Method for the preparation of a cyanobacteria extract comprising phycocyanin enriched in trace elements from natural trace element sources for the production of food supplements

Abstract

The invention relates to a method for preparing a cyanobacterium extract, preferably spirulina, comprising phycocyanin enriched in trace elements. The invention also relates to an extract obtained by the method according to the invention, to a composition comprising said extract and to the use thereof.

Description

[0001] PROCESS FOR PREPARING A CYANOBACTERIA EXTRACT COMPRISING PHYCOCYANIN ENRICHED WITH TRACE ELEMENTS FROM NATURAL SOURCES OF TRACE ELEMENTS FOR THE MANUFACTURE OF FOOD SUPPLEMENTS

[0002] TECHNICAL FIELD

[0003] The invention relates to the field of food supplements and more particularly to a process for preparing a cyanobacterium extract, preferably spirulina, comprising phycocyanin enriched with trace elements from natural sources of trace elements for the manufacture of food supplements.

[0004] It also relates to a composition comprising such an extract of cyanobacteria, preferably spirulina, for its use in the treatment of micro-nutritional deficiencies as well as its use as a food supplement.

[0005] PRIOR TECHNIQUE

[0006] Spirulina is a photosynthetic microorganism belonging to the cyanobacteria of the genus Arthrospira. Spirulina is known to be naturally rich in vitamins such as vitamins A, E, D, B1, B2, B3, B6, B7, B8 and K, trace elements such as zinc, selenium, manganese, iron, copper or chromium and minerals such as calcium, magnesium, sodium, potassium or phosphorus.

[0007] In recent years, studies have shown that spirulina can stimulate a large number of physiological mechanisms and strengthen immune defenses.

[0008] For example, the publication entitled “Impact of daily supplementation of Spirulina platensis on the immune system of naïve HIV-1 patients in Cameroon: A 12-months single-blind, randomized, multicenter trial” (Ngo-Matip et al. (2015), Nutrition Journal) showed that spirulina could effectively delay the progression of HIV by improving immune responses.

[0009] Furthermore, the publication "Effect of microalgae as iron supplements on irondeficiency anemia in rats" by Gao et al. describes that diets based on microalgae such as spirulina containing a high iron content allow better absorption of non-heme iron in anemic rats.

[0010] Spirulina has generated great interest among consumers, and many dietary supplements have been developed and marketed to treat various deficiencies in both humans and animals.

[0011] A dietary supplement is a concentrated source of nutrients, vitamins, mineral salts, and trace elements whose purpose is nutritional or physiological. It helps address nutritional and micronutritional deficiencies in order to prevent certain pathologies as part of a balanced diet. These dietary supplements are found in various galenic forms, for example, in the form of a green powder, tablets, or syrup.

[0012] Among these products, we find SPIRULINA BIO™ from NUTRIMEA Laboratories which mentions that the benefits of spirulina help to strengthen natural defenses and vitality, to promote the body's resistance and to maintain energy and tone.

[0013] PHYCO-BIOTECH Laboratories also offer the OPHYCURE™ range, which includes various food supplements based on spirulina enriched with synthetic mineral or organic trace elements.

[0014] We also find the product for dogs and cats from the FLORALPINA Laboratory which is recommended for tone, vitality, maintenance of immune defenses and helps combat anemia.

[0015] It appears that these food supplements contain either spirulina alone or spirulina enriched with trace elements of mineral or synthetic organic origin such as silica or magnesium stearate.

[0016] Cyanobacteria such as spirulina advantageously have the ability to complex different metals, particularly divalent metals, thanks to proteins such as phycocyanin.

[0017] This property exists naturally in cyanobacteria thanks to the internalization process which allows it to accumulate different metals within the photosynthetic apparatus.

[0018] As a result, various research projects have been carried out on the enrichment of cyanobacteria with trace elements in order to develop improved food supplements allowing bioavailability of active ingredients as well as optimum recommended daily intakes.

[0019] Cyanobacteria enrichment is usually performed on living cyanobacteria. This involves long enrichment and extraction processes involving many steps during which a significant amount of trace elements are not fixed inside the cyanobacteria.

[0020] Document FR2827301 describes a process for enriching whole photosynthetic microorganisms with biologically active molecules in an optimized culture medium for a time of between 1 and 12 hours, said biologically active molecules being able to be trace elements.

[0021] Document FR2929957 discloses a process for obtaining a phycocyanin loaded with divalent metals by the internalization of these metals by a cyanobacterium. The extraction of said metal-loaded phycocyanin is then carried out using conventional protein extraction means by a grinding and ammonium sulfate precipitation step. This document thus describes a two-step process with phycocyanin-trace element contact in the internal intracellular medium (i.e., a step of internalization of the metal in the cyanobacterium) followed by a step of extraction of the complex by grinding, releasing the phycocyanin-metal complex. Grinding is therefore subsequent to (intracellular) complexation.

[0022] Phycocyanin contained in cyanobacteria is extracted using different processes.

