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    • 专利摘要:
    Pseudomonas sp. CECT8437 is a cold-adapted bacterium isolated from a marine sediment sample collected from Deception Island (South Shetland Islands, Antarctica) that is noted for the highly mucous appearance of its colonies. An exopolysaccharide (EPS) is produced by this strain, which comprises glucose, galactose, fucose and uronic acid in a molar ratio of 2:1:1:0.3 approximately. The EPS shows the following weight percentages, approximately: 37.29% C, 6.17% H, 2.25% N, and 0.41 % S. Its molecular weight (MW) is higher than 2 x 106 Da. The EPS is useful for the following purposes: (i) cryoprotectant agent; (ii) emulsifier agent; (iii) thickening, stabilizing or structural agent; (iv) dermoprotective agent; and (v) agent for increasing skin elasticity. The EPS can be used in cosmetic compositions.
    公开号:ES2585398A2
    申请号:ES201690034
    申请日:2015-02-04
    公开日:2016-10-05
    发明作者:Mª Elena MERCADÉ GIL；Ornella CARRIÓN FONSECA；Mª Jesús MONTES LÓPEZ
    申请人:Universitat Autonoma de Barcelona UAB；Universitat de Barcelona UB；
    IPC主号:
    专利说明:

    DESCRIPTIONBacterial exopolysaccharide

    The present invention relates to the field of bacterial exopolymers, particularly exopolysaccharides (EPSs), which can be used as a source of oligosaccharides and sugar monomers.
    STATE OF THE TECHNIQUE
     10
    In nature, bacterial exopolymers have several biological functions, for example, as a reserve material for biodegradable compounds and trace metal nutrients, or as part of a protective structure against predators, desiccation, salinity, cytotoxic compounds and high or low temperatures.
     fifteen
    Bacterial exopolymers are polymers of high molecular weight (molecular weight, MW) that constitute up to 40-95% of the extracellular material (ME) that surrounds most microbial cells in marine environments, ME meaning all the components that bacteria they secrete to the extracellular environment. The majority of exopolymers produced by marine bacteria are EPSs, specifically 20 heteropolysaccharides that contain three or four different monosaccharides that can be pentoses, hexoses, amino sugars or uronic acids, arranged in groups of ten or less to form repetitive units. Organic or inorganic substituents may also be present, namely sulfate, phosphate, acetic acid, succinic acid and pyruvic acid. 25

    The presence of glucose, galactose and fucose residues in microbial polysaccharides is quite common, although their amounts vary. EPSs containing fucose are considered as interesting products for the pharmaceutical and cosmetic industry. The biological properties of this rare sugar enhances its use in drugs, eg as an anticarcinogenic or anti-inflammatory agent, and in cosmetic products as an anti-aging agent.

    One of the first EPS described with the sugars mentioned was collanic acid. Collanic acid is produced in small quantities by many 35 pathogenic bacteria of the Enterobacteriaceae family, eg Escherichia coli,
    Salmonella typhimurium and Enterobacter sp. o Serratia sp. The analysis of the EPS they produce shows a composition of glucose, galactose, fucose and glucuronic acid with a constant molar ratio of 1: 2: 2: 1.

    In recent years, the analysis of bacterial EPSs from polar environments has become an active field of research, but few of psychrophilic or psychrotolerant marine polar bacteria have been studied. EPSs have been found in Antarctic marine bacteria and winter Arctic ice, which modify the physical-chemical environment of bacterial cells, participate in cell adhesion to surfaces and favor water retention, nutrient concentration, retain and protect the Extracellular enzymes against cold denaturation and act as cryoprotectants.

    Pseudoalteromonas sp. SM20310, isolated from winter Arctic ice, secretes an EPS composed mainly of mannose, and traces of glucose, galactose, rhamnose, N-15 acetylglucosamine, N-acetylgalactosamine and xylose (Sheng-Bo et al., “Structure and ecological roles of a novel exopolysaccharide from the Arctic sea ice bacterium Pseudoalteromonas sp. SM20310 ”, 2012, Applied and Environmental Microbiology doi: 10.1128 / AEM.01801-12). Other cold-adapted bacteria isolated from Antarctica are Pseudoalteromonas haloplanktis TAC125, which synthesizes an EPS consisting of 20 mannose and glucose traces (Corsaro et al., “Influence of growth temperature on lipid and phosphate contents of surface polysaccharides from the Antarctic bacterium Pseudoalteromonas haloplanktis TAC 125 ”, Journal of Bacteriology, 2004, vol. 186. pp. 29-34), and Pseudoalteromonas arctica KOPRI 21653, with an EPS composed of galactose and glucose (Sung, JK et al.,“ Cryoprotective properties of exopolysaccharide 25 (P-21653) produced by the Antarctic bacterium, Pseudoalteromonas arctica KOPRI 21653 ”, Journal of Microbiology, 2007, vol. 45, pp. 510-514).

    More complex EPSs from Antarctic environments have also been described, including Pseudoalteromonas sp. CAM025, Pseudoalteromonas sp. CAM036, and other strains of the same genus. The EPS produced by Pseudoalteromonas sp. CAM025 is a sulfated heteropolysaccharide with high levels of uronic acids with acetyl groups, and its monosaccharide composition is estimated to be glucose, galactose, rhamnose and galacturonic acid. The strain Pseudoalteromonas sp. CAM036 also synthesizes a sulfated heteropolysaccharide with high levels of uronic acids with 35 acetyl groups, mainly composed of glucose, mannose, arabinose, acid
    galacturonic and N-acetylgalactosamine (Mancuso et al. "Production of exopolysaccharides by Antarctic marine bacterial isolates", Journal of Applied Microbiology, 2004, vol. 96, pp. 1057-1066). Other studies have confirmed that the chemical composition and molecular weight data can be very diverse among EPSs produced by Antarctic isolates (Mancuso Nichols et al. "Chemical 5 characterization of exopolysaccharides from Antarctic marine bacteria", Microbial Ecology, 2005, vol. 49 , 578-589), and it is remarkable that many of them have a high protein content.

