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Environmental Sustainability

, Volume 1, Issue 3, pp 267–278 | Cite as

Acid whey proteolysis to produce angiotensin-I converting enzyme inhibitory hydrolyzate

  • Carolina Villadóniga
  • Laura Macció
  • Ana María B. Cantera
Original Article
  • 257 Downloads

Abstract

α-Lactalbumin (ALA) and β-lactoglobulin (BLG) are the main proteins in acid whey and proteolytic digestion of these proteins provides a source of peptides that inhibit angiotensin-I converting enzyme (ACE-I). Enzymatic hydrolysis of acid whey proteins using cysteine peptidase enzyme extract (antiacanthain) from ripe Bromelia antiacantha Bertol fruit was investigated. The effect of pH on hydrolysis kinetics of ALA and BLG by antiacanthain was analysed at 50 °C. Antiacanthain had a high catalytic efficiency for ALA (1.3 × 104 M−1 s−1) and BLG (1.7 × 104 M−1 s−1) at pH 9.2. The effect of temperature and amount of antiancanthain (enzyme to substrate ratio or E/S) on acid whey hydrolysis were studied by measurement of degree of hydrolysis (DH) and protein degradation profile evaluated by tricine–sodium dodecyl sulfate-polyacrylamide gel electrophoresis (tricine–SDS-PAGE). Optimal acid whey hydrolysis was at pH 9.2, E/S = 1.0 (U/mg) and 50 °C. BLG was degraded faster than that of ALA, but after 30 min hydrolysis almost all BLG and ALA were degraded and new bands corresponding to peptides with a mass lower than 3.0 kDa appeared. Fractions of acid whey (DH = 12.3%), ALA (DH = 17.3%) and BLG (DH = 30.6%) hydrolyzates showed ACE-I inhibitory activity. Results also suggest that most inhibitory activity might be due to low molecular weight peptides from ALA, since 3 kDa permeate of ALA hydrolyzate showed the highest IC50 value (1.7 ± 0.4 µg/mL). Interestingly, low molecular weight peptides from both ALA and BLG had low immunoreactivity levels, monitored by latex agglutination tests, suggesting that these peptides could be safe for use by those with strong allergic responses. Results indicate that antiacanthain can be used as an enzymatic preparation for acid whey hydrolysis. The potential use of this hydrolyzate for manufacture of health-promoting products of high added value is discussed.

Keywords

Acid whey Bioactive peptides Plant endopeptidases Angiotensin converting enzyme inhibitor Hydrolysis 

Introduction

Acid whey (pH 4.5) is a by-product from cheese or casein preparation after milk acidification by lactic fermentation or isoelectric precipitation with addition of inorganic acids, respectively (Ganju and Gogate 2017). Worldwide dairy industries produce large quantities of whey (180–190 × 106 tons/year) (Baldasso et al. 2011), and most of this is discarded, resulting in serious problems due to high organic load and potential for damage to the environment.

Bovine acid whey, which is used to be processed as wastewater of high organic load, contains proteins with high nutritional value as well as those for production of bioactive peptides (Ryan and Walsh 2016). These proteins are mainly β-lactoglobulin (BLG) and α-lactalbumin (ALA), as well as serum albumin (BSA), immunoglobulin and minor proteins including lactoferrin and lactoperoxidase (Fox et al. 2017). These proteins from whey could then be used for manufacture of new dairy products, including bioactive peptides (antibiotics, antihypertensive molecules, antioxidants, opioids, etc.). In addition, dairy products are not of public concern and there is worldwide acceptance for their use in functional food or nutraceutical production (Yoshikawa 2015). Among potential bioactive peptides, angiotensin-I converting enzyme (ACE-I) inhibitory peptides are useful as therapeutic agents for high blood pressure control. Several antihypertensive peptides have been produced by enzymatic hydrolysis of casein and whey proteins (Jäkälä and Vapaatalo 2010), showing a lower in vitro activity and had much lower or no side effects compared with synthetic ACE-I inhibitors. In that these peptides have limited if any side effects (Hong et al. 2008), they can thus be used as food ingredients or as nutraceuticals for treatment and prevention of hypertension and related diseases.