[0023] Document FR3064269 describes in particular a so-called freeze-thaw extraction process using solvents such as phosphate buffers (pH7), a calcium chloride solution or sodium chloride. This process is long and requires more than 36 hours to allow degradation of the cell membranes and the release of phycocyanin.

[0024] Document FR2789399 discloses a process for manufacturing a micro-organism extract and in particular spirulina whose organoleptic and / or biochemical qualities are not degraded in particular by an increase in temperature in the process implemented, comprising the steps of:

[0025] - a) cultivation of said microorganism in a liquid culture medium adapted to the nutritional needs of said microorganism;

[0026] - b) physiological induction consisting of placing said culture in a particular liquid induction medium, such as sterilized seawater, in order to cause said microorganisms to produce an increased quantity of metabolites;

[0027] - c) cold grinding (at 2°C, maximum 4°C) of said culture so as to obtain a ground product thereof; and

[0028] - d) mixing said ground material in a separation medium and separating the insoluble part from the soluble part of said ground material by cold tangential filtration to obtain a crude extract.

[0029] Document W 02004070044 discloses photosynthetic microorganisms such as Arthrospira and Spirulina enriched in biologically active molecules by cold incubation with a NaCl solution comprising biologically active molecules, such as trace elements and bivalent metals, such as chromium, copper, iron, magnesium, manganese, molybdenum, selenium, zinc, the proportion of said molecules bound to the wall of the microorganism, or accumulated inside the microorganism, or integrated into one of the components of the cell of the microorganism, or bound to the constituents of the ground material of the microorganism, representing at least 50% of the quantity of said molecules used during the enrichment process.

[0030] US2008 / 124391 discloses a selenium-enriched spirulina extract. The selenium content of the Spirulina platensis used in the composition can be increased naturally by providing the growing algae with a selenium-supplemented production medium before harvesting and drying.

[0031] Document CN 1947792 discloses a liquid complex of phycocyanin and nano-selenium by a process that does not a priori involve a grinding step. Finally, document WO2015155224 is known, which describes a microalgae, spirulina, enriched with silicon in a water-soluble form. The enrichment is achieved by culturing microalgae to which silicon is subsequently added in the form of a solution or powder for a period of at least 12 hours. Harvesting a portion of the spirulina culture is carried out by sieving, centrifugation or flocculation, after an incubation time of between 12 hours and 3 days, then every day depending on the state of the culture and this for a period of 2 to 8 weeks, preferably for 3 weeks. Harvesting spirulina by sieving allows the elimination of silicon present in the culture medium and not integrated by the spirulina.

[0032] Such an enrichment process is therefore also complex and long and a significant quantity of silicon is not fixed by the spirulina.

[0033] Other processes use different technologies such as ultrasound, microwaves, high-pressure homogenization, pulsed electric fields or even supercritical CO2.

[0034] Another problem known in the prior art is the origin of the trace elements contained inside cyanobacteria.

[0035] Spirulina can be enriched with elements of different origin depending on the culture medium in which it is found. Thus, uncontrolled culture conditions can lead to the production of cyanobacteria containing elements dangerous to health such as toxic metals like lead and / or trace elements from mineral or synthetic organic sources that are poorly assimilated, particularly in the human body.

[0036] When formulating dietary supplements, it is also common to use trace elements in mineral form such as iron sulfate.

[0037] Alternatively, organic complexes comprising mineral trace elements complexed with amino acids such as iron bisglycinate can also be formulated.

[0038] These food supplements, formulated in this way, have the disadvantage of containing trace elements that are poorly bioavailable, meaning that the proportion of trace elements effectively absorbed by the body is low, which requires a significant intake for effective supplementation.

[0039] These dietary supplements, formulated in this way, also cause other side effects such as digestive system disorders in their users. Taking iron in addition to vitamin C can promote liver damage. An excess of trace elements and minerals can cause fatigue in detoxification organs such as the liver and kidneys.

[0040] TECHNICAL PROBLEM

[0041] In view of the above, a problem which the present invention aims to solve consists of developing a simple, rapid and ecological process making it possible to enrich in particular cyanobacteria, preferentially spirulina, with different varied and bioavailable trace elements from natural sources of trace elements, not having the drawbacks of the prior art.

[0042] TECHNICAL SOLUTION

[0043] The solution to this problem posed has as its first object a method for preparing a cyanobacterium extract comprising phycocyanin enriched in trace elements comprising the following steps: bringing into contact in an aqueous liquid solvent chosen from water, an aqueous solution of calcium chloride or an aqueous phosphate buffer having a pH between 7.8 and 8.2 at room temperature less than or equal to 40°C and greater than 4°C, a cyanobacterium, said cyanobacterium being chosen from the species of the genus Arthrospira or Spirulina, comprising phycocyanin, with an organic matrix, said matrix being a natural source of trace elements chosen from terrestrial plants, marine macro-algae or lichens, taken alone or in combination; grinding the cyanobacterium and the organic matrix allowing the co-extraction of the trace elements contained in the organic matrix by complexation with the phycocyanin of the cyanobacterium;and after possible filtration, recovery of a liquid extract of cyanobacteria comprising phycocyanin enriched in trace elements.;

[0044] Its second object is a cyanobacterium extract, preferably spirulina, comprising phycocyanin enriched in trace elements obtained by the process according to the invention.

