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Available online at https://www.meatjournal.ru/jour Original scientific article Received 05.08.2021 Accepted in revised 09.09.2021
FOR EXTRACTION OF BIOLOGICALLY ACTIVE Accepted or publication 25.09.2021
SUBSTANCES FROM THE RAW MATERIAL OF ANIMAL ORIGIN
DOI: https://doi.org/10.21323/2414-438X-2021-6-3-226-235
COMPARATIVE STUDY OF TECHNOLOGIES
Ekaterina R. Vasilevskaya, Marina A. Aryuzina, Evgeniya S. Vetrova*
V. M. Gorbatov Federal Research Center for Food Systems of Russian Academy of Sciences, Moscow, Russia
Keywords: extraction, pH, pancreas, low molecular weight proteins, porcine Abstract
Technologies of isolation and concentration of biologically active substances, developed in the middle of the 20th century, need adjustment and adaptation to modern conditions both to increase the activity of substances and for greater economic efficiency. The aim of the research is the comparison of dynamics of biologically active compounds extraction from porcines pancreas in two methods: the saline method based on 0.9% sodium chloride solution, and the acidic method based on 2.4% trichloroacetic acid solution. Also the purpose of research is to assess the possibilities for further optimization of technologies. The total protein concentration based on the biuret reaction in the samples taken during the extraction, as well as the calculation and analysis of the point degrees and rates of extraction are chosen as the controlled parameters. Local maxima of the protein yields into the extractant media at the 60th, 135th and 255th minute were recorded during saline extraction; and at the 75th and 135th minute during acid extraction. Also the proteomic profile of the extracts was studied. Wide range of compounds with molecular weight of less than 52 kDa was found in extracts based on physiological saline solution, and protein substances of whole presented range of molecular weights in trichloroacetic acid based extracts were considered. The predominance of low molecular weight protein fraction of interest was noted also in this method of extraction in comparison with the other methods of extraction. According to the UniProt database, we assume availability of probable compounds with a molecular weight of less than 30 kDa in the purified acidic extract. The presence of some proteins absent in the final saline extract was noted. The acidic erythrograms showed a weak degrading effect of both types of extracts on the membranes of rat erythrocytes, as well as the cytoprotective effect of acidic ultrafiltrates (less than 3 kDa). The obtained results prove a better efficiency of trichloroacetic acid extraction method used for obtaining a mixture of a wide range of compounds, including biologically active substances of low molecular weight.
Funding:
The research was supported by state assignment of V. M. Gorbatov Federal Research Centre for Food Systems of RAS, scientific research No. FNEN-2019-0008.
Introduction
The study and optimization of technologies for isolation of biologically active compounds from animal raw materials are today one of the most important aspects of modern biotechnology. Along with the fast development of the analytical chemistry and biochemistry spheres, the technological execution of methods, aimed to supervision and control of the observed processes, requires timely improvement to match the progress. In this regard, the permissible limits of sensitivity of the detection devices are improved, devices are designed with functional possibility to run combined experiments (for example, HPLC with a diode-matrix UV-visible spectrophotometric detector (DMD), fluorimetric MS-detector, capillary electrophoresis with DMD [1], low molecular weight electrophoresis combined with mass spectrometry of inductively coupled plasma [2]). The databases of discovered compounds are being replenished more and more (for example, refer to the UniProt protein database) [3]. The early studies, replicated on the currently obsolete equipment, were as accurate as
it was possible for that time. Technologies based on such researches are still used today. By the way, these technologies often feature high cost, low efficiency and high labor intensity. In order to potentially reduce the economic costs of production, as well as to reduce the volume of waste, the dynamics of the process flow shall be studied in order to identify possible ways to optimize the methods.
In the 1920s insulin, pancreatin, glucagon, obtained from animal pancreas extracts, were patented. Later, the development of technologies for production of certain medical preparations of enzymes and hormones, extracted from porcine pancreas, went on. Pigs were used as a source of a whole range of important biologically active substances, and in the 30s of the last century, traditional approaches to the extraction of some specific compounds or their mixtures from the pancreas were developed. Those achievements serve as the basis of modern technologies [4,5]. Due to the rapid development of the technological execution of necessary compounds isolation, it can be assumed that the potential of this raw material is not fully achieved.
