ES2813123A1 - Method for obtaining an extract with anti-hypertensive, antihyperlipidemic and antioxidant properties (Machine-translation by Google Translate, not legally binding) - Google Patents

Abstract

Method for obtaining an extract with anti-hypertensive, anti-hyperlipidemic and antioxidant properties. The present invention relates to a method for obtaining an extract of Arthrospira sp. comprising hatching Arthrospira sp. in an aqueous solution at a pH between 3 and 14 in the presence of the enzyme Alcalase {reg}; and separating the biomass from the solution obtained in step a) to obtain the extract. The aqueous extract thus obtained is a concentrate of low molecular weight amino acids and peptides easily assimilated by the body, and in polyphenols among other antioxidants. The extract has multiple therapeutic applications, being able to be used in the prevention and treatment of, for example, hypertension, anemia, malnutrition, hyperlipidemia, obesity and diabetes. (Machine-translation by Google Translate, not legally binding)

Description

DESCRIPTION

Method for obtaining an extract with anti-hypertensive, antihyperlipidemic and antioxidant properties.

TECHNICAL SECTOR

The present invention falls within the field of biotechnology and relates to an extract of Artrospira sp. with a peptide base, as well as its procedure for obtaining it. The extract is useful for preparing compositions with antihypertensive, antioxidant, and / or anti-hyperlipidemic activity for application in the food, cosmetic or pharmaceutical industry.

STATE OF THE ART

Microalgae are eukaryotic living organisms that present a considerable source of high-value biocomponents for various industrial fields. Cyanobacteria, on the other hand, are prokaryotic organisms and, although they do not have the same cellular structure, they share certain functional characteristics and therefore are also known as blue-green algae. Spirulina as an ingredient or dietary supplement is made from cyanobacteria of the genus Arthrospira, especially the species Arthrospira platensis and Arthrospira maxima (formerly known as Spirulina platensis and Spirulina maxima). These filamentous cyanobacteria are considered the major components of phytoplankton present in oceans and marine waters (Setchell & NLGardner. Spirulina maximum, pp 923, Published in: Geitler, L., Cyanophyceae. In: Kryptogamen-Flora von Deutschland, Osterreich und der Schweiz. Ed. 2. Rabenhorst, L. Eds. 1932, 14: 673-1196, i- [vi]. Leipzig: Akademische Verlagsgesellschaft).

Spirulina is an organism with a high protein content, around 60%, which, when cultivated under stress conditions, can reach up to 77% of its dry weight (Biotechnology, Agronomy, Society and Environment, 2017, 20 (3): 1- 9; J. of Aquatic Research, 2012, 40 (3): 763-771), and which also has components of diverse interest such as essential amino acids, minerals, antioxidants, vitamins (in a higher proportion vitamin B), essential fatty acids, carbohydrates and sterols, among others. It has also been shown to contain blue pigments in its structure, such as phycocyanin, which contribute to the increase in protein and iron content in its composition. Consuming spirulina has beneficial health properties (antiviral, anti-inflammatory, antimicrobial), in addition to the positive effect on both the animal and human immune systems. Several studies have demonstrated its innocuousness and safety in consumption and its minimal cytotoxicity, in addition to not producing toxins such as microcystins. For this reason, and due to its safety, this microorganism is classified as a Class A dietary supplement ingredient by the United States Pharmacopeia and National Formulary ( USP-NF). However, each producer must determine the possible contamination of spirulina with certain amounts of other microcystin-producing algae. Therefore, it has been seen the need to establish standards that identify the maximum allowable amounts of microcystins in commercial spirulina-based products. (Critical Reviews in Food Science and Nutrition, 2011, 51: 593-604).

The envelope structure of Gram (-) bacteria, like spirulina, is made up of two complex polymers: peptidoglycans and lipopolysaccharides. Peptidoglycans are made up of disaccharides and tetrapeptides. The latter join together and give rise to a rigid and covalent structure that surrounds the entire cell. For their part, lipopolysaccharides are composed of lipid A and a complex polysaccharide chain. The cell envelope makes it difficult to extract intracellular lipids, stored in the form of subcellular compartments called lipid bodies, lipid droplets or oleosomes (Appl. Biochem. Biotechnol. 2011, 164 (7): 1215-1224). These are formed by a nucleus of neutral lipids surrounded by a lipid monolayer or membrane enveloped by proteins (Proteomics 2011, 11 (21): 4266-4273). An effective lipid extraction requires the destruction of cellular and subcellular structures (J. Agric. Food Chem. 2012, 60 (47): 11771-11776).

There are different methods of extracting biocomponents from plant biomass. The most efficient ones include a biomass breakdown stage prior to the biocomponent recovery stage. The disintegration of the biomass notably increases the extractive yields, and when it causes the rupture of the cell membrane, it allows the recovery of intracellular biocomposites. The treatment of the biomass prior to the extraction of the peptides of interest has a great influence on the product obtained, different treatments generate different extracts with different properties. Different extractions with enzymatic assistance yield different extracts with different composition, which is a function of the type of biocatalyst and conditions of the bioprocess.

Thus, in a study carried out by Zhang and Zhang (Biotechnol Prog., 2013, 29 (5): 1230-8) extracts of peptides with antitumor activity are obtained by treating a spirulina biomass in two stages. In a first stage, proteins are extracted from the spirulina biomass by means of freezing / thawing cycles and the application of ultrasound, and in a second stage they are enzymatically hydrolyzed by means of a multiple enzymatic treatment with the enzymes trypsin, alcalase, papain, and pepsin.

On the other hand, the peptide Ile-Gln-Pro with antihypertensive activity is described (J. Agric. Food Chem. 2010; 58: 7166-7171), obtained by a method that comprises a first stage in which spirulina is subjected to cycles of freezing / thawing together with ultrasound to break the cells, and a subsequent step of enzymatic hydrolysis carried out with the enzyme alkalase.

The obtaining of peptides with antioxidant activity from spirulina has also been described (J. Microbiol Biotechnol. 2016; 26 (7): 1216-23). In this study, the hydrolysis of the cyanobacteria with the enzyme protease K and the subsequent extraction of the peptides by ultracentrifugation and gel chromatography is carried out.

Furthermore, the anticolesterolemic effect of spirulina is known (J. Nutr. Sci. Vitaminol 2010; 56 (1): 34-40) and more specifically of the entire molecule of C-phycocyanin obtained from said cyanobacteria (J. Nutr 2005 Oct; 135 (10): 2425-30).

Although spirulina is very rich in protein, its assimilation is not always good. The assimilation of some peptides and biocomponents of spirulina is affected by its greater or lesser capacity for degradation in the body. Thus, obtaining extracts based on peptides and amino acids resulting from the degradation of biomass, particularly by means of an enzymatic treatment prior to their recovery by solvent extraction, must represent a great advance in the design of processes for obtaining rich extracts in highly assimilable metabolites with high biological functionality.

Therefore, there is a need in the state of the art to continue developing extracts with new bioactive components generated from specific and selective degradations of spirulina biomass, as well as extraction methods for these, analyzing the possibility of obtaining yields. Extractives greater than currently available methods.

DESCRIPTION OF THE INVENTION

The present invention provides a method to extract biocomponents from a plant biomass, where said biomass is spirulina, through a step of enzymatic treatment of the biomass with the Alcalase® enzyme to selectively degrade specific components of its cell membrane. Subsequently, it can be completed with a second stage of extraction and recovery of its biocomponents and, optionally, a stage of concentration / purification and drying of the extract. Said method gives rise to obtaining an extract with particular properties, among which a much higher antihypertensive activity than that obtained in other spirulina enzymatic treatments stands out. Furthermore, the extract of the invention also has a wide range of activities: antioxidant, antihyperlipidemic (anti-hypertriglycerolémic and hypocholesterolemic).

The extract is very heterogeneous, containing high amounts of amino acids and low molecular weight peptides together with phenolic compounds, polyphenols and carbohydrates, and differs from other extracts due to its higher antihypertensive activity, as well as higher antioxidant and anti-oxidant activity / property. hyperlipidemic (antihypertriglyceridemic and anti-hypercholesterolemic). Obtaining an extract with greater effectiveness provides several advantages such as the need for a lower dose to obtain the same effect and the consequent reduction in possible side effects. Therefore, the extract is useful for preparing compositions with antihypertensive, antioxidant, and / or anti-hyperlipidemic activity for application in the food, cosmetic or pharmaceutical industry.

In a first aspect, the present invention describes a method for obtaining an extract of Arthrospira sp., Hereinafter called "method of the invention", characterized in that it comprises the following steps:

a) incubate the biomass of Arthrospira sp. in an aqueous solution at a pH between 3 and 14 in the presence of the Alcalase® enzyme and

b) separating the biomass from the solution obtained in step (a) to obtain the extract.

The term "extract" refers to the highly concentrated substance that is obtained from a bacterial biomass, algae, plant, seed or other object by various procedures, of which it preserves its properties. Said extract is made up of components of different nature, characteristic that allows its separation into different phases, for example: oily or organic, aqueous and residual biomass.

The genus Arthrospira comprises several species, including, but not limited to, A. platensis and A. maxima, formerly known as Spirulina platensis and Spirulina maxima. In a particular embodiment, the spirulina biomass is composed of the species A. platensis and A. maxima; more preferably of the species A. platensis. A. platensis is a levorotatory helix-wound multicellular filamentous cyanobacterium that grows naturally in tropical and subtropical lakes in Africa, Asia and South America with high pH and high concentrations of carbonate and bicarbonate; It is cultivated in the USA, Asia, Europe and South America. In the context of the present invention, a linear (non-helical) variety of A. platensis has been used. It can be started from both dry biomass and a wet biomass concentrate (for example, as obtained by decantation, centrifugation or flocculation of culture tanks) to obtain the aqueous extract. Furthermore, the origin of the biomass of Arthrospira sp. It can be both the cultivation of cyanobacteria and their extraction or collection from the natural environment.

The culture of the cyanobacterium Arthrospira sp. It can be carried out under conditions of gentle agitation of the culture medium, favoring contact with sunlight and photosynthesis reactions. Lighting is an important factor for the growth of spirulina, but it should not be maintained continuously 24 hours a day; a single filament of spirulina cannot withstand prolonged exposure to the sun as it is destroyed by photolysis and hence the need to shake the culture.

The water used to make the growing medium must be clean, or filtered, to remove contaminants. The water used can be fresh water or sea water. Drinking water can also be used but without containing too much chlorine. If seawater is used, which is very rich in magnesium, many precautions must be taken and previously analyzed. The culture medium can be obtained by dissolving chemicals in water; Chemicals including, but not limited to, sodium bicarbonate, salt, potassium nitrate, dipotassium sulfate, monoammonium phosphate, iron solution, and lime. The culture medium also has to contain nutrients to In cyanobacteria, the major nutritive element is carbon, which the culture medium absorbs spontaneously from the air in the form of carbon dioxide (CO2) when its pH is greater than 10. As the air contains very little CO2, its absorption corresponds at a maximum productivity (when the pH reaches 11) of 4 g of spirulina per day and per m3 of pond. However, it is possible to inject supplemental CO2 to increase productivity. If necessary, sugar can replace CO2 as a carbon source (0.5 kg of sugar = 1 kg of CO2). The preparation of the culture medium for the cultivation of spirulina is widely known in the state of the art and is routine practice for those skilled in the art.

In a particular embodiment, the biomass used in this invention is a linear variety of the cyanobacterium A. platensis, cultivated by ASN Leader SL with mountain water in southeastern Spain (Murcia), where daily analytical controls are carried out. This biomass is essentially pure and free of other contaminating microalgae or cyanobacteria. In another particular embodiment, the Arthrospira biomass is A. maxima.

Once the desired amount of Arthrospira sp. Biomass is obtained, the Alcalase® enzyme is added. Thus, in a first stage [stage (a)], the method of the invention comprises incubating the biomass of Arthrospira sp. in an aqueous solution at a pH between 3 and 14 in the presence of the Alcalase® enzyme, preferably at a pH between 4.5 and 8.5. In another more preferred embodiment for both the optimal extraction of hydrophilic and hydrophobic biocomponents, the enzyme-biomass suspension is at a pH of 6.5.

The incubation of the biomass together with the enzyme can be carried out in a wide range of temperatures. However, in a preferred embodiment of the method of the invention, step (a) is carried out at a temperature of between 5-80 ° C, preferably between 25-50 ° C. In a more preferred embodiment the temperature is 30 ° C.