[0045] Its third subject is a composition comprising, in a physiologically acceptable medium, an extract according to the invention.

[0046] Finally, its final object is the use of a composition according to the invention as a food supplement.

[0047] BENEFITS PROVIDED

[0048] The process according to the invention makes it possible to obtain in a single step a cyanobacterium, preferably spirulina, comprising phycocyanin enriched in trace elements from different organic matrices thanks to co-extraction.

[0049] The cyanobacterium extract, preferably spirulina, comprising phycocyanin enriched with trace elements thus obtained consists of a total of bioavailable active ingredients that are more easily assimilated by the human body than conventional food supplements containing spirulina alone and / or enriched with trace elements of mineral or synthetic organic origin.

[0050] This cyanobacterium extract, preferably spirulina, comprising phycocyanin enriched in trace elements can be purified in order to obtain a phycocyanin extract enriched in trace elements of high purity. The process according to the invention also allows a saving of time and a significant reduction in costs, particularly energy and industrial costs, for the formulation of food supplements comprising an extract of cyanobacterium, preferably spirulina, or phycocyanin enriched in trace elements obtained by the process according to the invention, namely advantageously a continuous eco-extraction.

[0051] Such food supplements comprising a cyanobacterium extract, preferably spirulina, comprising phycocyanin enriched with trace elements make it possible to meet the daily trace element requirements of the human body.

[0052] BRIEF DESCRIPTION OF THE DRAWINGS

[0053] The invention and the advantages resulting therefrom will be better understood upon reading the description and the non-limiting embodiments which follow, with regard to the appended figures in which:

[0054] Figure 1 is a graph representing the extraction yield following the implementation of a process according to the invention of phycocyanins having been co-extracted with different natural sources of trace elements in different solvents.

[0055] Figure 2 is a graph representing the purity of the phycocyanins thus extracted according to the absorbance ratio A620nm / A280nm.

[0056] Figure 3 is a graph showing the extraction yields of trace elements iron and magnesium in liquid medium.

[0057] DESCRIPTION OF EMBODIMENTS

[0058] The invention relates to a method for preparing a cyanobacterium extract comprising phycocyanin enriched in trace elements, comprising the following steps: bringing into contact, in an aqueous liquid solvent chosen from water, an aqueous solution of calcium chloride or an aqueous phosphate buffer having a pH between 7.8 and 8.2 at room temperature less than or equal to 40°C and greater than 4°C, a cyanobacterium, said cyanobacterium being chosen from the species of the genus Arthrospira or Spirulina, comprising phycocyanin, with an organic matrix, said matrix being a natural source of trace elements chosen from terrestrial plants, marine macroalgae or lichens, taken alone or in combination; grinding the cyanobacterium and the organic matrix allowing the co-extraction of the trace elements contained in the organic matrix by complexation with the phycocyanin of the cyanobacterium;and after possible filtration, recovery of a liquid extract of cyanobacteria comprising phycocyanin enriched in trace elements.;

[0059] The aqueous liquid solvent is chosen from water, an aqueous solution of calcium chloride (CaCl2), an aqueous phosphate buffer having a pH between 7.8 and 8.2, preferably pH 8, or any other solution allowing the stabilization of proteins in an aqueous medium for pHs ranging from 3 to 9. These pH values ​​correspond more particularly to values ​​allowing the phycocyanin to be kept stable, preferably at a pH between 4 and 8, more preferably 8.

[0060] Preferably, the aqueous calcium chloride solution used has a concentration of between 0.5% m / m and 2% m / m, more preferably 1.5% m / m.

[0061] Contact with the aqueous liquid solvent is carried out at room temperature less than or equal to 40°C and greater than 4°C, preferably less than or equal to 35°C, more preferably at a temperature between 19°C and 34°C.

[0062] Cyanobacteria refers to all photosynthetic prokaryotic organisms also called "blue algae" or microalgae.