FOR CITATION: Vasilevskaya, E.R., Aryuzina, M.A., Vetrova, E.S. (2021). Comparative study of technologies for extraction of biologically ac-
tive substances from the raw material of animal origin. Theory and practice of meat processing, 6(3), 226-235. https://doi. org/10.21323/2414-438X-2021-6-3-226-235
Among the variety of extraction methods used to obtain biologically active substances from porcine pancreas, one can distinguish method of saline solutions (sodium and calcium salts), method of acidic solutions based on weak and strong acids, method of alcoholic solution based on ethyl alcohol, incl. the method of bismaceration [6]. Each of the method presented above has its specific advantages. The main advantage of physiological saline extraction is the absence of aggressive agents and the proximity of the conditions to natural, which matters for the recipient organism. Acid extraction by trichloroacetic acid (TCA) provides an acidic reaction medium, which contributes to inactivation of the main proteolytic enzymes of the pancreas and inhibits autolysis. Alcohol extraction allows achieving a better degree of the active substance extraction due to the partial disintegration of tissues exposed to organic solvent (ethanol). The disadvantage of technologies that include the use of acids or organic solvents is the necessity to remove them from the obtained ready extract. Despite the obvious difficulties related to this task, those technologies are still often used in production of medical preparations based on an extract of the pancreas, since they allow obtaining a higher yield of specific compounds. Alcohol extraction technology, which is often used to obtain insulin, can be considered as potential for further study in the prospect of comparing it with the methods discussed in this article.
At this stage it is of scientific interest to compare the methods of saline extraction and acid extraction. First of all, this is reasoned by very different conditions of the process described above, namely — the acidity of the medium, duration of the extraction, nature of the extractants, and the purification stage. The technologies under consideration also allow running the process at low temperatures for a long time, which has a positive effect on the results of the analysis of the protein composition and provides a better yield of native low molecular weight compounds.
The aim of the article is to study and compare the saline and acidic method of extraction of biologically active compounds of protein range from porcine pancreas, as well as to determine the efficiency of the considered technologies for obtaining a highly active complex of substances rich in low molecular weight components.
The absence of aggressive conditions during extraction process in physiological saline medium implies a higher content of protein substances in the extracts, which are also rich in the low-molecular fraction, which was partially replenished during the extraction as a result of the action of proteases. A high activity of low molecular weight fraction in the acid ultrafiltrate is expected. This is primarily caused by decrease in proteolytic enzymes activity in an acidic medium, as well as by the additional stage of purification from ballast inactive proteins.
Objects and methods
The object of the study was porcine pancreas Sus scrofa, sampled at Limited Liability Company "Pushkinskiy Dvor" (Russia, Pushkino). The raw materials were subjected to
extraction by two methods: aqueous saline (physiological saline) and acidic (trichloroacetic acid), followed by study of obtained extracts and their ultrafiltrates.
The raw materials were frozen and stored at a temperature of minus (40 ± 2) °C with further thawing immediately before the study at a temperature of (22 ± 2) °C. Before extraction, the pancreas was also subjected to fine grinding with the help of a meat grinder (Kenwood, England): mesh of 100 holes with a diameter of 3 mm. The extraction scheme is shown below in Figure 1.
Saline extraction [7] was conducted in 0.9% sodium chloride solution by way of mixing of the finely chopped raw materials and an extractant in hydromodule in ratio of 1:5. The process lasted for 330 minutes. After completion of the extraction, the obtained solution was centrifuged in a Centrifuge CM-6M (ELMI, Latvia) at 3500 rpm for 8 min. at a temperature of (22 ± 2) °C. Then, the supernatant liquid, separated from the sediment, was saved for further analyses. To study the extraction process, samples were taken throughout the entire process: at 0, 5, 10, 15 minutes; and further every 15 minutes till to 330th min. Each sample was centrifuged for 5 minutes immediately after its sampling at a temperature of 4 °C on a Centrifuge 5427R (Eppendorf AG, Germany) at a speed of 3500 rpm; the supernatant liquid was frozen.
In case of acid extraction [8,9], the finely chopped raw material was loaded into the processor bowl together with an extractant — 2.4% trichloroacetic acid solution with a hydromodule of 1:3.15. The acid extraction technology also included the stage of the final extract purification from ballast proteins: primary centrifugation was run in a Centrifuge 5427R (Eppendorf AG, Germany) at 6,861 rpm for 10 minutes with cooling the extract to 4 °C; the process also included the precipitation with sodium hydroxide solution at pH 6.2-7.2. The final centrifugation was run at 3,500 rpm for 10 minutes on a Centrifuge CM-6M (ELMI, Latvia) at a temperature of (22 ± 2) °C. Extraction was conducted in the same laboratory installation, that was used for saline extraction, for 2 hours 15 minutes with sampling at 0, 5, 15 minute and then every 15 minutes till to 135 min. Each sample was centrifuged at 6,861 rpm for 10 minutes. when cooled to 4 °C. The supernatant liquid was frozen at a temperature of minus (40 ± 2) °C.
