AN INFLUENCE OF SPONTANEOUS MICROFLORA OF FERMENTED HORSEMEAT PRODUCTS ON THE FORMATION OF BIOLOGICALLY ACTIVE PEPTIDES

At present, different methods are used to accumulate functional peptides in meat raw materials, including the use of spontaneous microflora during autolysis, the use of the microbial enzymes (the application of starter cultures) and the use of the non-microbial enzymes (enzymes of animals and plant origin). Each method has its own specific characteristics of an impact on raw materials, which requires their detail study. This paper examines an effect of spontaneous microflora of fermented meat products from horsemeat on formation of biologically active peptides. Using the T-RFLP analysis, it was established that in air dried and uncooked smoked sausages produced with the use of the muscle tissue of horsemeat as a raw material, a significant proportion of microflora was presented by lactic acid microorganisms. The highest content of lactic acid microflora was observed in sample 1 (52.45 %), and the least in sample 3 (29.62 %). Sample 2 had the medium percent content of microflora compared to samples 1 and 3 — 38.82 %. It is necessary to note that about 25 % of microflora was unculturable; i.e., it had metabolic processes but did not grow on culture media. In the samples, the representatives of Actinobacteria and Pseudomonadales were found. Pathogenic and conditionally pathogenic microflora was not detected. Not only quantitative but also qualitative changes were observed in the studied samples. For example, in samples 1 and 2, the fractions of amilo-1,6-glucosidase, fast-type muscle myosin-binding-protein C; glucose-6-phosphate isomerase; fast skeletal muscle troponin I, phosphoglycerate kinase, pyruvate kinase and skeletal muscle actin were found, which were absent or reduced in sample 3. Therefore, in the studied product, good preservation of the main spectra of muscle proteins was observed, and the identified fractions, apparently, can be sources of new functional peptides. Not only quantitative but also qualitative changes were observed in the studied samples. For example, in samples 1 and 2, the C-terminal fragments of the myosin heavy chain were found, which were absent in sample 3. Also, the significant content of myoglobin was revealed in samples 2 and 3, and the myosin light chain was found in sample 1. Therefore, in the studied product, good preservation of muscle proteins myosin and myoglobin, which can be a source of new functional peptides, was observed. Based on the results of tandem mass-spectrometry, the proteins and natural short peptides present in the analyzed extracts were identified by the obtained masses. They belonged mainly to different peptides of equine myoglobin. Also, we identified several fragments, among which fast skeletal muscle troponin T and muscle creatine kinase were found. The obtained materials can be regarded as an experimental basis for the directed impact of starter cultures with a possibility to predict the protein and peptide composition of a finished product including with the aim of obtaining biologically active peptides. Irina M. Chernukha1, Il’ya N. Nikonov2, Natal’ya G. Mashentseva3, Daria L. Klabukova4,* Dmitrii A. Afanasev3, Leonid I. Kovalyov5, Larisa A. Ilina2 1 V.M. Gorbatov Federal Research Center for Food Systems of Russian Academy of Sciences, Moscow, Russia 2 «BIOTROPH» Limited, St. Petersburg, Russia 3 Moscow state university of food production, Moscow, Russia 4 Institute of Applied Biochemistry and Mechanical Engineering «Biochimmash», Moscow, Russia 5 Federal Research Centre «Fundamentals of Biotechnology» of the Russian Academy of Sciences,


Introduction
At present, many strategies were developed to improve the functional value of meat and meat products, which can be realized by addition of different functional compounds and well as by ante mortem modification of animal raw materials.The technology of functional and specialized products successfully uses the modification of the composition (amino-and fatty acids, control of sodium chloride), incorporation of functional elements and specialized modules (plant components (oils, extracts, fibers), soy protein, natural and synthetic antioxidants, lactic acid bacteria, fish oil and meat protein derivates (biologically active peptides).The modern strategy directed towards an increase in longevity due to a decrease in the risk of chronic noncommunicable diseases includes prediction, prevention, personalization and participativity (the principle of P4 medicine) [1].With that, the scientific forecast about an increase in an average life expectancy of humans is based on many directions; however, the central place is occupied by the development of effective and safe prophylaxis of socially significant diseases.In Russia, chronic noncommunicable diseases are a cause of 75 % of all deaths.With that, the cardiovascular diseases account for about 55 % [2].
