COMPARATIVE STUDY OF AUTOLYTIC CHANGES IN PORK AND BEEF MUSCLE TISSUE PROTEOME

I p a p e t presents tk s tu d ie s o n tk autotyttctraHsJormattoHS inpotkandbeefsamiples by proteomk methods. I c k n g e s nn tkptoteinltactionswetea.nalyzedbyone-dimensioila.ndtwo-dimensionalelectropktesiswitkfollowingidentifcca.tionby mass-sps^ctromu^trtcmietkods.^ecdiar^gs^s tnmuscte protetns tntkcourse ofautolyttc processes were found. ؟ or example, k̂(j tntenstty of tkeprotetn spots ofpyruvate ktnase, Y٣̂ŷĉ؛̂ :̂ n hght chatns â r̂ d adenylate ktnase tncreased d̂v̂ î :̂ r̂ g autotysts. I fragments ofmiyosinlightchainsappea.ted.lactivityoftkendogenousenzymes,suchascap>ain3>a.ndca.tkpsinDintkcoursea.utolysis wasatsostudted. Duringthefitst24 kutsa.ftera.TOm ialskughtet,tkactivityoftkseenzym iesteackdtkm ia.xim uilevela.nd thendecreased.lresults of tke performed investigations confirmed tkat potkand beef kave dt.erent rate ofautolysts but similar prots^omcc changes. процессов. Эти модификации включают окисление боковой цепи, образование поперечных связей и рас­ щепление белковых молекул и критически влияют на такие основные свойства мяса, как срок хранения, пи ­ щевая ценность, усвояемость и последствия для здо­ ровья [1]. За последние пять лет в области протеомики мяса наблюдался устойчивый рост научного интереса. Было проведено большое количество исследований с появ­ лением нескольких отдельных категорий. Открытие белков-маркеров для различных атрибутов качества мяса — еще одна важная область. Процессы посмерт­ ного превращение мышц привлекают особый интерес и представляет собой значительную серию событий, связанных с модификацией и разрушением белка [2]. Введение Белки являются важным нутриентом в рацио­ не человека. В качестве пищевых ингредиентов они в основном поступают из продуктов животного и ра­ стительного происхождения. Протеомика имеет в сво­ ем арсенале такие методы, которые способны охарак­ теризовать белковый состав продуктов, какие белки присутствуют в каждом типе ткани, а также позволя­ ет исследовать их различия в белковой композиции. Кроме того, протеомные методы позволяют отсле­ живать изменения протеома до и после убоя, а также оценивать влияние последующих процессов, таких как автолиз. В дополнение к посттрансляционным модификациям, которые происходят In vtvo, пищевые белки подвергаются широкому спектру послеубойных ДЛЯ ЦИТИРОВАНИЯ: Чернуха И.М., Ахремко А.Г. Сравнительное изучение автолитических изменений протеома мышечной ткани свинины и го­ вядины. Теория и практика переработки мяса. 2018; 3(3): 56-63. DOI 10.21323/2414-438Х-2018-3-3-56-63 FOR CITATION: Chernukha I.M., Akhremko A.G. Comparative study of autolytic changes in pork and beef muscle tissue proteome. Theory and practice of meat processing. 2018;3(3): 56-63. (In Russ.). DOI 10.21323/2414-438X 2 0 1 8 -3 -3 -5 6 -6 3


Выводы
Отмечена активность катепсина D, изменяющаяся в процессе посм ертны х превращений.Установлена корреляция меж ду активностью катепсина D и и з teomics.M a n y studies were carried out w ith appearance o f several in d ivid u al categories.D iscovery o f m arker proteins for different meat quality attributes is another im portant field.The processes o f muscle postm ortem changes attract particular interest and represent an im portant series o f events that are connected w ith protein m odification and destruction [2].
In the process o f muscle transform ation into meat, significant biochem ical/biophysical changes in muscles occur, and these changes directly influence meat quality characteristics [2,3].Therefore, the aim o f this research was to investigate the autolytic processes in p o rk and beef, and to compare them using the proteom ic methods.

