Innovative Food Science and Emerging Technologies 65 (2020) 102449

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Innovative Food Science and Emerging Technologies

journal homepage: www.elsevier.com/locate/ifset

Effect of enzymatic hydrolysis on molecular weight distribution, techno- T

functional properties and sensory perception of pea protein isolates
Verónica García Arteagaa,b, Marijose Apéstegui Guardiab, Isabel Muranyib, Peter Eisnerb,c,
Ute Schweiggert-Weiszb,
⁎

a
Center of Life and Food Sciences Weihenstephan, Technical University of Munich, Germany
b
Fraunhofer Institute for Process Engineering and Packaging IVV, Germany
c
ZIEL - Institute for Food & Health, Technical University of Munich, Germany

ARTICLE INFO ABSTRACT

Keywords: Pea protein isolate (Pisum sativum “Navarro”) was hydrolyzed with 11 proteolytic enzymes at different hydro-
Pea lysis times (15, 30, 60, and 120 min) to improve techno-functional and sensory properties. The degree of hy-
Protein isolate drolysis and changes within the molecular weight distribution were used as indicators for a reduced allergenic
Enzymatic hydrolysis potential. The highest degree of hydrolysis was reached by Esperase hydrolysates (9.77%) after 120 min of
Protein degradation
hydrolysis, whereas Chymotrypsin hydrolysates showed the lowest (1.81%). Hydrolysis with Papain, Trypsin,
Techno-functional properties
Bromelain, Esperase, Savinase, and Alcalase suggested an effective degradation of the 72 kDa-convicilin fraction.
Sensory analysis
Papain and Trypsin hydrolysates showed a degradation of the 50 kDa-mature vicilin after 15 min of hydrolysis.
Most hydrolysates showed a significant increase in protein solubility at pH 4.5 at all times of hydrolysis. Trypsin
hydrolysates showed the highest foaming (2271%) and emulsifying (719 mL/g) capacities. The bitterness of the
hydrolysates was strongly correlated (P < 0.05) with the degree of hydrolysis. In general, enzymatic hydrolysis
improved techno-functional properties indicating their potential usage as food ingredients.
Industrial relevance: Due to their high protein content, peas are becoming an attractive ingredient for the food
industry. However, pea protein isolates are often characterized by poor techno-functional and sensory proper-
ties. Enzymatic hydrolysis is known to change the molecular weight distribution of proteins. Consequently, the
techno-functional and immunogenic properties might be altered selectively. In this study, enzymatic hydrolysis
was applied, resulting in highly functional pea protein hydrolysates with a hypothesized reduction of main
allergens. The lower bitter perception highlights their high potential as valuable functional food ingredients.

1. Introduction are characterized by deficient techno-functional properties, in parti-

cular, their low foaming and emulsifying capacities, and by unpleasant
The use of protein-rich raw materials for food applications has be- sensory properties. Several approaches are described in the literature
come increasingly important in recent years. Within the legume family, for the alteration of protein structures in order to improve the techno-
peas (Pisum sativum L.) are an auspicious raw material due to the high functional as well as the sensory properties (Adler-Nissen and Olsen,
amounts of proteins as well as to their absence in the allergen list of 1979; Angioloni and Collar, 2013; Buchert et al., 2010; Raksakulthai
Official Journal of the European Union (O.J.E.U., 2011). The pea protein and Haard, 2003). Among them, enzymatic hydrolysis has shown to be
content ranges between 20 and 30% (Koyoro and Powers, 1987), and one of the most promising methods for the modification of tailor-made
the proteins are mainly composed of salt-soluble globulins (55–80% of protein preparations (Lqari et al., 2005; Meinlschmidt et al., 2016;
the total protein) and water-soluble albumins (18–25% of the total Polanco-Lugo et al., 2014; Schlegel et al., 2019). Proteolytic active
protein). The ratio of these storage proteins depends on genetic and enzymes cleave peptide bonds, resulting in a mixture of peptides of
environmental characteristics such as maturation, fertilizers, soil nu- different sizes and free amino acids (Wouters et al., 2016). Proteases are
trients and cultivation temperature (Barac et al., 2015.; Gueguen and classified as endopeptidases or exopeptidases depending on their me-
Barbot, 1988; Nikolopoulou et al., 2007). chanism of action and catalytic site. The efficiency of the enzymatic
Depending on the production conditions, pea protein isolates (PPI) hydrolysis mainly depends on the enzymes applied and hydrolysis

⁎
Corresponding author.
E-mail address: [email protected] (U. Schweiggert-Weisz).

https://doi.org/10.1016/j.ifset.2020.102449
Received 14 February 2020; Received in revised form 1 July 2020; Accepted 6 July 2020
Available online 10 July 2020
1466-8564/ © 2020 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/BY-NC-ND/4.0/).
V. García Arteaga, et al. Innovative Food Science and Emerging Technologies 65 (2020) 102449

