Antarctic psychrophilic bacteria extracellular polysaccharide with low temperature resistance and moisturizing function and preparation method and application thereof

Abstract

The present invention relates to an Antarctic psychrophilic bacteria extracellular polysaccharide with anti-low temperature and moisturizing functions, and a preparation method and application thereof. The extracellular polysaccharide EPS0-1 is composed of galactosamine, rhamnose, glucosamine, galactose, glucose, mannose, galactonic acid and gluconic acid in a molar ratio of 1.4:9.4:4.9:32:37:6.5:4.1:4.4; the weight-average molecular weight of the extracellular polysaccharide EPS0-1 is 4350-4360 kDa. The present invention also provides a preparation method of the extracellular polysaccharide EPS0-1. The extracellular polysaccharide EPS0-1 provided by the present invention has the property of anti-low temperature, can significantly reduce the freezing point of the solution, and improve the survival rate of microorganisms in the freeze-thaw cycle, and can be applied to strain cryopreservation and tissue and organ preservation and transportation. The extracellular polysaccharide EPS0‑1 also has hygroscopic and moisturizing properties. At the same time, within the dosage range of 250 to 750 μg / mL, it has no cytotoxicity to normal human liver cells LO2, indicating that it is safe and has no toxic side effects, and can be used to prepare frozen foods, cosmetics, pharmaceutical compositions and health products.

Description

Technical Field

[0001] The invention relates to an Antarctic psychrophilic bacteria extracellular polysaccharide with low temperature resistance and moisture retention functions and a preparation method and application thereof, belonging to the technical field of medicine and health food. Background Art

[0002] The unique geographical location and land-sea distribution make the marine and continental environments of Antarctica rich in diversity. The selection pressure of adverse environments such as low temperature, high salt, strong ultraviolet rays, and drought has made Antarctic microorganisms evolve a variety of strategies to resist these harsh conditions, such as macromolecular extracellular polymers with exopolysaccharides (EPS) as the main component. Psychrophilic EPS has the functions of improving the survival rate of microorganisms, reducing ice crystal damage, buffering temperature, pH or salt concentration changes, and transmitting carbon and energy. It protects and helps the aggregation and adhesion of bacteria, and helps the formation of biofilms and the absorption of nutrients.

[0003] In a narrow sense, psychrophilic bacteria refer to microorganisms that can grow and reproduce normally in the range of 0 to 20°C, and whose optimal growth temperature is less than or equal to 15°C. Due to the low temperature environment in which psychrophilic bacteria live, the large amount of EPS they release not only keeps the cells attached to the surface, but also plays a role in low-temperature protection in the cold environment, avoids physical cell damage, slows down the metabolic process, and creates a good growth environment for psychrophilic bacteria. The above functions of EPS are attributed to its structure and macromolecular association, which prevents the tetrahedral arrangement of water molecules and avoids their recrystallization or ice crystal nucleation. High molecular weight EPS has the ability to retain water, which is more conducive to hydrophobic interactions between non-polar residues and enhances adhesion. The presence of negatively charged residues on the EPS chain, such as sulfate and carboxyl, enables them to form a hydrated viscous three-dimensional network, thereby giving it anti-freezing adhesion and barrier properties. In addition, the EPS secreted by some psychrophilic bacteria can scavenge superoxide anions, that is, it has antioxidant activity and can be used as a cell cryoprotectant.

[0004] The macromolecular polysaccharides produced by psychrophilic bacteria have good low temperature resistance and large expansion performance, and can be widely used in food, cosmetics, medicine and other fields. Therefore, further research on the extracellular polysaccharides of Antarctic psychrophilic bacteria is needed. Summary of the invention

[0005] In view of the deficiencies in the prior art, the object of the present invention is to provide an Antarctic psychrophilic bacteria extracellular polysaccharide with low temperature resistance and moisturizing functions, and a preparation method and application thereof.

[0006] The technical solution of the present invention is as follows:

[0007] The invention discloses an Antarctic psychrophilic bacteria extracellular polysaccharide EPS0-1 with low temperature resistance and moisturizing functions. The extracellular polysaccharide EPS0-1 is an alpha-pyranose polysaccharide composed of galactosamine (GalN), rhamnose (Rha), glucosamine (GlcN), galactose (Gal), glucose (Glc), mannose (Man), galactonic acid (GalA) and gluconic acid (GlcA), and the molar ratios thereof are specifically GalN:Rha:GlcN:Gal, Glc:Man:GalA:GlcA=1.4:9.4:4.9:32:37:6.5:4.1:4.4; and the weight average molecular weight of the extracellular polysaccharide EPS0-1 is 4350-4360 kDa.

[0008] Preferably according to the present invention, the extracellular polysaccharide EPS0-1 contains a triple helical structure, side chains and branches.

