Relationship between Some Characteristics of WPC Hydrolysates and the Enzyme Complement in Commercially Available Proteinase Preparations

PII : S0958-6946(98)00121-6

Int. Dairy Journal 8 (1998) 819—827  1999 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0958-6946/99/$ — see front matter

Relationship between Some Characteristics of WPC Hydrolysates and the Enzyme Complement in Commercially Available Proteinase Preparations Maria Smytha1 and Richard J. FitzGeraldb* ? Teagasc, Dairy Products Research Centre, Moorepark, Fermoy, Co. Cork, Ireland @ Life Science Department, University of Limerick, Limerick, Ireland (Received 10 September 1997; accepted 23 October 1998) ABSTRACT Endoproteinase and exopeptidase activities were determined in nine commercially available enzyme preparations using synthetic fluorogenic substrates at pH 7.0 and 8.0 and at 37 and 50°C. Four Bacillus (i.e. Protamex, Alcalase 0.6 L, Neutrase and Neutral Protease), three Aspergillus (i.e. Corolase 7092, Fungal Protease and Flavourzyme) and two porcine pancreatic (i.e., PTN 3.0S powder and PTN 3.0S granulated) preparations were analysed. Considerable variations in the endoproteinase, elastase-like, chymotryptic and tryptic activities were observed. Flavourzyme contained high aminopeptidase, while Fungal Protease had high X-prolyl-dipeptidylaminopeptidase activity. Significant differences were observed in the characteristics of the WPC hydrolysates generated with the above preparations. Some trends were observed between enzyme complement and hydrolysate characteristics, e.g. enzyme preparations with high exopeptidase activities resulted in hydrolysates having high DH values and low levels of hydrophobic peptides. However, in general it was not possible to directly predict the characteristics of WPC hydrolysates from the endoproteinase and exopeptidase complement in the crude enzyme preparations.  1999 Elsevier Science Ltd. All rights reserved Keywords: endoproteinase; exopeptidase; WPC hydrolysates; commercial enzymes

INTRODUCTION

arations, very little information appears to be available on the enzyme complement, i.e., range of activities, in commercially available crude proteinase preparations from other sources. The objective of this study was therefore to identify and quantify some of the endoproteinase and exopeptidase activities in a range of commercial proteinase preparations of fungal (Aspergillus), bacterial (Bacillus) and mammalian (pancreatic) origin. Furthermore, some of the physicochemical properties of whey protein concentrate (WPC) hydrolysates generated with these enzymes were characterised.

Commercially available crude proteinase preparations are used extensively in the food industry to prepare protein hydrolysates (Godfrey, 1996). By carefully controlling the reaction conditions during the enzymatic digestion of milk proteins, hydrolysates with improved solubility and emulsifying characteristics (Chobert et al., 1988; Turgeon et al., 1992), improved foaming properties (Kuehler and Stein, 1974), reduced allergenic/antigenic potential (Asselin et al., 1989; van Beresteijn et al., 1994) increased gel strength (Ju et al., 1995), reduced bitterness levels (Vegarud and Langsrud, 1989) and increased bioactive peptide contents (Mullally et al., 1997) have been produced. Manipulation of hydrolysis reaction conditions can, to some extent, be used to define the characteristics of the final hydrolysate. However, the specificities of the enzyme complement in a proteinase preparation dictate the type of peptides produced and thus the properties of a specific food protein hydrolysate. Apart from Mullally et al. (1994, 1995), who analysed the proteolytic and peptideolytic activities in pancreatic proteinase prep-

MATERIALS AND METHODS Materials Enzymes: Commercial enzyme preparations were received as gifts from manufacturers. PTN 3.0S (porcine), Neutrase (Bacillus subtilis), Alcalase 0.6 L and Alcalase 2.4 L (Bacillus licheniformis), Protamex (Bacillus spp) and Flavourzyme (Aspergillus oryzae) were supplied by Novo Nordisk A/S (Bagsvaerd, Denmark), Fungal protease (Aspergillus oryzae) and Neutral protease (Bacillus subtilis) were obtained from Quest International Ireland Ltd. (Cork, Ireland). Corolase 7092 (Aspergillus) was supplied by Rohm GmbH (Darmstadt, Germany).

