Cryopreservation potential of Streptomyces lucensis and Streptomyces violaceus actinomycete collection strains as producers of glycosidase inhibitors

: The article presents a study into the effect of long-term, low-temperature (-80 and -150 °С) storage on the properties of Streptomyces lucensis RNCIM As-1743 and Streptomyces violaceus RNCIM As-1734 actinomycete collection strains acting as producers of glycosidase inhibitors. The titre (CFU in 1 cm 3 of the initial inoculum) and the inhibitory activity of strains were determined with respect to pancreatic α-amylase in the solutions obtained by Streptomyces culture on a corn starch hydrolysate. For Streptomyces, a high survival rate (91–100 %) was established after storage at temperatures of -80 and -150 °C using a 15 % glycerol solution in terms of a cryoprotector. Streptomyces violaceus strain was identified to be the most resistant to long-term storage at low temperatures. Its inhibitory activity turns to be completely retained after storage at temperatures of -80 and -150 °С. In Streptomyces violaceus strain, the maximum activity level of 2250±200 IU/cm 3 for an inhibitor of pancreatic α-amylase is observed on the 1st day of subculture, while Streptomyces lucensis RNCIM As-1743 demonstrates the highest activity value on the 3rd day to reach a value of 3660±200 IU/cm 3 following storage at a temperature of -80 °С. The studied Streptomyces strains are chromogenic. The most intense chromogenesis is noted during the culture of Streptomyces violaceus strain stored at a temperature of -150 °С. The cryopreservation of Streptomyces violaceus and Streptomyces lucensis actinomycete strains was established to provide high (10 7 –10 8 ) cell survival and preservation of their inhibitory activity at a high level when exposed to temperatures of -80 and -150 °С with a 15 % glycerol solution as a cryoprotector. Experimental data indicate the low-temperature storage method to be promising for Streptomyces lucensis and Streptomyces violaceus collection cultures.


INTRODUCTION
Actinobacteria of the Streptomyces genus manifest themselves as producers of more than half of the most common biologically active compounds applied in medicine, veterinary, agriculture and other industries. Thus, maintaining the viability of Streptomyces cultures during long-term storage constitutes an important research area and makes a significant theoretical and practical contribution to the problem of preserving an ever-increasing biological diversity.
For most microorganism collections, the preservation of strains in working condition with the retention of their valuable properties is carried out using periodic subculture methods, storage under a layer of mineral oil, in dry sterile soil or sand, in saline solutions, as well as in a lyophilised state or at low temperatures [1].
The primary method for long-term microorganism storage consists in cryopreservation. Among the advantages of cryogenic storage are the low probability of culture infection, preservation of microorganism properties in a stable state, low time and material costs, the possibility of using frozen cultures as a direct inoculum and eliminating the risk of genetic changes in cultures during storage. In addition, according to the published studies, microorganism cultures stored at low temperatures in a frozen state appear to be less damaged and demonstrate a higher level of viability as opposed to dried and lyophilised cultures [1,2]. The cryostability of microorganisms is determined by their taxonomic affiliation, physiological state, cell number, cryopreservation modes, the presence of a protective environment, as well as the temperature and heating rate. Different cryostability is observed in microbial organisms of not only different genera and species, but also strains. Gram-positive bacteria are known to be usually more resistant to freezing in contrast to gramnegative bacteria [3]. It is thought that microorganisms are most resistant to freezing at the end of the logarithmic growth stage or at the beginning of the stationary phase. According to a number of studies, increased cryoresistance is also characteristic of spore-forming microorganisms [4].
Protective substancesor cryoprotectorscan be applied in order to increase the resistance of cells to low temperatures. Two main cryoprotectant types can be distinguished. The first includes glycerin and dimethyl sulphoxide (DMSO) either easily to pass through the cell membrane and provide both intracellular and extracellular protection against freezing. The second type of cryoprotectors involves the following substances providing a protective effect on the outer surface of the cell membranes: sucrose, lactose, glucose, mannitol, sorbitol, dextran, polyvinylpyrrolidone and polyglycol. According to some studies, the first type of protectors (glycerin and DMSO) have been shown to be more effective and applicable for a wide range of bacteria [5]. In this case, the protective action of glycerin is explained by its prevention of excessive salt concentration levels causing a violation of biochemical processes in microorganism cells during freezing [3].
A distinctive feature of many Streptomyces consists in their chromogenetic ability by forming carotenoid pigments, black-brown melanins and blue-violet anthocyanins. Thus, the colour of the substrate and aerial mycelium is determined by a variety of pigments. Chromogenesis in microorganisms has a certain physiological significance through the protection of cells from natural ultraviolet radiation, participation in biochemical reactions and antibiotic effect [6]. Therefore, the study of stressful effects on Streptomyces bacteria appears to be of great practical importance.
Streptomyces lucensis RNCIM As-1743 and Streptomyces violaceus RNCIM As-1734 collection cultures of the All-Russian Research Institute of Food Additives (VNIIIPD) involves the producers of glycosidase inhibitorsbiologically active substances and potential food micro-ingredients [9]. Currently, no studies on effectiveness evaluation of the long-term storage at extremely low temperatures in relation to these actinobacteria were conducted.
The present study is aimed at the effect of low temperatures (-80 and -150 °С) on the viability of Streptomyces lucensis RNCIM As-1743 and Streptomyces violaceus RNCIM As-1734 actinomycetes and their preservation of inhibitory activity during storage.