[0063] By way of non-limiting examples, the cyanobacteria which can be used in the process according to the invention belonging to the genus Arthrospira and Spirulina are preferably chosen from the following species: Arthrospira amethystina, Arthrospira ardissonei, Arthrospira argentina, Arthrospira balkrishnanii, Arthrospira brevis, Arthrospira constricta, Arthrospira crassa, Arthrospira desikacharyiensis, Arthrospira gigantea, Arthrospira gomontiana, Arthrospira jenneri, Arthrospira joshii, Arthrospira khannae, Arthrospira laxissima, Arthrospira margaritae, Arthrospira massartii, Arthrospira miniata, Arthrospira pellucida, Arthrospira platensis, Arthrospira santannae, Arthrospira skujae, Arthrospira spirulinoides, Arthrospira tenuis, Spirulina abbreviate, Spirulina adriatica, Spirulina aeruginea, Spirulina agilis, Spirulina albida, Spirulina allansonii, Spirulina anjalensis, Spirulina attenuate, Spirulina baltica, Spirulina bayannurensis, Spirulina brévia rticu la ta,Spirulina brevis, Spirulina cabrerae, Spirulina caldaria, Spirulina californica, Spirulina cavanillesiana, Spirulina conica, Spirulina corakiana, Spirulina crassior, Spirulina duplex, Spirulina flavovirens, Spirulina gessneri, Spirulina gomontii, Spirulina gordiana, Spirulina gracilis, Spirulina innatans, Spirulina labyrinthiformis, Spirulina laxa, Spirulina laxissima, Spirulina légitima, Spirulina magnifica, Spirulina major, Spirulina mariae, Spirulina mediterranea, Spirulina meneghiniana, Spirulina minima, Spirulina mukdensis, Spirulina nodosa, Spirulina nordstedtii, Spirulina oceanica, Spirulina princeps, Spirulina pseudovacuolata, Spirulina regis, Spirulina robusta, Spirulina rosea, Spirulina schroederi, Spirulina sigmoidea, Spirulina socialis, Spirulina spirulinoides, Spirulina stagnicola, Spirulina subsala, Spirulina subtilissima, Spirulina tenurima, Spirulina tenuior, Spirulina tenuissima, Spirulina thermolis, Spirulina undulans, Spirulina vaginata,Spirulina versicolor and Spirulina weissii.,

[0064] According to a preferred embodiment, the cyanobacteria used in the method according to the invention belong to the genus Arthrospira, and are more preferably chosen from Arthrospira amethystina, Arthrospira ardissonei, Arthrospira argentina, Arthrospira balkrishnanii, Arthrospira brevis, Arthrospira constricta, Arthrospira crassa, Arthrospira desikacharyiensis, Arthrospira gigantea, Arthrospira gomontiana, Arthrospira jenneri, Arthrospira joshii, Arthrospira khannae, Arthrospira laxissima, Arthrospira margaritas, Arthrospira massartii, Arthrospira miniata, Arthrospira pellucida, Arthrospira platensis, Arthrospira santannae, Arthrospira skujae, Arthrospira spirulinoides, Arthrospira tenuis, even more preferably spirulina Arthrospira platensis.

[0065] According to another embodiment of the method according to the invention, other algae comprising phycocyanin belonging in particular to the genus Galdieria can be used, for example Galdieria sulphuraria, Galdieria daedala, Galdieria maxima, Galdieria partita.

[0066] An organic matrix is ​​any matrix consisting of organic elements such as proteins, sugars or lipids. Generally speaking, according to the invention, an organic matrix constitutes anything that is not a mineral matrix. In the context of the invention, it is understood that the organic matrix is ​​a natural source of trace elements in solid form which "traps" the trace elements which are therefore less available and more difficult to extract.

[0067] The organic matrix that can be used in the process according to the invention can belong to the plant kingdom or to the Fungi kingdom.

[0068] The first step of the process which is the subject of the invention consists of bringing into contact, in an aqueous liquid solvent chosen from water, an aqueous solution of calcium chloride (CaCl2) or an aqueous phosphate buffer having a pH between 7.8 and 8.2, preferably pH 8, a cyanobacterium preferably being spirulina with an organic matrix being chosen from terrestrial plants, marine macro-algae or lichens.

[0069] Preferably, the terrestrial plants are chosen from spinach, Brazil nut, reishi, turmeric root, camu camu berry, carob bean or cranberry and the marine macroalgae are chosen from ao-nori, wakame, dulse, kumbu, ulva also called sea lettuce, chondrus, nostoc or porphyra, more preferably ao-nori. These organic matrices are given as non-limiting examples and constitute the organic matrices among the richest in minerals and trace elements.

[0070] Preferably, the ratio of cyanobacteria - aqueous liquid solvent - organic matrix is ​​between 1:8:1 and 1:90:9.

[0071] In a preferred embodiment, the cyanobacteria used is spirulina, the aqueous liquid solvent is water and the organic matrix is ​​ao-nori. More preferably, the spirulina - water - ao-nori ratio is between 1:8:1 and 1:90:9.

[0072] Preferably, the cyanobacterium, preferably spirulina, used in the process according to the invention is in dried solid form, more preferably with a moisture content of less than 5% in the form of powder or flakes, or fresh in the form of paste or in the form of liquid extract, more preferably prepared directly after harvesting by concentration or dilution. The second step of the process which is the subject of the invention consists of carrying out grinding in order to allow the co-extraction of the trace elements contained in the organic matrix by complexation (post-grinding) with the phycocyanin contained in the cyanobacterium, preferably spirulina.