Over the last 10 years, extended studies on the substances of the protein and peptide nature in meat raw materials and finished meat products [3,4] have been carried out.These compounds are formed in the process of different technological processing and stipulate quality and functional characteristics as well as safety of foods.However, the complex investigations on the mechanisms of their biosynthesis and degradation at the molecular level are practically not highlighted.The strategy for studying meat proteins as potential sources of biopeptides consists in investigation of proteome regarding the presence of functional sequences and metabolites formed during the autolysis process, fermentation by starter cultures as well as hydrolysis of meat raw materials with the human gastrointestinal enzymes using the methods of applied proteomics and bioinformatics [5,6].
The current trends in food biotechnology and meat biochemistry are directed towards investigations regarding detection and identification of proteins and peptides characterizing quality characteristics in all raw material sources of animal and plant origin [7,8].At present, a great number of polypeptide substances containing about 2-30 amino acids, which exist initially in meat raw material or were formed during technological processing, have been extracted from meat raw materials, especially from beef, chicken and pork muscle proteins.A range of short peptides was revealed, which had hypotensive, opioid, antioxidant, antithrombotic and other biological effects that influenced a range of the most general pathogenetic mechanisms underlying the development of pathological processes [6,9,10].For example, meat proteins contain amino acid sequences with the hypotensive activity (chicken myosin, beef collagen α1 and pork troponin C), stimulators of the ubiquitin-mediated proteolysis (chicken myosin, beef collagen α1 and pork troponin C), antiamnesic and antithrombotic activities (beef collagen α1), antibacterial (chicken myosin), immunomodulatory (beef collagen α1) and opioid properties (beef collagen α1, pork troponin C), inhibitors of dipeptidyl peptidase IV (chicken myosin, beef collagen α1, pork troponin C), as well as regulators of the activity of the gastric mucosa (beef collagen α1).For example, myosin light chain has the sets of the amino acid sequences with the antimicrobial properties: connectin is rich in the peptides with the antithrombotic, antiamnesic, opioid, neuroprotective, immunomodulatory, antioxidant and hypotensive activities, as well as inhibitors of dipeptidyl peptidase IV and regulators of the activity of the gastric mucosa.Beef, chicken and pork actin carry the sequences -inhibitors of dipeptidyl peptidase IV.Collagen and elastin are the richest in the sequences having specific corrective properties due to the high content of glycine and proline.More than 220 functional peptides have been already identified in the above mentioned proteins.Presumably, the hypolipidemic effect is conditioned by an influence on expression of the genes responsible for lipid metabolism.It was shown that the pork elastin hydrolysate reduces the concentration of total cholesterol and atherogenic lipoprotein classes in blood serum of the rats with a model of hypercholesterolemia.This effect is assigned to peptides with the low ratio of methionine/glycine and lysine/ arginine, as well as formation of lysine bridges (desmosin and isodesmosin).In addition, it was demonstrated that the pork liver hydrolysate reduced the subcutaneous fat level due to suppression of the activity of liver enzymes -participants of lipogenesis.The chicken leg collagen hydrolysate has a positive effect in osteoporosis during menopause, chicken meat and bone hydrolysates facilitate better gut colonization with bifidobacteria and can be used as prebiotics; collagen hydrolysates stimulate proliferation of fibroblasts, neutrophils and monocytes, which is a part of immunocorrective action [11,12].
A number of methods are used for accumulation of functional peptides in raw meat including autolysis or direct hydrolysis, as well as fermentation (using spontaneous microflora, starter cultures or enzymes).
It is noted that genome of lactobacilli codes more proteases, peptidases, amino acid permeases and oligopeptide transport systems than lactococcus [17].For example, the oligopeptide transport system of L. lactis transports peptides, at least, up to 18 residues [18].