EXPERIMENTAL PART
The object of the study involves Streptomyces lucensis RNCIM As-1743 and Streptomyces violaceus RNCIM As-1734 actinomycete strains selected at VNIIIPD and deposited in the All-Russian collection of industrial microorganisms of the Research Institute for Genetics and Selection of Industrial Microorganisms [10, 11].
The studied actinobacteria strains were cultured before freezing on the slant starch-containing Czapek medium at a temperature of 29 °C for 7 days.
Cell separation was carried out by flushing the culture from a slant agar starch-containing Czapek medium using 10 cm 3 of nutrient fermentation medium. In order to increase the resistance of cells to the effects of low temperatures, a cryoprotector of 15 % glycerol solution was added to the nutrient medium for fermentation. In the study, bacterial suspensions were applied with a population density of 10 7 -10 8 CFU/cm 3 . Cryo-(Greiner Bio-One, Germany) and Eppendorf tubes were used for freezing spore suspensions at a temperature of -80 °С and -150 °С, respectively. All tubes were exposed to +4 °С for 2 h with subsequent placement in low-temperature freezers. Cells were frozen in stages: up to -30 °С at a controlled cooling rate of 1 °С/min followed by lowering the temperature to -150 °С at a rate of 15-30 °С/min.
The samples of Streptomyces actinomycete strains were frozen and stored in an MDF-1156ATN ULTRA-LOW temperature freezer (SANYO Electric Co. Ltd) at a temperature of -150 °С and in the "Station of low-temperature automated storage of biological samples at -80 °С" Unique Research Installation (LiCONiC Instruments, Liechtenstein) of the Departmental collection of beneficial microorganisms for agricultural purposes of the Research Institute of Agricultural Microbiology [12]. The storage duration comprises 9 months.
The recovery process for frozen cells was carried out by thawing at a temperature of 37 °C for 3 minutes. Then the cells were placed on Czapek agar medium in test tubes. After 7 days of incubation at a temperature of 29 °C, the viability of the cultures was determined and culturing was carried out.
The viable cell count (CFU/cm 3 ) of Streptomyces actinomycetes was determined by serial dilutions of thawed spore suspensions, followed by inoculation on Petri dishes with agar Czapek starch-containing medium. After exposing the cultures for 3 days at a temperature of 29 °C, the number of cultured microcolonies was calculated. Percentage of survived CFU of actinobacteria was determined relative to the number of CFU counted before cryopreservation.
The inhibitory activity of cells survived after storage was determined in the obtained solutions by culturing Streptomyces lucensis and Streptomyces violaceus using the colorimetric method in reference to pancreatic amylase (test enzyme). An amount of an inhibitor suppressing 1 unit of pancreatic α-amylase by 50 % at a temperature of 37 °C and pH = 7 was assigned as a unit of inhibitory activity [9]. Prior to determination, the intrinsic amylase of Streptomyces strains was inactivated in the solution by heat treatment at 98±1 °С. The inhibitory effect was studied in reference to pancreatic amylase (Sigma, USA, enzyme activity 34517 u/g) [14].
The chromogenesis property was evaluated using a UV-1800 spectrophotometer (Shimadzu, Japan) by determining the optical density of the solutions obtained by Streptomyces culturing on a starch hydrolysate at a wavelength of 560 nm.
Statistical processing data on inhibitory activity and viability was performed using the Microsoft Office Excel 2010.