In both cases, the extraction process was conducted by the laboratory unit in LDU (Labotex, Russia) along with cooling down to (4 ± 2) °C in order to avoid autolysis, at a stirring speed of 400 rpm. The supernatant liquids of the purified extracts obtained after centrifugation were frozen at a temperature of minus (40 ± 2) °C and stored for no more than a month.
To obtain a highly active low molecular weight fraction of the extracts, the extracts underwent ultrafiltration on filters with a permeability limit of 3 kDa (Amicon, Ireland) as the final stage of the technology process. The process was carried out on a Centrifuge 5427R (Eppendorf AG, Germany) at a speed of 11,481 rpm for 20 minutes at a temperature of 4 °C.
c
Sus scrofa pancreas
Preparation of raw materials: grinding to particles size of 10x5
Hydro module: 1:V. 15, Stirring speed: 400 rpm, Extraction time: 1V5 min.
Freezing for 24 hours at minus (40 ± 2) °C
1
Thawing at (22±2) °C
Grinding to particles size of 3x3 mm
Hydro module: 1:5, Stirring speed: 400 rpm, Extraction time: VV0 min.
Freezing of the supernatant liquid at temperature minus (40 ± 2) °C
Figure 1. Scheme of extractions based on saline and trichloroacetic acid Legend: CP1 — controlpoint fo r native pancreas sampling to obtain a comparative sample (CS); CP2 is the control point for sampling of extracts to study the dynamics of both extraction methods; CP3 — control point of the final extract sampling: after centrifugation ofthe eotal volume o f the extract — for saline extraction; after p urification and s econdary centrifugation — for acid extracti)n
In the samples taken during the extraction, the total protein concontration, the total proteolytic activity of enzymes, and the dynamics of tf yseld of protein compounds were analyzed by the proteomic profile. The mem-branotropic effect of biologically active compounds in primary purified extracts, as well as in their ultrafiltrates were studied.
Sample of the raw material (pancreas native — further PN) were prepared in a known way [7].
The total protein concentration and ultrafiltrates in the extracts were measured on the basis of the biuret reaction according to the Kingsley-Weixelbaum method [10]: 600 |l of the biuret reagent was incubated with the test sample, and after 10 minutes the optical density of the solution was
measured ai 540 nm by a photometer BioChem SA (HTI, USA).
On the basis of parameters of protein yield to the ex-tractant the basic characteristics of the process like degree and rate of extraction were determined.
The degree of extraction was determined as the ratio of the extracted substance (protein) mass t5 the total mass of raw material and was calculated by the following formula:
M
D=—x 100 % (1)
m
where
M — is the mass of the extracted substance, g; m — is the total mass of the initial raw material in the mixture, g.
The point rate of extraction was determined by change in protein concentration (extracted substance) along the vector of time. It was calculated at each moment of the extraction process. Calculations were conducted by the formula:
Ac dc , .
v= AT It (2)
where
c — is concentration of protein in extract, g/l;
t — is corresponding time of the extraction, min.
The total proteolytic activity of the final extracts was determined by Leilian-Folgard method, which included heating in a test flask up to 37 °C in a water bath of 20 ml of 5% alkaline casein solution with 10 ml of the test sample for 60 minutes. In a control flask, a solution of the same composition, immediately after mixing was precipitated first with a
0.2 N hydrochloric acid solution, and then with a 15% sodium sulfate solution. The precipitated fallout was filtered off. After one hour of sample exposure in a test flask, the same operations were carried out. Then 10 ml of the obtained filtrate was transferred into clean flasks and titrated with 0.1 N sodium hydroxide solution in presence of 1% cresol red solution till obtaining the saturated bright crimson color. The proteolytic capacity was calculated from the difference in the volumes of sodium hydroxide required for titration of the experimental sample and control sample.