However, proteolytic enzymes released by lactic acid bacteria were very different in different species and strains, which led to emergence of different groups of bioactive peptides [19,20].
In the majority of studies of bioactive substances from meat sources, the main attention was paid precisely to ACE inhibitory and antioxidant peptides.In fermented meat products a special attention is paid to the control of biogenic amines: tyramine, cadaverine, putrescine and histamine.Improper processing that favors contamination is a main cause of too high content of bioamine in meat; however, the literature also describes starter cultures that have an ability to synthesize biogenic amines, such as Lactobacillus curvatus [22].To prevent this risk, it is necessary to take into consideration selection of individual starter cultures having the aminooxidase activity and capable of bacteriocine synthesis with the aim of leveling undesirable effects of spontaneous fermentation.
This paper examines an effect of spontaneous microflora of the fermented meat products from horsemeat on the development of the biologically active peptides.

Materials and methods
At the first stage, an analysis of microflora by terminal restriction fragment length polymorphism (T-RFLP) was carried out in three naturally fermented meat products from horsemeat: • the national fermented product «Kazi» produced in Penzenskaya oblast.The composition of the product includes horsemeat and horse fat with addition of salt, sugar and garlic.Horsemeat was obtained from animals raised on pastures (lots 1 and 2; samples 1 and 2, respectively); • the uncooked smoked meat product «Balyk» from horsemeat produced by TS9213-028-54780900-2011 in an enterprise in the Moscow district.The composition of «Balyk» includes horsemeat, the nitrite-curing mixture, garlic, spices, sugar, sodium ascorbate (sample 3).For analysis of the product microflora, 3 samples of 1 g were taken from the inner part of the sausage in the sterile conditions 5 days after preparation.From these specimens, an average specimen was prepared by homogenization in a ceramic mortar.The DNA was recovered from the specimen by extraction with phenol/chloroform in a ratio of 1:1 and purification with the CTAB solution.
For DNA recovery, 0.5 g of the average specimen was transferred into an Eppendorf 1.5 ml tube with a screw cap.500 µl of buffer I (CTAB2 %; Tris-HCl 0.1M; EDTA-Na 2 20 mM; NaCl 1.4 M; pH 8.5) and 0.5 g of glass beads (Helicon, Russia) were added to the specimen.The specimen was heated at 65 o C for 15 min.and homogenized on a personal Vortex V-1 plus (Biosan, Latvia) at 3000 rpm for 15 min.;then, the heating process was repeated during 15 min.After that, the specimen was extracted at 14000 rpm for 10 min.in the centrifuge Mini Spin (Eppendorf, Germany) with 400 µl of phenol/chloroform mixture (1:1), then 400 µl of chloroform, each time transferring the supernatant into new Eppendorf 1.5 ml tubes.After that, DNA was settled in a centrifuge at 14000 rpm with 400 µl of 96 % ethanol in the presence of the sodium acetate solution to the final concentration of 0.3 М (Helicon, Russia) and dissolved in 100 µl of TE buffer (Tris-HCl 10 мM; EDTA-Na 2 1 mM) (Helicon, Russia).
The amplified fragment was isolated from the agarose gel using the 3М guanidine thiocyanate solution.To this end, an agarose block with the amplified DNA fragment was cut from the agarose gel and put into Eppendorf 1.5 ml tubes.100 µl of solution A (3М guanidine isothiocyanate, 20 mM EDTA-Na 2 , 10 mМ Tris-HCl (pH 6.8), 40 mg/ml of TritonX-100) (Helicon, Russia) was added to the block and heated to 65 °C until full dissolution of the agarose block.Then, the sample was mixed, 20 µl of solution B (1000 µl of solution А, 40 mg/ml of DNA sorbent Silica) (Helicon, Russia) were added and incubated at the room temperature for 10 min.with intermittent mixing.After that, the amplicon was settled with a sorbent in the centrifuge Mini Spin (Eppendorf, Germany) at 4000 rpm for 1 min.and the solution was fully removed.Similarly, the sediment of silica with DNA was washed with 100 µl of solution A, C (25 % C 2 H 5 OH, 25 % isopropanol, 100 mМ NaCl, 10 mМ TRIS-HCl, pH 8.0) (Helicon, Russia) and 70 % ethanol.After that, the sediment was dried and the DNA was eluted in 100 µl of 10 mМ Tris-HCl solution (pH 8.0) (Helicon, Russia) for 15 min at the room temperature.Then, the solution was centrifuged at 14000 rpm for 3 min.and the purified DNA preparation was transferred into new tubes.