[0009] The preparation method of the above-mentioned Antarctic psychrophilic bacteria extracellular polysaccharide EPS0-1 with anti-low temperature and moisturizing functions comprises the following steps:

[0010] (1) Preparation of seed solution: Pseudoalteromonas LP6-12-2 was inoculated into 2216E basal medium at an inoculum amount of 1.0-1.5%, and cultured at 8-15° C. and 150-180 r / min for 30-45 h to obtain seed solution;

[0011] (2) Preparation of fermentation broth: inoculating the seed liquid obtained in step (1) into 2216E improved medium at a volume of 1.0-1.5%, culturing at 8-15° C. and 150-180 r / min for 60-70 h, collecting the fermentation broth, centrifuging at 8000-9000 r / min for 15-20 min, removing the bacteria, and obtaining the fermentation broth supernatant;

[0012] (3) Polysaccharide precipitation: The fermentation supernatant obtained in step (2) is concentrated under reduced pressure at 50-60° C. to 20-25% of the original volume, cooled to room temperature, and 3-4 times the volume of the concentrate is added with 95% ethanol, and the mixture is allowed to stand at 4° C. for more than 12 h, and centrifuged at 7500-8500 r / min for 15-20 min to obtain a precipitate;

[0013] (4) Protein removal: add deionized water to the precipitate obtained in step (3), stir until the precipitate is dissolved, centrifuge at 11500-13000 r / min for 10-15 min, remove the precipitate and collect the supernatant, add Sevag reagent to the supernatant, mix thoroughly and shake vigorously, let stand and stratify, then recover the organic reagent to remove protein, repeat the operation 5-8 times, concentrate under reduced pressure at 55-65°C, remove the organic reagent, and obtain an extracellular polysaccharide solution;

[0014] (5) Dialysis and freeze drying: The extracellular polysaccharide solution obtained in step (4) is placed in a dialysis bag for dialysis, and deionized water is replaced every 8 hours for 48 hours, and then vacuum freeze-dried to obtain crude extracellular polysaccharides;

[0015] (6) crude product separation: the crude extracellular polysaccharide obtained in step (5) was dissolved in Tris-HCl buffer to prepare a polysaccharide solution with a concentration of 8 to 10 mg / mL, and then separated by DEAE-Sepharose Fast Flow anion exchange chromatography, eluted with Tris-HCl buffer at a flow rate of 0.5 mL / min, and collected in one tube every 10 minutes. The sugar-containing eluate was collected and vacuum freeze-dried to obtain the extracellular polysaccharide EPS-0;

[0016] (7) Purification: The extracellular polysaccharide EPS-0 obtained in step (6) is dissolved in deionized water to prepare a polysaccharide solution with a concentration of 4 to 6 mg / ml, and then concentrated and freeze-dried after dialysis to obtain the Antarctic psychrophilic bacteria extracellular polysaccharide EPS0-1 with anti-cold and moisturizing functions.

[0017] Preferably, according to the present invention, in step (1), the Pseudoalteromonas LP6-12-2 was deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on December 15, 2021, at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, with a deposit number of CGMCC NO.24106.

[0018] Preferably according to the present invention, in step (1), the composition of the 2216E basic culture medium is as follows: peptone 0.1-1% (w / v), yeast powder 0.05-0.2% (w / v), artificial sea salt 0.2-4% (w / v).

[0019] Preferably, according to the present invention, in step (2), the composition of the 2216E improved culture medium is as follows: peptone 0.1-1% (w / v), yeast powder 0.05-0.2% (w / v), artificial seawater salt 0.2-4% (w / v), glucose 0.5-2% (w / v), and sucrose 0.5-2% (w / v).

[0020] Preferably, in step (4), the Sevage reagent is a mixed solution of chloroform: n-butanol = 4:1-5:1, and the added amount is 18-22% of the volume of the supernatant.

[0021] The present invention also provides the use of the Antarctic psychrophilic bacteria extracellular polysaccharide EPS0-1 having the functions of anti-low temperature and moisturizing in the preparation of frozen foods, cosmetics, pharmaceutical compositions and health products.

[0022] The present invention also provides the use of the above-mentioned Antarctic psychrophilic bacteria extracellular polysaccharide EPS0-1 with low temperature resistance and moisture retention functions in the freezing preservation of bacterial strains and the preservation and transportation of tissues and organs.

[0023] The beneficial effects of the present invention are:

[0024] 1. The present invention provides an Antarctic psychrophilic bacteria extracellular polysaccharide EPS0-1 with anti-low temperature and moisturizing functions. The extracellular polysaccharide EPS0-1 has anti-low temperature function, can significantly reduce the freezing point of the solution, and improve the survival rate of microorganisms in the freeze-thaw cycle. It can be used in the frozen preservation of strains and the preservation and transportation of tissues and organs. In addition, the extracellular polysaccharide EPS0-1 also has a hygroscopic and moisturizing function. At the same time, within the dosage range of 250 to 750 μg / mL, it has no cytotoxicity to normal human liver cells LO2, indicating that it is safe and has no toxic side effects, and can be used to prepare frozen foods, cosmetics, pharmaceutical compositions and health products.

[0025] 2. The present invention provides a fermentation production method for the exopolysaccharide EPS0-1, identifies the structure of the exopolysaccharide EPS0-1, and provides a basis for laboratory research and industrial production, making up for the lack of research on exopolysaccharides of Antarctic bacteria in China. In addition, the preparation method provided by the present invention is simple in process, easy to operate, and convenient for large-scale production, and the yield can reach 153.9 mg / L. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a graph showing the properties and uniformity test results of the cold-resistant Antarctic psychrophilic bacteria exopolysaccharide EPS0-1;

[0027] In the figure: A is the iodine colorimetric reaction result of EPS-0; B is the UV scanning spectrum of EPS0-1; C is the high performance gel permeation chromatogram of EPS0-1.

[0028] Figure 2 This is the infrared spectrum of the extracellular polysaccharide EPS0-1.

[0029] Figure 3 This is a monosaccharide composition analysis diagram of the extracellular polysaccharide EPS0-1.

