WO2013121002A1

WO2013121002A1 - Microorganism-free extract from hypersaline aqueous environments and process for its preparation

Info

Publication number: WO2013121002A1
Application number: PCT/EP2013/053088
Authority: WO
Inventors: Caterina GÓMEZ CLAPERS; Josep FERRE ALEMANY; Jaume MIR MARTÍNEZ; Àngel LÁZARO AMORÓS; Àngela ROCA ALONSO; Anna UBACH FONT; Kristin BAUMANN
Original assignee: Arquebio, S. L.
Priority date: 2012-02-16
Filing date: 2013-02-15
Publication date: 2013-08-22

Abstract

It is provided an essentially microorganism-free extract from hypersaline aqueous environments, the extract comprising a total content of sugars from to 50% by weight based on the total weight of the dry extract, a total content of protein from 10 to 50% by weight based on the total weight of the dry extract, and a glycoprotein content from 10 to 30% by weight based on the total weight of proteins present in the dry extract, the extract having a conductivity value equal to or lower than 5 mS/cm at 20ºC. It is also provided a process for obtaining it, as well as their uses, and compositions comprising it.

Description

Microorganism-free extract from hvpersaline aqueous environments and process for its preparation

The present invention relates to an extract from hypersaline aqueous environments. The invention also relates to a process for obtaining the mentioned extract, to its use as moisturizing agent, as a blocking agent for sun protection against UV radiation, or as chelating agent, and to

compositions containing the extract. BACKGROUND ART

On the search for new active ingredients for cosmetic formulations, specifically to improve the skin health or appearance, not only plant and marine products have been studied lately but also inactivated microorganisms with their metabolic products and extracts.

Extremophiles are microorganisms that thrive in harsh condition

environments, like high or low temperatures, extreme pH, high pressures or high salinities. To overcome harsh environmental conditions, these

microorganisms have developed various strategies, like the accumulation of protective compounds into the cytoplasm, the secretion of molecules to the environment to protect the cell or the more specific generation of membrane channels to regulate the inner and outer pressure. Thus, microorganisms living in extreme environments are interesting for cosmetic industries due to the production of specific molecules for their protection (i.e. antarcticine, EP1402898; and ectoine, US6060071 ) that also help to protect skin. One interesting class of extremophiles is halophiles/halotolerant

microorganisms. These microorganisms live in ecosystems characterized by very high concentrations of salt, some times five times greater than the salt concentration of the ocean, such as the Dead Sea, the Great Salt Lake, Owens Lake or in evaporation ponds. Other physical characteristics related with the high salted environment are high temperatures, dryness and also high UV-radiation. The use of halophilic or halo-tolerant microorganisms or products extracted from the biomass of these microorganisms in cosmetic formulations has been described in several documents (e.g. WO2004103332, FR2590273, and US6849279). It must be noted that these documents disclose methods for obtaining compounds from a microorganism in which the first step is the recovery of the biomass from a natural environment or a culture growth, with the consequent discard of the liquid fraction.

Some examples of extracellular polymeric substances (EPS) isolated from the liquid phase of a high saline culture growth have been also described:

Raguenes et al. (2004), Anton et. al. (1988), Mishra et al. (2009). However the experiments of such examples were done in laboratory conditions and under artificial means induced by man. In other cases, it is known that some natural environments such as crystallizer ponds contain microorganisms that produce EPS, but details for a procedure to isolate them and the exact composition of the extracts that may be obtained is not disclosed: e.g. Oren (2010).

The composition of the extracts from high saline media described in reports of the prior art demonstrate that they can be diverse. Thus, Raguenes et al. (2004) described two EPS with different polysaccharide and protein

composition, being the content of this latter component not greater than 5% for the EPS obtained from the liquid phase. In both cases, however, the percentage of glycoprotein is not disclosed. In another example, Anton et al. (1988) reported an EPS having hexose as the major component, but the glycoprotein content in the extract is not defined. An assumption is made based on the presence of uronic acids which usually constitute the glucidic part of such glycoprotein. However, the characteristics of the polymer described in the results section of the original report indicate that The UV absorption spectrum of a 10 mg/mL solution did not absorb strongly at wavelengths specific for proteins'. Other examples can be found where only polysaccharides are obtained: e.g. Mishra et al. (2009).

With regard to the procedures to isolate and purify EPS from microorganisms from the liquid phase of the growth media, several literature examples describe operations that appear to be standard practice such as those indicated in Gutierrez et al. (2007) and other citations already mentioned above. It is generally known that the production and treatment of samples from microbial origin in high salt concentration and viscous media offer some difficulties. In the references given, the EPS were obtained through laboratory experiments which involved small scale operations and sometimes the use of solvents (e.g. ethanol) to induce the precipitation of the targeted substances, This latter option is representing an additional cost that may prevent the industrial implementation. Hence the potential application of these operations for a high scale and cost effective extractive process, such as the treatment of natural hypersaline aqueous environments from solar seawater salterns is not demonstrated.

In spite the teachings of the prior art, products comprising or derived from inactivated microorganisms, as well as metabolic products or extracts of thereof goes on being an active field of research, in the aim of providing new products with improved properties.