In the context of the present invention, the term "enzyme" refers to a protein responsible for catalyzing the biochemical reactions of metabolism, which depending on their catalytic action are classified as: oxidoreductases, transferases, hydrolases, lyases, isomerases and ligases In the present invention the enzyme used is a protease, which is a type of hydrolase, more specifically an alcalase (Serine endo-peptidase (mostly subtilisin A), EC 3.4.21.62).

To express the amount of enzyme that can be used in the method of the invention, the term "enzyme load" is used, which refers to the percentage of the enzyme in the solution. Thus, the enzyme load in the solution of step (a ) can be 0.3-10% v / w (volume of commercial Alcalase® solution referred to the total weight of spirulina biomass suspension in the enzyme solution), preferably 0.5-2%, and more preferably 1 % v / w. Alcalase® 2.4 L FG is a commercial preparation from Novozymes, based on a serine endoprotease that hydrolyzes internal peptide bonds. A protease (Subtilisin A) is the declared enzyme with an activity of 2.4 AU- A / g, this product not being GMO.

Once enough time has passed for the biomass incubation together with the enzyme (to extract the greatest number and quantity of hydrophilic and hydrophobic biocomponents to their respective phases, as identified for example by liquid chromatography analysis (HPLC )), the residual biomass is separated from the solution obtained in step (a) obtaining the extract of Arthrospira sp. [step b) of the method of the invention]. In a particular embodiment, the extract obtained comprises the aqueous phase or the oily phase of the solution obtained in step (a). To remove the less polar compounds present in the parts of the biomass, mainly lipids, phospholipids, steroids, vitamins and fat-soluble pigments or their degradation products, and other fat-soluble biocomponents, they are separated by adding different organic solvents, performing the corresponding distribution of the water-soluble biocomponents of the fat-soluble ones in the different phases formed in the extraction procedure. Thus, in a particular embodiment of the method of the invention, the method comprises an additional step comprising the addition of an organic extraction solvent between steps (a) and (b). In another more particular embodiment, the organic solvent is hexane, isopropanol or a mixture of both.

If the corresponding oily extract is not of interest, it is possible to avoid the addition of organic solvents after the enzymatic treatment, in which case only an aqueous solution is used to extract polar biocomponents from the aqueous phase, separating them from a small oily phase and of residual biomass. For this, any of the methods used in the state of the art can be used (separation of the aqueous, organic and residual biomass phases by a first stage of centrifugation or decantation or flocculation, and / or a filtering stage that can be under vacuum. or other known methods to separate / remove the residual biomass fraction remaining in suspension of the liquid phases, and followed by another stage of separation of the phases in different containers (Maniglia et al. Food Sci & Technol. 2014, 56: 269-277)).

Then, each of the extracts (aqueous and oily) can optionally be concentrated or purified by conventional techniques (chromatographic, etc.), and brought to dryness by any of the available and known techniques (lyophilization, rotary evaporator, under air current or nitrogen, etc). After the enzymatic treatment of the biomass, the extracts can be obtained using the Shoxlet method or by means of an accelerated solvent extraction (Food Chemistry, 2005, 93: 417-423). Before or after the enzymatic stage, an additional biomass treatment stage could also be applied by mechanical disruption techniques, ultrasound, microwaves, negative pressure cavitation, electrical pulses, etc., the effect of which is to disintegrate / separate the cellular components. rather than its breakdown at the molecular level. This last disintegration, therefore, facilitates the recovery / extraction of intracellular biocomponents, but not their transformation into molecular degradation products (eg, low molecular weight peptides). Once the biomass has degraded, for the recovery of biocomponents, Soxhlet distillation, autoclaving, osmotic shock, etc. can be used, and traditional solvents or "green" solvents such as supercritical fluids, subcritical water, ionic liquids, etc. can be used (Food Sci. and Technol. 2014, 56: 269-277) In a particular embodiment, the method comprises adding to the solution obtained in step (a) at least one organic solvent.

By "organic solvent" is understood the chemical agent outside the formulation, that is, the chemical compound that is not necessary in the final formulation but that is introduced during the production process in order to favor extraction. Organic solvents include alkanes such as hexane or toluene, ketones such as acetone, alcohols such as isopropanol, methanol, or ethanol, esters such as ethyl acetate, and other linear, cyclic, or branched hydrocarbons, substituted or not. , such as chloroform, and xylene, ethers such as dimethyl ether or petroleum ether, ionic liquids, organic acids and bases, and amides such as N, N-dimethyl formamide. In another preferred embodiment the organic solvent is hexane, isopropanol or any of their combinations. In particular, a 3: 2 (v / v) mixture of n-hexane / iso-propanol. Other methods may differ in the type, number and relative proportions of solvents used, whether or not they are miscible with each other, and may be only pure water or with various solutes: salts, buffer, etc., if you only want to extract hydrophilic components.

In another particular embodiment of the method of the invention, after step (b) of the method, successive processes of centrifugation, filtering, and / or decantation are carried out. Optionally, a step c) can be added for drying the extract by any known method, such as those based on evaporation or lyophilization.

In a second aspect, the present invention refers to an extract of Arthrospira sp., Hereinafter called "extract of the invention", obtained by the method described above, that is, the method of the invention. The term extract has been defined and explained in previous paragraphs of the present description.

In another preferred embodiment, said extract comprises at least one, two, three, four, five, six, seven or eight peptides selected from the list consisting of MKKIEAIIRPF (SEQ ID NO: 1), LPPL (SEQ ID NO: 2) , ALAVGIGSIGPGLGQGQ (SEQ ID NO: 3), TTAASVIAAAL (SEQ ID NO: 4), DFPGDDIPIVS (SEQ ID NO: 5), LELL (SEQ ID NO: 6), WKLLP (SEQ ID NO: 7) and / or CHLLLSM ( +15.99) (SEQ ID 8). In an even more particular embodiment, the extract of the invention comprises the peptides SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 , SEQ ID NO: 7 and SEQ ID NO: 8.

On the other hand, the present invention also refers to a composition, hereinafter "composition of the invention", comprising the peptides MKKIEAIIRPF (SEQ ID NO: 1), LPPL (SEQ ID NO: 2), ALAVGIGSIGPGLGQGQ (SEQ ID NO: 3), TTAASVIAAAL (SEQ ID NO: 4), DFPGDDIPIVS (SEQ ID NO: 5), LELL (SEQ ID NO: 6), WKLLP (SEQ ID NO: 7) and / or CHLLLSM (+15.99) ( SEQ ID 8) In a particular embodiment, said composition comprises an excipient and / or a vehicle The terms excipient and vehicle are defined in the following paragraphs.

It is a peptide-based extract from spirulina, which has antihypertensive, antioxidant and anti-hyperlipidemic (antihypertriglyceridemic and anti-hypercholesterolemic) capacity / activity which, in a particular embodiment, in addition to the peptides described above, comprises:

a) a minimum content of total polyphenols of 12 mg / g of extract,

b) a minimum total carbohydrate content of 226 mg / g extract, and / or

c) significant amounts of all amino acids including essential ones, with a minimum content of 45% w / w of total amino acids

In the present invention, "composition" is understood to mean the substance constituted by a combination of molecules, including the bioactive peptides of interest. The term "peptides" refers to natural molecules made up of linear chains of amino acids linked by peptide bonds. Amino acids are represented throughout the description and claims through the one letter code known in the state of the art. The term "antioxidant" refers to compounds that mitigate oxidation and that, when ingested, can be beneficial to human health. The antioxidants that are most abundant in the diet are ascorbic acid or vitamin C, polyphenols, vitamin E, and carotenoids, the first two being water-soluble and the last two fat-soluble. The term "polyphenol (s)" refers to natural molecules that are phenolic compounds with an aromatic ring and one or more hydroxyl groups, and are classified into "flavonoids" and "non-flavonoids". Flavonoids are polyphenols with 15 carbon atoms and two aromatic rings connected by a three-carbon bridge; they can have numerous substituents in their structure, and in nature they are mostly in the form of glycosides and to a lesser extent in the form of aglycones. The most common non-flavonoid structures are C6-C1 phenolic acids, which occur in nature as complex sugar esters. Polyphenols are secondary metabolites, which both in the form of (poly) phenolic aglycones and their conjugated sugars, exist in plants in concentrations of pM to mM. Both have protective functions, and important positive effects are attributed to them in chronic diseases. They modulate the activity of digestive enzymes, or act as antioxidants directly on ROS (reactive oxygen species) present in the lumen, or systemically (in organs, tissues or cells through their prior gastrointestinal absorption and distribution from the blood to such tissues). After their ingestion, they are transformed into the corresponding metabolites (phase II), and pass into the circulatory system, where their concentration in plasma is of the order nM. In the colon they are degraded by the local microbiota, giving small phenolic acids and aromatic catabolites that are absorbed into the circulatory system. The conjugated form of polyphenols is more polar (water soluble) and less absorbable (and bioavailable). In the large intestine, bacteria in the colon metabolize flavonoids, hydrolyzing their glycosidic bonds and thus promoting their absorption. Diets rich in polyphenols favor the proliferation of beneficial microbes and inhibit that of pathogenic organisms.

In a third aspect, the present invention relates to a pharmaceutical, food or cosmetic composition comprising the extract of the invention or the composition of the The invention, and optionally, further comprises at least one pharmaceutically, foodstuff or cosmetically acceptable excipient or vehicle, respectively.

The term "pharmaceutical composition" refers to a composition, as previously described, administered in solid or liquid form in any pharmaceutical format, such as, without limitation, tablets, capsules, sachets, syrups, sprays, injectables, ointments. , creams, lotions, suppositories, vials and ointments.

On the other hand "food composition" refers to a composition, as previously described, administered as a food or food supplement formulated as, for example, capsules, pills, syrups or drinks, etc. that provides nutrients to the subject who takes it, and can beneficially affect one or more functions of the body. It can be administered in the form of a drink or food, with or without prior preparation, and it can be used for human or animal consumption. Animals include, but are not limited to, pets, farm animals (cattle, sheep, pigs, goats), and fish farm animals, etc.

The term "cosmetic composition" refers to a composition, as previously described, administered as creams, ointments, lotions, emulsions, microemulsions, liposomes, sprays, liquids, compact or loose powders, anhydrous sticks, gels and oils, masks, soaps, shampoos, or other personal hygiene products and solar cosmetics.

The term "excipient" refers to a substance that helps the absorption of the pharmaceutical, food or cosmetic composition of the invention, stabilizes said compositions or helps their preparation, in the sense of giving it consistency or providing flavors that make it more pleasant. . Thus, excipients could have the function of holding the ingredients together, such as starches, sugars or celluloses, a sweetening function, a coloring function, a protective function of the drug, such as for example to isolate it from air and / or humidity. , function of filling a tablet, capsule or any other form of presentation, such as dibasic calcium phosphate, disintegrating function to facilitate the dissolution of the components and their absorption in the intestine, without excluding other types of excipients not mentioned in this paragraph. In order for the composition of the invention to have a pleasant taste, an essence can be added, such as essence of cinnamon, lemon, orange, tangerine or vanilla.

The "pharmaceutical, food or cosmetically acceptable vehicle" refers to those substances, or combination of substances, known in the pharmaceutical and / or cosmetic sector, used in the preparation of pharmaceutical and / or cosmetic forms of administration and includes, but is not limited to , solids, liquids, solvents or surfactants. The vehicle can be an inert substance. The vehicle is preferably at a concentration of 2.5 to 7% v / w with respect to the final composition. The function of the vehicle is to facilitate the incorporation of the product of the invention, as well as other compounds, to allow a better dosage and administration or to give consistency and shape to the pharmaceutical composition. When the presentation form is liquid, the vehicle is the diluent. These carriers may be known to those skilled in the art, for example, but not limited to, lysosomes, millicapsules, microcapsules, nanocapsules, sponges, millispheres, microspheres, nanospheres, milliparticles, microparticles and nanoparticles. The term "pharmaceutically, food, or cosmetically acceptable" refers to the fact that the compound to which it refers is allowed and evaluated so that it does not cause harm to the organisms to which it is administered.

In a fourth aspect, the present invention relates to the extract of the invention or the composition of the invention for use as a medicine. The term "drug", as used herein, refers to any substance used for the prevention, diagnosis, alleviation, treatment or cure of diseases in man and animals. Administration should be adjusted to a desirable dose of the composition of the invention and varies depending on the condition and weight of the subject, the severity of the disease, the form of the drug, the route and the period of administration, and may be chosen by the person skilled in the art.

