Received: 14 July 2019 Revised: 13 October 2019 Accepted: 17 December 2019

DOI: 10.1111/jfpe.13370

ORIGINALARTICLE

Optimization of the porcine liver enzymatic hydrolysis

conditions
1 1 1
José U. Maluf | Mônica L. Fiorese | Keiti L. Maestre |
1 2 3
Fernanda R. Dos Passos | Joana K. Finkler | Jessica F. Fleck | Carlos E. Borba1

1
Chemical Engineering Postgraduate
Program, State University of West Paraná,
UNIOESTE, Toledo, Paraná, Brazil
2
Fishing Resources and Fishing
Engineering Postgraduate Program, State
University of West Paraná, UNIOESTE,
Toledo, Paraná, Brazil
3
State University of West Paraná,
UNIOESTE, Toledo, Paraná, Brazil
Correspondence
Mônica L. Fiorese, Chemical Engineering
Postgraduate Program, State University
of West Paraná, UNIOESTE, Campus
Toledo, Faculdade St. 645, Jd. La Salle,
85903-000 Toledo, Paraná, Brazil. Email:
mlfiorese@gmail.com
Funding information
BRF S.A.; Coordination of Improvement of
Higher Education Personnel (CAPES), Grant/
Award Number: 001; State of Parana Research
Foundation (Araucária Foundation), Grant/
Award Number: 50027
Abstract
Worldwide porcine slaughterhouses produce significant amounts of by-products, such as
porcine liver, daily. Even though the liver presents a very restricted market demand
when raw, this product is composed of an exceptional nutritional content, with emphasis
on protein, being, therefore, characterized as a potential raw material to produce protein
hydrolysates. This article aimed to study the production of protein hydrolysate using the
commercial enzymes Alcalase 2.4L™ and Novo Pro-D™. For the development of this
research, a central composite rotatable design with 4 axial points was applied for each
enzyme. The factors evaluated were protein/water and enzyme/substrate ratios. The
best hydrolysis results indicate average percentages of hydrolysis degree of 27.5% for
both enzymes. The presence of low-molecular-weight peptides was also evidenced by
the electrophoresis technique. It is, therefore, con-cluded that there is positive viability
for the use of hydrolyzed porcine liver in differ-entiated food applications with an
aggregate market value.
Practical Applications
At present, the worldwide growing dissemination of food scarcity has become a
stim-ulus for the food industry to rethink the efficient use of raw materials to avoid
waste and maximize the use of existing nutritional resources. A potential way of
exploiting the nutritional value of the protein by-products with no commercial
applications is their transformation into protein hydrolysates by means of enzymatic
processes. Pro-tein hydrolysates have been in focus in the scientific community
with industrial appli-cations as beneficial products to the ingesting body, for acting
as either nutraceuticals or functional bioactive foods. Commercialization of this type
of prod-uct has become a growing market niche. Thus, this research, which
presents an inves-tigation regarding the conditions of the porcine liver hydrolysis,
fits within current issues, with innovative potential of obtainment of bioactive
peptides from by-products.

1 | I N T RO DU CT I O N Drummond, 2017). Porcine liver is considered to be a by-product of

swine slaughter, which is rich in proteins and preeminent nutrients,
The animal protein industry generates large amounts of by-products, such as Vitamins B12 and A (Nollet & Toldra, 2011; Seong et al.,
ranging from 33 to 49% of the weight of the living animal, depending 2015; Shimizu et al., 2006). The porcine liver can be commercialized

on the slaughtered species (Mullen, Alvarez, Zeugolis, Neill, & raw; however, it has a small consumer market. According to

J Food Process Eng. 2020;e13370. wileyonlinelibrary.com/journal/jfpe © 2020 Wiley Periodicals, Inc. 1 of 12

