© The Authors, 2021, Published by the Universidad del Zulia*Corresponding author: labjairo1@hotmail.com
Inuence of pH in the enzymatic hydrolyzate of concentrates from the shmeal industry
Inuencia del pH en el hidrolizado enzimático de concentrados de la industria de harina de pescado
Inuência do pH no hidrolisado enzimático concentrados da indústria de farinha de
peixe
Jairo Corral Palacios
1
*
María Hipatia Delgado-Demera
2
Carlos Alfredo Cedeño-Palacios
3
Rev. Fac. Agron. (LUZ). 2022, 39(1): e223904
ISSN 2477-9407
DOI: https://doi.org/10.47280/RevFacAgron(LUZ).v39.n1.04
Food Technology
Associate editor: Dra. Gretty Ettiene
1
Maestría en Ingeniería Química, Instituto de Posgrado,
Universidad Técnica de Manabí, Av. Urbina y Che Guevara,
Portoviejo, Manabí, Ecuador.
2
Departamento de Veterinaria. Facultad de Ciencias
Veterinarias. Universidad Técnica de Manabí, Ecuador.
3
Departamento de Procesos Químicos. Facultad de Ciencias
Matemáticas, Físicas y Químicas. Universidad Técnica de
Manabí, Ecuador.
Received: 12-02-2021
Accepted: 12-08-2021
Published: 16-12-2021
Published: 15
Abstract
The shmeal concentrate is a byproduct that has an important amount of
components useful for the food industry. However, if the shmeal concentrate
is not processed, it could cause an imbalance of the environment in which the
waste is discharged. The aim of this research was to evaluate the inuence of
the pH in the enzymatic hydrolysis of the shmeal industry for the production
of protein concentrates from stickwater as a primary commodity. Thus, the
pH values (5.32, 5.94 y 6.33) were examinated, also a proximal analysis to
hydrolysate and soluble sh was carried out. In order to determine protein,
moisture, fats and ashes the following methods were used, Kjeldhal method
for protein, rapid thermobalance method for moisture, Soxhlet methos
for fats, and ofcial INEN 0467 method for ashes. Protein concentration
analyzes were performed by the Bradford method, and subsequently, the
hydrolysis approximation was calculated and the amino acid composition
was determined by the reference method Waters UPLC. The results showed
that the pH of 6.33 allowed to achieve a better hydrolysis because a higher
hydrolysis approximation was obtained, thus also the results obtained from
the amino acid composition in the nal product demonstrate its potential use
as a food additive.
Keywords:
Protein
Stickwater
Amino acids
Food additive
Protein hydrolyzate
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2-5 |
Resumen
El concentrado de la industria de la harina de pescado es un
subproducto que contiene una gran cantidad de compuestos utilizables
en la industria alimentaria. Sin embargo, en el caso de no ser
procesados podría causar un desequilibrio en las propiedades físicas,
químicas y biológicas del entorno en el cual se realiza la descarga
de los desechos. El objetivo del estudio fue evaluar la inuencia del
pH en el hidrolizado enzimático de la industria de harina de pescado
para la elaboración de concentrados proteínicos a partir de muestras
de agua de cola como materia prima. Para la ejecución de esta
investigación se evaluaron tres valores de pH (5,32; 5,94 y 6,33). Se
realizó el análisis proximal al hidrolizado y al soluble de pescado.
La determinación de proteína se realizó por el método Kjeldahl,
humedad por el método rápido de la termobalanza, grasas por el
método Soxhlet y cenizas por el método ocial de la INEN 0467.
Se realizaron análisis de concentración de proteína por el método de
Bradford, posteriormente, se calculó la aproximación de hidrólisis y
se determinó la composición de aminoácidos mediante el método de
referencia Waters UPLC. Los resultados mostraron que el pH de 6,33
permitió alcanzar una mejor hidrólisis debido a que se obtuvo una
aproximación de hidrolisis más elevada, así también, los resultados
obtenidos de la composición de aminoácidos en el producto nal
demuestran su potencial uso como aditivo alimenticio.
Palabras clave: Proteína, agua de cola, aminoácidos, aditivo
alimenticio, hidrolizado proteico.