Membranotropic activity of biologically active compounds in extracts and ultrafiltrates was determined by constructing acidic erythrograms of erythrocyte membrane degradation [11, 12]. 10 ml of the test sample with a protein concentration of 1000 ng / ml on the basis of 0.9% sodium chloride solution that was introduced into a test flask. The control flask contained pure physiological saline. In both cases, 20 ^L of blood from rats' tails vein was added to the flasks, the contents were mixed for uniform contact of active compounds with erythrocytes and left to incubate at room temperature for 1 hour. After expiration of this time, 2 ml of both samples were transferred into clean flasks and mixed with 2 ml of 0.004 N sodium chloride-based hydrochloric acid solution. The dynamics of changes in optical density was recorded by SF-2000 spectrophotometer (Spektr, Russia) at 650 nm every 15 seconds from the moment of mixing until the end of hemolysis (when the optical density values reach a plateau), i. e. for at least 10 minutes.
The proteomic profile of the extracts was studied on the basis of electropherograms obtained by Laemmli method,
1. e. by one-dimensional denaturing electrophoresis in 12.5% polyacrylamide gel in presence of SDS. The process was run in a VE-10 chamber (Helicon, USA) at a temperature of (22 ± 2) °C at 60 V, until the samples reached the boundary of the separating gel, then at 130 V until the elec-trophoresis was completed. Standards (Fermentas, Lithuania) were used as comparison referential markers.
The analyzed samples were primary stained with Coo-massie Brilliant Blue G-250 solution. Later the stain was removed by 10% acetic acid.
To increase the sensitivity level of this method, silver staining was performed according to Bloom's method by alternate incubation of the samples in solutions of sodium hyposulfite, of silver nitrate and sodium carbonate.
Density diagrams were obtained by the software Image] (National Institutes of Health, USA) [13] based on results of studying the proteomic profile of extracts selected during the extraction process at control points. The results were visually presented on the basis of automatic densito-metric analysis of the scanned electropherograms. In order to avoid errors in study of various extractions electrophe-rogram, the same parameters were set up. The image was converted to 8-bit type, then an area of 1280x1938 pixels was selected on the track. The molecular weight of the fractions was determined by correlating of the corresponding peaks on the density diagram of the test sample with the peaks of the standards [14,15].
The software STATISTICA 10.0 was used for statistical processing of the total protein content. The final results were calculated and presented as "mean ± standard error" (M ± SE). Significant differences were checked using oneway analysis of variance ANOVA followed by Tukey's test. The deviations, when P values didn't exceed 0.05, were considered as statistically significant [16].
Results and discussion
When measuring the total protein concentration (Figure 2) in the samples taken during the saline extraction, a sharp increase in the values from 0 to 5 minutes was observed. And the further decrease in the protein content in the samples was observed at 90th min. (21.2 ± 2.7 g/l), at 165th min. (19.6 ± 0.4 g/l), at 240th min. (19.7 ± 0.2 g / l), at 270th min. (19.5 ± 0.3 g/l). The observed maximum concentration values are explained by release of a new fraction of compounds into the extractant, for example, at 60th min. (23.1 ± 0.9 g/l), which together with the concentration value for 135th min. (23.3 ± 0.6 g/l) was the highest throughout the entire process of extraction. Minor increases in concentration were also observed at 195th and 255th min. In general, it is necessary to note the smooth way of change in the samples protein content, starting from 5th min. of the process.
TCA extraction featured a smooth increase in total protein content up to 75th minute, where it reached a maximum and amounted to 15.6 ± 0.4 g/l. A further slight decrease in concentration led to coming of protein amount, released to extractant, to a plateau. This plateau values were was observed until the very end of the extraction process.
According to the diagram of dependence of the total protein concentration on time of extraction, in case of saline extraction a higher yield of protein into the extract-ant is evident throughout the entire process. From the first moments of the process, a sharp increase in the amount of protein was observed in comparison with a smooth increase of protein concentration in case of acid extraction. Both methods of extractions feature an oscillatory process of the protein content changing in the extracts.
