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Effects of combined microwave and enzymatic treatments on the hydrolysis and immunoreactivity of dairy whey proteins
International Dairy Journal 18 (2008) 918–922
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Effects of combined microwave and enzymatic treatments on the hydrolysis and immunoreactivity of dairy whey proteins ˜ as a, M. Luisa Baeza b, Rosario Gomez a, * F. Javier Izquierdo a, Elena Pen a b
´ cteos, Instituto del Frı´o, Jose´ Antonio Nova ´ is, 10, 28040 Madrid, Spain Departamento de Ciencia y Tecnologı´a de Productos La ´n, Doctor Esquerdo 46, 28007 Madrid, Spain ˜o Servicio de Alergia Hospital General Universitario Gregorio Maran
a r t i c l e i n f o
a b s t r a c t
Article history: Received 17 October 2007 Received in revised form 15 January 2008 Accepted 22 January 2008
The effects of microwave irradiation (MWI) treatment on the hydrolysis of a commercial bovine whey protein concentrate by Pronase, Chymotrypsin and five food grade enzymes (Papain, Corolases 7089 and PN-L 100, Alcalase and Neutrase) were analysed. Digestions were conducted for 5 min at 40 or 50 C depending on the enzyme. Proteolysis was measured with o-phthaldialdehyde, reverse-phase high performance liquid chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis. The residual immunochemical reactivity was assessed by an enzyme-linked immuno-sorbent assay using two pools of seven sera from children allergic to bovine milk. The MWI increased the degree of hydrolysis by all enzymes. Pronase showed the highest proteolysis under MWI followed by Papain and Alcalase and very low immunoreactivity was detected by using either pool of sera in the respective hydrolysates. Proteolysis of dairy whey proteins by either of these enzymes in combination with MWI treatment has the potential to more efficiently produce hypoallergenic dairy hydrolysates. Published by Elsevier Ltd.
1. Introduction The incidence of food allergies and the severity of related symptoms are increasing worldwide (Kimber et al., 2001; Wal, 1998). Allergy against cow’s milk (CMA) is the most common allergy among children younger than 2 years and 1–2% of newborn show an allergenic response to milk (Svenning, Brynhildsvoldb, Mollanda, Langsruda, & Vegaruda, 2000). Breast-feeding is the best form of nutrition for neonates, although mother’s milk may be replaced or supplemented with infant formulas when exclusive breast-feedings during the first months of life is not possible. Most formulas are based on hydrolysates from bovine milk proteins to correspond with infant nutritional requirements. The use of hydrolysates of bovine whey proteins (WP) by the food industry is increasing due to their properties that make them attractive as ˜ as, a source of essential amino acids in human nutrition (Pen Pre´stamo, & Go´mez, 2004). The major constituents of WP are blactoglobulin (b-Lg, 55–60%) and a-lactalbumin (a-La, 15–20%), but WP also contain other minor proteins such as bovine serum albumin (BSA), immunoglobulins, lactoferrin, phospholipoproteins and bioactive factors and enzymes (Smithers et al., 1996). Enzymatic proteolysis of WP and heat treatment are normally used in the production of partially or extensively hydrolysed
* Corresponding author. Tel.: þ34 91 549 23 00x294; fax: þ34 91 549 36 27. E-mail address: [email protected] (R. Gomez). 0958-6946/$ – see front matter Published by Elsevier Ltd. doi:10.1016/j.idairyj.2008.01.005
formulas to lower the content of b-Lg and other intact proteins, effectively reducing the antigenicity of milk proteins. However, residual allergenicity has been reported in several commercial preparations (Calvo & Gomez, 2002; Restani et al., 1995; Van Beresteijn, Meijer, & Schmidt, 1995) which could be due to inaccessibility of some sequential epitope to proteases, even in the denatured protein. Protein hydrolysis should be sufficient to remove epitope structures, since the short chain peptides are physiologically better than the elementary diets in which nitrogen components consist exclusively of a mixture of free amino acids. The absorption of amino acids from short chain peptides is more efficient than the equivalent amount of free amino acids (Siemensma, Weijer, & Bak, 1993). The peptides are less hypertonic than free amino acid mixtures, favouring the absorption of other dietary components (Adibi, 1989; Parrado, Bautista, & Machado, 1991). In addition, extensive hydrolysis makes the product less palatable (bitter) and can also reduce functional properties such as emulsion activity and stability (Van Beresteijn et al., 1994). Microwave irradiation (MWI) during the enzymatic hydrolysis could be an alternative to a conventional heating (CH) to reduce the antigenicity of milk proteins, since several studies on enhanced enzymatic proteolysis both in solution (Izquierdo, Alli, Gomez, Ramaswamy, & Yaylayan, 2005; Pramanik et al., 2002) and in gel (Juan, Chang, Huang, & Chen, 2005) under MWI have been reported. The peptide fragmentation in solution of several biologically active proteins, including cytochrome c, ubiquitin, lysozyme, myoglobin, and interferon a-2b, by endoproteases, trypsin or lysine
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C was achieved in minutes using MWI, in contrast to the hours required when CH was used (Pramanik et al., 2002). An enhanced initial rate of Pronase and a-Chymotrypsin hydrolysis of blactoglobulin has been reported by the use of MWI during enzymatic digestion (Izquierdo et al., 2005). The efficacy of MWI has also been demonstrated in gel digestion of lysozyme, albumin, conalbumin, and ribonuclease A by trypsin (Juan et al., 2005). According to these authors, the required time for their protein mapping was reduced from 16 h to as little as 5 min under MWI, when the protein bands (excised manually from the gel slabs) were subjected to in gel digestion with endoprotease trypsin under MWI. The potential advantages of MWI treatment have also been repor˜ o, Villamiel, Corzo, & ted for milk pasteurisation (Lo´pez-Fandin Olano, 1996) without adverse effects on flavour during cold storage (Valero, Villamiel, Sanz, & Martı´nez-Castro, 2000); preparation of samples for atomic absorption analysis (de la Fuente & Juarez, 1995); and acceleration of the rate-limiting step of amino acid analysis, which is the preparation of protein hydrolysates (Chen, Chiou, Chu, & Wang, 1987; Chiou & Wang, 1989; Marconi, Panfili, Bruschi, Vivanti, & Pizzoferrato, 1995). However, MWI application to enzymatic reactions of food proteins remains largely unexplored, as is its effect on immunoreactive properties of the hydrolysates. The aim of this study was to determine the possibility of reducing the immunoreactive properties of a commercial bovine whey protein concentrate (WPC) by enzymatic hydrolysis under MWI. 2. Materials and methods 2.1. Materials A commercial bovine WPC (PSNU-15400) with approximately 78% protein (60% of b-lactoglobulin) was purchased from Arla Foods Ingredients (Skanderborgvej, Denmark). Pronase from Streptomyces griseus (E.C. 3.4.24.31) and a-Chymotrypsin (type I-S) from bovine pancreas (E.C. 3.4.21.1) were obtained from Sigma Chemical Company (Alcobendas, Madrid, Spain). Papain from Carica papaya (E.C. 3.4.22.2) was obtained from Fluka Chemie GmbH (Buchs, Switzerland). Alcalase (E.C. 3.4.21.62) and Neutrase (E.C. 3.4.24.28) were purchased from Novozymes (Bagsvaerd, Denmark). Corolase 7089 (E.C. 3.4.24.28) and Corolase PN-L 100 (E.C. 3.4.21.63) were obtained from AB Enzymes (Darmstadt, Germany). 