    A common fact when analyzing many ME secreted by bacteria adapted to cold, is its complexity. Although bacterial ME characterization studies clearly demonstrate that EPSs are the largest chemical constituent, it is now clear that other molecules, specifically proteins, lipids, and nucleic acids are also present in the ME. An important finding is the presence of particulate structures called External Membrane Vesicles (VMEs) in ME 15 that increase the protein content of EPSs. VMEs occur during the course of the metabolism and normal cell growth of most Gram-negative bacteria. These are spherical vesicles of extruded lipid bilayer from regions of the bacterial outer membrane and contain lipopolysaccharide, periplasmic proteins, outer membrane proteins and phospholipids (Kulp A. et al., "Biological 20 functions and biogenesis of secreted bacterial outer membrane vesicles", Annual Review of Microbiology, 2010, vol. 64, pp. 163-184).

    The complexity of ME is clearly demonstrated in Antarctic bacteria of different genera, eg Pseudoalteromonas, Shewanella, Psychrobacter, Marinobacter and others. Most of the analyzed ME was composed of capsular heteropolysaccharides and large amounts of VMEs (Frias, A. et al. "Membrane vesicles: a common feature in the extracellular matter of cold-adapted Antarctic bacteria", Microbial Ecology, 2010, vol. 59 , pp. 476-486 (Errata in Microbial Ecology, 2010, vol. 60, p. 476). Many bacteria of the genus Pseudomonas and another 30 pathogenic bacteria produce large amounts of VMEs involved in the release of toxins from eukaryotic cells (Tashiro Y. et al, "Multifunctional membrane vesicles in Pseudomonas aeruginosa", Environmental Microbiology, 2011, doi: 10.1111 / j.1462-2920.2011.02632x) The presence of VMEs secreted by bacteria, as in most Pseudoalteromonas and other strains Gram-35 negatives, it is an inconvenience since they can hinder the extraction and purification of
    EPSs and at the same time increase their toxicity and / or antigenicity. The analysis of the components of the VMEs has shown that the vesicles contain a wide variety of virulence factors. These virulence factors include proteins, specifically adhesins, toxins, and enzymes as well as non-protein antigens such as lipopolysaccharides (Ellis TN et al., "Virulence and immunomodulatory roles of bacterial 5 outer membrane vesicles", Microbiology and Molecular Biology Reviews, 2010, vol .74, pp. 81-94). In addition, the small size of the VMEs, between 20-200 nm, complicates their separation from the EPSs that are simultaneously secreted by the bacteria.

    The genus Pseudomonas is one of the most studied sources of EPSs. Several 10 species of the genus Pseudomonas are capable of synthesizing a wide variety of exopolysaccharides. Many of them are alginate-type polymers, the main components being mannuronic acid and glucuronic acid. The gelan exopolysaccharide, composed mainly of glucose, rhamnose and glucuronate, is also synthesized by species of the genus Pseudomonas. When glucose 15 is used as a carbon source, Pseudomonas putida and Pseudomonas fluorescens synthesize an EPS composed of glucose, galactose and pyruvate. It has been described that the EPS produced by P. fluorescens Biovar II is composed of galactose, mannose, rhamnose, glucose, fucose, ribose, arabinose and xylose. The purified EPS of P. putida G7 contains the monosaccharides glucose, rhamnose, ribose, N-acetylgalactosamine and glucuronic acid. Similarly, some researchers have shown that the isolated EPS of Pseudomonas caryophylli CFR 1705 grown in a medium with lactose is composed of rhamnose, mannose and glucose. More recently, a heteropolysaccharide, composed of neutral galactose, glucose, mannose and rhamnose sugars and containing acetyl groups has been identified in a strain of 25 Pseudomonas oleovorans grown in pure glycerol or products rich in glycerol.

    Although molecular studies have shown that the presence of bacteria of the genus Pseudomonas is quite common in polar marine environments, up to the knowledge of the inventors, few of them from polar regions have been classified at the species level and EPSs have not been analyzed. of these strains (Reddy et al. "Psychrophilic pseudomonads from Antarctica: Pseudomonas antarctica sp. nov., Pseudomonas meridiana sp. nov. and Pseudomonas proteolytica sp.", Int. J. Syst. Evol. Microbiol., 2004, vol. 54 , pp. 713-719).
     35
    So it is interesting to find alternatives to bacterial EPSs, which are free of proteins, for proper and efficient use in industry.

    EXPLANATION OF THE INVENTION
     5
    One aspect of the present invention relates to a strain of Pseudomonas sp. deposited on 1.10.2013 with the number CECT8437 in the "Spanish Type Crops Collection", with address at: "Building 2 CUE. Science Park. University of Valencia. Calle Agtedín Escardino Professor, 9. 46980 Paterna (Valencia), Spain" . The extracellular matter of this strain is practically free of VME compared to 10 other Antarctic bacteria.

    Another aspect of the invention relates to a bacterial EPS isolated from a culture of Pseudomonas sp. CECT8437. In a particular embodiment, the EPS comprises glucose, galactose, fucose and uronic acid in a molar ratio of about 2: 1: 1: 0.3. In another particular embodiment, the EPS has an elementary composition, in weight percentages, of 37.29% C, 6.17% H, 2.25% N and 0.41% S. In another particular embodiment, the EPS has a Fourier transform infrared spectrum with (in cm-1): a band at 3400, a peak at 2930, a peak at 2985, a band between 1200 and 900, a band at 1720, a peak at 1640 and a peak at 1540. Cosmetic compositions comprising effective amounts of EPS and cosmetically acceptable ingredients or carriers are also parts of the present invention.

    Another aspect of the present invention relates to the use of the aforementioned EPSs for the following purposes: (i) cryoprotective agent; (ii) emulsifying agent; (iii) thickening, stabilizing or texturing agent; (iv) dermoprotective agent; and (v) agent to increase skin elasticity.