Restriction of amplicons was carried out with restriction enzymes HaeIII, HhaI and MspI (Fermentas, USA) at 37 °C for 2 hours.The total volume of the reaction mixture was 15 µl: 10 units of the restriction enzyme, 1.5 µl of buffer for restriction and up to 15 µl of deionized water.On completing restriction, DNA in the reaction mixture was settled with ethanol in an amount of 38 µl in the presence of 1.5 µl of 3M sodium acetate solution, then dissolved in 10 µl of SLS (BeckmanCoulter, USA) with addition of 0.2 µl of marker with the molecular weight of 600 bp (Beckman-Coulter, USA) and separated in the conditions of capillary electrophoresis (Frag4 program) with fluorescence detection using the automated sequencer CEQ8000 (Beckman-Coulter, USA).
Calculation of peak sizes and their areas was carried out using the Fragment Analysis software (Beckman Coulter, USA).The error of detection of peak areas at T-RFLP analysis is not more than 5 %.For identification of peaks, the T-RFLP patterns for three endonucleases (HaeIII, HhaI and MspI) were processed using Fragment Sorter (http:// www.oardc.ohio-state.edu/trflpfragsort/index.php).
The second stage of the work was analysis of the protein fractional composition in the studied samples by one-dimensional electrophoresis in 12.5 % polyacrylamide gel in the presence of sodium dodecyl sulfate (SDS) in a chamber VE-10 (Helicon, Russia) at a constant voltage of 60V.Upon reaching the front of the separating gel, the voltage was increased up to 130V and separation was continued for 2-2.5 hours [23].An amount of protein introduced into the gel was 20 µg for all samples.The protein concentration in the samples was detected by the biuret method using a spectrophotometer Bio-Chem SA (HTI, USA).As a standard for electrophoresis, we used a marker from Thermo, USA, which is a mixture of 11 recombinant proteins (250, 150, 100, 70, 50, 40, 30, 20, 15, 10 and 5 kDa).Staining was performed using Coomassie G-250.The protein composition was analyzed using the UniProt Protein Database (http://www.uniprot.org/) [25].
Identification of protein fractions on two-dimensional electrophoregrams (2DE) was performed after tryptic proteolysis [26] by MALDI-TOF MS and MS/MS mass-spectrometry using a MALDI-TOF mass-spectrometer Ultraflex («Bruker», Germany) with an UV-laser (336 nm) in the positive ion mode and a mass range of 500-8000 Da with their calibration according to the known trypsin autolysis peaks.
The obtained mass-spectra of tryptic peptides were analyzed by the Mascot software, Peptide Fingerprint option (Matrix Science, USA) with accuracy of МН+ mass detection of 0.01 %, with the use of the database of the National Center for Biotechnology Information (NCBI).
Mathematical and statistical processing of the results was carried out using Microsoft Excel 2010.

Results and discussion
When analyzing the samples of horsemeat sausage by T-RFLP, the following ratio of microflora was established (Table 1).
Large part of sausage microflora (52.45 % in sample 1, 38.82 % in sample 2 and 29.62 % in sample 3) was presented by lactic acid microorganisms.The high content of Lactobacillales in samples 1 and 2 was apparently conditioned by the spontaneous process of fermentation and an absence of preserving agents such as sodium nitrite and ascorbate.
The unculturable microorganisms accounted for about a quarter of microflora: 24.1 % in sample 1; 22.01 % in sample 2 and 25.17 % in sample 3.