2.2. Microwave and conventional heat treatment MWI treatment of samples was carried out in quadruplicate at 2450 MHz in an oven MDS-2000 (CEM Corporation, Buckingham, UK), as described previously in Villamiel, Corzo, Martı´nez-Castro, and Olano (1996). For all enzymatic reactions, 40% of available power (532 W) was used. A microwave transparent fibre-optic temperature probe was inserted into the thermowell of the sample vessel and exited the microwave cavity via a bulkhead connector. The temperature sensor is a phosphor material located at the tip of the probe which emits fluorescent light after temperature-dependent excitation is transmitted down the fibre-optic. The temperature of the apparatus was programmed to ensure that the reaction solution temperature was 40 or 50 C during 5 min of MWI treatment. The time to attain the desired temperature was less than 30 s in all cases. As a control, hydrolysis was performed under CH in a thermostatic water bath without MWI. Both MWI and respective CH treatments were conducted at the same temperature and for the same duration. 2.3. Hydrolysis experiments The WPC was dissolved in distilled water at a concentration of 50 mg mL1. The enzymatic digestions were carried out for 5 min
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using the seven enzymes separately. A total volume of 500 mL was used for the proteolysis reaction: 300 mL of 50 mM sodium phosphate at pH 8 for Pronase and Chymotrypsin, and of distilled water for the five food grade enzymes, with 100 mL enzyme (2 mg mL1) and 100 mL of substrate. The temperature during the treatment was 40 C when the WPC solution was used for the hydrolysis by Pronase, Chymotrypsin and Corolase PN-L 100 and 50 C for the hydrolysis with Alcalase, Neutrase and Corolase 7089. After incubation, the samples were heated at 80 C for 5 min in a water bath. Hydrolyses were performed under MWI and CH (controls) with untreated substrate. Incubation without enzyme was also performed. Each sample was replicated two times. The hydrolysates were stored at 20 C until analysis. 2.4. Assessment of proteolysis The extent of proteolysis was determined as a degree of hydrolysis, by quantification of hydrolysed peptide bonds using the o-phthaldialdehyde (OPA) method, as previously described in ˜ as et al. (2004). A volume (50 mL) of the hydrolysates was added Pen to 1 mL of OPA solution, swirled by inversion, incubated for 2 min at room temperature, and the absorbance at 340 nm was measured. Each sample of duplicate hydrolysis was analysed twice. Aliquots of the hydrolysates were also analysed by reversephase high performance liquid chromatography (RP-HPLC) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE). RP-HPLC was performed using an automated HPLC System consisting of a Consta metric 4100 pump, Spectra System AS 1000 autosampler, and Spectromonitor 5000 diode array detector (Thermo Separation products, Riviera Beach, FL, USA.) fitted with a Purospher Star column (5 mm particle size, 250 4.0 mm; Merck, Darmstadt, Germany). Operating conditions were column temperature, 25 C; flow rate: 0.8 mL min1; solvent A: 0.1% (v/v) trifluoroacetic acid (TFA) (sequential grade, Sigma, St. Louis, MO, USA) in acetonitrile (Scharlau, Barcelona, Spain) in Milli-Q water (Millipore, Saint-Quentin, Yvelines, France) (1:9); solvent B: 0.07% (v/v) trifluoroacetic in acetonitrile–Milli-Q (9:1). Elution was performed by applying 100% A for 5 min, then a linear gradient of 0–50% B over the following 45 min and to 70% B over the next 5 min and then maintained at 70% B for 5 min. Absorbance was recorded at 214 nm. Samples were diluted in urea (6 M) at a ratio of 1:1 (v/v). SDS-PAGE was performed using a Phast-SystemÒ with high density PhastGel mini-gels (Pharmacia, Du¨bendorf, Switzerland), with stacking (T7.5%, C2%) and separation gel (T20%, C2%) zones and a sample loading of 1 mL. The samples were lyophilised and dissolved in treatment buffer (2.5 mg mL1), consisting of 10 mM Tris– HCl, 1 mM EDTA at pH 8.0, SDS (2.5%), b-mercaptoethanol (5%) and bromophenol blue (0.01%). The bands were stained by silver staining using a modification of the method of Heukeshoven and Dernick (1985). The gels were immersed in sodium thiosulfate (0.02%, w/v) for 1 h, after fixing the bands and washing the gel. The staining solution consisted of silver nitrate (0.2%, w/v) and formaldehyde in Milli-Q water (0.072%, v/v). The developer solution contained sodium thiosulfate (0.0004%, w/v) and formaldehyde (0.050%, v/v). 2.5. Enzyme-linked immuno-sorbent assay The samples were diluted (0.5 mL mL1) in carbonate buffer (15 mM Na2CO3; 35 mM NaHCO3, pH 9.6), added (50 mL) to the wells of the microtiter plates (Nunc, Roskilde, Denmark) and then incubated for 18 h at 4 C. Residual free binding sites were blocked with 150 mL of 5% fish gelatin in phosphate buffered saline (PBS) (Sigma, Madrid, Spain) per well. Two pools of seven sera of patients allergenic to bovine milk were used; 50 mL of each pool was added separately to the wells and maintained for 1 h at 37 C. Two
0.5
β-Lg
negative controls (sera from two different nonallergic persons) were used. After four washes with PBS–Tween 20 (0.05%) containing 0.5% fish gelatin, goat anti-human peroxidase-labeled IgE (Sigma) was added to the plate, and incubated for 1 h at 37 C. After four washes, IgE content of samples bound to wells was followed by addition 100 mL of a freshly substrate-containing buffer (50 mM citrate, 125 mM phosphate, pH 5.0, 4.4 mM H2O2; 5 mM o-phenylenediamine dihydrochloride (OPD) (Sigma)), after incubation at room temperature in the dark for 15 min. The colorimetric reaction was stopped by addition of 0.05 mL of 2 M sulphuric acid per well and, then the absorbance at 495 nm determined on a microtiter automated enzyme-linked immuno-sorbent assay (ELISA) plate reader (3550 Microplate reader, Biorad, Richmond, CA, USA). Each sample of duplicate experiments was analysed twice by using each of the two pools of seven sera from children allergic to bovine milk.
α-La
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Absorbance 214 nm
920
A
B F
C
2.6. Statistical analysis
D
Data were analysed using SPSS for Windows (version 11.5, SPSS Inc. Chicago, IL, USA) following an analysis of variance (ANOVA) one way linear model. Mean comparisons were performed using the paired t test, and the significance level was established for P < 0.05. Data are shown as averages of duplicate analyses for each of the two hydrolysis experiments.
T
D
E 6
12
18
24
30
36
44
50
Retention time
3. Results and discussion 3.1. Proteolysis of bovine whey protein concentrate Fig. 1 compares the hydrolysis of WPC by Pronase, Chymotrypsin and five food grade enzymes, Papain, Corolases 7089 and PN-L, Alcalase and Neutrase, when CH and MWI were used during the enzymatic reactions. The highest hydrolysis was found for Pronase followed by Papain and Alcalase, irrespective of the heating procedure used. The degree of hydrolysis of WPC by all enzymes was significantly enhanced (P 0.05) when the digestion took place under MWI (Fig. 1). The RP-HPLC profiles of WPC solution (5%) in phosphate buffer 50 mM at pH 8.0 and distilled water pH 6.5 treated by MWI were studied. No differences were found by application of MWI, irrespective of treatment medium (results not shown). The chromatograms show the peak of a-lactalbumin (a-La) and blactoglobulin (b-Lg) eluting at retention times (RT) of 47.60 and 49.26 min, respectively, as shown in Fig. 2.