SUMMARY OF THE INVENTION

The inventors have surprisingly found that a microorganism-free extract obtained from hypersaline aqueous environments in solar salterns containing halotolerant/halophilic microorganisms, have advantageous cosmetic properties such as excellent moisturizing properties, as well as a good protecting effect against the harmful action of ultraviolet radiation and chelating properties.

Thus, a first aspect of the invention is the provision of an essentially microorganism-free extract from hypersaline aqueous environments, the extract comprising a total content of sugars from 10 to 50% by weight based on the total weight of the dry extract, a total content of protein from 10 to 50% by weight based on the total weight of the dry extract, and a glycoprotein content from 10 to 30% by weight based on the total weight of proteins present in the dry extract, the extract having a conductivity value equal to or lower than 5 mS/cm at 20°C. The presence of the mentioned glycoprotein fraction in the composition of the extract is a key feature compared to previous compounds obtained from other high saline media. Particularly, as it is shown in the examples, the cell-free extract of the invention shows a better moisturizing capacity compared with a

microorganism extract obtained from the same hypersaline aqueous environment.

The inventors have also found an easy and cost-effective process that allows to eliminate salts and microorganisms present in hypersaline aqueous environments in order to recover the mentioned extract with improved properties. It is considered a contribution to the art as up to now interesting molecules from salty environments were really difficult to isolate, basically due to the high salt concentration in the water and its complications with viscosity.

The process for obtaining the extract is based on the recovery of compounds from hypersaline aqueous environments after the removal of microorganisms and salts.

Accordingly, in a second aspect the invention relates to a process for the preparation of the microorganism-free extract of the invention, the process comprising the following steps:

a) collection of a liquid portion of a hypersaline aqueous environment with the following characteristics:

- conductivity superior to 10OmS/cm at 20°C,

- dry weight from 5 to 350 mg/mL,

- presence of microorganisms, and

- a total concentration of carotenoids equal to or higher than 0.3 μΜ,

b) removal of the particulate fraction from the liquid portion in order to obtain a liquid fraction, and

c) filtration and concentration of the liquid fraction in order to eliminate molecules below 50-150kDa and to reduce the salt content until the conductivity is equal to or lower than 5 mS/cm at 20°C,

to yield the extract in form of aqueous solution. Advantageously, as mentioned above, the process of the invention results in an essentially microorganism free extract with cosmetic and pharmaceutical applicability in a cost-effective manner.

The extract of the invention can also be defined by its preparation process. Thus, the essentially microorganism-free extract obtainable by the process of the invention as disclosed above is also considered part of the invention.

A third aspect of the present invention relates to the use of an effective amount of an extract as defined above as a skin and/or hair moisturizing agent. Such a moisturizing effect is understood as the effect of increasing or stabilizing the moisture content of a person's skin and/or hair. This aspect of the invention can also be formulated as a method, in particular a cosmetic method, for moisturizing the hair and/or skin, comprising administering an effective amount of an extract as defined above together with acceptable carriers or excipients appropriate for topical application, to the skin and/or hair of a subject in need of such a moisturizing effect.

Beside this, by the application of the extract as defined above on the skin and/or hair a protection against UV radiation is also achieved. Such a protection against UV radiation is understood as a shield around the skin and/or hair that helps decreasing the permeation of UV radiation and consequently ameliorates the appearance of the skin and/or hair, specifically the properties of the skin and/or hair. Accordingly, a forth aspect of the present invention relates to the cosmetic use of an effective amount of an extract as defined above as UV blocking agent against UV radiation of skin and/or hair. This aspect of the invention can also be formulated as a method, in particular a cosmetic method, for protecting the skin and/or hair against UV radiation, comprising administering an effective amount of an extract as defined above, together with cosmetically acceptable carriers or excipients appropriate for topical application, to the skin and/or hair of a subject in need of such a protecting effect.

It is also considered as part of the invention an extract as defined above for use in the protection of the hair and/or skin against the cumulative detrimental effect of UV radiation, i.e. against the aging process of the skin or the damage or harm of the hair produced by UV radiation.

A further aspect of the invention relates to the use of an effective amount of an extract as defined above as a chelating agent. This aspect of the invention can also be formulated as a method for quelating substrates, the method comprising putting into contact the extract as defined above with a substance susceptible of forming attractive interactions with the extract.

According to a another aspect, the present invention provides a composition comprising an effective amount of at least one extract as defined above, together with acceptable excipients or carriers appropriate for topical application to a person's skin and/or hair.

An important aspect of the invention is that the individual properties found in the extract and disclosed above (moisturizing, chelating, UV-protective) are given at the same time in a product, with the composition described, which was produced by natural means in hypersaline aqueous environments and obtained by an extractive cost-effective and scalable process from the liquid phase.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 represents the dynamic vapor sorption (DVS) profile of an extract from a saltern crystallizer pond from Delta de I'Ebre (Spain) located at 40° 35' 15 and 0° 41 ' 07 (upper line) compared to the DVS profile of hyaluronic acid (lower line). Discontinued lines represent relative humidity steps in the range from 5% to 95%.