[0073] The grinding is carried out at room temperature less than or equal to 40°C and greater than 4°C, preferably less than or equal to 35°C, more preferably at a temperature between 19°C and 34°C. The grinding is more preferably carried out wet at a pH between 4 and 8, at high shear at room temperature less than or equal to 40°C and greater than 4°C, preferably less than or equal to 35°C, more preferably at a temperature between 19°C and 34°C, for a duration between 1 minute and 60 minutes.

[0074] Advantageously, the co-extraction is carried out in a single step and allows the extraction of a compound or set of compounds from one matrix, in the state the organic matrix according to the invention, by a compound or set of compounds from another matrix, in the state the phycocyanin of the cyanobacterium, preferably spirulina, according to the invention. A compound extracted from one matrix promotes the transfer capacity of a compound from the other matrix.

[0075] Advantageously, co-extraction makes it possible to complex the phycocyanin contained in the cyanobacterium, preferably spirulina, with divalent cations contained in the second organic matrix. These divalent cations are preferably chosen from iron, magnesium, zinc, selenium, chromium, manganese, molybdenum and / or copper.

[0076] Preferably, the high shear grinding is carried out at rotation speeds of 9000 rpm (rotations per minute) allowing shear rates at a peripheral speed of between 14 and 20 m / s, for example more preferably 14 m / s and 15 m / s.

[0077] More preferably, high shear grinding is carried out wet at a pH of 8, at an ambient temperature less than or equal to 35°C, preferably at a temperature between 19°C and 34°C, for a duration of between 5 and 30 minutes.

[0078] High shear wet grinding at ambient temperature less than or equal to 40°C and greater than 4°C, preferably less than or equal to 35°C, more preferably at a temperature between 19°C and 34°C, can be transposed to continuous mode for shear rates of 14 to 20m / s and short contact times of 1 to 5 minutes. The shear rates in continuous mode take into account the flow rates used, the contact time and the shear rate. For contact times of 1 to 5 minutes and a volume of 1.2 liters (I) of extract with a shear rate of 14 to 20 m / s, a shear of 0.7 m / min / l to 5 m / min / l is induced. The contact time is adapted and advantageously a recirculation or a second high shear mill can be used in series.

[0079] Room temperature extraction allows for increased extraction yields and is of major interest in reducing phycocyanin extraction time. The short extraction time helps preserve quality and limits degradation.

[0080] This grinding allows the membranes of the cyanobacteria, preferably spirulina, to be reduced and broken, and thus the phycocyanin to be extracted using the aqueous liquid solvent.

[0081] A final step of the process according to the invention consists, after possible filtration, in recovering a liquid extract of cyanobacteria, preferably spirulina, comprising phycocyanin enriched in trace elements.

[0082] The liquid extract of cyanobacteria, preferably spirulina, comprising phycocyanin enriched with trace elements obtained can then be preserved in its entirety to promote the "totum" effect. It can then be formulated in different forms, liquid or solid.

[0083] Advantageously, the method according to the invention can be used continuously, namely continuous eco-extraction, in order to increase the yields of cyanobacterium extract, preferably spirulina, obtained, in particular for short extraction times of less than 5 minutes with grinding of approximately one minute.

[0084] It is thus possible to implement the process according to the invention industrially by using a high quantity from the start when bringing into contact according to a ratio of between 1:8:1 and 1:90:9 for the cyanobacterium, preferably spirulina, the aqueous liquid solvent and the organic matrix in order to obtain an extract of cyanobacterium, preferably spirulina, comprising phycocyanin enriched in trace elements in a continuous manner.

[0085] Alternatively, it is also possible to gradually add cyanobacteria, preferably spirulina, aqueous liquid solvent and organic matrix in a ratio of between 1:8:1 and 1:90:9 in order to obtain a cyanobacteria extract, preferably spirulina, comprising phycocyanin enriched in trace elements.

[0086] Preferably, the method is implemented by progressive addition of the cyanobacterium, preferably spirulina, and the organic matrix, in the aqueous liquid solvent. The solvent is stirred by the initiation of the grinding then the cyanobacterium, preferably spirulina, is incorporated into the medium initiating the grinding and the extraction of the phycocyanin. The organic matrix is ​​added concomitantly allowing the co-extraction of the active ingredients of interest. When the materials are fully incorporated, the grinding is maintained for a period of 5 to 30 minutes. In a particular embodiment of the method according to the invention, the liquid extract of cyanobacterium, preferably spirulina, comprising phycocyanin enriched in trace elements is filtered. Said filtration step is preferably carried out by centrifugation and / or by tangential membrane filtration.

[0087] This filtration step removes cellular debris and produces a liquid extract of phycocyanin enriched with pure trace elements.

[0088] Tangential filtration allows the removal of unwanted compounds with a size less than 10 kDa or even 100 kDa and advantageously in particular 25 kDa such as phenolic compounds or small proteins and possible extraction salts such as calcium chloride. A low cut-off threshold of 10 kDa allows the purification of small compounds less than 10 kDa. Choosing a higher threshold such as 100 kDa allows the removal of a higher quantity of compounds.

[0089] The different filtration modes considered can be used in a coupled or individual manner.