https://doi.org/10.1111/jfpe.13370
2 of 12
Srebernich, Gonçalves, and Domene (2017)), the powdered porcine
liver stands out as an excellent fortifier to be used in mixtures for
food preparations, which can be destined, for instance, to school
meals in soups, creams, cooked meat, and especially beans.
Among the technological alternatives for the meat processing
industry regarding the transformation of by-products are the biotech-
nological processes, specifically the enzymatic hydrolysis. The use
of enzymatic hydrolysis has grown considerably in recent years in
the industrial segment, not only in the meat processing industry, but
also in the pharmaceutical, food, and beverage industries (Martínez-
Alva-rez, Chamorro, & Brenes, 2015; Mora, Reig, & Toldrá, 2014;
Mullen et al., 2017; Toldrá, Mora, & Reig, 2016).
The attractiveness of enzymatic hydrolysis lies on the high effi-
ciency of enzymes to act as biocatalysts, allowing hydrolytic reac-
tions to occur under mild conditions, and consequently, with lower
energy requirements. Moreover, this process presents notable prod-
uct selectivity through simplified production routes, low generation of
undesirable bitter-tasting peptides (Shen, Guo, Dai, & Zhang, 2012),
lower environmental and physiological toxicity, and conse-quently,
high quality and productivity (Chapman, Ismail, & Dinu, 2018; Choi,
Han, & Kim, 2015; Fernandes, 2010; Kapoor, Rafiq, & Sharma,
2017; Madhavan, Sindhu, Binod, Sukumaran, & Pandey, 2017;
Roslan, Yunos, Abdullah, & Kamal, 2014; Sun, Zhang, Ang, & Zhao,
2018).
Protein hydrolysates can be obtained from different animal sources
and their by-products, such as fish viscera (Feltes et al., 2010; Roslan,
Mustapa Kamal, Md. Yunos, & Abdullah, 2015; Roslan et al., 2014; Silva,
Ribeiro, Silva, Cahú, & Bezerra, 2014); sardine viscera (Venturin et al.,
2017); blue whiting (Micromesistius poutassou) (Harnedy et al., 2018);
salmon backbone (Slizyte et al., 2016); bovine liver (Di Bernardini et al.,
2011); porcine liver (Shimizu et al., 2006); swine tissues (Damgaard,
Lametsch, & Otte, 2015); swine blood (Chang, Wu, & Chiang, 2007);
chicken blood (Zheng, Si, Ahmad, Li, & Zhang, 2018); swine plasma (Liu,
Kong, Xiong, & Xia, 2010); cattle plasma (Bah, Carne, McConnell, Mros,
& Bekhit, 2016); shrimp viscera (Katsuwonus pelamis) (Klomklao &
Benjakul, 2016); and boneless chicken carcasses (Oliveira, Franzen,
Terra, & Kubota, 2015). For rep-resenting potentially inexpensive
feedstocks, these by-products have become industrially relevant to the
meat industry, which has come to invest in attractive technology
solutions that are efficient and able to deliver products with high levels of
protein and essential amino acids (FAO, 2017; Mora et al., 2014; Toldrá
et al., 2016).
In the meat packing industry, the enzymatic hydrolysis is
employed for protein fragmentation, that is, a parent protein can be
hydrolyzed to produce a variety of different peptides with diverse
activities. The peptides generated may be bioactive or functional,
depending on the type of enzyme and the operating conditions
employed (Sánchez & Vázquez, 2017). Several studies (Chang et
al., 2007; Guo, Kouzuma, & Yonekura, 2009; Jang, Jo, Kang, & Lee,
2008; Liu, Xing, Fu, Zhou, & Zhang, 2016; Martínez-Alvarez et al.,
2015; Mullen et al., 2017; Sánchez & Vázquez, 2017; Villamil,
Váquiro, & Solanilla, 2017) showed that the ingestion of peptides
can improve health and highlight some of the major
MALUF ET AL.
bioactivities, such as antioxidant, antimicrobial, antihypertensive,
antithrombotic, immunomodulatory, lipid-lowering, and anticancer
(Kannan, Hettiarachchy, Marshall, Raghavan, & Kristinsson, 2011)
activities.
Enzymatic hydrolysis is very efficient in terms of process-related
costs, and the enzyme is one of the most important economic vari-
ables in the production of protein hydrolysates. In general, proteases
of microbial origin with high hydrolytic activity and substrate specific-
ity, in other words, high activity/cost ratio (Aspevik, Egede-Nissen, &
Oterhals, 2016) are employed in industrial processes.
Among the enzymes of microbial origin, the most widely used in the
production of protein hydrolysates, either commercially or for research
studies, is Alcalase 2.4L™, a bacterial endopeptidase devel-oped by the
company Novozymes, originated in the submerged fer-mentation
process with the microorganism Bacillus licheniformis (Alavi, Jamshidian,
& Rezaei, 2019; Bernardi et al., 2016; Corîci et al., 2011). This enzyme is
classified as a subtilisin, a serine protease that is known to operate
under moderate conditions of alkalinity (6–10) (Roslan et al., 2014)
compared to the neutral or acidic enzymes, and temperatures of 35–70
C (Schmidt & Salas-Mellado, 2009) with opti-mum temperature of 55 C
(Bhaskar, Benila, Radha, & Lalitha, 2008; See, Hoo, & Babji, 2011) and
60 C (Amiza, Nurul Ashikin, & Faazaz, 2011; Normah, Jamilah, Saari, &
Yaakob, 2005), acting in the peptide bonds contained in the hydrophobic
residues. The prominence of Alcalase 2.4L™ is found to be one of the
highly efficient enzymes for the protein hydrolysis due to its ability to
catalyze protein hydrolysis reaction with high degree of hydrolysis (DH)
in a relatively short period (Adler-Nissen, 1986; Aspmo, Horn, & Eijsink,
2005). Another enzyme that has presented application potential is Novo
Pro-D™ produced by genetically modified B. licheniformis in submerged
fermentation. The Novo Pro-D™ is also a subtilisin, but classified as a
serine-endoprotease, which acts by hydrolyzing the internal peptide
bonds (de Miranda 2012; Rojas, Siqueira, Miranda, Tardioli, & Giordano,
2014; Zhou et al., 2019).
The amino acid composition and DH during hydrolysis depend