conditions (time, temperature, pH) used, where the resulting cleavage enzymatic hydrolysis on the techno-functional properties such as pro-
products have a decisive influence on the hydrophobicity of the pep- tein solubility, emulsifying capacity, foaming capacity, and foam sta-
tides and thereby the techno-functional properties (Singhal, Karaca, bility as well as the sensory profile of PPI and its hydrolysates. An in-
Tyler, & Nickerson, 2016). Barac et al. (2011, 2012) studied the influ- dication of the degradation of the main pea allergens Pis s 1 and Pis s 2
ence of enzymatic hydrolysis (chymosin, Papain, and Streptomyces gri- was reached by determination of molecular weight distribution.
seus protease) on pea protein isolates. All hydrolysates showed an im-
provement in protein solubility (pH 5) and better emulsifying and 2. Materials and methods
foaming capacities. However, they focused only on the functional
properties for food application, and no attention was given regarding 2.1. Materials
the sensory perception of the hydrolysates.
Enzymatic hydrolysis also affects the peculiar sensory properties of Peas (Pisum sativum L. cultivar “Navarro”) were provided by
plant proteins such as the green, bitter, or astringent attributes (Adler- Norddeutsche Pflanzenzucht Hans-Georg-Lembke KG (Germany).
Nissen, 1986a; Saha and Hayashi, 2001). The extent of changes in Enzymes Alcalase® 2.4 L FG 1, Flavourzyme®, Neutrase®, Protamex®,
sensory properties is attributed to the degree of hydrolysis and, in and Savinase® 16 L were from Novozymes (Denmark); Trypsin,
particular, to the release of low molecular weight peptides constituted Bromelain, and Esperase® 8.0 L were obtained from Sigma-Aldrich
of hydrophobic amino acids. This release depends on the enzyme and (Germany); Chymotrypsin, Corolase® 7089 and Papain were from
the substrate used (Raksakulthai and Haard, 2003; Saha and Hayashi, Merck KGaA (Germany), AB Enzymes (Germany) and Carl Roth GmbH
2001). Humiski and Aluko (2007) demonstrated that Papain and α- (Germany), respectively. Broad Range™ Unstained Standard, 4–20%
Chymotrypsin hydrolysates from pea proteins were less bitter, while Criterion™ TGX Stain-Free™ Precast Gels and Coomassie Brilliant Blue
those hydrolyzed with Flavourzyme and Alcalase preparations resulted R-250 were purchased from Bio-Rad Laboratories GmbH (Germany).
in an increased bitterness. On the other hand, hydrolysis of soy protein Sodium dihydrogen phosphate, sodium dodecyl sulfate, sodium tetra-
isolates with Flavourzyme showed similar bitterness to the untreated borate decahydrate, o-phthaldialdehyde, and sodium monohydrogen
isolate (Meinlschmidt et al., 2016). phosphate were from Sigma-Aldrich (Germany). All chemicals used in
Although pea proteins are not included in the list of main allergens, this study were of analytical grade.
there is some evidence in the literature that also pea proteins, in par-
ticular, Pis s 1 (vicilin) and Pis s 2 (convicilin), exhibit an allergenic 2.2. Production of pea protein isolate
potential (Codreanu-Morel et al., 2019; Dreyer et al., 2014; Sanchez-
Monge et al., 2004). Sanchez-Monge et al. (2004) identified three major Peas were dehulled and split using an underflow peeler (Streckel &
pea allergens by immunodetection, immunoblot inhibition assays and Schrader KG, Germany) and separated using an airlift system (Alpine
cDNAs encoding of pea vicilin. These fractions are a 63 kDa convicilin Hosakawa AG, Germany). Subsequently, the split pea seeds were milled
(Pis s 2), a 47 kDa mature vicilin (Pis s 1), and its 32 kDa proteolytic using a pilot-plant impact mill (Alpine Hosakawa AG, Germany) with
fragment, which are recognized by the International Union of Im- 0.5-mm-sieve insertion. The isolation of pea protein was performed
munological Societies (IUIS) Allergen Nomenclature Sub-Committee. according to Tian, Kyle, & Small, (1999) with few changes. An aqueous
The results for pea albumin (2S) potential allergens such as PA1 alkaline extract of the pea flour was prepared in deionized water at a
(6.5 kDa) and PA2 (26 kDa) are ambiguous, and the proteins are yet not ratio of 1:8 (w/v) at pH 8.0 ± 0.1 using 3.0 mol/L NaOH under
recognized as allergens (Malley et al., 1976; Mierzeiewska, Mitrowska, constant stirring for 60 min. The protein extract was removed by means
Rudnicka, Kubicka, & Kostyra, 2008). Among the approaches to reduce of a decanter (3,300 rpm). For isoelectric precipitation, the protein
this allergenic potential, enzymatic hydrolysis has been investigated in extract was adjusted to pH 4.5 using 3.0 mol/L HCl. After 60 min, the
detail for different legume proteins such as peanut, soy, and lupin precipitated proteins were separated from the clear supernatant in an
(Kasera et al., 2015; Meinlschmidt et al., 2016; Schlegel et al., 2019). SC 20-disc separator (GEA Westfalia Separator Group GmbH, Germany)
but few data are available for pea proteins (Frączek et al., 2008; at 12,000 rpm. The isolate was neutralized with 3.0 mol/L NaOH,
Szymkiewicz and Jędrychowski, 2008). pasteurized (70 ± 2 °C) for 2 min and spray-dried.
As mentioned above, some studies have focused on the effect of
enzymatic hydrolysis on techno-functional and sensory properties of 2.3. Enzymatic hydrolysis of PPI
pea protein isolates and, to a lesser extent, on the mitigation of pea
allergenicity. However, only the simultaneous study of all effects of For enzymatic hydrolysis, a 9% (w/w) PPI dispersion was prepared
proteolysis will enable the production of highly functional and ap- in deionized water in a thermostatically controlled reactor with tem-
pealing food ingredients where changes in the molecular weight dis- perature and pH adjusted to the optimum conditions of each enzyme
tribution might have an effect on the reduction of allergens. (Table 1) according to product data sheet. The enzyme to substrate ratio
Therefore, the present study aimed to investigate the influence of (E/S) was chosen according to literature. After enzyme addition, the