* Corresponding author. Tel.: 353 61 202598; Fax: 353 61 331490; E-mail: [email protected]  Present address: National Cell and Tissue Centre, Dublin City University, Glasnevin, Dublin 9, Ireland. 819

820

M. Smyth, R. J. FitzGerald

Substrates: Aminomethylcoumarin (AMC) substrates (Suc-Leu-Leu-Val-Tyr-AMC, acetyl-Ala-Ala-Pro-AlaAMC, N-benzoyl-L-Arg-AMC, N-glutaryl-Phe-AMC, N-carbobenzoxy-Gly-Gly-Leu-AMC, Gly-Pro-AMC, Leu-AMC, Ala-AMC and Arg-AMC) were obtained from Bachem (Bubendorf, Switzerland). TAME (p-toluene—sulfonyl-L-arginine methyl ester) was obtained from Sigma Chemical Co. (Poole, England). Defatted WPC (Dietlac) was from Laiterie Triballot (France). Chemicals: Acetonitrile (HPLC grade) was obtained from Labscan Analytical (Dublin, Ireland). Trifluoroacetic acid (TFA) and dimethyl formamide (DMF) were from British Drug Houses Ltd. (Poole, England). Polypeptide molecular weight markers for sodium dodecyl-sulphate polyacrylamide gel electrophoresis (SDS-PAGE) were obtained from Pharmacia (Uppsala, Sweden). o-Phthaladialdehyde (OPA) and N-acetyl-Lcysteine (NAC) were obtained from Sigma Chemical Co. (Poole, England). All other reagents were of analytical grade.

et al. (1951), as modified by Polacheck and Cabib (1981), using bovine serum albumin as standard. The nitrogen content of WPC (N;6.38) was determined in duplicate by the micro-Kjeldahl procedure (AOAC, 1980). Preparation of WPC hydrolysates WPC (8%, w/v, protein) was dissolved in distilled water and pre-incubated at 37°C for 20 min and adjusted to pH 8.0 by addition of 0.5 N NaOH. Chloroform (1%, v/v) was included to prevent microbial growth (AdlerNissen, 1986). Commercial food- grade enzyme preparations, dissolved in water, were added to the reaction mixture at a final enzyme to substrate (E : S) ratio of 1 : 50 (w/w). The pH of the reaction mixture was maintained constant by continuous addition of 0.5 N NaOH using a pH-stat (Metrohm Ltd, Herisau, Switzerland). During hydrolysis, samples were withdrawn after 30 min and 8 h and the enzyme inactivated by heating at 85°C for 20 min. Hydrolysates were then cooled and stored at !20°C.

Methods Characterisation of hydrolysates Determination of enzyme activity Enzyme activities were assayed using a modification of the fluorogenic (AMC) assay (Zimmerman et al., 1977). Stock solutions of fluorogenic substrates (0.2 mM) were prepared as follows: Arg-AMC and Gly-Pro-AMC in distilled water; AMC in 0.02% DMF; Bz-Arg-AMC in 0.04% DMF; Glutaryl-Phe-AMC in 0.10% DMF, and Leu-AMC and Ac-Ala-Ala-Pro-Ala-AMC in 0.50% DMF. Substrate solution (100 kL) was pre-incubated with 800 kL of buffer (100 mM sodium phosphate, pH 7.0, or 100 mM bicine-NaOH, pH 8.0) at 37 or 50°C for 10 min. Commercial food-grade enzyme preparations (10 mg of powder mL\(aq)) were centrifuged at 14,400;g (Microcentaur, MSE, Sussex, UK) for 10 min, and 100 kL of supernatant were added to the substrate mixture. The substrate-enzyme mixture was incubated at 37 or 50°C for 30 min. The reaction was stopped by the addition of 1 mL of 1.5 M acetic acid, and fluoresence developed was measured (Perkin-Elmer 1000, spectrofluorometer Beaconsfield, Bucks, England) at excitation and emission wavelengths of 360 and 440 nm, respectively. One unit of activity was defined as the amount of enzyme which gave 1 kmol of AMC min\ mg\ protein. Gly-Pro-AMC was used to detect X-prolyl-dipeptidyl aminopeptidase (XPDA) activity, Leu-AMC to detect Pep N or general aminopeptidase activity, Ala-AMC to detect alanine aminopeptidase activity and Arg-AMC to detect aminopeptidase B activity. All assays were performed at least in triplicate on three separate occasions. Activities are reported as mean values. Variation about the mean activity value was generally less than mean $5%. Where larger variations occurred (i.e. for Flavourzyme and Protamex on Suc-Leu-Leu-Val-Tyr-AMC at pH 7.0 and 50°C, and for Flavourzyme on Leu-AMC at pH 8.0 and 37°C), assays were carried out in triplicate on five separate occasions. Substrate blanks containing the corresponding [DMF] for each substrate were used throughout. Determination of protein concentration Protein concentrations in the commercial enzyme preparations was determined by the method of Lowry