RESULTS AND DISCUSSION
Previous studies have proved the low temperature storage of Streptomyces to cause no adverse effect on the viability and inhibitory activity of cultures. The studied strains of Streptomyces lucensis RNCIM Аs-1743 and Streptomyces violaceus RNCIM Аs-1734 actinomycetes with a population density of 10 7 -10 8 CFU preserved a high viability and inhibitory activity with reference to pancreatic α-amylase after 4 months of storage in a 15% glycerol solution at a temperature of -12 and -18 °C [15]. As a result of these studies, the Streptomyces lucensis RNCIM As-1743 and Streptomyces violaceus RNCIM As-1734 collection strains were established to tolerate the freezing process at extremely low temperatures (-80 and -150 °C).
According to our data, high viability is retained in the cells of the studied cultures stored in a 15 % glycerol solution at temperatures of -80 and -150 °C after 9-month storage. Thus, after 9 months of storage at temperatures of -80 and -150 °C, the survival rate of the studied actinobacteria cells stored at a population density of 10 7 and 10 8 CFU was preserved throughout the entire storage time and amounted to 91-100 %. For Streptomyces lucensis RNCIM As-1743, the number of viable cells in frozen suspensions ranged from 1.94·10 8 (Table).
The viability study into Streptomyces lucensis RNCIM As-1743 and Streptomyces violaceus RNCIM As-1734 collection cultures established these Streptomyces actinomycete strains to be highly resistant to cryopreservation and remain viable under high stress conditions. The long-term, low-temperature resistance of the studied strains is apparently due to the biological characteristics of Streptomyces lucensis and Streptomyces violaceus.
Collection and industrial strain microorganisms are acknowledged to demonstrate population variability in addition to loss of cell viability during storage. In this case, the dominant phenotype is replaced by another with altered properties and productive activity, resulting in the loss of strain priority properties [7].
The studied Streptomyces strains manifest themselves as producers of a pancreatic α-amylase inhibitor. For this reason, maintaining a high level of biosynthetic activity of strains acting as producers of biologically active substances during low-temperature storage turns to be a prerequisite. Therefore, in addition to studies on the effect of cryopreservation on the viability and inhibitory activity of actinomycete cultures during long-term storage was evalu-ated. Throughout the storage period, inhibitory activity referenced to pancreatic α-amylase was monitored. Figures 1-3 demonstrate the data on inhibitory activity of Streptomyces strains stored at ultra-low temperatures of -80 and -150 °C when culturing on a corn starch hydrolysate.

