 187 of 3.3 m/s. Afterward, the membrane was subjected to the cleaning procedure described 188 above. 189 ACE inhibitory activity analysis 190 Raw hydrolysates, retentate and permeate streams were tested for their ACE inhibitory 191 activity. To this end, the spectrophotometric method proposed by Shalaby et al. (2006) 192 was employed. For determining the percentage of inhibition, the samples of retentate 193 hydrolysates were diluted 50 times with ultrapure water in order to reduce their absorbance 194 16 permeability) for the fouled membrane (○) increases after the alkaline cleaning stage (▲) 341 until attaining the permeability of the unfouled membrane (dotted line), which was 435 342 L/(h · m2 · bar) for the membrane of 50 kDa. According to the efficiency indices listed in 343 Table 2, the cleaning procedure assayed was efficient to restore the permeability of the 50 344 kDa, recovering 97% of the initial water flux. These results are in agreement with those of 345 Barlett et al. (1995), who reported that a NaOH treatment was effective to clean a ceramic 346 membrane after ultrafiltration of milk. This was attributed to the hydrolysis and 347 solubilisation of the protein deposits by the alkali solution. 348 Therefore, the filtration process proposed is an adequate stage which increases 30% of 349 ACE inhibitory activity of the goat milk casein hydrolysate. The permeate obtained could 350 be included directly in food formulas, but also could be stabilized by spray drying before 351 its use. According to previous studies (Espejo-Carpio et al., In press), the conditions which 352 maximize the yield of spray dried product would be an inlet air temperature of 170 ºC and 353 a feed flow of 4.35 mL/min. 354 Conclusions 355 The three stage process proposed in this work is appropriate to produce hydrolysates from 356 goat caseins with high ACE inhibitory activity. Initially the goat milk caseins were 357 satisfactorily separated by ultrafiltration through 0.14 m membrane. Afterward, the 358 hydrolysis of the casein fraction with the mixture of subtilisin and trypsin generated a high 359 ACE inhibitory hydrolysate (IC50 of 218.50 µg/mL). As a final stage, an ultrafiltration 360 process through a 50 kDa ceramic membrane allowed an improvement of 30% in the ACE 361 inhibitory activity. The final product could be employed directly in the formulation of 362 foods intended to control blood pressure or stabilized by spray drying before its use. 363 17 This work was funded by the project P07-TEP-02579 from the Consejería de Economía, 364 Innovación, Ciencia y Empleo of Junta de Andalucía, Spain. FJ Espejo-Carpio also 365 acknowledges the postdoctoral fellowship support from the Consejería de Economía, 366 Innovación, Ciencia y Empleo of Junta de Andalucía. 367 368 18 References 369 Adler-Nissen, J. (1986). Enzymic hydrolysis of food proteins (1st ed.). London: Elsevier 370 Applied Science Publishers. 371 Aluko, R.E. Technology for the production and utilization of food protein-derived anti372 hypertensive peptides: a review, Recent patents on biotechnology 1 (2007), pp. 260373 267. 374 Barlett, M., Bird, M.R., and Howell, J.A. 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Production of protein fractions through the 0.14 µm membrane: (a) 473 Observed flux of permeate (○) and predicted flux with the proposed model (line). 474 (b) Water flux against transmembrane pressure for the fouled membrane (○), alkali 475 cleaning stage (▲) and unfouled membrane (dotted line). 476  Figure 2. SDS-Page analysis of filtration streams. Lane 1: Standards; Lane 2: initial 477 milk (diluted 1:10); Lane 3: permeate; Lane 4: retentate (diluted 1:50). 478  Figure 3. Evolution of degree of hydrolysis (line) and ACE inhibitory activity (●) 479 for the hydrolysis of retentate with subtilisin (S), trypsin (T) and the mixture of 480 both (ST). ACE inhibitory activity was determined by triplicate. 481  Figure 4. Evolution of degree of hydrolysis (line) and ACE inhibitory activity (●) 482 for the hydrolysis of permeate with subtilisin (S), trypsin (T) and the mixture of 483 both (ST). ACE inhibitory activity was determined by triplicate. 484  Figure 5. Concentration of hydrolysate through the 50 kDa membrane: (a) 485 Observed flux of permeate (○) and predicted flux with the proposed model (line). 486 (b) Water flux against transmembrane pressure for the fouled membrane (○), alkali 487 cleaning stage (▲) and unfouled membrane (dotted line). 488 489 490 24 491 Table 1. Strategies proposed for studying the concentration process 492 Strategy First stage Second stage I 50 kDa 3 kDa II 15 kDa 3 kDa III 50 kDa 1 kDa IV 15 kDa 1 kDa 493 494 Table 2. Resistances and cleaning efficiency parameters. 495 Membrane AF, m2 Cleaning Stage RT (bar∙m2∙h/L) RF (bar∙m2∙h/L) %E Unfouled 7.66·10-4 0 0.14 µm 0.0450 After UF 3.25·10-3 2.48·10-3 99% NaOH 7.83·10-4 1.66·10-5 Unfouled 2.30·10-3 0 50 kDa 0.0094 After UF 5.24·10-3 2.94·10-3 97% NaOH 2.37·10-3 7.15·10-5 496 497 25 Table 3. Protein concentration and variation of IC50 for each filtration stage. 498 Strategy Membrane Stream Protein (g/L) Variation of IC50 I Feed 43.97 50 kDa Retentate 73.80 100.2% Permeate 27.16 -30.7% Feed 27.16 3 kDa Retentate 27.77 27.9% Permeate 30.32 3.4% II Feed 41.71 15 kDa Retentate 72.77 88.3% Permeate 33.07 -15.7% Feed 33.07 3 kDa Retentate 36.95 27.4% Permeate 32.66 -2.9% 499 500 501