Table 1
Enzymes preparations used for the hydrolysis of pea protein isolate and the respective hydrolysis conditions applied.
Enzyme E/S (%) T (°C) pH value (−) Activity Origin

Alcalase® 2.4 L FG 0.5 65 8 Serine Endoprotease Bacillus licheniformis

Bromelain 0.1 50 7 Cysteine Endoprotease Pineapple stem
Chymotrypsin 0.1 50 8 Serine Endoprotease Bovine pancreas
Corolase® 7089 0.5 50 7 Endoprotease Bacillus subtilis
Esperase® 8.0 L 0.5 65 8 Serine Endoprotease Bacillus sp.
Flavourzyme® 0.5 50 7 Endo- and exo-protease Aspergillus oryzae
Neutrase® 0.8 L 0.5 50 7 Metallo Endoprotease Bacillus amyloliquefaciens
Papain 0.1 65 7 Cysteine Endoprotease Papaya latex
Protamex® 0.5 65 7 Endoprotease Bacillus licheniformis and amyloliquefaciens
Savinase® 16 L 0.5 50 8 Serine Endoprotease Bacillus
Trypsin 0.1 50 8 Serine Endoprotease Bovine pancreas

E/S: enzyme to substrate ratio, T: temperature.

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suspension was continuously stirred and the temperature and pH were for 15 min at 15 °C (3 K30 Sigma Laborzentrifugen GmbH, Germany).
maintained constant. Aliquots of approximately 900 mL were trans- The supernatant was then filtrated in Whatman No.1 filter paper and
ferred to smaller reactor vessels after 15, 30, 60 and 120 min for en- frozen until analysis (−20 °C). The protein content was determined
zyme inactivation at 90 °C for 10 min. The hydrolysates were cooled to using the Dumas combustion method (AOACb, 2003). The protein so-
room temperature and neutralized to pH 7.0 ± 0.1. Aliquots of 5 mL lubility was analyzed for all four times of hydrolysis of each sample.
were stored at −20 °C for a minimum of 24 h until electrophoretic
analysis. The rest of the samples was lyophilized and ground for 10 s at 2.6.2. Foaming properties
7,500 rpm (Grindomix GM200, Retsch GmbH, Germany). The control The foaming capacity and foam stability were analyzed according to
samples were treated with the same conditions but without the addition Phillips et al. (1987) using a whipping machine (Hobart N50, Hobart
of the enzymes. The hydrolysis and controls were performed in dupli- GmbH, Germany). A 5% sample solution (w/w) was gently stirred for
cate. 15 min before whipping (580 rpm) for 8 min until the formation of
stable foam. Based on the relation of the foam volume before and after
2.4. Chemical composition whipping the foaming capacity was calculated. The foam stability was
assessed as the percent loss of foam volume after 60 min. The foaming
The dry matter and ash content of the samples were determined by properties of each sample were analyzed after 15 min and 120 min of
means of a thermogravimetric method (TGA 701, Leco Instruments, hydrolysis.
Germany). The protein content was determined according to the Dumas
combustion method (TruMac N, Leco Instruments, Germany) using the 2.6.3. Emulsifying capacity
average nitrogen-to-protein conversion factor of N x 6.25. All analyses The emulsifying capacity was determined according to Wang and
were performed in duplicate and according to AOAC Official Methods Johnson (2001). A 1% sample solution (w/w) and 125 mL of Mazola
(AOACa, 2003; AOACb, 2003). corn oil were placed in a reactor system (IKA®-Werke GmbH & Co. KG,
Germany). After 1 min homogenization at 11,000 rpm using an Ultra-
2.5. Determination of protein degradation Turrax (IKA-Werke GmbH & Co. KG, Germany), 10 mL/min oil were
added using a dispenser (IKA®- Werke GmbH & Co. KG, Germany),
2.5.1. Molecular weight distribution while measuring constantly the emulsion conductivity using a con-
The molecular weight distribution was analyzed by sodium dodecyl ductivity meter (LF 521 with electrode KLE 1/T, Wissenschaftliche-
sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under reducing Technische Werkstätten GmbH, Germany). The measurement was
conditions according to Laemmli (1970) with slight modifications. stopped as a phase inversion was reached (< 10 μS/cm) and the volume
Briefly, depending on protein content and dry matter, aliquots between of added oil was used to calculate the emulsifying capacity (mL oil/g
5.8 and 7.6 μL of the liquid hydrolyzed samples were suspended in 60% sample). The emulsifying capacity was analyzed in the samples hy-
(v/v) 2× Tris-HCl treatment buffer, 30% (v/v) phosphate buffer (pH 7) drolyzed for 15 min and 120 min, respectively.
and 10% (v/v) HPLC water to reach a protein concentration of 5 μg/μL.
The samples were heated at 95 °C for 5 min (300 rpm) prior to cen- 2.7. Sensory analysis
trifugation at 13,400 rpm for 3 min (Mini Spin Centrifuge, Eppendorf
AG, Germany). An aliquot of 3 μL was added into the gel pocket of the 2.7.1. Sample preparation
Bio-Rad 4–20% Criterion™ TGX Stain-Free™ Precast Gels. The Broad For sensory analysis, 2% solutions (w/w) of PPI and hydrolysates
Range™ Unstained Standard (Bio-Rad Laboratories, Germany) was used inactivated after 15 min and 120 min were prepared with tap water.
as a standard molecular weight marker. Gels were run for 30 min at The samples were adjusted to pH 7.0 with 1 mol/L NaOH and coded
200 V, 60 mA, and 100 W at room temperature. Staining of the gel was using three-digit random numbers. Water and plain crackers were
performed using 0.02% Coomassie Brilliant Blue R-250 solution. Fi- provided for palate cleansing in between.
nally, gel images were obtained with the Coomassie Blue Gel Doc™ EZ
Imager (Bio-Rad Laboratories, Germany). The SDS-PAGE was per- 2.7.2. Sample evaluation
formed in duplicate, with each sample being prepared two times in- The sensory evaluation was conducted according to DIN 10967-1-
dependently. 1999. For selection of the main attributes, a ten-member trained panel
evaluated attributes regarding retronasal aroma, taste and trigeminal
2.5.2. Degree of hydrolysis sensation of the PPI and its hydrolysates. Attributes selected by more
The degree of hydrolysis (DH) was analyzed according to Nielsen than five assessors were chosen for further sensory analysis such as pea-
et al. (2001). The DH was calculated based on the total number of like (3-s-butyl-2-methoxypyrazine), green (hexanal), earthy (geosmin),
peptide bonds per protein equivalent (htot), and the number of hydro- roasted (furaneol/acetylpyridine), cooked potato (3-(methylthio-) pro-
lyzed bonds (h) using the following equation: panal), salty, astringent, and bitter.
For sensory analysis, 20 mL of each sample were presented at room
DH = h/htot ·100%
temperature, in glass cups and random order. Six samples were pre-
The constant values used for α, β and htot factor were 1.0, 4.0, and sented per session. The panelists assessed the sample intensities of the
8.0, respectively, according to theoretical general values for un- attributes on a 0 (not noticeable) to 10 (strongly noticeable) ranging
examined raw material (Nielsen et al., 2001). The sample preparation scale. Furthermore, overall intensity (0 = not perceivable, 10 = very
was performed in duplicate with each preparation measured in tripli- strong perception) and hedonic scale (0 = dislike, 5 = neutral,
cate. 10 = like) were assessed. The results are presented as the mean values
among all panelists.
2.6. Techno-functional properties
2.8. Statistical analysis
2.6.1. Protein solubility
The protein solubility was performed according to Morr et al. All results, expressed as mean values ± standard deviation of at
(1985). A 3% (w/v) sample solution was prepared in 50 mL of 0.1 mol/ least two measurements (n = 2), were analyzed by one-way analysis of
L NaCl solution and adjusted to pH 4.5 and 7.0 using 0.1–1 mol/L variance (ANOVA). Additionally, a two-way ANOVA was used to ana-
NaOH or 0.1–1 mol/L HCl. After constant stirring for 1 h at room lyze the influence of pH and time of hydrolysis on the protein solubility.
temperature, the non-dissolved fraction was centrifuged at 13,650 rpm The mean values were compared using Tukey's post-hoc test. The

