Inuence of pH in the enzymatic hydrolyzate of concentrates from the shmeal industry

Inuencia del pH en el hidrolizado enzimático de concentrados de la industria de harina de pescado

Inuência do pH no hidrolisado enzimático concentrados da indústria de farinha de

peixe

Jairo Corral Palacios

María Hipatia Delgado-Demera

Carlos Alfredo Cedeño-Palacios

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

Maestría en Ingeniería Química, Instituto de Posgrado,

Universidad Técnica de Manabí, Av. Urbina y Che Guevara,

Portoviejo, Manabí, Ecuador.

Departamento de Veterinaria. Facultad de Ciencias

Veterinarias. Universidad Técnica de Manabí, Ecuador.

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 inuence 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 ofcial 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 inuencia 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 ocial 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 inuência 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 ocial

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 efuent

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 efcient 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 Inuence 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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3-5 |

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 Ofcial 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 Ofcial 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 inuencing 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 signicant 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

signicant 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 scientic 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

46 ± 2.50

5.94 2.64 ± 0.20

40 ± 4.40

6.33 1.22 ± 0.14

72 ± 3.10

a, b

Different letters symbolize statistically signicant 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

1.52

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 efcacy as a food additive or animal nutrition.

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