on various factors such as enzyme type, enzyme specificity, sub-
strate composition, and on the characteristics of the process, such
as protein/water and enzyme/protein ratios, pH, temperature, and
time of reaction. Thus, this study aimed to broaden the state of the
art regarding the knowledge of the basic principles of the factors that
influence the hydrolysis reaction, which are crucial to obtain
adequate process control which represent the maximum efficiency
of the raw material conversion into the desired product. The bio-
transformation of the porcine liver by-product in the production of
protein hydrolysates was performed by optimizing the hydrolysis
conditions of protein/water (%) and enzyme/protein (%) using the
experimental planning tool. The enzyme Alcalase 2.4L™ was
selected, as it is the most widely used in the industrial segment of
protein hydrolysates due to its high hydrolytic efficiency and low
cost, compared to the enzyme Novo-ProD™. Although the last still
lacks literature results, it was reported by Da Rosa et al. (2018) as
being similar to Alcalase 2.4L™, besides presenting high cleavage
possibility.
MALUF ET AL.
2 | MATERIALSANDMETHODS
2.1 | Feedstock
The porcine liver was donated by the company Brasil Food (BRF-Food),
Toledo Unit, Paraná. The sample collection took place after the slaughter, in
the stage of animal evisceration. The liver used in this study came from
animals of the WH species (genetics information owned by the company),
slaughtered after 190 days by carbon dioxide injection, ensuring the prin-
ciples of animal welfare. The by-product porcine liver was ground in a helical
mill, packed in polyethylene bags and stored in freezer (−18 ± 1 C) until
transported under refrigeration (2 ± 1 C) in thermal boxes to the Food Quality
Control Laboratory located at the West Paraná State Uni-versity (Campus
Toledo, Paraná, Brazil), for further use.
Total water ðgÞ = Water addition ðgÞ +
2.2 | Chemical composition of the porcine liver
The porcine liver was characterized regarding total protein (928.08)
(MAPA, 1999; AOAC, 2016), lipids (991.36) (MAPA, 1999; AOAC,
2016), mineral matter (920.153) (AOAC, 2016), and moisture
(950.46) (AOAC, 2016). The analyses were performed in triplicate.
Two enzymes were used in the hydrolysis process: Alcalase
2.4L™ (Novozymes Latino Americana Ltd, Paraná, Brazil), a serine
pro-tease of microbial origin (B. licheniformis) with endogenous
activity; and Novo Pro-D™, an alkaline protease produced by
genetically modi-fied B. licheniformis.
2.3 | Central composite rotatable design for
enzymatic hydrolysis of porcine liver
A central composite rotatable design (CCRD) was used to evaluate
the conditions protein/water (%) (X1) and enzyme/protein (%) (X2) in
the porcine liver enzymatic hydrolysis. The experimental conditions
employed for the enzymes Alcalase 2.4L™ and Novo Pro-D™ are
pres-ented in Table 1.
For the two-factor system, the polynomial equation (Equation 1)
represented by the degree of protein hydrolysis (dependent variable)
as a function of the terms of linear and quadratic effects was used,
where Y is the predicted response, b 0 is the intercept, b1 and b2 are
the linear terms, and b11 and b12 are the quadratic terms. Y1 is the
DH for the enzyme Alcalase 2.4L™ and Y2 for Novo Pro-D™.
coded levels of the central composite rotatable design for the enzymes Alcalase 2.4L™ and
Novo Pro-D™
T A B L E 1 Real values of the
independent variables at different
3 of 12
2 2
Y = b0 + b1X1b2X2 + b11X1 + b11X2 + b12X1X2: ð1Þ
The real values for the levels of each independent variable (X i)
were represented by the factors, defined from literature searches
regarding meat raw materials (Schmidt & Salas-Mellado, 2009), and
similar industrial processes from BRF-Foods.
The amount of water added to the hydrolysis reaction medium
was calculated from Equations (2) and (3):
protein ðgÞ
Water addition ðgÞ = ðX1Þ ð%Þ 2
raw material
Þ
ð 100 %
protein g ð Þ
− raw material ð%Þ ,
ð Þ
raw material ðgÞ moisture ð%Þ
,
100 ð%Þ
ð3Þ
where X1 = protein/water (%) (CCRD).
The hydrolysis was performed with 750 g of porcine raw
material for 60 min, under 100 rpm, the same pH of the raw material,
around 6.4, and temperatures of 60 C for Alcalase 2.4L™ and 64 C
for Novo Pro-D™. After the reaction time, the enzyme was
deactivated by heating the solution in a water bath at 85 C for 15
min (García-Moreno et al., 2014; Roslan et al., 2015; Verma, Chatli,
Kumar, & Mehta, 2017). The samples were then lyophilized to be
employed in molecular weight distribution and quantification of
amino acid composition.
The experimental data were evaluated by using the software
Statistica™ version 10 (Statsoft, Inc.), according to the variance analy-
sis (ANOVA) and the response surface analysis. The statistical signifi-
cance of the model was evaluated by the F test at a 5% level.
2.4 | Enzymatic kinetics of porcine liver
hydrolysate
Enzymatic kinetics was performed for the two enzymes in the condi-
tion of maximum response for protein/water (%) and enzyme/protein
(%). The samples were collected at 5, 10, 15, 20, 30, 45, 60, 120,
180, 240, 300, 360, 420, and 480 min and frozen for further quantifi-
cation of the DH (%).
 Independet variable symbol −1.41 −1 0 1 1.41
X
Protein/water % (p/p) (wt/wt) 1 8 10 15 20 22.1
Coded Enzyme/protein % (p/p) X2 0.3 0.5 1 1.5 1.7
level
Vari
(wt/wt)
able
a
Coded level: define the factor levels in an experimental design.
4 of 12 detected by spectrophotometry after the enzymatic hydro-lysis of the
sample, with pancreatin solution at 45 C for 24 hr, followed by
2.5 | Degree of hydrolysis (%) colorimetric reaction with diamino benzaldehyde acid in sulfuric acid
medium sheltered from light for 6 hr. Then, a solution

