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Enzymatic hydrolysis of the Eisenia andrei

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 Biocatalysis and Biotransformation

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Enzymatic hydrolysis of the Eisenia andrei

earthworm: Characterization and evaluation of its
properties

Mariano Rodrigues, Wagner Manica Carlesso, Daniel Kuhn, Tacilen

Altmayer, Maira Cristina Martini, Camila Durlo Tamiosso, Carlos Augusto
Mallmann, Claucia Fernanda Volken De Souza, Eduardo Miranda Ethur &
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Volken De Souza, Eduardo Miranda Ethur & Luclia Hoehne (2017): Enzymatic hydrolysis of
the Eisenia andrei earthworm: Characterization and evaluation of its properties, Biocatalysis and
Biotransformation, DOI: 10.1080/10242422.2017.1278754

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Biocatalysis and Biotransformation, 2017; Early Online: 110

RESEARCH ARTICLE

Enzymatic hydrolysis of the Eisenia andrei earthworm:

Characterization and evaluation of its properties

MARIANO RODRIGUES1, WAGNER MANICA CARLESSO1, DANIEL KUHN1,

TACIELEN ALTMAYER1, MAIRA CRISTINA MARTINI1, CAMILA DURLO TAMIOSSO2,
CARLOS AUGUSTO MALLMANN2, CLAUCIA FERNANDA VOLKEN DE SOUZA1,
EDUARDO MIRANDA ETHUR1 & LUCELIA HOEHNE1
1
Laboratorio de Biotecnologia de Alimentos, Programa de Pos-Graduacao em Biotecnologia, Centro Universitario
UNIVATES, Lajeado, Rio Grande do Sul, Brazil and 2Laboratorio de Analises Micotoxicologica, Universidade
Federal de Santa Maria UFSM, Santa Maria, Rio Grande do Sul, Brazil

Abstract
Some studies have carried out in order to retrieve proteins from the by-product of animal-processing industries. Earthworms
are rich in protein and usually are used in animal feed. Thus, this study aimed to optimize the hydrolysis process of Eisenia
andrei earthworms by employing Alcalase enzyme. Using the response surface methodology, we evaluated the following
conditions: temperature, hydrolysis time, stirring speed, and enzyme/substrate ratio. The optimal conditions for the
experimental design were determined through the analysis of the foaming and emulsifying properties, in vitro starch
digestibility, and antioxidant activity. The results demonstrate that the highest degree of hydrolysis (i.e., 92%) was obtained
under the following conditions: pH, 9.5; temperature, 25  C; hydrolysis time, 2.25 h; stirring speed, 200 rpm; and enzyme/
substrate ratio, 1.77%, using Alcalase enzyme. Evaluation of the amino acid composition under these conditions revealed
higher concentrations of aspartic acid, glutamic acid, and leucine. The in vitro protein digestibility of the hydrolysate was
approximately 73%. There were no significant improvements in either foam stability or emulsification after enzymatic
hydrolysis. Additional studies on the antioxidant activity are required. This bioproduct could potentially serve as a promising
supplementary food product.

Keywords: Biomass; worms; amino acids; hydrolysis

Introduction seen considerable growth. The use of earthworm

Earthworms, members of the class Oligochaeta, are
animals with a thinly pigmented cuticle and no
skeleton. They are hermaphrodites and produce ova
throughout the entire year. Earthworms have an
important role in the decomposition of litter material
and in the alteration of soil structure (Curry and
Schmidt 2007). Earthworms comminute litter
material, draw it into the soil and then mix these
litter particles with other soil constituents. The
burrowing behavior of earthworms creates holes
and pores, thereby stabilizing the soil (Kretzschmar
2004). Through its expansion into the waste
management industry, earthworm vermiculture has
biomass in the animal feed industry is also being
explored (Dynes 2003; Sogbesan and Madu 2008;
Sogbesan and Ugwumba 2008; Sinha et al. 2010;
Tiroesele and Moreki 2012). Earthworms are
important not only because they convert organic
material into nutrient-rich material but also because
they contain high protein levels (Guerrero 2006).
Earthworms contain 65% protein (on a dry weight
basis), 14% fat, 14% carbohydrates, and 3% ash
(Edwards and Bohlen 1996; Visvanathan et al. 2005;
Edwards and Lofty 2013).
Worm protein has a high concentration of lysine
and methionine. The arginine, lysine, glutamic acid,
and leucine content in worms are higher than in