Resumo
O concentrado da indústria da farinha de peixe é um subproduto
que contém uma grande quantidade de compostos utilizáveis na
indústria alimentar. Contudo, se não for processado, poderá causar
um desequilíbrio nas propriedades físicas, químicas e biológicas do
ambiente em que os resíduos são descarregados. O objectivo deste
estudo foi avaliar a inuência do pH no hidrolisado enzimático
na indústria de farinha de peixe para a produção de concentrados
de proteínas a partir de amostras de água de cola como matéria-
prima. Três valores de pH (5,32, 5,94 e 6,33) foram avaliados para
a execução desta investigação. Foi realizada uma análise proximal
do hidrolisado de peixe e peixes solúveis. A proteína foi determinada
pelo método Kjeldahl, a humidade pelo método de termobalanço
rápido, a gordura pelo método Soxhlet e as cinzas pelo método ocial
INEN 0467. A análise da concentração de proteínas foi realizada pelo
método de Bradford, depois a aproximação da hidrólise foi calculada
e a composição de aminoácidos foi determinada pelo método de
referência Waters UPLC. Os resultados mostraram que o pH de 6,33
permitiu alcançar uma melhor hidrólise porque foi obtida uma maior
aproximação à hidrólise. Além disso, os resultados obtidos a partir
da composição de aminoácidos no produto nal demonstram a sua
potencial utilização como aditivo alimentar.
Palavras-chave: Proteína, água de cola, aminoácidos, aditivo
alimentar, hidrolisado de proteínas.
Introduction
Currently the demand for shmeal has increased rapidly, especially
in some of the emerging aquaculture countries in Asia, such as China,
which is the largest producer and exporter of aquaculture and also
the largest. shmeal consumer worldwide (Han et al., 2018; Li et al.,
2019).
Fishmeal is a naturally balanced food ingredient, rich in protein,
energy and minerals (Jannathulla et al., 2019), however, during the
shmeal production process the raw material is subjected to a process
of pressing, obtaining a mass in which the suspended solids and sh
oil are separated by a centrifugation process, producing an efuent
commonly called tail water (Wu & Bechtel, 2012), if the by-product
is not used again in the plant, is usually discarded in the environment,
causing serious damage to the local fauna and ora (Babazadeh et al.,
2014). For this reason, it is necessary to nd an efcient method to
reuse the by-product and in turn give it added value, making the most
of its properties. Tail water evaporated to a thick syrup can provide
valuable amounts of protein, soluble solids, vitamins and minerals
(Mahdabi & Hosseini-Shekarabi, 2018). Currently, the by-products
of shmeal production are considered a raw material with a high
potential for the production of products instead of being considered a
waste (Mahdabi et al., 2018).
According to Wisuthiphaet et al. (2015), one way to use shmeal
by-products is for production of sh protein hydrolyzate. Although
this method is more time consuming and has a higher production
cost, the sh protein hydrolysates obtained by this method are more
nutritional and offer a wide range of applications including animal
nutrition or food additives.
The objective of this study was to evaluate the Inuence of pH in
the enzymatic hydrolyzate of concentrate from the shmeal industry
for the elaboration of protein concentrates from tail water samples
of the marine species Thunnus albacares, Katsuwonus pelamis and
Opisthonema libertate.
Materials and methods
Raw material
The raw material (tail water obtained from the scrap of tuna from
the marine species Thunnus albacares, Katsuwonus pelamis and
Opisthonema libertate), was collected from companies producing
shmeal in the city of Jaramijo-Manabí 0°58’34.9” South and
80°38’14.9” West.
Enzyme
The proteolytic enzyme Granozyme ACC® was used for
enzymatic hydrolysis. The details of the proteolytic enzyme used in
the present study are presented in table 1.
Table 1. Description of the Granozyme ACC® enzyme.
Specifications
Effective pH range
6 to 7,5
Effective temperature range
45 to 60°C
Shape
Smell
Density
Minimal Activity
Liquid
Typical of the enzyme
1.15 g.mL
-1
840 UHb.g
-1
Source: (Granotec, 2017)
Preparation of the enzymatic hydrolyzate
To obtain the enzymatic hydrolyzate, the tail water was passed
through a concentration process in a triple effect evaporating plant,
obtaining a liquid with a high concentration of organic matter called
concentrate or soluble sh, which must have 40 °Brix. Subsequently,
the soluble concentrate passed to a reactor where an 85 % potassium
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Corral et al. Rev. Fac. Agron. (LUZ). 2022, 39(1): e223901
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hydroxide solution was added, which allowed it to increase the pH
of the concentrate to a value that ranged between 5.5 and 7, (Hanna
HI99121 pH-meter). The selected enzyme was added when the
temperature in the reactor was 60 °C. Once the enzyme was added,
one (1) hour was waited, giving rise to the enzymatic process, in
which the proteins were hydrolyzed into peptides and amino acids
of lower molecular mass. Simultaneously with the hydrolysis, the
stabilizer (Xanthan Gum) was added in a dose of 3.5 g.kg
-1
of product
and potassium sorbate was used as preservative, in a dose of 2000
mg.kg
-1
of product, to achieve a better homogenization of these
with the hydrolyzed product. The Granozyme ACC® enzyme was
activated to perform its function and at the end of the reaction it must
be inactivated, either by temperature or pH. For which, the entry of
steam was opened to the jacket of the reactor to raise the temperature
of the hydrolyzate to 90 °C, maintaining it for 15 min. Subsequently,
the hydrolyzate was cooled to room temperature. Phosphoric acid
was added to the reactor, in a dose of 4 to 5 %, obtaining a nished
product with a pH lower than 4.5. Finally, the product was unloaded
in properly cleaned and sealed containers.