[0030] In the figure: A is the ion chromatogram of 16 monosaccharide standards; B is the ion chromatogram of EPS0-1;

[0031] In the figure, peaks 1-16 represent monosaccharides D-Fuc, D-GalN, D-Rha, D-Ara, D-GlcN, D-Gal, D-Glc, D-GlcNAc, D-Xyl, D-Man, D-Fru, D-Rib, D-GalA, L-GulA, D-GlcA and D-ManA, respectively.

[0032] Figure 4This is the EPS0-1 high-level structural analysis diagram.

[0033] In the figure: A is the detection of the triple helix structure of the polysaccharide, and the λmax of the Congo red-EPS0-1 complex changes with the NaOH concentration; B is the detection of the branching of the polysaccharide, and the UV-visible scanning spectrum of the iodine reagent-EPS0-1; C is the Cumulant autocorrelation function fitting curve of EPS0-1; D is the Regularization autocorrelation function fitting graph of EPS0-1.

[0034] Figure 5 The micromorphology and microstructure of EPS0-1.

[0035] In the figure: A is the AFM 2D and 3D image of EPS0-1; B is the SEM image of EPS0-1 (magnification from left to right are 500×, 2000× and 5000× respectively)

[0036] Figure 6 This is a graph showing the low-temperature protective effect of EPS0-1 on E.coli DH5α.

[0037] In the figure: A shows the freezing point lowering effect of extracellular polysaccharide EPS0-1; B shows the low-temperature protective effect of different concentrations of EPS0-1 on Escherichia coli (*p<0.05, **p<0.01, vs control group). DETAILED DESCRIPTION

[0038] The specific implementation process of the present invention is further described in detail below in conjunction with embodiments. The described embodiments are only part of the embodiments of the present invention, not all of the embodiments.

[0039] Unless otherwise specified, the experimental techniques and scientific terms used in the following examples have the same meanings as those generally understood by ordinary technicians. The experimental consumables and reagents involved are available through general commercial channels unless otherwise specified.

[0040] The Pseudoalteromonas sp. LP6-12-2, which efficiently produces extracellular polysaccharides as described in the example, was deposited at the General Microbiology Center of the China Culture Collection Administration on December 15, 2021, with the address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC NO.24106.

[0041] The composition of 2216E basic culture medium is as follows: peptone 0.1-1% (w / v), yeast powder 0.05-0.2% (w / v), artificial sea salt 0.2-4% (w / v).

[0042] The composition of the 2216E improved culture medium is as follows: peptone 0.1-1% (w / v), yeast powder 0.05-0.2% (w / v), artificial sea salt 0.2-4% (w / v), glucose 0.5-2% (w / v), and sucrose 0.5-2% (w / v).

[0043] Example 1. Preparation of low-temperature resistant Antarctic psychrophilic bacteria exopolysaccharide EPS0-1

[0044] A method for preparing Antarctic psychrophilic bacteria extracellular polysaccharide EPS0-1 with low temperature resistance and moisturizing function, comprising the following steps:

[0045] (1) Preparation of seed solution: Take out the frozen Pseudoalteromonas LP6-12-2 from a -80°C ultra-low temperature freezer, inoculate it into 100 mL of 2216E basal medium at a 1% inoculum amount, and culture it at 10°C and 150 r / min for 50 h to obtain a seed solution;

[0046] (2) Preparation of fermentation broth: The seed solution obtained in step (1) was inoculated into 100 mL of 2216E improved medium at a volume ratio of 1%, and cultured at 10° C. and 150 r / min for 60 h. The fermentation broth was collected and centrifuged at 8000 r / min for 15 min to remove the bacteria and obtain the fermentation broth supernatant;

[0047] (3) Polysaccharide precipitation: The fermentation supernatant obtained in step (2) was concentrated under reduced pressure at 60° C. to 25% of the original volume. After cooling, 3-4 times the volume of 95% ethanol was added to the concentrate, and the mixture was incubated at 4° C. overnight. The mixture was then centrifuged at 8000 r / min for 15 min to collect the precipitate.

[0048] (4) Protein removal: add sufficient deionized water to the precipitate obtained in step (3), stir for 2 h until the precipitate is fully dissolved, centrifuge at 12000 r / min for 10 min, remove the precipitate, and collect the supernatant; add 1 / 4 volume of Sevage reagent (chloroform: n-butanol = 5:1, V / V) to the supernatant, shake vigorously, repeat the operation 7 times until no obvious protein appears, and concentrate under reduced pressure at 60°C to remove the organic reagent to obtain an extracellular polysaccharide solution;

[0049] (5) Dialysis and freeze drying: The extracellular polysaccharide solution obtained in step (4) is placed in a 2000Da dialysis bag for dialysis, and deionized water is replaced every 8 hours for 48 hours. The dialyzed polysaccharide solution is placed in a freeze drying dish, frozen at -80°C, and then placed in a vacuum freeze dryer for drying to obtain crude extracellular polysaccharides;

[0050] (6) crude product separation: the crude extracellular polysaccharide obtained in step (5) was dissolved in a loading buffer (0.02 M Tris-HCl buffer, pH 7.8) to prepare a polysaccharide solution with a concentration of 10 mg / mL, and subjected to DEAE-Sepharose Fast Flow anion exchange chromatography, using Tris-HCl buffer for elution at a flow rate of 0.5 mL / min, collecting one tube every 10 min, collecting the sugar-containing eluate, and vacuum freeze-drying to obtain the extracellular polysaccharide EPS-0;

[0051] (7) Purification: The extracellular polysaccharide EPS-0 obtained in step (6) is dissolved in deionized water to prepare a polysaccharide solution with a concentration of 5 mg / ml, and then the extracellular polysaccharide solution is placed in a 300 kDa dialysis bag for sufficient dialysis. The dialyzed polysaccharide solution is placed in a freeze-drying dish, frozen at -80°C, and then placed in a vacuum freeze dryer for drying to obtain the Antarctic psychrophilic bacteria extracellular polysaccharide EPS0-1 with anti-cold and moisturizing functions.

