Developing the technology and evaluating the biological value of the peptone from secondary products of processing of fish raw material of the arctic region

http://www.iaeme.com/IJMET/index.asp 1880 editor@iaeme.com

International Journal of Mechanical Engineering and Technology (IJMET)

Volume 10, Issue 03, March 2019, pp. 1880-1893. Article ID: IJMET_10_03_191

Available online at http://www.iaeme.com/ijmet/issues.asp?JType=IJMET&VType=10&IType=3

ISSN Print: 0976-6340 and ISSN Online: 0976-6359

DEVELOPING THE TECHNOLOGY AND

EVALUATING THE BIOLOGICAL VALUE OF

THE PEPTONE FROM SECONDARY

PRODUCTS OF PROCESSING OF FISH RAW

MATERIAL OF THE ARCTIC REGION

Ludmila Kazimirovna Kuranova, Vlasimir Aleksandrovich Grokhovsky, Yulia

Viacheslavovna Zhivlyantseva and Vasily Igorevich Volchenko

Department of Food Production Technology

Institute of Natural Science and Technology

Federal State Educational Institution of Higher Education "Murmansk State Technical

University"

Murmansk, Russian Federation

ABSTRACT

The results of microbiological and toxicological tests have proved the safety of the

bone-muscular cod waste (BMW). Waste (humerus with meat cuts) from cod cutting

on fillets contains 18,95 % of a full-grade animal protein and insignificant amount of

fat (0.15 %). Thus, the possibility of using them as a protein raw material for

producing peptones has been proved. The technology of obtaining peptone from the

secondary fish raw material - bone-muscular waste from the cutting of cod fish - has

been developed and optimized. Using the cryoextrusion method in the technology of

peptone production at the stage of grinding waste is proposed. Using of the enzyme

(protosubtilin G3X) in hydrolysis is substantiated; proteolytic activity is established, it

is 560,77 μmol TYR / g, the optimal temperature of enzymatic hydrolysis of BMW is

(45±1) ˚С.

Using the theory of experiment planning and computer modeling, a series of works

was carried out to optimize the stage of enzymatic hydrolysis of fish wastes. Nearly

optimal hydrolysis parameters were found: enzyme concentration of 1,33 % to total

waste weight, duration of hydrolysis process of 3 hours. The chemical and

biochemical quality characteristics of the enzymatic peptone obtained by the

optimized technology have been researched. It was found that the mass fraction of

protein in the product is of 92,27 %, water is of 4,7 %, sodium chloride is of 2,6 %, fat

is of 0,3 %. The amino acid composition has been determined with the method of high-

performance liquid chromatography. It made it possible to calculate the biological

value of peptone. Tryptophan has been established to be the only limiting amino acid

Ludmila Kazimirovna Kuranova, Vlasimir Aleksandrovich Grokhovsky, Yulia Viacheslavovna

Zhivlyantseva and Vasily Igorevich Volchenko

http://www.iaeme.com/IJMET/index.asp 1881 editor@iaeme.com

in the peptone protein (score is 70 %); which characterizes the peptone as a

sufficiently balanced protein product, which can be recommended for use in food as a

complete protein additives to food.

Keywords: Bone-muscular cod waste, Protosubtilin, Enzymatic peptone, Amino acid

composition, Triptophan, Balanced protein product.

Cite this Article Ludmila Kazimirovna Kuranova, Vlasimir Aleksandrovich

Grokhovsky, Yulia Viacheslavovna Zhivlyantseva and Vasily Igorevich Volchenko,

Developing the Technology and Evaluating the Biological Value of the Peptone From

Secondary Products of Processing of Fish Raw Material of the Arctic Region,

International Journal of Mechanical Engineering and Technology, 10(3), 2019, pp.

1880-1893.

http://www.iaeme.com/IJMET/issues.asp?JType=IJMET&VType=10&IType=3

1. INTRODUCTION

The problem of providing the population of Russia with the foodstuffs with the increased

nutrition and biological value, balanced іn micronutrients and containing biologically active

substances positively influencing functions of organs and tissues of the person, is faced now

by national economy of Russia and it is acute and very urgent [1, 2].