The DH was determined according to the method described by

Hoyle and Merrit (1994)) and Baek and Cadwallader (1995).
Samples of hydrolyzed porcine liver were thawed and centrifuged
(Excelsa Baby I Centrifuge) at 2,600 rpm. Then, 20% trichloroacetic
acid was added in the ratio of 1:1 and held for 30 min. Subsequently,
the samples were filtered using a vacuum pump and Whatman
.
no. 40 filter paper. Finally, the absorbance of the samples were read
in a spectrophotom-eter (Thermo Fisher Scientific, model Spectronic
200E) at a wave-length of 750 nm. The results were expressed in
milligrams of albumin, according to Equation (4).

DH % p=p = Soluble protein ðmgÞ*100ð %Þ :

ð Þ

ð ÞðÞ Total protein mg

2.6 | Molecular weight distribution

This procedure was performed according to Laemmli's methodology

(Laemmli, 1970), where the Tris–HCl–glycine system buffer varied
from 4 to 17% Tris–tricine gradient gel. The samples (5 μL) were dis-
solved in 2% sodium dodecyl sulfate (SDS), 10% glycerol, 0.01%
−1
brom-ophenol blue, 0.0625 mol L Tris–HCl pH 6.8, and 5% β-
mercaptoethanol and heated for 5 min. Electrophoresis was con-
ducted using Mini PROTEAN 3 Cell electrophoresis system (Bio-Rad
−1
Laboratories, Hercules, CA) at 200 V with buffer (3 g L Tris-base,
−1 −1
14 g L glycine, and 1 g L SDS). After electrophoresis, the gel
pro-teins were stained with Coomassie Blue R-250 dye and
decolorized in a 50% methanol and 10% acetic acid solution. The
lmw marker from GE lifesciences, with molecular weight range
between 14.4 and 97 kDa was used as a control.