In a fifth aspect, the present invention refers to the extract of the invention or the composition of the invention for use in the treatment and / or prevention of any of the diseases selected from the list consisting of hypertension, anemia, malnutrition, hyperlipidemia (such as hypertriglycerollemia, hypercholesterolemia, and mixed hyperlipidemia), obesity and diabetes.

The term "treatment" refers to combating the effects caused as a consequence of the disease or pathological condition of interest in a subject which includes inhibiting the disease or pathological condition, ie, arresting its development; alleviating the disease. or the pathological condition, that is, causing regression of the disease or pathological condition or its symptomatology and stabilizing the disease or pathological condition.

The term "prevention" refers to preventing the onset of the disease, that is, preventing the disease or pathological condition from occurring in a subject, in particular, when said subject has a predisposition for the pathological condition, but has not yet been diagnosed as having it.

In the present invention, the disease that is more effectively treated and / or prevented than other extracts or compositions derived from spirulina is hypertension. The term "hypertension" also known as high blood pressure, is a disorder in which the blood vessels are persistently high, which can damage them. Normal blood pressure in adults is 120 mm Hg (systolic pressure) and 80 mm Hg (diastolic pressure). When the systolic pressure is equal to or greater than 140 mm Hg and / or the diastolic pressure is equal to or greater than 90 mm Hg, the blood pressure is considered high.

In addition to hypertension, other diseases that can be prevented and / or treated by the aqueous extract or the composition of the invention:

- Hyperlipidemia, which includes different metabolic disorders that lead to high levels of triglycerides and / or cholesterol in the bloodstream. They are classified as hypertriglyceridemia, hypercholesterolemia, and mixed hyperlipidemia.

• Type I hyperlipidemia, developed by lipoprotein lipase deficiency, causes an accumulation of chylomicrons (QM), resulting in an increase in plasma triglycerides.

• Hypercholesterolemia, which consists of the presence of high levels of cholesterol in the blood or, more specifically, a high concentration of low-density cholesterol (LDL), which is a factor that increases the risk of developing heart disease. In a preferred embodiment, the disease is hypercholesterolemia.

• Mixed hyperlipidaemia in which both cholesterol and triglycerides increase.

- Anemia is a disease characterized by an abnormal decrease in the number or size of red blood cells in the blood or in their level of hemoglobin.

- Malnutrition, which is that pathological state of different degrees of seriousness and of different clinical manifestations caused by the deficient assimilation of food by the body.

- Overweight, which refers to a pathology characterized by the subject having a body mass index (BMI) equal to or greater than 25. BMI is a measure of association between weight and height of an individual. The BMI has the following formula for its calculation: Mass (Kg) / height2 (m). Being overweight is characterized by a BMI between> 25 to <30.

- Obesity, which refers to a pathology characterized in that the subject has a BMI equal to or greater than 30. Obesity is classified at different levels, considering that subjects with a BMI> 40 suffer from morbid obesity. Other parameters used to determine if an individual suffers from central obesity are the absolute waist circumference (the subject suffers obesity when it is> 102 cm in men [central obesity] and> 88 cm in women) or the waist-hip ratio (the subject suffers obesity when it is> 0.9 for men and> 0.85 for women). An alternative way to determine obesity is to measure the percentage of body fat (the subject is obese when he has approximately> 25% body fat in a man and approximately> 30% body fat in a woman).

- Diabetes, preferably gestational diabetes or type 2 diabetes Mellitus. Type 2 diabetes Mellitus is characterized by a relative deficit in tissue production and sensitivity to insulin and, therefore, a deficient peripheral use of glucose.

In a sixth aspect, the present invention relates to the extract of the invention or the composition of the invention for use as an antioxidant agent.

In a seventh aspect, the present invention relates to the extract of the invention or the composition of the invention for its use as an anti-hypertensive or anti-hyperlipidemic agent.

In an eighth aspect, the present invention refers to the extract of the invention or the composition of the invention to prepare a protein concentrate that is easily assimilated.

Throughout the description and claims the word "comprise" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and characteristics of the invention will emerge partly from the description and partly from the practice of the invention. The following examples and figures are provided by way of illustration, and are not intended to be limiting of the present invention.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1. Chromatogram of the gradient analysis by HPLC-ELSD of the oily extract of A. platensis biomass obtained with the assistance of the Alcalase® biocatalyst. Extraction conditions: 1% v / w of Alcalase® enzyme at pH 6.5 and 30 ° C for 24h. The eluted peaks were computed from 3 min., At lower times the solvent is eluted. The internal standard (hexadecane) elutes at 9 min.

Figure 2. Influence of the pH of the biomass degradation step with Alcalase® in the oily extract obtained using 1% v / w of enzyme, at 40 ° C for 4h. AU, arbitrary units: variation of the total peak area in the HPLC-ELSD chromatogram.

Figure 3. Influence of the temperature of the step of degradation of the biomass with Alcalase® in the oily extract obtained: variation of the total area of peaks in the HPLC-ELSD chromatogram. Conditions: pH 6.5 and 1% (v / w) of enzymatic load during the enzymatic treatment of 4 or 24 hours.

Figure 4. Influence of the enzymatic load (v / p) of the biomass degradation step with Alcalase® in the oily extract obtained: variation of the total area of peaks in the HPLC-ELSD chromatogram, at pH 6.5 and 30 ° C, and at different times of enzymatic treatment (0-24h). The control test (0% enzyme load) was performed with the same protocol, but using milli-Q water instead of the enzyme solution buffered at 30 ° C.

Figure 5. Chromatogram of the gradient analysis by HPLC-ELSD of the aqueous extract of A.platensis biomass, obtained with the assistance of the Alcalase® biocatalyst.

Extraction conditions: 1% v / w of Alcalase® enzyme at pH 6.5 and 30 ° C for 24h. The peak identification limit was 7500 u.a. of area. The eluted peaks were computed from 2.39 min, at lower times the solvent is eluted.

Figure 6. Chromatogram of the gradient analysis by HPLC-ELSD of the aqueous extract of A. platensis biomass, obtained with the assistance of the Control biocatalyst (without enzyme). Extraction conditions: milli-Q water instead of buffered enzymatic solution, 30 ° C and 24h. The peak identification limit was 7500 UA of area. The eluted peaks were computed from 2.39 min. , at lower times the solvent is eluted.

Figure 7. Influence of the pH of the biomass degradation process with Alcalase® in the recovery of polar components of spirulina (variation of the total area of peaks in the HPLC-ELSD chromatogram of the aqueous extract). A) Effect of pH at 4 h after enzymatic treatment. B) Kinetics of the extraction at the optimum value of pH 6.5. Other conditions: 1% (v / p) enzymatic load and 40 ° C. u Arbitrary units of area.

Figure 8. Influence of the temperature of the step of enzymatic degradation of the biomass with Alcalase® in the aqueous extract obtained: variation of the total area of peaks in the HPLC-ELSD chromatogram. Conditions: 1% v / w of Alcalase® at pH 6.5 for 24h (variation of the total area of peaks).

Figure 9. Influence of the enzymatic load of the biomass degradation step with Alcalase® in the aqueous extract obtained: variation of the total area of peaks in the HPLC-ELSD chromatogram. Conditions: pH 6.5 and 30 ° C for 24h. The control extraction (0% enzymatic load) was carried out with the same extraction protocol, but using milli-Q water instead of the enzymatic solution buffered at 30 ° C.

Figure 10. Evolution of the area of the different chromatographic peaks of the aqueous extract obtained with Alcalase® under optimal extraction conditions. Conditions: pH = 6.5, 30 ° C and 1% (v / p) biocatalyst. A) retention time (Tr) = 3-5 min .; B) Tr = 5.5min; C) Tr = 11-14min; D) Tr = 20-22min .; E) Tr = 28min .: F) Tr = 68min. The peak identification limit was 7500 UA of area. The identification and follow-up in time of the first two peaks (Tr = 3-5.5 min) was carried out with an injection volume of 5 µl of sample, while the rest of the peaks were identified with injections of 20 µl of sample. .

Figure 11. Comparative study of the oil extracts (enzymatic and control) obtained in their respective optimal conditions. Variation of the total area of peaks in the HPLC-ELSD analyzes weighted by its corresponding yield, that is, equivalent area extracted for each gram of initial spirulina biomass. Conditions: 24h of enzymatic treatment in their respective optimal conditions (pH 6.5, 1% v / w and 30 ° C for Alcalase®; pH 6.0, 1% v / w and 30 ° C for Flavourzyme®; pH 7.0 , 1% v / w and 30 ° C for Ultraflo®; pH 6.5, 2% v / w and 40 ° C for Vinoflow®). The control extraction (0% enzymatic load) was carried out with the same protocol, but using milli-Q water instead of the enzymatic solution buffered at 30 ° C.

Figure 12. Comparative study of the aqueous extracts (enzymatic and control) obtained in their respective optimal conditions. Variation of the total area of peaks in the HPLC-ELSD analyzes weighted by its corresponding yield, that is, equivalent area extracted for each per gram of initial spirulina biomass. Conditions: 24h of enzymatic treatment in their respective optimal conditions (pH 6.5, 1% v / w and 30 ° C for Alcalase®; pH 6.0, 1% v / w and 30 ° C for Flavourzyme®; pH 7.0 , 1% v / w and 30 ° C for Ultraflo®; pH 6.5, 2% v / w and 40 ° C for Vinoflow®; pH 6.5, 1% v / w and 30 ° C for Lipozyme®; pH 6, 5.1% v / w and 30 ° C for Celluclast®). The control extraction (0% enzymatic load) was carried out with the same protocol, but using milli-Q water instead of the enzymatic solution buffered at 30 ° C.

Figure 13. Micrographs taken at 15,000 magnification. A) Dry commercial A. platensis biomass not treated with enzyme or extracted; B) biomass extracted with Alcalase® under the optimal conditions determined for this extractive process (pH 6.5, 1% v / p Alcalase® and 30 ° C for 24h).

Figure 14. Micrographs taken at 200,000 magnification. A) Dry commercial biomass not enzyme treated or extracted; B) extracted with Alcalase® under the optimal conditions determined for this extractive process (pH 6.5, 1% v / p Alcalase® and 30 ° C for 24h).

Figure 15. Total concentration of amino acids extracted in different extractive methods in their respective optimal conditions. The optimal conditions were: pH 6.5, 1% v / p and 30 ° C for Alcalase®; pH 6.0, 1% v / w and 30 ° C for Flavourzyme®; pH 7.0, 1% v / w and 30 ° C for Ultraflo®; pH 6.5, 2% v / w and 40 ° C for Vinoflow®, pH 6.5, 1% v / w and 30 ° C for Lipozyme®; pH 6.5, 1% v / w and 30 ° C for Celluclast®. Milli-Q water was added to the control extract instead of buffered enzymatic solution, 30 ° C and 24h. The control method is the same as the enzymatic ones, but using milli-Q water instead of the buffered enzyme solution.

Figure 16. Chromatograms of the aqueous biomass extracts of A. platensis obtained with different enzymatic processes in their respective optimal conditions and without enzyme (control), analyzed by LC ESI-MS MS in positive ionization mode. The optimal conditions were: pH 6.5, 1% v / p and 30 ° C for Alcalase®; pH 6.0, 1% v / w and 30 ° C for Flavourzyme®; pH 7.0, 1% v / w and 30 ° C for Ultraflo®; pH 6.5, 2% v / p and 40 ° C for Vinoflow®. Milli-Q water was added to the control extract instead of buffered enzymatic solution, 30 ° C and 24h.

EXAMPLES

The invention is based on a specific enzymatic step, in which the biocatalyst acts to specifically and selectively degrade biomass. The enzymatic degradation of biomass makes it possible to recover and extract a greater amount of intracellular biocomponents and different degradation products depending on the biocatalyst / bioprocess used. The extracted components are both aqueous and oily (or hydrophilic and hydrophobic). The quantity and composition of both extracts are a consequence of both the choice of the biocatalyst and the effect of variations in the values of the parameters that influence the degradation stage of the biomass. This enzymatic stage prior to the recovery of aqueous and oily spirulina biocomponents, facilitates their extraction, maximizing extractive yields for optimal values of said parameters. On the other hand, regardless of the yields of extracted material, the specific biocomposites obtained as enzymatic degradation products are specific to the type of enzyme used, and to their operating conditions during the treatment of biomass with the biocatalyst, since they are the The result of a specific and selective enzymatic action on cellular components of the biomass, and of the extension of said action depending on the operating conditions of the degrading enzymatic process.