Degree of hydrolysis (DH) The degree of hydrolysis was determined by the OPA method described by Church et al. (1983), using NAC as a reducing agent, as described by Garcia Alvarez-Coque et al. (1989). Molecular mass distribution of peptides in ¼PC hydrolysates Gel permeation HPLC was performed on a Spherogel-TSK 2000SW (Beckman Instruments Ltd., High Wycome, UK) gel permeation column (600; 7.5 mm) fitted to a Waters HPLC system (Millipore, Harrow, UK) comprised of a Model 150 pump, Model 717 autosampler and a Model 441 absorbance detector set to operate at 214 nm (Smyth and FitzGerald, 1997). The detector was interfaced with a Minichrom2+ data handling package (V.G. Data System, Manchester, UK). The mobile phase consisted of 30% (v/v) acetonitrile containing 0.1% (v/v) TFA. Hydrolysates were diluted in mobile phase to 0.25% (w/v) protein, filtered through 0.2 km syringe filters (Whatman, Maidestone, England) and 20 kL applied to the column. The flow rate was 1.0 mL min\ and chromatography was carried out at room temperature. A calibration curve was prepared from the average retention volume of standard proteins and peptides. The calibration standards used were: bovine serum albumin (67,500 Da), b-lactoglobulin (36,000 Da), ribonuclease A (13,700 Da), cytochrome C (13,000 Da), aprotinin (6500 Da) and bacitracin (1400 Da), which were from Sigma Chemical Co. (Poole, Dorset, UK), and His-Phe-Arg-Trp (764.8 Da), Leu-TrpMet-Arg (604.8 Da), Arg-Pro-Pro (404.4 Da), Leu-Phe (292.8 Da), Asp-Glu (262.2 Da) and Tyr-Gly (240.1 Da), which were from Bachem (Bubendorf, Switzerland). Reversed-phase high performance liquid chromatography (RP-HP¸C) of ¼PC hydrolysates Peptides in WPC hydrolysates were separated by RPHPLC on a Phenomenex (Phenomenex Ltd., Macclesfield, Chesire, England) C18 column (250;3.2 mm, 5 km), equilibrated with solvent A (0.1% TFA in H O)  and eluted with a linear gradient to 80% solvent B (60%

Enzymatic hydrolysis

acetonitrile, 40% H O, 0.1% TFA) during the first  50 min, to 90% in the next 10 min, to 100% in the next 5 min and the column re-equilibrated to 0% solvent B for the last 20 min. Runs were conducted at room temperature using a Shimadzu HPLC system (Shimadzu Corp., Analytical Instruments Nakagyo-ku, Kyoto, Japan) at a flow rate of 0.3 mL min\ and the absorbance of the column eluate monitored at 214 and 280 nm. The injection volume was generally 50 kL and the concentration of peptide material applied was approx. equivalent to 0.1 mg protein mL\. All samples were filtered through a 0.2 km filter prior to application to the C18 column. Gel electrophoresis Discontinuous SDS-PAGE was performed on the hydrolysates using the method of Schagger and von Jagow (1987). RESULTS Commercially available proteinases were incubated with a range of synthetic fluorogenic substrates in order to determine the specificity and to quantify the level of a range of endoproteinase and aminopeptidase activities in a given preparation. Activities were determined at pH 7.0 and 8.0. The activities obtained on incubation at 37 or 50°C are summarised in Tables 1 and 2, respectively. Endoproteinase activity Suc-Leu-Leu-Val-Tyr-AMC was used to determine endoproteinase activity. With the exception of Corolase 7092 and PTN 3.0 S (powder), all the preparations had highest activity on this substrate at 37°C and pH 8.0. Highest overall activity on this substrate was observed with Alcalase 0.6 L; it produced 13,740 kmol AMC min\ mg\ protein at 37°C and pH 8.0 (Tables 1 and 2). Lowest activity on this substrate was observed in the Neutrase preparation. The hydrolytic activity on SucLeu-Leu-Val-Tyr-AMC in the different preparations can be ranked as follows: Alcalase 0.6 L'Protamex'Flavourzyme'PTN 3.0S (granulated)'Corolase 7092' PTN 3.0S (powder)'Neutral Protease'Fungal Protease. All enzyme preparations tested could hydrolyse AcAla-Ala-Pro-Ala-AMC, the level of activity being highly dependent on the temperature and pH of the assay (Tables 1 and 2). Four of the preparations, i.e., Fungal Protease, Flavourzyme, Neutrase and PTN 3.0S (granulated) displayed highest activity at 37°C and pH 7.0, with activities ranging from 0.6 to 101.7 kmol AMC min\ mg\ protein (Table 1). On the other hand, Protamex, Alcalase 0.6 L and Neutral Protease showed highest activity at 50°C and pH 8.0, ranging from 1.8 to 16.6 kmol AMC min\ mg\ protein (Table 2). For Corolase 7092, highest activity (i.e., 10.5 kmol AMC min\ mg\ protein, Table 2) was observed at 50°C and pH 7.0. The level of Ac-Ala-Ala-Pro-Ala-AMC hydrolysing activity in the different preparations can be ranked as follows: PTN 3.0S (granulated)'Alcalase 0.6 L'Corolase 7092'Neutrase'Protamex'Flavourzyme' Neutral Protease'PTN 3.0S (powder)'Fungal Protease. Surprisingly, a higher level of activity was observed in the granulated than in the powdered form of PTN 3.0S. Furthermore, granulated PTN 3.0S showed highest