ACE-I inhibitory peptides were previously obtained in vitro using peptidases, including gastrointestinal digestive enzymes (Mullally et al. 1997), microbial enzymes (Hernández-Ledesma et al. 2006) and by fermentation processes (Fitzgerald and Murray 2006). Plant peptidases have also been used, e.g. cardosin peptidases extracted from Cynara cardunculus flowers. These peptidases were used to hydrolyze bovine whey protein concentrates to produce new and very effective ACE-I inhibitory peptides (Tavares and Malcata 2013). Other examples are peptidases from Arctium minus flowers (Cimino et al. 2015) and Maclura pomifera fruit (Bertucci et al. 2015).

The aim of this study was autochthonous plant peptidases with focus on ripe Bromelia antiacantha Bertol. fruit also known as “wild banana”. This native fruit is a source of cysteine peptidases (Vallés et al. 2007; Vallés and Cantera 2018) that can remove soft tissue (Macció et al. 2013). Herein we report the release of ACE-I inhibitory peptides by hydrolysis of ALA, BLG and acid whey using a partially purified proteolytic extract from ripe B. antiacantha fruit (antiacanthain). The sustainable potential for using this proteolytic extract in the dairy industry is discussed.

Materials and methods

Materials

Bovine ALA, BLG, and BSA as well as ACE-I from rabbit lung, papain from papaya latex, o-phthalaldehyde (OPA), azocasein, l-leucine, tricine, SDS-PAGE Ultra-Low Range Molecular Weight Marker, hippuryl–histidyl–leucine (Hip–His–Leu) and iodoacetamide (IAA) were purchased from SIGMA. Trichloroacetic acid (TCA) was purchased from Rhone Poulenc. Latex particles of 0.3 µm diameter were purchased from Prolabo Ltd. Specific anti-ALA and anti-BLG rabbit IgG were prepared as described previously (Villadóniga et al. 2007).

Acid whey preparation

Acid whey was prepared by isoelectric precipitation of casein from bovine milk. Fresh pasteurized skim milk (Conaprole, Uruguay) was acidified to pH 4.5 with 6 M HCl. Then, it was incubated at 20 °C for 30 min and then filtered through cheesecloth and centrifuged at 4000g for 30 min at 4 °C to separate acid whey from precipitated casein. Acid whey was stored at − 20 °C until use.

Preparation of proteolytic extract

Proteolytic crude extract was prepared as described by Macció et al. (2013). Briefly, pulp from ripe B. antiacantha Bertol. fruits was homogenized in a domestic blender and centrifuged (30 min at 16,000g and 4 °C). The supernatant was precipitated with four volumes of cold acetone according to Vallés and Cantera (2018) and the pellet was dried and stored at − 20 °C. When used, the pellet was dissolved in buffer (0.1 M sodium acetate, pH 4.5; 0.1 M sodium phosphate, pH 7.5 or 0.1 M sodium borate, pH 9.2) and activated with 15 mM cysteine (final concentration) for 15 min at 4 °C. This partially purified extract was termed antiacanthain.

Determination of proteolytic activity

Proteolytic activity was determined using azocasein as substrate according to a modification of the method Andrews and Asenjo described by Fullana et al. (2017). Briefly, reaction mixtures containing 0.34 mL antiacanthain (1/200 dilution in 0.2 M phosphate buffer, pH 7.5), 0.34 mL of 1% (w/v) azocasein solution and 0.34 mL 0.2 M phosphate buffer, pH 7.5, was incubated for 10 min at 37 °C. The reaction was stopped by addition of 0.34 mL 10% (w/v) TCA, then centrifuged (20 min at 20,600g and room temperature) and supernatant analyzed at 337 nm with a UV-1800 spectrophotometer (Shimadzu, Japan). One enzymatic unit (U) was defined as the amount of enzyme, which generates an increment of one absorbance unit at 337 nm per min. Protein concentration was determined by Bradford (1976) using BSA as standard.