Proximal analysis
The proximal analysis of the enzymatic hydrolyzate and the
soluble sh was carried out using the following methods: moisture
was determined by the rapid thermobalance method, according to
the procedure described in the Ofcial Mexican Standard for the
determination of moisture in food (NMX, 1982), the percentage of
proteins was calculated using the Kjeldahl method according to the
methodology described by the Association of Ofcial Analytical
Chemists (AOAC, 2005), fats were determined using the Soxhlet
method described in the Ecuadorian Technical Standard 0466 (INEN,
1980a) and ashes were determined by means of the Ecuadorian
Technical Standard 0467 (INEN, 1980b).
Determination of protein concentration and hydrolysis
approximation
The protein concentration was determined by the method of
Bradford (1976), the reading was carried out with a microplate reader
(iMark Microplate Absorbance Reader-catalog # 168-1130) at an
absorbance of 595 nm. Protein concentration was calculated using the
standard curve for bovine serum albumin. The results were expressed
in mg.mL
-1
.
The hydrolysis approximation was determined by the
following formula:
Hydrolysis approximation = (1 - (A / B)) X 100 Equation 1
Where A: is the protein concentration after hydrolysis and B: is
the protein concentration before subjecting the concentrate to the
hydrolyzing action of the enzyme.
Composition of amino acids
The amino acid composition was determined using the Waters
UPLC reference method for amino acid analysis (Waters, 2021),
using ultra-performance liquid chromatography (UPLC), for which
an ultra-high-performance liquid chromatograph (Waters Acquity
UPLC), equipped with a Waters AccQ-TagTM Ultra column (2.1
mm x 100 mm), a mobile phase A AccQ-TagTM Ultra Eluent A1,
a mobile phase B AccQ-TagTM Ultra Eluent B and the ow rate
was 0.7 mL.min
-1
. The sample and column temperatures were 20
and 55 °C, respectively. The amino acids determined were: arginine,
histidine, isoleucine, lysine, methionine, phenylalanine, threonine
and valine. These amino acids were selected because they are within
the nutritional requirements of essential amino acids necessary for the
marine species to which the product is directed (shrimp).
Experimental design and statistical analysis
A completely randomized design (DCA) was used with three
treatments and three repetitions for each one. Each treatment
consisted of a pH value with a different degree of alkalization at the
beginning of each hydrolyzate (5.32, 5.94 and 6.33). For statistical
analysis the mean values of the Bradford analysis and the hydrolysis
approximation were obtained from three replicates and were used for
statistical analysis. The difference between the means of the treatments
was calculated using Tukey’s multiple range test (p≤0.05) using the
statistical software SPSS version 23.0 (IBM SPSS Statistics, 2015).
Data were expressed as means ± standard deviation (SD).
Results and discussion
Proximal composition of sh soluble and hydrolyzate
The results of the proximal composition of the tail water (AC) and
the hydrolyzate with each of the different pH´s are shown in tables 2
and 3.
Table 2. Proximal composition of the sh soluble.
Fish soluble
pH Humidity (%) Protein (%) Ash (%) Fat (%)
5.32 54.08 ± 1.35 32 ± 0.02 10.98 ± 0.11 1.70 ± 0.22
5.94 54.44 ± 1.31 33 ± 0.01 10.25 ± 0.23 1.55 ± 0.32
6.33 53.98 ± 1.29 32 ± 0.04 10.66 ± 0.15 1.57 ± 0.06
The data represent the mean ± SD
Table 3. Proximal composition of the hydrolyzated.