Correspondence: Lucelia Hoehne, Centro Universitario UNIVATES, Avenue Avelino Tallini, 171, Room 406-8, Lajeado, Rio Grande do Sul, Brasil. Tel: +55
51 3714 7000/5524. Fax: +55 51 3714 7001. E-mail: luceliah@univates.br

(Received 29 May 2016; revised 14 November 2016; accepted 7 December 2016)

ISSN 1024-2422 print/ISSN 1029-2446 online 2017 Informa UK Limited, trading as Taylor & Francis Group
DOI: 10.1080/10242422.2017.1278754
2 M. Rodrigues et al.
fishmeal. The level of tryptophan in worms is
approximately four times higher than in blood
powder and approximately seven times higher than
in beef liver (Xiang et al. 2006). Worm protein has
been shown to be a nutritionally acceptable protein
source for animals and is used as an additive to
produce pellet feeds in the United States of America,
Canada, and Japan (Li et al. 2010). Moreover,
preliminary evidence suggests that replacing fishmeal
with Eisenia foetida and Eudrilus eugeniae earthworm
species has increased the growth performance
(2030%) of a number of fish species including
tilapia (Sayed 1999), vundu catfish, (Sogbesan and
Madu 2008), rainbow trout (Stafford and Tacon
1985), common carp (Nandeesha et al. 1988), and
others (Morillo et al. 2013; Mombach et al. 2014).
Thus, earthworms have the nutritional potential to
be used as a food supplement (McInroy 1971;
Yoshida and Hoshii 1978; Guerrero 1983; Cayot
et al. 2009).
Hydrolysis of worm proteins could be applied in
the food industry. These hydrolyzed proteins have
low molecular weights and are rich in peptides and
free amino acids that enhance functional properties
(Kristinsson and Rasco 2000). The cleavage of
peptide bonds through protein hydrolysis can be
carried out by either enzymatic or chemical pro-
cesses. Chemical processes, including alkaline or
acid hydrolysis, tend to be difficult to control and
yield poor results in terms of the nutritional quality
of the proteins and the conservation of amino acids
(Shahidi et al. 1999). The extreme conditions
required by chemical hydrolysis could be avoided
by performing enzymatic hydrolysis instead, which
has milder condition requirements. Usually, enzym-
atic processes avoid side reactions and do not
decrease the nutritional value of the protein source
(Maldonado et al. 1998). Additionally, enzymes have
substrate specificity, which permits the development
of protein hydrolysates with better-defined chemical
and nutritional characteristics (Castro et al. 2011).
Some studies have made progress in applying
biological enzymatic processes to produce animal
protein hydrolysates (Nilsang et al. 2005; Guerard
et al. 2007). Several studies provide examples of
enzymatic protein hydrolysates with enhanced prop-
erties using fish, chicken, and pig protein hydrolys-
ates (Cui et al. 2009; Benhabiles et al. 2012; Pagan
et al. 2013; Dong et al. 2014; Valencia et al. 2014;
Duarte et al. 2015); however, there are few reports in
the literature on earthworm hydrolysates (Lin et al.
2010).
Studies have investigated the optimization of
enzymatic hydrolysis conditions using the response
surface methodology (Nilsang et al. 2005; Guerard
et al. 2007). This methodology has a number of
advantages, including fewer required experiments,
improved statistical interpretation and a reduced
time requirement for the overall analysis (Jatinder
et al. 2006; Deepak et al. 2008; Pandiyan et al. 2014;
Demim et al. 2014; Chenna et al. 2016).
The aim of this study was to optimize the
conditions for the enzymatic hydrolysis of the
Eiseina andrei earthworm. The hydrolysis process
was carried out using Alcalase enzyme and the effects
of temperature, hydrolysis time, stirring speed and
enzyme/substrate ratio were examined using the
response surface methodology. The conditions and
functional properties of the bioproduct with the
highest degree of hydrolysis were evaluated.
Materials and methods
Material
Alcalase enzyme (2.4 L) supplied by Novozymes
Latin America Ltd., from Araucaria PR, was used
as the enzyme for the hydrolysis process. All other
reagents were obtained from Sigma-Aldrich and
were of analytical grade.
Earthworms culture
Eisenia andrei earthworm cultures were developed by
Tecnovates Laboratories at the Centro Universitario
UNIVATES. The earthworms (about 10,000 ani-
mals adults) were put in three commercial plastic
boxes (80  39  62 cm). Half of the boxes volume
was filled with 50% soil, 20% fruit peels, 10% dry
leaves, and 20% cow manure. The duration of the
vermiculture process was 60 days and was conducted
under controlled temperature and humidity. Adult
earthworms were used in all experiments.
Physicochemical characterization of the earthworms
For physicochemical characterization of the earth-
worms, about 25 g of adult animals were randomly
selected from the plastic boxes, immersed in water
for 24 h. After pooled samples were submitted to the
moisture, ash, protein, and lipid analysis according
to the established methodology (AOAC 2000). For
moisture analysis, sample was put in the oven at
105  C for 3 h. For ash analysis, samples were put in
the oven at 450  C for 3 h. For protein analysis,
samples were submitted at the Total Kjeldahl nitro-
gen process using the correction factor 6.25 for meat.
For lipid analysis, samples were submitted to
gravimetric method, using Soxhlet extraction with
petroleum ether for 8 h.