ALA and BLG proteolysis kinetics

Hydrolysis kinetics of ALA and BLG by antiacanthain or papain (as control) were done at 50 °C and different pHs (pH 4.5, 7.5 and 9.2 using buffers: 0.1 M sodium acetate, 0.1 M sodium phosphate, and 0.1 M sodium borate, respectively). A total of 450 μL of substrate prepared at different concentrations (0.5–12 mg/mL) was mixed with 50 µL (2 U) of antiacanthain or papain. Aliquots (50 μL) were taken every 30 s (for 3 min) and reactions were stopped with addition of 100 μL 10% (w/v) TCA and then centrifuged at 6000g (10 min at 4 °C). The reaction was followed by estimation of primary amino groups released by action of antiacanthain on substrate with time. The supernatant (20 µL) was added to 1 mL of OPA reagent (25 mL 100 mM sodium tetraborate, 2.5 mL 20% (w/v) SDS, 1 mL 4.0% (w/v) OPA dissolved in methanol, 100 µL β-mercaptoethanol and distilled water up to 50 mL). After mixing and incubation at room temperature for 2 min, absorbance at 340 nm was measured as described by Church et al. (1983). Primary amino group concentration was estimated from a standard curve of absorbance at 340 nm versus l-leucine concentration (mM). The initial velocities, defined as the increase in product concentration (amino group released) with reaction time (M/s), were plotted versus substrate concentrations and kinetics parameters were determined with Hanes–Woolf linear plots using OriginPro 2016 (OriginLab Corporation, Northampton, MA, USA).

Enzymatic hydrolysis of whey protein

Hydrolysis of acid whey was done with antiacanthain and reaction conditions were analyzed and optimized to achieve extensive degradation of ALA and BLG. Enzyme–substrate ratio (E/S) ranging from 0.2 to 1.0 U/mg (at pH 9.2 and 30 °C) were used to analyse the effect of amount of antiacanthain on hydrolysis. The effect of temperature on hydrolysis was also analyzed in the range from 30 to 50 °C (at pH 9.2 and E/S = 1.0). All experiments were done for 120 min and under agitation at 200 rpm. Aliquots of hydrolyzates were collected at variable time intervals for analysis. The progress of hydrolysis was evaluated by measurement of degree of hydrolysis (DH), defined as the percentage of cleaved peptide bonds using the formula: \(DH(\% ) = 100 \times n/n_{tot}\) where n is hydrolysis equivalent formed during the enzymatic action that was estimated from increase in primary amino groups using OPA, as described in “ALA and BLG proteolysis kinetics”. The total number peptide bonds present is \(n_{tot}\) and was determined for a given sample by acid hydrolysis at 110 °C, for 20 h, in 6 N HCl, in sealed tubes. Acid hydrolyzate was centrifuged at 8000g for 10 min at room temperature and supernatants were used for primary amino group estimation as described in “ALA and BLG proteolysis kinetics”. In parallel, 90 µL hydrolyzate aliquots were mixed with 10 µL 100 µM IAA and incubated at 40 °C for 15 min. The samples were stored at − 20 °C until electrophoretic analysis.

Acid whey, ALA (1.3 mg/mL) and BLG (3.3 mg/mL) were hydrolyzed at pH 9.2 under selected E/S and temperature conditions. Aliquots were taken at different times, reactions were stopped by heating (15 min at 95 °C), and were centrifuged at 16,000g and 4 °C for 10 min. Supernatants were ultrafiltered using a 3 kDa cut-off membrane (Amicon Ultra-4 3K; Millipore, USA), and both 3 kDa retentate and 3 kDa permeate were stored at − 20 °C until analysis for ACE-I inhibitory activity. Hydrolyzates peptide concentrations were estimated by OPA assay as described in “ALA and BLG proteolysis kinetics”.

Electrophoretic analysis—tricine–SDS-PAGE

Tricine SDS-PAGE was performed according to Schägger (2006) in a Mini-Protean Tetra Cell (BioRad Laboratories Inc, Hercules, CA, USA) apparatus. Samples were prepared as follows: 100 µL of sample was mixed with 33 µL of reducing sample buffer (4×) and the mixture was heated for 30 min at 37 °C. Samples (5–7 µL) were loaded onto a gel system including a separating gel (T = 16.5%, C = 3%, 6 M Urea) overlaid with a spacer gel (T = 10% and C = 3%) and stacking gel (T = 4% and C = 3%). The power supply was initially set at 30 V for samples to enter the stacking gel and then increased to 90 V (at constant voltage) for running the separating gel. After electrophoresis, gels were stained with Coomassie Brilliant Blue G-250. Once desired staining was achieved, gels were scanned and images were analyzed using ImageJ software for Windows (Schneider et al. 2012). The MW of peptides in hydrolyzates was calculated with reference to the migration of SDS-PAGE Ultra-Low Range Molecular Weight Markers.

