Cryoprotective exopolysaccharide from pseudoalteromonas sp, cryoprotective composition comprising same, and method for producing same

Abstract

The present invention relates to a cryoprotective exopolysaccharide and a cryoprotective composition containing same, and more specifically, to a cryoprotective exopolysaccharide derived from a strain of Pseudoalteromonas sp. isolated from the Ross Sea. The exopolysaccharide and the cryoprotective composition containing same according to the present invention exhibit excellent cell viability at low temperatures and are thus useful for protecting or preserving biological samples in refrigerated and frozen states.

Description

Extracellular polysaccharide having antifreeze activity derived from Pseudoalteromonas strain, antifreeze composition containing same, and method for producing same

[0001] The present invention relates to an extracellular polysaccharide having antifreeze ability and an antifreeze composition containing the same, and more particularly, to an extracellular polysaccharide derived from a Pseudoalteromonassp. strain isolated from the Antarctic Ocean.

[0002]

[0003] In extreme climates like Antarctica, freezing and thawing cycles occur continuously, and exposure to subzero temperatures causes ice crystals to form within cells in living organisms. Structural damage caused by ice crystals leads to excessive water loss, protein denaturation, metabolic disturbances, and the accumulation of reactive oxygen species (ROS), ultimately leading to cell death (Zhu M et al., Frontiers in Genetics. 13:870446, 2022).

[0004] It is known to use cryoprotective agents (CPAs) such as dimethyl sulfoxide (DMSO) or glycerol to prevent cell death during freezing and to prevent the formation of ice crystals within cells (Yong SH et al., Plant Biotechnology Reports. 17:625-635, 2023).

[0005] CPA helps protect cells from freezing conditions by inducing cellular dehydration, inhibiting ice crystal formation, and lowering the freezing point, promoting vitrification. High concentrations of CPA are commonly used to achieve these effects, but high concentrations have been reported to cause significant cellular toxicity (Awan M et al., Regenerative Medicine. 15:1463-1491, 2020).

[0006] Meanwhile, many bacteria living in marine environments secrete viscous extracellular hydrocarbon polymers called exopolysaccarides (EPS). EPS protects microorganisms from cold environments, and its viscosity increases at low temperatures due to hydrogen bonding between numerous hydrophilic side chains, which is known to inhibit ice crystal formation (Korber et al., 1982. Diversity and distributions. 11:3-23).

[0007] However, the extraction and purification of plant- and animal-derived EPS is complex and time-consuming. Furthermore, seasonal variations affect the yield and production costs of plant- and animal-derived EPS, making large-scale production and utilization difficult. Therefore, EPS produced by microorganisms isolated from cold marine environments are being extensively studied to develop new cryoprotectants. EPS produced by microorganisms isolated from cold marine environments have advantages such as low cost, low toxicity, high selectivity, inactivity at extreme temperatures, and biodegradability. However, research on extracellular polysaccharides from Antarctic microorganisms is still insufficient, and the need for the development of new antifreeze agents with excellent antifreeze properties remains a pressing issue.

[0008] Accordingly, the inventors of the present invention isolated an extracellular polysaccharide from a Pseudoalteromonas strain isolated from the Antarctic Ocean, and measured cell viability using this in a freeze-thaw experimental environment targeting a HaCaT cell line. As a result, they confirmed that cell viability could be dramatically increased, and thus completed the present invention.

[0009]

[0010] Summary of the invention

[0011] The purpose of the present invention is to provide a Pseudoalteromonassp. RosPo-2 strain (KCTC 15865BP).

[0012] Another object of the present invention is to provide an extracellular polysaccharide (p-CY02) derived from Pseudoalteromonassp. RosPo-2 strain (KCTC 15865BP) having antifreeze activity against cells.

[0013] Another object of the present invention is to provide an anti-freezing composition comprising the extracellular polysaccharide.

[0014] Another object of the present invention is to provide a method for producing the extracellular polysaccharide.

[0015] To achieve the above purpose, the present invention provides a Pseudoalteromonas sp. RosPo-2 strain having the deposit number KCTC 15865BP.

[0016] The present invention also produces a Pseudoalteromonas sp. RosPo-2 strain having the deposit number KCTC 15865BP, and 2.04x10 6 Da to 1.35x10 7 An extracellular polysaccharide having a molecular weight of Da is provided.

[0017] The present invention also provides an anti-freezing composition comprising the extracellular polysaccharide as an effective ingredient.

[0018] The present invention also provides a method for producing an extracellular polysaccharide produced by a Pseudoalteromonas sp. RosPo-2 strain, comprising the steps of a) culturing the Pseudoalteromonas RosPo-2 strain; and b) obtaining an extracellular polysaccharide from the cultured strain.