2.7 | Amino acid composition

The amino acid compositions of the porcine liver and protein hydro-
lysates were quantified by acid hydrolysis with 6 N hydrochloric acid,
3% (wt/vol) phenol, at 110 C for 24 hr. After hydrolysis, deriva-
tization occurred with the solution (4:4:2) of 0.2 N sodium acetate
trihydrate, high-performance liquid chromatograph (HPLC)-grade
methanol and 99–100% triethylamine. Amino acid identification was
performed by HPLC reverse phase column, with injection of 30 μL,
−1
detection at 254 nm, pH 6.40, flow of 1 mL min , 58 C, Eluent A
with 0.14 N sodium acetate, 0.14 N acetonitrile, and 0.14 N
triethylamine buffer and eluent B acetonitrile 60% (vol/vol) according
to the methodology proposed by White, Hart, and Fry (1986) and
Hagen, Frost, and Augustin (1989). The tryptophan con-tent was
 MALUF ET AL.

of sodium nitrite was added and after 30 min the transmittance was
read at 590 nm in a spectrophotometer with quartz cuvettes.

3 | RESULTSANDDISCUSSION

3.1 | Chemical composition of porcine liver

The chemical composition of the raw porcine liver is reported in

Table 2.
From the results presented in Table 2, it can be verified that the
values found agree with the study of (Honikel, 2011), in which the
author reported variations in the porcine liver composition for protein
(18.9–21.6%) and lipids (2.4–6.8%).
Seong et al. (2015) evaluated the aspects of the chemical and
nutritional composition of chicken by-products, such as liver, gizzard,
stomach, small intestine, cecum, and duodenum. The results indicated
that the approximate range (minimum and maximum) of these by-
products were moisture (76.68–83.23%), fat (0.81–4.53%), protein
(10.96–17.70%). Although liver and gizzard presented higher levels of
protein, these values are lower than those found in the present study.
It should be noted that factors such as feeding, climate, and
time of growth of the animal to be slaughtered influence the
constitution of the nutrients present in the organ, however, the
values found are similar to those of this research.
In this context, by comparing the protein content found in the
meat by-products with the values present in the animal muscle, for
example, bovine loin (21%), porcine loin (22.2%) (De Castro
Cardoso Pereira & Dos Reis Baltazar Vicente, 2013), porcine chops
(17.3%), and duck meat (19.3%) (Ockerman & Basu, 2004), it is
noted that these values are similar to those found in by-products of
animal origin, especially the liver.
Thus, the potential presented by raw porcine liver as a source of
protein of high biological value, able to meet daily nutrient require-
ments is verified, with the possibility of being used in hydrolytic
indus-trial processes to obtain new high-value products, because
biopeptides are obtained from enzymatic hydrolysis.

3.2 | Porcine liver enzymatic hydrolysis

The results of the variables used in the CCRD regarding the produc-
tion of porcine liver protein hydrolysates using the enzymes Alcalase

T A B L E 2 Chemical composition of the raw porcine liver

Composition Raw porcine livera

Ashes (%) 1.4

Lipids (%) 2.0

Total protein (%) 19.7

Moisture (%) 74.3

a
Analysis performed in triplicate, mean value expressed.
MALUF ET AL. 5 of 12

2.4L™ and Novo Pro-D™ are presented in Table 3. The results of
the analysis of variance concerning the DH are reported in Table 4.
Within the range studied, linear (X 1) and (X2) variables were sig-
nificant (p value <.05) for the two enzymes used. Results of the response
surface of the model (Figure 1a) for the enzyme Alcalase 2.4L™ were
evaluated by the ANOVA and F test (Table 4). The F value calculated for
a 95% confidence interval (p value <.05) was Fc (2;5;0.05) = 11.09, which
the model can represent 95.44% of the response variability, demon-
strating that the regression model is significant for both enzymes.
The equation obtained by the statistical analysis of the regression
model with the coded variables is presented in Equations (3) and
(4), respectively.
Y1 = 18:66 + 3:80ðX1Þ + 2:58ðX2Þ, ð3Þ

was greater than the tabulated value (F tab2;5;0.05 = 5.79). The

2 Y2 = 18:81 + 7:26ðX1Þ + 7:56ðX2Þ:
regressions obtained provided R of 0.8607, indicating that the model ð4Þ

can explain 86.07% of the response vari-ability (Y 1). While for the
enzyme Novo Pro-D™ (Figure 2a), the F value calculated for a 95% The DH (Table 3) ranged from 10.93 to 27.86% (wt/wt), with Assay 4
confidence interval (p value <.05) was Fc (2;5;0.05) = 18.54, which was as the best response for both enzymes (20% X 1 and 1.5% X2), providing
higher than the tabulated F (F tab 2;5;0.05 = 5.79) (Barros Neto, Bruns, & results of 27.86 and 27.30% (wt/wt) for Y 1 and Y2, respec-tively. Table 5
Scarminio, 2010). In addition, presents an overview of the hydrolytic ability of