[0052] The yield of the Antarctic psychrophilic bacteria extracellular polysaccharide EPS0-1 with low temperature resistance and moisturizing function prepared in this embodiment can reach 150.0-170.0 mg / L, which is suitable for large-scale industrial production.

[0053] Example 2: Analysis of properties of the low-temperature-resistant Antarctic psychrophilic bacteria exopolysaccharide EPS0-1

[0054] The process of identifying the properties of the Antarctic psychrophilic bacteria extracellular polysaccharide EPS0-1 with low temperature resistance and moisturizing function in the present invention is as follows:

[0055] 1. Iodine colorimetric reaction, BCA method for protein detection, UV spectrum scanning and sugar content analysis

[0056] a. Iodine color reaction: add 1 mL of positive control (2 mg / mL soluble starch solution), EPS0-1 (0.2 mg / mL) and negative control (dH 2 O) and add 5 μL of iodine solution, mix well and observe. Figure 1 As shown in A.

[0057] Depend on Figure 1 As shown in A, the positive control starch solution turned blue, while the extracellular polysaccharide EPS0-1 and the negative control dH 2 O had no obvious color change, only a light yellow color of diluted iodine solution, which indicated that the extracellular polysaccharide EPS0-1 did not contain starch.

[0058] b. Protein detection by BCA method: Using bovine serum albumin as a positive control, the BCA method was used to detect whether the extracellular polysaccharide EPS0-1 (1 mg / mL) contained protein. The results showed that no obvious color reaction was observed in the extracellular polysaccharide EPS0-1 sample. This indicates that the extracellular polysaccharide EPS0-1 does not contain protein.

[0059] Place the cuvette containing 1 mg / mL extracellular polysaccharide EPS0-1 solution in a UV spectrophotometer, set the wavelength range to 200-400 nm, and perform UV scanning. The results are as follows: Figure 1 As shown in B.

[0060] Depend on Figure 1 B shows that there are no obvious absorption peaks at 260nm and 280nm in the scanning spectrum, indicating that the sample does not contain nucleic acids, polypeptides, proteins and other substances. The polysaccharide content was detected by the phenol-sulfuric acid method, and the sugar content of the extracellular polysaccharide EPS0-1 was calculated according to the formula of the glucose standard curve. The results showed that the sugar content of the extracellular polysaccharide EPS0-1 was 91.5% ± 1.78%.

[0061] c. Uniformity test of extracellular polysaccharide EPS0-1: Prepare the extracellular polysaccharide EPS0-1 into a 5 mg / mL solution, centrifuge at 12000rpm for 10 min, take the supernatant, filter with a 0.22μm microporous filter membrane, and then transfer the filtrate to an injection bottle for high-performance gel permeation chromatography. The chromatographic conditions are differential detector; mobile phase is water and 50% methanol solution; RID-20A chromatographic column: SB-804HQ gel column (8×300mm); flow rate is 0.5mL / min, column temperature is 40℃; injection volume is 5μL, the results are as follows Figure 1 As shown in C.

[0062] Depend on Figure 1 C shows that the extracellular polysaccharide EPS0-1 has only one component, that is, a single relatively symmetrical elution peak at 6.73 min, and the extracellular polysaccharide EPS0-1 is tasteless and odorless, easily soluble in water, but the solubility is small (<10 mg / mL), and the viscosity of the saturated sugar solution is high; it is insoluble in organic reagents such as methanol, dimethyl sulfoxide, benzene, ethanol, chloroform, and acetone.

[0063] 2. Infrared spectroscopy analysis of extracellular polysaccharide EPS0-1

[0064] The extracellular polysaccharide EPS0-1 was analyzed by infrared spectroscopy using the ATR method: 50 mg of extracellular polysaccharide EPS0-1 was placed in the sample pool of the Nicolet iS50 Fourier transform infrared spectrometer, and the ATR mode was used at 4000-400 cm -1 The scanning conditions are: scanning times 32, resolution 4, automatic atmospheric background subtraction, and the results are as follows: Figure 2 shown.

[0065] Depend on Figure 2 It can be seen that the extracellular polysaccharide EPS0-1 has characteristic peaks of sugars at 3304 and 2926 cm -1 The bands at 1724 and 1245 cm-1 are the characteristic bands of CH stretching vibration in OH and methylene, respectively. -1 The absorption peaks at 1648, 1544 and 1375 cm -1 There are absorption peaks at 1130, 1037 and 1003 cm -1 The presence of absorption peaks indicates that the extracellular polysaccharide EPS0-1 is composed of pyranoside; the absorption peak at 847cm-1 is caused by the CH scissor vibration of α-pyranose, which is a typical α-configuration absorption peak, that is, the extracellular polysaccharide EPS0-1 is an α-configuration polysaccharide.

[0066] The above results showed that the extracellular polysaccharide EPS0-1 was an α-configuration polysaccharide containing uronic acid, amino and sulfate groups.