[0090] Advantageously, the cyanobacterium extract, preferably spirulina, comprising phycocyanin enriched with filtered trace elements is an extract comprising phycocyanin enriched with pure trace elements free from cellular debris.

[0091] The liquid extract of cyanobacteria, preferably spirulina, comprising phycocyanin enriched with recovered trace elements, filtered or not, advantageously filtered, can be reduced to solid powder by atomization or freeze-drying.

[0092] According to another embodiment, a lyophilization or atomization step is carried out on the liquid extract of phycocyanin enriched with pure trace elements thus reduced to solid powder.

[0093] Advantageously, in order to reduce energy costs, the dry matter content of the cyanobacterial extract, preferably spirulina, must be high before drying to reduce the amount of water to be removed.

[0094] Once dried by conventional drying processes such as freeze-drying or spray-drying, the cyanobacterium extract, preferably spirulina, comprising phycocyanin, filtered or not, advantageously filtered, in powder form has a low water content of less than 5% which allows it to have higher microbiological stability and thus to be stored at room temperature unlike non-stabilized liquid products, which need to be kept at positive cold.

[0095] The cyanobacterium extract, preferably spirulina, comprising phycocyanin in powder form can also be used in different galenic forms in order to formulate food supplements.

[0096] The invention also relates to a cyanobacterium extract, preferably spirulina, comprising phycocyanin enriched in trace elements obtained by the process according to the invention. The organic matrix used being a solid natural source of trace elements chosen from terrestrial plants, marine macroalgae or lichens used and the co-extraction and complexation of the trace elements with the phycocyanin contained in the cyanobacterium being carried out after grinding at room temperature, the cyanobacterium extract comprising phycocyanin enriched in trace elements thus obtained by the process according to the invention is different (in terms of quality (types) and quantity of trace elements) from those obtained with the processes of the prior art, the process implemented and the sources of trace elements being different.

[0097] The cyanobacterium extract, preferably spirulina, comprising phycocyanin enriched with trace elements, filtered or not, can be used as is or in combination with one or more excipient(s) suitable for the formulation of food supplements.

[0098] Such an extract can also be used in the treatment of micronutritional deficiencies likely to cause metabolic, degenerative and / or autoimmune pathologies.

[0099] The invention also relates to a composition comprising such an extract according to the invention in a physiologically acceptable medium.

[0100] Physiologically acceptable medium means a medium proportionate to a reasonable benefit / risk ratio, comprising excipients known and commonly used in the field of food supplements such as binders, disintegrating agents, bulking agents, dispersing agents, agglomerating agents, lubricants, wetting agents, surfactants, emulsifiers, thickeners, sagging agents, flavoring agents, sweetening agents, coloring agents, film-coating agents, stabilizers and / or preservatives.

[0101] A person skilled in the art will take care to choose these possible excipients and their quantity in such a way that they do not harm the interesting properties of the compositions according to the invention.

[0102] Examples of excipients include cellulose, preferably microcrystalline cellulose, and silicon dioxide.

[0103] In a preferred embodiment, the composition according to the invention is in a form suitable for oral administration, preferably in the form of a capsule, tablet, lozenge, powder, chewing gum, capsule, oral solution, oral suspension or syrup.

[0104] The composition according to the invention can also be used in the treatment of micro-nutritional deficiencies likely to cause metabolic, degenerative and / or autoimmune pathologies.

[0105] Micronutrient deficiencies are defined as micronutrient deficiencies such as deficiencies in minerals and trace elements essential to the human body.

[0106] These micronutrient deficiencies are likely to cause: metabolic pathologies such as fibromyalgia, cardiovascular diseases, diabetes, diseases of the blood system; degenerative pathologies such as cancers, age-related macular degeneration (AMD), neurodegenerative diseases such as Alzheimer's disease or Parkinson's disease; and / or autoimmune pathologies such as osteoporosis, rheumatic diseases such as rheumatoid arthritis and spondylitis.

[0107] Finally, the invention has as its final object the use of a composition as a food supplement.

[0108] EXAMPLES

[0109] The present invention will now be illustrated by means of the following examples:

[0110] Example 1: Process for the preparation according to the invention of a spirulina extract of phycocyanin enriched in trace elements by co-extraction

[0111] Two organic matrices were tested using the process of the invention in order to carry out a co-extraction of the phycocyanin contained in spirulina (Arthrospira platensis) and trace elements from the natural source of trace elements chosen from: spinach, a natural terrestrial plant source; or ao-nori, a marine macro-algae.

[0112] These two natural sources of trace elements were brought into contact with different aqueous liquid solvents such as water, an aqueous phosphate buffer at pH 8 or an aqueous solution of calcium chloride and with spirulina.

