STUDY OF THE BIOFUNCTIONAL PROPERTIES OF CEDAR PINE OIL WITH THE USE OF IN VITRO TESTING CULTURES

Cedar pine (Pinus sibirica) nuts are an environmentally friendly natural product that contains a unique set of useful biologically active substances. Due to its composition, pine nuts and their derivative products are widely used in a comprehensive therapy and prevention program for a lot of diseases. The objects of the study were cedar oil and oil emulsions (the cedar oil concentration was 1.0, 5.0 and 10.0%). The antimicrobial properties were determined using the diffusion method and by measuring the optical density. The prebiotic properties were estimated according to the ability to stimulate the bifidobacteria growth. The antioxidant activity was determined using the fluorescent ORAC method. The antihypertensive activity was estimated according to the ability to inhibit the angiotensin-1-converting enzyme. All the studied experimental oil emulsion samples, regardless of a pressing method (cold or heat), showed high antimicrobial characteristics without suppressing only Candida albicans EMTC 34 and Proteus vulgaris ATCC 63 from the studied 10 strains of the main testing cultures. The prebiotic properties of the emulsions obtained with the addition of cedar oil have been determined. The number of cells of the bifidobacteria cultivated in nutrient media with the addition of cedar oil (with the concentration from 5.0%) is almost 3 times as large as the amount when cultivated without it. Antioxidant cedar oil properties have been revealed. The hypotensive characteristics of cedar oil can be observed even at a concentration of 5.0%, the percentage of inhibition of the angiotensin-1-converting enzyme is up to 69%. With an increase in the concentration of cedar oil to 10.0% inhibition increases to 70%. The carried out studies of the functional properties of experimental cedar oil samples have confirmed its high quality and an opportunity to use it as the basis of biologically active food supplements and dietary, medical-preventive and sports nutrition.


INTRODUCTION
Cedar pine has always been highly demanded by the local population in any place of its growth thanks to extremely useful qualities.The most consumed pine nuts are the fruits of Korean pine (Pinus koraiensis), cedar pine (Pinus sibirica), umbrella pine (Pinus pinea) and chilgoza pine (Pinus gerardiana) [1,2].As raw materials, cedar is very convenient for a non-waste product manufacturing process [3][4][5][6].The more northern the latitudes of cedar pine habitats, the higher the value of cedar products.The high demand for pine nuts has resulted in an increase in their world output, according to FAO, China, Korea, Pakistan and Russia (Siberia and the Far East) are the main exporting countries [7].
Traditionally, it is believed that the most valuable cedar product is cedar oil, which is produced from cedar kernels [8,9].The oil yield depends on a pressing method (cold and heat pressing, solvent extraction), but about 45 to 65 g of butter per 100 g of nuts is usually reported about [10,11].Cedar nut oil is a high-quality natural and ecologically friendly food supplement [12,13] that is necessary for the normal functioning of the human body [14,15].
The vitamin E content in pine nut oil is several times more than that in olive oil.The use of cedar oil in the diet makes it possible to restore working capacity and cope with psychoemotional disorders.The beneficial effect of cedar oil in the complex of both therapeutic and preventive measures is well known [22][23][24][25][26][27][28].The limited consumption of oil, as part of dietary nutrition, is recommended for faster satiation [29].
The clear antioxidant effect of both nuts themselves and their derivative products has been noted.The study [12] of rats has found an increase in the antioxidant capacity in the blood of the animals that used Far-Eastern pine oil.Lin et al. [30] derived four peptides from deodar cedar nuts with pronounced antioxidant properties that were enhanced to 90% by an impulse action and an electric field.The ability of cedar oil to reduce cellular activity is planned to be used in the inhibition of cancer metastases [1,31].
Cedar needles, which are widely represented in traditional Chinese medicine, primarily with their antimicrobial properties, as well as their biologically active characteristics, are of particular interest to researchers [32].A lot of types of cedar needles are added into functional foods and food supplements to enhance a nutritional and / or pharmaceutical effect [33,34].If needles, with their antimicrobial properties, are also noted for their biologically active characteristics, nuts are probably noted for the similar effect, too, i.e., in addition to their nutritional and functional properties, they also have antimicrobial characteristics.
The study was aimed at the in vitro biofunctional properties (antibacterial, antioxidant, prebiotic, etc.) of the oil derived from the nuts of cedar pine that grows in the Kemerovo region as the basis of biologically active food supplements and dietary, therapeutic, preventive and sports nutrition.