821

activity at pH 7.0 and 37°C (i.e., 101.7 kmol AMC min\ mg\ protein, Table 1) whereas the powdered form of PTN 3.0S had highest activity at pH 8.0 and 37°C (i.e., 0.8 kmol AMC min\ mg\ protein, Table 1). Trypsin activity, measured by hydrolysis of N-benzoyl-Arg-AMC, was observed only in the powdered form of PTN 3.0S (31.9—63.7 kmol AMC min\ mg\ protein, Tables 1 and 2). Highest activity was observed at 37°C and pH 8.0. Surprisingly, no trypsin activity was observed in the granulated form of PTN 3.0S (Tables 1 and 2). Quantification of trypsin activity in both PTN 3.0S preparations using TAME as substrate gave similar results to those obtained with the fluorometric assay (data not shown). As expected, little or no trypsin-like activity was observed in the bacterial proteinase preparations tested. A low level of chymotrypsin activity, as measured by the hydrolysis of glutaryl-Phe-AMC, was observed only in PTN 3.0S (powder); highest activity was observed at 37°C and pH 7.0 (Table 1). No activity on Z-Gly-Gly-Leu-AMC was observed in the Neutrase, Neutral Protease and Corolase 7092 preparations. Low activities were observed in Protamex, PTN 3.0S (granulated), Fungal Protease and Flavourzyme (0.2—1.2 kmol AMC min\ mg\ protein, Tables 1 and 2)). The PTN 3.0S (powder) and Alcalase 0.6 L preparations displayed relatively high activity with Z-Gly-Gly-Leu-AMC, i.e., 68.9 and 26.7 kmol AMC min\ mg\ protein, respectively, when assayed at 50°C and pH 8.0 (Table 2). Aminopeptidase activity Four substrates were used to determine aminopeptidase activity in the commercial enzyme preparations. Little or no aminopeptidase activities were detected in Corolase 7092, Protamex, Alcalase 0.6 L, Neutrase, PTN 3.0S (granulated) or Neutral Protease. Highest levels of XPDA activity were observed in the Fungal Protease preparation, i.e. 30.02 kmol AMC min\ mg\ protein at 50°C and pH 8.0 (Table 2). Flavourzyme produced 0.25 kmol AMC min\ mg\ protein at 37°C pH 8.0 (Table 1). Flavourzyme had the highest level of pepN activity (640 kmol AMC min\ mg\ protein, 37°C, pH 8.0, Table 1), alanine aminopeptidase-like activity (170.5 kmol AMC min\ mg\ protein, 50°C, pH 8.0, Table 2) and aminopeptidase B activity (179.60 kmol AMC min\ mg\ protein, 50°C, pH 7.0, Table 2). Significantly lower levels of these three aminopeptidases were found in the Fungal Protease preparation (Tables 1 and 2). Trace levels of Arg-AMC activity were observed in the powdered form of PTN 3.0S (Tables 1 and 2). Characterisation of WPC hydrolysates WPC hydrolysates were generated using the nine enzyme preparations at 37°C and pH 8.0. Degree of hydrolysis (DH) The DHs obtained and the molecular mass distribution of WPC hydrolysates on incubation for 30 min or 8 h with the range of commercial food-grade enzyme preparations are shown in Table 3. Under similar conditions (i.e., pH, temperature, E : S and incubation time), the degree to which WPC was hydrolysed varied, depending