Fig. 2. RP-HPLC profile of peptides obtained by Papain and Alcalase hydrolysis of whey protein concentrate (WPC) under conventional heating (CH) and microwave irradiation (MWI). (A) WPC in distilled water, pH 6.6, (B) Papain hydrolysates under CH, (C) Papain hydrolysates under MWI, (D) Alcalase hydrolysates under CH, (E) Alcalase hydrolysates under MWI. F: Peptide fraction eluting between 22–32 min for the papain hydrolysate. T and D: Peptide fractions eluting between 26–28 min and 34–40 min, respectively, for the alcalase hydrolysates.
The RP-HPLC profiles of enzymatic hydrolysates showed the activity of all enzymes (with the exception of Chymotrypsin) on bLg and a-La. The peptide profiles of the samples digested by Pronase, Chymotrypsin, Corolases 7089 and PN-L and Neutrase, during the MWI did not differ significantly from the samples hydrolysed under CH. Only a slight increase of peaks in the WPC hydrolysed in combination with MWI was observed (results not shown). These results suggest that the specificity of these enzymes did not change when the MWI was applied. No qualitative changes were found in the peptide profile when the WPC was digested by Papain and Alcalase during the MWI
Micromols amino groups mg -1protein
5 4,5 4
Control (CH)
3,5
MWI
3 2,5 2 1,5 1 0,5 0 Pronase
Chymotrypsin
Papain
Corolase 7089
Corolase PNL
Alcalase
Neutrase
Enzymes Fig. 1. Degree of hydrolysis determined by the o-phthaldialdehyde method in enzymatic hydrolysates from commercial bovine whey protein concentrate (WPC). The experiments were performed at 40 C for Pronase, Chymotrypsin and Corolase PN-L and at 50 C for Papain, Alcalase, Neutrase and Corolase 7089, under conventional heating (CH) and microwave irradiation (MWI). The values are the mean and standard deviation of duplicate analysis of samples from duplicate experiments.
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1,2
Absorbance at 495 nm
compared with the hydrolysates performed under CH (Fig. 2). However, quantitative differences in the relative abundance of some peaks were obtained. Thus, an increase in the fraction (F) eluting between 22 and 32 min was noticed for the Papain hydrolysates performed under MWI compared with CH hydrolysates. Also, a lower proportion of the hydrophobic peptide eluted at >32 min (Fig. 2B and C). The MWI also increased the fractions T and D eluting between 26–28 and 34–40 min, respectively, in the Alcalase hydrolysates. The results suggest a possible positive effect of the MWI on the sensory characteristics of the hydrolysates obtained by Papain, since a low ratio of hydrophobic to hydrophilic peptide is related to high palatability (Clemente, 2000). Different patterns of peptides were observed by SDS-PAGE after the hydrolysis of WPC by the seven proteases. The b-Lg and a-La bands appeared with low intensity in the hydrolysates of WPC by Pronase and Alcalase performed under CH (results not shown). In contrast, no protein bands were observed in the respective digestions performed under MWI that, however, presented bands of apparent molecular masses <3.4 kDa, which was not observed in the CH hydrolysates. All Papain hydrolysates only showed three bands with molecular masses ranging between 9.0 and 1.4 kDa; these were of very low relative intensity when the enzymatic treatment was performed under MWI, suggesting that these peptides were digested under these conditions. The electrophoretograms for Chymotrypsin showed the a-La band in all cases. The b-Lg band only appeared when the enzymatic treatment was performed under CH and, then only with low intensity. No other differences induced by MWI were observed, presenting the CH and MWI hydrolysates three additional bands of about 8.0, 4.0 and 2.5 kDa (results not shown) These results suggest that Chymotrypsin significantly hydrolysed b-lactoglobulin regardless of the heating treatment used, whereas a-lactalbumin seemed to be resistant to the chymotryptic action in all cases. The b-Lg and a-La bands appeared in the hydrolysates by Corolases 7089, PN-L and Neutrase. Control Corolase 7089 hydrolysate presented three additional well-resolved bands of about 10, 9 and 6 kDa. Neither of these bands appeared in the MWI hydrolysates, suggesting that the highest proteolysis occurred under MWI with production of smaller peptides, since low intensity of the a-La band was also observed, in agreement with the highest degree of hydrolysis observed by the OPA method (Fig. 1). There were no important qualitative changes in the peptide profile of Corolase PNL and Neutrase hydrolysates induced by MWI. The increase in proteolysis observed in the present study when MWI was applied during the digestion could be related to either increased susceptibility of the substrate or increased activity of the proteases under MWI. Our finding of the enhancement of the enzymatic proteolysis under MWI is in good agreement with data previously described by our group for kinetic parameters for Pronase and a-Chymotrypsin hydrolysis of bovine b-Lg (Izquierdo, Alli, Yaylayan, & Gomez, 2007). Higher catalytic effectiveness (KcatK1 m ) values were obtained in both enzymatic digestions of the protein performed under MWI in comparison with the values in the respective CH digestions. The Michaelis–Menten constant (Km) for either enzyme was reduced under MWI, which suggests the highest substrate-enzyme affinity at these conditions (Izquierdo et al., 2007). Furthermore, Pramanik et al. (2002) reported acceleration of enzymatic processes for protein mapping by trypsin digestion. These authors reported the effectiveness of this treatment, since the peptide fragmentation of several biologically active proteins was achieved in minutes using MWI, in contrast to the hours required by CH treatment. The potential advantages of MWI treatment have also been reported for acceleration of protein hydrolysis in the preparation of samples for amino acid analysis (Chen et al., 1987; Chiou & Wang, 1989; Marconi et al., 1995). Bohr and Bohr (2000a, 2000b) described the unfolding, folding and denaturation
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Control (CH) MWI Untreated WPC
1 b 0,8 a
a
0,6
a
0,4
a
a 0,2
b
b
a aa
b
b b
0 Pronase Chymotrypsin
Papain
Corolase 7089
Corolase PNL
Alcalase
Neutrase
Untreated WPC
Enzymes Fig. 3. Residual immunochemical reactivity determined by an ELISA assay (expressed as absorbance units at 495 nm) of enzymatic hydrolysates from whey protein concentrate (WPC). CH: conventional heating; MWI: microwave irradiation. The values are the mean and standard deviation of duplicate analysis of samples from duplicate experiments, when two pools of seven sera each one from allergic children to bovine milk were used. Different letters above the bars for each enzyme indicate significant differences P 0.05.
