WO2015117985A1 - Exopolysaccharide bactérien - Google Patents

Exopolysaccharide bactérien Download PDF

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WO2015117985A1
WO2015117985A1 PCT/EP2015/052249 EP2015052249W WO2015117985A1 WO 2015117985 A1 WO2015117985 A1 WO 2015117985A1 EP 2015052249 W EP2015052249 W EP 2015052249W WO 2015117985 A1 WO2015117985 A1 WO 2015117985A1
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eps
exopolysaccharide
agent
pseudomonas
acid
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PCT/EP2015/052249
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English (en)
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Mª Elena MERCADÉ GIL
Ornella CARRIÓN FONSECA
Mª Jesús MONTES LÓPEZ
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Universitat De Barcelona
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Priority to ES201690034A priority Critical patent/ES2585398B1/es
Publication of WO2015117985A1 publication Critical patent/WO2015117985A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/72—Cosmetics or similar toiletry preparations characterised by the composition containing organic macromolecular compounds
    • A61K8/73—Polysaccharides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
    • A23L29/20—Foods or foodstuffs containing additives; Preparation or treatment thereof containing gelling or thickening agents
    • A23L29/269—Foods or foodstuffs containing additives; Preparation or treatment thereof containing gelling or thickening agents of microbial origin, e.g. xanthan or dextran
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/60—Sugars; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/99—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from microorganisms other than algae or fungi, e.g. protozoa or bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/08—Anti-ageing preparations
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/38—Pseudomonas

Definitions

  • the present invention relates to the field of bacterial exopolymers, particularly of exopolysaccharides (EPSs), that can be used as a source of oligosaccharides and sugar monomers.
  • EPSs exopolysaccharides
  • bacterial exopolymers serve various biological functions, for example, as a reserve material of biodegradable compounds and trace metal nutrients, or as part of a protective structure against predators, desiccation, salinity, cytotoxic compounds and high or low temperatures.
  • Bacterial exopolymers are high molecular weight (MW) polymers that constitute up to 40- 95% of the extracellular matter (EM) surrounding most microbial cells in marine environments, understanding by EM all components that the bacteria secrete to the extracellular environment.
  • Most exopolymers produced by marine bacteria are EPSs, specifically heteropolysaccharides containing three or four different monosaccharides that may be pentoses, hexoses, amino sugars or uronic acids, arranged in groups of ten or less to form repeating units.
  • Organic or inorganic substituents namely sulfate, phosphate, acetic acid, succinic acid and pyruvic acid, may also be present.
  • Fucose-containing EPSs are seen as interesting products for pharmaceutical and cosmetic industries.
  • the biological properties of this rare sugar do potentiate its use in pharmaceuticals, e.g. as anticarcinogenic or antiinflammatory agents, and in cosmetic products as anti-aging agents.
  • EPSs have been found in Antarctic marine bacteria and in Arctic winter sea ice, modifying the physicochemical environment of bacterial cells, participating in cell adhesion to surfaces and retention of water, favoring the sequestration and concentration of nutrients, retaining and protecting extracellular enzymes against cold denaturation and acting as cryoprotectants. Pseudoalteromonas sp.
  • SM20310 isolated from Arctic sea ice, secretes an EPS composed mainly of mannose, and traces of glucose, galactose, rhamnose, N- acteylglucosamine, 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 Applied and Environmental Microbiology, 2012, doi:10.1 128/AEM.01801 -12).
  • Other cold-adapted bacteria isolated from the Antarctica are Pseudoalteromonas haloplanktis TAC125, which synthesizes an EPS consisting of mannose and traces of glucose (Corsaro et al., "Influence of growth temperature on lipid and phosphate contents of surface polysaccharides from the Antarctic bacterium
  • EPSs More complex EPSs have also been described from Antarctic environments, including Pseudoalteromonas sp. CAM025, Pseudoalteromonas sp. CAM036, and other strains from 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. Pseudoalteromonas sp.
  • CAM036 strain also synthesizes a sulfated heteropolysaccharide with high levels of uronic acids with acetyl groups, composed mainly of glucose, mannose, arabinose, galacturonic acid and N-acetyl galactosamine (Mancuso et al., "Production of exopolysaccharides by Antarctic marine bacterial isolates", Journal of Applied Microbiology, 2004, vol. 96, pp. 1057-1066). Other studies confirmed that chemical composition and MW data were very diverse among EPSs produced by
  • OMVs are produced during the course of normal metabolism and cell growth of most analyzed Gram-negative bacteria. These are spherical bilayered lipid vesicles extruded from regions of the bacterial outer membrane and they contain lipopolysaccharide, periplasmic proteins, outer membrane proteins and phospholipids (A. Kulp et al., "Biological functions and biogenesis of secreted bacterial outer membrane vesicles", Annual Review of Microbiology, 2010, vol. 64, pp. 163-184).