T A B L E 3 Experimental design for the degree of hydrolysis variable response with enzyme Alcalase 2.4L™ (Y 1) and Novo Pro-D™ (Y2) in 1 hr

Coded level Real level Response

Run (X1) (X2) Protein/water (%) (wt/wt) Enzyme/protein (%) (wt/wt) Y1 Y2

1 −1 0.5 10 0.5 12.43 12.56

2 1 0.5 20 0.5 22.47 15.57

3 −1 1.5 10 1.5 17.05 17.53

4 1 1.5 20 1.5 27.85 27.30

5 −1.41 1 8 1 15.70 11.07

6 1.41 1 22 1 22.34 22.49

7 0 0.3 15 0.3 12.29 10.93

8 0 1.7 15 1.7 19.75 20.40

9 0 1 15 1 18.12 18.85

10 0 1 15 1 18.60 18.65
11 0 1 15 1 19.36 19.04

TABLE4 Analysis of variance for the degree of hydrolysis (Y1 and Y2)

Degrees of
Model freedom Sum-of-squares Mean squares F value p Value R2 Adjacent R2
Y1 Regression 3 118.41 39.48 0.8607 0.7214
Linear 1 114.67 114.67 293.32 .0034
Square 1 3.60 3.60 9.20 .0936
Interaction 1 0.14 0.14 0.36 .6052
Residual 5 37.75 7.55
Lack of fit 3 27.80 9.27 23.71 .0407
Total 7 205.19
Y2 Regression 3 117.36 39.12 0.9544 0.9088
Linear 1 104.50 104.50 2,747.50 .0004
Square 1 1.44 1.44 37.92 .0254
Interaction 1 11.42 11.42 300.38 .0033
Residual 5 2029.29
Lack of fit 3 11.19 3.73 98.10 .0101

Total 7 2,247.18
6 of 12 MALUF ET AL.

F I G U R E 1 Response surface (a) and Pareto chart (b) for the porcine liver hydrolysate for the enzyme Alcalase 2.4L™

F I G U R E 2 Response surface (a) and Pareto chart (b) for the porcine liver hydrolysate for the enzyme Novo Pro-D™

different enzymes in meat raw materials. Thus, these results demon-
strate that the hydrolytic process of this study was good, as it
resulted in a higher DH compared to the literature. It also showed
that the vol-ume of water and enzyme added in the process was
low, which, from an industrial point of view, is an important factor, as
it does not entail considerable costs regarding the acquisition of
enzyme and energy for the product drying phase, thus contributing
to the economic viability of the process.
Using the software Statistica, the contour plot regarding the X 1
and X2 effects for Y1 and Y2 (Figures 1 and 2b) showed a positive
effect for Y1 and Y2, with X1 as the most significant. On the other
hand, the interaction between the variables had no significant effect
within a 95% confidence interval (p value <.05). The greatest
influence on the enzymatic hydrolysis process of porcine liver was
provided by the variable X1, presenting better contour for the Novo
Pro-D™ enzyme.
According to the response surface plots, the linearity of the
model was verified, as the higher X1 and X2, the higher Y1 and Y2. If
X1 is 22 and X2 is 1.7, the DH will be approximately 25% for the
enzyme Alcalase 2.4L™, whereas for Novo Pro-D™, if X 1 is 22 and
X2 is 1.7, the DH is 27%. Thus, the directly proportional behavior
between Y1 and Y2 reinforce the linearity of the model.
MALUF ET AL. 7 of 12

T A B L E 5 Overview of the hydrolytic ability of different enzymes and raw materials

Temperature Degree of
Raw material Enzyme pH ( C) T (hr) hydrolysis (%) References
Tilapia frame protein isolate Pepsin 2 37 4 23.46 Lin, Alashi, Aluko, Sun Pan, and Chang
(2017)

Tilapia Alcalase 2.4L™ 7.5 60 2 20.2 Roslan et al. (2015)

Sardine Alcalase 2.4L™ 8 50 2 13.7–14.9 García-Moreno et al. (2014)
Blue whiting (Micromesistius Alcalase 2.4L™ 7 50 4 29.13–32.58 Harnedy et al. (2018)
poutassou)
Porcine liver Papain 6.5 50 6 1.18–19.12 Verma et al. (2017)

Porcine liver Alcalase 2.4 L™ 8.0 50 6 1.22–23.56 Verma et al. (2017)