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V. García Arteaga, et al. Innovative Food Science and Emerging Technologies 65 (2020) 102449

Fig. 1. Molecular weight distribution of the untreated pea protein isolate (PPI) and PPI hydrolysates (Flavourzyme, Neutrase, Savinase, Chymotrypsin, and Alcalase)
obtained at different times of hydrolysis as determined by SDS-PAGE under reducing conditions. M = molecular weight standard, indicated in kilo Dalton (kDa).

relationship among DH, protein solubility, and bitterness was analyzed protein content of 83%, dry matter of 95%, and ash content of 6%.
using the Pearson correlation coefficient. All statistical analyses were Complete data can be found in Table A-1 in the Mendeley dataset
performed using OriginPro 2018b and were considered statistically (García Arteaga et al., 2020).
significant at P < 0.05.
3.1. Effects of enzymatic hydrolysis on protein degradation
3. Results and discussion
3.1.1. Molecular weight distribution
The reference PPI showed 83%of protein, 92% of dry matter, and The molecular weight distribution was analyzed in order to in-
5% ash content. The hydrolyzed PPI solutions showed an average vestigate the effect of enzymatic hydrolysis on the pea proteins and on

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V. García Arteaga, et al. Innovative Food Science and Emerging Technologies 65 (2020) 102449

Fig. 2. Molecular weight distribution of the untreated pea protein isolate (PPI) and PPI hydrolysates (Protamex, Papain, Esperase, Bromelain, Trypsin, and Corolase)
obtained at different times of hydrolysis as determined by SDS-PAGE under reducing conditions. M = molecular weight standard, indicated in kilo Dalton (kDa).

the main allergens. Protein fractions of the PPI ranged from 97 to 7 kDa 3.1.1.1. Pis s 2 degradation. Pis s 2 (~72 kDa) was degraded almost
(Figs. 1 and 2), which is in accordance to pea SDS-PAGE profiles completely after 15 min of hydrolysis with Savinase, Alcalase, Papain,
available in literature (Barac et al., 2012). Enzymatic hydrolysis Esperase, Bromelain, and Trypsin. Hydrolysis with Neutrase and
showed an influence on molecular weight distribution, especially re- Corolase showed no effect on this protein fraction; whereas
garding the high molecular weight fractions (Figs. 1 and 2). hydrolysis with Flavourzyme, Chymotrypsin, and Protamex showed a