Introduction
Proteins are im portant nutrients in hum an diet.A s food ingredients, they m ain ly come from the products o f anim al and plant origin.Proteom ics has such methods in its ar senal that can characterize protein com position o f p ro d ucts, determine what proteins are present in each tissue type, and study the differences in the protein com position.Moreover, the proteom ic m ethods allow tracing changes in a proteome before and after slaughter, as w ell as assess ing an effect o f the subsequent processes such as autolysis.In addition to the post-translational m odifications, w hich occur in vivo, food proteins are subjected to a broad spec tru m o f the postm ortem processes.These m odifications include side-chain oxidation, form ation o f cross-links and cleavage o f protein molecules, and critica lly affect such m ain meat properties as shelf life, nu tritional value, assim ilab ility and consequences for health [1].D u rin g the last five years, a stable grow th o f the scien tific interest has been observed in the field o f meat pro-and reached 6.04 by the 9th day.The water holding capac ity decreased from 67.29 % to 66.83 % by the 6th day o f autolysis and then began to rise.
The investigation of the effect o f the autolyTic processes on changes in the po rk protein profile by the m ethod o f one-dim ensional electrophoresis showed that the fractions higher than 100 kD a increased in the staining intensity o f the bands by the 120th hour o f autolysis, but the num ber o f the fractions was stable.The intensity of the fractions w ith the m olecular weight in the range o f 90-100 kD a increased from the first day to the fifth.In the range from 50 kD a to 70 k D a the intensity o f bands was constant; however, on the 5th day, two m in o r bands at the 80 k D a region ap peared.A n increase in the intensity o f protein bands w ith a m olecular weight o f 30-32 k D a was noticed; in addition, degradation o f proteins w ith a m olecular weight o f lower than 15 k D a took place under the effect o f endogenous en zymes.
Changes in the ratio o f the major and m in o r protein fractions were observed.
It was shown in the analysis of the one-dim ensional electropherograms o f the protein profile in the skeletal muscles o f Bos Taurus that the concentrations o f the major and m in o r protein fractions increased by the second day, and, then, the num ber o f the fractions decreased m arkedly by the 9th day as 32 protein bands were observed on the 1st day and 23 bands on the 9th day.Presumably, it is associ ated w ith changes in the p H value, w hich is moved towards the alkaline side by the 9th day (from 5.65 to 6.04); this p H value is close to the optim al activity o f proteinasescalpains, under w hich im pact protein molecules degrade [6,7].
Figure 1 and Figure 2 present the fragments o f the tw o dim ensional electropherograms (2DE) o f p o rk and beef proteins, respectively, in w hich the deform ation o f the p ro tein fractions is m ost pronounced.
A n increase in the intensity o f the protein pyruvate kinase, m yosin lig h t chain 15 kD a, m yosin lig h t chain 20 k D a and adenylate kinase was observed from the 1st day to the 5th day o f the experim ent.The same trend to wards changes in the spot intensity was found in the beef samples (Figure 2), w ith exception for the pyruvate k i nase fraction, w h ich was sim plified to short peptides by the 9th day o f autolysis under the action o f endogenous enzymes.
To obtain more detailed inform ation about changes in proteins, tw o-dim ensional electrophoresis was carried out w ith the follow ing identification by mass spectrom etry M A L D I -T O F M S and M S /M S (Table 1).
The obtained results o f the proteom ic investigations allow observing sim ilar mechanisms o f protein fraction deformation, but w ith different rate; these changes occur over a longer period o f tim e in beef muscles than in pork muscles.It is associated w ith the fact that beef muscle fi bers are larger; consequently, the autolyTic processes occur slower.
w hich corresponds to rigor m ortis.The process of autoly sis occurred at a temperature o f 4±2 °C for 12 days in a vacuum container.2) po rk M. longissimus dorsi taken from the meat processing plant in the city o f Rostov-on-Don.The samples were stored at a temperature o f 2±2 °C in the conditions o f the com m ercial refrigerator for 120 hours.The samples for investigations were taken at 24 h, 72 h and 120 h.
The water holding capacity was measured by the Grau and H am m m ethod (filter paper press method).M oisture in meat raw materials was determined by the m ethod of specimen dryin g w ith sand u n til the constant mass at a temperature o f (103±2) °C according to G O S T R51479-99 (ISO 1442-97)«Meat and meat products.M ethod for de term ination o f moisture content».
p H measurement was carried out b y the potentiometric m ethod on the p H -ionom eter «Expert» according to G O S T R51478-99 (ISO 2917-74)«Meat and meat products.Reference m ethod for measurement o f pH».
A s a m ain proteom ic technology, tw o-dim ensional electrophoresis (2DE) by O 'Farrel was used w ith isoelectrofocusing in the am pholine gradient (IEF-PAG E); p ro tein detection in 2 D E was carried out by staining w ith Coom assie R-250 and silver nitrate.To perform the com puter densitometry, tw o-dim ensional electropherograms being in a wet state were used.Their complete digital im ages and/or images o f in d ivid u al fragments were obtained by scanning using an Epson scan in the mode o f 300 dpi, 48 b it C o lo r [4,5].
The calpain 3 and cathepsin D were quantified by im m u nochem ical analysis, the E L IS A m ethod on an im m unoas say analyzer Im m unoChem (H T I, U S A ) using com m ercial kits (C lo ud -C lo ne Corp., China).Sample preparation was carried out by the follow ing method: the muscle tissue was hom ogenized in a m ortar w ith addition of the phosphatebuffered saline (pH 7.4 1X), quantitatively transferred to Eppendorf, centrifuged at 5000g for 5 min., supernatant was taken and an analysis was carried out.