Porcine liver Alcalase 2.4 L™ 6.4 60 1 12.29–27.85 In this work

Porcine liver Novo Pro-D™ 6.4 64 1 10.93–27.30 In this work

F I G U R E 3 Maximum response of protein hydrolysis for the enzyme Alcalase 2.4L™ where (a) X 1 (%) constant and X2 (%) varying
and (b) X2 (%) constant and X1 (%) varying

enzyme/protein ratio was fundamental to obtain DH and conse-

quently the protein content
3.3 | Study in the regions of maximum protein Regarding the enzyme Alcalase 2.4L™, it was observed in Figure
hydrolysis response 3a that when the protein/water ratio is 20% (wt/wt), there is an increase

It was not possible to obtain an optimized condition by the CCRD,

but rather an indication of the best relations with the maximum
response, thus, the best conditions for the hydrolytic process
response of the DH was determined for each enzyme. These assays
were performed by fixing the protein/water ratio factor (20, 22, 24
and 26% [wt/wt]) and varying the enzyme/protein ratio factor (0.5,
1.0, 1.5, 2.0, 3.0, and 6.0). The tests regarding Alcalase 2.4L™
ranged from 23 to 46 and Novo Pro-D™ from 47 to 70.
Figure 3a shows that the enzyme/protein ratio of 1.5% (wt/wt)
was the one with the highest DH for Alcalase 2.4L™ and adopted as
reference for this analysis. Therefore, it is verified that the variable
in DH, which was adopted as reference for the kinetics. However, for
the variable enzyme/protein ratio, the best DH was 6.0% while the
vari-ation of the DH was not significant when compared to an
enzyme/pro-tein ratio of 1.5%, which justifies the choice of the
enzyme/protein ratio of 1.5% for the kinetic experiment, because the
enzyme repre-sents a material of high value for the process.
According to Sadana (1991), an increase in the velocity
decreases the size of the droplets formed, thereby increasing the
interfacial area, which leads to an extension of the hydrolysis. For
protein/water ratios higher than 24%, Figure 3b shows that for
Alcalase 2.4L™, the rheol-ogy of the product presented difficulties in
maintaining a homoge-neous velocity of the sample movement,
generating regions with lower speeds, which lead to less favorable
conditions for a hydrolyzed product uniformity.
Figure 4a shows that for the enzyme Novo Pro-D™, the protein/
water ratio of 22% produced the highest DH, while Figure 4b shows
that an enzyme/protein ratio of 3.0% provided the best response.
Thus, this condition was adopted for the kinetics study
8 of 12 MALUF ET AL.

F I G U R E 4 Maximum response of protein hydrolysis for the enzyme Novo Pro-D™ where (a) X1 (%) constant and X2 (%) varying and (b)
X2 (%) constant and X1 (%) varying

F I G U R E 5 Kinetics of the enzymatic hydrolysis varying X1 (%) for both enzymes, (a) Alcalase 2.4L™ (b) Novo Pro-D™

3.4 | Kinetics of the enzymatic hydrolysis for the enzyme would be more favorable to be applied in industrial
enzymes processes.

The kinetics was developed under the condition of maximum

response of protein hydrolysis. Figure 5a,b shows the kinetic curves
for the hydrolysis condition X1 at 20% (wt/wt) and X2 at 1.5% (wt/wt)
at 62 C and pH of 6.4. It is observed that in the hydrolytic process
(Figure 5a,b), the reactions occurred with a high rate up to 60 min.
After that, there is a reduction in the reaction speed, reaching the
stationary phase in 550 min (Alcalase 2.4L™) and
300 min (Novo Pro-D™) with DH of 34 and 31%, respectively. How-
ever, a 9-hr (550 min) hydrolysis time would be economically unfeasible
to the industry due to the process time as well as changes that could
arise in the hydrolysate, such as degradation of bioactive principles and
bitterness (Shen et al., 2012). Thus, the Novo Pro-D™
3.5 | Molecular weight distribution
Figure 6 shows the protein profile of the porcine liver both raw and
hydrolyzed for the best condition of the CCRD after 1 and 4 hr for both
enzymes. It was verified that there was hydrolysis in all the sam-ples
considering the formation of peptides with low molecular weight (14
kDa), therefore the cleavage of the peptide bonds happened. The
distribution of the peptides was similar and compatible with the DH
observed for the different enzymes. The formation of bioactive pep-tides,
which occur at low molecular weight, is likely to happen. Studies have
shown that the enzyme Alcalase 2.4L™ can produce low
MALUF ET AL. 9 of 12

ence of essential amino acids both balanced and similar to those of

muscle proteins. When comparing amino acid levels with those of

F I G U R E 6 Molecular weight distribution. Lane 1, control; Lane

2, Novo Pro-D™ in 1 hr; Lane 3, Novo Pro-D™ in 4 hr; Lane 4,
Alcalase 2.4L™ in 1 hr; and Lane 5, Alcalase 2.4L™ in 4 hr

molecular weight peptides when there is a high DH (Lalasidis, Boström,

& Sjöberg, 1978; Liaset, Lied, & Espe, 2000; Roslan et al., 2015). This
behavior was also observed for the enzyme Novo Pro-D™.