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V. García Arteaga, et al. Innovative Food Science and Emerging Technologies 65 (2020) 102449

reduction of this fraction with longer times of hydrolysis. Furthermore, highest DH after 120 min (9.77%) followed by Alcalase (9.24%) and
Flavourzyme, Neutrase, Chymotrypsin, and Corolase hydrolysates Savinase (8.62%) hydrolysates after 120 min of hydrolysis. Trypsin
showed one neo-band at around 55 kDa, which has been previously hydrolysates showed lower DH with 7.59% after 120 min of proteolysis,
described by Le Gall et al. (2005) as a possible cleaved-peptide of while Chymotrypsin hydrolysates showed the lowest DH with 1.81%
convicilin. after 120 min. As mentioned in the previous section, different results
within the same protease family might be due to substrate specificity,
3.1.1.2. Pis s 1 degradation. Pis s 1 (~50 kDa) was completely however, the presence of a Pisum sativum Trypsin inhibitor (PSTI II)
hydrolyzed by Papain and Trypsin within the first 15 min of could have an influence on the hydrolysis with Trypsin and Chymo-
hydrolysis, whereas Esperase and Bromelain cleaved this fraction only trypsin (Pouvreau et al., 1998), thus reducing their proteolytic me-
after 60 and 120 min, respectively. Alcalase, Protamex, and Savinase chanism of action. Another explanation for the low DH of Chymotrypsin
reduced the Pis s 1-intensity by approximate 71%, 38%, and 20%, hydrolysates might be the low amounts of methionine and tryptophan
respectively, after 120 min of hydrolysis. The mature Pis s 1 vicilin is in pea proteins reducing the enzyme-substrate interactions (Hedstrom
composed by different polypeptides such as vicilin ⍺β (30–36 kDa) and et al., 1992).
vicilin-γ (12–16 kDa), and the breakdown of mature Pis s 1 could result Although Papain and Bromelain showed noticeable changes in the
in an increase of these fractions. The vicilin ⍺β has been also described molecular weight distribution, the DH values of 5.04% and 3.57% were
as one major pea allergen (Sanchez-Monge et al., 2004). Except for all unexpectedly low after 120 min. A reason could be an unstable and
Papain hydrolysates and Esperase 120-min hydrolysate, all other weak reaction of the OPA reagent with cysteine, as postulated by Chen
enzymes were unable to hydrolyze the vicilin ⍺β fraction, which et al. (1979). Hydrolysates from Protamex, Corolase, Flavourzyme, and
might indicate a preservation of the allergenic potential of this Neutrase showed a lower increase in the DH with 4.15%, 4.65%, 4.70%,
protein fraction. and 5.16% after 120 min of hydrolysis, respectively.
Different results within the same protease family might be due to
substrate specificity. From the cysteine endopeptidases, Papain shows a 3.2. Effects on techno-functional properties
preference for bulky hydrophobic residues, whereas Bromelain shows a
preference for polar amino acids in both P1 and P1′ position (Cstorer According to the molecular weight distribution and DH, the hy-
and Ménard, 1994; Rowan, 2013). The electrophoretic results from this drolysates with the most changes in the electrophoretic profile (Papain,
study suggest that the PPI probably had more of the hydrophobic re- Trypsin, Esperase, Bromelain, and Alcalase hydrolysates) and hydro-
sidues such as leucine or glycine, which enabled Papain to cleave lysates with the least changes (Chymotrypsin hydrolysates) are shown
peptide bonds within the protein efficiently. Similarly, hydrolysates in tables and figures of further sections. Complete data can be found in
from serine proteases showed different degradation patterns suggesting Tables B-1 and B-2 in the Mendeley dataset (García Arteaga et al.,
different substrate specificities. Furthermore, as pea protein composi- 2020).
tion depends on the botanical variety, time of harvest, and environ-
mental conditions, further studies of the PPI “Navarro,” such as amino 3.2.1. Protein solubility
acid profile and protein fractioning, are necessary to understand the Enzymatic hydrolysis promotes the interaction of hydrophilic
mechanism of action of these enzymes. A comparison of electrophoretic groups with water molecules by decreasing peptide size, hence in-
results in this study with those from literature is difficult as different creasing protein solubility (Wouters et al., 2016). Consequently, an
pea varieties and enzyme conditions have been used. increase in protein solubility could be attributed to changes in the
Comparable to the results of Le Gall et al. (2005), the PA2 albumin protein structures, the release of smaller peptides and hydrophilic
fraction (26 kDa) showed resistance to proteolysis in all hydrolysates amino acids as well as changes in the electrostatic forces (Lam et al.,
except for Papain hydrolysate. The complete and partial degradation of 2016).
Pis s 2 and Pis s 1, respectively, indicates that enzymatic hydrolysis Protein solubility was analyzed at pH 4.5 (general isoelectric point
might represent an effective method to destroy the main allergens of of pea proteins) and pH 7.0 as well as after the four different hydrolysis
pea proteins. times. The PPI showed a low protein solubility of 2% at pH 4.5, while
the protein solubility at pH 7.0 was 51%. Fig. 3 shows the protein so-
3.1.2. Degree of hydrolysis lubility of the different hydrolysates. Except for Chymotrypsin at
The DH was analyzed with the OPA reagent, which forms a complex 15 min and 30 min, all the hydrolysates improved protein solubility
with free primary α- and ε-amino groups, which is then photometrically significantly at pH 4.5. Esperase hydrolysates showed the highest pro-
detected. The DH increased significantly with longer times of hydrolysis tein solubility at pH 4.5 and pH 7.0, with 71% and 78%, respectively,
(Table 2). Among the serine proteases, Esperase hydrolysate showed the after 120 min. Trypsin, Savinase, and Alcalase hydrolysates followed