The unculturable forms of bacteria have the metabolic activity but do not grow on culture media.The transition to the unculturable form occurs at an exposure to unfavorable factors; however, when entering the human body, they can be re-cultivated (revived), which explains a presence of natural focal diseases.Therefore, information about their presence in meat products is important for assessment of product safety and quality.
In addition, several other microorganisms were detected such as Microbacterium, Pseudomonas sp., Burkholderia sp., including Burkholderia cepacia, and Flavobacterium spp.
Pathogenic microorganisms and coliforms were not revealed.
The results of the performed electrophoretic investigations showed the differences between the studied samples, which electrophoregrams are presented in Fig. 1.
When analyzing the results of protein fractionation in the studied samples, it was found that samples 1 and 2 were similar in the protein profile and had only insignificant differences (towards a decrease in sample 2) in the quantity of proteins in the zones 38-42, 23 and 15 kDa when staining СВВ G-250, which gives high linearity of bonding with proteins.Sample 3 was clearly different from them, which can be explained by the differences in their compositions and production technology.It contained less protein fractions, and some of them disappeared practically completely.Staining by more sensitive method (with silver nitrate) confirmed the revealed changes and allowed detection of a range of other fractions that presented in the minor quantity and changed their quantity.
They included the fractions with the molecular weight of 132, 128, 75, 90, 53, 45, 36, 28, 20 and 16 kDa.The proteins of these fractions are the main components that generate short peptides.Eight of them were identified by timeof-flight mass-spectrometry.For example, in samples 1 and 2, the fractions of amilo-1,6-glucosidase, fast-type muscle myosin-binding-protein C; glucose-6-phosphate isomerase; fast skeletal muscle troponin I, phosphoglycerate kinase, pyruvate kinase and skeletal muscle actin were found, which were absent or reduced in sample 3. The results of identification are presented in Table 2.
Based on the results of the tandem mass-spectrometry, the proteins, which natural short peptides existed in the analyzed extracts were identified by the obtained masses.Largely, they all belonged to different peptides of equine myoglobin (Table 3).
Samples 1 and 2 were similar in terms of spectra, which can be explained by the similarity of their composition and the conditions of the fermentation process; they had identical mass peaks, while sample 3 had more prominent differences.
A set of the same myoglobin peptides in the first two samples was absent in sample 3; however, its own peptide  was revealed.The troponin T peptide was present in all three samples.Moreover, extended sequences of unknown proteins were detected.In analysis in manual mode, we were able to reveal that the peptide with m/z 2000.9168 is the peptide of 14-28 amino acid sequence of creatine kinase M of the muscle type; this peak is in all types of studied sausages.

Conclusions
In analysis of microflora in three naturally fermented meat products from horsemeat (air dried sausage «Kazi» and uncooked smoked sausages «Balyk» by T-RFLP, it was established that lactic acid microorganisms prevailed over other groups of microorganisms.With that, unculturable microorganisms accounted for about a quarter of microflora.
In addition, several other microorganisms were detected such as Microbacterium, Pseudomonas spp., Burkholderia sp., and Flavobacterium spp.Pathogenic microorganisms were not revealed in the samples.
The study of these products by one-dimensional electrophoresis showed the differences in the protein profile.The sausage sample «Balyk» contained less protein fragments than the «Kazi» samples.
The performed comparative proteomic study of three sausage types demonstrated quantitative differences by several fractions.Based on the results of the tandem massspectrometry, the proteins and natural short peptides existed in the analyzed samples were identified.Largely, they all belonged to different peptides of equine myoglobin.
In general, good preservation of muscle proteins myosin and myoglobin, which can be a source of functional peptides, was observed.The differences in product protein profiles were, apparently, conditioned by the technological peculiarities of production and spontaneous course of the fermentation process.
Therefore, the spontaneous microflora of fermented meat products, especially upon prevalence of lactic acid microorganisms can affect the development of the biologically active peptides in meat, which is confirmed by the results of the study presented in this paper.Moreover, the applied approach and search algorithm can be used for search and identification of short natural peptides in sausage samples.