RU2497945C2 - STRAIN OF UNICELLULAR ALGAE Dunaliella salina IPPAS D-295-PRODUCER OF BIOLOGICALLY ACTIVE SUBSTANCES HAVING ANTIOXIDANT ACTIVITY - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: strain of unicellular algae Dunaliella salina IPPAS D-295-producer of biologically active substances having antioxidant activity was deposited in the culture collection of microalgae of the Institute of Plant Physiology n.a. K.A.Timiryazev RAS (IPP RAS (IPPAS)) under registration number IPPAS D-295 and can be used to produce biologically active substances having antioxidant activity and antagonistic activity against opportunistic pathogenic bacteria.

EFFECT: invention enables to increases the yield of antioxidant substances.

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Description

The invention relates to biotechnology and can be used in the microbiological industry to obtain biologically active substances with antioxidant activity.

At present, the search for substances that stimulate the body's defenses against aggressive environmental influences, intensified as a result of the activation of man-caused human activity, does not stop. One of the groups of drugs of considerable interest is antioxidant substances that can suppress the processes of free radical oxidation of lipids and, as a result, affect the processes of aging, carcinogenesis, atherogenesis, toxic damage to tissues and organs, etc. (Ames BN, Shigenaga MK, HagenTM Oxidants, antioxidants and the degenerative diseases of aging. Proceedings of the National Academy of Sciences of the United States of America, 1993. V.90. P.7915-7922. Poli, G. Liver damage due to free radicals. British Medical Bulletin, 1993. V. 49 (3). P.604-620). Unicellular algae can be one of the sources of substances with antioxidant properties.

Known thermophilic strain Dunaliella bardawil Masazir CCAP 19/38, isolated from Lake Masazir on the Absheron peninsula of the Republic of Azerbaijan (patent EA 201000466 A1, IPC C12N 1/12 A61K 36/02 A23K 1/00 C12R 1/89). The strain is designed to produce biomass as an additive in feed for poultry, livestock, fish farming and sericulture, and can also be used in pharmacology and cosmetology.

The closest analogue is the algae strain Dunaliella salina Teod. CALU - 834 isolated from the coastal zone of Sasyk estuary (patent SU 1324627 A1, (51) 4 A23K 1/00, A01H 13/00, C12N 1/12), which is used in the microbiological industry as a producer of protein and β-carotene, in quality of feed, vitamin and biostimulating additives in the diet of animals, as well as for waste disposal.

The technical result to which the invention is directed is to obtain a new strain - a producer of biologically active substances with antioxidant activity.

The specified technical result is achieved by a new strain of unicellular algae Dunaliella salina IPPAS 295, which was isolated from the brine of the Razval lake (average salinity level - 290.9 g / l), Sol-Iletsk, Orenburg region.

The resulting strain was deposited in the Collection of unicellular algae (IPPAS) Institute of Plant Physiology. K.A. Timiryazev RAS under the number IPPAS 295.

Isolation was carried out in the following way: samples of natural water with a volume of 0.1 ml were sown on an OPS liquid mineral medium of the following composition: NaCl — 116 g, MgSO ₄ × 7 H ₂ O — 50 g, KNO ₃ — 2.5 g, K ₂ HPO ₄ - 0.2 g, NaHCO ₃ - 1.0 g, tap water - 1 l. To isolate a pure culture after biomass growth, a suspension of algae (0.1 ml) was placed on an OPS agar medium: NaCl - 116 g, MgSO ₄ × 7 Н ₂ О - 50 g, KNO ₃ - 2.5 g, K ₂ HPO ₄ - 0.2 g, NaHCO ₃ - 1.0 g, agar-agar - 10 g, tap water - 1 l, individual colonies of algae were obtained by mechanical dissociation with a Drygalsky spatula.

When algae colonies reached the sizes visible to the naked eye, they were transferred under sterile microbiological loop conditions to the OPS liquid mineral medium.

The identification of the strain was carried out on the basis of the study of its cultural and morphological characters in accordance with the criteria given in the monograph Masyuk N.P. “Morphology, systematics, ecology, geographical distribution of the genus Dunaliella Teod. and prospects for practical use ”- Kiev: Naukova Dumka, 1973. - S.172-182.