Figure 2. Diagram ofthedependence of the total protein concentration in acid and saline extracts on the duration of the process
25,00
m ~ 20,00
■¡u o
^ 15,00
is
o
£ 10,00
ö <u o Ö
o O
5,00
0,00
-5,00
k
• Protein concentration in saline extraction —■— Protein concentration in acid extraction
1 15 30 4 5 6 0 75 9 01 51 >0 135 150 1 51 01 52 0 225 2^ 02 >5 2' 02 5 300 3 5 330 34
Time, min
As result of processing the data on total protein concentration in the extracts, the degree (D) and point rate (v) oC extraction were calculated eor both methods. Diagram of the dependence of rate of proteins release into tire extract-ant are shown below in Figure 3. It is necessary to note the high rate of saline extraction till the 5th minute, in contrast to TCA extraction, in which the rate gradually increased up to the f5th minute and then began to decrease unceitical-ly. In both cases, the procrss showed an oscillatory change in the rate wrthin the range oe 0 g/(l*min), i. e. in general the release of protein into the extractanttook approximately constaat values with minor deviations. Thus, there was a slight decrease in rate of extraction nt the 165th end 270* minutes in sefine extraction, and at ahe 90th minate in eaee of TCA extraction.
The data on degrer of extraction are ehown above in the Table 1. It is ne crssa-y to note the correlation with the data on the total protein ct-ntent in the extracts. The final degree af extraction of the protrin componene in the sal-ne variant exceeded 2.25 times tha vrlue oO D eor acid extrac-
tion. It proves that the saline solution is more efficient as an extractant in terms of this regard. The highest degree of extraction (Table 1, light green line) was observed from the 30th till 150th minute, and st the 75th minute. (Table 1, red line) for extracts based on 0.9% sodium chloride solution and TCA, respectively.
The study of proteolytic activity of enzymes in the final extracts showed a higher value in the case of saline solution extraction and amounted to 94 , 2276 Uaml in comparison wfih 33,74 8 U/ml for the TCA-based sample. Tlie data indicate the best enzymatic actmty in the saline extract, which is primarily associated with mild environmental conditions (neutral pH, the absence of denaturing agents that can negatively affect the active centers of rnzymos, etc.). The low proteolytic capacity of tl e aci d extract indicatea an insufficie nt t egree of purification oh rctive enzymee irom th e inhibitory action of TCA, even after the sompletion ol additional stages of purilication. The reraon may also be the removal of enzymes hrastirn togeth er with the ballast proteins
o
PL,
4, 5
4 H 3,5 3
2,5 2 1,5 1
0,5 H 0
-0,5
15 30 45 60 75 90 105 120 135 150
•Rate of extraction in saline extraction
•Rate of extraction in acid extraction
5 180 195 210 225 240;
5 270 285 300 315 330 345
Time of extraction, min
Figure 3. Diagram of the change in the point r ate of extraction for saline extraction and for acid extraction
Table 1. Extraction rate of protein-peptide fraction in the process of saline and acid extraction
Saline extraction
D, % Time, min 1.30 0 11.17 5 10.63 10 10.40 15 11.11 30 11.03 45 11.33 60 10.61 75 10.34 90 10.55 105 10.56 120 11.25 135 10.73 150
D, % Time, min 9.40 165 9.90 180 10.20 195 9.69 210 y.oi 225 y.DV 240 y.OO y.lt 255 270 9.06 285 9.1/ 300 9.23 315 9.62 330
Acid extraction
D, % 0.27 0.70 2.52 3.91 4.10
Time, min 0 5 15 30 45
precipitated at the stage of neutralization and centrifu-gation. It is necessary to keep in mind that a decrease in proteolytic activity may be a positive factor, indicating that many large and medium-sized proteins, that avoided the undesirable process of autolysis, are preserved in their native form.
To obtain information on the biological activity of the low molecular weight fraction, ultrafiltration was concluded. The dala on tle totalprotein aencantration en the final investigated extracts and their ultrafiltcates are presented below in the Table 2.
4.01
4.86
3.66 3.98 3.97 4.28
60 75 90 105 120 135
Table 2. Total protein concentration in extracts before and after ultrafiltration
Extraction variation Total protein concentration, g / l
Final extract Tl X -L ft £ Ultrafiltrate is. AÎ _i_ A 1
Saline Acidic 23.3 ± 0.6 10.8±0.1 6.03 ± 0.1 8.04 ±0.2
The results of the fnalysis of erythcograms obtained witg acid inhibiiion oO rat blood erythrocytes after its in-cubaiion with extracts (Figure 4) and their low-molecular-weight fractions (Figure 5) proved a short-term hemolysis and a tendency for a smooth decline in all samp les.