Example 1

Methodological description

Enzymatic treatment of spirulina biomass

The cellular disruption of the biomass (4 g) was carried out in duplicate or triplicate in a 25 mL reactor where spirulina was at a concentration of 0.2 g / ml in a 0.1 M buffer. pH value studied was used sodium acetate or sodium phosphate buffer. Although the enzymatic degradation of biomass can be carried out using different biomass concentrations in the reactor, the one used corresponds to the compromise concentration to have the highest volumetric productivity. The biomass concentration value used corresponds to the concentration range to have the highest possible amount of biomass per ml of aqueous solution, which allows a completely dispersed suspension to be formed and in such a way that there are minimum limitations of matter transfer and to enable contact. among those involved in the enzymatic and extractive degradation process. The enzymatic degradation process of the biomass was carried out in an incubator (Stuart SI50) at controlled temperature and magnetic stirring (500 rpm). During the reaction, 0.5 mL aliquots were collected in duplicate to monitor the enzymatic reaction for up to 24-48 h.

Extraction of Biocomponents by Solvents

To each aliquot (0.5 ml) collected of the enzymatic reaction medium, 1 ml of a solvent mixture composed of hexane and isopropanol was added in a 3: 2 (v / v) ratio. The mixture was vortexed for 1 minute and then centrifuged for 15 minutes at 10,000 rpm. The organic phase or upper phase was carefully extracted into an eppendorf. The operations of adding the mixture of hexane and isopropanol, and subsequent extraction of the oil phase, were carried out two more times using 0.25 ml of the solvent mixture. The three volumes of organic phases recovered after each of the three solvent additions were pooled and homogenized in the vortex. The aqueous phase recovered after the three solvent extractions was filtered. Both extracts (aqueous and oily) were stored at 4 ° C until analysis by HPLC-ELSD.

Analysis of the extracts

The study of the influence of the parameters of pH, temperature, time and enzymatic load in the oily extracts, was carried out by means of analysis of the solutions in chloroform of aliquots of the different oily phases by HPLC-ELSD ( High pressure liquid chromatography with an evaporative light scattering detector), following the variation of the total area of the significant chromatographic peaks. An example of a chromatogram is shown in Figure 1. The peak eluted at 9 min. corresponds to the hexadecane used as an internal standard. The ELSD detector used is universal, although it does not display highly volatile compounds such as isopropanol or difficult to nebulize (high molecular weight, etc.).

The analyzes of the oily extracts were carried out by high pressure liquid chromatography (HPLC, High-Performance Liquid Chromatography), in an equipment brand Merck-Hitachi Ltd. (Germany and Japan) coupled to a Kromasil column of silica 5pm, 250x4. 6 mm. and with an evaporative light scattering detector (ELSD, Evaporative Light Scattering Detector) from SEDERE model SEDEX 55. The samples were filtered through a nylon filter (15mm x 0.45pm, from Analisis Vinicos SA, Spain). pl of sample dissolved in chloroform (HPLC quality 99% pure), analyzed at a flow rate of 1.5 ml / min. for 30min. A gradient mobile phase was used where mobile phase A was n-Hexane (HPLC grade 96%) and mobile phase B contained Hexane / Isopropanol / Ethyl Acetate (80:10:10). From an A: B phase ratio of 1:99, the phase ratio was increased linearly to 98: 2 over 20 minutes. These conditions were maintained for 3 minutes, from which the initial conditions (1:99) were returned in 1 min, which were maintained isocratically for 9 minutes.

The study of the influence of the same parameters of the enzymatic stage in the aqueous extracts was carried out by means of HPLC-ELSD analysis, evaluating variations in the total area of all the significant peaks. Examples of chromatograms are shown in Figures 5-6.

The analyzes of the aqueous extracts were carried out in the same HPLC equipment, coupled to a Kromasil C18 5u column, 250x4.6 mm. 10 μl of a solution of the extract in water (50 mg / ml) was injected and analyzed at a flow of 1.5 ml / min for 83 minutes in a gradient using 100% MiliQ Water as phase A and a mixture of MiliQ as phase B. Acetonitrile: MiliQ Water at 80:20. Isocratic conditions of 96% A and 4% B were maintained for five minutes, followed by a linear increase to 40% B in 60 min and a linear increase to 95% B in one minute. The proportions were maintained for seven minutes and returned to initial conditions in 10 minutes. All analyzes were replicated 2-3 times.

The aqueous extracts obtained after 24 hours of enzymatic treatment of the biomass were studied from the lyophilized extracts obtained on a larger scale.

Extractive process on a larger scale

The extractive process on a larger scale was prepared by performing the cellular disruption of the biomass (10 g) in a 250 ml reactor where spirulina was at a concentration of 0.2 g / ml in a 0.1 M buffer. the pH value studied was used sodium acetate or sodium phosphate buffer. The enzymatic degradation process of the biomass was carried out for 24 hours. in an incubator (Stuart SI50) at controlled temperature and magnetic stirring (500 rpm). These samples were extracted using a 3: 2 (v / v) hexane / isopropanol mixture. To do this, after adding the solvent mixture in the same biomass / solvent ratio as for the small-scale process, the resulting mixture was kept under magnetic stirring at 500 rpm at room temperature for 10 min, after which it was centrifuged in a refrigerated centrifuge. at 10 ° C at 14,000 rpm for 30 min. After phase separation occurred in this process, the upper (oily) phase was carefully separated. Next, this extractive step was repeated by adding to the remaining material in the centrifuge tube (residual biomass aqueous phase) 90 mL of the 3/2 (v / v) hexane / isopropanol solvent mixture, then repeating the steps of stirring, centrifugation and removal of the oil phase. After successive solvent extraction steps, the liquid aqueous phase remaining in the centrifuge tube was filtered on filter paper and transferred to a separatory funnel, leaving the residual biomass at the bottom of the centrifuge tube. The aqueous phase was lyophilized for 4 days. The two volumes obtained of the oil phase were combined and filtered, taking them to dryness in a rotary evaporator and finally under a stream of nitrogen. The two dry extracts (aqueous and oily) were stored until use at -70 ° C. The control extract was obtained with the same protocol except that milli-Q water was used instead of the enzyme solution in buffer.

Determination of extractive yield

The weight yield of the aqueous extracts was determined from the lyophilized dry weight values of each aqueous extract on a 4-digit precision balance. For this, the aqueous phases were lyophilized for 4 days and the oily phases were rotavaporated at 30 ° C to eliminate the solvent, and subsequently treated for 4 hours under a stream of nitrogen until the complete elimination of traces of solvent. From the dry weight values of the aqueous extracts obtained, the value of the weight corresponding to its content in salts of the buffer and that of the enzyme added in the Alcalase® solution was subtracted, the total amount subtracted being less than 1% w / p. The residual biomass was also dried under a stream of nitrogen.

Analysis of residual biomass

The morphological changes produced in the biomass by the different extractive methods were studied with a Jeol Jem 1010 (100Kv) Yokyo Japan transmission electron microscope. Digital camera attached to the Orius SC200 from Gatan Inc. Pleasanton, California. Digital Micrograph Image Acquisition Software v 3.4. All samples were previously treated for fixation with aldehydes, washing, osmification, dehydration, inclusion in durcupan resin with a final polymerization in an oven at 60 ° C for 48h.

Amino acid composition analysis of aqueous extracts

The quantitative analysis of the amino acid mixtures was carried out according to the chromatographic procedure developed by Spackman, Moore and Stein in 1958 (Fed Proc. 1958, 17 (4): 1107-1115) and based on the basic principle of operation in flow mode continuous, on a Biochrom 30 Series amino Acid Analyzer, with a reproducibility> 0.5 CV at 10 nmol. Biochrom 30 used classical amino acid analysis methodology based on ion exchange liquid chromatography and a continuous post-column reaction with ninhydrin to obtain a qualitative and quantitative analysis, with a sensitivity of ~ 10 pmol,

The corresponding solutions of the different dry aqueous extracts (1-2.6 mg / mL) were prepared in triplicate in hydrolysis tubes, and a determined volume of a solution of known norleucine concentration (used as internal standard) was added. Known amounts of standard and internal standard were analyzed to calibrate the analyzer.

Identification of biocomponents of the aqueous extracts by LC ESI-MS MS The different aqueous extracts (enzymatic and control) were analyzed by LC ESI-MS MS in positive ionization mode, in order to identify their respective biocomponents.

Prior to analysis, all samples were cleaned using Millipore C18 stationary phase tips. LC ESI-MS MS analyzes were performed on an Ultimate 3000 nanoHPLC (Dionex, Sunnyvale, California) coupled to an AmaZon Speed ion trap mass spectrometer (Bruker Daltonics, Bremen, Germany). The analytical column used was C18 PepMap reversed phase silica based 75 µm * 15 cm, 3 µm particle size and 100 µm pore size (Dionex, Sunnyvale, California). The trap column was a C18 PepMap (Dionex, Sunnyvale, California), 5 pm particle diameter, 100 A pore size, connected in line with the column. analytics. The pump charge eluted a solution of 0.1% trifluoroacetic acid in 98% water / 2% acetonitrile (ScharLab, Barcelona, Spain) at 30 ^ l / minute. The nanpump provided a flow rate of 300 nl / minute and operated under gradient elution conditions, using 0.1% formic acid (Fluka, Buchs, Switzerland) in water as mobile phase A, and 0.1% formic acid in 80% acetonitrile / 20% water as mobile phase B. The elution gradient was performed according to the following scheme: in isocratic mode with 96% A: 4% B for five minutes, a linear increase to 40% B in 60 minutes, a linear increase to 95% B in one minute, isocratic conditions of 95% B for seven minutes and return to initial conditions in 10 minutes. 5 ^ l of the extract solutions (4 ^ g / ^ l) were injected, the recorded wavelengths being 214 and 280 nm. In a second analysis, 5 ^ l of extract solutions of 10 ^ g / ^ l were injected. The LC system was connected by a CaptiveSpray source (Bruker Daltonics, Bremen, Germany) to the ion trap mass spectrometer operating in positive mode with a capillary voltage set at 1400 V. Automatic data acquisition allowed to obtain sequentially, the scan complete MS spectra (m / z 350-1500), and MS CID spectra of the eight most abundant ions. In the analysis of the 10 ^ g / ^ l samples, the complete MS spectra scan was 100-1000 m / z. The exclusion dynamics was applied to prevent the same m / z from its isolation for 1 minute after its fragmentation.

For peptide identification, MS and MS / MS data obtained for individual HPLC fractions were processed with DataAnalysis 4.1 (Bruker Daltonics, Bremen, Germany). For the identification of peptides, the MS / MS spectra (in the form of generic Mascot files) were analyzed against a composite database obtained from NCBInr (National Center for Biotechnology Information, as a protein database for searches. sequences from GenBank CDS translations, PDB, Swiss-Prot, PIR, and PRF) and contained 68623 protein entries from both Spirulina and Arthrospira. Database searches were done using a licensed version of Mascot v.2.6.0 (Matrix Science, London, UK) (Electrophoresis, 1999, 20 (18): 3551-67). The search parameters were set as follows: methionine oxidized as a variable modification, without enzyme restriction. Peptide mass tolerance was set at 0.3 Da in MS mode and 0.4 Da in MS / MS mode, no breakdown losses were allowed. In most cases, a precision of ± 0.1-0.2 Da was obtained in both MS and MS / MS spectra.

In addition, in the case of the Alcalase® sample, all the MS and MS / MS spectra obtained in the analysis of the sample were taken and analyzed using the tool "de novo" from the Peaks program (Bioinformatics Solutions, Inc). This program allows, like Mascot, to face the MS / MS spectra obtained in the analysis of the samples against a database of sequence entries, in this case of Artrhospira-Spirulina. This "de novo" analysis mathematically analyzes the spectral data looking for the best sequence solution that explains the spectrum. It is an unconditional analysis. The table includes only those MS and MS / MS spectra and their corresponding de novo interpretations. The confidence column has the values only equal to or greater than 80 to prevent very doubtful sequences from entering. The interpretation has a series of conditioning factors that make the identities indistinguishable: I and L; K and Q; F and M (ox).

Determination of total carbohydrate content

The total carbohydrate content of the lyophilized aqueous extracts was determined by the phenol-sulfuric method (Analytical Chemistry, 1956, 28 (3): 350-356). The extracts were resuspended in milliQ water at a concentration of 0.2 g / l and analyzed in duplicate, the results being expressed as the mean value calculated with their standard deviation.