ACE-I inhibition assay

ACE-I inhibitory activity of fractions (3 kDa retentate and 3 kDa permeate) were measured using the assay of Cushman and Cheung (1971) with some modification. Briefly, 20 µL of each fraction was mixed with 0.1 mL of 5 mM Hip–His–Leu (prepared in 0.1 M potassium phosphate buffer, pH 8.3 containing 0.3 M NaCl) and 5 mU of ACE-I, incubated at 37 °C for 30 min and the reaction was stopped with the addition of 0.1 mL 1 M HCl. Hippuric acid formed was extracted with 1.0 mL ethyl acetate by vortex mixing for 60 s. After centrifuging for 10 min at 3000g, a 750 µL aliquot of the ethyl acetate phase was transferred to a clean tube and ethyl acetate was heat evaporated at 95 °C. Hippuric acid present was dissolved in 1.0 mL distilled water and absorbance at 228 nm was measured. The percentage of ACE-I inhibition was calculated using the following equation: \(ACE - I\,inhibition\,(\% ) = 100 \times \frac{{(A - B) - (C - D)}}{{(A - B)}}.\)

Where A is the absorbance of a solution containing ACE-I but without sample; B is the solution with ACE-I previously inactivated by adding HCl without sample; C is the absorbance in the presence of ACE-I and sample and D is the absorbance with ACE-I previously inactivated with HCl and containing the sample.

Inhibition effectiveness coefficient (IEC), defined as the ratio of percentage of ACE-I inhibition to peptide concentration was calculated and IC50 values in hydrolyzates fractions with the highest IEC were determined. IC50 values (concentration of sample that causes 50% inhibition of ACE-I activity) were determined from the non-linear curve fit of ACE-I inhibitory activity (%) versus logarithmic peptide concentration using OriginPro 2016.

Latex agglutination test

Affinity purified polyclonal anti-ALA and anti-BLG IgG produced in rabbits were adsorbed onto latex spheres as described previously by Villadóniga et al. (2007). The assay was performed on 12 black circle test cards, using serial twofold dilutions of acid whey hydrolyzates fractions (3 kDa retentate and 3 kDa permeate) prepared in 0.1 M glycine buffer (pH 8.2, supplemented with 0.15 M NaCl). 25 µL samples were mixed with 18 µL latex reagent, and gently mixed (100 rpm for 3 min at room temperature) and agglutination ability was evaluated by eye. Negative controls using only buffer as sample were also done. Sensitivities of latex reagents were established using 100 µg/mL antigen solutions (ALA or BLG). Twofold dilutions of antigens were assayed until no agglutination was detected. The highest reciprocal dilution of antigen solution detected as positive was defined as the titer for an antibody.

Statistical analysis

All data were analyzed using one-way analysis of variance (ANOVA) complemented with Tukey’s test. Differences were reported as statistically significant when p < 0.05. The statistical program used was from OriginPro 2016.

Results and discussion

ALA and BLG proteolysis kinetics

ALA and BLG undergo pH-dependent structural changes (O’Mahony and Fox 2013) that could affect enzyme access to catalytic sites. As such, we analyzed the effect of pH on the hydrolysis kinetics of both proteins by antiacanthain, and papain, a model enzyme. Results presented in Figs. 1 and 2 show that both enzymes exhibit Michaelis–Menten behavior, and apparent kinetic parameters are shown in Table 1.
Fig. 1

Kinetics of ALA hydrolysis by antiacanthain (a) and papain (b) at 50 °C and different pHs

Fig. 2

Kinetics of BLG hydrolysis by antiacanthain (a) and papain (b) at 50 °C and different pHs

Table 1

Kinetic parameters for antiacanthain, papain and cardosins (A and B) for ALA and BLG hydrolysis at different pHs and temperatures

Peptidase

Substrate

T (°C)

pH

KMap (M)

KCatap (s−1)

kCat/KM (M−1 s−1)

References

Antiacanthain

ALA

50

4.5

7.2 × 10−5

9.5 × 10−1

2.1 × 104

 

ALA

50

7.5

2.1 × 10−4

2.0

9.8 × 103

 

ALA

50

9.2

1.8 × 10−4

2.2

1.3 × 104

This study

BLG

50

7.5

3.6 × 10−4

2.0

7.4 × 103

 

BLG

50

9.2

3.4 × 10−4

5.4

1.7 × 104

 

Papain

ALA

50

4.5

3.9 × 10−4

1.4

4.1 × 103

 

ALA

50

7.5

3.5 × 10−4

1.1

3.0 × 103

This study

ALA

50

9.2

2.7 × 10−4

1.1

4.6 × 103

 