Hydrolysis conditions
The worms were immersed in water for 24 h, washed
in ultrapure water and stored in a freezer at
10.5  C. The sample was ground in a Britain
Black 200 W mixer and then transferred to a reactor.
Buffer solution was added to the reactor to control
the pH value during hydrolysis. The hydrolysis
reaction was carried out in an MA 830 Marconi
refrigerated incubator to control the temperature and
agitation conditions. The effects of pH, temperature,
hydrolysis time, stirring speed, and enzyme/substrate
ratio on the degree of hydrolysis of Eisenia andrei
earthworm using Alcalase enzyme were determined
using the central composite rotatable design
(CCRD) with k 5, which generated 45 treatment
combinations. Five levels of each variable were
chosen; the upper and lower limits of each variable
were consistent with those described in the literature
and with our own previous experience (Chae et al.
1998; Rossi et al. 2009; Schmidt and Sallas-Mellado
2009). In the statistical model, Y denotes the degree
of hydrolysis (%); x1 (pH 7.25)/0.95; x2 (tem-
perature 47.5)/9.5; x3 (hydrolysis time 2.25)/
0.74; x4 (stirring speed 100)/42; and x5
(enzyme/substrate ratio 4.25)/1.58. The actual
levels of the coded settings, treatment combinations,
and responses are shown in the supplementary data.
Each measurement was made in triplicate (25 g of
adult animals for each test was used). This design is
represented by the following second-order polyno-
mial regression model (Equation 1):
X X X protein immediately after the addition of enzyme
Y 0 i xi ii x2i ij xi xj 1 and 10 mL of 20% TCA, final protein represents the
where Y response variable, 0 constant,
i coefficient for the linear effect, ii coefficient
for the quadratic effect, ij coefficient for the
interaction effect, and xi and xj the coded levels of
variables Xi and Xj, respectively. Equation (1) was
used to plot the surfaces of these variables.
The test factors were coded according to
Equation 2:
 