Analysis of antihypertensive activity

In vitro evaluation of the inhibitory activity of ACE (angiotensin converting enzyme I), according to the protocol described by Sentandreu and Toldrá (Nat. Protoc. 2006, 1: 2423-2427). The renin-angiotensin system constitutes the major blood pressure regulating system in the human body. In this system, ACE transforms the decapeptide angiotensin I into the vasoconstrictor peptide angiotensin II and, in turn, deactivates the vasodilator bradykinin. In addition, this enzyme plays an important role in the control of blood pressure. The IC50 value (concentration of spirulina extract necessary to inhibit 50% of ACE activity) is used to express the in vitro ACE inhibitory capacity of the extract.

ACE activity was determined by following the generation of fluorescence due to the release of the fluorescent product o-aminobenzoylglycine (Abz-Gly) from the non-fluorescent substrate Abz-Gly-Phe (NO2) -Pro. The ACE inhibitory activity of the samples was measured in triplicate. The emitted fluorescence was determined every minute for 30 minutes at emission and excitation wavelengths of 355 and 405 nm, respectively, on a Synergy HT fluorimeter microplate (Biotek, Winooski, VT, USA). The IC50 value is determined by dose-response curves in which the protein concentration range (0-0.8 mg / ml) was distributed along a logarithmic scale. and using the fit function to a sigmoidal non-linear regression curve in GraphPad Prism 4.00 (Graphpad Software Inc., San Diego, CA, USA).

Analysis of anti-hyperlipidemic activity: inhibition of pancreatic lipase The activity of the extracts in inhibiting the activity of pig pancreatic lipase was carried out based on the method previously described by Lee et al., Slightly modified (Biochim Biophys Acta 1993; 1169: 156-164). Briefly, p-nitrophenyl butyrate (PNPB, from Sigma) was dissolved in acetonitrile giving a stock solution of 13.33 mM concentration, which was stored at -20 ° C. Pig pancreatic lipase was dissolved in Milli-Q water at an Anal concentration of 15 mg / ml.

All reactions were carried out with a substrate concentration of 0.4 mM PNPB in 0.061 M Tris-HCl buffer, pH 8.5 at 37 ° C for 5 minutes. The increase in absorbance of p-nitrophenol released in the presence of 1.5 mg / ml of porcine pancreatic lipase was measured at 400 nm in a UV-vis spectrophotometer (Jasco V-630 Spectrophotometer) for 3 minutes. Subsequently, the reaction rate was determined under identical conditions in the presence of a concentration of 0.75 mg / ml of the corresponding spirulina extract. The difference between the enzymatic activity of the assay without extract and with extract was defined as the inhibitory activity of the extract. The activity test was carried out in triplicate for each extract, the results were averaged and expressed with their standard deviations.

Analysis of anti-hyperlipidemic activity: inhibition of the pancreatic cholesterol esterase enzyme

The ability of the extracts to inhibit pancreatic cholesterol esterase was determined, using the method described by Pietsch and Gütschow (J. Med. Chem. 2005; 48: 8270-8288). The enzyme plays an essential role in hydrolyzing cholesterol esters of the diet. Generally, the hydrolysis of cholesterol esters takes place in the lumen of the small intestine mediated by pancreatic cholesterol esterase (CEase), producing cholesterol. The inhibitory capacity of some compounds makes it possible to control the bioavailability of cholesterol from cholesterol esters, and thus limit the absorption of free cholesterol into the bloodstream.

The buffer solution contained 100 mM sodium phosphate and 100 mM NaCl, pH 7.0. A fresh stock solution of CEase (0.075 mg / ml) was prepared in the buffer and stored at 0 ° C until use. A stock solution of taurocholic acid (TC, 8.6 mM) was prepared in the buffer solution and kept at 4 ° C until its use. The stock solution of PNPB (13.33 mM) was prepared in acetonitrile. In the cuvette, the enzyme extracts (75 pg / ml) were incubated at 25 ° C with a mixture containing 5.16 mM taurocholic acid, 0.2 mM PNPB in 0.1 M sodium phosphate buffer, 100 mM NaCl at pH 7.0. The reaction was initiated by the addition of porcine pancreatic cholesterol esterase (2.5 pg / ml). The absorbance variation of the mixtures was measured in a UV-vis spectrophotometer at 405 nm.

Analysis of antioxidant activity

Antioxidant analysis methods are classified into two types: i) those based on a hydrogen atom transfer reaction (HAT) and ii) those based on a reaction with electron transfer (ET). Generally, in HATs the antioxidant and the substrate compete for thermally generated peroxide radicals in the decomposition of azo compounds (eg, ORAC method). ET methods measure the antioxidant's ability to reduce to an oxidant, which changes color as it is reduced. The color change correlates with the antioxidant concentration of the sample. Among the ET tests are those for the determination of total phenols by Folin-Ciocalteu reagent (FCR) and some for the determination of Trolox equivalence antioxidant capacity (TEAC). FCR is used to quantify the reducing capacity of antioxidants and the ORAC method to quantify the capacity to scavenge peroxide radicals.

It is common to find different values for different antioxidant capacity techniques. This is one of the reasons why it is recommended to measure the antioxidant capacity by more than one method and that is why they are expressed in TEAC. For a comprehensive study of antioxidants in a sample, in addition to these commonly accepted tests (ABTS, ORAC and FCR), it is recommended to carry out specifically validated tests for it (J Agric Food Chem. 2005, 53 (6): 1841-56) .

All aqueous extracts were analyzed by ABTS, ORAC and FCT.

• ABTS test.

The method described by Re et al. (Biol. Med. 1999; 26, 1231-1237), which measures the ability of antioxidant compounds to sequester a stable radical cation (ABTS '+). The preformed monocation radical of 2,29-azinobis- (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS ^ 1) is generated by oxidation of ABTS with potassium persulfate and is reduced in the presence of hydrogen-donating antioxidants. The influences of both the antioxidant concentration and the duration of the cationic radical absorption inhibition reaction are considered to determine the antioxidant activity. The results are expressed by the TEAC value ( trolox equivalent antioxidant capacity), which is the concentration (mM) of the solution a standard reference (Trolox) with antioxidant capacity equivalent to that of a 1 mM solution of analyte investigated. Results are expressed as TEAC values (mmol trolox / g extract). The antiradical activity with ABTS was determined in quadruplicate, being the reproducibility of the ABTS method of 5%.

• Analysis of antioxidant activity by determining the sequestration of hydroxyl radicals ( Hydroxyl radicalscavenging assay, ( ORAC-Fluorescein) Assay).

The method described by Dávalos et al. (J. Agric. Food Chem. 2004; 52, 48-54). The hydroxyl radical (OH *) is a radical generated in the human body, which plays an important role in tissue damage with inflammatory processes of diseases caused by oxidative stress.

In both analyzes (ABTS and ORAC) a SynergyTM HT multimodal microplate reader with automatic sample dispenser and temperature control from Biotek Instruments (VT, USA) was used.

Biotek Gen5TM was the software used for data analysis. Each 96-well plate was tested four times, with four levels of standards for calibration and eight replicates per blank or control. The reaction was initiated by the automatic addition of 60 ql of the ABTS radical in the solution or of the AAPH for the ABTS and ORAC tests, respectively. The antiradical activity with ABTS was determined in a standard way after 10 minutes of reaction. For the ORAC method the values were taken after 180 minutes of reaction.

The antiradical activity with the ABTS was determined in quadruplicate and the ORAC test in triplicate. The reproducibility of some of the samples in ORAC, specifically the control sample, is higher than normal (approx. 10%). The reproducibility of the ABTS method is usually 5%.

• Determination of the total content of polyphenols by FCT

The Folin-Ciocalteu method (Nature Protocols, 2007; 2 (4): 875-877) was used, which determines the content of polyphenols and other antioxidants. Although aromatic amines, phenols, high levels of sugars and ascorbic acid interfere with the determination, this method reduces the interference of ascorbic acid by 15%.

The lyophilized aqueous extracts were resuspended (10g / l) in 95% (v / v) methanol / water, and were subjected to an ultrasound bath (20 kHz) for 10 minutes to better dilute them in the solution. The results are expressed as the mean value of values obtained in two replications with their standard deviation.

Example 2.

Optimization of the enzymatic treatment parameters for the oil extract

The optimization of the enzymatic treatment of spirulina biomass was carried out by studying the effect of different reaction parameters on the recovery of oily spirulina biocomponents. The study allowed to determine the optimal values of the pH of the medium, temperature and biocatalyst load in the enzymatic reaction for the recovery of hydrophobic spirulina biocomponents.

The spirulina biomass used in the study was a commercial lyophilized powder for food use supplied by the company ASN Spirulina Leader SL. All solvents used both in the extraction and in the analyzes were HPLC grade supplied by Scharlau. Both the hexadecane and the salts for the preparation of the solutions with purity of more than 99% were supplied by Sigma-Aldrich (Madrid, Spain).

The enzymes used in the study were: Alcalase, commercially known as Alcalase® 2.4 L FG (EC 3.4.21.62), Flavourzyme® protease; Ultraflo® Max endoglucosidase, and Vinoflow® Max exoglucanase, Lipozyme® 100L lipase and Celluclast® Cellulase. All of them were supplied by Novozymes.

The study of all the parameters was carried out by means of analysis of the different oil phases by HPLC-ELSD, following the variations in the total area of the major peaks. An example chromatogram is shown in Figure 1. The peak eluted at 9 min. corresponds to hexadecane used as an internal standard. All the experiments were carried out in duplicate or triplicate, giving the mean value of the values obtained and their error calculated as the standard deviation.

- Effect of pH on enzyme-assisted extraction

The effect of the pH of the enzymatic treatment on the recovery of oily biocomponents with six different commercial enzymes was carried out with an enzyme load of 1% (v / p) (volume of enzyme solution per total weight of reaction mixture: biomass and buffer) at 40 ° C. The study was carried out in duplicate, taking aliquots of each biomass-enzyme mixture after 4 h. The optimal pH values were determined considering the Total area values of chromatographic peaks obtained in the analysis of the oil extract (Figure 2).

For optimal obtaining of the oily extract with the two proteases studied (Alcalase® and Flavourzyme®), the optimal pH values were 6.5 and 6.0, respectively (Figure 2). These pH values made it possible to obtain the maximum values of the total area of peaks with enzymatic treatments of 4 h. In the case of Ultraflo®, an optimum pH value of 7.0 was found. In the case of Vinoflow®, only the optimum pH value of 6.5 yields the maximum amount of biocomponents extracted with 4h of enzymatic treatment, suggesting that at this pH value the degradation of the biomass is faster. For the enzymes Lipozyme® and Celluclast®, the optimal extraction is obtained at pH 6.5.

In summary, the optimal pH values determined were pH 6 to obtain the oily extract with Flavouzyme®, pH 6.5 for the extraction with Alcalase®, Vinoflow®, Lipozyme® and Celluclast®, and pH 7.0 for that of Ultraflo ®. These values are within the range of operating conditions declared by the manufacturer Novozymes Ltd (Denmark) for these enzyme preparations.

The optimal values of temperature and biocatalyst loading were then determined at the respective optimal pH values of the different extractive enzymatic processes.

- Study of temperature

This study was carried out at the respective optimal pH values of each enzyme in the range 25-50 ° C (Figure 3).

The effect of the temperature of the biomass degrading stage (stage a) in the extractive process was studied by taking aliquots of the biomass-enzyme mixture after 4 h and 24 h of treatment. Figure 3 shows the total peak area values of the HPLC-ELSD chromatograms of the oil phase obtained with Alcalase®.

Considering the experimental error of the set of values taken at 4h and 24h, an optimum temperature value of 30 ° C was determined for the extractions with the two proteases (Alcalase® and Flavourzyme®), for Ultraflo® (with p-glucanase and xylanase, along with other secondary ones such as cellulase, hemicellulase, and pentosanase), for Celluclast® cellulase and Lipozyme® lipase. This value corresponds to the lowest temperature at which the maximum value for the total area of peaks of oily biocomponents extracted with these five different 24-hour enzymatic treatments can be obtained. Although for some enzymes the values at short times (4h) are higher at another temperature, at long times the values at those temperatures are lower than those at 30 ° C, indicating a degradation of the products at said temperatures over long periods of time. However, at a mild temperature of 30 ° C the products susceptible to oxidation (polyunsaturated fatty acids, vitamins, antioxidants, etc.) are better preserved. In the case of Vinoflow®, the maximum concentration of biocomponents extracted in the oil phase can be reached at 40 ° C or higher (considering the experimental error). The use of higher temperatures favors the kinetics of the biomass degrading enzymatic process, reducing the time necessary to obtain the maximum extraction of biocomponents, but this entails an increase in energy expenditure and reduces both the stability of labile products and the operational stability of the biocatalyst. For this reason, a decrease in the total area observed is observed when the enzymatic treatment of the biomass is prolonged from 4h to 24h by increasing the temperature of the process with some of these enzymes (Alcalase® at 50 ° C, Flavourzyme® at 40- 50 ° C, Ultraflo® and Vinoflow® at 50 ° C). Taking into account the decrease in enzymatic stability with increasing temperature, and the thermolability of the compounds to be extracted, the temperature of 40 ° C is more favorable compared to higher temperatures for Vinoflow®.