0.03 0.02 0.01 —

0.03 0.14 0.01 —

1,693.00 3.30 — — 0.70

pH 8.0

— 0.02 — —

12,158.00 8.20 — — 14.80

pH 7.0

— 0.42 — —

13,740.00 10.50 0.01 0.01 24.70

pH 8.0

Alcalase 0.6 L

— — — —

— 8.40 — — —

pH 7.0

— — — —

— 1.50 — — —

pH 8.0

Neutrase

0.01 0.01 — —

137.60 0.90 — — —

pH 7.0

0.01 0.01 0.01 —

253.00 1.40 — — —

pH 8.0

Neutral protease

0.01 0.01 0.01 0.01

303.30 1.10 — — —

pH 7.0

0.01 0.01 0.01 0.01

27.70 9.70 — — —

pH 8.0

Corolase 7092

0.01 20.40 0.05 2.12

15.40 0.60 — — 0.20

pH 7.0

0.02 35.60 0.06 1.45

16.00 0.50 — — 0.10

pH 8.0

Fungal protease

0.15 511.20 7.80 61.90

206.00 3.10 0.02 0.02 1.10

pH 7.0

0.25 640.00 12.90 48.30

372.00 2.80 — — 0.14

pH 8.0

Flavourzyme

0.01 0.01 — 2.01

220.00 0.80 63.70 0.16 65.90

0.13 0.02 0.01 —

0.12 0.02 0.01 —

1,118.00 5.20 — — 1.00

pH 8.0

0.01 0.03 — —

7,131.00 14.20 — — 18.30

pH 7.0

0.01 0.03 — —

7,734.00 16.60 — — 26.70

pH 8.0

Alcalase 0.6 L

0.01 — 0.11 —

0.10 1.30 — — —

pH 7.0

0.01 0.01 — —

0.30 1.90 — — —

pH 8.0

Neutrase

0.04 0.01 0.02 0.01

214.00 1.20 — — —

pH 7.0

0.24 0.01 0.01 0.01

206.30 1.80 — — —

pH 8.0

Neutral protease

— 0.01 0.01 0.02

24.40 10.50 — — —

pH 7.0

0.01 — — 0.08

35.30 0.80 — — —

pH 8.0

Corolase 7092

30.02 30.05 0.10 1.40

7.60 0.30 — — 0.20

pH 7.0

0.06 28.10 0.06 3.54

1.60 0.20 — — —

pH 8.0

Fungal protease

0.16 115.80 14.90 179.60

78.00 2.30 0.02 0.03 0.82

pH 7.0

0.06 287.70 170.50 170.50

40.60 0.10 0.02 0.01 0.08

pH 8.0

Flavourzyme

0.04 0.01 — 0.43

120.00 0.10 37.20 0.13 45.40

0.03 0.02 — 3.96

110.60 0.10 31.90 0.05 68.90

pH 8.0

PTN 3.0S (powder) pH 7.0

AMC, 7-amino-4-methyl coumarin; Ac, acetyl; Bz, benzoyl; Suc, succinyl; Z, carbobenzoxy; —, no significant activity (i.e. activity values (0.01 kmol AMC min\ mg\ protein).

Aminopeptidase substrates Gly-Pro-AMC Leu-AMC Ala-AMC Arg-AMC

Endoproteinase substrates Suc-Leu-Leu-Val-Tyr-AMC 1,108.00 Ac-Ala-Ala-Pro-Ala-AMC 3.50 Bz-Arg-AMC — Glutaryl-Phe-AMC — Z-Gly-Gly-Leu-AMC 0.70

pH 7.0

Protamex

Enzyme activity (kmol AMC min\ mg\ protein)

Table 2. Endoproteinase and Aminopeptidase Activities (kmol AMC min\ mg\ protein) in Commercial Enzyme Preparations Determined at pH 7.0 and 8.0 at 50°C

0.01 0.01 — 0.28

284.00 — 38.10 0.20 31.30

pH 8.0

PTN 3.0S (powder) pH 7.0

AMC, 7-amino-4-methyl coumarin; Ac, acetyl; Bz, benzoyl; Suc, succinyl; Z, carbobenzoxy; —, no significant activity (i.e. activity values (0.01 kmol AMC min\ mg\ protein).

Aminopeptidase substrates Gly-Pro-AMC Leu-AMC Ala-AMC Arg-AMC

Endoproteinase substrates Suc-Leu-Leu-Val-Tyr-AMC 506.00 Ac-Ala-Ala-Pro-Ala-AMC 2.40 Bz-Arg-AMC — Glutaryl-Phe-AMC — Z-Gly-Gly-Leu-AMC 0.40

pH 7.0

Protamex

Enzyme activity (kmol AMC min\ mg\ protein)

Table 1. Endoproteinase and Aminopeptidase Activities (kmol AMC min\ mg\ protein) in Commercial Enzyme Preparations Determined at pH 7.0 and 8.0 at 37°C

0.02 0.05 — —

335.40 18.60 — — 0.03

pH 8.0

0.11 0.14 0.12 0.01

238.80 27.00 — — 0.03

pH 7.0

0.07 0.02 0.01 0.01

230.10 44.40 — — 0.03

pH 8.0

PTN 3.0S (granulated)

0.03 0.05 0.01 0.01

259.00 101.70 — — 0.70

pH 7.0

PTN 3.0S (granulated)

822 M. Smyth, R. J. FitzGerald

823

Enzymatic hydrolysis

Table 3. The Degree of Hydrolysis (DH%) Obtained and the Molecular Mass Distribution of WPC Hydrolysates after 30 min and 8 h Incubation with a Range of Commercial Food Grade Enzyme Preparations Enzyme preparation

Protamex Protamex Alcalase 0.6 L Alcalase 0.6 L Neutrase Neutrase Neutral Protease Neutral Protease Corolase 7092 Corolase 7092 Fungal Protease Fungal Protease Flavourzyme Flavourzyme PTN 3.0S (powder) PTN 3.0S (powder) PTN 3.0S (granulated) PTN 3.0S (granulated)