  • vesicles contain a wide variety of virulent factors. These virulent factors include proteins, namely adhesins, toxins, and enzymes as well as non-protein antigens like lipopolysaccharides (T.N. Ellis et al., "Virulence and immunomodulatory roles of bacterial outer membrane vesicles",
  • the Pseudomonas genus is one of the most studied sources of EPSs.
  • Various species of Pseudomonas genus are able to synthesize a wide variety of exopolysaccharides. Many of them are from the polymer class alginates, being the main components mannuronic acid and glucuronic acid.
  • gellan exopolysaccharides mainly composed by glucose, ramnose and glucuronate are synthesized by Pseudomonas species. Having glucose as carbon source, Pseudomonas putida and Pseudomonas fluorescens synthesize an EPS composed of glucose, galactose and pyruvate.
  • EPS produced by P. fluorescens Biovar II is composed of galactose, mannose, rhamnose, glucose, fucose, ribose, arabinose and xylose.
  • the purified EPS from P. putida G7 contains
  • An aspect of the present invention relates to a strain of Pseudomonas sp. deposited on 1 .10.2013 with number CECT8437 in the "Coleccion Espafiola de Cultivos Tipo", with address at: "Edificio 2 CUE. Pare Cientific. Universitat de Valencia. Calle Catedratico Agustin Escardino, 9. 46980 Paterna (Valencia), Spain”. The extracellular matter of this strain is virtually free of OMVs when compared with other Antartic bacteria.
  • EPS isolated from a culture of Pseudomonas sp. CECT8437.
  • the EPS comprises glucose, galactose, fucose and uronic acid in a molar ratio of 2:1 :1 :0.3 approximately.
  • the EPS has an elementary composition, in weight percentage, of 37.29% C, 6.17% H, 2.25% N and 0.41 % S approximately.
  • 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 the EPS and cosmetically acceptable ingredients or carriers, are also part of the present invention.
  • Another aspect of the present invention relates to the use of the above-mentioned EPSs 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 EPSs of the present invention can be partial or completely hydrolized chemically, mechanically (e.g. by sonication) or enzimatically, in conditions analogous to those known in the art, to produce oligosaccharides of different lengths, with features different from those of the native ones.
  • the respective obtained hydrolized products are also part of the present invention; they can be used as a source of oligosaccharides and sugar monomers like L-fucose, useful in cosmetics, pharmaceuticals, and dietary supplements.
  • the hydrolyzation is chemical; preferably by treatment with an acid; and more preferably by treatment with sulfuric acid, hydrochloric acid, or trifluoroacetic acid.
  • hydrolyzation with sulfuric acid can be done by treatment at 100-120 °C for 0.5 to 8 h, preferably at 100-120 °C for 0.5 to 2 h.
  • These hydrolyzation treatments yield mono- and/or oligosaccharides that are frequently derivatized into alditol acetates, trimethylsilylated methyl glycosides, and trimethylsilylated (-)-2-butyl glycosides.
  • the invention's EPS is new with respect to other EPSs produced by else cold-adapted bacteria, Pseudomonas species or other bacteria reported so far.
  • the invention's EPS has an emulsifying activity against different food and cosmetic oils which is much higher than that of commercial emulsifiers, namely xanthan gum, arabic gum or Span 20. It forms highly stable emulsions against the cosmetic oil cetiol V, exhibiting pseudoplastic flow behavior, low thixotrophy and yield stress, and confers significant cryoprotection for the strain itself as well as for other bacteria, including E. coli, indicating a universal cryoprotectant role.
  • the cryoprotective activity of the EPS showed a clear dose-response relation at -20 °C and -80 °C unlike what was observed when the membrane stabilizer Fetal Bovine Serum (FBS) is added to the cells.
  • FBS membrane stabilizer Fetal Bovine Serum
  • EPSs Due to the wide composition diversity and different physical-chemical properties, EPSs have emerged as new, industrially important polymeric materials, which are gradually becoming economically competitive with natural gums produced by marine algae and plants.