Table 2
Degree of hydrolysis of pea protein isolate (PPI, 0 min) and PPI hydrolysates after 15, 30, 60 and 120 min of hydrolysis.
Degree of hydrolysis (%)

0 min 15 min 30 min 60 min 120 min

a a,b b,c c
Flavourzyme 2.36 ± 0.16 2.96 ± 0.46 3.41 ± 0.21 3.88 ± 0.05 4.70 ± 0.24d
Neutrase 2.36 ± 0.16a 4.12 ± 0.15b 4.14 ± 0.34b 4.73 ± 0.15c,d 5.16 ± 0.14d
Savinase 2.36 ± 0.16a 5.40 ± 0.57b 6.45 ± 0.81b,c 7.44 ± 1.04c,d 8.62 ± 1.07d
Chymotrypsin 2.36 ± 0.16a 1.43 ± 0.70a 1.50 ± 0.80a 1.74 ± 0.75a 1.81 ± 0.70a
Alcalase 2.36 ± 0.16a 7.15 ± 0.59b 7.77 ± 0.69b 8.40 ± 0.99b,c 9.24 ± 0.28c
Protamex 2.36 ± 0.16a 2.79 ± 0.69a 2.92 ± 0.71a 3.21 ± 0.80a 4.15 ± 0.67a
Papain 2.36 ± 0.16a 4.41 ± 0.44b 4.67 ± 0.35b 4.81 ± 0.33b 5.04 ± 0.37b
Esperase 2.36 ± 0.16a 5.96 ± 0.19b 7.05 ± 0.76b,c 8.15 ± 0.75c 9.77 ± 0.51d
Bromelain 2.36 ± 0.16a 2.29 ± 1.28a 2.71 ± 1.05a 2.48 ± 0.43a 3.57 ± 0.87a
Trypsin 2.36 ± 0.16a 3.29 ± 0.48a 4.72 ± 0.80a,b 6.08 ± 0.88b,c 7.59 ± 1.67c
Corolase 2.36 ± 0.16a 3.38 ± 0.41a,b 3.94 ± 0.19b,c 4.26 ± 0.67b,c 4.65 ± 0.45c

Results are expressed as means ± standard deviation (n = 4). Means with different letters within one row indicate significant differences (Tukey, P < 0.05).

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Fig. 3. Protein solubility of the untreated pea pro-

tein isolate (PPI) and PPI hydrolysates at different
pH and hydrolysis times. Results are expressed as
means ± standard deviation (PPI n = 2,
Hydrolysates n = 4). Means with different letters
within each enzyme indicate significant differences
with the untreated PPI (Tukey, P < 0.05).