[0019]

[0020] Figure 1 shows the results of measuring the EPS production and viscosity of 10 strains. Figure 1a is a graph showing the results of measuring the extracellular polysaccharide production in dry weight after culturing in MBC medium at 15℃ for 3 days to select microorganisms with excellent extracellular polysaccharide production among microorganisms isolated from the Ross Sea. Figure 1b is a graph showing the shear stress and shear rate of 0.3% (w / v) EPS. RosPo-7, -11, -13, and -16 showed only slight changes in shear stress and shear rate and are not shown in this graph.

[0021] Figure 2 is a graph showing the comparison of cryoprotective activity in HaCaT cells against various concentrations of EPS produced by RosPo-1 and RosPo-2 strains and DMSO containing 0.8% p-CY02. In Figure 2a, HaCaT cells were suspended in various concentrations of EPS produced by RosPo-1 and RosPo-2 strains, frozen at -80°C for 1 hour, and then thawed at 37°C for 5 minutes. In Figure 2b, HaCaT cells were frozen at -80°C for 1 hour in DMSO alone or in combination with 0.8% (w / v) p-CY02, and then thawed at 37°C for 5 minutes. "Non-freezing" means that there is no cell death because the freezing process does not occur in the medium. "Non-freezing" means HaCaT cells in DMEM; "PBS" means HaCaT cells in phosphate-buffered saline (PBS); "10% DMSO" refers to HaCaT cells suspended in DMEM containing 10% DMSO, and the freezing and thawing conditions were identical. Error bars represent the standard deviation of the mean of three independent experiments. The percentage of damaged cells was determined by LDH cytotoxicity fluorometry. Asterisks indicate statistical comparisons between the control and treatment groups (** p < 0.05).

[0022] Figure 3 shows the results of calculating the molecular weight of p-CY02 produced by the RosPo-2 strain using the Pullulan standard calibration curve.

[0023] Figure 4A shows the results of confirming the composition of p-CY02 produced by the RosPo-2 strain by GS / MS, and Figure 4B shows the results of confirming the binding pattern of the composition of EPS by GC / MS using the partially methylated alditol acetate (PMAA) analysis method.

[0024] Figure 5 shows the results of a phylogenetic analysis based on 16S rRNA gene sequences showing the relationships between Pseudomonas sp. RosPo-2 (PP396838) and other Pseudomonas species. Bootstrap values ​​(>60%) based on 1,000 replicates are indicated for the corresponding branches (maximum likelihood method, neighbor-joining method, and maximum-parsimony method). Asterisks indicate bootstrap values ​​less than 60%. Filled circles indicate conserved nodes with bootstrap values ​​≥70% for the maximum likelihood method, neighbor-joining method, and maximum-parsimony methods. Pseudoalteromonas bacteriolytica IAM14595T (D89929) was used as an outgroup (scale bar, 0.01 substitution per nucleotide position).

[0025] Figure 6 shows a three-dimensional response surface diagram for the activity of the following combinations of salts for p-CY02 production (g / L). Figure 5a: Na2SO4 and KCl, Figure 5b: CaCl2 and KCl, Figure 5c: CaCl2 and Na2SO4.

[0026] Figure 7 is a graph showing the production amount of p-CY02 produced by culturing the RosPo-2 strain.

[0027]

[0028] Detailed description of the invention and preferred embodiments

[0029] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains, unless otherwise defined herein. Generally, the nomenclature used herein is well known and commonly used in the art.

[0030] The present invention screened 66 strains producing mucus in the Ross Sea, Antarctica, using solid marine agar plates, and among them, strains producing exopolysaccharides were isolated. Among these, strains with excellent exopolysaccharide (EPS) production and antifreeze activity against HaCaT cells were isolated and subjected to 16S rRNA sequence analysis. As a result, a novel Pseudoalteromonas sp. RosPo-2 strain producing exopolysaccharides was isolated from a seawater sample from the Ross Sea.

[0031] The extracellular polysaccharide produced by the above Pseudoalteromonassp. RosPo-2 strain was confirmed to have high antifreeze activity during the initial selection process, and as a result, the effect of the extracellular polysaccharide produced by the RosPo-2 strain on the viability of HaCaT cells at low temperatures was confirmed to exhibit excellent antifreeze activity.

[0032] Accordingly, the present invention relates, from one aspect, to a Pseudoalteromonas (Pseudoalteromonas sp.) RosPo-2 strain having a deposit number of KCTC 15865BP. The Pseudoalteromonas RosPo-2 strain of the present invention was classified as a strain of the genus Pseudoalteromonas, which is abundant in Antarctica, through phylogenetic analysis using 16S rRNA sequences (Fig. 5). The RosPo-2 strain showed high homology with Pseudoalteromonas tetraodonis IAM 14160T (99.86%), Pseudoalteromonas isachenkonii KMM 3549T (99.85%), Pseudoalteromonas undina NCIMB 2128T (99.49%), and Pseudoalteromonas espejiana NCIMB 2127T (99.49%) (Fig. 5).