The storage of the unicellular algae strain Dunaliella salina IPPAS 295 is carried out on an OPS liquid mineral medium: NaCl - 116 g, MgSO ₄ × 7 H ₂ O - 50 g, KNO ₃ - 2.5 g, K ₂ HPO ₄ - 0.2 g, NaHCO ₃ - 1.0 g, tap water - 1 l, at a temperature of 20 ° C for 2 months under natural light. Longer storage is carried out by periodically (optimally - once every 2 months, maximum - once every 5 months) reseeding on fresh liquid mineral medium OPS.

Conditions and media for the propagation of a strain of unicellular algae Dunaliella salina IPPAS 295: inoculation of the initial culture on an OPS liquid mineral medium, followed by cultivation under round-the-clock illumination with fluorescent lamps (5000 lx) at 20-25 ° C for 10 days.

The strain of unicellular algae Dunaliella salina IPPAS 295 is characterized by the following cultural, morphological, physiological characteristics and biological properties.

Cultural properties.

On a liquid medium, OPS is characterized by growth in the form of a green suspension. The degree of purity of the culture is axenic. The absence of bacteria was checked by seeding on solid nutrient media (meat-peptone agar, hungry agar and MPA with 8% NaCI content), the absence of other algae was monitored by light phase contrast microscopy.

Morphological features

The cells have a wide oval shape, with an enlarged posterior and slightly narrowed anterior end, 11 × 8 μm in size (8-15 × 5-13 n = 33). The pyrenoid is large, round, cup-shaped, radially symmetric green chromatophore. Cells with two elastic, homodynamic flagella, of equal length, approximately equal to the length of the cell, making oars-like movements from front to back.

When stored for more than 5 months without reseeding, cells are immobilized, lose their green color, deform, form cysts.

Physiological properties

The optimal abiotic conditions for the maximum yield of biomass of the strain of unicellular algae Dunaliella salina IPPAS 295 are mineralization of 93 g / l, the presence of mixing, a temperature of 20-25 ° C, round-the-clock illumination with fluorescent lamps of 5000 lux. The source of CO ₂ uses air with periodic mixing (16 hours with an interval of 8 hours) on a LAB-PU-01 LOIP mixing device (Russia) with a platform oscillation frequency of 50 rpm. Under optimal cultivation conditions, growth rate, µ: 0.28 million / ml per day.

The practical application of the unicellular algae strain Dunaliella salina IPPAS 295 is determined by its ability to produce biologically active substances with antioxidant activity. The resulting strain is promising for creating on its basis a biologically active additive (BAA), used for direct use in writing as a prophylactic or to create functional foods.

The production of antioxidant substances.

The production of antioxidant substances by the strain of unicellular algae Dunaliella salina IPPAS 295 was determined by the ability of its cell extract to inhibit lipid peroxidation, the rate of which was judged by the intensity of chemiluminescence recorded in chemiluminomer XL-003 (Russia) (Vladimirov Yu.A., Lopukhin Yu.M. ., Molodenkov MN, Klebanov GI // Bull. Expert biol. 1982. No. 4. P.101-102; Farkhutdinov PP, Likhovskikh VA Chemiluminescent methods for the study of free radical oxidation in biology and medicine, Ufa, 1995.54 p.).

The extract of cells of the unicellular algae strain Dunaliella salina IPPAS 295 reduced the intensity of iron-induced lipid peroxidation in vitro, which was reflected in the chemiluminescence parameters of the standard chemiluminescent system: decrease in maximum luminosity by 68%, decrease in light sum of a slow flash by 75%, compared to the control .

The production of antioxidant substances by the unicellular algae strain Dunaliella salina IPPAS 295 is also confirmed in vivo.

Thus, suppression of lipid peroxidation processes in blood serum and in the liver in rats was observed with oral administration of the extract of cells of the unicellular algae strain Dunaliella salina IPPAS 295. The sum of the slow flash of the chemiluminescence curve in the blood serum of animals treated with the cell extract of the unicellular algae strain Punasella sal , decreased by 27%, which indicates a decrease in the ability of blood lipids to undergo oxidation in the experimental group of rats. The decrease in the maximum luminosity of blood serum, that is, the amount of peroxide radicals formed (ROO ^. ) In the process of free radical oxidation, was 44% in the group of experimental animals. Similarly, the parameters of chemiluminescence, reflecting the intensity of lipid peroxidation in the liver, changed. The luminosity of the slow flash reduction in the experimental group was 50% lower than in the control group, and the maximum luminosity was 51%.

Examples of specific performance.