Xi  X0
xi 2 2695) and a Multi- Fluorescence detector (Waters
D Xi
where xi is the coded value and Xi is the actual value
of the ith independent variable, X0 is the actual value
at the central point, and DXi is the step change value.
After the hydrolysis process, the reaction was
terminated by adding 6.25% trichloroacetic acid
(TCA). The amount of soluble protein was deter-
mined using methods described by Lowry et al.
(1951).
All statistical experimental designs and analyses
were carried out using Statistica 7.0 software
Enzymatic hydrolysis of the E. andrei earthworm 3
(StartSoft Inc., Tulsa, OK). The statistical analysis
of the model was performed using analysis of
variance (ANOVA), including the Fishers F test
(overall model significance), its associated probabil-
ity P(F), correlation coefficient R, and the determin-
ation of coefficient R2, which measures the goodness
of fit of the regression model. The Students t value
was used to estimate the coefficients and their
associated probabilities, P(t). For each variable, the
quadratic model was represented as a contour plot.
The error calculation was based on replications of
the central point.
Determination of the degree of hydrolysis
The method for determining the degree of hydrolysis
(DH) was adapted from Sathivel et al. (2003). The
DH (%) was defined as the percentage of soluble
protein in trichloroacetic acid (TCA). A sample
aliquot (10 mL) was mixed with 10 mL of 20% TCA
and then centrifuged at 5000  g for 15 min at 25  C.
The soluble nitrogen in the supernatant and the total
nitrogen were determined using the Kjeldahl
method. The degree of hydrolysis was calculated
using Equation 3:
Degree of hydrolysis%
Final protein hydrolysed  Initial protein
 100
Total N
3
where initial protein represents the amount of
amount of soluble protein after hydrolysis and total
N represents the amount of nitrogen in the hydro-
lyzed protein solution.
Determination of amino acids
The earthworm hydrolysate obtained under optimal
experimental design conditions was analyzed using a
Waters HPLC system (Milford, MA) equipped with
a separation module with an autoinjector (Waters
2475) for simultaneous analysis of amino acids. This
methodology, previously developed by Pereira et al.
(2008), was improved upon by adding an initial step
that included the derivation of cysteine via carbox-
ymethylation using iodoacetic acid (IDA), in accord-
ance with Pereira et al. (2015).
Evaluation of functional properties
Foaming and emulsifying properties, in vitro starch
digestibility and antioxidant activity of the
4 M. Rodrigues et al.
earthworm hydrolysate obtained under optimal
experimental conditions were assessed.
Foaming properties
Foaming properties were assayed according to
methods described by Okezie and Bello (1988).
The foam volume immediately after whipping and
after 30 min of rest was used to determine the
foaming stability (Equation 4).
Foam stability %
Foam volume after standing for 30 min
 100
Foam volume immediately after mixing
4
Emulsifying properties
The emulsifying properties were assessed using
methods described by Silva-Sanchez et al. (2004).
Protein dispersions (1%, w/v) were prepared in
0.05 M phosphate buffer (adjusted to a pH of 39 with
0.1 M HCl or 0.1 M NaOH). 10 mL of earthworm
hydrolysate was dispersed in 10 mL of corn oil. The
mixtures were homogenized for 1 min. Emulsifying
activity was defined as follows (Equation 5):
Emulsifying activity %
Height of emulsified layer 5 published by Cayot et al. (2009) using earthworm
 100
Height of the contents of the tube
In vitro protein digestibility
In vitro protein digestibility was assessed according to
methods described by Babiker and Tinay (1992). A
0.2 g sample was placed into a 50 ml centrifuge tube,
and 15 mL of 0.1 M HCl containing 1.5 mg pepsin
was added. The tube was then incubated at 37  C for
3 h. The suspension (supernatant) was neutralized
with 0.5 M NaOH, treated with 4 mg of pancreatin
in 0.2 M phosphate buffer (pH 8.0) containing
0.005 M sodium azide and incubated at 37  C for
24 h. After incubation, the sample was treated with
10 mL of 10% trichloroacetic acid and centrifuged at
5000  g for 20 min at room temperature. The
nitrogen content was determined using the
Kjedahl method. Digestibility was calculated using
Equation 6:
Protein digestibility %
Nsupernatant  Nenzyme 6 under the following conditions: pH, 8.2; tempera-
 100
Nsample
where Nsupernatant is the nitrogen content of the
suspension, Nenzyme is the nitrogen content of pepsin
and pancreatin, and Nsample is the nitrogen content
of the initial sample.
Antioxidant activity
Antioxidant activity was assessed by calculating the
percent discoloration using the method described by
Brand-Williams et al. (1995). Di(phenyl)-(2,4,6-
trinitrophenyl) iminoazanium (DPPH) radical
scavenging activity (%) was calculated using
Equation 7:
Antioxidant activity %
 