Fig. 2 represents the dynamic vapor sorption (DVS) profile of an extract from a saltern crystallizer pond from Alicante (Spain) located at 37° 59' 00 and 0° 42' 14 (upper line) compared to the DVS profile of hyaluronic acid (lower line). Discontinued lines represent relative humidity steps in the range from 5% to 95%.

Fig. 3 represents the dynamic vapor sorption (DVS) profile of an extract from a saltern crystallizer pond from Delta de I'Ebre (Spain) (upper line) compared to the DVS profile of a microorganisms pellet from the same pond (lower line). Discontinued lines represent relative humidity steps in the range from 5% to 95%. Fig. 4 represents the dynamic vapor sorption (DVS) profile of an extract from a saltern crystallizer pond from Alicante (Spain) (upper line) compared to the DVS profile of a microorganisms pellet from the same pond (lower line).

Discontinued lines represent relative humidity steps in the range from 5% to 95%.

Fig. 5 is a UV-visible spectrum of an extract from a saltern crystallizer pond from Alicante (Spain) (upper line) compared to hyaluronic acid (medium line). Distillated water was used as reference (lower line).

Fig. 6 represents percentage uptake of several metal ions (Fe²⁺, Zn²⁺, and Ni²⁺) ions by an extract from a saltern crystallizer pond from Alicante (Spain) after 24 hours compared with distilled water. DETAILED DESCRIPTION OF THE INVENTION

For the purposes of the present disclosure, the term "hypersaline aqueous environments" will be used to refer to hypersaline aqueous media found in crystallizer ponds from solar seawater salterns, characterized by a sodium chloride composition of at least 35% (w/v) and microbial communities with at least 70% hyperhalophilic square archaeon Haloquadratum walsbyi.

For the purposes of the present disclosure, the term "effective amount" means an amount that is sufficient to obtain the expected effect.

For the purpose of the invention, the term "cosmetic" is intended to denote a use intended, principally, to provide and aesthetic and/or comfort effect, in particular, to ameliorate the appearance of the skin and/or hair, specifically the properties of the skin and/or hair.

For the purpose of the invention, the term "cosmetically acceptable excipients or carriers" refers to that excipients or carriers suitable for use in contact with human skin without undue toxicity, incompatibility, instability, allergic response, among others.

For the purposes of the present disclosure, the term "cosmetic use" intends to exclude any use having a therapeutic effect. For the purposes of the present disclosure, the terms "essentially free of microorganisms" or "essentially microorganism-free" are understood to mean having a content of microorganisms equal to or lower than 100 colony forming units (cfu) per g of dry extract.

Conductivity of the extract solution (i.e. the extract of the invention before being submitted to a drying process) is used as a measure of the content of salt. The control of this parameter during the process for preparing the extract of the invention, allows getting a final product with a conductivity equal to or lower than 5 mS/cm at 20°C, particularly with a conductivity equal to or lower than 2 mS/cm at 20°C, and more particularly with a conductivity from 0.1 to 1 mS/cm at 20°C. In order to have an indication of the content of salts of the dry extract, a solution can be reconstituted (1 -10 mg/ml in distilled water) in order to be able to measure the conductivity, nevertheless the control of this parameter during the process assures the absence of salts in the final product before drying, and so also in the dry extract.

For the purposes of the present disclosure, the term "cosmetically acceptable medium" is understood to mean a physiologically acceptable medium, that is to say a medium compatible with the skin and which does not produce feelings of discomfort (redness, tightness, tingling, and the like) after application to the skin which are unacceptable to the user. As an example, the cosmetically acceptable medium can be an aqueous medium which consists exclusively of water or of a mixture of water and a cosmetically acceptable solvent, such as monoalcohols, polyalcohols, oils or glycol ethers, which can be used alone or as a mixture.

All the percentages mentioned herein are in weight, unless otherwise stated.

As mentioned above, the extract of the invention comprises a total content of sugars from 10 to 50% by weight based on the total weight of the dry extract, a total content of protein from 10 to 50% by weight based on the total weight of the dry extract, and a glycoprotein content from 10 to 30% by weight based on the total weight of proteins present in the dry extract, the extract having a conductivity value equal to or lower than 5 mS/cm at 20°C. In an embodiment, the extract of the invention comprises a total content of sugars from 20 to 30% by weight based on the total weight of the dry extract, a total content of protein from 30 to 40% by weight based on the total weight of the dry extract, and a glycoprotein content from 20 to 30% by weight based on the total weight of proteins present in the dry extract. The content of sugars or proteins, as well as of including glycoproteins, is determined by conventional methods known to those skilled in the art, such as the ones described in the examples. As mentioned above, the microorganism-free extract of the invention is obtained from hypersaline aqueous environments containing

halotolerant/halophilic microorganisms. The hypersaline aqueous

environment has the following characteristics:

- Conductivity superior to 100 mS/cm at 20°C;

- Dry weight from 5 to 350 mg/mL, more particularly from 10 to 50 mg/mL.