The need for protein is an evolutionary dominant in human nutrition, which is due to the

need to provide the optimal physiological level of essential amino acids. The quality of the

protein – its biological value – is determined by the presence in it of a complete set of

essential amino acids in a certain ratio. Essential amino acids are represented by amino acids

with carbon branch chain – leucine, isoleucine and valine, aromatic – phenylalanine,

tryptophan and aliphatic – threonine, lysine and methionine. Since the body synthesizes

cysteine and tyrosine from methionine and phenylalanine, respectively, then the presence in

food in sufficient quantities of these two amino acids reduces the need for essential

precursors. Partially nonessential amino acids include arginine and histidine, as they are

synthesized rather slowly in the body. A young and growing organism is particularly in need

of histidine. The absence of at least one essential amino acid in food causes a negative

nitrogen balance, disruption of the central nervous system, growth stop and severe clinical

consequences such as vitamin deficiency. Lack of one essential amino acid leads to

incomplete assimilation of others [3].

There is a need to develop new technologies for the development of non-traditional

nutrition for the fishing industry — protein products - peptones, which can be successfully

used as a protein component of foodstuff, feed of farm animals, birds, and aquaculture

facilities, nutritional and diagnostic microbiological environments, pharmaceutical and

cosmetic products [4, 5]. Peptons are large protein fragments that are formed during

hydrolysis. It is a protein-containing product, the amount of "total" protein in which is not less

than 85 %, and "true" protein – not less than 75 %, i. e. protein isolate [3]. The creation of

new types of products based on peptones will partly solve not only the problem of

environmental pollution, but also the problem of protein deficiency.

It is known that enzymatic hydrolysis of fish proteins forms a mixture of free amino acids,

di -, tri-and oligopeptides, the number of polar groups and the solubility of peptone increases,

and therefore changes the functional characteristics of proteins, improving their functional

properties and biological value. This is important if the peptone is used as a food ingredient

[6].

Developing the Technology and Evaluating the Biological Value of the Peptone From Secondary

Products of Processing of Fish Raw Material of the Arctic Region

http://www.iaeme.com/IJMET/index.asp 1882 editor@iaeme.com

The pace of modern life dictate their own conditions and, unfortunately, people do not

always manage to eat the right, balanced food, observe the regime, take sports. In nature,

there are only a few equivalent sources of high-grade protein of the highest biological value:

milk, egg, meat, fish and soy. The finiteness of natural resources leads to the fact that the

increase in the volume of animal protein produced lags behind the growing needs of the

population.

Today, the volume of fish catch in Russia in absolute terms is 4.2 million tons. However,

up to 28 % of the catch (or more than 1 million tons) is not used for food purposes. Due to the

development of new industrial projects in the Arctic Region, a significant increase in traffic

volume along the Northern Sea Route can be currently observed. To protect the environment

against polluting emissions from shipping, now, requirements to the quality of marine fuel are

being updated and tightened, new environmental regulations worked out. The strictest

regulations are applied to some air pollutant Emission Control Areas (ECA). In Europe, ECA

include Baltic Sea and Northern Sea areas, in the Northern America – Pacific and Atlantic

coast areas [7]. One of the most significant examples of intensive anthropogenic load on the

natural landscape is the metallurgical complex “Severonikel” (Murmansk Region). The

metallurgical complex is located on the Kola Peninsula in close proximity to the

administrative border of the Russian Federation with Finland [31]. Deposition of heavy

metals with the atmospheric precipitation in the form of acid rain is the main way of their

ingress from atmospheric air into soil and surface water bodies [8].

In this regard, of particular importance and relevance there are the studies aimed at the

development of safe food protein products from hydrobionts, involving in the processing of

secondary products formed in the processes of traditional processing of hydrobionts, in

particular, bone-muscular waste from cutting cod fish species on fillets. Atlantic cod (Gadus

morhua) is a traditional object of fishing in the Arctic region. The chemical composition of

cod is well studied. The average protein content in the muscle tissue of cod varies within 16–

18% [9, 10]. The component composition of tissues is characterized by low fat content (less

than 1 %). Waste from cutting into fillets include: skin cover and scales, fins, rib bones,

vertebral bone, head, entrails and tail. Fish waste is an important reserve of food raw

materials, which is often underestimated [11, 12]. Currently, in the literature there are works

devoted to the study of their chemical and biochemical composition [4, 13, 14]. However, in-

depth study of the properties and possibilities of deep processing of bone-muscular waste

from cutting of traditional raw materials was practically not carried out.

