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ACTA

Acta Sci. Pol., Technol. Aliment. 6(1) 2007, 5-16

SPECIES IDENTIFICATION OF MEAT

BY ELECTROPHORETIC METHODS

Magdalena Montowska, Edward Pospiech

Agricultural University of Pozna ı

Abstract. Electrophoretic methods can be used to identify meat of various animal species.

The protein electrophoresis, especially the IEF of the sarcoplasmic proteins, is a well-

established technique for species identification of raw fish and is used in the control of

seafood authenticity. However, in the case of the analysis of heat-processed fish, the

method is applicable only to those species which possess characteristic patterns of the

heat-stable parvalbumins. Heat-denatured fish muscle proteins may be solubilised by urea

or sodium dodecylsulfate (SDS) and separated by urea-IEF or SDS-PAGE, respectively.

The comparison of these two methods allowed to conclude that, basically, each of them

can be used for species identification of heated fishery products. However, extensively

washed products may be preferentially analysed by the SDS-PAGE, because most of the

parvalbumins are washed out leaving mainly myosins. On the other hand, the IEF method

may be preferred for the differentiation of closely related species rich in parvalbumins

isoforms. It is evident from the literature data that species-specific protein separations

yield proteins of low molecular weight made up of three light chains of myosin (14-23

kDa), troponin (19-30 kDa) and parvalbumin (about 12 kDa). Investigations showed that

the SDS-PAGE method can be used to identify meats of: cattle, sheep, lambs, goats, red

deer and rabbits. The technique allowed researchers to identify the following myofibrillar

and sarcoplasmic muscle proteins: myosin and actin, ŋ -actinin, tropomyosin, troponin.

SDS-PAGE allowed the identification of myofibrillar proteins taking into account their

molecular weights which was not possible with the assistance of the PAGIF because too

many protein bands were obtained. It was possible to obtain differences in the separation

of proteins characteristic for certain species, e.g. beef, resulting from the presence of sin-

gle myofibrillar proteins.

Key words: IEF, SDS-PAGE, species identification

INTRODUCTION

In recent years, species identification of meat has been gaining in practical impor-

tance. This has been caused, on the one hand, by a lack of confidence of consumers

Corresponding author – Adres do korespondencji: Mgr Magdalena Montowska, Institute of Meat

Technology of Agricultural University of Pozna ı , Wojska Polskiego 31, 60-624 Pozna ı , Poland,

e-mail: magmont@au.poznan.pl

M. Montowska, E. Pospiech

regarding the origin of marketed products and, on the other hand, by the concern of

producers to ensure the safety of the consumers’ products. Unfortunately, without an

effective system of control of food articles available on the market, consumers cannot

be certain of their appropriate quality as well as the absence of the harmful effect of

these articles on the human organism. The need for meat species identification derives

from the lack of confidence regarding the statement of the manufacturer concerning the

meat species used in a given product, occurrence of allergies to the protein of certain

animal species as well as some religious considerations (Hindus do not eat beef, follow-

ers of Islam and Judaism – pork) as well as the prohibition of trading of meat derived

from animal species under protection or threatened with extinction.

It is necessary to control meat products which were subjected during their manufac-

ture to various technological processes causing significant property changes of the ap-

plied tissues and raw materials. It is not uncommon to add to such products various

plant raw materials which may, additionally, contribute to difficulties in the process of

their identification. It may also be necessary to determine the percentage composition of

an article manufactured using different meat species.

The optimal identification method should allow the identification of meat species,

both in raw materials and in processed meat products containing animal - and plant

derived components. It is also important that the applied method should make it possi-

ble to identify meat species in products subjected to thermal processes. Furthermore, the

employed method should be sensitive and simple.

Methods applied currently for the identification of muscle tissues of unidentified

species are frequently based on different indices and, therefore, the results obtained with

the assistance of these methods cannot be compared in practice. The essence of these

methods is to show the presence of typical constituents or their proportions characteris-

tic for the muscle tissue of a given species.

The following methods can be employed to determine the species of meat, its com-

position and origin:

– electrophoretical,

– immunological,

– chromatographic,

– genetic.

The presented article discusses examples of the species identification of products of

animal origin with the assistance of electrophoretic methods.

THE ESSENCE OF ELECTROPHORETIC METHODS

The electrophoretic methods are based on the separation of proteins in the electric

field following their extraction from the muscle tissue and later placed on special media.

Initially, starch gels were employed and later polyacrylamide and agarose ones. At the

present time, the electrophoretic separation can be conducted on polyacrylamide gels

(PAGE), on polyacrylamide gels containing a denaturing agent (sodium dodecyl sulfate)

(SDS-PAGE) or by isoelectric focusing (IEF) on the agar or polyacrylamide gel

(PAGIF).