with 56%, 65%, and 59%, respectively, at pH 4.5 and 73%, 68%, and
64% at pH 7.0, respectively. Serine endopeptidases such as Alcalase, Table 3
Esperase, Savinase hydrolyze peptide bonds with tyrosine, phenylala- Foaming properties of the pea protein isolate (PPI) and PPI hydrolysates after
nine or leucine at the carboxyl side (Mahajan and Badgujar, 2010) 15 min and 120 min of hydrolysis.
which might have a positive effect on protein solubility (Adler-Nissen,
Time (min) Foaming capacity (%) Foam stability (%)
1986b; Molina Ortiz and Wagner, 2002). However, Chymotrypsin
showed the lowest protein solubility at both pH 4.5 and pH 7.0, which Alcalase 15 1940 ± 35a 81 ± 6a
might be attributed to previously discussed reasons in Section 3.1.2. 120 1806 ± 60b 80 ± 6a
Moreover, Flavourzyme hydrolysates showed the second lowest Papain 15 2119 ± 72a 97 ± 1a
120 2101 ± 167a 97 ± 2a
protein solubility at pH 7.0, followed by Protamex, Neutrase, and
Esperase 15 2237 ± 124a 90 ± 5a
Corolase. Papain (45%) and Bromelain (40%) improved significantly 120 1859 ± 78b 74 ± 12b
the solubility at pH 4.5, especially after 120 min, while at pH 7.0 their Bromelain 15 1710 ± 19a 87 ± 5a
hydrolysates showed no significant difference to the PPI. Protein solu- 120 1830 ± 80b 81 ± 16a
bility correlated strongly with the DH after 15 min and 120 min. Trypsin 15 2065 ± 122a 93 ± 2a
120 2271 ± 19b 95 ± 3a
Chymotrypsin 15 1619 ± 11a 88 ± 4a
120 1831 ± 18b 79 ± 3a
3.2.2. Foaming properties
Proteins are known as good foaming agents that distribute homo- Results are expressed as means ± standard deviation (n = 4). Means with
geneously fine air cells, especially if they have a low molecular weight, different letters within each enzyme indicate significant differences in each
a highly hydrophobic surface, and a low electrostatic repulsion as well experiment (Tukey, P < 0.05).
as a low surface tension (Barac et al., 2015; Lam et al., 2016; Zayas,
1997).
The method used in this study considers that a sample is able to Alcalase hydrolysates (15 min: 1939%, 120 min: 1806%), and Papain
form a foam, only when no liquid remains visible in the whipping bowl hydrolysates (15 min: 2119%, 120 min: 2101%). Flavourzyme hydro-
directly after whipping. According to this method, the untreated PPI lysates showed the lowest foaming capacity of 1614% and 1611% at
was unable to properly form foam, probably due to its higher molecular 15 min and 120 min of hydrolysis, respectively, followed by Corolase,
weight and unfolded structure. On the other hand, all hydrolysates Neutrase, Protamex, and Bromelain hydrolysates.
showed a significant improvement of the foaming capacity and foam Horiuchi et al. (1978) suggested that the foam stability of enzymatic
stability (Table 3). This might have been caused by changes in the hydrolysates improves with an increase in the hydrophobic surface of
molecular peptide size and surface hydrophobicity. The improvement the protein molecules rather than with the release of hydrophobic
of protein solubility is known to impact the surface hydrophobicity amino acids. In our study, Papain hydrolysates showed the highest foam
(Molina Ortiz and Wagner, 2002), and although the solubility was stability (97%) after 15 min and 120 min of hydrolysis. On the other
correlated with the degree of hydrolysis, there were no significant hand, Neutrase hydrolysates showed the lowest foam stability after
correlations between the foaming capacity and the degree of hydrolysis. 15 min (19%) and 120 min (12%) of hydrolysis, followed by Fla-
Thus, to some extent, the average hydrophobicity of the released pep- vourzyme hydrolysate (22%) after 120 min and Protamex hydrolysate
tides might have played an essential role in foaming capacity (Lam (34%) after 15 min of hydrolysis. These results suggest that the higher
et al., 2016). Trypsin hydrolysates showed the highest foaming capacity hydrolyzed isolates might have formed peptides with larger hydro-
of 2271% after 120 min of hydrolysis, followed by the samples obtained phobic surfaces resulting in higher stabilities.
after 15 min of Esperase hydrolysis (2237%). However, the foaming
capacity decreased significantly after a 120 min treatment with 3.2.3. Emulsifying capacity
Esperase (1859%). A similar tendency was observed for the foaming Emulsions are dispersions of two immiscible liquid phases, which
capacity of Savinase hydrolysates (15 min: 2013%, 120 min: 1798%), are generally unstable due to high interfacial tension. Proteins have the

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V. García Arteaga, et al. Innovative Food Science and Emerging Technologies 65 (2020) 102449

Fig. 4. Emulsifying capacity of the untreated pea

protein isolate (PPI) and PPI hydrolysates after
15 min and 120 min of hydrolysis. Results are ex-
pressed as means ± standard deviation (PPI n = 2,
Hydrolysates n = 4). Means with different letters
within each enzyme indicate significant differences
with the untreated PPI (Tukey, P < 0.05).