BLG

50

7.5

3.1 × 10−4

8.8 × 10−1

3.1 × 103

 

BLG

50

9.2

2.8 × 10−4

1.1

4.2 × 103

 

Cardosin A

ALA

55

5.2

3.9 × 10−5

1.0 × 10−1

2.6 × 103

Barros and Malcata (2002)

ALA

55

6.0

8.0 × 10−6

6.0 × 10−3

7.5 × 102

BLG

55

5.2

3.1 × 10−4

2.0 × 10−3

6.0

BLG

55

6.0

7.3 × 10−4

0

0

Cardosin B

ALA

55

5.2

3.8 × 10−5

1.6 × 10−3

4.2 × 102

ALA

55

6.0

2.0 × 10−5

8.4 × 10−2

4.2 × 103

BLG

55

5.2

3.1 × 10−4

7.0 × 10−3

2.3 × 101

BLG

55

6.0

4.7 × 10−5

3.0 × 10−3

6.4 × 101

Analysis of results suggests that, unlike ALA hydrolysis by papain (Fig. 1b), hydrolysis by antiacanthain (Fig. 1a) showed pH-dependent kinetic behavior. Antiacanthain was more efficient at pH 4.5 and 9.2 (highest values for \(k_{cat} /K_{M;}\) Table 1). However, antiacanthain showed a higher affinity (lower \(K_{M} ap\) value) for ALA at pH 4.5 compared with pH 9.2. At pH 9.2, however, antiacanthain had the highest hydrolysis reaction rate (\(k_{cat} ap\)), likely due to higher reactivity of thiol groups under alkaline pH conditions. According to the established cysteine peptidase reaction mechanisms, alkaline pH conditions may favor nucleophilic attack by the thiolate ion of the catalytic cysteine on the carbonyl carbon of the peptide bond (Polgár 2013). Neither antiacanthain nor papain hydrolyzed BLG at pH 4.5 (data not shown), probably due to formation of octomers by BLG between pH 3.5 and 5.1 that prevent enzyme accessibility to catalytic sites (Cheison and Kulozik 2017). Both enzymes hydrolyzed BLG, but at a faster rate at pH 9.2 compared with pH 7.5 (Fig. 2a, b), and with similar affinity but with different catalytic constants (higher at pH 9.2 rather than at pH 7.5) as shown in Table 1. Papain showed a similar behavior, but the efficiency for ALA and BLG hydrolysis was lower than found for antiacanthain. Catalytic efficiency of antiacanthain for ALA and BLG hydrolysis was also higher than that of cardosins, the C. cardunculus peptidases noted above (Table 1).

The concentrations of ALA (9.0 × 10−5 M) and BLG (1.8 × 10−4 M) in acid whey (Dupont et al. 2013) were lower than antiacanthain \(K_{M} ap\) values (Table 1) independent of reaction pH. At this substrate concentration, reaction velocity depends on antiacanthain \(k_{cat} /K_{M}\). According to the determined values of \(k_{cat} /K_{M}\), hydrolysis of both ALA (1.3 × 104 M−1 s−1) and BLG (1.7 × 104 M−1 s−1) would be more efficient at pH 9.2. At pH 4.5 ALA hydrolysis would be more efficient (\(k_{cat} /K_{M}\) = 2.5 × 104 M−1 s−1) but BLG was resistant to hydrolysis. This condition could be evaluated for BLG purification from acid whey.

Acid whey protein hydrolysis

After optimal pH conditions for ALA and BLG hydrolysis by antiacanthain were determined, i.e., pH 9.2, hydrolysis of acid whey was analyzed at this pH and the effect of E/S and reaction temperature were evaluated. An enzymatic preparation of antiacanthain (dissolved in 0.1 M borate buffer pH 9.2), showing a specific activity of 102 ± 18 U/mg was used. The extent of hydrolysis was evaluated by the evolution of DH with reaction time (Fig. 3a, b). Hydrolysis curves showed a fast increase of DH at initial reaction times and a slower increase in DH at later times (Fig. 3a, b). This was consistent with the current model for enzymatic hydrolysis of whey proteins (Gonzalez-Tello et al. 1994).
Fig. 3

DH of acid whey hydrolyzates by antiacanthain at pH 9.2. a E/S (U/mg) effect on DH at 30 °C; b temperature effect on DH at E/S 1.0 (U/mg)