STUDY OBJECTS AND METHODS
The experimental studies were carried out at the Research Institute of Biotechnology of the Kemerovo State University.
The objects of the study were the samples of the oil made with the use of two methods (cold and heat pressing) from nut kernels of cedar pine that grows on the territory of the Kemerovo Region (Tashtagol District, the crop of 2016 and 2017) and 1.0%, 5.0% and 10.0% O/W emulsions of this cedar oil.
Testing cultures: opportunistic and pathogenic strains of microorganisms: Pseudomonas aeruginosa ATCC 9027 is an opportunistic bacterium that induces nosocomial infections in humans; Candida albicans EMTC 34 is a microscopic fungus, the causative agent of opportunistic human infections; Alcaligenes faecalis EMTC 1882 is an opportunistic bacterium that induces itraabdominal infections, septicemia and meningitis in humans; Leuconostoc mesenteroides EMTC 1865 is an opportunistic bacterium that induces infectious diseases in humans; Escherichia coli ATCC 25922 is an opportunistic bacterium that induces gastroenteritis in humans; Enterococcus casseliflavus EMTC 1866 is a pathogenic bacterium that induces sepsis in humans; Salmonella enterica ATCC 14028 is a pathogenic bacterium that induces gastroenteritis in humans; Staphylococcus aureus ATCC 25923 is a pathogenic bacterium that induces pneumonia, meningitis, osteomyelitis, endocarditis, infectious toxic shock and sepsis in humans; Bacillus mycoides EMTC 9 are permanent contaminating agents of food raw materials and food products that induce food toxic infections in humans; Proteus vulgaris ATCC 63 is an opportunistic bacterium that induces intestinal infections in humans; as well as the Bifidobacterium adolescentis MC 42 bifidobacteria strain and the MDCK1 canine kidney epithelial cell line.
Sample preparation.The shell was preliminarily separated from the pine nut kernel, Sample No. 1 is from the crop of 2016, Sample No. 2 is from the crop of 2017.To prepare the samples of seeds from the cedar pine layers different in height (upper, middle and lower), 3 single samples weighing 100 g each were taken from each batch.
To obtain particles of 2 mm in size, the pine nut kernel was grinded in an electric mill for each of the pressing methods (cold or heat).In the case of cold pressing, the pine nut kernel was put under a UP-20 hydraulic press (Nizhny Novgorod, Russia) and the oil was pressed out by means of a gradual increase in the load.Heat pressing is washing the heated pine nut kernels with hot water and then the heat pressing thereof.
The 1.0%, 5.0% and 10.0% cedar O/W emulsions were prepared by adding cedar oil (1 g, 5 g or 10 g) slowly to water (100 g) in the presence of an emulsifier (0.01 g, 0.05 and 0.10 g of soya lecithin, respectively) stirring continuously (the stirring rate is 500 rpm).
The antibacterial (antimicrobial) properties of the in vitro oil obtained from pine nuts were determined in terms of the growth of the opportunistic and pathogenic test strains of microorganisms using the diffusion method and a method based on the measurement of optical density.
Diffusion method [35,36].The testing culture was inoculated on a dense nutrient medium as lawn.The microorganisms were grown at the values of pH and temperature optimal for each test strain for 24 hours.The culture liquid was centrifuged at 7000 rpm for 10 minutes and the supernatant was separated.To separate the cells, the supernatant was filtered through Millex-GV filters (0.22 μm, Nihon "Millipore", USA).The antimicrobial activity was estimated by measuring the inhibition zones with respect to the testing culture of a microorganism [37].The paper discs were dipped in the emulsions (cedar oil) / suspensions (a proteinvitamin complex, a carbohydrate-mineral complex) containing the tested food ingredients squeezing out the excesses.The discs were put on the agar with a testing culture observing the rules of asepsis.The disks were arranged so that the distance between their centers was not less than 24 mm.After placing the discs on the agar, they were pressed with a sterile needle or pincers until they completely contacted with the surface of the medium.
After 15 minutes after placing the discs, the cups were inverted and incubated at the pH and temperature optimal for each test strain for 24 hours.After incubation, the diameter of the zones of complete incubation was measured (according to the observation with the naked eye), including the diameter of the disk, to the nearest whole millimeter using a caliper, ruler or stencil designed for these purposes.
Optical density measurement.The method for determining antimicrobial activity based on the measurement of optical density is as follows.To estimate the antibacterial effect of the food ingredients, the testing cultures were co-incubated with the studied ingredients in 96-well culture plates.