Hydrolysis time (h) 0.5 8 0.5 8 0.5 8 0.5 8 0.5 8 0.5 8 0.5 8 0.5 8 0.5 8

Degree of hydrolysis (DH, %) 3.9 8.7 3.3 7.9 3.6 7.4 6.3 11.6 4.9 13.9 4.3 11.4 7.1 24.3 8.6 11.5 4.0 5.5

Molecular mass distribution* (kDa) '10

10—5

(5

43.22 16.21 36.84 12.84 38.54 15.42 24.44 9.40 46.95 18.64 45.77 19.57 60.14 28.77 6.85 1.45 34.68 15.39

7.74 6.23 11.51 5.73 10.60 6.58 6.16 3.82 6.77 5.12 5.90 4.30 3.83 3.40 5.95 2.52 7.24 5.66

49.04 77.55 61.64 81.42 50.58 78.00 69.40 86.79 46.23 76.23 48.33 76.10 36.02 67.81 87.20 96.04 58.07 78.94

* , Values are areas within a defined molecular mass distribution, expressed as % of total area of a chromatogram at 214 nm.

on the enzyme preparation used. In general, low DH values (approx. 5%) were observed following 30 min incubation of WPC with most of the preparations (Table 3). However, Neutral Protease, Flavourzyme and PTN 3.0S (powder) gave DH values of 6.1, 7.1 and 8.6%, respectively, following 30 min incubation at 37°C. With the exception of the granulated form of PTN 3.0S, the DH values obtained were significantly higher following 8 h incubation for all the enzyme preparations tested. The Flavourzyme preparation caused the most extensive hydrolysis of WPC following 8 h incubation, giving a DH value of 24.3%. Based on the DH value reached following incubation of WPC for 8 h at 37°C and pH 8.0, the hydrolytic efficiency of the enzyme preparations under the experimental conditions (Table 3) could be ranked in the following order: Flavourzyme'Corolase 7092'Neutral Protease'PTN 3.0S (powder)' Fungal Protease'Protamex'Alcalase 0.6 L'Neutrase'PTN 3.0S (granulated). Molecular mass distribution of peptides in WPC hydrolysates The molecular mass distributions of the WPC hydrolysates changed during the incubation period, showing a general decrease in high molecular mass and a concomitant increase in low molecular mass material for the longer incubation time. Lowest levels of high molecular mass material ('10 kDa) were observed in both the 30 min and 8 h PTN 3.0S (powder) digests of WPC (Table 3). A high level of high molecular mass material ('10 kDa) was present in the Flavourzyme digests, even though this enzyme preparation caused the most extensive hydrolysis of WPC. The SDS-PAGE profiles of selected hydrolysates are shown in Fig. 1. Unhydrolysed material was evident in the WPC hydrolysates prepared with Corolase 7092 for 30 min and 8 h and with Neutrase for 30 min (Fig. 1, lanes 3, 4 and 8). The 8 h digest with Neutrase contained

Fig. 1. SDS-PAGE profile of WPC hydrolysates generate produced with selected commercial enzyme preparations. Lane 1, Horse myoglobin polypeptide molecular mass standards, Lane 2, unhydrolysed WPC; Lane 3, 30 min Corolase 7092 hydrolysate (1 mg); Lane 4, 8 h Corolase 7092 hydrolysate (1 mg); Lane 5, 30 min Alcalase 0.6 L hydrolysate (1 mg); Lane 6, 8 h Alcalase 0.6 L hydrolysate (1 mg); Lane 7, 8 h Neutrase hydrolysate (1 mg) and Lane 8, 30 min Neutrase hydrolysate (1 mg).

no unhydrolysed material (Fig. 1, lane 7). On the other hand, Alcalase 0.6 L hydrolysates contained no unhydrolysed WPC even after 30 min digestion (Fig. 1, lanes 5 and 6). Reversed-phase HPLC of WPC hydrolysates Representative reversed-phase HPLC profiles for five WPC hydrolysates at 30 min and 8 h are given in Fig. 2. In general, the elution profile obtained depended on the enzyme preparation used and on the duration of incubation. Following 30 min incubation, a considerable amount of unhydrolysed material was present in the

824

M. Smyth, R. J. FitzGerald

Fig. 2. Reversed-phase (C18) HPLC chromatograms of WPC hydrolysates produced with selected commerical enzyme preparations, (a) 30 min Flavourzyme, (b) 8 h Flavourzyme, (c) 30 min Corolase 7092, (d) 8 h Corolase 7092, (e) 30 min Neutrase, (f ) 8 h Neutrase, (g) 30 min Alcalase 0.6 L, (h) 8 h Alcalase 0.6 L, (i) 30min Protamex and ( j) 8 h Protamex hydrolysates.

hydrolysates generated with Flavourzyme and Fungal Protease. This is represented by a large peak of material eluting at 65 min (Fig. 2a and data not shown, respectively). Lower levels of unhydrolysed material were observed following 30 min incubation with Corolase 7092, Neutrase and to a lesser extent with the granulated form of PTN 3.0S (Fig. 2c, e and data not shown, respectively).