Hydrolyzates of acid whey (8.9 ± 0.3 mg/mL) were prepared at pH 9.2 and 30 °C with different amounts of antiacanthain. E/S ratios between 0.2 and 1.0 U/mg for digestion of acid whey protein showed an increase in DH with E/S (Fig. 3a). The effect of temperature on acid whey hydrolysis (at pH 9.2 and E/S = 1.0 U/mg) was evaluated at 30, 40 and 50 °C and result are shown in Fig. 3b. An increase in temperature led to a significant increase in reaction velocity and DH value up to 12.3 ± 0.3%, and reached a plateau after 30 min incubation at 50 °C (Fig. 3b). These results suggested that temperature, apart from effects on antiacanthain kinetics, could affect substrate structure resulting in higher DH values. Whey proteins are quite resistant to hydrolysis in their native state due to globular structure and low flexibility (O’Mahony and Fox 2013). Ca2+ dissociates from ALA favoring denaturation at pH 9.0 and 50 °C (Boye et al. 1997). This temperature is also close to the denaturation temperature of BLG at 57 °C (Haug et al. 2009). As such, BLG hydrolyzable bonds could be more available for enzymatic action thereby explaining observed increase in DH values.

Optimal acid whey hydrolysis was at pH 9.2, E/S = 1.0 (U/mg) and 50 °C. These conditions resulted in highest DH and therefore were used to generate peptides from antiacanthain. Structure–activity relationships reported for ACE-I inhibitory peptides is not clearly established but molecular size(s) are considered to be less than 1000 Da (Dziuba and Darewicz 2007). Extensive hydrolysis of the substrate was sought for production of inhibitory peptides. Primary specificity studies showed that antiacanthain, as in other plant cysteine peptidases, has a broad substrate specificity with preference for hydrophobic residues at P1 and P2 positions (Macció et al. 2013). Then, hydrolysis of acid whey with antiacanthain could result in the formation of many low molecular weight peptides with hydrophobic residues at C-ter position.

Degradation profiles of acid whey proteins and purified ALA and BLG, hydrolyzed under optimal conditions, were analyzed by Tricine SDS-PAGE (Fig. 4). Digested protein band profiles and densitograms of acid whey hydrolyzates show that at initial stages BLG hydrolysis was faster than that of ALA. However, after 30 min hydrolysis, almost all BLG and ALA were degraded. New bands corresponding to peptides with a mass lower than 3.0 kDa appeared (indicated by arrows on the densitogram, Fig. 4a). Profiles of purified ALA and BLG hydrolyzates (Fig. 4b, c, respectively) were similar to those obtained for acid whey suggesting that slower hydrolysis of ALA in acid whey is not related to competition with BLG. Purified ALA and BLG had similar hydrolysis curves (DH versus time plots) as that of acid whey (data not shown). DH values of 17.3 ± 1.9% and 30.6 ± 3.2% for ALA and BLG were obtained after 30 min of reaction and were maintained constant until final reaction time (120 min). These results confirmed that BLG was hydrolyzed to a greater degree than ALA.
Fig. 4

Tricine SDS-PAGE and desitograms of acid whey (a); ALA (b) and BLG (c) hydrolyzates obtained with antiacanthain (E/S = 1.0 U/mg) at pH 9.2, 50 °C and different reaction times. M molecular weight markers ultra-low range: 26.6 kDa (1), 17.0 kDa (2), 14.2 kDa (3), 6.5 kDa (4), 3.5 kDa (5) and 1.1 kDa (6)

Other fruit cysteine endopeptidases from plants of the Bromeliaceae family have been used for hydrolysis of acid whey protein. Preferential degradation of BLG compared with that of ALA at alkaline pH was also found in acid whey hydrolyzates prepared from papain (Lieske and Konrad 1996). In another study, ALA and BLG degradation with papain was achieved in 15 min at pH 7, but at a temperature of 70 °C (Peñas et al. 2006). Hydrolysis of acid whey with B. hieronymi Mez fruit peptidases was shown to be rapid (30 min) for ALA at pH 6.5 even at low temperature (30 or 40 °C). However, only slow and partial degradation of BLG occurred and also required 50 °C temperature (Bruno et al. 2010). The presence of 0.5 mM EDTA and reaction at acid pH could cause destabilization of ALA. This is likely due to loss of Ca2+, which normally stabilizes structure by chelation and explains increased susceptibility to hydrolysis.