of b-Lg by MWI treatment, structural changes were also reported by de Pomerai et al. (2003) for BSA in solution, that probably favour the hydrolysis of proteins by proteases. The potential effect of MWI on enzymes has been described by Porcelli et al. (1997) who reported that 10.4 GHz caused conformational changes in two enzymes (S-adenosylhomocysteine and 50 -methylthioadenosine phosphorylase), detected by fluorescence spectroscopy and circular dichroism spectroscopy. These authors suggested that the MWI treatments induced structural rearrangements in the enzymes/proteins which were not related to temperature. 3.2. Immunochemical properties of the hydrolysates ELISA was used to assess the residual immunochemical reactivity in the digests obtained from WPC through combined enzymatic and MWI treatments. An important decrease in immunoreactivity was observed in hydrolysates obtained by combining MWI and Pronase, Papain or Alcalase and, to a lesser extent, Corolase 7089, in comparison to untreated WPC and enzymatic treatment alone when two pools of seven sera were used (Fig. 3). In contrast, enhanced reactivity was observed in the MWI hydrolysates by the other three enzymes compared to the respective CH digestions. These results are consistent with the residual a-La and b-Lg contents and possibly with the size of the peptides in these enzymatic digests (data not shown). The combined enzymatic and MWI treatments, compatible with retention of enzyme activity and reversibility of structural modifications of proteins, may have a practical relevance by decreasing their immunoreactivity, as reported by several authors for enzymatic hydrolysates from b-Lg (Bonomi et al., 2003), bovine WP ˜ as, Pre´stamo, Baeza, Martı´nez-Molero, & Go´mez, 2006; Pen ˜ as (Pen ˜ as, Snel, Floris, Pre´stamo, & Go´mez, 2006) and et al., 2006b; Pen ˜ as et al., 2004, 2006a; Pen ˜ as, Pre´stamo, Polo, & soybean WP (Pen Go´mez, 2006) performed under high pressure. The unfolded proteins formed under MWI could represent an ideal substrate for the action of proteases, making some epitopes more accessible to enzymatic hydrolysis. 4. Conclusions MWI treatment enhanced the enzymatic hydrolysis of bovine WPC. Pronase and Papain showed the highest proteolysis under
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MWI followed by Alcalase. The b-Lg was hydrolysed by Papain under all conditions, whereas this protein was only completely hydrolysed by Pronase, Alcalase and Chymotrypsin under MWI. Both proteins were resistant to hydrolysis by Corolase PN-L and Neutrase for 5 min of digestion, irrespective of the heating procedure applied. A decrease of immunochemical reactivity was also found for all hydrolysates compared to untreated WPC and the lowest antigenicity was found in those obtained by combined treatment of MWI and Pronase, Papain or Alcalase, concomitant with a considerable hydrolysis of a-La and b-Lg and production of small peptides. Thus, in general, MWI enhances WPC hydrolysis, and depending upon the choice of enzymes, reduced the residual antigenicity of the hydrolysates. Proteolysis of bovine WPC by either of the three enzymes in combination with MWI treatment has the potential to more efficiently produce hypoallergenic dairy hydrolysates. Acknowledgements The ‘‘Comisio´n Interministerial De Ciencia y Tecnologı´a’’ through the Project No AGL2000-1497 supported this work. Dr. Nieves Corzo (Instituto Fermentaciones Industriales) is acknowledged for the kind counselling on the microwave equipment. References Adibi, S. A. (1989). Glycyl dipe´ptides: new substrates for protein nutrition. Journal of Laboratory and Clinical Medicine, 113, 665–673. Bohr, H., & Bohr, J. (2000a). Microwave-enhanced folding and denaturation of globular proteins. Physical Review E – Statistical Physics, Plasmas, Fluids, and Related Interdisciplinary Topics, 61, 4310–4314. Bohr, H., & Bohr, J. (2000b). Microwave enhanced kinetics observed in ORD studies of a protein. Bioelectromagnetics, 21, 68–72. Bonomi, F., Fiocchi, A., Frokiaer, H., Gaiaschi, A., Iametti, S., Poiesi, C., et al. (2003). Reduction of immunoreactivity of bovine b-lactoglobulin upon combined physical and proteolytic treatment. Journal of Dairy Research, 70, 51–59. Calvo, M. M., & Gomez, R. (2002). Peptidic profile, molecular mass distribution and immunological properties of commercial hypoallergenic infant formulas. Milchwissenschaft, 57, 187–190. Chen, S. T., Chiou, S. H., Chu, Y. H., & Wang, K. T. (1987). Rapid hydrolysis of proteins and peptides by means of microwave technology and its application to amino acid analysis. International Journal of Peptide and Protein Research, 30, 572–576. Chiou, S. H., & Wang, K. T. (1989). Peptide and protein hydrolysis by microwave irradiation. Journal of Chromatography, 491, 424–431. Clemente, A. (2000). Enzymatic protein hydrolysates in human nutrition. Trends in Food Science and Technology, 11, 254–262. de la Fuente, M. A., & Juarez, M. (1995). Determination of phosphorus in dairy products by sample wet digestion in a microwave oven. Analytica Chimica Acta, 309, 355–359. Heukeshoven, J., & Dernick, R. (1985). Simplified method for silver staining of proteins in polyacrylamide gels and the mechanism of silver staining. Electrophoresis, 6, 103–112. Izquierdo, F. J., Alli, I., Gomez, R., Ramaswamy, H. S., & Yaylayan, V. (2005). Effects of high pressure and microwave on pronase and a-chymotrypsin hydrolysis of blactoglobulin. Food Chemistry, 92, 713–719. Izquierdo, F. J., Alli, I., Yaylayan, V., & Gomez, R. (2007). Microwave-assisted digestion of b-lactoglobulin by pronase, a-chymotrypsin and pepsin. International Dairy Journal, 17, 465–470. Juan, H. F., Chang, S. C., Huang, H. C., & Chen, S. T. (2005). A new application of microwave technology to proteomics. Proteomics, 5, 840–842.
Kimber, I., Pichowski, J. S., Betts, C. J., Cumberbatch, M., Basketter, D. A., & Dearman, R. J. (2001). Alternative approaches to the identification and characterization of chemical allergens. Toxicology in Vitro, 15, 307–312. ˜ o, R., Villamiel, M., Corzo, N., & Olano, A. (1996). Assessment of the Lo´pez-Fandin thermal treatment of milk during continuous microwave and conventional heating. Journal of Food Protection, 59, 889–892. Marconi, E., Panfili, G., Bruschi, L., Vivanti, V., & Pizzoferrato, L. (1995). Comparative study on microwave and conventional methods for protein hydrolysis in food. Amino Acids, 8, 201–208. Parrado, J., Bautista, J., & Machado, A. (1991). Production of soluble enzymatic protein hydrolysate from industrially defatted undehulled sunflower meal. Journal of Agricultural and Food Chemistry, 39, 447–450. ˜ as, E., Pre´stamo, G., Baeza, M. L., Martı´nez-Molero, M. I., & Go´mez, R. (2006). Pen Effects of combined high pressure and enzymatic treatments on the hydrolysis and immunoreactivity of dairy whey proteins. International Dairy Journal, 16, 831–839. ˜ as, E., Pre´stamo, G., & Go´mez, R. (2004). High pressure and the enzymatic Pen hydrolysis of soybean whey proteins. Food Chemistry, 85, 641–648. ˜ as, E., Pre´stamo, G., Polo, F., & Go´mez, R. (2006). Enzymatic proteolysis under Pen high pressure of soybean whey: analysis of peptides and the allergen Gly m1 in the hydrolysates. Food Chemistry, 99, 569–573. ˜ as, E., Restani, P., Ballabio, C., Pre´stamo, G., Fiocchi, A., & Go´mez, R. (2006a). Pen Assessment of the residual immunoreactivity of soybean whey hydrolysates obtained by combined enzymatic proteolysis and high pressure. European Food Research and Technology, 222, 286–290. ˜ as, E., Restani, P., Ballabio, C., Pre´stamo, G., Fiocchi, A., & Go´mez, R. (2006b). Pen Evaluation of the residual antigenicity of dairy whey hydrolysates obtained by combination of enzymatic hydrolysis and high pressure treatment. Journal of Food Protection, 69, 1707–1712. ˜ as, E., Snel, H., Floris, R., Pre´stamo, G., & Go´mez, R. (2006). High pressure can Pen reduce the antigenicity of bovine whey hydrolysates. International Dairy