  • EPSs derived from natural resources have a competitive advantage, due to their biodegradability and often biocompatibility.
  • the invention's EPS contains no detectable amounts of OMVs, therefore excluding potentially toxic proteins or antigenic compounds, namely lipopolysaccharides.
  • the absence of OMVs will favor its obtention and purification processes, all of which makes it desirable for particular uses.
  • CECT8437 EPS is therefore an attractive material for industrial and medical applications as a thickening, stabilizing, binding or structure creation agent.
  • Pseudomonas sp. CECT8437's EPS is a slime polysaccharide which is loosely bound to the cell surface and, unlike capsular polysaccharides, mainly excreted to the extracellular medium. It is remarkable that the EM of this strain does not contain significant amounts of OMVs, contrary to what is observed in the extracellular matter of several cold-adapted Antarctic bacteria. The presence of proteins and other compounds mainly derived from OMVs in EPSs can be antigenic or toxic. The fact that the invention's strain is free of OMV means a definite technological advantage over other EPSs extracted from Pseudomonas or else other Gram-negative bacteria. The commercial value of the invention's EPS mainly relies on this feature.
  • the EPS of the present invention is found to be a more efficient emulsifier against several food oils and n-hexadecane than other bacterial EPSs or plant gums of the art, namely xanthan gum and arabic gum.
  • the EPS demonstrates higher emulsification activity against the cosmetic oil cetiol V than the commercial emulsifier used as a positive control, Span 20.
  • the Pseudomonas sp. CECT8437 EPS was observed to form long-term, stable emulsions with pseudoplastic behavior.
  • the main properties of the EPS namely its ability to form a long-term stable emulsion against different food and cosmetic oils and its universal cryoprotective activity, make it a promising alternative to commercial polysaccharides of the art.
  • FIG 1 Infrared spectrum of the EPS produced by Pseudomonas sp. CECT8437 (cf.
  • FIG 2 1 H-NMR spectrum of the EPS produced by Pseudomonas sp. CECT8437 (cf. Example 6).
  • FIG 3 Emulsifying activities (A 540 ) of the EPS, xanthan gum and arabic gum against olive, sunflower and corn oils, and n-hexadecane after 24 h at room temperature.
  • A Olive oil.
  • B Sunflower Oil.
  • C Corn oil.
  • D n-Hexadecane.
  • FIG 4 Rheogram of emulsions with 2% of EPS from Pseudomonas sp. CECT8437 and a mixture of water and cetiol V (1 :2; v/v).
  • Shear stress (SS) is represented by a black line.
  • Viscosity (V) is represented by a grey line.
  • SR Shear Rate (cf. Example 9).
  • FIG 5 Backscattering (BS) profiles of the emulsion with 2% of EPS and a mixture of water and cetiol V (1 :2; v/v) as a function of sample height (SH), analyzed at days 0 and 30 of storage at room temperature. Black line: day 0. Grey line: day 30. (cf. Example 1 1 ).
  • FIG 6 Transmission electron microscopy images of wild-type cells of Pseudomonas sp. CECT8437 (A), and non-EPS producing mutant cells (B) (cf. Example 12).
  • FIG 9 Transmission electron microscopy micrographs of ultrathin sections from cold- adapted Antarctic bacteria after high pressure-freezing and freeze-substitution (HPF-FS). Bars are 200 nm.
  • A Pseudomonas sp. CECT8437.
  • B Pseudoalteromonas sp.
  • C
  • D Marinobacter sp.
  • E Psychrobacter sp. NF23.
  • F Shewanella sp. M7 (cf. Example 13).
  • FIG 10 Elasticity (EL) of the skin treated with placebo or 0.2% of EPS. Skin elasticity is expressed as the % of the net-elasticity at day 9 with respect to day 0 and the control.
  • White column placebo.
  • Black column 0.2% of EPS.
  • FIG 1 1 Transepidermal water loss (TEWL) of the skin treated with placebo or 1 % of EPS after the exposure to SLS.
  • TEWL is expressed as the percentage of TEWL after the treatment with SLS with respect to the initial TEWL.
  • White column placebo.
  • Black column 1 % of EPS.
  • the monosaccharide and uronic acid analysis of the EPS of the invention reveals that it comprises glucose, galactose, fucose and an uronic acid in a molar ratio of 2:1 :1 :0.3 (cf. Examples 2, 3).