- Presence of microorganisms, particularly of bacillus, coccus and square microorganisms (being Haloquadratum walbsbyi at least 70% of them); and

- Total concentration of carotenoids equal of higher than 0.3 μΜ.

Conductivity was measured with a conductimeter at a temperature of 20°C.

Dry weight was measured by first filtering 10mL of a liquid sample through a 0.45 μιτι diameter porous glass fiber filter pre-weighed. Then, the filter was heated for 24h at 100°C and the weight difference was taken as the dry weight measure.

The presence of microorganisms was determined by optic microscopy using stained slides.

Carotenoids were determined by centrifuging 50ml_ of a sample of the hypersaline aqueous environment at 15000xg for 15min and then mixing the pellet with a methanol: chloroform (2:1 , V/V) solution. The concentration of carotenoids was obtained measuring the absorbance at 490nm of the chlorophorm phase using pure chloroform as a blank. The pH of hypersaline aqueous environments can vary depending on their particular chemical composition, particularly on their salt composition. This parameter is not critical for the purpose of the invention. As an instance, the pH of hypersaline aqueous environments can be from 6.5 to 1 1 .5, particularly from 6.9 to 7.6.

In order to obtain the extract of the invention, first a liquid fraction of a saline pond with the above mentioned characteristics is collected. The sample can be stored in a tank/bag.

Then, the particulate fraction is removed from the liquid portion in order to obtain a liquid fraction. Particularly, the removal of the particulate fraction is carried out by centrifugation. This step can be carried out at a laboratory scale by centrifuging the sample in batch (discontinuous) at a centrifuge speed from 12000xg to 18000xg for 15 to 40 min. As an instance, this step can be carried out at 15800xg (12000 rpm) for 20 min. Alternatively, removal of the particulate fraction can be achieved in a continuous process by using a continuous centrifuge (e.g. disk stack, tubular bowl) or a filtration process with a 0,2-0.45 μιτι, preferably a 0,45 μιτι, diameter porous filter (i.e. a porous filter with a pore size equal to the specified values in diameter, wherein the pore size also indicates the diameter of the smallest particle retained during filtration).

Subsequently, a filtration and concentration process is carried out in order to eliminate molecules below 50-150kDa (small proteins, DNA and salts), particularly below 100kDa. Such a filtration/concentration process can be carried out by conventional methods known to those skilled in the art, such as by diafiltration or ultrafiltration. The filtration/concentration process can be carried out by using a hollow fiber with a particle cutoff of 50-150kDa, particularly of 100kDa. The term "particle cutoff' referred hereinbefore and hereinafter is intended to means the particle size of particles (expressed in kDa) with which the hollow fiber membrane can exhibit a blocking rate of 80% or higher, particularly of 90% or higher, or more particularly of 95% or higher. In a particular embodiment, the filtration/concentration process is carried out by diafiltration.

During the filtration process the sample volume is concentrated and the salt content is reduced until the conductivity is equal to or lower than 5 mS/cm at 20°C. As mentioned above, the control of the conductivity during the process allows getting a final extract with the required conductivity. Preferably, the salt content is reduced until the conductivity is equal to or lower than 2 mS/cm at 20°C, more preferably until a value from 0.1 to 1 mS/cm at 20°C. Accordingly, in a preferred embodiment, the extract of the invention is free of salts, namely their conductivity is reduced until a value equal to or lower than 2 mS/cm at 20°C, preferably until a value from 0.1 to 1 mS/cm at 20°C. Once the salt content and the viscosity of the sample have been reduced, optionally, an additional filtration step can be carried out using a 0.2-0.45 μιτι, preferably a 0.45 μιτι, diameter porous filter in order to assure a total elimination of microorganisms in the final step. Accordingly, in a preferred embodiment the process of the invention further comprises and additional step d) of filtration using a 0.2-0.45μηη, preferably a 0.45 μιτι, diameter porous filter.

The extract obtainable by this process, either including or not the optional step mentioned above, is also considered part of the invention.

Accordingly, in another preferred embodiment, the extract of the invention is free of microorganisms. Advantageously, the extract of the invention is a cleaner extract (without DNA or microorganisms) than microorganism preparations obtained from hypersaline aqueous environments known in the prior art.

Also optionally, if required, finally the extract can undergo a drying step yielding a final hygroscopic, water soluble, amorphous, light pink colored powder. Advantageously, the dry extract is a stable and easy to handle product. Accordingly, in another preferred embodiment, the process of the invention further comprises submitting the extract of either step c) or step d) to an additional step e) of drying.

The dry extract obtainable by this process is also considered part of the invention. So, in a preferred embodiment, the extract of the invention is a dry extract. The drying step can be carried out by conventional methods known to those skilled in the art, such as by lyophilization, by spray-drying or even in an oven to eliminate the water. As an instance, the lyophilization step can be carried out freezing the extract at -80°C overnight before being introduced into the lyophilizer. During this step the sample thickness is preferably set at a value lower than 1 cm. Within these conditions, 24-48 hours are enough to dry all the frozen liquid extract.