The IEF method consists in the separation on a gel which is characterised by the pH

gradient resulting from the addition of ampholyte. Individual proteins move towards the

Acta Sci. Pol.

Species identification of meat by electrophoretic methods

pH value which is in agreement with their isoelectric point (Ip). The PAGE electropho-

resis is also referred to as native electrophoresis due to the absence of a denaturing

agent. In this method, the protein separation depends on the electrical charge and sizes

of protein molecules. Individual proteins move towards the anode or cathode depending

on their charge. In the case of the SDS-PAGE, protein molecules with a negative charge

imparted onto them by the SDS move only towards anode with the speed depending,

with few exceptions, primarily on their molecular weight. There is a linear correlation

between the distance travelled by the proteins and the value of the decimal logarithm of

their molecular weight which makes it possible to determine the molecular weight of

proteins. With the assistance of two-dimensional electrophoresis (2-DE), it is possible to

identify meat of various related species of fish, birds and mammals. The obtained pro-

tein separation is visible with naked eye (in the case of proteins containing colour com-

ponent) or following appropriate staining. In the latter case, the most common dyes

include: Coomasie blue, silver salts or enzymatic staining [Hofmann 1997].

APPLICATION OF THE IEF METHOD

Protein separation with the assistance of the IEF method is influenced by many fac-

tors, among others, by: meat pH and meat defects, age and gender of animals, way of

nutrition, rearing conditions, meat storage conditions, activity of native or microbiologi-

cal proteases as well as the occurrence of polymorphism. It is possible to carry out sepa-

ration focusing on a single constituent, e.g. myoglobin which is a species specific mus-

cle dye independent of the above-mentioned factors and whose chains are relatively

short. Employing myoglobin as an indicator of species, the same separation can be ob-

tained by examining different muscles of the same animal but also by different animals

of the same species. It was demonstrated that in the case of meats of such animal spe-

cies as: cattle, horses, pigs, sheep, roe deer, kangaroos, camels, brown bears, rabbits,

hares, chickens, ducks and ostriches the set of myoglobin bands is characteristic and

they can be identified on the basis of myoglobin patterns [Hofmann 1997]. In the case

of closely related animals, e.g. pig and wild boar, roe deer and fallow deer, the myoglo-

bin patterns are so similar that they rule out possibilities of their identification. The IEF

technique can be employed for the identification of related animals with low myoglobin

content, for example, a chicken and turkey, using pseudo-peroxidase staining with the

aid of a mixture composed of odianizidine and hydrogen peroxide. Some to several

minutes after the treatment, red-brown bands appear on the gel. The IEF method based

on the myoglobin analysis is also effective in the situation of mixtures composed of

different species. Effective electrophoretic separation is possible when fresh meat, mus-

cle fluid as well as lyophilised muscle extracts derived from animals of the same species

are used. The band distribution is always identical and the conditions of storage, freez-

ing and de-frosting do not influence the results [Hofmann 1997].

When the IEF method is used for the species identification of meats subjected to

thermal treatment, the obtained results are influenced by the height of the temperature

during the technological process. Although high temperature does not affect the reloca-

tion of the isoelectric points, the bands are not sharp and this may make the assessment

more difficult. Therefore, it is recommended to conduct the analysis on one gel onto

which the examined samples as well as the reference sample should be placed. In the

Technologia Alimentaria 6(1) 2007

M. Montowska, E. Pospiech

case when the tissue was heated to less than 100°C, pseudo-peroxidase staining is suffi-

cient. However, when the applied temperature exceeded 100°C, one of the possibilities

is staining with silver compounds. Staining with silver compounds turned out effective

in the case of the identification of: cattle, swine, sheep, horse and roe deer semi-

processed and processed meats as well as in the case of processed articles consisting of

muscle tissues derived from different animal species, provided the proportion of a given

tissue is sufficient, e.g. pork and beef can be identified in mixtures subjected to thermal

treatment if their proportions exceed 10% [Hofmann 1997].

APPLICATION OF THE PAGE AND SDS-PAGE METHODS

PAGE electrophoresis can be used for the protein identification of meats derived

from: pigs, cattle, horses, sheep, fish, reindeer, moose, goats and bears. The method can

be employed for the identification of meats of related animal species, including wild

animals, provided that the examined material was not subjected to thermal treatments.

The identification of meat additives of different animal species in processed products

heated to the temperature of 70°C is possible on condition that the examined proteins

are dissolved in a 6 M solution of guanidine chloride and employment of isoelectric

focusing followed by enzymatic staining [Pyz-Łukasik 1998].