The night broth cultures were resuspended in a nutrient medium corresponding to the species of microorganisms bringing the number of microorganisms to an inoculation dose of ~10 5 CFU/ml.A cell suspension and the studied food ingredients were simultaneously added in the wells in an amount of 1/10 of the total volume.Control is a liquid nutrient medium with no cedar oil added.The reference drug (Control 1) is the antibiotic ciprofloxacin (10 μg/ml).The total volume of suspension in a well is 200 μl.The number of repetitions is 2. The incubation was carried out at a temperature corresponding to the optimal growth temperature for each test strain using a shaker (580 rpm).After 24 hours, the optical density (OD) was measured using a multireader at a wavelength of 595 nm.The presence of antimicrobial properties was estimated by a change in OD as compared with the control [38].In the wells where the cell growth stopped or slowed down, OD was lower than that in the wells with the normal growth of microorganisms.
To estimate the prebiotic properties of in vitro oil derived from pine nuts, the ability to stimulate selectively the growth of protective populations (bifidobacteria) of normal intestinal microflora was determined; as the test strain, Bifidobacterium adolescentis MC 42 was used.The test strain was inoculated in Petrie dishes followed by thermostating under anaerobic conditions (using an anaerobic culture apparatus and anaerobic agent) into the agarized selective culture media with the addition of a certain amount of a food ingredient and / or the dilution thereof.The Petrie dishes were thermostated at (37 ± 1)°C for (72 ± 3) h under anaerobic conditions.After incubation, the number of the colonies grown in the Petrie dishes was counted.
The in vitro antioxidant activity of the test oil samples obtained from pine nuts was estimated using the ORAC fluorescence method with the generation of a peroxyl radical in the reaction medium using a BioTek Synergy 2 microplate photometerfluorometer (USA).The strategy for determining antioxidant activity in vitro in the culture test model included the incubation of MDCK1 canine kidney epithelial line cells in the presence of a potential antioxidant agent, the further effect of a free radical oxidation initiator and methods for detecting radical activity in a cell.The free radicals in the cultured cells were detected fluorimetrically according to the fluorescence intensity of dichlorodihydro-fluorescein diacetate (DCFH-DA) fluorochrome that penetrates into living cells where, being affected by cellular enzyme systems, a non-fluorescing compound is formed, which, in turn, being affected by free radicals, turns into fluorochrome.
The passage of 100 µl of cell suspension with the cytosis of 10 6 cells/ml was provided in the wells of sterile black 96-well plates.The edge wells of the plate were filled with a cell-free culture medium to provide the same moisture conditions in all the experimental wells of the plate.The plate was incubated in a thermostat for 12...24 h to allow the cells to precipitate and attach to the surface of the culture plastic.Then the contents of the wells were aspirated and 100 μl of potential antioxidant agent solutions (emulsions) at concentrations of 1.0%, 5.0% and 10.0% was added to each well.As the control, the cells cultured in 100 μl of a saline solution without the addition of food ingredients were used.The plate was incubated in a thermostat for 2 hours, then the contents of the wells were aspirated and 100 μl of a working DCFH-DA solution (30 min) was added, after which the solution was aspirated and the peroxide oxidation was induced by adding 100 μl of a working 2,2'-azobis 2-amidino-propane) dihydrochloride (AAPH) solution into the wells.
The fluorescence intensity was determined at an excitation wavelength of 485 nm and an emission wavelength of 528 nm using a Synergy 2 spectrophotometer-fluorometer immediately after the addition of AAPH ("0") in 30 minutes, 60 minutes and 90 minutes.
The anti-hypertensive (hypotensive) activity of the in vitro food ingredients derived from pine nuts was determined in vitro according to their ability to inhibit the angiotensin-1-converting enzyme (ACE), a key link in the renin-angiotensin system that regulates human blood pressure.The most sensitive method for determining the ACE inhibitory activity of a substance is a method with the use of internally-quenched ACE substrates.As an internally-quenched substrate, o-aminobenzoylphenylalanyl-arginyl-lysyl(dinitrophenyl)-proline was used, the measurements were carried out using a BioTek Synergy 2 microplate photometer-fluorometer (USA).The reaction time was 30 minutes at 37°C.