  • FIG 3 shows the measured emulsifying activities of the EPS under neutral conditions against three different food oils and n-hexadecane.
  • the EPS exhibits a high emulsifying capacity for olive, sunflower and corn oils, with absorbance at the wave length of 540 nm (A 540 ) of 1 .28, 1.15 and 0.66, respectively.
  • the EPS showed a higher emulsifying activity than the commercial emulsifier xanthan gum and arabic gum, with the exception of sunflower oil, for which the EPS showed the same emulsifying activity as xanthan gum.
  • the EPS also demonstrated a higher emulsifying capacity against n-hexadecane than the positive controls.
  • the EPS emulsifying activity was also tested against the cosmetic oil cetiol V using another emulsifying protocol, and compared with the commercial cosmetic emulsifier Span 20. Stable emulsions with a creamy consistency were formed with 2% of EPS, whereas 6% of Span 20 was needed to achieve the same emulsification grade (cf. Example 7).
  • ZP Zeta potential
  • exopolysaccharides which stabilize emulsions by forming an extended network in the continuous phase.
  • the EPS consequently becomes highly viscous, wherein droplet movements and encounters are reduced.
  • EPS production by the marine psychrophilic bacterium Colwellia psych rerythraea strain 34H is about tenfold higher at -8 °C than at -4 °C.
  • the EPS of the invention increases the survival rates at freezing temperatures of Pseudomonas sp. CECT8437 and other species cells (cf.
  • Example 12 This indicates the benefits of the EPS of the invention for use as
  • cryoprotectant Unless specified, the protocols cited in the following examples are accessible in the textbook C.A. Reddy et al. (eds.), "Methods for General and Molecular Microbiology", 3rd Edition, ASM Press, Washinton, USA (hereinafter the "textbook"), and feasable for the person having average skills in 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 on the minimal medium MM1 : 20 g/l glucose; 0.5 g/l bacto-peptone; 0.1 g/l yeast extract; 0.4 g/l citrate; 7 g/l NaN0 3 ; 2 g/l K 2 HP0 4 ; 0.7 g/l NaNH 5 P0 4 ⁇ 4 H 2 0; 0.1 g/l MgS0 4 ⁇ 7 H 2 0; 0.018 g/l FeS0 4 ⁇ 7 H 2 0; 1 ml trace elements. The culture was incubated at 1 1 °C on an orbital shaker at 150 rpm for 120 h.
  • EPS recovery cells were removed from culture by centrifugation (6,000 rpm, 25 min, 4 °C). The cell-free supernatant was reserved and pellets were washed three times with a Ringer solution (Scharlau) and centrifuged (40,000xg, 20 min, 4 °C) to remove the EPS adhered to the cell surface. Washing supernatants were pooled with the first culture supernatant and subjected to a tangential flow filtration process through 0.22 ⁇ membranes (Millipore).
  • the total uronic acid content of EPS was 2.40 ⁇ 0.33% of the total weight of the EPS determined by the methahydroxydiphenyl method (N. Blumenkrantz, N et al. "New method for quantitative determination of uronic acids", Analytical Biochemistry, 1973, vol. 54, pp. 484-489). No galacturonic or glucuronic acid were detected by HPLC technique.
  • the EPS protein content determined by the Bradford assay (BioRad) was less than 2% of the total weight of the obtained EPS.
  • the EPS sample of Example 1 was hydrolyzed with 1 % H 2 S0 4 at 1 1 1 °C for 0.5 h.
  • the hydrolysate was used to identify and quantify the constituent monosaccharides by High-Perfomance Liquid Chromatography (HPLC) using Aminex HPLC Carbohydrate Analysis Columns HPX-87P (300 x 7.8 mm) and HPX-87C (300 x 7.8 mm) (BioRad). MilliQ water at 85 °C was used as the eluent, and detection was carried out with a Waters 2414 Refractive Index Detector (Waters). 15 commercial sugars and amino sugars were used as patterns for monosaccharide identification.
  • HPLC High-Perfomance Liquid Chromatography
  • Results showed the presence of the following neutral sugars: glucose in a 17.04 ⁇ 0.32%, galactose in a 8.57 ⁇ 1 .15%, and fucose in a 8.21 ⁇ 1 .12%, related to the total weight of the obtained EPS.
  • the molar ratio of these sugars and the uronic acid is 2:1 :1 :0.3 and remains constant for the EPS.