This implies the purpose of this work, which is associated with the study of bone-

muscular waste from cutting cod for its subsequent use in the creation of innovative

technologies for processing hydrobionts — development of peptone technology, optimization

of process parameters, the study of the quality of the fish peptone, the study of its biological

value and directions of use.

The possibility of using peptones obtained from secondary fish raw materials will allow to

preserve valuable protein products in the food ration, reduce the volume of non-recyclable

waste and expand the range of products from hydrobionts used for food, feed and medical

purposes.

2. OBJECTS, MATERIALS AND METHODS OF RESEARCH

The objects of research were: bone-muscular cod waste (BMW); obtained by cutting fish into

a carcass by separating the head with the nape (cod caught by the Public Joint-Stock

Company "Murmansky Trailing Fleet" in the fishing areas of the Barents sea, was frozen and

delivered to the port of Murmansk, where it was stored for 1 month at a temperature not

Ludmila Kazimirovna Kuranova, Vlasimir Aleksandrovich Grokhovsky, Yulia Viacheslavovna

Zhivlyantseva and Vasily Igorevich Volchenko

http://www.iaeme.com/IJMET/index.asp 1883 editor@iaeme.com

higher than minus 18°C); protosubtilin G3X – industrial enzyme preparation – a product of

bacteria strain Bacillus subtilis; pepton (PBMW), obtained in the process of enzymatic

hydrolysis of BMW according to the technology developed by the authors [15].

Chemical reagents, amino acid standards for chromatography and standard samples for

atomic absorption spectroscopy were purchased from Sigma Aldrich (Germany) for chemical

analysis [30].

Chemical and biochemical methods accepted in scientific researches are used in the work.

The mass fraction of water, lipids, protein, amine nitrogen, minerals of raw materials was

determined by standardized methods. The protein content was determined by the Kjeldahl

method using equipment consisting of two elements: BLOCK–DIGEST–12 for sample

mineralization and automatic distillation unit PRO–NITRO A (J.P. Selekta, Spain). The fat

content was determined by the Soxhlet method using the Selecta DET/GRAS extractor

(Spain). Amino acid composition of the peptone was determined by hydrolysis of the sample

with hydrochloric acid or alkali upon heating [16], subsequent modification of the resulting

amino acids with phenylisothiocyanate, separation of phenylthiocarbamyl amino acid

derivatives on a column with reversed phase followed by registration with SPD-20AV

spectrophotometric detector on liquid chromatograph LC-20 Prominence of Shimadzu (Japan)

[32].

The content of lead, cadmium, arsenic, mercury was determined by atomic absorption

spectrometry [17] on the atomic absorption spectrometer Shimadzu AA-6300 (Japan).

Proteolytic activity of the enzyme preparation was determined by the method based on

spectrophotometric determination of dissolved amino acids and peptides formed during

enzymatic hydrolysis of casein [18].

The degree of hydrolysis was determined by calculation as the ratio of the mass fraction of

amine nitrogen to the mass fraction of total nitrogen in the hydrolyzate [19].

Amino acid score (AAS) [19, 21] was calculated by the formula:

AAS= 𝑚1

𝑚2

∙100 , % (1)

where:

m1 – content of essential amino acid in 1 g of peptone protein, mg/g of protein,

m2 – content of essential amino acid in 1 g of reference protein, mg/g of reference protein.

When microbiological control of the samples we tested (according to standard

microbiological techniques) for the mesophyll aerobic and optional-anaerobic

microorganisms microorganisms (QMAFAnM), the presence of coliform bacteria (coliforms)

of the genus Staphylococcus aureus, the presence of pathogenic organisms, including of the

genus Salmonella, Listeria monocytogenes, yeast and mold fungi.