ability to reduce the tension between the two phases by redirecting 3.3. Effects on sensory properties
their amphiphilic residues towards the water and oil phase resulting in
smaller droplets. The retronasal aroma of the PPI resulted in attributes such as pea-
The PPI showed an emulsifying capacity of 467 mL/g (Fig. 4). After like (4.2), green (2.9), earthy (1.6), roasted (2.3), and cooked potato
15 min of hydrolysis, Chymotrypsin hydrolysate showed the highest (3.8), whereas the main taste attributes were salty (2.1), astringent
emulsifying capacity with 727 mL/g, followed by Flavourzyme (1.9), and bitter (3.0) with an overall intensity of 4.9 and a preference
(715 mL/g) and Trypsin (711 mL/g) hydrolysates. Savinase (499 mL/g) indication (hedonic) of 4.3. Compared to the PPI aroma profile, bit-
and Esperase (529 mL/g) hydrolysates showed a slight increase in the terness was the only attribute with a significant change after 15 min
emulsifying capacity after 15 min of hydrolysis but it decreased with and 120 min (Fig. 5). Complete data can be found in Tables C-1 and C-2
longer hydrolysis times (120 min). Papain, Bromelain, and Alcalase in the Mendeley dataset (García Arteaga et al., 2020).
hydrolysates were not significantly different from the PPI. Hydrolysates The bitter intensity of the Savinase and Alcalase hydrolysates
from Neutrase and Corolase improved the emulsifying capacity sig- (15 min of hydrolysis) increased significantly to 6.7 and 6.5, respec-
nificantly after both times of hydrolysis, ranging from 592 mL/g to tively, compared to the untreated PPI (3.0); however, with longer hy-
641 mL/g. Protamex hydrolysates showed only a significant increase in drolysis times (120 min), the bitterness of those samples was reduced to
emulsifying capacity after 15 min of hydrolysis with 645 mL/g. 6.5 and 5.4, respectively. On the other hand, the bitterness of Esperase
Negative correlations between the degree of hydrolysis and emul- hydrolysates increased significantly to a score of 6.4 only after 120 min
sifying capacity have been reported in the literature (Achouri et al., of hydrolysis. After 15 min of hydrolysis, Bromelain (2.4), Protamex
1998; Adler-Nissen and Olsen, 1979; Klost and Drusch, 2019). Thus, the (2.5), Trypsin (2.6), and Papain (2.7) hydrolysates showed lower bitter
molecular protein size might influence protein-protein and protein-oil intensities compared to the PPI followed by Chymotrypsin (3.5),
interactions. Peng et al. (2016) suggested that higher molecular weight Corolase (3.5) and Neutrase (3.7) hydrolysates. The lowest bitterness in
and bigger hydrodynamic diameter of the proteins might improve the samples (2.2) was obtained by hydrolysis with Chymotrypsin and
emulsifying capacity. They also suggested that heat treatment and Protamex after 120 min of hydrolysis followed by Neutrase (2.4) and
larger peptide sizes increase surface hydrophobicity, promoting hy- Corolase (2.4). These results suggested a strong correlation between the
drophobic interactions between protein-oil droplets, which result in bitterness and the DH. The correlation between the DH and the for-
higher emulsifying capacity. However, Barac et al. (2012) suggested mation of bitter peptides has been extensively studied (Adler-Nissen
that high molecular weight aggregates decreased emulsifying capacity and Olsen, 1979; Meinlschmidt et al., 2016; Saha and Hayashi, 2001;
but formed more stable emulsions. Sun, 2011), where the cleavage of peptide bonds and release of small
The present study showed a weak negative correlation between the peptides with hydrophobic amino acid residues leads to an increase in
degree of hydrolysis and emulsifying capacity. The weakness of this bitterness.
correlation was mainly due to trypsin hydrolysates as they showed The highest overall intensity after 15 min of hydrolysis was ob-
higher emulsifying capacity compared to other highly hydrolyzed served in Alcalase (5.9) and Savinase (5.7) hydrolysates; whereas
samples. One explanation might be that the trypsin hydrolysates Papain (4.1) and Trypsin (4.1) hydrolysates showed the lowest overall
maintained the protein fractions between 35 kDa and 22 kDa. These intensity. However, after 120 min of hydrolysis, Esperase hydrolysate
two protein fractions might provide an amphiphilic character to the showed the highest overall intensity of 6.9 followed by Savinase (5.9),
trypsin hydrolysates since the hydrolysates without these fractions Alcalase (5.4), and Trypsin (5.0) hydrolysates. The high overall in-
presented lower emulsifying capacity. However, the emulsifying capa- tensity results suggest that the panelist perceived this intensity as an
city of trypsin hydrolysates was also higher compared to some of the increase in bitterness. Accordingly, Esperase (2.9, 1.9), Savinase (1.7,
less hydrolyzed samples. Therefore, trypsin might have facilitated the 1.8), and Alcalase (1.6, 2.0) hydrolysates were the least favorite among
unfolding of hydrophobic side chains of the pea proteins, promoting the panelist after 15 min and 120 min of hydrolysis, respectively. After
optimal interaction with the oil. 15 min of hydrolysis, Protamex hydrolysate (5.5) was the favorite

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V. García Arteaga, et al. Innovative Food Science and Emerging Technologies 65 (2020) 102449

Fig. 5. Retronasal aroma and taste profile of the untreated pea protein isolate (PPI) and PPI hydrolysates after 15 min (A) and 120 min (B) of hydrolysis. Results are
expressed as means (n = 10).

sample among the panelists, followed by Chymotrypsin hydrolysate molecular weight peptides and an increase in low molecular weight
(4.8), Bromelain hydrolysate (4.6), and Trypsin hydrolysate (4.2). peptides. This was particularly evident in the Papain and Trypsin hy-
These results suggest that bitterness is an important factor influencing drolysates. Although these electrophoretic results might indicate a de-
the acceptance by the panelist (Fig. 6). gradation of the main pea allergens, the SDS-PAGE gives only an in-
dication of molecular changes, and further immunological studies are
4. Conclusions necessary to evaluate a possible reduction in the allergenic potential.
Most enzymes improved the techno-functional properties of the PPI,
The present study aimed to investigate the effect of hydrolysis of PPI especially protein solubility at pH 4.5 and foaming capacity. Regarding
with different enzyme preparations on the techno-functional and sen- sensory properties, only bitterness changed significantly after enzy-
sory properties as well as on the degradation of potential allergens matic hydrolysis. This increase in bitterness might affect their usage as
through changes in the molecular weight distribution. Of the 11 en- a food ingredient; therefore, ongoing studies such as the combination of
zyme preparations investigated by SDS-PAGE, only Papain, Trypsin, enzymes and fermentation of hydrolysates are being considered to re-
Esperase, Bromelain, and Alcalase hydrolysates showed major changes duce bitterness while maintaining improved techno-functional proper-
in the molecular weight distribution with a degradation of high ties.

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V. García Arteaga, et al. Innovative Food Science and Emerging Technologies 65 (2020) 102449

Fig. 6. Hedonic evaluation of the untreated pea protein iso-

late (PPI) and PPI hydrolysates after 15 min and 120 min of
hydrolysis. Results are expressed as means ± standard de-
viation (n = 10). Means marked with an asterisk (*) indicate
significant differences between the individual sample and the
untreated PPI (Tukey, P < 0.05).

Author agreement statement functional properties of enzymatically modified soy protein. Functionality and protein
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Society.
The authors hereby declare that this manuscript is original, has not Angioloni, A., & Collar, C. (2013). Impact of high hydrostatic pressure on protein ag-
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with chymosin. International Journal of Molecular Sciences, 12(12), 8372–8387.
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interests or personal relationships that could have appeared to influ- M. (2012). Functional properties of protein hydrolysates from pea (Pisum sativum, L)
seeds. International Journal of Food Science and Technology, 47(7), 1457–1467.
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