Example 1

The culture of the strain of unicellular algae Dunaliella salina IPPAS 295 was grown on a liquid mineral medium of OPS of the following composition:

sodium chloride 116 g, magnesium sulfate 7-water 50 g, potassium nitrate 2.5 g, disubstituted potassium phosphate 0.2 g, sodium bicarbonate 1.0 g, tap water 1000 ml.

The prepared medium was sterilized at 1 atm. for 20 min, cooled to 20-25 ° C and poured into sterile vials in a volume of 50 ml.

A 5 ml volume of a culture of a unicellular algae strain Dunaliella salina IPPAS 295 was introduced into the prepared medium with a sterile pipette. Crops were grown under round-the-clock illumination with fluorescent lamps (5000 lux) at t20-25 ° C for 10 days.

A cell extract was prepared from the grown culture. For this, the algae was concentrated by centrifugation for 20 min at 3000 rpm (cell concentration 3 × 10 ⁷ cells / ml), the resulting precipitate was diluted with distilled water to a final NaCl concentration of 0.85%, frozen at a temperature of -20 ° C, and then thawed.

The presence of antioxidant substances was determined in the cell extract. For this, a standard chemiluminescent system was prepared consisting of AppliChem lecithin (Germany) diluted in phosphate buffer. The composition of the phosphate buffer: potassium phosphate disubstituted 2.72 g, potassium chloride 7.82 g, distilled water 1 l, pH 7.45.

An extract of cells in an amount of 2 ml was added to 20 ml of a standard chemiluminescent system and placed in a cuvette chamber of chemiluminomer XL-003 (Russia). Glow was induced by adding 1 ml of a 50 mM FeSO ₄ * 7H ₂ O solution with constant stirring, which led to the oxidation of the unsaturated fatty acids that make up the lipids, accelerated lipid peroxidation and was recorded as a flash of chemiluminescence.

When evaluating the results, several basic parameters of chemiluminescence were determined: the height of the fast flash, depending on the content of lipid hydroperoxides in the studied system; the height of the slow flash, reflecting the ability of lipids to peroxidation; maximum LPO intensity after Fe ²⁺ administration.

At the same time, the light sum of the slow flash was estimated, defined as the area under the curve in the graph from the beginning of the slow flash to the maximum value, revealing the number of branching side chains, i.e. the amount of peroxide radicals formed (ROO ^. ) per 1 ion of Fe ²⁺ .

The effect of quenching chemiluminescence was determined as the percentage of decrease in the height of the slow flash and other parameters of chemiluminescence relative to the control standard chemiluminescent system that does not contain algae cell extract.

For comparison, we used the drug "Bioastin" (NPO "Source of Longevity", Russia) containing the extract of alga cells Haematococcus pluvialis Flotow, 1844.

The addition of an algae cell extract to the standard chemiluminescent system led to a decrease in all the basic parameters of chemiluminescence (spontaneous luminosity, slow flash light sum, fast flash height, slow flash height, maximum luminosity), which indicated the ability of the unicellular algae Dunaliella salina IPPAS 295 to produce antioxidant substances .

According to the results of a quantitative comparison of the level of antioxidant activity of different drugs, the most significant and reliable were the differences in the light sum parameters of a slow flash and the maximum luminosity of a standard chemiluminescent system.

The data obtained are presented in table 1.

Table 1 Changes in the chemiluminescence parameters of a standard chemiluminescent system under the influence of antioxidants Chemiluminescence parameters
samples Spontaneous luminosity, conventional units Light sum of a slow flash, conv. Quick flash, conv. Maximum luminosity, arb. The control 0.71 ± 0.04 4.60 ± 0.23 0.89 ± 0.04 1.14 ± 0.06 Bioastin 0.37 ± 0.02 1.53 ± 0.08 0.37 ± 0.02 0.37 ± 0.02 Dunaliella salina IPPAS 295 Cell Extract 0.37 ± 0.02 1.17 ± 0.06 0.42 ± 0.02 0.37 ± 0.02

As can be seen from table 1, the decrease in maximum luminosity in the presence of the cell extract of the strain of unicellular algae Dunaliella salina IPPAS 295 was 68%, compared with the control, indicating a decrease in the intensity of iron-induced lipid peroxidation.

At the same time, under the action of the extract of cells of the unicellular algae strain Dunaliella salina IPPAS 295, a decrease in the light sum of the slow flash by 75% was registered, which turned out to be more pronounced than under the influence of the Bioastin preparation (67%).