Statistical analysis.
All the experiments were carried out n-fold, n = 5.The data were processed using mathematical statistics standard methods.The homogeneity of the sampling effects was checked using Student's t-test.The differences between the averages were considered significant if the confidence interval was less than 5% (p ≤ 0.05).

RESULTS AND DISCUSSION
The previous studies of the nut kernels of the crop of 2016-2017 of the cedar pines that grow in the Kemerovo region confirmed the nutritional value of the samples studied [39,40].Oil was derived from the cedar kernels by heat and cold pressing as well as emulsions with the addition thereof.
Table 1 shows the results of the determination of antimicrobial properties of the examples of cedar oil emulsions using the diffusion method.All the experimental samples of cedar oil obtained both by cold and heat pressing are characterized by high antimicrobial activity against all the tested strains except for Candida albicans EMTC 34 and Proteus vulgaris ATCC 63.It should be noted that the antimicrobial properties are determined by cedar oil concentration (up to a concentration of 5.0%).The further increase in the content of cedar oil is not followed by a significant increase in the diameter of inhibition zones of pathogenic test strains.The antimicrobial activity of cedar oil in a liquid nutrient medium was determined for the samples of 5.0% O/W cedar emulsions.The results are shown in Fig. 1.
The analysis of the data (Fig. 1) shows that the results of determining the antimicrobial activity of cedar oil in a liquid nutrient medium are consistent with the data obtained using the diffusion method (Table 1).Cedar oil shows high antimicrobial activity with regard to the test strains of Pseudomonas aeruginosa ATCC 9027, Alcaligenes faecalis EMTC 1882, Leuconostoc mesenteroides EMTC 1865, Escherichia coli ATCC 25922, Enterococcus casseliflavus EMTC 1866, Salmonella enterica ATCC 14028, Staphylococcus aureus ATCC 25923 and Bacillus mycoides EMTC 9.
Figures 2-4 show the diagrams of a change in the number of bifidobacteria cells in the nutrient media that contain the oil derived from pine nuts.
The oil emulsions obtained with the addition of cedar nut oil have prebiotic properties since the number of cells of the bifidobacteria cultivated in nutrient media with oil (Fig. 2-4) increases compared to the amount of Bifidobacterium adolescentis cultivated in a nutrient medium with no addition of cedar oil.When cultivating bifidobacteria for 48 hours in a nutrient medium that contains cedar oil (5.0 and 10.0% emulsions in water), the number of cells increases up to 3.0 times compared to the control.
Figures 5-7 show the results of the determination of the antioxidant properties of the test cedar oil samples.Cedar oil shows pronounced antioxidant properties; there is a decrease for all the test samples in the intensity of fluorescence after 90 minutes of cell incubation (from 100% to 81.8%).Antioxidant activity grows with an increase in the concentration of ingredients up to 5.0%.The further increase in the concentration of cedar oil is followed by a slight increase in antioxidant activity.
Table 2 shows the results of the determination of the antihypertensive activity of the in vitro oil obtained from pine nuts.All the test emulsion samples with oil obtained from pine nuts have shown hypotensive properties.An oil emulsion, at a concentration of 5.0% already, has the highest ability to inhibit the angiotensin-1-converting enzyme, approximately by 70%, the further increase in the oil content practically does not change the inhibitory characteristics.The hypotensive properties of cedar oil are probably due to the presence of vitamins E and PP in its composition.29.2 ± 0.9 68.2 ± 0.9 69.7 ± 1.2 Oil (heat pressing) 28.9 ± 1.2 69.5 ± 0.9 70.8 ± 0.8 CONCLUSIONS According to the results of the study of the oil produced from the nuts of cedar pine that grows in the Kemerovo region, all the test samples obtained by both cold and heat pressing are characterized by high antimicrobial activity in relation to all the test strains, except for Candida albicans EMTC 34 and Proteus vulgaris ATCC 63.The antimicrobial characteristics depend on the concentration of cedar oil and can be observed at a concentration of up to 5.0%.
Pine cedar oil, regardless of a production method, has a pronounced prebiotic effect.The number of the Bifidobacterium adolescentis cells cultivated in nutrient media with cedar oil exceeds the amount when cultivated in a nutrient medium without cedar oil from 1.7 to 3.1 times.The highest value (the difference is about 3 times) of the yield of bifidobacterial cells was observed during the cultivation in a nutrient medium supplemented with cedar oil for 48 hours (a 5.0% and 10.0% emulsion in water).The in vitro study of the antioxidant effect of cedar oil using the cultured MDCK1 cells has shown that cedar oil is characterized by pronounced antioxidant properties after 90 minutes of cell incubation for all the test samples (a decrease in fluorescence intensity).There was a decrease in antioxidant activity with an increase in the concentration of ingredients up to 5.0% for all the samples.The further increase in the concentration of food ingredients results in a slight increase in antioxidant activity.
The determination of antihypertensive activity of in vitro cedar oil shows that all the test samples have revealed antihypertensive characteristics.Cedar oil in a 5.0% emulsion inhibits angiotensin-1 by 68.9%, in a 10.0% emulsion -up to 70.2%, which is explained by the presence of vitamins E and PP therein.
The confirmation in vitro of the availability of biofunctional properties of the oil obtained from the nuts of cedar pine that grows in the Kemerovo region allows it to be used as the basis of not only biologically active food supplements, but also of dietary, therapeutic, preventive and sports nutrition.