  • Example 4 Elemental analysis The elemental composition of the EPS was analyzed using a Thermo EA 1 108 elemental organic analyzer (Thermo Scientific), working in the standard conditions recommended by the instrument supplier (helium flow at 120 ml/min, combustion furnace at 1 ,000 °C, chromatographic column oven 156 at 60 °C, oxygen loop 10 ml at 157 kPa). Results showed a content of 37.29 ⁇ 1 .07% C; 6.17 ⁇ 0.10% H; 2.25 ⁇ 0.21 % N; and 0.41 ⁇ 0.16% S.
  • Thermo EA 1 108 elemental organic analyzer (Thermo Scientific)
  • the MW of the EPS was determined by size-exclusion chromatography (SEC) in an HPLC Waters 2695 apparatus (Waters) equipped with an ultrahydrogel 500 column (7.8 x 300 mm; Waters) and a differential refractive index detector (Waters 2414). 0.1 M NaN0 3 was used as the eluent at room temperature. Dextran standards (Sigma-Aldrich) of MW ranging from 8.8 x 10 3 to 2 x 10 6 Da were used to calibrate the column for MW estimation. Results showed a MW higher than the maximum MW dextran standard available 2 x 10 6 according to Sigma.
  • Thermo Scientific Thermo Scientific.
  • the spectra were obtained in a 100 ⁇ x 100 ⁇ area, with a resolution of 4 cm “1 and 64 scans.
  • the infrared spectra of the EPS were acquired with a Thermo FT-IR Microscope 173 IN10MX (Thermo Scientific), using a MCT detector cooled by liquid nitrogen. Units are wavenumber in cm "1 .
  • the Fourier Transform Infrared (FT-IR) spectrum of the EPS can be interpreted as follows: the broad and intense band around 3400 represents O-H stretching of hydroxyls and bound water; C-H stretching peaks of CH2 and CH3 appear at 2930 and 2985, respectively; the well-defined envelope found between 1200 and 900 represents skeletal C-0 and C-C vibration bands of carbohydrates; the band at 1720 may be attributed to the C-0 stretching of carbonyls in acyl groups; the C-0 stretching peak of amide 1 at 1640 and N-H stretching peak of amide 2 at 1540 are characteristic of proteins (FIG 1 ).
  • Example 7 Emulsifying activity against food, cosmetic oils and n-hexadecane
  • Emulsions were allowed to stand undisturbed at room temperature for 24 h. After that period, 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).
  • EPS emulsifying activity was also tested against the cosmetic oil cetiol V (Fagron), and compared to the commercial emulsifier Span 20 (Croda).
  • cetiol V Cosmetic oil cetiol V
  • Span 20 commercial emulsifier
  • a mixture of water and the hydrophobic compound cetiol V was prepared in the proportion 1 :2 v/v respectively.
  • the invention's EPS or Span 20 was added to different samples of the mixture at concentrations ranging from 1 % to 12%, and 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% of EPS, whereas 6% of Span 20 was needed to achieve the same emulsification grade.
  • IKA Ultra-turrax T10 basic homogenizer
  • the stability of the EPS emulsion was tested under several conditions of pH, temperature and salinity, giving that emulsions remained stable for more than four months at a pH ranging from 5 to 8, temperatures of 4 °C to 42 °C and NaCI concentrations up to 1 M.
  • Example 8 Particle size analysis.
  • LD90% value indicates that 90% (volume distribution) of the measured particles possess a diameter equal to or lower than the given value.
  • Emulsions with 2% of EPS and a mixture of water and cetiol V (1 :2; v/v) revealed LD10% values ⁇ 4.57 ⁇ , LD50% ⁇ 10.66 ⁇ and LD90% ⁇ 20.36 ⁇ , respectively, indicating that emulsion particles were in the micron range, with the major peak around 12 ⁇ .
  • Example 9 Rheological measurements. Rheological measurements of the emulsions with 2% of EPS, and a mixture of water and cetiol V (1 :2; v/v) were performed on a Haake RheoStress 1 rheometer (Thermo).
  • Example 10 Zeta potential measurements.
  • the zeta potential (ZP) of the emulsions with 2% of 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 ZP was calculated from the electrophoretic mobility using the Helmholtz-Smoluchoswski equation (cf. S.R. Deshiikan et al., "Modified booth equation for the calculation of zeta potential", Colloid and Polymer Science, 1998, vol. 276, pp. 1 17-124).
  • the processing was run by the software included in the system.