[0033]

[0034] The present invention also relates, from another aspect, to an extracellular polysaccharide produced by Pseudoalteromonassp. RosPo-2 strain having the deposit number KCTC 15865BP.

[0035] In the present invention, the extracellular polysaccharide is 2.04x10 6 Da to 1.35x10 7 It can be characterized by having a molecular weight of Da.

[0036] In the present invention, the main constituent sugars of the extracellular polysaccharide are glucose and galactose, and the molar ratio can be characterized as being about 7.57:1.

[0037] In the present invention, the extracellular polysaccharide may be characterized by a glycosyl linkage analysis result in which 4-linked glucopyranose and 4-linked N-acetyl glucosapyranose are the main components and have a repeating structure, and these components are the components forming the main chain.

[0038]

[0039] In the present invention, 66 strains producing mucus were isolated from seawater samples from the Ross Sea in the Antarctic, and 10 strains with a dry weight of crude extracellular polysaccharide of 1 g / L or more were reselected. Then, viscosity characteristics such as shear stress and shear rate were measured for the purified extracellular polysaccharides of the 10 strains, and two strains, RosPo-1 and Rospo-2, were finally selected. To measure the cryoprotective activity of the extracellular polysaccharides produced by the two strains, the viability of HaCaT cells, widely used in tissue engineering research, was confirmed and measured, and as a result, the extracellular polysaccharide produced by the RosPo-2 strain showed a higher cryoprotective effect on HaCaT cells.

[0040]

[0041] From another aspect, the present invention relates to an anti-freezing composition comprising the extracellular polysaccharide as an effective ingredient.

[0042] In the present invention, the anti-freezing composition preferably has an extracellular polysaccharide content of 0.1 to 1.0 (w / v)%, more preferably 0.8 (w / v)%, but is not limited thereto.

[0043] The cryoprotective composition of the present invention can be used for the purpose of protecting and preserving biological samples, including cells, embryos, tissues derived from humans or animals, microorganisms, plants, etc., while the sample is kept in a refrigerated or frozen state, thereby maintaining the activity of the sample.

[0044] The above-described anti-freezing composition may contain, in addition to the extracellular polysaccharide of the present invention, conventional commercially available BSM (balanced salt media), non-electrolytes, citric acid, magnesium chelate, or high molecular weight anions, in order to maintain the optimal refrigerated preservation condition of the biological sample. In addition, the above-described anti-freezing composition may contain, but is not limited to, a buffer, mannitol, glutathione, or glutamic acid, in order to prevent acidosis, intracellular free radical generation, and contracture that may occur during refrigerated preservation of the biological sample, in addition to the extracellular polysaccharide.

[0045] The anti-freezing composition of the present invention may additionally include an anti-freezing agent such as DMSO, glycerol, propylene glycol, ethylene glycol, propanediol, dimethylformamide, or acetamide, in addition to the extracellular polysaccharide of the present invention, in order to maintain an optimal freezing state of a biological sample.

[0046] The anti-freezing composition of the present invention may additionally include penetrants such as polyvinyl alcohol, polyvinyl pyrrolidine, plant-derived anti-freezing proteins, carboxymethylcellulose, serum albumin, hydroxyethyl starch, ficoll, dextran, gelatin, dairy products, lipid carriers, lecithin, etc., but is not limited thereto.

[0047] The method of preserving a biological sample by refrigerating or freezing it using the anti-freezing composition of the present invention can be performed using techniques commonly known in the art.

[0048] In the present invention, “anti-freezing ability” means the ability to protect cells from damage caused by ice crystal formation at low temperatures, and is used with the same meaning as “cryoprotection” or “cryoprotection activity.”

[0049]

[0050] In another aspect, the present invention relates to a method for producing an extracellular polysaccharide produced by a Pseudoalteromonas RosPo-2 strain, comprising the steps of: a) culturing the Pseudoalteromonas RosPo-2 strain in a medium to obtain a culture solution; and b) extracting the extracellular polysaccharide from the culture solution.

[0051] In the present invention, the medium may be characterized by containing one or more salts selected from the group consisting of KCl, Na2SO4, and CaCl2.

[0052]

[0053] [Example]

[0054] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

[0055]

[0056] Experimental Example 1: Screening of extracellular polysaccharide-producing strains

[0057] During the Antarctic expedition cruise of the Korean icebreaker Araon (December 2012–February 2013), marine samples of various types (seawater, sediment, invertebrates, and benthic organisms) were collected from multiple locations in the Ross Sea (location: 73.0–77.0°S, 167°E–163°W). Seawater samples were collected at various depths using a Niskin bottle system equipped with a Conductivity-Temperature-Depth (CDT) sensor, and bacteria present in the seawater were concentrated using a 0.2 μm filter. The concentrated bacteria were then diluted in sterilized seawater. Deep-sea sediment samples were collected using a box-core sampler, and the sediments were suspended in sterilized seawater. Invertebrates and benthic organisms were collected from the sea floor using a dredge, and the samples were washed five times with sterilized seawater. The samples were then homogenized using a homogenizer and suspended in sterilized seawater. The pretreated marine samples were cultured on marine agar plates (MA; BD DIFCO, USA) at 15°C for 7 days for the initial screening of EPS-producing strains. EPS-producing strains were screened based on colony adhesiveness and ropiness characteristics.