Hidrolizated
pH Humidity (%) Protein (%) Ash (%) Fat (%)
5.32 49.05 ± 1.27 34.90 ± 0.05 14.55 ± 0.46 1.20 ± 0.34
5.94 49.60 ± 1.25 35.00 ± 0.07 14.20 ± 0.53 0.90 ± 0.68
6.33 50.30 ± 1.29 35.60 ± 0.11 13.55 ± 0.22 1.05 ± 0.01
The data represent the mean ± SD
It can be seen that in all three cases there was a decrease in the
percentage of fat and moisture, and an increase in the percentage
of ash and protein (component of greater nutritional interest). The
results obtained in the hydrolyzate are different from those reported
in previous studies carried out by Nilsang et al. (2005), Souissi et
al. (2007); Ovissipour et al. (2009); See et al. (2011); and Taheri et
al. (2013). These variations could be due to the source of the raw
material used, the production process or even the season in which
the sh were caught. In this regard, Šližyte et al. (2014) indicated
that this aspect can be an inuencing factor in the quality of the nal
product.
Protein concentration and hydrolysis approximation
The results of the protein concentration and hydrolysis
approximation are shown in table 4. The statistical analysis showed
that there are no statistically signicant differences (p≤0.05) in the
protein concentration and in the hydrolysis approximation, at values
of pH 5.32 and 5.94. However, it can be observed that there is a
signicant difference (p≤0.05) for a pH of 6.33, these results suggest
that the pH has an important effect at the time of hydrolyzing. These
results agree with the studies carried out by Baez-Suarez et al. (2016)
This scientic publication in digital format is a continuation of the Printed Review: Legal Deposit pp 196802ZU42, ISSN 0378-7818.
Rev. Fac. Agron. (LUZ). 2022, 39(1): e223904. January - March. ISSN 2477-9407.
4-5 |
and Zapata et al. (2019). In addition, it can be observed that the
hydrolyzate prepared with a pH of 6.33 had a higher hydrolysis
approach, generating a reduction in concentration, which indicates
that the hydrolysis with this treatment was more effective.
Table 4. Protein concentration and hydrolysis approximation.
pH Protein concentration
mg.mL
-1
Hydrolysis approximation
(%)
5.32 2.38 ± 0.12
a
46 ± 2.50
a
5.94 2.64 ± 0.20
a
40 ± 4.40
a
6.33 1.22 ± 0.14
b
72 ± 3.10
b
a, b
Different letters symbolize statistically signicant differences (p <0.05).
The data represent the mean ± SD
Amino acid composition
The results of the composition of essential amino acids (EAA)
of the enzymatic hydrolyzate using the soluble sh as raw material
for its elaboration are shown in table 5. It can be seen that the
hydrolyzate using the Granozyme ACC® enzyme had a lower
EAA composition than that obtained by Bhaskar et al. (2008) and
Ovissipour et al. (2012). These results are as expected, because these
studies have used viscera or blood that contain a higher percentage
of protein to obtain the hydrolyzate. However, the composition of
amino acids obtained meets all the nutritional requirements of EAA
of the marine species Marsupenaeus japonicus (Teshima et al.,
2002), which is a promising result for the use of this type of by-
products in the supplement industry. nutritional.
Table 5. Essential amino acid composition of the hydrolyzate.
EAA
Hidrolizated
g.100g
-1
Bhaskar
et al.
(2008)
g.100g
-1
Ovissipour et
al. (2012)
g.100g
-1
EAA request
(Marsupenaeus
japonicus)
g.100g
-1
Arginine 1.80 10.82 8.81 1.40-1.80
Histidine 2.32 2.06 8.45 0.50-0.70
Isoleucine 1.40 3.6 6.93 1.10-1.40
Lisine 1.92 7.07 1.87 1.70-2.00
Methionine
Phenylalanine
Threonine
Valine
1.36
1.36
1.52
1.53
2.02
3.53
4.02
4.79
1.48
3.85
5.9
8.93
0.60-0.80
1.30-1.60
1.10-1.40
1.20-1.50
EAA: essential amino acids; sample number: 25
Conclusions
The enzymatic hydrolysis of the soluble sh had a better
performance at a pH of 6.33; With the implementation of this value,
the performance in obtaining large amounts of protein with this by-
product as raw material could be improved, generating added value
to it.
Soluble sh has proven to be a promising source of nutritional
supplements for marine species to take advantage of a by-product
that would otherwise be discarded causing environmental pollution.
However, future studies in various marine species are necessary to
determine its efcacy as a food additive or animal nutrition.
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