Therefore, the values at 4h and 24h indicated that the optimum temperature could be 30-40 ° C for some enzymes. Based on the thermolability of the biocomponents (antioxidants, etc.) and considerations to minimize the energy consumption of the process, the temperature of 30 ° C was selected as optimal for the enzymatic processes with Alcalase®, Ultraflo®, Flavourzyme®, Lipozyme® and Celluclast®, and 40 ° C for Vinoflow®.

- Study of the enzymatic load

The biocatalyst load used in the degrading stage is an important parameter of the extractive process, since it affects the biodegradation rate of the biomass prior to the extraction of biocomponents with solvents, and this determines the value of the extractive yield. The optimal value of the enzyme load must be carefully determined. Although the use of a high enzymatic load reduces the time necessary to achieve the desired degradation of the biomass, this also means increasing the cost of the biocatalyst batch, and in the case of proteases it can favor self-destruction via autolysis of the biocatalyst.

The results obtained for the extraction with Alcalase® at different loads of the biocatalyst are shown in Figure 4. This study was carried out in the 0-2% v / p load interval for all the enzymes studied, except for Vinoflow® (0- 4% v / w). The results obtained in the different enzymatic extractions were compared with the obtained in the control experiment without enzyme, where instead of the enzymatic solution an equivalent amount of aqueous solution was used (Figure 4).

An optimal enzyme load value of 1% v / p was determined for all cases, except for Vinoflow®, which was 2% v / p, taking into account that either the use of higher enzymatic loads yielded lower area values or the An increase in this did not justify the use of a greater quantity of biocatalyst.

In general, the total area of peaks increases when the duration of the enzyme treatment is prolonged in the interval 0-24h with all the enzyme loads studied (Figure 4). Only in the case of Vinoflow® with 1% (v / p) enzyme does the reaction slow down after 4h, so that the amount of biocomponents recovered is practically unchanged. Also, the total area of extracted products does not increase when increasing the treatment time with Vinoflow® from 4h to 24h using the highest enzymatic load studied (4% v / p Ultraflow®). This suggests that an excessive enzyme concentration may favor the subsequent degradation of the extracted products. In addition, the recovery of oily products increases when the enzymatic load is increased from 0.5% to 1% (v / p) when the two proteases and Ultraflo® are used, but it decreases dramatically when higher loads of them are used (eg. , 2% v / w). This effect may be due to an excessive biodegradation of the biomass in the presence of an excessive concentration of enzyme. As a result, a further biodegradation of the extracted products can take place, reducing their concentration in the oily extract. In the case of proteases, the lower extractive yields of oily biocomponents may be due to rapid enzyme inactivation via autolysis favored at high enzyme concentrations.

It should be noted that the extractions with a previous degrading stage by Alcalase®, Flavourzyme®, Ultraflo® and Vinoflow® (24h of enzymatic treatment in their respective optimal operating conditions) provide significant increases in the recovered amounts of oily biocomponents, compared to those of the Extraction process without enzymatic treatment of biomass (Figure 4). This result proves the favorable contribution of the spirulina biomass degradation stage by the studied enzymes, for the extraction of the oily components.

The optimal values of enzymatic load determined are 1% v / p for all the biocatalysts studied, except for Vinoflow® (2% v / p).

In conclusion, for each enzymatic extraction, its optimal process conditions were determined, depending on the pH, temperature and time of exposure to the enzyme. (pH 6.5, 1% v / w and 30 ° C for Alcalase®, Lipozyme® and Celluclast®; pH 6.0, 1% v / w and 30 ° C for Flavourzyme®; pH 7.0, 1% v / p and 30 ° C for Ultraflo®; pH 6.5, 2% v / p and 40 ° C for Vinoflow®; the duration in all cases is 24h). The extracts of the control experiment without enzyme were obtained by adding milli-Q water instead of the buffered enzyme solution, at 30 ° C and 24h.

Example 3

Optimization of the enzymatic treatment for the aqueous extract of Alcalase®. Its comparison with other enzymatic extractions and control without enzyme.

The optimization of the enzymatic treatment of the spirulina biomass was carried out, prior to the extraction by the solvent mixture hexane / isopropanol 3: 2 (v / v) for the recovery of hydrophilic spirulina biocomponents, studying the effect of different parameters of the enzymatic reaction in the aqueous extract. This study allowed to determine the optimal values of the pH of the medium, temperature, biocatalyst load and duration of the enzymatic reaction to obtain the aqueous extract of spirulina.

The study of all the parameters was carried out by taking aliquots of the enzyme-biomass mixture at the indicated time, and analyzing the different aqueous phases extracted by HPLC-ELSD, and by comparing the total area of the significant peaks of each chromatogram. An example of a chromatogram obtained with Alcalase® is shown in Figure 5 (in Figure 6, control without enzyme). The first peak eluted up to 2.39 min. corresponds to the solvent and was not computed. In the initial study of optimization of the variables of the enzymatic processes, the values of total area of peaks in their respective chromatograms of analysis by HPLC-ELSD were compared, to determine the optimal operating conditions that allow to obtain the highest recovery rate of hydrophilic biocomponents.

The aqueous extracts obtained with the two proteases, the two (endo- and exo-) glucanases, cellulase and lipases used in this study, must contain different biocomponents, as a result of the different types of reactions catalyzed by the different biocatalysts. In all cases, the total sums of area values of the significant peaks were also comparatively analyzed.

The following shows the determination of the optimal conditions of the extraction process with Alcalase® based on pH, temperature, enzymatic load and time of exposure to the enzyme on the aqueous extract after extraction with solvents.

- Effect of pH on extraction assisted by Alcalase®

The effect of the pH of the enzymatic treatment of biomass in the recovery of aqueous biocomponents was carried out at 40 ° C with an enzymatic load of 1% (v / p) (units in volume of enzyme solution per total weight of reaction mixture: biomass and buffer). The aqueous extracts post-extraction with solvents obtained at different pH values of the enzymatic stage (stage (a)) were studied for different times of enzymatic treatment.

Figure 7 represents the variation of the total area of chromatographic peaks with pH for the extraction with Alcalase®. The results obtained on the effect of pH in the total area of the significant chromatographic peaks obtained after 4h of enzymatic action, are represented in Figure 7A. The highest initial speed (4h) of extraction of biocomponents with Alcalase® is obtained at pH 6.5 (Figure 7A). At pH 6.5 the enzymatic degradation process is the fastest, and consequently the maximum total area value of chromatographic peaks is obtained at this pH for extractions with short enzymatic treatment (4h, Figure 7A).

Also, the evolution of the enzymatic extraction with the duration of stage a) (enzymatic disintegration of the biomass) for the optimum pH value was determined during the first 48h. The results obtained with Alcalase® are represented in Figure 7B. Similar to the case of Alcalase®, the rest of the enzymatic extractions studied require 24 hours of enzymatic treatment of the spirulina biomass to obtain the maximum amount of biocomponents in the aqueous extract extracted with solvents. A longer duration of stage a) (enzymatic), for example 48 hours, yields a lower amount of biocomponents, possibly due to an enzymatic degradation of part of these (peptides by Alcalase® protease),

- Study of temperature

The effect of the temperature of step a) on the extraction of polar components was studied for 24 h of enzymatic treatment of the biomass. The study was carried out at the corresponding optimal pH value of the Alcalase® enzyme, and an enzyme load of 1% (v / p) in the 30-50 ° C range.

Figure 8 shows the total area values of chromatographic peaks of the corresponding aqueous extracts obtained with Alcalase®. In order to identify the temperature that allows the extraction of the greatest amount of hydrophilic components from For each gram of spirulina, Figure 8 represents the total area value referred to the total amount of aqueous extract obtained from 1 g of spirulina, calculated considering the total area obtained with the injected amount of extract (see below the determination extractive yields).

Considering the experimental error, 30 ° C or a lower temperature turns out to be the optimum temperature for extraction with Alcalase®. The temperature to obtain the maximum recovery of biocomponents in the aqueous extract is relatively low. Vitamins, antioxidants and other thermolabile components of the extracts can be preserved relatively well at these temperatures (30-40 ° C). Compared with higher temperatures, at these mild temperatures the energy cost and the denaturation of the extracts is reduced, while the operational stability of the biocatalysts used increases. In fact, the decrease observed in the extraction of biocomponents at higher temperatures (Figure 8) may be due to an undesirable enzymatic degradation of the biocomponents when the process is thermally accelerated and / or to the decrease in the operational stability of the enzyme.

- Study of the enzymatic load

The enzyme load value used must be carefully selected. Among other reasons, an optimal load of biocatalyst must be identified, so as to reduce the time to complete the desired degradation of the biomass to extract its biocomponents, without excessively increasing the cost of the biocatalyst (J Am Oil Chem Soc, 2009, 86 (12): 1235-1240). The catalyst load used affects the biodegradation rate of the biomass, prior to the solvent extraction stage, and determines the extractive yield at a given time.

The results obtained with different loads of Alcalase® (0-2% v / p enzyme) are represented in Figure 9. The results were compared with those obtained in the control extraction, in which an equivalent volume of milli- Q instead of the enzyme solution (0% enzyme loading in Figure 9).

The extraction of polar biocomponents increases when the enzymatic load used increases from 0.0% to 1% (v / p) of Alcalase®, but decreases dramatically when using higher loads (eg, 2% v / p; Figure 9) .

The decrease in the recovery of products when using loads higher than a certain value, suggests that with high concentrations of enzyme an excessive biodegradation of the biomass is favored. As a result, the extracted components are the result of a higher degree of biodegradation of the products extracted at loads. minors. In the case of proteases, the decrease in the extractive yields of biocomponents may also be due to a certain degree of enzymatic autolysis, favored at high concentrations of proteolytic enzymes.

An individual analysis of the evolution of the area of the different chromatographic peaks (Figure 10) indicates that some product (s) is (are) extracted almost completely with only 4h of enzymatic treatment (retention time (Tr ) = 68min, Figure 10E), while other products require longer times (24h) for stage a) (those eluted at Tr = 3-5min .; 5.5 min; 11-14min .; 20-22min .; 28min ., Figures 10 AE). These results suggest that some of the extracted biocomponents are either intracellular or may be formed by the enzymatic degrading action on cellular components (eg, peptides and / or sugars, etc).

The individual inspection of the temporal evolution of the area of the different peaks of extracted products in the aqueous extract reveals the existence of two types of extractive kinetics for the different products. These results suggest that products located in the most accessible place in the cell (eg, extracellular region) need a short time for step a) of enzymatic degradation of biomass. But, the complete extraction of the two types of biocomponents requires a duration of the enzymatic stage of 24 hours. The duration of the enzyme treatment stage is an important parameter. In this process, 24 hours are necessary to obtain the maximum extraction of intra- and extracellular components from the spirulina biomass.

Finally, it was determined that the optimal conditions of the enzymatic treatment for Alcalase® are: temperature of 30 ° C, pH 6.5, 24h and enzyme load 1% v / p.

The studies of examples 2 and 3 revealed that both the amounts of biocomponents recovered in the aqueous phase (determined based on chromatographic areas), and the composition of the different extracts, vary according to these parameters (type and load of enzyme, temporal extension of its action, temperature and working pH). Also, it was determined that the optimal operating conditions for the biomass enzymatic degradation stage are identical for the recovery of both hydrophilic and hydrophobic biocomponents (aqueous and oily extracts, respectively).

Example 4

Yields by weight of extracts under optimal conditions

The extractive processes were carried out on a larger scale (10 g of A. platensis biomass) and the corresponding oily and aqueous extracts obtained in their respective conditions were brought to dryness to determine their weight. The different enzyme extracts and the control (without enzyme) were prepared (in duplicate) in their respective optimal conditions: pH 6.5, 1% v / p and 30 ° C for Alcalase®, Lipozyme® and Celluclast®; pH 6.0, 1% v / w and 30 ° C for Flavourzyme®; pH 7.0, 1% v / w and 30 ° C for Ultraflo®; pH 6.5, 2% v / p and 40 ° C for Vinoflow®. Control extracts without enzyme were obtained by adding milli-Q water instead of the buffered enzyme solution, at 30 ° C and 24h.