[0058] The isolated strains were inoculated into Marine Broth (MB; DB DIFCO, USA) and cultured at 15°C and 120 rpm for one day. Glycerol was added to the culture medium to obtain a 20% glycerol solution, and each strain was stored at -80°C until the next experiment. Further screening for EPS-producing strains was performed according to the method of Kim et al. (Kim et al., A novel exopolysaccharide (p-CY01) from the Antarctic bacterium Pseudoalteromonas sp. strain CY01 cryopreserves human red blood cells. Biomaterials Science. 11: 7146-7157, 2023).

[0059]

[0060] Experimental Example 2: Cell Culture Conditions and EPS Purification

[0061] For seed culture, glycerol stocks of 66 strains were inoculated into MB medium, respectively, and cultured at 15°C and 120 rpm until the optical density at 600 nm (OD 600 nm) reached 3. The seed was inoculated at a concentration of 5% (v / v) into 25 mL of MB medium containing 2.0% (w / v) glucose (MBG) and cultured at 120 rpm and 15°C for 72 h (main culture). The sample cultured in 25 mL of MBG medium was separated into cells and culture medium by centrifugation (12,000 × g, 25°C for 10 min). The cells were washed three times with deionized water (DW) to recover the EPS attached to the cell surface.

[0062] The culture medium used for washing and DW were mixed, and two volumes of chilled ethanol were added to extract EPS from the mixture. The EPS floating on the solution was harvested, and then centrifuged to remove the remaining supernatant from the EPS. The precipitated EPS was dissolved in 25 mL of DW. This EPS solution was dialyzed for approximately 16 h using a 10 kDa dialysis tube (Thermo Fisher Scientific, USA). The dialyzed EPS solution was lyophilized, and the dry weight was measured (crude EPS).

[0063] Purification prior to cryoprotective activity assay was performed according to the method of Kim and Yim (Kim SJ, Yim JH., Cryoprotective properties of exopolysaccharide (P-21653) produced by the Antarctic bacterium, Pseudoalteromonas arctica KOPRI 21653. Journal of Microbiology. 45: 510-514, 2007). Crude EPS was dissolved in deionized water and treated with protease (500 units / L) at 37°C for 1 h. The mixture was then centrifuged at 12,000 × g for 10 min at 25°C. The crude EPS solution from which proteins had been removed was reprecipitated by adding a 3.0% (w / v) solution of cetylpyridinium chloride (CPC; Sigma, St. Louis, MO). After centrifugation at 12,000 × g and 25°C for 10 min, the supernatant was removed. The precipitated EPS-CPC complex was redissolved in 10.0% (w / v) NaCl. EPS was separated from the EPS-CPC complex solution with 2 volumes of chilled ethanol. The EPS was redissolved in deionized water (DW) and dialyzed using a 100 kDa Viva-Flow (Sartorius, Germany) against deionized water until the conductivity of the wastewater became 0, and the mixture was lyophilized.

[0064]

[0065] Experimental Example 3: Rheological Properties

[0066] The rheological properties of EPS from various strains were measured using the method described by Kim et al. (Kimet et al., 2023. A novel exopolysaccharide (p-CY01) from the Antarctic bacterium Pseudoalteromonas sp. strain CY01 cryopreserves human red blood cells. Biomaterials Science. 11: 7146-7157). Rheological behavior, including shear rate versus shear stress, was measured at 0.3% (w / v) EPS. This approach facilitated the comparison and verification of the relative properties of each EPS. The viscosities of the solutions were measured using an LTV rotating spindle viscometer (DV-II, Brookfield, USA) equipped with a spindle (No. S18).

[0067]

[0068] Experimental Example 4: Cryoprotective activity of Antarctic Ocean strains

[0069] To measure the cryoprotective activity of EPS produced by RosPo-1 and RosPo-2 strains, the HaCaT cell line, which is sensitive to freezing-induced damage and widely used in tissue engineering studies, was used. HaCaT cells (5 × 10 5Cells (10 cells / mL / vial) were suspended in Dulbecco's Modified Eagle Medium (DMEM) supplemented with different concentrations [0.1 to 1.0% (w / v)] of each EPS. These mixtures were frozen at -80°C for 1 h and rapidly thawed in water at 37°C for 5 min. Cell viability, based on the cryoprotective properties of each EPS, was measured according to the manufacturer's instructions for the CyTox-ONETM Homogenate Membrane Integrity Assay Kit (Promega, USA). The percentage of cell viability was calculated using the following formula:

[0070]

[0071] Afterwards, HaCaT cells (5Х10 5 cells / mL / vial) were suspended in DMEM supplemented with 0.8% (w / v) EPS (p-CY02) produced by the RosPo-2 strain or different concentrations [1.0–5.0% (v / v)] of DMSO alone. The freeze-thaw cycles and cryoprotection property measurement methods used were the same as those described herein.