Рис. 3. Влияние температуры криогенного консервирования на динамику ингибиторной активности штамма Streptomyces lucensis ВКПМ Ac-1743 в процессе рекультивирования
As a result of the studies, a high level of biosynthetic activity is established to be preserved in both strains after 9-month storage at temperatures of -80 and -150 °C. Streptomyces violaceus strain was identified to be the most resistant to long-term storage at low temperatures, as opposed to Streptomyces lucensis. As referenced to pancreatic α-amylase, the inhibitory activity of Streptomyces violaceus is completely retained at temperatures of -80 and -150 °C; this contrasts with the Streptomyces lucensis strain, in which with the inhibitory activity decreased by 78 % by the end of the storage period at a temperature of -150 °С as compared to the value prior to cryopreservation.
At a storage temperature of -150 °C, the inhibitory activity of the Streptomyces violaceus strain remained at a higher level as compared to the temperature of -80 °C. In Streptomyces violaceus strain, the maximum activity level for an inhibitor of pancreatic α-amylase is observed on the 1st day of reculturing and comprises (2250±200) IU/cm 3 after storage at -150 °С, while Streptomyces lucensis RNCIM As-1743 demonstrates the highest activity on the 3rd day of reculturing to reach a value of 3660±200 IU/cm 3 following storage at a temperature of -80 °С. The Streptomyces lucensis cells reduced following cryopreservation at a temperature of -150 °С is characterised by low activity glycosidase inhibitor production on the 1st-2nd day of subculture equal to 600 ± 70 IU/cm 3 . Unlike the strain of Streptomyces violaceus stored under similar conditions, the Streptomyces lucensis strain stored on a Czapek dense nutrient medium for a long time (9 months) at a temperature of +4 °С without subculture is also noted to almost completely lose the ability to synthesise a glycosidase inhibitor (see Fig. 2, 3).
According to the obtained data, the reaction of the Streptomyces lucensis strain to the lowtemperature effect (-150 °С) is represented by the almost complete loss of the inhibitor synthesis ability.
A sharp increase in inhibitory activity of producers exposed to abiotic stress on the first day of subculture is apparently due to the fact that, when the temperature drops from negative to positive (from -80 and -150 to +37 °С compared to rise from +4 to +37 °С when thawing cultures after storage), the restoration of biochemical reactions in response to stress exposure is sharply activated. During the first days following anabiosis, the culture synthesises its own amylases to assimilate the carbon source (glucose, maltose and dextrins representing products of starch hydrolysis in starch hydrolysate) followed by a secondary metabolite, amylolytic enzyme inhibitor, is produced under conditions of carbon starvation. The inhibitor activity decreases with increasing duration of fermentation, possibly due to its structural modification. This is acknowledged for the studied glycosidase inhibitors synthesised by streptomycetes during the fermentation of carbohydrate-containing media The adaptation to culture conditions turns out to be slower, when subculturing the studied streptomycetes stored at a temperature of +4 °С.
The Streptomyces strains under study are recognised to be chromogenic. In relation to the presented experiments, the "chromogenic" indicator is possible to be used as a criterion for assessing the adaptation of Streptomyces culture to external conditions. In other words, when the temperature moves from negative to positive, the restoration of biochemical reactions in response to stressful effects is intensified. The synthesis of secondary metabolites is activated, including pigments and enzyme inhibitors.
In accordance to the results of the studies, following 7 days of streptomycetes culture incubation on Czapek dense agar medium in test tubes at a temperature of 29 °С, a clear difference is observed in the staining of aerial mycelium strains. For Streptomyces lucensis RNCIM As-1743, the aerial mycelium pigment is white with a greyish tint, while the staining of Streptomyces violaceus RNCIM As-1734 is pink-violet (Fig. 4).
After the first culture on agar medium of cells was reduced following storage at low temperatures (-80 and -150 °C), a more intense chromogenesis was observed in Streptomyces violaceus RNCIM As-1734 strain. The colour of the aerial mycelium of the strain takes on a dark purple hue (tubes 4-6 in Fig. 4). The effect of low temperatures probably results in a partial destruction of proteins, with the products composing a substrate for tyrosinase catalysing the oxidation of phenols. More intense chromogenesis of the Streptomyces violaceus RNCIM As-1734 culture is probably due to higher tyrosinase activity compared to Streptomyces lucensis RNCIM As-1743 strain [14]. The most intense pigment diffusion into the medium is noted after storage of the conidia of Streptomyces violaceus strain at a temperature of -150 °C.
In order to determine the pigment concentration varying, as proved by studies, in dependency with the storage temperature and the duration of streptomycete culturing after removal from the anabiosis, the optical density of solutions was measured as characterised by an increase at the maximum absorption of the synthesised pigment by the end of the culture process: after storage at a temperature of 80 °C, the optical density increased from 0.42 AU on the 1st day to 1.5 AU at the end of the process; after storage at a temperature of -150 °C, an increase from 0.85 to 1.9 AU, respectively, is detected. The data obtained indicate an increase in the concentration of chromogenic substances in the culture fluid. Apparently, the actinorhodin pigment is synthesised, inherent to the cultures of Streptomyces coelicolor, S. violaceoruber and S. lividans, with the absorption maximum ranging from 540 to 640 nm [18,19]. This pigment changing colour from red to blue in dependence to the conditions of microorganism culturing is used by researchers as an indicator of changes in pH of the medium. The results of the studies demonstrated the pH to vary from 7 to 4.5 for the culture fluid where benzoisochromanquinone pigments are synthesised as characteristic for actinomycetes with a generic affiliation to Streptomyces [20, 21]. 1 2 3 4 5 6 (2 5) and -150 °C (3, 6) -80 °C (2, 5) и -150 °C (3, 6)

ФИЗИКО-ХИМИЧЕСКАЯ БИОЛОГИЯ / PHYSICOCHEMICAL BIOLOGY
According to the study results, the intensely pigmented Streptomyces violaceus RNCIM As-1734 strain is more resistant to temperature changes; this is additionally confirmed by the above results of studies on the effect of ultra-low temperatures on the strain inhibitory activity (see Fig. 1).
A study of the viability of Streptomyces lucensis RNCIM As-1743 and Streptomyces violaceus RNCIM As-1734 collection cultures identified these strains of Streptomyces actinomycetes to be highly resistant to cryopreservation and remain viable and biosynthetic activity under high stress conditions. The resistance of the studied Streptomyces strains to low-temperature exposure for a long time is apparently due to the biological characteristics of these actinobacteria.

CONCLUSION
The cryopreservation study of Streptomyces violaceus and Streptomyces lucensis actinomycete strains showed that the process supports high (10 7 -10 8 ) cell survival and preservation of inhibito-ry activity at a high level under the exposure to temperatures of -80 and -150 °С using a 15 % glycerol solution as a cryoprotector.
The Streptomyces violaceus RNCIM As-1734 strain is identified to be the most resistant to storage at low temperatures, retaining both viability and complete inhibitory activity at the studied temperatures. A higher level of the inhibitory activity of the strain is determined to be preserved at a storage temperature of -150 °C, as opposed to -80 °C.
The most intense pigment diffusion into the medium is noted following storage of the Streptomyces violaceus strain at a temperature of -150 °C.
The obtained experimental data indicate that low-temperature storage of Streptomyces collection cultures supported by the Research Institute for Genetics and Selection of Industrial Microorganisms is a promising method.
The investigated modes of freezing and storage guarantee the preservation of the viability and functional activity of the studied cultures.