Figure 4. Acidie erythrograms oe erythroeytss intcbatirn with th- raiginal sxtracts. Legend: A-E — curve of incubation of eryttrocytcs with TCA extract; S-E — curveol incubation oferythrocytes with saline extract; C — curve of incubation of erythrocytes with saline soluSion without the cample
1,1 1
0,9
^
S 0,8 Ö <U
Ü 0,7
CS
u
Uo,6 o
0,5 0,4 0,3
LM S-E O LM A-E C
0 50 100 150 200 250 300 350 400 450 500 550 600
Time, s
Figure 5. Acidif etcthrograms olincubation olerythrorytes with ultrafiltrate extracts. Legend:LM A-E — ircubation curve of erythroayteswith low malecular weight fraction oe TCA extrect;hM S-E — incubation curve of erythrorytet withlow molecelar waight fraction of saline extrnct; C — curve ol incubalion of erythrocytes with saline withaut a sample
Analysis of the diagram typical for incubation of erythrocytes with primary extracts (Figure 4) revealed particularly low optical densities for the TCA extract, which is explained by the presence in the sample of a large number of biologically active compounds that can penetrate through the membrane and accelerate the process of its degradation. In addition, this hemolysis curve is also spanned below the control sample curve, which confirms the overall negative effect of the extract on biological membranes. The results of determining the optical density of erythrocytes in the case of incubation with a saline extract were similar to the values in the control sample at the very beginning of the process and at the end of hemolysis. The decline in the diagram, that indicates the process of direct cells destruction, was found below the control curve and almost coincided with the values for the acid extract, which indicates a high degree of membrane degradation. Based on the results of the analysis, it can be concluded that the primary extracts are unable to provide cytoprotective effect on biological membranes, which is more profound in the TCA-based sample. In this regard, it is possible to assume the possible presence of highly active compounds in the samples, capable to transport substances through the membrane, which indirectly leads to its destruction.
A similar process was observed in the case of saline ultrafiltrate (Figure 5). The incubation diagram with erythrocytes also coincided with the control curve, which proves a weak membranotropic activity of the low molecular weight fraction in this sample. An obvious difference was observed when considering the acid ultrafiltrate curve. High values of the optical density of the eample based on TCA confirmed the cytoprotective effect of biologically active substances released into the extract, as well as an increase of erythrocyte membrane preservation level being exposed to degrading agent — hydrochlorie acid.
Analysis of the proteomic profile of the final extaacts (refer to Figure 6) showed the range of protein compounds on electropherogram at the final point of saline extraction (the 330th min. — Figure 6, No. 1) and track of the raw material (Figure 6, No. 2) corresponding to the pancreas sample, which was not subjected to extraction. Compounds within the range of 52 kDa and less were found in the extr acts, which were discussed in detail in our previour researcla [7]. It should be noted that the fractional bands in this extract are more saturated than the primary electropherogram of acid extraction (Figure 6, No. 3, 4). In case of acid extraction only a few slightly expressed protein fractions were observed in the range of more than 250 kDa, 69-70 kDa, 52 kDa, 22-23 kDa and 15 kDa. The visual analysis of the native track of purified acid extract foun d no difference from the track of extraction 135 minutes long. The staining with silver (Figure 6, No. 5) revealed a wide profile of protein compounds in whole presented range of molecular weights. In the track of the purified extract (refer to Figure 6, No. 6), some medium-molecular fractions were absent:
70-95 kDa, 32-48 kDa; the fraction 15-16 kDa beceme less saturated, and the intensity of the low-molecular fraction of weight of less than 8 kDa increased slightly.
ST №1 №2 JVgJ JN»4 ST
250 150 100 70
50 40
30 20
10
zm
Xiguee 6. One-dimensional electropherogram of porcine pancreas extracts Legend: ST — molecular weights standards: 250, 150, 100, 70 50, 40,30,f0 P5, 10 and 5 kDa; No. 1 — track of the endpoint of saline extraction; No. 2 — traek of the sample nct subjected to axtraction (PN); No.r — track oa the endpoint of acid extraction; No. 4 — track
of purified acidic extract; No. 5 — silver-stained aria extracticn endpoint track; No.6 — silver staine d track oX a purified acid extract. Significant positions of fractional bands or changes in fractional composition arr marked rra
The densitnmetric study of native electropherograms of acid extrac-s in -he ImcgeJ softwere brought little information, sinee only 5 weak peaks were identified at the endpoint of extraction and in the purified extract too (Figure 7,66o. 3, 4). After silver staininx, a greater number of fractions were found on the cotresponding tracks diagram within entire presented range of molexular weights. This range teaaured prevalence of fractions over S00 kDa, 83-90 kDa, 70-75 kDa, 43-65 kDa, 18-32 SDa,14-16 kDa, less than 11 kDa. After purification oa the sxtract fome of the protein aompoelndi were removad, tnd that providad better separaeion of the pexkc in thesv specified ranges of massea. Thus, peaks at 137-M0 kDa, 110 kDa, 100 kDa, 70 kDa, 52 kDa, 41-42 kD a, 27-31 kDa, and 21 2Da wfre clearly distinguishable. The absent peaks in the density diagram (49-50 kDa, 33-34 kDa, 16 and 11 kDa) most likely corresponded to the removed ballast proteins.