[0072]

[0073] Experimental Example 5: Measurement of the molecular weight of EPS produced by RosPo-2 strain

[0074] The molecular weight of EPS produced by the RosPo-2 strain was measured by gel permeation chromatography (GPC; Waters Alliance 2695 high-performance liquid chromatography (HPLC) system, 2414 refractive index detector, and Shodex GPC columns (804 and 805)). 5 μL p-CY02 (2 mg / mL) was added to the HPLC, and DW was used as the mobile phase. The mobile phase flow rate was set to 0.4 mL / min. The molecular weight was calculated using a pullulan standard calibration curve (Shodex, Japan).

[0075]

[0076] Experimental Example 6: Analysis of the composition and binding patterns of EPS produced by the RosPo-2 strain.

[0077] Analysis of the composition of EPS produced by the RosPo-2 strain was performed using the Trimethylsilation Derivatization analysis method using GC / MS. 1 mg of the sample and standard sugar were each dissolved in 1 ml of HCl ~1.25 M methanol and reacted at 80°C for 16-18 h, then concentrated while removing methanolic-HCl using nitrogen gas. 500 μl of a (Me-OH: Prydine: Acetic unhydride = 4:1:1) solution was added to the dried sample and standard sugar to induce Re-N-acetylation, followed by reaction at 100°C for 1 h. After drying the sample using nitrogen gas, the dried and concentrated sample was dissolved in 0.5 ml of hexane, and 1 μl of it was loaded onto GC-MS for analysis. The analysis was performed using an Elite-5MS low-bleed capillary column (30 m × 0.25 m; Perkin-Elmer) in FID mode on a Clarus 500 (Perkin-Elmer, Waltham, MA, USA) gas analyzer. The analysis conditions were as follows: after sample introduction, the temperature was maintained at 120°C for 1 min, then increased by 2°C / min to 180°C, and then increased by 20°C / min to 230°C. At this time, the retention time and fragmentation patterns of each monosaccharide peak were analyzed in real time using a Clarus 500 mass spectrometer (Perkin-Elmer, Waltham, MA, USA). The TMS derivatives of each monosaccharide were determined by comparing the retention time and fragmentation patterns of the peaks with those of standard monosaccharides.

[0078] Analysis of the binding pattern of EPS produced by the RosPo-2 strain was performed using the partially methylated alditol acetate (PMAA) analysis method based on methylation and acethylation. 5 mg of the sample was placed in a screw-capped tube, and 1 ml of DMSO was added to sufficiently dissolve the sample. 0.5 ml of iodomethane was added, and the atmosphere was purged with nitrogen gas. The methylation process was performed with sufficient stirring at room temperature. 1 ml of a 1:1 chloroform: DW solution was added to the methylated sample, stirred, and centrifuged to remove the supernatant. 0.5 ml of distilled water was added, and the stirring, centrifugation, and supernatant removal processes were repeated three more times, followed by drying using nitrogen gas. 0.5 ml of 2 M TFA was added to the methylated sample, and hydrolysis was performed at 100°C for 2 hours. The residual TFA was evaporated using nitrogen gas, and 0.2 ml of 0.26 M NaBD4 solution was added, gently stirred, and reduced at 40°C for 90 minutes. The excess NaBD4 was stopped by adding 0.2 ml of acetic acid, and the sample was dried using nitrogen gas. The process of adding 0.5 ml of methanol, stirring, and drying to remove the remaining borate was repeated three times. After adding 0.3 ml of acetic anhydride and 0.3 ml of pyridine, stirring was performed, acetylation was performed at 100°C for 20 minutes, and drying was performed using nitrogen gas. 1 ml of dichloromethane was added to the dried sample, stirred, and 1 ml of distilled water was added. After stirring again, the dichloromethane layer was stored at -20°C and analyzed using GC-MS.

[0079]

[0080] Experimental Example 7: Microbial Identification

[0081] The RosPo-2 strain was identified using 16S rRNA gene sequencing, and phylogenetic analysis was performed according to the method described by Baek et al. (Baek et al., 2015. Psychroserpens jangbogonensis sp. nov., a psychrophilic bacterium isolated from Antarctic marine sediment. International journal of systematic and evolutionary microbiology. 65: 183-188). The 16S rRNA gene sequence of the RosPo-2 strain (1,399 bp, SEQ ID NO: 1) was compared with the sequence available in the EzTaxon database (www.eztaxon.org). The 16S rRNA gene sequence was aligned to the closest phylogenetic lineage using the RDP II online aligner (http: / rdp.cme.msu.edu / index.jsp). The phylogenetic tree was reconstructed using the neighbor-joining method based on the Jukes-Cantor distance, the maximum parsimony method, and the maximum likelihood method with the MEGA 6 program. The robustness of the phylogenies generated by the three tree-building algorithms was confirmed by bootstrap analysis based on 1000 random sequence resamples.