The SDS-PAGE (Sodium Dodecyl Sulphate – Polyacrylamide Gel Electrophoresis)

method makes it possible to analyse proteins which dissolve poorly in solvents other

than SDS solutions. This method can be applied to analyse proteins which do not dis-

solve in urea-containing solutions. The method can be utilized for protein quality analy-

ses, for example, proteins of various species of fish or for the quantitative analysis

which takes into account different degrees of binding of the applied dye by individual

proteins. This method is not very convenient because the obtained results can be influ-

enced by many factors, among others, by: age, way of animal nutrition, stress, meat

quality deviations as well as the lack of standards since the lyophilized meat extract

which might be used as the reference can yield different results than the extract from

fresh meat [Minkiewicz et al. 2004].

EXAMPLES OF SPECIES IDENTIFICATION OF MAMMALIAN MEAT

Identification techniques of meats derived from different animal species are based

on the examination of muscle extracts. The employed techniques use either muscle drip

or squeezed juice which contain most of the soluble meat proteins and even some struc-

tural proteins released into them either as a result of the applied technological processes

or factors affecting the animals during the short period before slaughter. Among the

electrophoretic methods, the most suitable for the comparison of proteins from different

animal species are: PAGE, SDS-PAGE and PAGIF [Parisi and Aguiari 1985].

Investigations carried out by Parisi and Aguiari [1985] showed that the SDS-PAGE

method can be used to identify meats of: cattle, sheep, lambs, goats, red deer and rab-

bits. The technique allowed researchers to identify the following myofibrillar and sar-

coplasmic muscle proteins: myosin and actin, ŋ -actinin, tropomyosin and troponin.

Acta Sci. Pol.

Species identification of meat by electrophoretic methods

SDS-PAGE allowed the identification of myofibrillar proteins taking into account their

molecular weights which was not possible with the assistance of the PAGIF because too

many protein bands were obtained. It was possible to obtain differences in the separa-

tion of proteins characteristic for certain species, e.g. beef, resulting from the presence

of single myofibrillar proteins.

Hofmann [1985] obtained somewhat different results using the SDS-PAGE method.

The aim of his experiments, in which muscles and their water extracts were used, was to

identify and compare several muscles derived from cattle, pigs and horses. Separations

of the muscle proteins of these three animal species showed identical bands. Distinct

differences were observed in the intensity of the stained bands. These differences were

also observed between the protein separations of different muscles derived from the

same species. A considerable variability in the intensity of the myoglobin bands derived

from different muscles of the same species confirmed their wide diversification with

regard to the myoglobin content. On the basis of his experiments Hofmann [1985] con-

cluded that the SDS-PAGE method cannot be employed for the species identification of

muscles derived from cattle, pigs and horses. However, simultaneously, he conducted a

number of studies with the aim to check possibilities of the identification of different

animal species, i.e. cattle, pigs, horses, red deer, roe deer, fallow deer and wild boar

using the isoelectric focusing on the polyacrylamide gel (PAGIF). Protein bands in the

swine and wild boar separations were situated almost at the same positions but differed

with regard to their intensity. No apparent differences were found between separations

of the roe deer and fallow deer, while those of the red deer and roe deer differed with

regard to the intensity of bands which were less intensively dyed in the upper range of

the separation. Bearing in mind the results of the above experiments, it is not at all cer-

tain if the IFE is capable of differentiating closely related species. It was further found

that different muscles derived from the same species can yield different separations. In

experiments conducted on other species which were not closely related (rabbit, duck,

chicken), the obtained separations were more diversified and there were no problems

with their species identification [Hofmann 1985].

In further investigations, the IEF of soluble proteins of: cattle, pigs, wild boar, horse,

roe deer, fallow deer, rabbit and chicken was employed for the species identification on

the basis of the determination and comparison of the bands of myoglobin which is a

species specific muscle dye and whose chains are relatively small with regard to their

molecular weight. The separations of myoglobin bands can be observed without gel

staining. In order to achieve greater diversity in the distribution of myoglobin bands, an

ampholyte with a narrow pH 5-9 was applied. Moreover, attempts were made to achieve

better separations by the application of higher protein concentrations using squeezed

juice instead of the muscle extract and employing 1 mm thick gels. The determined

species yielded characteristic separations of two or more bands of myoglobin. Different

muscles gave identical separations. Wild boar separations were poorly visible because

the employed extracts derived from frozen meat stored for three years. The remaining

samples were either fresh or frozen for a short period of time. The obtained myoglobin

separations were distinctly characteristic for the determined species [Hofmann 1985].

Technologia Alimentaria 6(1) 2007

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