    The EPSs of the present invention can be hydrolyzed, partially or completely, chemically, mechanically (eg by sonication) or enzymatically, under conditions analogous to those known in the art, to produce oligosaccharides of different lengths, with different characteristics of those of the natives. The respective hydrolyzed products obtained are also part of the present invention; they can be used as a source of oligosaccharides and sugar monomers such as L-fucose, useful in cosmetic products, pharmaceuticals and
    Food supplements. In a particular embodiment the hydrolyzing is chemical; preferably by treatment with an acid; and more preferably by treatment with sulfuric acid, hydrochloric acid or trifluoroacetic acid. For example, hydrolyzing with sulfuric acid can be done by treatment at 100-120 ° C for 0.5 to 8 hours, preferably at 100-120 ° C for 0.5 to 2 hours. These 5 hydrolyzing treatments produce mono- and / or oligosaccharides that are frequently derivatized into alditol acetates, trimethyl silylated methyl glycosides, and silylated (-2) -butyl trimethyl glycosides.

    The EPS of the invention is new with respect to other EPSs produced by other cold-adapted bacteria 10, Pseudomonas sp. or other bacteria reported to date.

    The EPS of the invention has an emulsifying activity against different food and cosmetic oils that is far superior to that of commercial emulsifiers, specifically xanthan gum, gum arabic or Span 20. It forms highly stable emulsions against cetiol V cosmetic oil, exhibiting a behavior of pseudoplastic flow, low thixotropy and pour point, and confers significant cryoprotection for the strain itself as well as for other bacteria, including E. coli, indicating a universal cryoprotective role. The cryoprotective activity of EPS showed a clear dose-response relationship at -20 ° C and -80 ° C, unlike what was observed when the Fetal Bovine Serum (SBF) membrane stabilizer was added to the cells.

    Due to the wide diversity of composition and different physicochemical properties, EPSs have emerged as important new industrially polymeric materials, which are gradually becoming economically competitive with natural gums produced by seaweed and plants. In addition, EPSs derived from natural resources have a competitive advantage, due to their biodegradability and often biocompatibility.
     30
    The EPS of the invention does not contain detectable amounts of VMEs, thus excluding potentially toxic proteins or antigenic compounds, specifically lipopolysaccharides. In addition, the absence of VMEs favors its procurement and purification processes, all of which make it desirable for particular uses. Thus, the EPS CECT8437 is an attractive material for medical applications such as thickening, stabilizing, bonding or texturing agent.

    The EPS of Pseudomonas sp. CECT8437 is a silt-type polysaccharide that is weakly bound to the cell surface and, unlike capsular polysaccharides, is primarily excreted into the extracellular medium. It should be noted that the ME of this strain does not contain significant amounts of VMEs, contrary to what is observed in the extracellular material of several cold-adapted Antarctic bacteria. The presence of proteins and other compounds derived mainly from VMEs in EPSs can be antigenic or toxic. The fact that the strain of the invention is free of VMEs means an obvious technological advantage over other EPSs extracted from Pseudomonas or other Gram-negative bacteria. The commercial value of the EPS of the invention is based primarily on this property.

    It has been found that the EPS of the present invention is a more efficient emulsifier against various food oils and n-hexadecane hydrocarbon than other bacterial EPSs or plant gums described in the prior art, mainly xanthan gum and gum arabic. Similarly, the EPS demonstrates an emulsifying activity against the cosmetic oil cetiol V superior to the commercial emulsifier used as a positive control, Span 20. In addition, it was observed that the EPS of Pseudomonas sp. CECT8437 forms long-term stable emulsions with pseudoplastic behavior. These results, together with their stability, highlight the potential applications of the EPS of Pseudomonas sp. CECT8437 as an emulsifying agent, with the additional attractions of sustainable production, non-toxicity and biodegradability.

    The main properties of EPS, specifically its ability to form 25 long-term stable emulsions against different food and cosmetic oils and its universal cryoprotective activity, make it a promising alternative to the commercial polysaccharides described in the prior art.

    Throughout the description and the claims the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. In addition, the word "comprises" covers the case "consists of". For those skilled in the art, other objects, advantages and features of the invention will be derived partly from the description and partly from the practice of the invention. The following examples and drawings are provided by way of illustration, and are not intended to be limiting of the present invention. In addition, the present invention covers
    all possible combinations of particular and preferred embodiments indicated herein.
    BRIEF DESCRIPTION OF THE DRAWINGS
     5
    FIG 1: Infrared spectrum of the EPS produced by Pseudomonas sp. CECT8437 (cf. Example 6).

    FIG 2: 1 H-NMR spectrum of EPS produced by Pseudomonas sp. CECT8437 (cf. Example 6). 10

    FIG 3: Emulsifying activities (A540) of EPS, xanthan gum and gum arabic against olive, sunflower and corn oils, and n-hexadecane after 24 hours at room temperature. A: Olive oil B: Sunflower oil. C: Corn oil. D: n-Hexadecane. Dotted: EPS. Diagonal: Xanthan gum. Flat lines: Gum arabic. (cf. Example 15 7).

    FIG 4: Rheogram of emulsions with 2% EPS of Pseudomonas sp. CECT8437 and a mixture of water and cetiol V (1: 2; v / v). The shear stress (SS, shear stress) is represented by a black line. The viscosity (V) is represented by a gray line. SR = shear rate (cf. Example 9).

    FIG 5: Backscattering profiles (BS) of the emulsion with 2% EPS and a mixture of water and cetiol V (1: 2; v / v) depending on the sample height (SH, sample height), analyzed at days 0 and 30 of storage at room temperature. Thick line: 25 day 0. Gray line: day 30 (cf. Example 11).