[0082]

[0083] Experimental Example 8: Plackett-Burman Design

[0084] The Plackett-Burman design was used to identify essential components that significantly affect p-CY02 production (Plackett RL, Burman JP. 1946. The design of optimum multifactorial experiments. Biometrika. 33: 305-325). Based on the design, 14 nutrients (C6H5FeO7, NaCl, MgCl26H2O, Na2SO4, CaCl22H2O, KCl, NaHCO3, KBr, SrCl2, H3BO3, Na2SiO3, NaF, NH4NO3, and Na2HPO4) were tested at two concentration levels [low level (-1) and high level (+1)] to identify components with negative effects.

[0085]

[0086]

[0087] To determine the factors that significantly affect EPS production, selected components (C6H5FeO7, NaCl, MgCl2, Na2SO4, CaCl2, KCl, KBr, H3BO3, NH4NO3, Na2HPO4) were re-examined using the Plackett-Burman design method.

[0088]

[0089]

[0090]

[0091]

[0092] Experimental Example 9: Central Composite Design

[0093] A central composite design was applied to optimize the concentrations of previously selected nutrients through experiments using a Plackett-Burman design (Myers RH, Montgomery DC, Anderson-Cook CM. 2016. Response surface methodology: process and product optimization using designed experiments, pp. Ed. John Wiley & Sons.). The variables and experimental conditions for the central composite design are shown in [Table 5] and [Table 6] below.

[0094]

[0095]

[0096] To predict the optimal concentration, a second-order polynomial model was designed to describe the relationship between independent variables such as nutrients and the response: Y = β0+ ∑β i X i + ∑β ij X i X j + ∑β ii X j 2 , where Y is the predicted reaction (p-CY02 production), and β0, β i , β ij and β ii are the constants and regression coefficients of the model, and X i and X j represents an independent nutrient component. Minitab (v. 14.1; Minitab, Inc., USA) statistical software was used for experimental design and regression analysis of the obtained data. A p value less than 0.05 was considered statistically significant.

[0097]

[0098] Example 1: Microbial isolation and screening

[0099] In the same manner as described in Experimental Example 1, 66 strains producing slime substances were isolated on a Marine agar plate using a loop touch test, and EPS-producing strains were screened based on colony viscosity and viscous properties (primary selection). Subsequently, the microorganisms selected in the first round were cultured in MBG medium, and crude EPS was extracted using cooled ethanol. Among the microorganisms selected in the first round, 10 strains were re-selected based on the crude EPS dry weight of 1 g / L or more (secondary selection; Fig. 1a). After purifying the EPS produced from the microorganisms selected in the second round, the viscosity of the purified EPS was measured (Fig. 1b).

[0100] As a result, EPS produced by RosPo-1, -2, -3, -6, -18, and -19 showed a shear rate-dependent increase in shear stress, and in particular, EPS produced by RosPo-1 and RosPo-2 showed a remarkably rapid increase (Fig. 1b).

[0101]

[0102] For final screening, the cryoprotective activity of adherent cells, including HaCaT cells, was measured using EPS produced by RosPo-1 and RosPo-2 strains (Fig. 2a). HaCaT cells were treated with various concentrations [0.1–1.0% (w / v)] of each EPS. Comparison of the viability between the control group [non-freezing, PBS] and the EPS-treated groups revealed that both EPS concentrations increased the viability of HaCaT cells in a concentration-dependent manner. In the case of EPS produced by RosPo-1 strain, the highest viability was observed at 0.8% EPS (55.9 ± 2.0%), whereas a lower viability (51.9 ± 10.6%) was observed at 1.0% EPS compared to 0.8% EPS produced by RosPo-1 strain. And in the case of EPS produced by RosPo-2 strain, the highest survival rate was observed at 0.8% EPS (68.7 ± 0.9%), whereas a lower survival rate (55.1 ± 10.4%) was observed at 1.0% EPS compared to 0.8% EPS produced by RosPo-2 strain (Fig. 2a). As a result of comparative analysis of the cryoprotective activity of EPS produced by RosPo-1 and RosPo-2 strains, it was observed that EPS produced by RosPo-2 strain showed a higher cryoprotective effect on HaCaT cells than EPS produced by RosPo-1 strain. Therefore, RosPo-2 strain was finally selected for further study of cryoprotective properties on HaCaT cells, and EPS produced by RosPo-2 strain was named "p-CY02".

[0103]

[0104] Example 2: Measurement of molecular weight of EPS produced by RosPo-2 strain

[0105]

[0106] As a result, the molecular weight of p-CY02 was 2.04 x 10 6 Da to 1.35 x 10 7 It was calculated as Da (Fig. 3).