ACE-I inhibitory activity of hydrolyzate fractions

ACE-I inhibitory activity in ultrafiltration fractions of hydrolyzates obtained at 30, 60 and 120 min were significantly increased compared with samples at initial times (hydrolysis reaction time 0 min) indicating that antiacanthain was able to release sequences with ACE-I inhibitory activity in acid whey proteins (Table 2). The 3 kDa permeates of 30 min hydrolyzates were the most active fractions (highest IEC) independent of the substrate (Table 2). This is consistent with findings that the most effective ACE-I inhibitors are mainly di or tripeptides (Hong et al. 2008). Generally, longer reaction times are necessary for other enzymes to produce whey hydrolyzates with percentages of ACE-I inhibition similar to those obtained for antiacanthain hydrolyzates. Abubakar et al. (1998) obtained cheese whey hydrolyzates with ACE-I inhibitory activities between 57 and 96% after hydrolysis 24 h using gastrointestinal peptidases, proteinase K and actinase F. Hydrolyzates of acid whey with ACE-I inhibitory activity of 91% and 77% (Silvestre et al. 2012) were also obtained after 5 h hydrolysis with pancreatin and papain, respectively. Results suggest a good potential for use of antiacanthain for preparation of hydrolyzates with bioactive properties of biotechnological interest.
Table 2

ACE-I inhibition, peptide concentration and IEC of ultrafiltration fractions (Amicon Ultra membranes 3 kDa cut-off) of acid whey (9.8 mg/mL), ALA (1.3 mg/mL) and BLG (3.3 mg/mL) hydrolyzates, respectively

Fraction

Time (min)

ACE-I inhibition (%)

Peptide concentrationA (mM)

IEC

AWHR

0

4.60 ± 0.04

1.300 ± 0.006

3.5

AWHR

30

93.00 ± 0.04

1.400 ± 0.017f

66.4

AWHR

60

87.00 ± 0.08

1.400 ± 0.005f

62.1

AWHR

120

84.00 ± 0.15

1.400 ± 0.001

60.0

AWHP

0

0.75 ± 0.75

0.160 ± 0.004

4.5

AWHP

30

89.00 ± 0.05a

0.530 ± 0.008

167.9

AWHP

60

92.00 ± 0.40a

0.700 ± 0.009g

131.4

AWHP

120

93.00 ± 0.35a

0.710 ± 0.004g

131.0

ALAHR

0

46.00 ± 2.50

1.100 ± 0.005

41.8

ALAHR

30

67.00 ± 0.10

0.610 ± 0.001

109.8

ALAHR

60

86.00 ± 0.50b

0.540 ± 0.001

159.3

ALAHR

120

82.00 ± 0.50b

0.480 ± 0.003

170.8

ALAHP

0

2.50 ± 0.50

0.072 ± 0.001h

34.7

ALAHP

30

100.00 ± 0.10c

0.071 ± 0.001h

1408.5

ALAHP

60

96.00 ± 2.50c

0.130 ± 0.002

738.5

ALAHP

120

100.00 ± 6.00c

0.160 ± 0.001

625.0

BLGHR

0

0

1.400 ± 0.003i

0

BLGHR

30

82.00 ± 0.20

1.500 ± 0.003i

54.7

BLGHR

60

80.00 ± 0.05d

1.200 ± 0.023

66.7

BLGHR

120

100.00 ± 0.50d

1.400 ± 0.001i

71.4

BLGHP

0

0

0.530 ± 0.001

0

BLGHP

30

94.00 ± 0.05

0.095 ± 0.002

989.5

BLGHP

60

100.00 ± 0.06e

0.300 ± 0.001

333.3

BLGHP

120

100.00 ± 0.05e

0.450 ± 0.001

222.2

Values with the same superscript do not have significant differences

AWHR acid whey hydrolyzate retentate, AWHP acid whey hydrolyzate permeate, ALAHR ALA hydrolyzate retentate, ALAHP ALA hydrolyzate permeate, BLGHR BLG hydrolyzate retentate, BLGHP BLG hydrolyzate permeate