3.6 | Amino acid composition

Amino acid compositions are shown in Table 6. It is verified that

both porcine liver and protein hydrolysates (Y1 and Y2) showed high
levels of glutamic acid and aspartic acid. In general, there was no
significant difference for cysteine between the raw material and the
hydroly-sates. Protein hydrolysate with the Novo Pro-D™ enzyme
had higher tryptophan content than porcine liver and protein
hydrolysate with the enzyme Alcalase 2.4L™.
Protein hydrolysates showed a higher content for the amino
acids glutamic acid, aspartic acid, leucine, and lysine for both
enzymes, reaching values of 1.98, 1.51, 1.41, and 1.27%,
respectively, for Alcalase 2.4L™ and 2.18, 1.67, 1.53, and 1.37%,
respectively, for Novo Pro-D™.
All essential and nonessential amino acids were detected in bal-
anced amounts for both porcine liver and protein hydrolysate (Y1
and Y2), with percentages of essential amino acids of 50.22, 51.05,
and 50.33%, respectively. (Anderson, 1988), in his characterization
study of porcine by-products, found varied levels of essential amino
acids, but similar to those of the muscular tissues of the porcine.
Also, Aristoy (2011) found, in by-products of animal origin, the pres-
 D™, hydrolysis rates were similar and relatively high. However, from
T A B L E 6 Total amino acid composition (%) of raw porcine liver the response surface and Pareto chart, it was found that, within the
and protein hydrolysate in 1 hr
conditions evaluated, the protein/water factor was more significative
Protein for the enzyme Alcalase 2.4L™, while for Novo Pro-D™, the
hydrolysate enzyme/protein ratio was the most significative fac-tor. Regarding
Raw porcine industrial application, the best condition for the porcine liver would
Total amino acid liver Y1 Y2 be a 1.5% (wt/wt) enzyme/protein and 20% (wt/wt) protein/water

Essential ratio, 62 C, pH 6.4, 5 hr, and enzyme Novo

Histidine 0.67 0.53 0.58

Arginine 0.97 0.70 0.77

Threonine 0.84 0.65 0.73

Valine 1.20 0.95 1.03

Methionine 0.44 0.35 0.38

Isoleucine 0.91 0.69 0.76

Leucine 1.82 1.41 1.53

Phenylalanine 0.98 0.76 0.82

Lysine 1.66 1.27 1.37

Tryptophan 0.30 0.25 0.39

Nonessential
Aspartic acid 2.10 1.51 1.67

Glutamic acid 2.57 1.98 2.18

Serina 0.83 0.63 0.75

Glycine 1.08 0.86 0.98

Alanine 1.17 0.92 0.99

Proline 0.92 0.66 0.81

Tyrosine 0.58 0.38 0.56

Cysteine 0.34 0.31 0.31

Total essential amino acid 9.79 7.56 8.36
content

Total nonessential amino acid 9.59 7.25 8.25

content
Total amino acid content 19.88 14.81 16.61

Percentage of essential amino 50.52 51.05 50.33

acids

FAO, it is found that it meets the demand of the daily recommenda-

tions established by FAO (2017).

4 | CONCLUSIONS

The use of the response surface methodology approach made it

possible to identify the factors relevant to the hydrolytic process. For
both enzymes used in the processes, Alcalase 2.4L™ and Novo Pro-
10 of 12
Pro-D™ The enzyme Novo Pro-D™ showed to be more efficient,
presenting a higher DH (28%), in less process time when compared
to the commercially used for industrial processes, Alcalase 2.4L™.
When it comes to molecular weight distribution, the values obtained
were low for both enzymes (<14 kDa), which may repre-sent the
formation of bioactive compounds that show beneficial physiologic
effects for the living organisms. Thus, we conclude that the porcine
liver can be employed in the development of new products.
ACKNOWLEDGMENTS
The authors acknowledge the financial support from the company
Brasil Food (BRF-Food), Toledo Unit, Paraná. And the Coordination
of Improvement of Higher Level Personnel, Brazil (Capes), Finance
Code 001, Araucária Foundation of Support to the Scientific and
Technological Development of the State of Paraná.
ORCID
Mônica L. Fiorese https://orcid.org/0000-0001-5250-7178
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