A sample at t 30 7
1  100
A sample at t 0
where Asample is the absorbance of the sample.
Results and discussion
Physicochemical characterization of the earthworms
Results regarding the characterization of E. andrei
are (% in dry weight): protein, 70.95  0.31; ash,
6.17  0.01; carbohydrate, 10.25  0.10, and lipids
12.63  0.10. The protein content obtained in the
current study was similar to or exceeded those
reported by other studies, e.g., 48% (Isea et al.
2008) using worm flour with other ingredients and
68% (Ferruzzi 2001) using earthworm flour.
Ash and lipid content were consistent with those
powder. These results confirm that earthworm
hydrolysates are rich in protein and provide evidence
for its nutritional potential as a food supplement
(McInroy 1971; Yoshida and Hoshii 1978; Guerrero
1983; Cayot et al. 2009).
Hydrolysis process
The experimental design and the degree of hydroly-
sis of the E. andrei earthworm using Alcalase enzyme
are presented in the supplementary data. The degree
of hydrolysis varied from 4.90 to 43.93% depending
on the hydrolysis conditions. This condition-
dependent variation demonstrates the importance
of optimization for productive enzymatic hydrolysis
of the E. andrei earthworm.
Treatment 13 (pH, 6.3; temperature, 57  C;
hydrolysis time, 2.99 h; stirring speed, 58 rpm; and
enzyme/substrate ratio, 2.67%) produced the lowest
degree of hydrolysis (4.90%). The highest degree of
hydrolysis, 43.93% (treatment 20), was obtained
ture, 38  C; hydrolysis time, 1.52 h; stirring speed,
142 rpm; and enzyme/substrate ratio, 5.83%. This
result indicates that increases in pH, stirring speed,
and enzyme/substrate ratio and decreases in tem-
perature and hydrolysis time provides favorable
conditions for the hydrolysis of the E. andrei
 Enzymatic hydrolysis of the E. andrei earthworm 5
Table 1. Effect and coefficient estimates by the regression model The model clearly reveals significant interactions
for optimization of degree of hydrolysis of E. andrei earthworm by
between pH and temperature (p x1.x2 0.0105), pH
Alcalase enzyme.
and hydrolysis time (p x1.x3 0.0268), pH and
Independent
variables stirring speed (p x1.x4 0.0055), pH and enzyme/
(parameter) Effect Coefficient () t value p Value substrate ratio (p x1.x5 0.0139), temperature and
Intercept (0) 19.2922 19.2922 38.76 0.0007 enzyme/substrate ratio (p x2.x5 0.0106), hydrolysis
x1** 11.0992 5.5496 41.52 0.0006 time and enzyme/substrate ratio (p x3.x5 0.0270),
x1.x1** 3.2755 1.6378 11.69 0.0072 and stirring speed and enzyme/substrate ratio
x2** 13.6717 6.8358 51.15 0.0004
x2.x2* 2.6193 1.3097 9.35 0.0112 (p x4.x5 0.0024). Therefore, treating these variables
x3* 2.1722 1.0861 8.13 0.0148 separately may not reflect their real influence on the
x3.x3 0.1190 0.0595 0.42 0.7123 degree of hydrolysis of the E. andrei earthworm by
x4** 3.0178 1.5089 11.29 0.0077
x4.x4 0.0258 0.0129 0.09 0.9351 Alcalase enzyme. These data suggest that the one-
x5* 2.4099 1.2049 9.01 0.0121 variable-at-a-time approach may not be the most
x5.x5* 2.1472 1.0736 7.66 0.0166
x1.x2* 3.0048 1.5024 9.66 0.0105
effective to study enzymatic hydrolysis.
x1.x3* 1.8607 0.9304 5.98 0.0268 Whenever possible, the model was simplified by
x1.x4** 4.1631 2.0815 13.39 0.0055 the elimination of statistically insignificant terms.
x1.x5* 2.6113 1.3056 8.40 0.0139
x2.x3 0.7040 0.3520 2.26 0.1519 After simplification, the quadratic model was
x2.x4 0.8526 0.4263 2.74 0.1113 reduced to the following (Equation 8):
x2.x5* 2.9930 1.4965 9.62 0.0106
x3.x4 0.3423 0.1711 1.10 0.3858 Y 19:2922 5:5496x1 1:6378x1 :x1  6:8358x2
x3.x5* 1.8539 0.9270 5.96 0.0270
x4.x5** 6.2989 3.1494 20.25 0.0024
1:3097x2 :x2:  1:0861x3 1:5089x4 1:2049x5
 1:0736x5 :x5  1:5024x1 :x2 0:9304x1 :x3
x1, x2, x3, x4 and x5 are the coded values of variables pH,
temperature ( C), hydrolysis time (h), stirring speed (rpm), and 2:0815x1 :x4 1:3056x1 :x5  1:4965x2 :x5
enzyme/substrate ratio (%), respectively. 0:9270x3 :x5  3:1494x4 :x5
*Statistically significant at 95% of confidence level.
**Statistically significant at 99% of confidence level. 8
where Y is the predicted response to the degree of
hydrolysis (%), x1 is the pH, x2 is the temperature
earthworm using Alcalase enzyme. In regard to the ( C), x3 is the hydrolysis time (h), x4 is the stirring
pH effect, a similar result was reported in a study by speed (rpm), and x5 is the enzyme/substrate
Lin et al. (2010), where the authors investigated the ratio (%).
enzymatic hydrolysis of this earthworm using The statistics of the model were analyzed using
Alcalase in the experimental design. The authors Fishers test for the ANOVA; the data are presented
verified that a pH value of 8.0 was most effective for in Table 2. The computed F value (6.69) was highly
this process. significant (p 0.00001). The determination coeffi-
The significance of each regression coefficient was cient (R2 0.85) implies that the sample variation of
determined using the t values and p values listed in 85% for the degree of hydrolysis is attributed to the
Table 1. The p values suggest that the positive independent variables and can be explained by the
linear effects of pH (p x1 0.0006), stirring speed model. The R value (0.92) signified a high degree of
(p x4 0.0077) and the enzyme/substrate ratio correlation between the experimental results and the
(p x5 0.0121) were significant. The negative linear theoretical values predicted by the model equation
effects of temperature (p x2 0.0004) and hydrolysis indicating that the process model (Equation 8) serves
time (p x3 0.0148) were also significant. These data as an excellent representation of the degree of
indicate that increases in pH, stirring speed and hydrolysis of the E. andrei earthworm by Alcalase
enzyme/substrate ratio and decreases in temperature enzyme.
and hydrolysis time yield a high degree of hydrolysis The contour shapes shown in Figure 1 are plotted