Alcalase 0.6 L, Protamex and PTN 3.0S (powder) hydrolysates contained essentially no unhydrolysed material (Fig. 2g, i and data not shown, respectively). Following further hydrolysis, i.e., up to 8 h, a significant amount of unhydrolysed material was still present in the Flavourzyme WPC hydrolysate (Fig. 2b), slightly lower levels were present in the 8 h Corolase 7092 hydrolysate

Subtilisin and Neutral proteinase Endoproteinase (Subtilisin Carlsberg) Alkaline Protease Neutral metalloproteinase (Zn) Endoproteinase

Bacillus

Bacillus licheniformis

Bacillus subtilis

Bacillus subtilis

Aspergillus

Aspergillus oryzae

Aspergillus oryzae

Porcine pancreas

Porcine pancreas

Protamex

Alcalase 0.6 L

Neutrase

Neutral Protease

Corolase 7092

Fungal Protease

Flavourzyme

PTN 3.0S

PTN 3.0 S

*EC, Enzyme Commission; —, Data not supplied.

Trypsin

Endoproteinase (enzyme complex) Exopeptidase Trypsin

Endoproteinase/ Exopeptidase Endoproteinase

Type of activity

Source

Enzyme preparation

Anson method with haemoglobin as substrate

Anson method with haemoglobin as substrate Folin method using casein substrate Anson method with haemoglobin as substrate Anson method with haemoglobin as substrate Folin method using soy protein isolate as substrate Leucine-p-nitroanilide Anson method with haemoglobin as substrate

Anson method with haemoglobin as substrate Anson method with hemoglobulin as substrate

Actvity determination

40—55°C

— 40—55°C

7.0 6.0—9.0 6.0—9.0

45—50°C

40—60°C

45°C/55°C

45—60°C

45—55°C

55—70°C

—

Temperature range

5.0—7.0

4.0—7.0

5.5—9.5

5.5—8.0

5.5—7.5

6.5—8.5

5.5—7.5

pH range

3.2.11.11 (Aminopeptidase) 3.4.21.4 (Trypsin) and minor constituent 3.4.21.1 (Chymotrypsin) 3.4.21.4 (Trypsin) and minor constituent 3.4.21.1 (Chymotrypsin)

—

—

—

—

3.4.24.28 (Neutral protease)

3.4.21.62 (Subtilisin) 3.4.24.28 (Neutral protease) 3.4.21.62 (Subtilisin)

EC* number

Table 4. Summary of Details Provided by the Manufacturers on the Characteristics of Commercially Available Crude Proteinase Preparations