APeptide concentration in ACE-I assay

Standardized 30 min hydrolyzates were prepared, and 3 kDa permeates obtained were freeze-dried and dissolved at different concentrations to determine IC50 values for ACE-I inhibitory activity. The highest ACE-I inhibitory activity in 3 kDa permeates was derived from ALA (IC50 = 1.7 ± 0.4 µg/mL), followed by BLG (IC50 = 11.7 ± 1.8 µg/mL) and acid whey (IC50 = 35.8 ± 1.5 µg/mL). IC50 values of BLG and acid whey hydrolyzates were of the same order as those reported by Tavares et al. (2011) for whey protein hydrolyzate (IC50 = 24 µg/mL) prepared for 7 h using a crude extract of C. cardunculus flowers. The antiacanthain hydrolyzate of the purified ALA prepared in 30 min was superior to that obtained from thermolysin hydrolyzate of purified ALA prepared in 3 h for which IC50 value of 45 µg/mL was determined (Otte et al. 2007).

Immunoreactivity of acid whey hydrolyzates

Epitope destruction by hydrolysis was determined as a reduction of antigen level (immunoreactivity) that could be monitored by latex bead agglutination tests. Remaining immunoreactivity was used to estimate residual reactivity of allergenic proteins to hydrolysis.

Latex agglutination reagents were prepared in order to monitor ALA and BLG levels of 3 kDa permeates and retentates of acid whey hydrolyzates. ALA reagent showed a detection limit (sensitivity) of 98 ng/mL and BLG reagent, 390 ng/mL. The 3 kDa permeates from unhydrolyzed acid whey and hydrolyzates prepared at 0, 30, 60 and 120 min showed lower ALA and BLG levels than the detection limit of latex bead reagents. Acid whey hydrolyzate 3 kDa retentates showed remaining ALA and BLG levels that decreased with hydrolysis time (Table 3). Production of safe products suitable for use by persons prone to allergic responses will require that results be confirmed by a quantitative immunoassay e.g. ELISA plate assay.
Table 3

ALA and BLG remaining immunoreactivity (RI) of 3 kDa retentates of acid whey hydrolyzates, obtained by antiacanthain (E/S = 1.0) at pH 9.2 and 50 °C, determined by latex agglutination test

Sample

Anti-ALA

Anti-BLG

Immunoreactivity (mg/mL)

RI (%)

Immunoreactivity (mg/mL)

RI (%)

Unhydrolyzed acid whey

3.2

12.3

AWH 0 min

4.0 × 10−1

12.5

1.5

12.2

AWH 30 min

5.0 × 10−2

1.6

8.0 × 10−1

6.5

AWH 60 min

2.5 × 10−2

0.8

8.0 × 10−1

6.5

AWH 120 min

1.2 × 10−2

0.4

4.0 × 10−1

3.3

AWH acid whey hydrolyzate

Conclusions

Antiacanthain was able to hydrolyze ALA and BLG and kinetic parameters were evaluated and compared with those for papain. Antiacanthain was shown to have good catalytic efficiency for hydrolysis of ALA and BLG at pH 9.2, and had higher catalytic activity than papain. Hydrolysis treatment of acid whey proteins at 50 °C and pH 9.2 for 30 min combined with ultrafiltration generated a product with good ACE-I inhibitory activity. Preliminary evaluation of ALA and BLG immunoreactivity indicated that hydrolysis treatment of acid whey resulted in a reduction in allergenic epitopes but results should be confirmed by ELISA plate assays.

These products could be interesting for the valorization of acid whey and plant peptidases and as an improved alternative treatment for dairy industry effluent that normally leads to environmental damage. Further studies are required to identify and characterize the peptides responsible for ACE-I inhibitory activity. In order to better understand the extent of potential application and importance of these peptides as ingredients in functional antihypertensive foods, it is essential to evaluate both gastrointestinal degradation resistance and to characterize in vivo inhibitory activity of ACE-I in animal models.

Notes

Acknowledgements

This work was supported by a Grant from DINACYT (PDT 74/10), CSIC (UdelaR) (Grant no. C278-348). C. Villadóniga (partially) and L. Macció were supported by PDT 74/10. C. Villadóniga received a doctoral fellowship from ANII (Grant no. POS_NAC_2010_1_2_2438).

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Copyright information

© Society for Environmental Sustainability 2018

Authors and Affiliations

  1. 1.Laboratorio de Enzimas HidrolíticasFacultad de Ciencias, Universidad de la RepúblicaMontevideoUruguay
  2. 2.Cátedra de Bioquímica, Facultad de Química, Universidad de la RepúblicaMontevideoUruguay

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