[0067] Example 3: Analysis of monosaccharide composition of the Antarctic psychrophilic bacteria exopolysaccharide EPS0-1 with anti-cold and moisturizing functions

[0068] Determination of monosaccharide composition: The monosaccharide composition of the extracellular polysaccharide EPS0-1 was determined by ion chromatography, which was carried out in the following steps:

[0069] (1) Monosaccharide standards and preparation: 16 monosaccharide standards were prepared into 10 mg / mL standard solutions. Each monosaccharide standard solution was further prepared with 5 mg / mL standard as a standard. The mass of different monosaccharides can be determined according to the absolute quantitative method, and then the molar ratio can be calculated according to the molar mass of the monosaccharide; the 16 monosaccharides are D-Fuc, D-GalN, D-Rha, D-Ara, D-GlcN, D-Gal, D-Glc, D-GlcNAc, D-Xyl, D-Man, D-Fru, D-Rib, D-GalA, L-GulA, D-GlcA and D-ManA.

[0070] (2) Sample preparation: The extracellular polysaccharide EPS0-1 and each monosaccharide sample (10 mg) were weighed and placed in an ampoule, and trifluoroacetic acid (3 M, 10 mL) was added and hydrolyzed at 120 °C for 3 h. The acid-hydrolyzed sample solution was transferred to a tube, blown dry (nitrogen blower), and 5 mL of ddH 2 O vortex to mix, dilute 10 times, centrifuge at 12000 rpm / min for 5 min, and take the supernatant for IC analysis.

[0071] (3) The extracellular polysaccharide EPS0-1 and each monosaccharide sample were subjected to ion chromatography analysis. Ion chromatography conditions: electrochemical detector, chromatographic column: Dionex CarbopacTM PA20 (3*150mm); mobile phase A: ddH 2 O, B is 15mM NaOH, C is 15mM NaOH & 100mM NaOAC; 0.3mL / min flow rate; 5μL injection volume; 30℃ column temperature, the results are as follows Figure 3 shown.

[0072] In the ion chromatogram, the retention time of each monosaccharide absorption peak in the extracellular polysaccharide EPS0-1 was compared with the retention time of the 16 standard peaks to determine the monosaccharide composition of the extracellular polysaccharide EPS0-1; based on the molar concentration and peak area of ​​the monosaccharide standard and internal standard, the molar mass of different monosaccharides in the sample was calculated, and then the molar ratio of the monosaccharides was calculated. Figure 3 It can be seen that the monosaccharide peaks 2, 3, 5, 6, 7, 10, 13, and 15 of the extracellular polysaccharide EPS0-1 are respectively galactosamine hydrochloride (GalN), rhamnose (Rha), glucosamine hydrochloride (GlcN), galactose (Gal), glucose (Glc), mannose (Man), galacturonic acid (GalA), and glucuronic acid (GlcA), with a molar ratio of 1.4:9.4:4.9:32:37:6.5:4.1:4.4. This shows that the extracellular polysaccharide EPS0-1 is an acidic heteropolysaccharide composed of eight monosaccharides such as galactose and glucose. In addition, the extracellular polysaccharide EPS0-1 contains a small amount of amino sugars and uronic acid, which is consistent with its infrared spectroscopy analysis results.

[0073] Example 4: Advanced structural analysis of the Antarctic psychrophilic bacteria exopolysaccharide EPS0-1 with anti-cold and moisturizing functions

[0074] 1. The triple helix structure of polysaccharide was determined by Congo red experiment: 2 mg / mL of extracellular polysaccharide EPS0-1 solution and Congo red reagent (100 μmol / L) were mixed in a 1:1 ratio, and then NaOH solution (1 mol / L) was added to maintain the final NaOH concentration of the sample at 0, 0.05, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, 0.50 mol / L. In this process, the maximum absorption wavelength λmax (400-700 nm) at each NaOH concentration was recorded by ultraviolet spectrophotometer, and Congo red reagent without extracellular polysaccharide EPS0-1 was used as a control. When the polysaccharide has a triple helix structure conformation, it can combine with Congo red to form a special complex. The maximum absorption wavelength of the complex is red-shifted compared with Congo red within a certain NaOH concentration range, that is, the maximum absorption wavelength becomes larger. When the concentration of NaOH is greater than a certain concentration, the alkaline conditions destroy the hydrogen bonds between molecules, the maximum absorption wavelength drops sharply, and the triple helix structure of the polysaccharide disintegrates into a single helix and becomes an irregular coil structure. Figure 4 As shown in A.

[0075] Depend on Figure 4 A shows that within the range of 0-0.5M NaOH concentration, the λmax of the Congo red-EPS0-1 complex decreases with the increase of NaOH concentration. Under the initial conditions, Congo red is added to the extracellular polysaccharide EPS0-1 solution to form a Congo red-polysaccharide complex solution. During this process, the color of the solution changes from red to purple, and its λmax is significantly greater than the λmax of Congo red, indicating that a red shift occurs, with a red shift value of 15nm; after adding NaOH, λmax drops sharply, indicating that low concentration NaOH destroys the hydrogen bonds between extracellular polysaccharide EPS0-1 molecules, that is, the polysaccharide triple helix depolymerizes; when the NaOH concentration is greater than 0.1mol / L, λmax tends to be stable, indicating that the triple helix structure has disintegrated, and the disintegrated extracellular polysaccharide EPS0-1 polysaccharide chain can no longer combine with Congo red to form a complex. This shows that there is a triple helix structure in the EPS0-1 polysaccharide chain, and the internal binding of the helix structure is weak, and a low concentration of alkali can destroy the structure.