[0113] The following different co-extractions were carried out:

[0114] 20 g of Spirulina - 20 g of spinach - 400 mL of water;

[0115] 20 g of Spirulina - 20 g of Ao nori - 400 mL of water;

[0116] 20 g of Spirulina - 20 g of spinach - 400 mL of Phosphate buffer pH=8;

[0117] 20 g of Spirulina - 20 g of Ao nori - 400 mL of Phosphate buffer pH=8;

[0118] 20 g of Spirulina - 20 g of spinach - 400 ml of Calcium Chloride Solution (CaCI2) at 1.5% m / m;

[0119] 20 g of Spirulina - 20 g of Ao nori - 400 mL of 1.5% m / m calcium chloride (CaCI2) solution.

[0120] The results obtained for each of these co-extractions are illustrated in Figures 1 to 3.

[0121] For each co-extraction, the spirulina and the natural source of trace elements were ground using a high shear mill allowing rapid and fine grinding.

[0122] The grinding is carried out for a period of 10 minutes and is advantageously carried out at a temperature less than or equal to 40°C and greater than 4°C. The initial temperature is 19°C and can reach 34°C at the end of grinding without heat treatment (heating or cooling). Since the temperature is likely to vary during grinding due to heating due to grinding, the application of cold can be used to maintain the temperature less than or equal to 40°C for a period greater than 10 minutes.

[0123] The spirulina extract containing phycocyanin enriched with trace elements obtained after this grinding is a liquid with a viscous consistency.

[0124] This extract can first be filtered by centrifugation using a centrifugal clarifier in order to separate the solid and liquid parts comprising respectively the cellular debris and the aqueous liquid solvent containing the phycocyanin.

[0125] In a second step and optionally, the liquid extract obtained can be purified by using tangential ultrafiltration.

[0126] This optional step can be repeated several times if necessary in order to concentrate the liquid extract of phycocyanin enriched with trace elements.

[0127] The concentration can be increased to a defined phycocyanin titration ranging from 1 g to 150 g / l. Re-concentration from 24 g / l to 115 g / l eliminates many impurities and increases the mass purity from 73% to 88.9%. This purity is correlated with an A620 / A280 index increased from 1.4 to 2.6 (for a measured concentration of 115 g / L), i.e. by more than 110%.

[0128] Such extracts can be packaged in liquid or solid form after freeze-drying or atomization. This last step allows the stabilization of the extracts.

[0129] Lyophilized liquids showed no freeze-drying hindrance for phycocyanin concentrations of 1 to 40 g / l. Atomized liquids did not require atomization support for phycocyanin concentrations of 1 to 115 g / l. Drying can be carried out without prior adjustment and without addition, allowing a reduction in operating costs and higher final purity.

[0130] As illustrated in Figure 1, phycocyanin extraction yields vary depending on the natural source of trace elements used in the co-extractions carried out. Two forms of phycocyanins were measured: C-phycocyanin and allo-phycocyanin. Total phycocyanin represents the sum of the latter two.

[0131] The implementation of the process according to the invention advantageously increases the extraction yield of phycocyanin when the natural source of trace elements used is ao-nori.

[0132] The extraction yield of phycocyanins obtained is greater than 90% when the extraction solvent used is water or phosphate buffer at pH 8.

[0133] Extractions carried out in the presence of calcium chloride show similar results for both natural sources of trace elements used. The purity of the phycocyanins thus extracted was also measured and is illustrated in Figure 2.

[0134] A higher purity is observed in the presence of ao-nori compared to spinach, which is correlated with a higher extraction of phycocyanins. The phycocyanin / impurity ratio is then less favorable with the use of spinach compared to ao-nori.

[0135] Finally, the specific trace element extraction yield was measured for iron (Fe) and magnesium (Mg) as shown in Figure 3.

[0136] The extraction of trace elements contained in these extracts was then measured. The measured extraction yield of trace elements increased by more than 186% in liquid medium compared to the quantity of trace elements available in spirulina alone.

[0137] The iron and magnesium contained in the plant and algal matrices are extracted and measured in the liquid medium. These trace elements are favorably extracted in the presence of phycocyanin. The liquid and solid extracts are not separated, but dried and ground into a powder. The observed enrichment in iron increases by more than 150% and in magnesium by more than 115%, compared to the contents measured in spirulina alone. The spirulina used has an initial iron and magnesium content of 31 mg and 167 mg per 100 g of dry spirulina.

[0138] Conclusion :

[0139] The implementation of the method according to the invention makes it possible to demonstrate that co-extraction allows the extraction of trace elements from the organic matrix by the phycocyanin of spirulina. This thus results in a high enrichment of phycocyanin in trace elements from natural sources, making it possible to obtain a spirulina extract comprising phycocyanin enriched in trace elements that is more easily assimilated by the human body and more quickly than the techniques known in the prior art.