of the E. andrei earthworm using Alcalase enzyme. on the basis of the model equation and show the
Second-order effects of pH (p x1.x1 0.0072), interactions among the variables for the optimal
temperature (p x2.x2 0.0112), and enzyme/sub- degree of E. andrei earthworm hydrolysis. Each
strate ratio (p x5.x5 0.0166) were also highly sig- contour curve represents a number of combinations
nificant. The highly significant effect of variables on of two tested variables, with the other variables
the second-order model indicates that they can act as maintained at their respective zero level. The analysis
limiting factors and that even small variations in their of the contour shapes for pH, temperature, hydroly-
values will impact the degree of hydrolysis to a sis time, stirring speed, enzyme/substrate ratio, and
considerable extent. degree of E. andrei hydrolysis reveals that there are
6 M. Rodrigues et al.
Table 2. Analysis of variance (ANOVA) for the model regressiona.
Source SS Df MS F value p Value
Model 4609.72 20 230.48 6.69 0.00001
Residual 826.73 24 34.45
Total 5436.45 44
a
R 0.92; R2 0.85.
SS: sum of squares; Df: degrees of freedom; MS: mean square.
mutual interactions among the studied variables.
Figure 1(A) shows the degree of E. andrei hydrolysis
using different pH values and temperatures.
Maximal hydrolysis was obtained using lower incu-
bation temperatures and higher pH values during the
hydrolysis process.
Figure 1(B,E) show the contour plots of pH
against hydrolysis time and temperature against
hydrolysis time. These plots demonstrate that the
degree of E. andrei hydrolysis by Alcalase can be
increased using a pH above 9.0, a temperature below
30  C, and hydrolysis times between 0.5 and 4 h.
Figure 1(C) shows the contour of pH against stirring
speed, where the highest values of both variables led
to an increase in the degree of hydrolysis. Figure
1(D) shows the contour of pH against enzyme/
substrate ratio. These results suggest that an enzyme/
substrate ratio between 1.6 and 8% and in a high pH
environment yields the optimum degree of E. andrei
earthworm hydrolysis using Alcalase.
According to the optimized mathematical model,
the optimal levels for the five hydrolysis parameters
obtained at the maximum point of the polynomial
model were calculated to be the following: pH, 9.5;
temperature, 25  C; hydrolysis time, 2.25 h; stirring
speed, 200 rpm; and enzyme/substrate ratio, 1.77%.
Under these conditions, the model predicts a max-
imum degree of hydrolysis response of 89%. In order
to verify the predicted results, experiments were
performed under the optimized parameters, and the
experimental value obtained was 92% (mean of three
experiments). The excellent correlation between the
predicted and measured values of these experiments
validates the response model and the existence of an
optimal condition.
Amino acid composition
A comparison of the amino acid compositions (g/kg
of protein) of hydrolyzed earthworms and hydrolyzed
fish are presented in Table 3.
The results revealed that enzymatic hydrolysates
are rich in Asp, Glu, and Leu. In comparison to fish
(Benhabiles et al. 2012), the earthworm hydrolysates
had high concentrations of all amino acids. In
comparison, the amino acid composition of
hydrolysed muscle from the highly proteinaceous
marine fish Collichthys niveatus is approximately 10
100 times greater than those obtained in this study
(Shen et al. 2012).
Amino acid levels obtained from enzymatic
hydrolysis of chickpea protein isolates compared to
earthworm were in accordance with Ghribi et al.
(2015). When compared to the enzymatic hydrolysis
of chicken bone, amino acid levels of earthworms
were lower or similar; however, the chicken bone
extract was prepared by heating the chicken at
130  0.5  C for 120 min followed by filtrating,
standing, defatting, and concentrating (Dong et al.
2014). This step was not necessary for the experi-
ments described in this paper. The amino acid
profiles of hydrolysates in the current study were
characterized by assessing the essential amino acid
content, which is more nutritionally beneficial (FAO/
WHO 2007).
Functional properties
Based on previous studies on foaming and emulsi-
fication, hydrolysis causes changes in protein struc-
ture, thereby impacting surface characteristics and
functional properties. Proteins are used effectively in
food emulsions (Lam and Nickerson 2013); how-
ever, the emulsifying capacities of the hydrolysates
decrease with increasing amounts of protein hydroly-
sis (Fainerman and Miller 1998). In this study, there
were no significant improvements in either foam
stability or emulsification after enzymatic hydrolysis.
These results are in accordance with those reported
by Govindaraju and Srinivas (2006). It is likely that
hydrolysis causes reductions in emulsification, foam-
ing capacity, and stability. These results can be
explained by decreases in film viscoelasticity because
of the involvement of smaller peptides at the inter-
face due to hydrolysis. Moreover, due to charge
repulsions, peptides with low molecular weights can
neither unfold nor reorient at the interface (Severin
and Xia 2006).
The in vitro protein digestibility results suggest
that protein hydrolysates can be suitable nutritional
supplements in various foods. In the current study,
protein digestibility was found to be 72.57  1.06%
under optimal experimental conditions. These values
are in accordance with those reported in other
studies using different hydrolysate samples. Using
sheep visceral protein hydrolysate, Bhaskar et al.
(2007) obtained 74.6  1.3% in vitro protein digest-
ibility under optimal conditions. Foh et al. (2011)
reported in vitro starch digestibility results of 80%
using fish protein hydrolysate and concentrate.
Abdul-Hamid et al. (2002) reported in vitro protein
 Enzymatic hydrolysis of the E. andrei earthworm 7