[0076] 2. The degree of polysaccharide branching was determined using the iodine-potassium iodide test: the polysaccharide solution was mixed with an iodine reagent (0.02% I 2 and 0.2% KI, w / v) and mixed evenly, and the spectrum was scanned in the range of 300-600 nm. If the maximum absorption was at 565 nm, it means that the polysaccharide has fewer branches and / or shorter side chains; otherwise, it means that the polysaccharide contains longer side chains and / or more branches. The results are as follows: Figure 4 As shown in B.

[0077] Depend on Figure 4B shows that the scanning spectrum of iodine reagent-EPS0-1 shows that its maximum absorption peak is not at 565nm, but at 350nm, indicating that the extracellular polysaccharide EPS0-1 has more side chains and / or longer branches.

[0078] 3. Dynamic light scattering DLS analysis of the particle size and molecular uniformity of extracellular polysaccharide EPS0-1: Dynamic light scattering is used to detect the interference enhancement or interference attenuation of scattered light generated by Brownian motion of particles (macroscopically manifested as light fluctuations). The autocorrelation function can describe the principle of light fluctuation characteristics, compare the sample particle size and change trend, and judge the sample uniformity. Add 1 mg / mL of extracellular polysaccharide EPS0-1 solution to a clean colorimetric cup, and use a dynamic light scattering instrument to detect the particle size and sample molecular uniformity of extracellular polysaccharide EPS0-1. Repeat three times for each sample, and scan 10 times for each repeat. The results are as follows: Figure 4 As shown in C.

[0079] Depend on Figure 4 C shows that the Cumulant autocorrelation function fitting curve of the extracellular polysaccharide EPS0-1 is smooth and falls back to the baseline smoothly, indicating that the data is reliable; the curve shows a single decay trend and the decay time is long, indicating that the particle size of the extracellular polysaccharide EPS0-1 component is large, the component is relatively single, and there is almost no influence of other small particle components.

[0080] 4. Calculate the values ​​of different components or sizes of extracellular polysaccharide EPS0-1 by using the Regularization autocorrelation function. Figure 4 As shown in D.

[0081] Depend on Figure 4 D shows that there are two sizes of components in the extracellular polysaccharide EPS0-1 particles. There is a smaller particle component 1 between 1 and 10 nm, and a large particle component 2 between 10 and 1000 nm. However, component 1 and component 2 are of the same size and are bound in a certain form. This indicates that there are different degrees of binding of extracellular polysaccharide EPS0-1 polysaccharides, resulting in larger aggregates in the extracellular polysaccharide EPS0-1 solution.

[0082] 5. Analysis of the ultrastructure of extracellular polysaccharide EPS0-1 using atomic force microscopy (AFM) and scanning electron microscopy

[0083] Use ddH 2O to dissolve the exopolysaccharide to prepare a 1 mg / mL exopolysaccharide EPS0-1 solution, and the solution was continuously diluted to a final concentration of 0.1 mg / mL. After filtering through a 0.22 μm filter, 10 μl of the sample was dropped onto a freshly cut mica slide and dried at room temperature. The AFM image of the exopolysaccharide EPS0-1 was obtained by scanning in tapping mode using a Bruker TESPA-V2 scanning probe microscope equipped with a Z-scanner. The setting parameters were: the spring constant was 42 N / m, the resonance frequency was approximately 320 kHz, and the scanning area was set to 4 μm 2 The resolution is 512 × 512 points. The AFM images of the extracellular polysaccharide EPS0-1 were obtained using JPKSPM Data Processing software (version 6.1.111, Germany). Figure 5 As shown in A.

[0084] The freeze-dried extracellular polysaccharide EPS0-1 sample was fixed on the SEM sample column with double-sided tape and gold was sprayed using a sputtering coater under vacuum. After gold spraying, the sample column was placed in the SEM. Microscopic images of the extracellular polysaccharide were obtained under the conditions of 5kV acceleration voltage and 500×, 2000×, and 5000× magnification. The results are shown in Figure 5 As shown in B.

[0085] Depend on Figure 5 A shows that the extracellular polysaccharide EPS0-1 component does not show obvious chain shape in the solution, but presents larger polysaccharide particles. The height of the extracellular polysaccharide EPS0-1 is 2.0-5.5nm, which is higher than the height of a single polysaccharide chain of 0.1-1.0nm, indicating that they have generated intermolecular and intramolecular aggregation.

[0086] Depend on Figure 5 B shows that the exopolysaccharide EPS0-1 is a sheet-like and rod-like structure (500× and 2000×), and its surface is smooth and dense (5000×), indicating that the exopolysaccharide EPS0-1 has a high degree of cross-linking and a compact structure. The results of dynamic light scattering analysis, atomic force microscopy and scanning electron microscopy all show that the polysaccharide chains of the exopolysaccharide EPS0-1 undergo different degrees of cross-linking in the solution, which supports the results of the Congo red experiment, that is, the exopolysaccharide EPS0-1 has a triple helical conformation.

[0087] Example 5: Anti-cold function of Antarctic psychrophilic bacteria exopolysaccharide EPS0-1 with anti-cold and moisturizing functions

[0088] 1. The lowering of the freezing point of the solution helps to prevent the formation of ice crystals, thereby playing a role in low-temperature protection. Based on this property, the extracellular polysaccharide EPS0-1 was tested for low-temperature resistance. The specific method is: add extracellular polysaccharide EPS0-1 with a final concentration of 10μg / mL, 100μg / mL and 1000μg / mL to 15mmol / L NaCl solution, respectively, and use 15mmol / L NaCl solution as the control group, then place it in an ice salt bath, and measure the freezing point of the solution. The results are as follows: Figure 6 As shown in A.