All the extracts were dried (in a rotary evaporator + nitrogen stream) or lyophilized, determining their dry weight on a precision scale (4 digits). Table 1 shows the yield values in dry weight of the different phases (oily, aqueous and residual). The yields were calculated as the percentage of weight of the dry extract (or dry residual biomass) with respect to the weight of the dry biomass used. spirulina.

All the enzyme-assisted biocomponent recovery systems provided higher extractive yields of the water and oil phases than the control system without enzyme. The highest yield in weight of oily extract was obtained with Vinoflow®, while, for the aqueous extract, the extractive systems that use proteolytic biocatalysts (Alcalase® and Flavourzyme®) gave the highest yield in dry weight; although the highest yield in the aqueous extract was that of Alcalase®. The enzymatic extraction process with Alcalase® allowed to increase 1.5 and 1.9 times the weight of the oily and aqueous extracts, respectively, with respect to those obtained in the control without enzyme (Table 1).

Table 1 . - Yields in weight of the Extraction under optimal conditions _____

EXTRACTION YIELDS (%)

Enzyme PHASE

Organic Aqueous Residual Biomass Alcalase® 6.7 ± 0.1 36.5 ± 0.1 47.7 ± 0.4 Flavourzyme® 6.4 ± 0.1 31.8 ± 0.1 47.8 ± 1.0

Ultraflo® 5.9 ± 0.1 19.7 ± 0.1 61.4 ± 1.2

Vinoflow® 8.1 ± 0.2 26.3 ± 0.1 57.1 ± 0.3

Lipozyme® 5.0 27.0 66.6

Celluclast® 4.9 26.1 69.3

Control 4.7 ± 0.2 19.2 ± 0.2 70.1 ± 0.4 Alcalase® * na 42.9 56.1 Flavourzyme® * na 41.8 56.8

Control * n.d. 49.0 50.2

* Yields of the extraction of biocomponents from stage a), without using solvents post-enzymatic treatment. n.d .: not determined.

When the recovery of the aqueous extract is carried out without adding the hexane / isopropanol solvent mixture, the weight yields of the aqueous phases and residual biomass vary with respect to the extraction using these solvents (Table 1, indicated as *), such that greater weight of aqueous phase is obtained without the use of these solvents. A fact that is particularly notable in the case of the control extraction without enzyme. This is probably due to the difficulty of separating the oily material well, which, by not withdrawing to the hexane phase, remains firmly adhered to the residual biomass and partially emulsified in the aqueous phase. Unlike what happens using the hexane / isopropanol mixture to extract the biocomponents, the aqueous phases of the extractions with Alcalase® and Flavouzyme® recovered without the use of these solvents have less weight than their respective control extraction without enzyme. This is due to the extraordinary increase in the recovered weight of the aqueous phase of the solvent-free control extraction compared to the same control extraction using solvents for the recovery of biocomponents. These extracts without the addition of organic solvents also have yield losses by weight due to the same reasons as the other extracts (filtration operations, etc.).

For the extracts recovered with the solvent mixture from the weight yields of the oily and aqueous extracts (Table 1), the units of total area of peaks of the HPLC-ELSD analyzes of the different optimal extracts (enzymatic and control without enzyme) referred to 1 gram of biomass of initial A. platensis. The calculated value is indicative of the total amount of biocomponents extracted from 1 g of dry spirulina biomass in the different extractive processes. The comparative study of the values obtained for the different extractive processes is represented in Figures 11 and 12 for the aqueous and oily extracts, respectively.

Figure 11 represents the different values of total area of peaks of oily biocomponents corresponding to one gram of biomass extracted with the different enzymatic treatments in their respective optimal conditions of operation. All the extractive processes after a step a) of enzymatic digestion of the biomass make it possible to obtain the largest amount of oily biocomponents detected by HPLC-ELSD, indicating that the action of all the enzymes facilitates the recovery of intracellular oily biocomponents. Vinoflow® provides the highest recovery of biocomponents detected by HPLC-ELSD.

Comparison of the values obtained for the different aqueous extracts (Figure 12) indicates that the best enzymatic method (1% v / p Alcalase®, pH = 6.5, 30 ° C and 24h) allows to increase the recovery of polar biocomponents per gram of spirulina 2.5 times with respect to that obtained with simple solvent extraction (control), which is a consequence of the fact that the Alcalase® method increases the weight of the aqueous extract by 90% (1.90 times greater% p / p) and the different composition of the extracts obtained with the different enzymes (see Examples 6 and 7). The amount of hydrophilic biocomponents detected by HPLC-ELSD, extracted from each gram of spirulina with Alcalase® is 3 times greater than that obtained with the other protease (Flavourzyme®). For the aqueous extract, not all enzyme systems increase the amount of biocomponents extracted, the amounts obtained with Vinoflow®, Lipozyme® and Celluclast® being similar to that obtained in the control without enzyme.

These results demonstrate the superiority of the Alcalase® extractive process for the recovery of hydrophilic spirulina biocomponents.

Example 5

Morphological analysis of the biomass.

The highest net extraction of biocomponents and the least amount of residual biomass) obtained by treatment with Alcalase®, was also visualized by transmission electron microscopy (TEM) of the residual biomass, being that of Alcalase® the one that was visualized more disintegrated and translucent, indicating greater disappearance of its biocomponents, with respect to the other cases (Figures 13-14).

The visualization of the residual biomass under the electron microscope corroborates the extractive superiority of the process components with Alcalase® with respect to other enzymatic processes studied and to the control without enzyme.

Example 6

Composition analysis in amino acids of the aqueous extracts obtained under optimal conditions of the extractive processes

The quantitative analysis of the amino acid mixtures was carried out according to the procedure described in example 1. Figure 15 represents the total content of moles of amino acids extracted per g of biomass with each extractive system.

The aqueous extract obtained with Alcalase® has a higher content of total amino acids (45% w / w lyophilized extract) than the control extract without enzyme (34% w / w) and than those of the other enzymes (13-17%).

The extract obtained with the Alcalase® method is particularly rich in Glu, Asp and Leu; The increase in the recovery of Tyr moles stands out (3.7-10.8 times higher than with the other methods). The Alcalase® method allows to recover up to 7.2 and 10.8 times more moles of Arg and Tyr, respectively, and for the other elements the Alcalase® method is 2.6-3.3 times higher than the method of protease (Flavourzyme®).

Example 7

Identification of biocomponents of the aqueous extracts by LC ESI-MS MS The biocomponents of the different aqueous extracts separated with solvents (enzymatic and control) were analyzed and identified by LC ESI-MS MS in positive ionization mode, using the methodology described in the Example 1. The conditions for obtaining the extracts were determined as optimal: pH 6.5, 1% v / p and 30 ° C for Alcalase®, Lipozyme® and Celluclast®; pH 6.0, 1% v / w and 30 ° C for Flavourzyme®; pH 7.0, 1% v / w and 30 ° C for Ultraflo®; pH 6.5, 2% v / w and 40 ° C for Vinoflow®; the extract of the control without enzyme was obtained by adding milli-Q water at 30 ° C instead of the buffered enzymatic solution and 24h.

The chromatographic profiles obtained with the Bruker AmaZon trap of the equipment used are represented in Figure 16, where the variation in signal intensity received by the detector (a system based on dinodal conversion (Daly detector)) is represented. The different chromatograms indicated that all the extracts were composed of a significant number of biocomponents. It was also observed that all the extractive enzyme systems gave a greater number and quantity of biocomponents in the aqueous extract than the non-enzymatic control (Figure 16).

The chromatographic column used resolves by hydrophobic interaction with the products, eluting those with lower polarity at longer times. In the control extract, three peaks are observed that do not appear in the rest of the extracts: two narrow bands at 46 and 63 min. medium intensity, and a wide and high band at high retention times (66-94 min .; Figure 16). The greater degradation of cellular material observed by TEM in the case of the enzymatic extracts compared to the control extract (non-enzymatic) corresponded to the fact that the enzyme extracts presented a greater number of biocomponents in the analysis by LC ESI-MS MS, there are several zones of the chromatogram with biocomponents, which do not appear in those of the control extract (especially the elution time zone of 50-70 min., Figure 16). The different enzyme extracts presented different biocomponents (Figure 16). The greater disappearance of the cellular material observed by TEM in the case of the Alcalase® extract, corresponded to the fact that this Alcalase® extract presented a greater number of biocomponents in the analysis by HPLC-MS-MS, with several areas of the chromatogram with biocomponents, which do not appear in the other extracts. (Major peaks at 53 min., 60 min. and 80 min., Figure 16). The Flavourzyme® chromatogram shows peaks at retention times between 35 and 45 min. nonexistent in the comatograms of the rest of the extracts (Figure 16).

According to Table 2, 8 different peptides have been detected in the Alcalase® extract from those detected in the rest of the extracts (enzymatic or control). Specifically, it has at least 11 families of exclusive peptides, corresponding to different proteins of Arthrospira sp. with its corresponding biological activity: MKKIEAIIRPF (SEQ ID NO: 1), (nitrogen regulatory protein P-II [Arthrospira platensis NIES-39]); LPPL (SEQ ID NO: 2), no assignment; ALAVGIGSIGPGLGQGQ (SEQ ID NO: 3), (AtpH [Arthrospira platensis HN01]); TTAASVIAAAL (SEQ ID NO: 4), (ATP synthase c chain [Arthrospira platensis NIES-39]); DFPGDDIPIVS (SEQ ID NO: 5), (Full = Elongation factor Tu; Short = EF-Tu), LELL (SEQ ID NO: 6), WKLLP (SEQ ID NO: 7) and CHLLLSM (+15.99) (SEQ ID NO : 8); the last three unassigned. In an even more particular embodiment, the extract of the invention comprises the peptides SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 , SEQ ID NO: 7 and SEQ ID NO: 8

Table 2 . -Peptide sequence, retention time ( Rt, minutes), calculated and experimental molecular mass ( Da), and mark or score of the peptides identified in the aqueous extracts of A. platensis using LC ESI-MS MS.

Extract ^RT Peptide Sequence Mr. M. Exp

_min. I know . ID: Da Calc. _Da Marca

Example 8

Determination of the bioactive properties of aqueous extracts

A comparative study of the antihypertensive, antihyperlipidemic (anti-hypertriglycerolémic and anti-cholesterolemic), and antioxidant properties of the aqueous enzymatic and control extracts was carried out.

Analysis of anti-hypertensive activity

The anti-hypertensive activity values of all the extracts are summarized in Table 3. The treatment with Alcalase® shows the highest ACE inhibitory activity, being 3.7 times higher than that of the control extract. The subsequent extraction with solvents allows to increase this activity against the control even more, up to 7.3 times.

Table 3. - Anti-hypertensive activity. In vitro evaluation of the inhibitory capacity of ACE ( angiotensin converting enzyme

^IC 50 A enzyme extracted * control _{(mg /} A / A _{ml) (ml)}

Alcalase® (protease) 0.050 ± 0.006 7.3 ± 0.9 7.3 Flavourzyme® (protease) 0.236 ± 0.012 1.3 ± 0.1 1.3

Ultraflo® (endoglucanase) 0.178 ± 0.029 1.1 ± 0.2 1.1 Vinoflow® (exoglucanase) 0.404 ± 0.029 0.7 ± 0.0 0.6 Celluclast® (cellulase) 0.216 ± 0.019 1.2 ± 0, 1 1.2 Lipozyme® (lipase) 0.255 ± 0.054 1.1 ± 0.2 1.0

Control ( Without enzyme) 0.189 ± 0.028 1.0 ± 0.2 1.0

Alcalase® ( protease) *** 0.105 ± 0.004 4.1 ± 0.2 3.7

Flavourzyme® ( protease) *** 0.397 ± 0.063 1.1 ± 0.2 1.0

Control ( No enzyme) *** 0.446 ± 0.052 1.1 ± 0.1 1.0

* The anti-hypertensive activity extracted from 1 gr biomass, calculated as the product of the inverse of the IC50 value by the weight obtained from the corresponding extract from 1 gr of spirulina (yield (%) / 10 g biomass extracted).