During saline extraction (Figure 7, No. 1) and analysis of the sample not subjected to extraction (Figure 7, No. 2), a large number of similar peaks were noted: a profound peak at 52 kDa, a row of clear minor peaks with varying degrees of manifestation within the range 32-47 kDa, at 30 kDa, the peaks at 24-25 and 29 kDa were expressed better in case of the saline extraction, as well as the low molecular weight fraction of 10-15 kDa which was better visible too.
Figure 7. Histogramsof the density of the protein fractions ofthe pancreas extraction. Legend:ST — molecufar weight standards: 250,150,100,70 50,40, 30, 20 15, 10 and 5 kDa; No. 1 — track of the endpoint of saline extraction f330 min);No.S — track oS the sample not subjocted ti extraction (PN); No. 3 — track of the endpoinf of acid extraction (135 min); No. 4 — track of purified acidic extract; No. 5 — silver stained acid extraction endpoint track;
No.6 — silver stfined track of a pufified fcid extract. Significant chanf es inthe fractional composiOion of ths extracfs are marked red
Analysis of electropherogram and corresponding density diagram by ImageJ software showed a wide range of compounds in all types of extracts. In case of acid extraction the high molecular weight proteins prevailed, which was delected by silver staining. Hie saturation of the protein bands prevailed in the saline extract and the track of the initial PN sample, which peotein bands correlated with the previously presented datt on the total proUein content.
The analysis of dynamics op the biologically active protein subotances extraction from porcinc panereas showed high values ol the total protein eoncentration during; saline extractioo along with a wide range of compounds througheut the entire process. In case oC acidic extract- eha protein conte nt valuee were l ower; however, the froteomic profile of the samples tukon during the process was characterized by a greater variety, of fractions -within the whole presented range of molecular weights. First of all, this may be explained by dependence of solubility [17, 18] and activity of many enzymes [1y] secroted by the pordno pancreas on the acidity of the medium. Thus, as the p H approaches the isoelectric point of a protein, its solubility decreases when it reaches ite mmimum at pI value [20]. This indiea-tor has long been established for many hydrolytic enzymes of the panareas of cattla and pige. It hae been determined that most of the main enzymes yre secreted by the organ under normal iionditiong in the lorm of pancreatic fluid. To the maximum ettent, isoenzymes op a-amylase I and II, for eyample,precipitate at pH 6.5 and 6.1i trypsin at pH 10.2-10.8, chymotrypain at pH 8.1, ribonuclease at pH 9.6 [6,21-23].In this regard the acidic environment created bye TCA provides belter solubility and, accordingly, a greater variety of compounds in extracts. In saline samples, due to
physiological solution. a neutral reaction of the medium is ettabliehed, which leads, to varying degrees1 to a decrease inathe yield of some compounds into the extractant. The lowir concentration o- total vrotein in atid extracts samples is also associated with inhibition of many digestive enzymes, since the maximum activity oft pancreatic lipase is observed at pH 8-9, trypein 7-9, chymotrypsin 7.8-8.0, pancreatic elastase 8.5, eaoboxypeptidase A 8.0 [9,24-27]. Thus, e higher yield of pancoeatic proteins, moet of which are enzymes [28] t is observed in case of neutaal or slightly alkaline medium, since thesa pH valued account for maximum activity of tnzymes ; this phenomena was ills o observed in case cf saline extraction. On the other hend, high values of the protein concintration in these extracts can be caused by mild extraction conditions as those mild conditions providad extraction of active substcncee together with biologically active compounds of ballast proteins. The ballast proteins were removed from the extract in case of add extraction by an additionel purification stage.