[0089] Depend on Figure 6 A shows that low concentration of extracellular polysaccharide EPS0-1 (10 μg / mL) has no effect on the freezing point of the solution, and the cooling speed is faster than that of the control group, reaching the freezing point 20s earlier than that of the control group. With the increase of EPS0-1 concentration, the freezing point of the solution is also decreasing. When the concentration of extracellular polysaccharide EPS0-1 reaches 100 μg / mL and 1000 μg / mL, the freezing point decreases by 2°C and 4°C respectively compared with the control group. This shows that extracellular polysaccharide EPS0-1 can play a role in lowering the freezing point of the solution.

[0090] 2. Extracellular polysaccharide EPS0-1 can significantly improve the low temperature resistance of E.coli DH5α.

[0091] Specifically, when 0.01% (w / v) extracellular polysaccharide EPS0-1 was added to E. coli DH5α, the survival rate of E. coli DH5α was 73.0% after one freeze-thaw cycle; after four freeze-thaw cycles, its survival rate was still 43.0%; when 0.5% (w / v) extracellular polysaccharide EPS0-1 was added to E. coli DH5α, the survival rate of E. coli DH5α after four freeze-thaw cycles reached 88.4%, and its freeze-thaw protection effect was equivalent to that of EPS and 30% glycerol at the same concentration; when 1% (w / v) extracellular polysaccharide EPS0-1 was added to E. coli DH5α, the survival rate after four freeze-thaw cycles reached 89.5%, which was slightly higher than 0.5% (w / v), and was equivalent to the anti-low-temperature effect of 30% glycerol. Taking E. coli DH5α as a control, the results are as follows: Figure 6 As shown in B.

[0092] Depend on Figure 6 B shows that the extracellular polysaccharide EPS0-1 plays a major role in low-temperature protection; when the concentration of the extracellular polysaccharide EPS0-1 reaches 0.5% (w / v), it has a very high protection efficiency.

[0093] Example 6: Moisturizing function of Antarctic psychrophilic bacteria exopolysaccharide EPS0-1 with anti-low temperature and moisturizing functions

[0094] The hygroscopicity and moisture retention of the extracellular polysaccharide EPS0-1 were determined as follows:

[0095] The prepared saturated sodium carbonate solution and saturated ammonium sulfate solution were placed in different desiccators, and the relative humidity in the equilibrium desiccators was 43% and 81% respectively. Then the hygroscopicity of the extracellular polysaccharide EPS0-1, sodium alginate and hyaluronic acid was analyzed by weight method. Then, the dry environment was formed by silica gel, and the moisture retention of the extracellular polysaccharide EPS0-1, sodium alginate and hyaluronic acid was determined.

[0096] The results showed that in an environment with a humidity of 43%, the moisture absorption rate of the extracellular polysaccharide EPS0-1 reached 11.4% within 2 hours, and then gradually increased, reaching a maximum of 20.5% at 24 hours; although it was slightly lower than the hyaluronic acid's 25.7% moisture absorption rate during the same period, it was significantly higher than the 8.70% moisture absorption rate of sodium alginate. In an environment with a humidity of 81%, the moisture absorption effect of the extracellular polysaccharide EPS0-1 was equivalent to that of hyaluronic acid within 2 hours, with a moisture absorption rate of 16.5%; at 36 hours, the moisture absorption rate of the extracellular polysaccharide EPS0-1 reached a maximum of 33.1%. In a dry environment, the moisturizing rates of the extracellular polysaccharide EPS0-1, sodium alginate and hyaluronic acid gradually decreased, and the moisturizing capacity of the extracellular polysaccharide EPS0-1 was between hyaluronic acid and sodium alginate.

[0097] Example 7: Cytotoxicity detection of the Antarctic psychrophilic bacteria exopolysaccharide EPS0-1 with anti-cold and moisturizing functions

[0098] The cytotoxicity of extracellular polysaccharide EPS0-1 was studied using the human normal liver cell line LO2. The specific method is as follows:

[0099] Take 2mg / mL extracellular polysaccharide EPS0-1 mother solution and prepare it into 250, 500, 750 and 1000μg / mL extracellular polysaccharide EPS0-1 solution. Add 200μL of extracellular polysaccharide EPS0-1 solution to each well of the 96-well plate with LO2 cells. Set 6 replicates for each concentration and culture for 24h. Remove the culture medium, add culture medium at 100μL / well, add CCK-8 enhanced solution at 10μL / well, mix well, incubate (37℃, 4h), and detect absorbance at 450nm. Cell viability (%) = OD 样品-空白 / OD 阴性对照-空白 × 100%, LO2 cells were used as control.

[0100] The results showed that after LO2 cells were treated with 250, 500, and 750 μg / mL extracellular polysaccharide EPS0-1 solution for 24 hours, the survival rate of LO2 cells was slightly improved compared with the control, but there was no significant difference. After LO2 cells were treated with 250, 500, and 750 μg / mL extracellular polysaccharide EPS0-1 solution for 24 hours, 1000 μg / mL extracellular polysaccharide EPS0-1 solution had a certain effect on promoting the proliferation of LO2 cells compared with the control, increasing by 21.0%. This shows that the extracellular polysaccharide EPS0-1 is not toxic to normal hepatocytes.

[0101] In summary, the extracellular polysaccharide EPS0-1 prepared by the present invention is a large molecular weight heteropolysaccharide with amino and uronic acid, has good low temperature resistance, moisture absorption and moisturizing functions, and is non-toxic to normal hepatocytes cultured in vitro, so it can be developed and applied as an antifreeze agent, cosmetic and pharmaceutical additive.