Granulate

Powder

Powder

Powder

Liquid

Powder

Liquid

Liquid

Granulate

Form

Enzymatic hydrolysis 825

826

M. Smyth, R. J. FitzGerald

(Fig. 2d) while trace amounts of unhydrolysed material was present in the 8 h Protamex and Fungal Protease hydrolysates (Fig. 2j and data not shown, respectively). DISCUSSION In general, the specificity and level of enzymatic activities in commercially available food-grade enzyme preparations are not well characterised. The details supplied by the manufacturers of the nine proteinases used in this study are summarised in Table 4, which shows that overall enzyme activities are generally determined using non-specific substrates while little or no information is supplied on secondary activities. It is also apparent that the various enzyme preparations are quoted as having broad pH and temperature ranges. Enzyme activities in the present study were assayed at pH 7.0 and 8.0, and at 37 and 50°C. With the exception of Neutrase, all the enzyme preparations tested hydrolysed Suc-Leu-Leu-Val-Tyr-AMC (Tables 1 and 2). Neutral proteinase, the main activity in Neutrase (Table 4), cleaves at the amino side of hydrophobic residues (Ward, 1983). It is expected, therefore, that neutral proteinase should hydrolyse Suc-Leu-LeuVal-Tyr-AMC at the Val-Tyr and the Leu-Leu bonds, releasing Tyr-AMC and Leu-Val-Tyr-AMC, respectively. However, the lack of significant aminopeptidase activity in Neutrase (Tables 1 and 2) may explain the observed apparent lack of hydrolysis. The apparent specificity differences between Neutrase (Novo Nordisk A/S) and Neutral Protease (Quest International), both Bacillus subtilis preparations (Table 4), may be clarified by determining activity with Suc-Leu-Leu-Val-Tyr-AMC in the presence of exogenous aminopeptidase. Furthermore, the apparent absence of activity with Neutrase may be due to a lack of calcium in the assay, Neutrase is specifically described as being a metalloproteinase (Table 4). Hydrolysis of Suc-Leu-Leu-Val-Tyr-AMC may also be indicative of the presence of chymotrypsin or chymotypsin-like activity. The activity values obtained with the PTN 3.0S preparations at 37°C and pH 7.0 are in general agreement with those previously reported (Mullally et al., 1994). However, the reason for the observed differences in temperature optimum between the powder and granulated forms of PTN 3.0S on this substrate are unclear (Tables 1 and 2). In general, there were significant differences in the substrate activity profiles for the two forms of PTN 3.0S (Tables 1 and 2), differences which may relate to modifications in the manufacturer’s production protocols, e.g in zymogen activation (Mullally et al., 1995). Hydrolysis of Z-Gly-Gly-Leu-AMC is indicative of the presence of subtilisin- and chymotrypsin-like activity. The high level of subtilisin activity observed in Alcalase 0.6 L is in agreement with previous reports (Ottesen and Svenden, 1970; Ward, 1983; Adler-Nissen, 1993). Hydrolysis of Z-Gly-Gly-Leu-AMC with PTN 3.0S (powder) probably arises from the chymotryptic side activity present in this porcine pancreatic preparation (Table 4). The presence of elastase or elastase-like activity, as indicated by the hydrolysis of Ac-Ala-Ala-Pro-AlaAMC, in Corolase 7092, i.e., an Aspergillus proteinase, has not been reported previously. In agreement with the present study, elastase has been observed in alkalophilic Bacillus (Tsai et al., 1988) and in pancreatic endoproteinase preparations (Mullally et al., 1995).

It is expected that the DH achieved during an hydrolysis reaction would be related to the level and specificity of the enzyme activities used. For example, the high DH attained during WPC hydrolysis with Flavourzyme may, in part, be attributed to the presence of high levels of general aminopeptidase activity (Table 2). However, under the experimental conditions described herein, it is clear that it is not possible to directly relate the level and specificity of activities in a particular crude enzyme preparation to the DH attainable during an hydrolysis reaction. For example, incubation of WPC with Fungal Protease, PTN 3.0S (powder) or Neutral Protease for 8 h at 37°C, pH 8.0 resulted in similar final DH values, i.e., 11.4, 11.5 and 11.6%, respectively (Table 3). Therefore, similar final DH values can be attained using proteinase preparations having significantly different specificities and levels of activity (Tables 1 and 2). It is also not possible to relate DH with the molecular mass distribution profiles for WPC hydrolysates generated with crude enzyme preparations. The 8 h hydrolysate produced with Flavourzyme had a DH of 24.4%; however, a considerable amount ('28%) of the peptide material was '10 kDa. On the other hand, the PTN 3.0S (powder) 8 h WPC hydrolysate had a DH of 11.5% but only 1.45% of the peptide material was '10 kDa (Table 3). While the WPC hydrolysates produced with Flavourzyme may have a good flavour profile due to the debittering effect of its exopeptidase activities (Tan et al., 1993), these hydrolysates may not be suitable for protein fortification of low osmolality food products due to high levels of free amino acids (Silk et al., 1975). WPC hydrolysates produced with Neutral Protease, for example, may find application in reduced or hypoallergenic foods due to the low levels of high ('10 kDa) molecular mass material (Thibault, 1991). As with the molecular mass distribution profiles, it is difficult to relate the reversed-phase profiles of the different WPC hydrolysates (Fig. 2) to the level and specificity of the activities in the crude enzyme preparations. However, the high levels of hydrophobic peptides (having a retention time '50 min) present in the 8 h WPC hydrolysates produced with Neutrase and Alcalase 0.6 L (Fig. 2f and h) would indicate that these hydrolysates may have a bitter taste (Tan et al., 1993). Considerable variation in the enzyme complement was observed for the different commercial crude enzyme preparations. Knowledge of the range of activities present in commercial preparations is a useful starting point when designing new hydrolysate products. For example, some trends were observed between enzyme complement and WPC hydrolysate characteristics, e.g., enzyme preparations with high exopeptidase activities (Flavourzyme) resulted in hydrolysates having high DH values and low levels of hydrophobic peptides. However, based on the results obtained in this study, it does not appear possible to directly relate the characteristics of WPC hydrolysates, i.e., DH, molecular mass distribution and reversed-phase chromatography profiles, to the enzyme complement present of crude commercial enzyme preparations. ACKNOWLEDGEMENTS Funding from the EU AIR programme project ‘Chemical Composition and Structure of Food Constituents:

Enzymatic hydrolysis

Defining Allergenic Potential’ (AIR-CT94-0970) is gratefully acknowledged.

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