All the embodiments of the process of the invention contemplate all the combinations providing all the embodiments of the extract of the invention and combinations thereof.

As mentioned above, the extract of the invention can be used as a skin and/or hair moisturizing agent, namely it is useful for increasing and/or stabilizing the moisture content of the skin and/or hair. Also, it can be used as a UV blocking agent for sun protection against UV radiation of skin and/or hair. Preferably, the use of the extract of the invention is a cosmetic use, and so the extract of the invention is used in cosmetic compositions. Due to the mentioned properties, the extract of the invention can also be useful as an acceptable excipient or carrier in pharmaceutical compositions for topical use.

Accordingly, in one embodiment, the composition of the invention is a cosmetic composition. By the application of the cosmetic composition of the invention an increasing or stabilizing effect on the moisture content of the skin and/or hair is achieved ameliorating their appearance and mechanical properties, which imparts to these compositions a high practical value from the cosmetic viewpoint. This effect is strengthened by the additional protection against UV radiation provided by the extract of the invention when acting as a UV blocking agent.

The cosmetic composition of the invention comprises one or more

cosmetically acceptable excipients or carriers, wherein at least one of the excipients is an extract of the invention. Accordingly, the cosmetic

compositions of the invention may contain, in addition to the extract of the invention, water and additives customarily used in cosmetics. These additives are, for example, thickening agents, perfumes, preservatives, antioxidants, stabilizers, emulsifiers, vegetable or mineral oils, antiseptic agents, acidifying or alkalinizing agents, vitamins, anti-UV agents, surfactants, solvents, pH- stabilizing agents, silicones and the like. The compositions in accordance with the invention may be provided in the form of a solution, suspension, emulsion, paste, ointment, gel, cream, lotion, serum, mask, powder, soap, surfactant-containing cleanser, oil and spray. Other characteristic use forms in cosmetology are lipsticks, lipsalve sticks, mascara, eyeliner, blusher, powder foundation, emulsion foundation and wax foundation, and also suncare and after-sun preparations.

The active concentration of the extract of the invention in the cosmetic composition according to the invention amounts from 0.05 to 5% by weight, preferably 0.1 to 2% by weight, based on the total weight of the composition.

In another embodiment, the composition of the invention is a pharmaceutical composition comprising at least one pharmaceutically active ingredient and one or more pharmaceutically acceptable excipients, wherein at least one of the excipients is an extract as defined above.

Additionally, the extract of the invention can be used as a chelating agent. As it is shown in Example 6, the extract of the invention has the ability of chelating some ions. In particular, it has shown this property with niquel, zinc and iron, in order of decreasing uptake. As such, it can be useful in producing nutritional supplements, fertilizers, in chemical analysis, as water softeners, commercial products such as shampoos and food preservatives, medicine, heavy metal detox, and other industrial applications. In particular, the extract of the invention can be useful to trap or remove non desirable metal ions, such as heavy metal ions or other toxic ions.

Some chemical species, such as ferrous iron (Fe²⁺) can facilitate the production of ROS (reactive oxygen species) within animal and human systems, and thus, the ability of substances to chelate iron can be valuable for antioxidant properties. Therefore minimizing ferrous ion may afford protection against oxidative damage by inhibiting production of ROS and molecular damage [Giilgin, I., "Antioxidant activity of food constituents: an overview", 20††, Arch. Toxicol, DOI 10.1007/s00204-01 1 -0774-2]. Others, such as Ni²⁺ are well known ions by its toxicity and capacity of generating oxidative damage in different tissues [Das, K.K., et al., "Nickel, its adverse effects & oxidative stress", Indian J. Med. Res., 2008, Vol. 128, pp 412-425]. Thus, by virtue of its chelating property, the extract of the invention can also have some antioxidant properties. Accordingly, in an embodiment the invention provides the use of the extract of the invention as an antioxidant, or to uptake and remove toxic ions. This embodiment can also be formulated as a method for use of the extract of the invention as an antioxidant, or to uptake and remove toxic ions.

Another practical application of the extract of the invention in view of its chelating property can be its use as a carrier for topically administered compounds not intended to penetrate skin. Accordingly, in an embodiment the use of the extract as defined above is as a carrier for topically administered compounds. This embodiment can also be formulated as a method for chelating topically administered compounds not intended to penetrate skin, the method comprising adding an effective amount of an extract as defined above to a composition comprising the compounds being to be topically administered.

In a particular example, the extract of the invention can be used in

pharmaceutical compositions as a carrier for compounds useful in the treatment of several skin diseases, such as atopic dermatitis. This application is especially interesting as it combines the chelating property of the extract together with the moisturizing property and the effect of blocking agent for sun protection against UV radiation of the skin and/or hair.