Throughout tine entire experiment, in both methods the oscillatory nature of the degree and rate of protein extraction from pavoreac wae observed. De crease in these values at some moments of thee process is expMned by a decrease in amount e! proteins released to the extractant oi by their fermentative breakage ai e result of autolysis caused by action ol extracted proteolytic enzymes [20-32]. Decrease in proteasts acUivity can Ice achieued by dropping; down of the process temperance (which was done in this research), as well as by acidifying the extractants ae indicated above. When using saline as an extraceant, an instant increase in the extraction rate was obeerced due to abnenve ol obvious inhibitory agents. For obvious reasons the extraction
of TCA was accompanied by smooth increase in rate of protein release into the extractant. Based on the obtained results, it is possible to note the greater dependence of the extraction method efficiency on availability of inhibiting factors, as well as on the type of the extractant.
The fraction of interest with molecular weight of less than 30 kDa in the final saline extract was researched in early works [7]. Using the UniProt database [3] the following low molecular weight compounds were presumably detected in the purified acid extract: chymotrypsin C (28.9 kDa), a member of family of chymotrypsin-like elastases 1 (28.8 kDa) and 2A (28.7 kDa), glutathione S — transferase omega-1 (27.4 kDa), kininogenase (27.2 kDa), proglucagon (21 kDa), secretin (14.6 kDa), trefoil factor 2 (13.8 kDa), somatostatin (12.7 kDa), colipase (12.1 kDa), inhibitor of serine protease of Kazal-type 4 (9.6 kDa) and 1 (6 kDa), precursor of pancreatic hormone (7.3 kDa) etc. Due to smaller amount of compounds in the saline extract, many of the above specified types of substances were not found there.
Conclusion
The study of dynamics of extraction based on 0.9% sodium chloride solution showed a higher concentration of total protein throughout the entire process with a wide range of compounds with molecular weight of less than 52 kDa. In TCA extraction, the protein content in the analyzed samples was lower than in the first extraction method; however, analysis of the proteomic profile of the sam-
ples showed a greater variety of compounds in the entire presented range of molecular weights. The low molecular weight fraction prevailed in acid extracts both before ultrafiltration (according to results of electropherograms study) and after ultrafiltration (based on analysis of protein content in the ultrafiltrates).
Using of the drawn acidic erythrograms enabled to detect the capability, peculiar for primary extracts, to destroy the biological membranes. The low molecular weight fraction of the acid extract possessed the highest cytoprotec-tive effect, which proved the presence of highly active compounds in the extract.
When comparing the saline and acid extraction methods, the applicability of both modifications for obtaining a mixture of proteins with different molecular weights is noted. In this case, greater efficiency is observed in extraction with trichloroacetic acid. In result of this method it is possible to obtain a complex of low-molecular-weight highly active biological compounds.
It is necessary to note that the processes, considered in this work, require a comprehensive approach and are the subject of further extensive research. It is necessary not only to trace the dynamics of the process, but also to select target markers-compounds, which could be used to determine the efficiency of extraction, and also to study the potential for isolating of some specific compounds while maintaining of their high activity. The study of other ex-tractants, for example, alcohol or sulfuric acid, is also considered a promising direction of researches.
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AUTHOR INFORMATION
Ekaterina R. Vasilevskaya — candidate of technical sciences, researcher, Experimental clinic-laboratory of biologically active substances of animal origin, V. M. Gorbatov Federal Research Center for Food Systems of Russian Academy of Sciences. 26, Talalikhina str., 109316, Moscow, Russia. Tel.: +7-676-95-11(128), E-mail: e.vasilevskaya@fncps.ru ORCID: https://orcid.org/0000-0002-4752-3939
Marina A. Aryuzina — senior laboratory assistant, Experimental clinic-laboratory of biologically active substances of animal origin, V. M. Gorbatov Federal Research Center for Food Systems of Russian Academy of Sciences. 26, Talalikhina str., 109316, Moscow, Russia Tel.: +7-495-676-95-11(207), E-mail: m.aryuzina@fncps.ru ORCID: https://orcid.org/0000-0002-6886-496X
Evgeniya S. Vetrova — senior laboratory assistant, Experimental clinic-laboratory of biologically active substances of animal origin, V. M. Gorbatov Federal Research Center for Food Systems of Russian Academy of Sciences. 26, Talalikhina str., 109316, Moscow, Russia. Tel.: +7-915-027-83-89, E-mail: jozefina-veter@yandex.ru ORCID: http://orcid.org/ 0000-0003-2219-5964 * corresponding author
All authors bear responsibility for the work and presented data. All authors made an equal contribution to the work.
The authors were equally involved in writing the manuscript and bear the equal responsibility for plagiarism. The authors declare no conflict of interest.