Journal, 16, 969–975. de Pomerai, D. I., Smith, B., Dawe, A., North, K., Smith, T., Archer, D. B., et al. (2003). Microwave radiation can alter protein conformation without bulk heating. FEBS Letters, 543, 93–97. Porcelli, M., Cacciapuoti, G., Fusco, S., Massa, R., d’Ambrosio, G., Bertoldo, C., et al. (1997). Non-thermal effects of microwaves on proteins: thermophilic enzymes as model system. FEBS Letters, 402, 102–106. Pramanik, B. N., Mirza, U. A., Ing, Y. H., Liu, Y. H., Bartner, P. L., Weber, P. C., et al. (2002). Microwave-enhanced enzyme reaction for protein mapping by mass spectrometry, a new approach to protein digestion in minutes. Protein Science, 11, 2676–2687. Restani, P., Velona, T., Plebani, A., Ugazio, A. G., Poiesi, C., Muraro, A., et al. (1995). Evaluation by SDS-PAGE and immunoblotting of residual antigenicity in hydrolysed protein formula. Clinical and Experimental Allergy, 25, 651–658. Siemensma, A. D., Weijer, W. J., & Bak, H. J. (1993). The importance of peptide lengths in hypoallergenic infant formulae. Trends in Food Science and Technology, 4, 16–21. Smithers, G. F., Ballard, F. J., Copeland, A. D., De Silva, K. J., Dionysius, D. A., Francis, G. L., et al. (1996). Symposium: advances in dairy foods processing and engineering. Journal of Dairy Science, 79, 1454–1459. Svenning, C., Brynhildsvoldb, J., Mollanda, T., Langsruda, T., & Vegaruda, G. E. (2000). Antigenic response of whey proteins and genetic variants of b-lactoglobulindthe effect of proteolysis and processing. International Dairy Journal, 10, 699–711. Valero, E., Villamiel, M., Sanz, J., & Martı´nez-Castro, I. (2000). Chemical and sensorial changes in milk pasteurised by microwave and conventional systems during cold storage. Food Chemistry, 70, 77–81. Van Beresteijn, E. C. H., Meijer, J. G. M., & Schmidt, D. G. (1995). Residual antigenicity of hypoallergenic infant formulas and the occurrence of milk-specific IgE antibodies in patients with clinical allergy. Journal of Allergy and Clinical Immunology, 96, 365–374. Van Beresteijn, E. C. H., Peeters, R. A., Kasper, J., Meijer, R. J., Robben, A. J., & Schmidt, D. G. (1994). Molecular mass distribution, immunological properties and nutritive value of whey protein hydrolysates. Journal of Food Protection, 57, 619–625. Villamiel, M., Corzo, N., Martı´nez-Castro, I., & Olano, A. (1996). Chemical changes during microwave treatment of milk. Food Chemistry, 56, 385–388. Wal, J. M. (1998). Strategies for assessment and identification of allergenicity in (novel) foods. International Dairy Journal, 8, 413–423.
Contents lists available at ScienceDirect
International Dairy Journal journal homepage: www.elsevier.com/locate/idairyj
Effects of combined microwave and enzymatic treatments on the hydrolysis and immunoreactivity of dairy whey proteins ˜ as a, M. Luisa Baeza b, Rosario Gomez a, * F. Javier Izquierdo a, Elena Pen a b
´ cteos, Instituto del Frı´o, Jose´ Antonio Nova ´ is, 10, 28040 Madrid, Spain Departamento de Ciencia y Tecnologı´a de Productos La ´n, Doctor Esquerdo 46, 28007 Madrid, Spain ˜o Servicio de Alergia Hospital General Universitario Gregorio Maran
a r t i c l e i n f o
a b s t r a c t
Article history: Received 17 October 2007 Received in revised form 15 January 2008 Accepted 22 January 2008
The effects of microwave irradiation (MWI) treatment on the hydrolysis of a commercial bovine whey protein concentrate by Pronase, Chymotrypsin and five food grade enzymes (Papain, Corolases 7089 and PN-L 100, Alcalase and Neutrase) were analysed. Digestions were conducted for 5 min at 40 or 50 C depending on the enzyme. Proteolysis was measured with o-phthaldialdehyde, reverse-phase high performance liquid chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis. The residual immunochemical reactivity was assessed by an enzyme-linked immuno-sorbent assay using two pools of seven sera from children allergic to bovine milk. The MWI increased the degree of hydrolysis by all enzymes. Pronase showed the highest proteolysis under MWI followed by Papain and Alcalase and very low immunoreactivity was detected by using either pool of sera in the respective hydrolysates. Proteolysis of dairy whey proteins by either of these enzymes in combination with MWI treatment has the potential to more efficiently produce hypoallergenic dairy hydrolysates. Published by Elsevier Ltd.
1. Introduction The incidence of food allergies and the severity of related symptoms are increasing worldwide (Kimber et al., 2001; Wal, 1998). Allergy against cow’s milk (CMA) is the most common allergy among children younger than 2 years and 1–2% of newborn show an allergenic response to milk (Svenning, Brynhildsvoldb, Mollanda, Langsruda, & Vegaruda, 2000). Breast-feeding is the best form of nutrition for neonates, although mother’s milk may be replaced or supplemented with infant formulas when exclusive breast-feedings during the first months of life is not possible. Most formulas are based on hydrolysates from bovine milk proteins to correspond with infant nutritional requirements. The use of hydrolysates of bovine whey proteins (WP) by the food industry is increasing due to their properties that make them attractive as ˜ as, a source of essential amino acids in human nutrition (Pen Pre´stamo, & Go´mez, 2004). The major constituents of WP are blactoglobulin (b-Lg, 55–60%) and a-lactalbumin (a-La, 15–20%), but WP also contain other minor proteins such as bovine serum albumin (BSA), immunoglobulins, lactoferrin, phospholipoproteins and bioactive factors and enzymes (Smithers et al., 1996). Enzymatic proteolysis of WP and heat treatment are normally used in the production of partially or extensively hydrolysed
* Corresponding author. Tel.: þ34 91 549 23 00x294; fax: þ34 91 549 36 27. E-mail address: [email protected] (R. Gomez). 0958-6946/$ – see front matter Published by Elsevier Ltd. doi:10.1016/j.idairyj.2008.01.005
formulas to lower the content of b-Lg and other intact proteins, effectively reducing the antigenicity of milk proteins. However, residual allergenicity has been reported in several commercial preparations (Calvo & Gomez, 2002; Restani et al., 1995; Van Beresteijn, Meijer, & Schmidt, 1995) which could be due to inaccessibility of some sequential epitope to proteases, even in the denatured protein. Protein hydrolysis should be sufficient to remove epitope structures, since the short chain peptides are physiologically better than the elementary diets in which nitrogen components consist exclusively of a mixture of free amino acids. The absorption of amino acids from short chain peptides is more efficient than the equivalent amount of free amino acids (Siemensma, Weijer, & Bak, 1993). The peptides are less hypertonic than free amino acid mixtures, favouring the absorption of other dietary components (Adibi, 1989; Parrado, Bautista, & Machado, 1991). In addition, extensive hydrolysis makes the product less palatable (bitter) and can also reduce functional properties such as emulsion activity and stability (Van Beresteijn et al., 1994). Microwave irradiation (MWI) during the enzymatic hydrolysis could be an alternative to a conventional heating (CH) to reduce the antigenicity of milk proteins, since several studies on enhanced enzymatic proteolysis both in solution (Izquierdo, Alli, Gomez, Ramaswamy, & Yaylayan, 2005; Pramanik et al., 2002) and in gel (Juan, Chang, Huang, & Chen, 2005) under MWI have been reported. The peptide fragmentation in solution of several biologically active proteins, including cytochrome c, ubiquitin, lysozyme, myoglobin, and interferon a-2b, by endoproteases, trypsin or lysine