    FIG 6: Wild-type transmission electron microscopy images of Pseudomonas sp. CECT8437 (A), and non-EPS (B) producing mutant cells (cf. Example 12). 30

    FIG 7: Survival rates (SUR) of E.coli ATCC 10536 cultures frozen at -20 ° C with EPS or SBF. Survival rates are expressed as a percentage of viable cells with respect to non-frozen cells (n = 3). Baseline brand: SBF. Diagonal framework: EPS (cf. Example 12). 35

    FIG 8: Survival rates (SUR) of E.coli ATCC 10536 cultures frozen at -80 ° C with EPS or SBF. Survival rates are expressed as a percentage of viable cells compared to non-frozen cells (n = 3). Baseline brand: SBF. Frame in frames: EPS (cf. Example 12).
     5
    FIG 9: Transmission electron microscopy micrographs of ultrafine sections of cold-adapted Antarctic bacteria after high pressure fixation and cryosubstitution (High pressure-freezing and freeze-substitution, HPF-FS). The bars are 200 nm. A: Pseudomonas sp. CECT8437. B: Pseudoalteromonas sp. C: Shewanella sp. NF22. D: Marinobacter sp. E: Psychrobacter sp. NF23. F: Shewanella sp. M7 (cf. Example 13). 10

    FIG 10: Elasticity (EL) of the skin treated with placebo or 0.2% EPS. Skin elasticity is expressed as% of net elasticity on day 9 with respect to day 0 and control. White column: placebo. Black column: 0.2% EPS.
     fifteen
    FIG 11: Transepidermal water loss (TEWL) of the skin treated with placebo or with 1% EPS after exposure to SLS. TEWL is expressed as a percentage of TWEL after won SLS treatment with respect to the initial TEWL. White column: placebo. Black column: 1% EPS.
     twenty
    DETAILED DESCRIPTION OF PARTICULAR EMBODIMENTS

    Pseudomonas sp. CECT8437 is a cold-adapted bacterium that was isolated from a sample of marine sediment collected on Deception Island (South Shetland Islands, Antarctica); The highly mucous appearance of its colonies is remarkable. The molecular weight of the EPS of the invention was evaluated by size exclusion chromatography (SEC) using conventional dextran standards of molecular weight between 8.8x103 to 2x106, the molecular weight of the EPS of the invention being greater than 2 x 106 Da (cf. Example 5). The analysis of monosaccharides and uronic acid of the EPS of the invention reveals that it comprises glucose, galactose, fucose and uronic acid in a molar ratio of 2: 1: 1: 0.3 (cf. Examples 2, 3).

    The emulsifying capacity of the EPS of Pseudomonas sp. CECT8437 was tested against several hydrophobic compounds, specifically food oils, cosmetics and hydrocarbons. FIG 3 shows the emulsifying activities of the low EPS
    Neutral conditions against three different food oils and n-hexadecane. The EPS exhibits a high emulsifying capacity for olive, sunflower and corn oils, with an absorbance at a wavelength of 540 nm (A540) of 1.28, 1.15 and 0.66, respectively. In all cases, EPS showed a higher emulsifying capacity than commercial xanthan gum and gum arabic emulsifiers, with the exception of sunflower oil, with which EPS showed the same emulsifying activity as xanthan gum. The EPS also demonstrated a superior emulsifying capacity against n-hexadecane at positive controls. The emulsifying activity of EPS was also tested against cetiol V cosmetic oil using another emulsification protocol, and compared with the commercial cosmetic emulsifier 10 Span 20. Stable emulsions were formed with a creamy consistency with 2% EPS, while it required 6% of Span 20 to achieve the same degree of emulsification (cf. Example 7).

    The pseudoplastic behavior of the EPS of Pseudomonas sp. CECT8437 (cf. 15 Example 9) makes it an attractive material for industrial and medical applications as a thickening, stabilizing, binding or texturing agent, similar to other bacterial EPS currently marketed.

    The zeta potential (PZ) represents the electrical charge of the emulsion particle surface, representing an important parameter that allows the prediction of the emulsion's physical stability. If the value of the PZ is> | 25 |, the particle suspensions will tend to stabilize since the repulsion forces will exceed those of attraction. On the other hand, if the PZ is <| 25 |, the particles will join until flocculation since the attractive forces will be stronger. The PZ value of the emulsions with 25% EPS and a mixture of water and cetiol V (1: 2; v / v) was 0.1 ± 0.4 mV, which indicated that the emulsion particles had a weak surface charge (cf. Example 10).

    The results of Turbiscan demonstrate that the emulsion of the EPS of the invention with 30 cetiol V was highly stable, and no variation of backscattering was detected that indicated destabilization phenomena such as sedimentation, cremation, flocculation or coalescence (cf. Example 11). This stability is not due to the electrostatic charge of the particles, represented by the low PZ value shown by the EPS. It is likely that the stability of the EPS emulsions of Pseudomonas sp. CECT8437 is due to steric stabilization, a common process among exopolysaccharides of
    high molecular weight, which stabilize the emulsions forming an extensive network in the continuous phase. The EPS consequently becomes highly viscous, so that the movements of the drops and encounters are reduced.

    Several studies have shown that the production of EPS in bacteria at low temperatures is a mechanism of adaptation to cold temperatures. It has been described that Pseudoalteromonas sp. CAM025 produced 30 times more EPS at -2 ° C and 10 ° C compared to 20 ° C (cf. CA Mancuso Nichols et al., "Production of exopolysaccharides by Antartic marine bacterial isolates", Journal of Applied Microbiology, 2004, vol. 96, pp. 1057-1066). The production of EPS by the psychophilic marine bacteria 10 Colwellia psychrerythraea strain 34H is about 10 times higher at -8 ° C than at -4 ° C. The EPS of the invention increases survival rates at freezing temperatures of Pseudomonas sp. CECT8437 and cells of other species (cf. Example 12). This indicates the benefits of the EPS of the invention for use as a cryoprotectant. fifteen

    Unless specified, the protocols cited are accessible in the textbook C. A. Reddy et al. (eds.), "Methods for General and Molecular Microbiology", 3rd Edition, ASM Press, Washington, USA (hereinafter, the "textbook"), and feasible for the person with average knowledge of the art. In general, three measurements were made, and the error estimate is the standard error of the mean.
    Example 1: EPS production