Claims

CLAIMS 1. Process for preparing a cyanobacterium extract comprising phycocyanin enriched in trace elements, characterized in that it comprises the following steps: bringing into contact in an aqueous liquid solvent chosen from water, an aqueous solution of calcium chloride or an aqueous phosphate buffer having a pH between 7.8 and 8.2, at room temperature less than or equal to 40°C and greater than 4°C of a cyanobacterium, said cyanobacterium being chosen from the species of the genus Arthrospira or Spirulina, comprising phycocyanin, with an organic matrix, said matrix being a natural source of trace elements chosen from terrestrial plants, marine macroalgae or lichens, taken alone or in combination; grinding of the cyanobacterium and the organic matrix allowing the co-extraction of the trace elements contained in the organic matrix by complexation with the phycocyanin of the cyanobacterium;and after possible filtration, recovery of a liquid extract of cyanobacteria comprising phycocyanin enriched in trace elements.; 2. Process according to claim 1, characterized in that the cyanobacterium belonging to the genus Arthrospira and Spirulina, preferably to the genus Arthrospira, is chosen from the following species: Arthrospira amethystine, Arthrospira ardissonei, Arthrospira argentina, Arthrospira balkrishnanii, Arthrospira brevis, Arthrospira constricta, Arthrospira crassa, Arthrospira desikacharyiensis, Arthrospira gigantea, Arthrospira gomontiana, Arthrospira jenneri, Arthrospira joshii, Arthrospira khannae, Arthrospira laxissima, Arthrospira margaritae, Arthrospira massartii, Arthrospira miniata, Arthrospira pellucida, Arthrospira platensis, Arthrospira santannae, Arthrospira skujae, Arthrospira spirulinoides, Arthrospira tenuis, Spirulina abbreviata, Spirulina adriatica, Spirulina aeruginea, Spirulina agilis, Spirulina albida, Spirulina allansonii, Spirulina anjalensis, Spirulina attenuate, Spirulina baltica, Spirulina bayannurensis, Spirulina breviarticulata, Spirulina brevis,Spirulina cabrerae, Spirulina caldaria, Spirulina californica, Spirulina cavanillesiana, Spirulina conica, Spirulina corakiana, Spirulina crassior, Spirulina duplex, Spirulina flavovirens, Spirulina gessneri, Spirulina gomontii, Spirulina gordiana, Spirulina gracilis, Spirulina innatans, Spirulina labyrinthiformis, Spirulina laxa, Spirulina laxissima, Spirulina légitima, Spirulina magnifica, Spirulina major, Spirulina mariae, Spirulina mediterranea, Spirulina meneghiniana, Spirulina minima, Spirulina mukdensis, Spirulina, nodosa, Spirulina nordstedtii, Spirulina oceanica, Spirulina princeps, Spirulina pseudovacuolata, Spirulina régis, Spirulina robusta, Spirulina rosea, Spirulina schroederi, Spirulina sigmoidea, Spirulina socialis, Spirulina spirulinoides, Spirulina stagnicola, Spirulina subsala, Spirulina subtilissima, Spirulina tenerrima, Spirulina tenuior, Spirulina tenuissima, Spirulina thermalis, Spirulina undulans, Spirulina vaginata, Spirulina versicolor and Spirulina weissii, preferably the spirulina Arthrospira platensis.

3. Method according to claim 1 or 2, characterized in that the cyanobacteria, preferably spirulina, is in dried solid form or fresh in the form of paste or in the form of liquid extract.

4. Method according to one of the preceding claims, characterized in that the terrestrial plants are chosen from spinach, Brazil nut, reishi, turmeric root, camu camu berry, carob bean, cranberry and the marine macro-algae are chosen from aonori, wakame, dulse, kumbu, ulva, chondrus, nostoc, porphyra, preferably aonori.

5. Method according to one of the preceding claims, characterized in that the grinding is carried out at room temperature less than or equal to 40°C and greater than 4°C.

6. Method according to claim 5, characterized in that the grinding is carried out wet at a pH between 4 and 8, at high shear, at ambient temperature less than or equal to 40°C and greater than 4°C, for a duration between 1 minute and 60 minutes.

7. Method according to one of the preceding claims, characterized in that the phycocyanin enriched in trace elements is complexed with divalent cations chosen from iron, magnesium, zinc, selenium, chromium, manganese, molybdenum and / or copper.

8. Method according to one of the preceding claims, characterized in that it comprises a filtration step carried out by centrifugation and / or tangential filtration on a membrane.

9. Method according to one of the preceding claims, characterized in that the liquid extract of cyanobacteria, preferably spirulina, comprising recovered trace element-enriched phycocyanin is reduced to solid powder by atomization or freeze-drying.

10. Extract of cyanobacterium, preferably spirulina, comprising phycocyanin enriched in trace elements obtained by the process according to one of claims 1 to 9. 1 1. Composition comprising, in a physiologically acceptable medium, an extract according to claim 10.

12. Composition according to claim 11, characterized in that it is in a form suitable for oral administration, preferably in the form of capsules, tablets, lozenges, powder, chewing gum, capsules, oral solution, oral suspension or syrup.

13. Composition according to claim 11 or 12, for its use in the treatment of micro-nutritional deficiencies likely to cause metabolic, degenerative and / or autoimmune pathologies.

14. Use of the composition according to claim 11 or 12 as a food supplement.

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