In determining the optimal parameters of enzymatic hydrolysis, a second-order rotatable

composite plan was used for two factors [22]. Mathematical data processing was performed

using DataFit version 9.1

2.1. Statistical analysis

The experiments were repeated three times, and the data obtained were subjected to single-

factor analysis of variance (ANOVA) using Origin Pro 8.0. Differences between averages

were considered significant at p  0,05.

3. RESEARCH RESULTS AND DISCUSSION

Developing the Technology and Evaluating the Biological Value of the Peptone From Secondary

Products of Processing of Fish Raw Material of the Arctic Region

http://www.iaeme.com/IJMET/index.asp 1884 editor@iaeme.com

Currently, there are many technologies for obtaining peptones. As a raw material, any natural

proteins full of amino acid composition are used, the sources of which are blood and its

components; tissues of animals and plants; waste of dairy and food industries; food and low-

value food products obtained in the processing of various species of animals, birds, etc. [23].

At the same time, there is another promising source that can be used for the production of

peptones – bone-muscular waste generated during the cutting of cod fish species in the sea.

According to the existing standards of fishing waste should be frozen and delivered to the

shore for disposal, but due to the closure of plants for the production of fish meal, the issue of

its processing has not yet been resolved.

The authors conducted a comprehensive study of the quality of waste obtained from

cutting cod. Analysis of the chemical composition found that the protein content in the waste

is 20.2 % (due to a significant number of cuts of meat), fat content - 0.31 %.

The content of toxic elements in the bone-muscular cod waste was: lead - less than 0.05

mg/kg, cadmium - less than 0.005 mg/kg, arsenic - less than 0.05 mg/kg, mercury - 0.028

mg/kg, copper - 0.83 mg / kg.

Microbiological studies have established the absence of dangerous microorganisms for the

human body: coliform bacteria, Staphylococcus aureus, pathogenic Salmonella and Listeria.

The results of chemical and microbiological tests indicate the safety of bone-muscular cod

waste, and thus confirm the possibility of using them as additional raw materials in the

production of food. Taking into account the results of their own research and literature data,

the authors developed a technology for the production of peptones from bone-muscular cod

waste by enzymatic hydrolysis.

The basic technological scheme of production of peptone from bone-muscular waste is

reduced to the following operations: thawing of bone-muscular waste, crushing, fermentation,

purification from the bone residue and non-hydrolyzed protein by acidification and

alkalization with gradual filtration, drying.

One of the stages of the technological process is the production of ground minced mass.

To this end, traditionally the raw material is subjected to defrosting and grinding. This is quite

time-consuming and lengthy process, during which there is a decrease in protein content due

to the loss of cellular juice. To reduce energy and labor costs, the method of grinding raw

materials by cryoextrusion on the layout of the plunger with a die, 50 mm in diameter, cooled

to the temperature of the raw material was used. The method of cryoextrusion is a crushing of

frozen raw materials by punching through a cooled die hole, and cutting the fibers of the

muscle tissue of the raw materials by the ice crystals [24]. The use of cryo-grinding eliminates

the defrosting of raw materials, which avoids the loss of raw materials and preserve its

properties.

Studying the manufacturing process of enzymatic peptone, it is impossible to take into

account all the conditions affecting the fermentation process. Important factors affecting the

hydrolysis process are the temperature and duration of the process, as well as the quality and

quantity of the enzyme administered.

For protein hydrolysis as an enzyme preparation, various enzymes can be used. So

Zubtsov V. A and others [25] carried out pepsin and trypsin hydrolysis of animal products.

Gastric mucosa and pancreas were used as the enzyme source. Sultanov and others [26]

conducted pepsin hydrolysis, as raw materials they uses fish (sprat, pollock, salmon) or

feeding meal. E. Dimova and others [27] carried out the hydrolysis of the skin and blood of a

calf by using alkaline proteases of bacterial origin (Bacillus subtilis strain DY). Antipova L.

V. and others [28] conducted hydrolysis of secondary products of cutting pond fish using the

Developing the technology and evaluating the biological value of the peptone from secondary products of processing of fish raw material of the arctic region

The results of microbiological and toxicological tests have proved the safety of the bone-muscular cod waste (BMW). Waste (humerus with meat cuts) from cod cutting on fillets contains 18,95 % of a full-grade animal protein and insignificant amount of fat (0.15 %).

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