[0107]

[0108] Example 3: Analysis of the composition and binding patterns of EPS produced by the RosPo-2 strain.

[0109] The compositional analysis of extracellular polysaccharides was performed using GC / MS. The analysis was performed using a Clarus 500 (Perkin-Elmer, USA) with a mass-selective detector and electron impact ionization mode. As shown in Figure 4A, the major constituent sugars of p-CY02 were glucose and galactose, with glucose being the predominant sugar component, and the molar ratio of glucose:galactose was approximately 7.57:1.

[0110] The binding pattern of each constituent was confirmed by searching the retention time and fragmentation pattern of each PMAA in the Library such as the CCRC Spectral Database for PMAA's through the PMAA GC-MS analysis. The binding pattern of the constituent sugar was characterized by the main components having a repeating structure, including 4-linked glucopyranose and 4-linked N-acetyl glucopyranose as major peaks in the glycosyl linkage analysis of p-CY02 shown in Fig. 4B, and the minor peaks included terminally linked galactopyranose, terminally linked glucopyranose, 4-linked galactopyranose, 4-linked mannopyranose, and 6-linked N-acetyl galactosapyranose.

[0111]

[0112] Example 4: Microbial identification

[0113] Microorganisms were identified using the same method as in Experimental Example 6, and the 16S rRNA gene sequences were compared. As a result, the selected RosPo-2 strain (1399 bp, SEQ ID NO. 1.) was closely related to Pseudoalteromonas tetraodonis IAM 14160T (Accession No.: AF214730; 99.86% 16S rRNA gene sequence similarity), Pseudoalteromonas isachenkonii KMM 3549T (AF316144; 99.85%), Pseudoalteromonas undina NCIMB 2128T (X82140; 99.49%), and Pseudoalteromonas espejiana NCIMB 2127T (X82143; 99.49%), and in all phylogenetic trees generated using the 16S rRNA gene sequences, RosPo-2 and P. isachenkoniiKMM3549T (AF316144) formed a strong clade, suggesting that the RosPo-2 strain was influenced by Pseudoalteromonassp. RosPo-2 (PP396838) (Fig. 5).

[0114]

[0115] Example 5: Antifreeze activity of EPS (p-CY02)

[0116] After setting the cell culture conditions and purifying EPS in the same manner as in Experimental Example 2, the cryoprotective activity was measured in the same manner as in Experimental Example 4. HaCaT cells were treated with 0.8% p-CY02 and various concentrations of DMSO alone or with various concentrations (1.0 to 5.0%, w / v), and the viability was measured (Fig. 2b).

[0117] As a result, the survival rates of the DMSO only treatment group and the p-CY02 and DMSO combination treatment group increased as the DMSO concentration increased, and the survival rate of the p-CY02 and DMSO combination treatment group increased significantly compared to the DMSO only treatment group. Specifically, the survival rate increased 23.6, 1.6, and 1.7 times more in the 1%, 3%, and 5% (v / v) DMSO and p-CY02 combination treatment groups, respectively, than in the DMSO only treatment group.

[0118]

[0119] Example 6: Plackett-Burman design

[0120] As in the Plackett-Burman design and first-order model described in Experimental Example 7, 14 nutrients were prepared at two concentrations (-1, +1) to remove nutrients that negatively affect p-CY02 production. The polynomial model that explains the correlation between the 14 nutrients and the predicted p-CY02 production is as follows:

[0121]

[0122]

[0123] As a result, the p values ​​of NH4NO3, MgCl2, and H3BO3 were > 0.05, suggesting that these components were not significant factors affecting p-CY02 production compared to other factors. The effects of NaF, SrCl2, NaHCO3, and Na2SiO3 on p-CY02 production were negative, and the effects of C6H5FeO7, NaCl, Na2SO4, CaCl2, KCl, KBr, and Na2HPO4 were all positive with p values ​​< 0.05. The seven compounds with positive effects on p-CY02 production and the three compounds with insignificant effects were used in the second Plackett-Burman design.

[0124]

[0125] To identify the 10 nutrients (C6H5FeO7, NaCl, MgCl2, Na2SO4, CaCl2, KCl, KBr, H3BO3, NH4NO3, Na2HPO4) that most significantly influence p-CY02 production, the 10 nutrients selected through a preliminary Plackett-Burman design were retested using the Plackett-Burman design. The 10 selected nutrients were prepared at two concentrations (-1, +1), and the polynomial model describing the correlation between the 10 nutrients and the predicted p-RosPo-2 production was as follows:

[0126]

[0127] As a result, KCl, Na2SO4, and CaCl2 showed positive effects (0.1275, 0.0642, and 0.0517, respectively), whereas Na2HPO4 and C6H5FeO7 showed negative effects (-0.1633 and -0.1908, respectively). Factors significant at the 95% level (p < 0.05) were considered to have a significant effect on p-CY02 production. The remaining compounds (NH4NO3, KBr, NaCl, H3BO3, and MgCl2) showed no significant differences (p > 0.05).