    Pseudomonas sp. CECT8437 was grown in MM1 minimum medium: 20 g / l glucose; 25 0.5 g / l bacto-peptone; 0.1 g / l yeast extract; 0.4 g / l citrate; 7 g / l NaNO3; 2 g / l K2HPO4; 0.7 g / l NaNH5PO4 · 4H2O; 0.1 g / l MgSO4 · 7H2O; 0.018 g / l FeSO4 · 7 H2O; 1 ml trace elements. The culture was incubated at 11 ° C on an orbital shaker at 150 rpm for 120 h. To obtain the EPS, the cells were removed from the culture by centrifugation (6000 rpm, 25 min, 4 ° C). The cell-free supernatant was reserved 30 and the sediments were washed three times with a Ringer solution (Scharlau) and centrifuged (40000xg, 20 min, 4 ° C) to remove the EPS adhered to the cell surface. The supernatants of the washes were combined with the first culture supernatant and subjected to a tangential flow filtration process through 0.22 μm membranes (Millipore). The filtrate was subjected to a dialysis process with sterile distilled water 1:10 (v / v) through 10000 Da (Millipore) membranes to
    remove salts, pigments and other components from the culture medium and to obtain a concentrated and purified EPS. The resulting product was lyophilized.
    Example 2: Characterization of protein content and uronic acids.
     5
    The total uronic acid content of the EPS was 2.40% ± 0.33% of the total EPS weight determined by the metahydroxidiphenyl method (N. Blumenkrantz et al., "New method for quantitative determination of uronic acids", Analytical Biochemistry, 1973, vol. 54, pp. 844-489). Neither galacturonic acid nor glucuronic acid was detected by the HPLC technique. The protein content of the EPS determined by the Bradford 10 test (BioRad) was less than 2% of the total weight of the EPS obtained.
    Example 3: Preparation of the hydrolyzed product and analysis of monosaccharides
    In order to analyze the composition of neutral sugars, the EPS sample of Example 1 was hydrolyzed with 1% H2SO4 at 111 ° C for 0.5 hour. The hydrolyzate was used to identify and quantify the constituent monosaccharides by High Performance Liquid Chromatography (HPLC) using the Aminex HPLC columns for HPX-87P (300 x 7.8 mm) carbohydrate analysis and HPX-87C (300 x 7.8 mm) (BioRad). MiliQ water at 85 ° C was used as eluent, and the detection was carried out with a Waters 2414 refractive index detector (Waters). 15 commercial sugars and amino sugars were used as standards for the identification of monosaccharides. The results showed the presence of the following neutral sugars: glucose at 17.04 ± 0.32%, galactose at 8.57 ± 1.15%, and fucose at 8.21 ± 1.12%, in relation to to 25 total weight of the EPS obtained. The molar ratio of these sugars and uronic acid is 2: 1: 1: 0.3 and remains constant for the EPS.
    Example 4: Elementary Analysis  30
    The elementary composition of the EPS was analyzed using a Thermo EA 1108 organic elemental analyzer (Thermo Scientific), working under the standard conditions recommended by the instrument supplier (helium flow at 120 ml / min, combustion furnace at 1000 ºC, furnace the chromatographic column at 60 ° C, oxygen loop 10 ml at 157 kPa). The results showed a content of 37.29% 35
    ± 1.07 Carbon; 6.17% ± 0.10 Hydrogen; 2.25% ± 0.21 Nitrogen; and 0.41% ± 0.16 Sulfur.
    Example 5: Size Exclusion Chromatography  5
    The molecular weight (PM) of the EPS was determined by (size-exclusion chromatography, SEC) on a Waters 2695 HPLC kit equipped with an Ultrahydrogel 500 column (7.8 x 300 mm; Waters) and a differential refractive index detector (Waters 2414). 0.1 M NaNO3 was used as eluent at room temperature. Dextran (Sigma-Aldrich) PM patterns of between 8.8 x 103 and 2 x 106 Da were used to calibrate the column for PM estimation. The results showed a molecular weight greater than the dextran standard with the highest available molecular weight 2 x 106 Da according to Sigma.
    Example 6: FT-IR and NMR spectroscopy 15
    All FT-IR spectra were obtained in the transmission mode, using a Thermo diamond compression cell (Thermo Scientific). The spectra were obtained in an area of 100 μm x 100 μm, with a resolution of 4 cm-1 and 64 scanners. twenty

    The infrared spectra of the EPS were acquired with a Thermo 173 IN10MX FT-IR microscope (Thermo Scientific), using an MCT detector cooled with liquid nitrogen. In the Fourier Transform Infrared (FT-IR) infrared spectrum, the wide and intense band around 3400 cm -1 represents the O-H bond of hydroxyl and bound water; the peaks of the CH2 and CH3 bonds appear at 2930 cm-1 and 2985 cm-1, respectively; well-defined signals between 1200 and 900 cm-1 represent the C-O skeleton and the C-C vibration bands of carbohydrates; The band at 1720 cm-1 can be attributed to the C-O bonds of the carbonyls of the acyl groups; The C-O peak of amide 1 to 1640 30 cm -1 and the N-H peak of amide 2 to 1540 cm -1 are typical of proteins (FIG 1). This protein detection is in line with the low protein content detected by the Bradford assay and is probably due to cell lysis during the process of obtaining and purifying the EPS. In any case, the high speed centrifugation process is validated as one of the best methods for
    avoid cell lysis in obtaining EPSs from bacterial cultures (textbook page 366, chapter 15.3.8).

    Proton Nuclear Magnetic Resonance (1H-NMR) spectra were obtained in 99.96% D2O (Euriso-top) at 25 ° C using a Varian VNMRS500 5 spectrometer (500 MHz, Varian Ltd). The results are in line with the carbohydrate composition of the biopolymer, with marked peaks of the predominant glycan component (FIG 2). Detailed analysis of the EPS spectrum was not possible due to the complexity of the sample, but the presence of sugars can be observed in the region of anomeric protons (4.2-5.5 ppm) and in the region of ring protons (3.2-10 4 ppm). The intense signals at 1.2 ppm in the EPS spectrum could correspond to the methyl H6 protons of the fucose.