In comparison with the control, a decrease was noted in other indicators of LPO intensity: spontaneous luminosity by 48% and fast flash by 53%, which indicated a decrease in the content of lipid hydroperoxides in the studied system.

According to the results of changing the chemiluminescence parameters of the standard chemiluminescent system, the strain of unicellular algae Dunaliella salina IPPAS 295 is capable of producing antioxidant substances, the level of which is comparable to the known analogues.

Example 2

In order to determine how the antioxidant effect will be realized in the conditions of the whole organism, an experiment was conducted on animals. Animals - male rats weighing 220-250 g were kept in standard vivarium conditions on a standard diet and were of the same age.

Daily at the same time for 10 days, under ether raush anesthesia in compliance with aseptic and antiseptic rules, 10 test animals were injected with a probe 0.5 ml of the extract of the cells of the strain of unicellular algae Dunaliella salina IPPAS 295, prepared analogously to example 1, and A placebo preparation (0.5 ml of a 0.85% NaCl solution) was administered to 10 control animals under a rash anesthesia probe.

Animals were withdrawn from the experiment by inhalation of a lethal dose of ether on the 11th day of the experiment.

Animal studies were carried out in accordance with the draft Federal Law No. 97802163-2 "On the Protection of Animals from Cruelty" adopted by the State Duma of the Russian Federation on December 1, 1999, and also by order of the USSR Ministry of Health No. 755 of 08/12/1977, "Rules for working with using experimental animals. "

Table 2 shows the data on the effect of the extract of cells of the unicellular algae strain Dunaliella salina IPPAS 295 on the intensity of lipid peroxidation in rat blood serum by chemiluminescence indices reflecting lipid peroxidation processes.

In rat serum of the experimental group, a decrease in all indicators reflecting the LPO intensity was observed compared with the control group; the most significant and significant differences were the light sum of the slow flash and the maximum luminosity of rat serum. The luminosity of the slow flash reduction in the experimental group was 27% lower than in the control group, and the maximum luminosity was 44%. The obtained data demonstrate a significant decrease in the intensity of lipid peroxidation in blood serum in experimental animals that received the extract of cells of the strain of unicellular algae Dunaliella salina IPPAS 295.

table 2 Indicators of chemiluminescence, reflecting the intensity of lipid peroxidation in rat serum, conventional units Options
chemiluminescence The control Experience Mann-Whitney test p Light amount of slow flash 1.83 ± 0.27 1.34 ± 0.13 thirty ≤0.01 Maximum luminosity 0.61 ± 0.22 0.34 ± 0.03 thirty ≤0.01

Given the high activity of metabolic processes in the liver, along with an assessment of the intensity of peroxidation in blood serum, we studied the chemiluminescence indices in rat liver homogenates.

Table 3 presents the indicators of chemiluminescence in the homogenates of rat liver of the experimental and control groups.

The luminosity of the slow flash reduction in the experimental group was 50% lower than in the control group, and the maximum luminosity was 51%. The obtained data demonstrate a significant decrease in the intensity of lipid peroxidation in the liver in experimental animals that received the extract of cells of the strain of unicellular algae Dunaliella salina IPPAS 295.

Table 3 Chemiluminescence indices reflecting the intensity of lipid peroxidation in rat liver homogenates, conv. Chemiluminescence parameters The control Experience Mann-Whitney test R Light amount of slow flash 19.64 ± 7.65 9.82 ± 3.04 28 > 0.05 Maximum luminosity 5.33 ± 2.08 2.62 ± 0.53 thirty ≤0.01

From the results of evaluating the intensity of chemiluminescence in blood serum and liver, it follows that the cell extract of the strain of unicellular algae Dunaliella salina IPPAS 295 contains antioxidant substances and suppresses lipid peroxidation in the body.

Thus, the results obtained indicate that the isolated strain of unicellular algae Dunaliella salina IPPAS 295 is a producer of antioxidant substances and can be used as the basis for the development of biologically active additives used for direct use in writing as a prophylactic or for creating functional foods .

Claims (1)

The strain of unicellular algae Dunaliella salina, deposited in the collection of microalgae cultures of the Institute of Plant Physiology K.A. Timiryazev RAS (IPRAS) under the registration number IPPAS D-295 is a producer of biologically active substances with antioxidant activity.