Throughout the description and claims the term "comprise" and variations of the term, such as "comprising", are not intended to exclude other technical features, additives, components, or steps. The term includes the expression "consist of and variations thereof. Additional objects, advantages and features of the invention will become apparent to those skilled in the art upon examination of the description or may be learned by practice of the invention. The following examples are provided by way of illustration, and they are not intended to be limiting of the present invention. Furthermore, the present invention covers all possible combinations of particular and preferred embodiments described herein. EXAMPLES

EXAMPLE 1 . Obtaining of an extract derived from the microorganism free fraction of a saltern crystallizer pond from Delta de I'Ebre (Spain)

A 500 mL sample was collected from a Delta de I'Ebre saline pond (Spain) located at 40° 35' 15 and 0° 41 ' 07. The sample had the following

characteristics:

- pH: 7.00

- Conductivity: 100.3 mS/cm at 20°C

- Dry weight: 25 mg/mL

- Present microorganisms: bacillus, coccus and square microorganisms

- Total concentration of carotenoids: 1 μΜ.

The sample was centrifuged at 15800xg (12000 rpm) for 20 min using a Sorvall ST-16 centrifuge with a FIBERL/^'te™ F15-6X 100 rotor. The pellet (microorganisms) was discarded and the supernatant was diafiltrated and concentrated using a 100 kDa polysulfone Hollow Fiber. Eight volumes of deionized water were required for the elimination of salts of the sample, which resulted in a final conductivity of about 3 mS/cm at 20°C. An additional filtration step using a 0.45 μιτι diameter porous filter was carried out to assure the total elimination of microorganisms from the extract. Finally, the extract was lyophilized to yield 254 mg of a hygroscopic, water soluble, amorphous, light pink colored powder.

The protein and sugar composition was evaluated dissolving the dry extract in distilled water to make 1 mg/mL solution. In this solution, the total protein and glycoprotein concentration, as well as the total sugar content were measured.

Results, shown in Table 1 , are expressed as percentage of protein and sugar present in the dry extract.

Table 1

% content (w/w) final extract

Total protein 1 1

Total sugar 13 Glycoprotein content is approximately 13% of the total amount of protein present in the extract. The total protein content of the extract was quantified by the Bradford Method as described in Bradford M.M. "A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle protein- dye binding", Anal. Biochem. 1976, Vol. 72, pp. 248-254. Mucin type VII was used as reference.

The glycoprotein content of the extract was determined by the Periodic Acid Shift Assay as described in Mantle M. "A colorimetric assay for glycoprotein based on the periodic acid/Schiff stain", Biochemical Society transactions, 1978, Vol. 6, pp. 607-609. Mucin type VII was used as reference.

The content of neutral sugars was determined by the phenol/sulphuric assay as described in "Current Protocols in Food Analytical Chemistry" 2001 , E1 .1 .1 -E1 .1 .8. Glucose was used as reference.

EXAMPLE 2. Obtaining of an extract derived from the microorganism free fraction of a saltern crystallizer pond from Alicante (Spain)

An 830 ml_ sample was collected from Torrevieja saline pond in Alicante (Spain) located at 37° 59' 00 and 0° 42' 14. The sample had the following characteristics:

- pH: 7.31

- Conductivity: 108.4 mS/cm at 20°C

- Dry weight: 14 mg/mL

- Present microorganisms: bacillus, coccus and square microorganisms

- Total concentration of carotenoids: 0.3 μΜ.

The sample was centrifuged at 15800xg (12000 rpm) for 20min using a Sorvall ST-16 centrifuge with a FIBERL/^'te™ F15-6X 100 rotor. The pellet (microorganisms) was discarded and the supernatant was diafiltrated and concentrated using a 100kDa polysulfone Hollow Fiber. Ten volumes of deionized water were required for the elimination of salts of the sample, which resulted in a final conductivity of about 1 mS/cm at 20°C. An additional filtration step using a 0.45μηη diameter porous filter was added to assure the total elimination of microorganisms from the extract. Finally, the extract was lyophilized to yield 130 mg of a hygroscopic, water soluble, amorphous, light pink colored powder.

The protein and sugar composition was evaluated dissolving the dry extract in distilled water to make 1 mg/mL solution. Protein and glycoprotein

concentration was measured in this solution, as well as the total sugar content. Methods used were Bradford and Periodic Acid Shift reaction for the total protein and glycoprotein content, respectively, and phenol/sulphuric assay for sugars, as mentioned in example 1 .

Results, expressed as the percentage of protein and sugar present in the dry extract, are shown in Table 2.

Table 2

% content (w/w) final extract

Total protein 38

Total sugar 40

Glycoprotein content is approximately 26% of the total amount of protein present in the extract.

EXAMPLE 3. Moisturizing properties

In order to know whether the extract had moisturizing properties, DVS

(dynamic vapor sorption) studies were developed. This technique allows measuring, through gravimetric changes with a water sorption analyzer (Q5000SA Sorption Analyzer, TA Instruments), the kinetics of solvent (water) absorbed by a sample while varying the environment relative humidity. The weight variation of a 10 mg dry extract of the invention or hyaluronic acid (cosmetic grade; MW: 1 .2x10⁶ Da) was monitored in the range of a relative humidity between 5% and 95% by sampling water adsorption isotherms at 25 °C. Prior to the measurements, the samples were equilibrated at 30 °C and 0% relative humidity until the sample weight remained constant.