    Example 7: Emulsifying activity against food oils, cosmetics and n-hexadecane 15

    To test the emulsifying activity of EPS, a described protocol was carried out (cf. Gutierrez et al., "Emulsifying and metal ion binding activity of a glycoprotein exopolymer produced by Pseudoalteromonas sp. Strain TG12", Applied and Environmental Microbiology, 2008, vol. 74, pp. 4867-4876). Xanthan gum and Arabic gum (Sigma-Aldrich) were used as positive controls. The emulsions were prepared as described below: 20 mg of the analyzed emulsifier (EPS, xanthan gum and gum arabic) were added to 5 ml of 0.1 M PBS (0.02% w / v), and then mixed with 0.8 ml of n-hexadecane (Sigma-Aldrich), olive oil, sunflower oil or corn oil using an Ultra-turrax T10 basic 25 (IKA) homogenizer at speed 5 for 1 min. The emulsions were allowed to stand at room temperature for 24 h. After this time, the emulsifying activity (A540) was determined by measuring the turbidity of the lower aqueous layer using a UV-1800 spectrophotometer (Shimadzu) at 540 nm. Emulsifying activities were compared under neutral conditions (0.1 M PBS, pH 7). Samples of the corresponding food oils or the n-hexadecane and phosphate buffered saline solution (PBS) without the EPS were used as a control, and the A550 values of these controls were subtracted from those of the EPS, rubber Xanthan and gum arabic to obtain its final emulsifying activities.
     35
    The emulsifying activity of EPS against the cosmetic oil cetiol V (Fagron) was also tested, and compared with the commercial emulsifier Span 20 (Croda). For this purpose, a mixture of water and the hydrophobic compound cetiol V was prepared in the ratio 1: 2 v / v respectively. Then, the EPS of the invention or Span 20 was added to different samples of the mixture at a concentration of between 1% and 5 12%, and the emulsions were prepared using an Ultra-turrax T10 basic homogenizer (IKA) at speed 5 for 2 min. Stable emulsions with a creamy consistency were formed with 2% EPS, while 6% of Span 20 was needed to achieve the same degree of emulsification.
     10
    The stability of the EPS emulsion was tested under various conditions of pH, temperature and salinity, resulting in the emulsions being stable for more than four months at pH between 5 and 8, temperatures from 4 ° C to 42 ° C and NaCl concentrations up to 1 M.
     fifteen Example 8: Analysis of particle size
    The particle size analysis of the emulsions with 2% EPS, and a mixture of water and cetiol V (1: 2; v / v) was carried out by laser diffractometry (DL) using a Mastersizer Hydro 2000MU ( Malvern instruments) obtaining volume 20 of particle distribution. For the DL analysis the diameters 10, 50 and 90% were used: for example, a value of DL90% indicates that 90% (volume distribution) of the measured particles have a diameter equal to or less than the given value. Emulsions with 2% EPS and a mixture of water and cetiol V (1: 2; v / v) revealed LD10% values ≤ 4.57 μm ± 0.001, LD50% ≤ 10.66 μm ± 0.006 and LD90% ≤ 20.36 μm ± 25 0.03, respectively, indicating that the emulsion particles were in the micrometer range, with the main peak around 12 μm.
    Example 9: Rheological measures  30
    The rheological measurements of emulsions with 2% EPS, and a mixture of water and cetiol V (1: 2, v / v) were performed on a Haake RheoStress 1 rheometer (Thermo Scientific) equipped with cone and plate geometry (diameter plate 35 mm, cone angle 2). All measurements were carried out at 25 ° C. Continuous shear studies were performed to evaluate the shear stress (Pa) based on the speed of shear (s-1). This study was carried out with a shear rate of 0-100 s-1
    for 3 min, 100 s-1 for 1 min and turn to 0 s-1 for 3 min. The resulting shear stress and viscosity were measured. In rheological studies, the emulsion flow curve with 2% EPS exhibited pseudoplastic flow behavior, and low thixotropy and pour point (FIG 4). However, the flow curves of water and cetiol V without EPS showed a Newtonian behavior (data not shown), indicating that pseudoplasticity was due to the presence of EPS in the emulsion. In addition, the estimated viscosity of the emulsion was 4.46 ± 0.007 Pa · s.
    Example 10: Measurement of potential zeta 10
    The zeta potential (PZ) of the emulsions with 2% EPS and a mixture of water and cetiol (1: 2; v / v) was determined using a Zetasizer Nano ZS (Malvern Instruments) at 25 ° C. The PZ was calculated from electrophoretic mobility using the Helmholtz-Smoluchoswski equation (cf. SR Deshiikan et al. "Modified booth equation for the 15 calculation of zeta potential", Colloid and Polymer Science, 1998, vol. 276, pp. 117-124). The data processing was performed with the software included in the system. The PZ value of the emulsions with 2% EPS and a mixture of water and cetiol V (1: 2; v / v) was 0.11 ± 0.4 mV, which indicated that the emulsion particles had a weak surface charge twenty
    Example 11: Stability of the emulsion
    The stability of emulsions with 2% EPS and a mixture of water and cetiol V (1: 2; v / v) was evaluated by measuring the variations in backscattering using a 25 Turbiscan Lab Expert (Formulaction), based on dispersion multi angle laser light. Due to the opacity of the samples, only backscattering (BS) profiles were used to assess the physicochemical stability of the emulsions. The measurements were carried out at 25 ° C with the freshly prepared emulsion (day 0) and one month later after storage at room temperature (day 30). 30

    The emulsion stability of EPS was also studied under various conditions of temperature, salinity and pH. For pH stability tests, mixtures with 2% EPS, water with a pH between 5 and 9 and cetiol V (1: 2; v / v) were prepared and stored at room temperature. For temperature stability tests, mixtures with 2% EPS, water adjusted to pH 7 and cetiol V (1: 2; v / v) and
    They were stored at temperatures between 4 ° C and 42 ° C. Finally, for salinity stability studies, emulsions with 2% EPS and water mixtures containing up to 1 M NaCl and cetiol V (1: 2; v / v) were prepared and also stored at room temperature. The stability of the emulsions was evaluated over a period of 4 months, observing changes in their macroscopic appearance. 5