** List of units of activity extracted with each enzymatic method and with the control

*** Values of the aqueous extraction of step a), without using solvents after enzymatic treatment.

Analysis of anti-hyperlipidemic activity: inhibition of pancreatic lipase

Hyperlipidemia includes different metabolic disorders that lead to

hypertriglyceridaemia and / or hypercholesterolemia in the bloodstream. In the long run, everything

this leads to vascular complications (microangiopathy, diseases

cardiovascular, cerebrovascular and / or metabolic syndrome).

All enzyme extracts have a higher inhibitory capacity than the

control extract, being 35-62 times higher than those (Table 4). The two excerpts

obtained with proteases (Alcalase® and Flavourzyme®) were found to have the highest activity

inhibitory, which was similar for both extracts.

Regarding the extractive processes, the two protease processes (Alcalase® and

Flavourzyme®) were found to extract the highest inhibitory activity, being 106 and 94 times

higher than that obtained with the control extract.

Table 4. - In vitro inhibitory activity of pig pancreatic lipase.

Enzyme _(Aab ^A Inhibition A ^{ex tra c to} A ^{ex tra id a}

_{s / s) *} (AAbs x L / s. g (AAbs x L / s. g A / Acontrol (%) extract) biomass)

Without Inhibitor 0.368 ± 0.008 - - - -

Alcalase®

(protease) 0.256 ± 0.007 30.3 ± 1.9 0.149 ± 0.019 54.5 ± 6.8 106 Flavourzyme®

(protease) 0.254 ± 0.008 31.1 ± 2.1 0.152 ± 0.020 48.3 ± 6.4 94 Ultraflo®

(endoglucanase) 0.282 ± 0.003 20.7 ± 0.7 0.115 ± 0.013 22.6 ± 2.6 44 Vinoflow®

(exoglucanase) 0.304 ± 0.009 17.5 ± 2.2 0.085 ± 0.021 22.4 ± 5.6 44 Celluclast®

(cellulase) 0.366 ± 0.008 0.5 ± 2 0.002 ± 0.020 0.6 ± 5.2 1 Lypozime®

(lipase) 0.368 ± 0.008 0.0 ± 4.6 0.000 ± 0.020 0.0 ± 5.4 0 Control

(Without enzyme) 0.366 ± 0.012 0.5 ± 3.0 0.003 ± 0.011 0.5 ± 2.1 1 * units of activity as absorbance increase per second, for 750 mg / l extract in the cuvette

** List of units of activity extracted with each enzymatic method and with the control

Analysis of anti-hyperlipidemic activity: inhibition of the pancreatic cholesterol esterase enzyme

Results are presented as inhibition (%) of pancreatic cholesterol esterase (Table 5).

All the enzyme extracts showed greater inhibitory activity than the control, being that obtained with Alcalase 16 times greater than that of the control and 1.1-3.5 times greater than that of the rest of the enzyme extracts. For each gram of biomass, the Alcalase® method allows the extraction of 26 times more hypocholesterolemic products than the control without enzyme.

Table 5 . - Hypocholesterolemic activity determined by inhibition of pancreatic cholesterol esterase.

Extract A extracted

A Inhibition (AAbs x (AAbs x L / sec A / Acontrol Enzyme

(Aabs / sec) * (%) L / sec. g x g biomass) **

abstract)

Without Inhibitor 0.515 ± 0.007 - - - -Alcalase®

0.432 ± 0.006 16.0 ± 1.2 1.11 ± 0.17 0.404 ± 0.063 26 (protease)

Flavourzyme® 0.439 ± 0.008 14.7 ± 1.6 1.01 ± 0.20 0.322 ± 0.064 21 (protease)

Ultraflo®

0.491 ± 0.006 4.5 ± 1.2 0.32 ± 0.17 0.063 ± 0.034 4

(endoglucanase)

Vinoflow®

0.452 ± 0.011 12.2 ± 2.1 0.84 ± 0.24 0.221 ± 0.063 14

(exoglucanase)

Control

0.509 ± 0.006 1.0 ± 1.2 0.08 ± 0.17 0.015 ± 0.033 1

(Without enzyme)

* Activity ( A) in units of activity as an increase in absorbance per second for 75 mg / l extract.

** List of units of activity extracted with each enzymatic method and with the control

Analysis of antioxidant activity

The antioxidant activity of the lyophilized extracts was determined by three different methods: by the ABTS test, which measures the ability of antioxidant compounds to sequester a stable radical cation (ABTS '+), by the ORAC test, in which the Hydroxyl radical sequestration ( ORAC-Fluorescein) Assay), by analysis of total polyphenols by FCR.

A) ABST and ORAC methods

The results of the ABTS and ORAC methods are expressed by the TEAC value ( Trolox Equivalent Antioxidant Capacity), which is the concentration of the standard reference solution (Trolox, mM) with antioxidant capacity equivalent to that of a 1 mM solution of analyte under investigation. . The results are expressed as mmol Trolox per g extract.

The results obtained are collected in Table 6, where it can be seen that all the extracts have antioxidant activity. It is common to find different values for different antioxidant capacity techniques and that is why they are expressed in TEAC, as in this case. This is one of the reasons why it is recommended to measure the antioxidant capacity by more than one method. By the two methods used, the extracts have antioxidant activity, and the Alcalase® process allows extracting more antioxidant activity than the control. By the ORAC method, the Alcalase® and Ultraflo® extracts have higher antioxidant activity values than the rest. In the Alcalase® process, for each gram of spirulina a higher value of antioxidant activity is extracted than with the other processes by the ORAC method, this being 4.4 times greater than the amount obtained with the control process, and 1.6 times greater than with the other protease. The antioxidant capacity extracted with Alcalase®, determined by the ABTS method, turned out to be 1.5 times greater than that obtained with the control extraction, and 1.4 times greater than that of Flavourzyme®.

Table 6. - Antioxidant Activity determined by ABTS and ORAC methods.

ABTS ORAC *

TOTAL TOTAL EXTRACT R ** EXTRACT R ** EXTRACTED EXTRACTED

Enzyme

TEAC TEAC TEAC TEAC

(pmol / g (pmol / g (pmol / g (pmol / g

Biomass extract) Biomass extract) lyophilized) lyophilized)

Alcalase®

19.7 ± 0.5 7.19 ± 0.18

(protease) 1.5 462 ± 19 169 ± 7 4.4

Flavourzyme®

15.5 ± 0.5 4.93 ± 0.16

(protease) 1.0 325 ± 14 107 ± 4 2.8

Ultraflo®

24.1 ± 0.6 4.75 ± 0.12

(endoglucanase) 1.0 269 ± 12 53 ± 2 1.4

Vinoflow®

16.4 ± 0.6 4.31 ± 0.16 0.9 260 ± 5 68 ± 1

(exoglucanase) 1.8

Celluclast® 31.5 ± 0.8 8.22 ± 0.22 1.7 - - -Lipozyme® 28.7 ± 0.4 7.75 ± 0.10 1.6 - - -Control (Without

23.4 ± 0.9 4.78 ± 0.12

enzyme) 1.0 199 ± 16 38 ± 3 1.0

* Kinetic values obtained at 180 min.

** Ratio of units of activity extracted with each enzymatic method and with the control without enzyme

B) Determination of the total content of polyphenols.

The Folin-Ciocalteu (FCT) method described in Example 1 was used, which determines the content of polyphenols and other antioxidants. The results shown in Table 7 indicate that all enzymatic methods increase the recovery capacity of polyphenols. From each gram of spirulina, with Alcalase® you obtain 2.3 times more weight of polyphenols than that of the control and 1.4-2.1 times more than with the other enzymes (1.12 times more than with Flavourzyme) .

Table 7 . - Polyphenols recovered in the different extractive processes

Enzyme ^Polyphenols Polyphenols R *

_{(mg / g extract)} (mg / g biomass)

Alcalase® (protease) 12.1 ± 0.1 4.16 ± 0.23 2.3 Flavourzyme® (protease) 9.3 ± 0.0 2.95 ± 0.01 1.6 Celluclast® (cellulase) 8 .2 ± 0.1 2.15 ± 0.02 1.2 Ultraflo® (endoglucanase) 11.0 ± 0.1 2.16 ± 0.02 1.2 Vinoflow® (exoglucanase) 8.0 ± 0.1 2.11 ± 0.04 1.1 Lipozyme® (lipase) 7.3 ± 0.1 1.98 ± 0.03 1.1 Control 9.6 ± 0.0 1.84 ± 0.01 1.0 * Ratio of the weight of polyphenols extracted per gram of spirulina with each enzymatic method and with the control method without enzyme

Determination of total carbohydrate content

The total carbohydrate content of the lyophilized aqueous extracts was determined using the phenol-sulfuric method described in Example 1. The results shown in Table 8 indicate that Flavourzyme® is the enzyme that allows the extraction of the greatest amount of carbohydrates. 2.1 and 1.6 times more carbohydrates are extracted from each gram of biomass with this enzyme than with the control process and with Alcalase®, respectively. The Alcalase® extracts 1.3 times more carbohydrates than the control.

Table 8 . - Carbohydrates recovered in the different extractive processes.

^Carbohydrate Enzyme Carbohydrates

_{(mg / g extract)} (mg / g biomass) ^{R *}

Alcalase® (protease) 226.3 ± 13.0 82.6 ± 4.7 1.3 Flavourzyme® (protease) 425.5 ± 25.7 135.3 ± 8.2 2.1 Celluclast® (cellulase) 308 , 2 ± 13.9 80.4 ± 3.6 1.2 Ultraflo® (endoglucanase) 122.0 ± 17.7 24.0 ± 3.5 0.4 Vinoflow® (exoglucanase) 337.0 ± 4.2 88.6 ± 1.1 1.4 Lipozyme® (lipase) 327.6 ± 7.8 88.4 ± 2.1 1.4 Control 337.0 ± 43.8 64.6 ± 8.4 1.0 * Ratio of the weight of carbohydrates extracted per gram of spirulina with each enzymatic method and with the control method without enzyme

Claims (17)

1. Method for obtaining an extract of Arthrospira sp. characterized in that it comprises the following stages: a) incubate the biomass of Arthrospira sp. in water or in an aqueous solution buffered at a pH between 3 and 14 in the presence of the enzyme Alcalase®; Y b) separating the biomass from the solution obtained in step a) to obtain the extract. 2. Method according to claim 1, wherein the method comprises an additional step comprising the addition of an organic extraction solvent between steps a) and b). 3. Method according to claim 2, wherein the organic solvent is hexane, isopropanol or a mixture of both. 4. Method according to any one of claims 1 to 3, wherein the extract obtained in step b) comprises the aqueous phase or the oily phase of the solution obtained in a). 5. Method according to any one of claims 1 to 4, wherein Arthrospira sp. it is A. platensis or A. maximum. 6. Method according to any one of claims 1 to 5, wherein step (a) is carried out at a temperature of 30 ° C. 7. Method according to any one of claims 1 to 6, wherein the aqueous solution of step (a) is at a pH of 6.5. 8. Method according to any one of claims 1 to 7, wherein the enzyme load in the solution of step (a) is 1% v / w. 9. Method according to any one of claims 1 to 8, wherein after step (b) the separation of the aqueous, oily and residual biomass phases is carried out, by means of centrifugation or decantation. 10. Extract of Arthrospira sp. obtained by a method according to any one of claims 1 to 9. 11. Extract according to claim 10, wherein said extract comprises at least 1, 2, 3, 4, 5, 6, 7 or 8 peptides selected from the list consisting of MKKIEAIIRPF (SEQ ID NO: 1), LPPL (SEQ ID NO: 2), ALAVGIGSIGPGLGQGQ (SEQ ID NO: 3), TTAASVIAAAL (SEQ ID NO: 4), DFPGDDIPIVS (SEQ ID NO: 5), LELL (SEQ ID NO: 6), WKLLP (SEQ ID NO: 7) and CHLLLSM (+15.99) (SEQ ID NO: 8). 12. Pharmaceutical, food or cosmetic composition comprising an extract according to claim 10 or 11 and, optionally, further comprising at least one pharmaceutical, food or cosmetically acceptable excipient or vehicle, respectively. 13. An extract according to any one of claim 10 or 11 for use as a medicament. 14. Extract according to claim 10 or 11 for use in the treatment and / or prevention of a disease selected from the list consisting of hypertension, anemia, malnutrition, hyperlipidemia, obesity and diabetes. 15. Extract according to claim 10 or 11, or the composition according to claim 12, for use as an anti-hypertensive or anti-hyperlipidemic agent. 16. Extract according to claim 10 or 11, or the composition according to claim 12, for use as an antioxidant agent. 17. Use of the extract according to claim 10 or 11, or the composition according to claim 12, to prepare an easily assimilated protein concentrate.