[0102] The above description is only an embodiment of the preferred scheme used in the present invention, but the implementation mode of the present invention is not limited by the above embodiment. Any modifications, equivalent substitutions and improvements made within the principle and spirit of the present invention are within the protection scope of the present invention.

Claims

1. An Antarctic psychrophilic bacteria extracellular polysaccharide EPS0-1 with low temperature resistance and moisturizing function, characterized in that: The extracellular polysaccharide EPS0-1 is an α-pyranose polysaccharide composed of galactosamine, rhamnose, glucosamine, galactose, glucose, mannose, galactosonic acid and gluconic acid, and the molar ratio thereof is specifically galactosamine: rhamnose: glucosamine: galactose: glucose: mannose: galactosonic acid: gluconic acid = 1.4: 9.4: 4.9: 32: 37: 6.5: 4.1: 4.4; the weight average molecular weight of the extracellular polysaccharide EPS0-1 is 4350-4360 kDa.

2. The Antarctic psychrophilic bacteria exopolysaccharide EPS0-1 with low temperature resistance and moisture retention function as claimed in claim 1, characterized in that: The extracellular polysaccharide EPS0-1 contains a triple helical structure, side chains and branches.

3. The method for preparing the Antarctic psychrophilic bacteria extracellular polysaccharide EPS0-1 with low temperature resistance and moisture retention function according to claim 1, characterized in that: The steps include: (1) Preparation of seed solution: Inoculate Pseudoalteromonas LP6-12-2 into 2216E basal medium at a rate of 1.0-1.5%, and culture at 8-15°C and 150-180 r / min for 30-45 h to obtain seed solution; (2) Preparation of fermentation broth: inoculate 1.0-1.5% of the volume of the seed liquid obtained in step (1) into 2216E improved medium, culture at 8-15°C and 150-180 r / min for 60-70 h, collect the fermentation broth, centrifuge at 8000-9000 r / min for 15-20 min, remove the bacteria, and obtain the fermentation broth supernatant; (3) Polysaccharide precipitation: The fermentation supernatant obtained in step (2) is concentrated under reduced pressure at 50-60°C to 20-25% of the original volume, cooled to room temperature, and 3-4 times the volume of the concentrate is added with 95% ethanol. The mixture is allowed to stand at 4°C for more than 12 h, and centrifuged at 7500-8500 r / min for 15-20 min to obtain a precipitate. (4) Protein removal: add deionized water to the precipitate obtained in step (3), stir until the precipitate is dissolved, centrifuge at 11500-13000 r / min for 10-15 min, remove the precipitate and collect the supernatant, add Sevag reagent to the supernatant, mix thoroughly and shake vigorously, let stand and stratify, then recover the organic reagent to remove the protein, repeat the operation 5-8 times, concentrate under reduced pressure at 55-65°C, remove the organic reagent, and obtain an extracellular polysaccharide solution; (5) Dialysis and freeze drying: The extracellular polysaccharide solution obtained in step (4) is placed in a dialysis bag for dialysis, and deionized water is replaced every 8 hours for 48 hours. The solution is then vacuum freeze-dried to obtain crude extracellular polysaccharides. (6) Crude product separation: The crude extracellular polysaccharide obtained in step (5) was dissolved in Tris-HCl buffer to prepare a polysaccharide solution with a concentration of 8-10 mg / mL, and then separated by DEAE-Sepharose Fast Flow anion exchange chromatography, eluted with Tris-HCl buffer at a flow rate of 0.5 mL / min, and collected in one tube every 10 minutes. The sugar-containing eluate was collected and vacuum freeze-dried to obtain the extracellular polysaccharide EPS-0; (7) Purification: The extracellular polysaccharide EPS-0 obtained in step (6) is dissolved in deionized water to prepare a polysaccharide solution with a concentration of 4-6 mg / ml. After dialysis, the solution is concentrated and freeze-dried to obtain the Antarctic psychrophilic bacteria extracellular polysaccharide EPS0-1 with anti-cold and moisturizing functions.

4. The preparation method according to claim 3, characterized in that: In step (1), the Pseudoalteromonas LP6-12-2 was deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on December 15, 2021, at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC NO.24106.

5. The preparation method according to claim 3, characterized in that: In step (1), the composition of the 2216E basal medium is as follows: 0.1-1% w / v peptone, 0.05-0.2% w / v yeast powder, and 0.2-4% w / v artificial seawater.

6. The preparation method according to claim 3, characterized in that: In step (2), the composition of the 2216E improved culture medium is as follows: 0.1-1% w / v peptone, 0.05-0.2% w / v yeast powder, 0.2-4% w / v artificial seawater, 0.5-2% w / v glucose, and 0.5-2% w / v sucrose.

7. The preparation method according to claim 3, characterized in that: In step (4), the Sevage reagent is a mixed solution of chloroform: n-butanol = 4:1~5:1, and the amount added is 18~22% of the volume of the supernatant.

8. Application of the Antarctic psychrophilic bacteria extracellular polysaccharide EPS0-1 with low temperature resistance and moisture retention function described in claim 1 in the preparation of frozen foods, cosmetics, pharmaceutical compositions and health products.

9. Application of the Antarctic psychrophilic bacteria extracellular polysaccharide EPS0-1 with low temperature resistance and moisture retention function described in claim 1 in strain freezing preservation and tissue and organ preservation and transportation.

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