    The stability of the emulsion was evaluated from% BS at days 0 and 30 in samples stored at room temperature. As can be seen in FIG 5, the profiles of the emulsion at days 0 and 30 were extremely similar, and no phenomena of coalescence, flocculation, cremation, or sedimentation were observed, 10 indicating a long-term stability of the EPS emulsion. . In addition, the emulsions remained stable for more than four months at pH between 5 and 8, temperatures of 4 ° C to 42 ° C and NaCl concentrations of up to 1 M.
    Example 12: Cryoprotective Activity 15
    Obtaining a non-EPS mutant strain: A non-EPS mutant strain was obtained by ultraviolet light mutagenesis to assess the influence of EPS on cell preservation when subjected to various freezing temperatures. To confirm that the mutant strain did not produce EPS, a staining with ruthenium tetraoxide was used to reveal the polysaccharides around the bacterial cells. For this purpose, the cells were subjected to chemical fixation with 5% glutaraldehyde in 0.1 M cacodylate buffer at pH 7.3 and 4 ° C for a period of one night. After being washed for 10 min with 0.1 M cacodylate buffer at pH 7.3 five times, the samples were incubated twice with 25 0.25% ruthenium tetraoxide and 0.25% potassium ferrocyanide in 0.1 M cacodylate buffer at pH 6.8 for 1 h in darkness at 4 ° C. The samples were washed five times for 15 min with miliQ water at 4 ° C and kept in 0.1 M cacodylate at pH 6.8 and 4 ° C until high pressure cryofixation. For cryosubstitution, a solution containing: 1% osmium tetraoxide, 0.5% uranyl acetate and 3% glutaraldehyde 30 in methanol was used. The cryosubstitution began at 72h at -90 ° C, followed by heating to 4 ° C with a ramp of 5 ° C / h, at which point the temperature was maintained for 4 h. Once the process was finished, the samples were kept at room temperature and in darkness at 4 ° C. They were then washed twice with methanol for 2 h and washed three times with acetone for 15 min. Finally, the 35 samples were infiltrated in Epon: methanol 1: 3, 2: 2 and 3: 1 for 3 h at each step and
    embedded in Epon. Ultrafine sections were obtained using the UCT ultramicrotome (Leica Mycrosystems), and stained with 2% uranyl acetate and lead citrate. The samples were observed with an electronic transmission microscope TEM Tecnai Spirit Twin (FEI) 120Kv. In the wild cells (wt) of Pseudomonas sp. CECT8437, a dyed polymeric material was clearly observed around the 5 cells (FIG 6A), while in the mutant cells this layer was not observed at all (FIG 6B) confirming that the mutant strain was not capable of producing EPS.

    Cryoprotection assays with Pseudomonas sp. CECT8437: Wild (wt) and mutant (mt) cells of Pseudomonas sp. CECT8437 in Tryptone 10 Soybean Agar (TSA; Oxoid) plates at 10 ° C for 5 days to reach confluent growth. Suspensions of wt and mt cells were prepared in a Ringer solution and adjusted to an optical density of 0.6 (540 nm). 1 ml aliquots were prepared and centrifuged at 12,000 rpm for 30 min. The supernatants were discarded, and the cell pellets were frozen at -20 ° C and -80 ° C. After one week, the samples were thawed, and the sediments were resuspended in 1 ml of Ringer's solution. Cell viability was determined by the serial dilution method, and the survival rate was expressed as the percentage of viable cells with respect to non-frozen cells (Table 1). It is demonstrated that the wt strain exhibited significantly higher survival rates than the mt strain at all 20 temperatures tested.

    -------------------------------------------------- -------------------------------------------------- ----------------
    Table 1: Survival rates of wild and mutant strains after freezing the cells at -20 ° C and -80 ° C, expressed as the percentage of viable cells 25 with respect to non-frozen cells (n = 8)

     Wild strain Mutant strain
     -20 ° C  75.19 ± 8.06 40.27 ± 5.97
     -80ºC  93.53 ± 12.18 49.94 ± 7.43
    -------------------------------------------------- -------------------------------------------------- ----------------
    Cryoprotection assays with E.coli .: Escherichia coli ATCC 10536 was grown in 30 Tryptone and Soy Broth (TSB) to an optical density of 0.6 (540 nm). 0.1 ml aliquots were mixed with different concentrations of EPS between 0% and 10%, to a final volume of 1 ml. The samples were frozen at -20 ° C and -80 ° C for one week, they were thawed and the number of viable cells was determined by the serial dilution method. The survival rate was expressed as the percentage of viable cells with respect to non-frozen cells. Fetal bovine serum (SBF) was used as a control because it is used as a membrane stabilizer in freezing procedures. As shown in FIG 7 and FIG 8, EPS 5 also conferred cryoprotection on E.coli cells, with a clear dose-response relationship at all temperatures tested and a maximum survival rate (35.68% at -20 ºC and 64.13% at -80 ºC) with an EPS concentration of 10%, and without the addition of any cryoprotective agent that could penetrate the cells, such as dimethylsulfoxide (DMSO) or glycerol. This dose-response relationship was not observed with the SBF, and the survival rates obtained at any concentration of SBF were below 0.4% and considered void in the graphs.