Figure 1. Contour plot for the effect of (A) pH  temperature, (B) pH  hydrolysis time, (C) pH  stirring speed, (D) pH  enzyme/
substrate ratio, (E) temperature  hydrolysis time, (F) temperature  stirring speed, (G) temperature  enzyme/substrate ratio, (H)
hydrolysis time  stirring speed, (I) hydrolysis time  enzyme/substrate ratio, and (J) stirring speed  enzyme/substrate ratio on
hydrolysisdegree of E. andrei earthworm by Alcalase enzyme. The variables that are not plotted are fixed at zero level in all of the 10 graphs.
8 M. Rodrigues et al.
Table 3. Amino acid composition from hydrolyzed earthworms amino acids, and had significant protein digestibility
and fishes.
in vitro. This bioproduct is promising as a supple-
Hydrolyzed Hydrolyzed mentary food product. Further studies are required
(wet weight) from from
Amino acid earthworms fishesa with other enzymes and conditions.
Aspartic acid (Asp) 4.3 0.1
Glutamic acid (Glu) 5.2 0.3
Serine (Ser) 2.4 0.06
Glycin (Gly) 1.7 0.6 Acknowledgements
Histidine (His) 1.4 0.05
Arginine (Arg) 2.3 0.21 This research was supported by UNIVATES, who
Threonine (Thr) 1.7 0.06 provided scholarships and additional financial sup-
Alanine (Ala) 1.9 1.44 port for this study. We thank Novozymes Latin
Proline (Pro) 1.2 0.37
Tyrosine (Tyr) 1.2 0.15 America Ltd. for donating the enzyme.
Valine (Val) 2.0 0
Isoleucine (Ile) 1.9 0.64
Leucine (Leu) 3.3 1.34
Phenylalanine (Phe) 1.7 0.46 Declaration of interest
Lysine (Lys) 2.7 0.42
b
Total amino acid content 35.3 The authors declare that there are no conflicts of
a
Benhabiles et al. [56]. interest regarding the publication of this paper.
b
Not informated.
All the results are expressed in g kg1.