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C was achieved in minutes using MWI, in contrast to the hours required when CH was used (Pramanik et al., 2002). An enhanced initial rate of Pronase and a-Chymotrypsin hydrolysis of blactoglobulin has been reported by the use of MWI during enzymatic digestion (Izquierdo et al., 2005). The efficacy of MWI has also been demonstrated in gel digestion of lysozyme, albumin, conalbumin, and ribonuclease A by trypsin (Juan et al., 2005). According to these authors, the required time for their protein mapping was reduced from 16 h to as little as 5 min under MWI, when the protein bands (excised manually from the gel slabs) were subjected to in gel digestion with endoprotease trypsin under MWI. The potential advantages of MWI treatment have also been repor˜ o, Villamiel, Corzo, & ted for milk pasteurisation (Lo´pez-Fandin Olano, 1996) without adverse effects on flavour during cold storage (Valero, Villamiel, Sanz, & Martı´nez-Castro, 2000); preparation of samples for atomic absorption analysis (de la Fuente & Juarez, 1995); and acceleration of the rate-limiting step of amino acid analysis, which is the preparation of protein hydrolysates (Chen, Chiou, Chu, & Wang, 1987; Chiou & Wang, 1989; Marconi, Panfili, Bruschi, Vivanti, & Pizzoferrato, 1995). However, MWI application to enzymatic reactions of food proteins remains largely unexplored, as is its effect on immunoreactive properties of the hydrolysates. The aim of this study was to determine the possibility of reducing the immunoreactive properties of a commercial bovine whey protein concentrate (WPC) by enzymatic hydrolysis under MWI. 2. Materials and methods 2.1. Materials A commercial bovine WPC (PSNU-15400) with approximately 78% protein (60% of b-lactoglobulin) was purchased from Arla Foods Ingredients (Skanderborgvej, Denmark). Pronase from Streptomyces griseus (E.C. 3.4.24.31) and a-Chymotrypsin (type I-S) from bovine pancreas (E.C. 3.4.21.1) were obtained from Sigma Chemical Company (Alcobendas, Madrid, Spain). Papain from Carica papaya (E.C. 3.4.22.2) was obtained from Fluka Chemie GmbH (Buchs, Switzerland). Alcalase (E.C. 3.4.21.62) and Neutrase (E.C. 3.4.24.28) were purchased from Novozymes (Bagsvaerd, Denmark). Corolase 7089 (E.C. 3.4.24.28) and Corolase PN-L 100 (E.C. 3.4.21.63) were obtained from AB Enzymes (Darmstadt, Germany). 2.2. Microwave and conventional heat treatment MWI treatment of samples was carried out in quadruplicate at 2450 MHz in an oven MDS-2000 (CEM Corporation, Buckingham, UK), as described previously in Villamiel, Corzo, Martı´nez-Castro, and Olano (1996). For all enzymatic reactions, 40% of available power (532 W) was used. A microwave transparent fibre-optic temperature probe was inserted into the thermowell of the sample vessel and exited the microwave cavity via a bulkhead connector. The temperature sensor is a phosphor material located at the tip of the probe which emits fluorescent light after temperature-dependent excitation is transmitted down the fibre-optic. The temperature of the apparatus was programmed to ensure that the reaction solution temperature was 40 or 50 C during 5 min of MWI treatment. The time to attain the desired temperature was less than 30 s in all cases. As a control, hydrolysis was performed under CH in a thermostatic water bath without MWI. Both MWI and respective CH treatments were conducted at the same temperature and for the same duration. 2.3. Hydrolysis experiments The WPC was dissolved in distilled water at a concentration of 50 mg mL1. The enzymatic digestions were carried out for 5 min
919
using the seven enzymes separately. A total volume of 500 mL was used for the proteolysis reaction: 300 mL of 50 mM sodium phosphate at pH 8 for Pronase and Chymotrypsin, and of distilled water for the five food grade enzymes, with 100 mL enzyme (2 mg mL1) and 100 mL of substrate. The temperature during the treatment was 40 C when the WPC solution was used for the hydrolysis by Pronase, Chymotrypsin and Corolase PN-L 100 and 50 C for the hydrolysis with Alcalase, Neutrase and Corolase 7089. After incubation, the samples were heated at 80 C for 5 min in a water bath. Hydrolyses were performed under MWI and CH (controls) with untreated substrate. Incubation without enzyme was also performed. Each sample was replicated two times. The hydrolysates were stored at 20 C until analysis. 2.4. Assessment of proteolysis The extent of proteolysis was determined as a degree of hydrolysis, by quantification of hydrolysed peptide bonds using the o-phthaldialdehyde (OPA) method, as previously described in ˜ as et al. (2004). A volume (50 mL) of the hydrolysates was added Pen to 1 mL of OPA solution, swirled by inversion, incubated for 2 min at room temperature, and the absorbance at 340 nm was measured. Each sample of duplicate hydrolysis was analysed twice. Aliquots of the hydrolysates were also analysed by reversephase high performance liquid chromatography (RP-HPLC) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE). RP-HPLC was performed using an automated HPLC System consisting of a Consta metric 4100 pump, Spectra System AS 1000 autosampler, and Spectromonitor 5000 diode array detector (Thermo Separation products, Riviera Beach, FL, USA.) fitted with a Purospher Star column (5 mm particle size, 250 4.0 mm; Merck, Darmstadt, Germany). Operating conditions were column temperature, 25 C; flow rate: 0.8 mL min1; solvent A: 0.1% (v/v) trifluoroacetic acid (TFA) (sequential grade, Sigma, St. Louis, MO, USA) in acetonitrile (Scharlau, Barcelona, Spain) in Milli-Q water (Millipore, Saint-Quentin, Yvelines, France) (1:9); solvent B: 0.07% (v/v) trifluoroacetic in acetonitrile–Milli-Q (9:1). Elution was performed by applying 100% A for 5 min, then a linear gradient of 0–50% B over the following 45 min and to 70% B over the next 5 min and then maintained at 70% B for 5 min. Absorbance was recorded at 214 nm. Samples were diluted in urea (6 M) at a ratio of 1:1 (v/v). SDS-PAGE was performed using a Phast-SystemÒ with high density PhastGel mini-gels (Pharmacia, Du¨bendorf, Switzerland), with stacking (T7.5%, C2%) and separation gel (T20%, C2%) zones and a sample loading of 1 mL. The samples were lyophilised and dissolved in treatment buffer (2.5 mg mL1), consisting of 10 mM Tris– HCl, 1 mM EDTA at pH 8.0, SDS (2.5%), b-mercaptoethanol (5%) and bromophenol blue (0.01%). The bands were stained by silver staining using a modification of the method of Heukeshoven and Dernick (1985). The gels were immersed in sodium thiosulfate (0.02%, w/v) for 1 h, after fixing the bands and washing the gel. The staining solution consisted of silver nitrate (0.2%, w/v) and formaldehyde in Milli-Q water (0.072%, v/v). The developer solution contained sodium thiosulfate (0.0004%, w/v) and formaldehyde (0.050%, v/v). 2.5. Enzyme-linked immuno-sorbent assay The samples were diluted (0.5 mL mL1) in carbonate buffer (15 mM Na2CO3; 35 mM NaHCO3, pH 9.6), added (50 mL) to the wells of the microtiter plates (Nunc, Roskilde, Denmark) and then incubated for 18 h at 4 C. Residual free binding sites were blocked with 150 mL of 5% fish gelatin in phosphate buffered saline (PBS) (Sigma, Madrid, Spain) per well. Two pools of seven sera of patients allergenic to bovine milk were used; 50 mL of each pool was added separately to the wells and maintained for 1 h at 37 C. Two
0.5
β-Lg
negative controls (sera from two different nonallergic persons) were used. After four washes with PBS–Tween 20 (0.05%) containing 0.5% fish gelatin, goat anti-human peroxidase-labeled IgE (Sigma) was added to the plate, and incubated for 1 h at 37 C. After four washes, IgE content of samples bound to wells was followed by addition 100 mL of a freshly substrate-containing buffer (50 mM citrate, 125 mM phosphate, pH 5.0, 4.4 mM H2O2; 5 mM o-phenylenediamine dihydrochloride (OPD) (Sigma)), after incubation at room temperature in the dark for 15 min. The colorimetric reaction was stopped by addition of 0.05 mL of 2 M sulphuric acid per well and, then the absorbance at 495 nm determined on a microtiter automated enzyme-linked immuno-sorbent assay (ELISA) plate reader (3550 Microplate reader, Biorad, Richmond, CA, USA). Each sample of duplicate experiments was analysed twice by using each of the two pools of seven sera from children allergic to bovine milk.