  • the ZP value of emulsions with 2% of 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.
  • Example 1 1 Emulsion Stability.
  • the stability of the emulsions with 2% of EPS and a mixture of water and cetiol V (1 :2; v/v) was assessed by measuring the variations in backscattering using a Turbiscan Lab Expert (Formulaction), based on multiangle laser light scattering. Due to the opacity of the samples, only backscattering (BS) profiles were used to evaluate the physicochemical stability of the emulsions. Measurements were performed at 25 °C with the freshly prepared emulsion (day 0) and one month later after storage at room temperature (day 30).
  • EPS emulsion stability was also studied under several conditions of temperature, salinity and pH.
  • pH stability tests 2% of EPS, and mixtures of water at pH ranging from 5 to 9 and cetiol V (1 :2; v/v) were prepared and stored at room temperature.
  • temperature stability assays mixtures with 2% of EPS, water adjusted to pH 7 and cetiol V (1 :2; v/v) were prepared and stored at temperatures ranging from 4 °C to 42 °C.
  • emulsions with 2% of EPS and mixtures of water containing up to 1 M NaCI 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 by observing changes in their macroscopic characteristics.
  • the emulsion stability was evaluated from % BS at days 0 and 30 in samples stored at room temperature. As can be observed in FIG 5, emulsion profiles at days 0 and 30 were extremely similar, and no coalescence, flocculation, creaming, or sedimentation phenomena were observed, indicating a long-term stability of the EPS emulsion.
  • emulsions remained stable for more than four months at a pH ranging from 5 to 8, temperatures of 4 °C to 42 °C and NaCI concentrations up to 1 M.
  • Example 12 Crvoprotective activity Obtaining a non-EPS-producing mutant strain: A non-EPS-producing mutant strain was obtained by mutagenesis with ultraviolet light to evaluate the influence of the EPS in the preservation of the cells when submitted to several freezing temperatures. To confirm that the mutant strain did not produce EPS a ruthenium tetraoxide staining was used to reveal polysaccharides around bacterial cells. For that purpose, cells were submitted to chemical fixation with 5 % glutaraldehyde in 0.1 M cacodylate buffer at pH 7.3 and 4 °C for an overnight period.
  • samples were twice incubated with 0.25% ruthenium tetraoxide and 0.25% potassium ferrocianyde in 0.1 M cacodylate buffer at pH 6.8 for 1 h in darkness at 4 °C. Samples were washed five times for 15 min with milliQ water at 4 °C and kept in 0.1 M cacodylate at pH 6.8 and 4 °C until cryofixation by High Pressure Freezing. For freeze substitution, a solution containing 1 % osmium tetroxide, 0.5% uranyl acetate and 3% glutaraldehyde in methanol was used.
  • the freeze substitution started at 72 h at -90 °C, followed by a warming up to 4 °C with a slope of 5 °C/h, at which point the temperature was maintained for 4 h.
  • samples were kept at room temperature and in darkness at 4 °C. Afterwards they were twice washed with methanol for 2 h and washed three times with acetone for 15 min. Finally, samples were infiltrated with Epon: methanol 1 :3, 2:2 and 3:1 for 3 h in each step and embedded in Epon.
  • Pseudomonas sp. CECT8437 cells were grown on Tryptone Soya Agar (TSA; Oxoid) plates at 10 °C for 5 days to reach a confluent growth. Suspensions of wild type and mutant 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.
  • TSA Tryptone Soya Agar
  • E.coli ATCC10536 was grown on Tryptone 279 Soya Broth (TSB, Oxoid) to an optical density of 0.6 (540 nm). 0.1 ml aliquots were mixed with different EPS concentrations ranging from 0% to 10%, to a final volume of 1 ml. Samples were frozen at -20 °C and -80 °C for one week, thawed and the number of viable cells was determined by the dilution plating method. Survival rate was expressed as the percentage of viable cells with respect to unfrozen cells. Fetal bovine serum (FBS) was used as control because it is used as a stabilizer of membranes in freezing procedures.
  • FBS Fetal bovine serum
  • the EPS also conferred cryoprotection to E.coli cells, with a clear dose-response relation in all assayed temperatures and a maximal survival rate (35.68% at -20 °C and 64.13% at -80 °C) with an EPS concentration of 10%, and without the addition of any cryoprotective agent that could penetrate the cells, as dimethylsulfoxide (DMSO) or glycerol.