digestibility results ranging from 7.9 to 92.1% using
tilapia protein hydrolysate powder and Alcalase.
Antioxidative peptides have been purified from
many protein hydrolysates, but the majority come
from vegetable samples such soybean (Moure et al.
2006), rice bran (Parrado et al. 2006), canola
(Cumby et al. 2008), Chinese leek (Hong et al.
2014), and sweet potato (Zhang et al. 2014). There
is little information concerning the antioxidant
properties of animal hydrolysates (Bougatef et al.
2010). Studies have shown that it is possible to assess
the antioxidant properties of animal hydrolysates by
measuring the reducing power of the raw material,
metal-chelating activity, and b-carotene bleaching
inhibition activity, among others (Bougatef et al.
2010; Mokni et al. 2015). In the present study, the
antioxidant activity was assessed using methods
described by Brand-Williams et al. (1995). The
assessment revealed that there was no antioxidant
activity during the enzymatic hydrolysis of the
earthworm. Further studies are required to investi-
gate antioxidant activities in vivo using several
measurements.
Conclusions
Eisenia andrei can be used to obtain hydrolyzed
products. The highest degree of hydrolysis (i.e.,
92%) was obtained under the following conditions:
pH, 9.5; temperature, 25  C; hydrolysis time, 2.25 h;
stirring speed, 200 rpm; and enzyme/substrate ratio,
1.77%, using Alcalase as the enzyme. The biopro-
duct was rich in protein value, contained essential
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