α-La
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Absorbance 214 nm
920
A
B F
C
2.6. Statistical analysis
D
Data were analysed using SPSS for Windows (version 11.5, SPSS Inc. Chicago, IL, USA) following an analysis of variance (ANOVA) one way linear model. Mean comparisons were performed using the paired t test, and the significance level was established for P < 0.05. Data are shown as averages of duplicate analyses for each of the two hydrolysis experiments.
T
D
E 6
12
18
24
30
36
44
50
Retention time
3. Results and discussion 3.1. Proteolysis of bovine whey protein concentrate Fig. 1 compares the hydrolysis of WPC by Pronase, Chymotrypsin and five food grade enzymes, Papain, Corolases 7089 and PN-L, Alcalase and Neutrase, when CH and MWI were used during the enzymatic reactions. The highest hydrolysis was found for Pronase followed by Papain and Alcalase, irrespective of the heating procedure used. The degree of hydrolysis of WPC by all enzymes was significantly enhanced (P 0.05) when the digestion took place under MWI (Fig. 1). The RP-HPLC profiles of WPC solution (5%) in phosphate buffer 50 mM at pH 8.0 and distilled water pH 6.5 treated by MWI were studied. No differences were found by application of MWI, irrespective of treatment medium (results not shown). The chromatograms show the peak of a-lactalbumin (a-La) and blactoglobulin (b-Lg) eluting at retention times (RT) of 47.60 and 49.26 min, respectively, as shown in Fig. 2.
Fig. 2. RP-HPLC profile of peptides obtained by Papain and Alcalase hydrolysis of whey protein concentrate (WPC) under conventional heating (CH) and microwave irradiation (MWI). (A) WPC in distilled water, pH 6.6, (B) Papain hydrolysates under CH, (C) Papain hydrolysates under MWI, (D) Alcalase hydrolysates under CH, (E) Alcalase hydrolysates under MWI. F: Peptide fraction eluting between 22–32 min for the papain hydrolysate. T and D: Peptide fractions eluting between 26–28 min and 34–40 min, respectively, for the alcalase hydrolysates.
The RP-HPLC profiles of enzymatic hydrolysates showed the activity of all enzymes (with the exception of Chymotrypsin) on bLg and a-La. The peptide profiles of the samples digested by Pronase, Chymotrypsin, Corolases 7089 and PN-L and Neutrase, during the MWI did not differ significantly from the samples hydrolysed under CH. Only a slight increase of peaks in the WPC hydrolysed in combination with MWI was observed (results not shown). These results suggest that the specificity of these enzymes did not change when the MWI was applied. No qualitative changes were found in the peptide profile when the WPC was digested by Papain and Alcalase during the MWI
Micromols amino groups mg -1protein
5 4,5 4
Control (CH)
3,5
MWI
3 2,5 2 1,5 1 0,5 0 Pronase
Chymotrypsin
Papain
Corolase 7089
Corolase PNL
Alcalase
Neutrase
Enzymes Fig. 1. Degree of hydrolysis determined by the o-phthaldialdehyde method in enzymatic hydrolysates from commercial bovine whey protein concentrate (WPC). The experiments were performed at 40 C for Pronase, Chymotrypsin and Corolase PN-L and at 50 C for Papain, Alcalase, Neutrase and Corolase 7089, under conventional heating (CH) and microwave irradiation (MWI). The values are the mean and standard deviation of duplicate analysis of samples from duplicate experiments.
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1,2
Absorbance at 495 nm
compared with the hydrolysates performed under CH (Fig. 2). However, quantitative differences in the relative abundance of some peaks were obtained. Thus, an increase in the fraction (F) eluting between 22 and 32 min was noticed for the Papain hydrolysates performed under MWI compared with CH hydrolysates. Also, a lower proportion of the hydrophobic peptide eluted at >32 min (Fig. 2B and C). The MWI also increased the fractions T and D eluting between 26–28 and 34–40 min, respectively, in the Alcalase hydrolysates. The results suggest a possible positive effect of the MWI on the sensory characteristics of the hydrolysates obtained by Papain, since a low ratio of hydrophobic to hydrophilic peptide is related to high palatability (Clemente, 2000). Different patterns of peptides were observed by SDS-PAGE after the hydrolysis of WPC by the seven proteases. The b-Lg and a-La bands appeared with low intensity in the hydrolysates of WPC by Pronase and Alcalase performed under CH (results not shown). In contrast, no protein bands were observed in the respective digestions performed under MWI that, however, presented bands of apparent molecular masses <3.4 kDa, which was not observed in the CH hydrolysates. All Papain hydrolysates only showed three bands with molecular masses ranging between 9.0 and 1.4 kDa; these were of very low relative intensity when the enzymatic treatment was performed under MWI, suggesting that these peptides were digested under these conditions. The electrophoretograms for Chymotrypsin showed the a-La band in all cases. The b-Lg band only appeared when the enzymatic treatment was performed under CH and, then only with low intensity. No other differences induced by MWI were observed, presenting the CH and MWI hydrolysates three additional bands of about 8.0, 4.0 and 2.5 kDa (results not shown) These results suggest that Chymotrypsin significantly hydrolysed b-lactoglobulin regardless of the heating treatment used, whereas a-lactalbumin seemed to be resistant to the chymotryptic action in all cases. The b-Lg and a-La bands appeared in the hydrolysates by Corolases 7089, PN-L and Neutrase. Control Corolase 7089 hydrolysate presented three additional well-resolved bands of about 10, 9 and 6 kDa. Neither of these bands appeared in the MWI hydrolysates, suggesting that the highest proteolysis occurred under MWI with production of smaller peptides, since low intensity of the a-La band was also observed, in agreement with the highest degree of hydrolysis observed by the OPA method (Fig. 1). There were no important qualitative changes in the peptide profile of Corolase PNL and Neutrase hydrolysates induced by MWI. The increase in proteolysis observed in the present study when MWI was applied during the digestion could be related to either increased susceptibility of the substrate or increased activity of the proteases under MWI. Our finding of the enhancement of the enzymatic proteolysis under MWI is in good agreement with data previously described by our group for kinetic parameters for Pronase and a-Chymotrypsin hydrolysis of bovine b-Lg (Izquierdo, Alli, Yaylayan, & Gomez, 2007). Higher catalytic effectiveness (KcatK1 m ) values were obtained in both enzymatic digestions of the protein performed under MWI in comparison with the values in the respective CH digestions. The Michaelis–Menten constant (Km) for either enzyme was reduced under MWI, which suggests the highest substrate-enzyme affinity at these conditions (Izquierdo et al., 2007). Furthermore, Pramanik et al. (2002) reported acceleration of enzymatic processes for protein mapping by trypsin digestion. These authors reported the effectiveness of this treatment, since the peptide fragmentation of several biologically active proteins was achieved in minutes using MWI, in contrast to the hours required by CH treatment. The potential advantages of MWI treatment have also been reported for acceleration of protein hydrolysis in the preparation of samples for amino acid analysis (Chen et al., 1987; Chiou & Wang, 1989; Marconi et al., 1995). Bohr and Bohr (2000a, 2000b) described the unfolding, folding and denaturation
921
Control (CH) MWI Untreated WPC
1 b 0,8 a
a
0,6
a
0,4
a
a 0,2
b
b
a aa
b
b b
0 Pronase Chymotrypsin
Papain
Corolase 7089
Corolase PNL
Alcalase
Neutrase
Untreated WPC
Enzymes Fig. 3. Residual immunochemical reactivity determined by an ELISA assay (expressed as absorbance units at 495 nm) of enzymatic hydrolysates from whey protein concentrate (WPC). CH: conventional heating; MWI: microwave irradiation. The values are the mean and standard deviation of duplicate analysis of samples from duplicate experiments, when two pools of seven sera each one from allergic children to bovine milk were used. Different letters above the bars for each enzyme indicate significant differences P 0.05.