Results and discussion
The biochem ical mechanisms leading to changes in the muscle tissue depend on pH .In the p o rk samples, a change in p H was noticed after 24 h, 72h and 120 h; w ith that, the water binding capacity gradually decreased from 92.05 % to 87.66 % by 120 h o f autolysis.
In case o f beef autolysis, the p H value o f 5.65 moved to wards the acid side by the 6th day up to the m in im a l value o f 5.15, and then began to grow towards the alkaline side   The obtained results did not show a significant change o f calpain 3 in pork, w hich can be explained by the m in o r role o f calpain 3 in activation o f the processes o f bioch em i cal changes in po rk skeletal muscle proteins.O u r results correspond to the data o f Geesink et al. [8] obtained in the experiment on calpain 3 knockout mice, w ho did not re veal differences in the postm ortem tissue proteolysis com pared to the control.

Conclusions
The activity o f cathepsin D that changes du rin g post m ortem transform ations was observed.The correlation between the activity o f cathepsin D and changes in the proteome o f the studied skeletal muscles was found.It was e m p irica lly confirm ed by the proteom ic investiga tions that autolytic processes in p o rk and beef proceeded w ith different rates but by sim ila r m echanism s: during autolysis, the breakdow n o f h igh m olecular w eight p ro teins was pra ctica lly id en tical in all p o rk samples; m ore over, the changes in the related groups o f m uscle proteins such as pyruvate kinase, m yosin lig h t chains and adenyl ate kinase to o k place.
Significant changes in calpain 3 were not revealed dur ing 5 days (in pork) or 9 days (in beef) after slaughter.
It is know n that postmortem autolyrtic changes in muscles proceed under an im pact o f the proteolytic en zymes -calpains and cathepsins, associated w ith catalysis o f continuous breakdown o f the m ain structural elements o f muscle fibers, in clud ing the actom yosin complex.W ith that, exopeptidases are m ost active at the last stages o f p ro tein m olecule disintegration; under the action o f endopeptidases m any new term inal groups are formed, w hich was m entioned above.Therefore, a study on the dynam ics of changes in cathepsin D and calpain 3 in the p o rk and beef muscle tissue was o f undoubted interest.
The calpain system, w hich includes about eight related subgroups and calpain-like proteins, is the most important w ith respect to the meat ageing processes.U nder the effect o f enzymes, the structure and properties of meat proteins change.Cathepsins localized m ainly in lysosomes are released upon activation by the acid reaction o f the cell environment and cause destruction o f high molecular weight proteins.
These data correlate w ith the p H values: cathepsin D being a carboxyl endopeptidase that cleaves lo w m o lec ular w eight peptides b u ilt fro m 5 and m ore am ino acid residues, proteins -tro po nin, tropom yosin, connectin, exhibits the m a xim u m activity at р Н 2.8-5.0,w hile p ro teoglycans at р Н 5.0-6.0.

Figure 1 .Figure 2 .
Figure 1.2DE fragments with reealed signs of the autolyrtic changes in the studied pork samples by the 1st, 3rd and 5th days of autolysis M/ C: Score -an indicator of matches or scoring; Match peptides -the number of matched peptides; Coverage -the percentage of the complete amino acid sequence of a protein covered by identified peptides.** Mm/pI (exp.)-scores obtained by the results of the electrophoretic mobility on 2DE, Mm/pI (calc.)-estimates made on the basis of the data on the amino acid sequence with consideration for signal peptide removal, but without regard for other post-synthetic modifications *** Predicted on the basis of transcripts or genes.**** Characterized by the Mascot program by analogy with the corresponding protein, gene or transcript in the other mammalian species (cow, wild camel); in the column «Numbers in Protein NCBI», the references to the information for a corresponding animal are given.***** msms -indication on the confirming identification by tandem mass-spectrometry; in parentheses, the number of sequenced tryptic peptides is indicated.