    Example 13. Ultrastructure of the extracellular and EPS material of Pseudomonas sp. CECT8437 and other cold-adapted Antarctic bacteria. fifteen

    All strains were grown in trypticase soy agar (TSA, Oxoid) and incubated for 3 days at 15 ° C. Random bacterial colonies were selected for examination by transmission electron microscopy (MET) followed by high pressure fixation and cryosubstitution (High -pressure freezing and freeze substitution, HPF-FS) (textbook, 20 pages 66, 67, chapter 4.2.4). Ultra-thin sections were cut with a Leica UCT ultramicrotome and mounted on Formvar carbon-coated grilles. The sections were subsequently stained with 2% (w / v) aqueous uranyl acetate and lead citrate and examined in a Tecnai Spirit (FEI Company) electron microscope at an acceleration voltage of 120 kV. As shown in FIG 9, all 25 extracellular materials of cold-adapted Antarctic bacteria that were analyzed, with the exception of Pseudomonas sp. CECT8437, appeared as a net-shaped mesh composed of a capsular polymer around the cells and large amounts of outer membrane vesicles (VMEs, see arrows).
     30
    Example 14: In vivo tests of elasticity and dermoprotective properties in humans

    Tests were carried out with an efficiency of 0.2%, 1% and 5% EPS on human volunteers between 24 and 64 years, for 9 days. 35

    All volunteers were instructed to avoid using any topical products on the right forearm during the 48 hours prior to the experiment. To obtain reliable measurements, the volunteers acclimatized for 15 min in a heated room, 23 ºC and 50% relative humidity, before carrying out the tests.
     5
    To study the effect of EPS on skin properties, five 4 cm2 areas were delimited in the right forearm of each volunteer: four areas for topical applications of the formulations (three different concentrations of EPS and placebo) and one area not Treated as a control. On day 0, the elasticity and transepidermal water loss (TEWL) in each area were measured. 10 Next, 20 µl of each sample was applied daily for 7 days. TEWL was measured in triplicate on days 2, 4 and 9, measuring the elasticity in triplicate on days 0 and 9.

    Skin elasticity was evaluated with a Cutometer® SEM 575 device (Courage & 15 Khazaka). Volunteers treated with 0.2% EPS showed a 13.28% increase in skin elasticity compared to those treated with placebo (FIG 10).

    The dermoprotective effect of EPS was also evaluated. To do this, after finishing all 20 skin measures, all previously treated areas were contacted with 2% of the sodium lauryl sulfate surfactant (LSS) for 2 h, and the TEWL was evaluated 2 h 30 min after exposure to the LSS.

    In order to evaluate the skin's protective function, a Tewameter® 25 TM 300 device (Courage & Khazaka) was used to measure the TEWL. Volunteers treated with 1% EPS showed a lower percentage of TEWL (154.28%) after the application of the irritant LSS, compared to those treated with placebo (181.83%; FIG 11). This shows that EPS is a dermoprotective agent that strengthens the skin against external aggressions. 30
    权利要求:
    Claims (15)
    [1]

    1. A strain of Pseudomonas sp. with deposit number CECT 8437.

    [2]
    2. An exopolysaccharide isolated from a culture of Pseudomonas sp. with 5 deposit number CECT8437.

    [3]
    3. The exopolysaccharide according to claim 2, comprising glucose, galactose, fucose and uronic acid in a molar ratio of about 2: 1: 1: 0.3.
     10
    [4]
    4. The exopolysaccharide according to any of claims 2-3, comprising the following percentages by weight, approximately: 37.29% C, 6.17% H, 2.25% N and 0.41% S.

    [5]
    5. The exopolysaccharide according to any of claims 2-4, with a transformed Fourier infrared spectrum comprising, in cm -1, a band at 3400, a peak at 2930, a peak at 2985, a band between 1200 and 900, a band at 1720, a peak at 1640 and a peak at 1540.

    [6]
    6. Use of the exopolysaccharide defined in any of claims 2-5, as a cryoprotective agent.

    [7]
    7. Use of the exopolysaccharide defined in any of claims 2-5, as an emulsifying agent.
     25
    [8]
    8. Use of the exopolysaccharide defined in any of claims 2-5, as a thickening, stabilizing or texturing agent.

    [9]
    9. Use of the exopolysaccharide defined in any of claims 2-5, as a dermoprotective agent. 30

    [10]
    10. Use of the exopolysaccharide defined in any of claims 2-5, to increase skin elasticity.

    [11]
    11. A cosmetic composition comprising a cosmetically effective amount of the exopolysaccharide defined in any of claims 2-5, and cosmetically acceptable ingredients or carriers.

    [12]
    12. A hydrolyzed product obtained by hydrolyzing the exopolysaccharide defined in any one of claims 2-5, the chemical and / or mechanical and / or enzymatic hydrolyzing being.

    [13]
    13. The hydrolyzed product according to claim 12, wherein the hydrolyzing is chemical. 10

    [14]
    14. The hydrolyzed product according to claim 13, wherein the chemical hydrolyzing is carried out by treatment with an acid.

    [15]
    15. The hydrolyzed product according to claim 14, wherein the acid is selected from the group consisting of sulfuric acid, hydrochloric acid and trifluoroacetic acid.
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    同族专利:
    公开号 | 公开日
    WO2015117985A1|2015-08-13|
    ES2585398R1|2017-01-05|
    ES2585398B1|2017-10-27|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    FR2681601B1|1991-09-25|1993-12-24|Elf Sanofi|POLYSACCHARIDE, ITS APPLICATIONS, OBTAINING IT BY FERMENTATION, PSEUDOMONAS STRAIN PRODUCING IT.|
    ES2390033B1|2010-11-30|2013-10-31|Lipotec S.A.|EXOPOLISACÁRIDO FOR THE TREATMENT AND / OR CARE OF SKIN, MUCOSAS, HAIR AND / OR NAILS.|EP3295927A1|2016-09-19|2018-03-21|Institut Univ. de Ciència i Tecnologia, S.A.|Uses of an exopolysaccharide-protein complex obtained from a bacterium|
    EP3295926A1|2016-09-19|2018-03-21|Institut Univ. de Ciència i Tecnologia, S.A.|Exopolysaccharide-protein complex, a method of preparing said complex and uses thereof|
    KR101816802B1|2017-05-11|2018-01-11|한국해양과학기술원|Cryoprotective Agent Containing Exopolysaccharide from Pseudoalteromonas sp. CY01|
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