The water retention profiles of extracts of the invention from different ponds were compared to a control (hyaluronic acid). The extracts were obtained as disclosed in Examples 1 and 2 from the following ponds:

(A) saltern crystallizer pond from Delta de I'Ebre (Spain),

(B) saltern crystallizer pond from Alicante (Spain))

As it shown in Figures 1 (for pond A) and 2 (for pond B), both extracts present an increased DVS profile compared to hyaluronic acid, retaining more water at a relative humidity between 65 and 95%.

The higher retention of water shown by the extracts either of pond A or of pond B compared to hyaluronic acid is indicative of the higher moisturizing properties of the extract of the invention.

EXAMPLE 4. Comparison between the extract of the invention and a lysed microorganism extract from different ponds Initially discarded pellet (microorganisms) samples from Example 1 (pond A) and Example 2 (pond B) were lysed in distillated water and recovered through centrifugation (same conditions as in Example 1 ). Salts were removed by dialysis or diafiltration with more than 8 volumes of distillated water. DVS assay for the microorganism preparation was carried out as in Example 3.

The results depicted in Figures 3 and 4 show that the extract of the invention (obtained as in Examples 1 and 2, respectively) has a better DVS profile than a microorganism preparation (a common preparation used in cosmetic industry) from the same pond. Based on these results, it can be stated that the extract of the invention has unexpectedly high moisturizing properties.

As mentioned in Example 2, from a 830 mL sample from pond (B), 130 mg of liofilized extract were obtained. However for the microorganism pellet, from a 1000 mL sample of the same pond the final microorganism pellet only yield 16 mg. This fact is indicative of the higher efficiency of the process for obtaining the extract compared to the process for obtaining the microorganism pellet. Additionally, the extract of the invention the extract of the invention is a cleaner extract (without DNA or microorganisms).

EXAMPLE 5. Protection against UV radiation

Hyaluronic acid is a natural molecule present in the skin that is considered as a natural UV filter because it minimizes effects of UV irradiation such as generation of ROS and other responses [Averbeck, M, et. al. "Differential Regulation of Hyaluronan Metabolism in the Epidermal and Dermal

Compartments of Human Skin by UVB Irradiation", Journal of Investigative Dermatology, 2006, Vol. 127, pp. 687-697]. The ability of the extract to act as blocking agent for sun protection against UV radiation was assessed by comparison with hyaluronic acid. Thus, to prove a possible UV effect protection of the extract of the invention, a spectrophotometric scan was performed from 200 to 800 nm.

Starting from the lyophilized extract from a crystallizer pond from Alicante (Spain), obtained as in Example 2, a 0.1 % (w/v) extract aqueous solution was prepared. Similarly, a hyaluronic acid aqueous solution with the same concentration was also prepared. Distillated water was used as a reference.

UV-visible spectrum of the extract compared to hyaluronic acid (Fig. 5) shows that the extract of the invention presents a higher absorption in the UV range of 280-300 nm than hyaluronic acid. This result is indicative of the capacity of the extract of the invention to block UV radiation in the range tested.

EXAMPLE 6. Metal adsorption capability by the extract (chelating properties) To check the chelating properties of the extract, some metallic ions were tested, particularly Ni²⁺, Zn²⁺, and Fe²⁺. The metal salts used (0.1 mM) were: nickel sulfate, zinc sulfate, and ferric sulfate, and the buffer solution was MES (2-(/V-morpholino) ethanesulfonic acid) at pH 4.0. Starting from the lyophilized extract from a crystallizer pond from Alicante (Spain), obtained as in Example 2, a 1 % (w/v) extract aqueous solution was prepared. Metal-binding analyses were made by placing the mentioned extract solution (1 ml_) in a dialysis tube and this in a flask containing 200ml_ of each appropriate metal-salt buffered solution. The solutions were shaken in an orbital shaker (100 rpm) at 37°C for 24h. The extract ion uptake from the buffered solution was calculated by measuring the ions in the solution at time zero and those remaining after 24h by inductively coupled plasma optical emission spectrometry (Perkin-Elmer, Optima 4300DV). Controls were made by placing distilled water (1 ml_) in a dialysis tube with the various metal-salt solutions. Samples were diluted 1 :10 prior its analysis. The results depicted in Figure 6, representing the percentage uptake of several metal ions (Ni²⁺, Zn²⁺, and Fe²⁺) ions by the extract of the invention after 24 hours compared with distilled water, are indicative of the marked chelating ability of the extract of the invention. EXAMPLE 7. COSMETIC PREPARATION (CREAM)

This example illustrates a cream comprising the extract of Example 2:

"csp" means adding a quantity sufficient to achieve the desired percentage (i.e. 100%).

Several compositions can be prepared as the cream above but comprising a 2, 3, 4 or 5% w/w of the extract of either Example 1 or Example 2. EXAMPLE 8. COSMETIC PREPARATION (LOTION)

This example illustrates a lotion comprising the extract of Example 2:

COMPONENTS % w/w

Cosmetic oil 35 Lactic acid 0,5

Citric acid 4

Extract 1

Water csp 100

Preservative 1

"csp" means adding a quantity sufficient to achieve the desired percentage (i.e. 100%). Several compositions can be prepared as the lotion above but comprising a 2, 3, 4 or 5% w/w of the extract of either Example 1 or Example 2.