Fig. 2 .
Fig. 2. Dynamics of the number of Bifidobacterium adolescentis MC 42 (lgN) cells in the nutrient medium that contains cedar oil (1.0% emulsion in water) after 24 h, 48 h and 72 h of fermentation: (1) oil (cold pressing); (2) oil (heat pressing); (3) control (a culture medium that does not contain any potential antioxidant agents).The data are expressed as a mean ± standard deviation (n = 5).

Fig. 3 .
Fig. 3. Dynamics of the number of Bifidobacterium adolescentis MC 42 (lgN) cells in the nutrient medium that contains cedar oil (5.0%emulsion in water) after 24 h, 48 h and 72 h of fermentation: (1) oil (cold pressing); (2) oil (heat pressing); (3) control (a culture medium that does not contain any potential antioxidant agents).The data are expressed as a mean ± standard deviation (n = 5).

Fig. 4 .
Fig. 4. Dynamics of the number of Bifidobacterium adolescentis MC 42 (lgN) cells in the nutrient medium that contains cedar oil (10.0%emulsion in water) after 24 h, 48 h and 72 h of fermentation: (1) oil (cold pressing); (2) oil (heat pressing); (3) control (a culture medium that does not contain any potential antioxidant agents).The data are expressed as a mean ± standard deviation (n = 5).

Fig. 5 .
Fig. 5. Results of in vitro determination of the antioxidant effect of cedar oil on the cultured MDCK1 cells for 30 min: (1) oil (cold pressing);(2) oil (heat pressing).The data are expressed as a mean ± standard deviation (n = 5).

Fig. 6 .
Fig. 6. Results of in vitro determination of the antioxidant effect of cedar oil on the cultured MDCK1 cells for 60 min: (1) oil (cold pressing);(2) oil (heat pressing).The data are expressed as a mean ± standard deviation (n = 5).

Fig. 7 .
Fig. 7. Results of in vitro determination of the antioxidant effect of cedar oil on the cultured MDCK1 cells for 90 min: (1) oil (cold pressing);(2) oil (heat pressing).The data are expressed as a mean ± standard deviation (n = 5).
The present study has been carried out under the Federal Target Program "Research and Development in Priority Development Areas of Scientific and Technological Complex of Russia for 2014-2020 (Agreement No. 14.577.21.0255, the unique identifier of the Agreement is RFMEFI57717X0255).

Table 1 .
Results of the determination of antimicrobial properties of the examples of cedar oil emulsions using the diffusion method

Table 2 .
Results of the determination of the antihypertensive activity of the in vitro oil obtained from pine nut kernels