of b-Lg by MWI treatment, structural changes were also reported by de Pomerai et al. (2003) for BSA in solution, that probably favour the hydrolysis of proteins by proteases. The potential effect of MWI on enzymes has been described by Porcelli et al. (1997) who reported that 10.4 GHz caused conformational changes in two enzymes (S-adenosylhomocysteine and 50 -methylthioadenosine phosphorylase), detected by fluorescence spectroscopy and circular dichroism spectroscopy. These authors suggested that the MWI treatments induced structural rearrangements in the enzymes/proteins which were not related to temperature. 3.2. Immunochemical properties of the hydrolysates ELISA was used to assess the residual immunochemical reactivity in the digests obtained from WPC through combined enzymatic and MWI treatments. An important decrease in immunoreactivity was observed in hydrolysates obtained by combining MWI and Pronase, Papain or Alcalase and, to a lesser extent, Corolase 7089, in comparison to untreated WPC and enzymatic treatment alone when two pools of seven sera were used (Fig. 3). In contrast, enhanced reactivity was observed in the MWI hydrolysates by the other three enzymes compared to the respective CH digestions. These results are consistent with the residual a-La and b-Lg contents and possibly with the size of the peptides in these enzymatic digests (data not shown). The combined enzymatic and MWI treatments, compatible with retention of enzyme activity and reversibility of structural modifications of proteins, may have a practical relevance by decreasing their immunoreactivity, as reported by several authors for enzymatic hydrolysates from b-Lg (Bonomi et al., 2003), bovine WP ˜ as, Pre´stamo, Baeza, Martı´nez-Molero, & Go´mez, 2006; Pen ˜ as (Pen ˜ as, Snel, Floris, Pre´stamo, & Go´mez, 2006) and et al., 2006b; Pen ˜ as et al., 2004, 2006a; Pen ˜ as, Pre´stamo, Polo, & soybean WP (Pen Go´mez, 2006) performed under high pressure. The unfolded proteins formed under MWI could represent an ideal substrate for the action of proteases, making some epitopes more accessible to enzymatic hydrolysis. 4. Conclusions MWI treatment enhanced the enzymatic hydrolysis of bovine WPC. Pronase and Papain showed the highest proteolysis under
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MWI followed by Alcalase. The b-Lg was hydrolysed by Papain under all conditions, whereas this protein was only completely hydrolysed by Pronase, Alcalase and Chymotrypsin under MWI. Both proteins were resistant to hydrolysis by Corolase PN-L and Neutrase for 5 min of digestion, irrespective of the heating procedure applied. A decrease of immunochemical reactivity was also found for all hydrolysates compared to untreated WPC and the lowest antigenicity was found in those obtained by combined treatment of MWI and Pronase, Papain or Alcalase, concomitant with a considerable hydrolysis of a-La and b-Lg and production of small peptides. Thus, in general, MWI enhances WPC hydrolysis, and depending upon the choice of enzymes, reduced the residual antigenicity of the hydrolysates. Proteolysis of bovine WPC by either of the three enzymes in combination with MWI treatment has the potential to more efficiently produce hypoallergenic dairy hydrolysates. Acknowledgements The ‘‘Comisio´n Interministerial De Ciencia y Tecnologı´a’’ through the Project No AGL2000-1497 supported this work. Dr. Nieves Corzo (Instituto Fermentaciones Industriales) is acknowledged for the kind counselling on the microwave equipment. References Adibi, S. A. (1989). Glycyl dipe´ptides: new substrates for protein nutrition. Journal of Laboratory and Clinical Medicine, 113, 665–673. Bohr, H., & Bohr, J. (2000a). Microwave-enhanced folding and denaturation of globular proteins. Physical Review E – Statistical Physics, Plasmas, Fluids, and Related Interdisciplinary Topics, 61, 4310–4314. Bohr, H., & Bohr, J. (2000b). Microwave enhanced kinetics observed in ORD studies of a protein. Bioelectromagnetics, 21, 68–72. Bonomi, F., Fiocchi, A., Frokiaer, H., Gaiaschi, A., Iametti, S., Poiesi, C., et al. (2003). Reduction of immunoreactivity of bovine b-lactoglobulin upon combined physical and proteolytic treatment. Journal of Dairy Research, 70, 51–59. Calvo, M. M., & Gomez, R. (2002). Peptidic profile, molecular mass distribution and immunological properties of commercial hypoallergenic infant formulas. Milchwissenschaft, 57, 187–190. Chen, S. T., Chiou, S. H., Chu, Y. H., & Wang, K. T. (1987). Rapid hydrolysis of proteins and peptides by means of microwave technology and its application to amino acid analysis. International Journal of Peptide and Protein Research, 30, 572–576. Chiou, S. H., & Wang, K. T. (1989). Peptide and protein hydrolysis by microwave irradiation. Journal of Chromatography, 491, 424–431. Clemente, A. (2000). Enzymatic protein hydrolysates in human nutrition. Trends in Food Science and Technology, 11, 254–262. de la Fuente, M. A., & Juarez, M. (1995). Determination of phosphorus in dairy products by sample wet digestion in a microwave oven. Analytica Chimica Acta, 309, 355–359. Heukeshoven, J., & Dernick, R. (1985). Simplified method for silver staining of proteins in polyacrylamide gels and the mechanism of silver staining. Electrophoresis, 6, 103–112. Izquierdo, F. J., Alli, I., Gomez, R., Ramaswamy, H. S., & Yaylayan, V. (2005). Effects of high pressure and microwave on pronase and a-chymotrypsin hydrolysis of blactoglobulin. Food Chemistry, 92, 713–719. Izquierdo, F. J., Alli, I., Yaylayan, V., & Gomez, R. (2007). Microwave-assisted digestion of b-lactoglobulin by pronase, a-chymotrypsin and pepsin. International Dairy Journal, 17, 465–470. Juan, H. F., Chang, S. C., Huang, H. C., & Chen, S. T. (2005). A new application of microwave technology to proteomics. Proteomics, 5, 840–842.
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