[0128] In conclusion, KCl, Na2SO4, and CaCl2, which showed significant effects at the 95% level compared to other compounds while exhibiting positive effects, were found to significantly affect p-CY02 production. These compounds were ultimately selected for use in the central composite scheme. The concentrations of the remaining compounds were maintained at the concentrations in the MB medium.

[0129]

[0130]

[0131] Example 7: Central composite design

[0132] As described in Experimental Example 8, three nutrients (KCl, Na2SO4, and CaCl2) selected through the Plackett-Burman design were tested at five concentrations (1.68, +1, 0, -1, -1.68) as in [Table 5] of Experimental Example 8, and the central composite design matrix of the nutrients along with the predicted and observed values ​​for p-CY02 production is as in [Table 6] of Experimental Example 8.

[0133] The polynomial model describing the correlation between the three components and the predicted p-CY02 production is as follows:

[0134]

[0135] The statistical significance of the second-order polynomial for the experimental data was assessed by the F value and ANOVA (analysis of variance), and the model was shown to be statistically significant at the 95% confidence level (p < 0.05), as shown in [Table 9] below.

[0136]

[0137]

[0138] As a result of measuring the main effects and interaction effects of the three factors KCl, Na2SO4, and CaCl2 at different concentrations, the optimal concentrations of KCl, Na2SO4, and CaCl2 were determined to be 1.43940, 4.32163, and 3.27066 g / L, respectively, from the three-dimensional response surface diagram (Fig. 6).

[0139] To verify the results of the central composite design, Pseudoalteromonassp. RosPo-2 was cultured in the optimized medium (fructose 15.0 g / L, yeast extract 20.0 g / L, C6H5FeO70.1 g / L, NaCl 19.45 g / L, MgCl2·6H2O 5.9 g / L, KCl 1.43940 g / L, Na2SO4 4.32163 g / L, CaCl2·2H2O 3.27066 g / L, KBr 0.08 g / L, H3BO3 0.022 g / L, NH4NO3 0.0016 g / L, Na2HPO4 0.008 g / L). As a result, the average concentration of the produced p-CY02 was 2.21 ± 0.025 g / L, which was in good agreement with the predicted value (2.24639 g / L), and the yield of p-CY02 was 1.64 times higher than that of the MBG medium (1.35 ± 0.120 g / L) (Fig. 7).

[0140]

[0141] The extracellular polysaccharide according to the present invention and the anti-freezing composition containing the same exhibit excellent cell viability at low temperatures, and are therefore useful for protecting or preserving biological samples in a refrigerated or frozen state.

[0142]

[0143] While specific aspects of the present invention have been described in detail above, it will be apparent to those skilled in the art that these specific descriptions merely represent preferred embodiments and are not intended to limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

[0144]

[0145] [Accession number]

[0146] Name of depositor: Korea Research Institute of Bioscience and Biotechnology, Biological Resource Center (KCTC)

[0147] Accession number: KCTC15865BP

[0148] Date of acceptance: 20240403

[0149]

[0150] Electronic file attached.

[0151]

Claims

1. Pseudoalteromonassp. RosPo-2 strain with accession number KCTC 15865BP.

2. Exopolysaccharide produced by Pseudoalteromonassp. RosPo-2 strain with deposit number KCTC 15865BP.

3. In paragraph 2, 2.04x10 6 Da to 1.35x10 7 An extracellular polysaccharide characterized by having a molecular weight of Da.

4. An extracellular polysaccharide according to claim 2, characterized in that the main constituent sugars are glucose and galactose.

5. An extracellular polysaccharide according to claim 4, characterized in that the molar ratio of glucose and galactose is about 7.57:

1.

6. An extracellular polysaccharide characterized in that it has a structure in which a main chain composed of 4-linked glucopyranose and 4-linked N-acetyl glucosapyranose is repeated in the second paragraph.

7. An anti-freezing composition comprising the extracellular polysaccharide of any one of claims 2 to 6 as an active ingredient.

8. An anti-freezing composition according to claim 7, characterized in that the content of the extracellular polysaccharide is 0.1 to 1.0 (w / v)%.

9. An anti-freezing composition according to claim 7, characterized in that it further comprises DMSO.

10. A method for producing an extracellular polysaccharide produced by Pseudoalteromonassp. RosPo-2 strain, comprising the following steps: a) a step of culturing the Pseudoalteromonassp. RosPo-2 strain of paragraph 1; and b) A step of obtaining an extracellular polysaccharide from a cultured strain.

11. A method according to claim 10, characterized in that the strain is cultured in a medium containing one or more salts selected from the group consisting of KCl, Na2SO4, and CaCl2.

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