REFERENCES CITED IN THE APPLICATION 1 . EP1402898

2. US6060071

3. WO2004103332

4. FR2590273

5. US6849279

6. Raguenes G. et al. "A Novel Exopolymer-Producing Bacterium,

Paracoccus zeaxanthinifaciens subsp. payriae, Isolated from a Kopara Mat Located in Rangiroa, an Atoll of Freeh Polynesia", Current

Microbiology 2004, Vol. 49, pp. 145-154.

7. Anton J. et. al. "Production of an Extracellular Polysaccharide by

Haloferax mediterranei' , Applied and Environmental Microbiology 1988,

Vol. 54, pp. 2381 -2386.

8. Mishra A. et al., "Isolation and characterization of extracellular polymeric substances from micro-algae Dunaliella salina under salt stress",

Bioresource Technology 2009, Vol. 100, pp. 3382-3386.

9. Oren A. "Thoughts of the 'missing link' between saltworks biology and solar salt quality", Global Nest Journal 2010, Vol. 12, pp.417-425.

10. Gutierrez T. et al. "Glycoprotein emulsifiers from two marine Halomonas species: chemical and physical characterization", Journal of Applied Microbiology 2007, Vol. 103, pp. 1716-1727.

1 1 . Bradford M.M. "A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle protein-dye binding", Anal. Biochem. 1976, Vol. 72, pp. 248-254. 12. Mantle M. "A colorimetric assay for glycoprotein based on the periodic acid/Sch iff stain", Biochemical Society transactions, 1978, Vol. 6, pp. 607-609.

13. "Current Protocols in Food Analytical Chemistry" 2001 , E1 .1 .1 -E1 .1 .8. 14. Giilgin, I., "Antioxidant activity of food constituents: an overview", 201 1 ,

Arch. Toxicol, DOI 10.1007/s00204-01 1 -0774-2.

15. Das, K.K., et ai, "Nickel, its adverse effects & oxidative stress", Indian J.

Med. Res., 2008, Vol. 128, pp 412-425

16. Averbeck, M, et. al. "Differential Regulation of Hyaluronan Metabolism in the Epidermal and Dermal Compartments of Human Skin by UVB

Irradiation", Journal of Investigative Dermatology, 2006, Vol. 127, pp. 687-697.

Claims

1 . An essentially microorganism-free extract from hypersaline aqueous environments, the extract comprising a total content of sugars from 10 to 50% by weight based on the total weight of the dry extract, a total content of protein from 10 to 50% by weight based on the total weight of the dry extract, and a glycoprotein content from 10 to 30% by weight based on the total weight of proteins present in the dry extract, the extract having a conductivity value equal to or lower than 5 mS/cm at 20°C.

2. The extract according to claim 1 , wherein the total content of sugars is from 20 to 30% by weight based on the total weight of the dry extract, the total content of protein is from 30 to 40% by weight based on the total weight of the dry extract, and the glycoprotein content is from 20 to 30% by weight based on the total weight of proteins present in the dry extract.

3. The extract according to any one of claims 1 -2, wherein the conductivity value is from 0.1 to 1 mS/cm at 20°C.

4. The extract according to any one of claims 1 -3, which is free of

microorganisms.

5. The extract according to any one of claims 1 -4, which is a dry extract.

6. A process for preparing an extract according to any one of claims 1 -4 comprising the following steps:

- conductivity superior to 10OmS/cm at 20°C,

- dry weight from 5 to 350 mg/mL,

- presence of microorganisms, and

- a total concentration of carotenoids equal to or higher than

0.3 μΜ,

to yield the extract in form of aqueous solution.

7. The process according to claim 6, further comprising and additional step d) of filtration using a 0,2-0.45μηη or lower diameter porous filter.

8. The process according to any one of claims claim 6-7, wherein the extract is in form of a solid extract, the process further comprising submitting the extract of either step c) or step d) to an additional step e) of drying.

9. Use of an effective amount of the extract according to any one of claims 1 - 5, as a skin and/or hair moisturizing agent.

10. Cosmetic use of an effective amount of the extract according to any one of claims 1 -5, as a blocking agent for sun protection against UV radiation of skin and/or hair.

1 1 . Use of an effective amount of an extract according to any one of claims 1 - 5, as a chelating agent.

12. The use according to claim 1 1 , wherein the chelating agent is acting as an antioxidant, or to uptake and remove toxic ions.

13. A composition comprising an effective amount of at least one extract as defined in any one of claims 1 -5, together with acceptable excipients or carriers appropriate for topical application to a person's skin and/or hair.

14. The composition according to any one of claims 1 1 -12, which is a cosmetic composition.

15. The composition according to any one of claims 1 1 -12, which is a pharmaceutical composition comprising at least one pharmaceutically active ingredient and one or more pharmaceutically acceptable excipients, wherein at least one of the excipients is an extract as defined above.