  • DMSO dimethylsulfoxide
  • glycerol glycerol
  • Example 13 Ultrastructure of the extracellular matter and EPS from Pseudomonas sp. CECT8437 and other cold adapted Antarctic bacteria. All strains were grown on trypticase soy agar (TSA, Oxoid) and incubated for 3 days at 15 °C. Bacterial colonies were selected randomly for examination by transmission electron microscopy (TEM) following high-pressure freezing and freeze substitution (HPF-FS) (textbook, pages 66,67, chapter 4.2.4). Ultrathin sections were cut with a Leica UCT ultramicrotome and mounted on Formvar carbon-coated grids.
  • TSA trypticase soy agar
  • HPF-FS high-pressure freezing and freeze substitution
  • Sections were post-stained with 2% (w/v) aquous uranyl acetate and lead citrate and examined in a Tecnai Spirit electron microscope (FEI Company) at an acceleration voltage of 120 kV. As shown in FIG 9, all analyzed extracellular matter from cold-adapted Antarctic bacteria, with exception of Pseudomonas sp. CECT8437, appeared as a netlike mesh composed of a capsular polymer around cells and large numbers of outer membrane vesicles (OMVs, see arrows).
  • OMVs outer membrane vesicles
  • Example 14 In vivo tests of elasticity and dermoprotective properties in humans
  • a Tewameter® TM 300 device (Courage & Khazaka) was used to measure the TEWL. Volunteers treated with 1 % of EPS showed a lower percentage of TEWL (154.28%) after the application of the irritant agent SLS compared to those treated with the placebo (181 .83%; FIG 1 1 ). This demonstrates that the EPS is a dermo-protective agent that strengthens the skin against external aggressions.
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Abstract

La présente invention concerne Pseudomonas sp. CECT8437 qui est une bactérie adaptée au froid isolée à partir d'un échantillon de sédiment marin collecté sur l'île de la Déception (îles Shetland du Sud, Antarctique) qui se caractérise par l'aspect hautement muqueux de ses colonies. Un exopolysaccharide (EPS) est produit par cette souche, qui comprend du glucose, du galactose, du fucose et de l'acide uronique selon un rapport molaire d'environ 2:1:1:0,3. L'EPS présente les pourcentages en poids suivants, d'environ : 37,29 % de C, 6,17 % de H, 2,25 % de N, et 0,41 % de S. Sa masse moléculaire (MM) est supérieure à 2 x 106 Da. L'EPS est utile aux fins suivantes : (i) agent cryoprotecteur; (ii) agent émulsifiant; (iii) agent épaississant, stabilisant ou structurant; (iv) agent dermoprotecteur; et (v) agent permettant d'augmenter l'élasticité de la peau. L'EPS peut être utilisé dans des compositions cosmétiques.
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EP3295927A1 (fr) 2016-09-19 2018-03-21 Institut Univ. de Ciència i Tecnologia, S.A. Usages d'un complexe de protéine exopolysaccharide obtenu à partir d'une bactérie
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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3295926A1 (fr) 2016-09-19 2018-03-21 Institut Univ. de Ciència i Tecnologia, S.A. Complexe protéine exopolysaccharide, procédé de préparation ledit complexe et ses utilisations
EP3295927A1 (fr) 2016-09-19 2018-03-21 Institut Univ. de Ciència i Tecnologia, S.A. Usages d'un complexe de protéine exopolysaccharide obtenu à partir d'une bactérie
WO2018050912A2 (fr) 2016-09-19 2018-03-22 Institut Univ. De Ciència I Tecnologia, S.A. Utilisations d'un complexe exopolysaccharide-protéine obtenu à partir d'une bactérie
WO2019029833A1 (fr) 2016-09-19 2019-02-14 Institut Univ. De Ciència I Tecnologia, S.A. Complexe exopolysaccharide-protéine, son procédé de préparation et ses utilisations
EP3401389A1 (fr) * 2017-05-11 2018-11-14 AlteroBiotech Inc. Agent cryoprotecteur contenant de l'exopolysaccharide à partir de pseudoalteromonas sp. cy01
FR3120308A1 (fr) * 2021-03-02 2022-09-09 Universite De Nantes Utilisation d’exopolysaccharides de micro-algues a titre d’agents texturants
WO2022184745A1 (fr) * 2021-03-02 2022-09-09 Nantes Université Utilisation d'exopolysaccharides de micro-algues a titre d'agents texturants

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