WO2024133379A1

WO2024133379A1 - 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

Info

Publication number: WO2024133379A1
Application number: PCT/EP2023/086822
Authority: WO
Inventors: Jean-Marie SANTANDER; Maryline ABERT VIAN; Jean-Baptiste MAZZITELLI; Farid Chemat
Original assignee: Cyanophy Laboratoire; Avignon Universite
Priority date: 2022-12-23
Filing date: 2023-12-20
Publication date: 2024-06-27
Also published as: FR3143953A1

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

METHOD FOR PREPARING A CYANOBACTERIA EXTRACT COMPRISING PHYCOCYANIN ENRICHED WITH TRACES ELEMENTS FROM NATURAL SOURCES OF TRACES ELEMENTS FOR THE MANUFACTURE OF FOOD SUPPLEMENTS

TECHNICAL AREA

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

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

PRIOR ART

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, Bl, B2, B3, B6, B7, B8 and K, in trace elements such as zinc, selenium, manganese, iron, copper or chromium and minerals such as calcium, magnesium, sodium, potassium or phosphorus.

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

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

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.

As spirulina arouses great interest among consumers, numerous food supplements have been developed and put on the market in order to treat various deficiencies in both humans and animals.

A food supplement is a concentrated source of nutrients, vitamins, mineral salts and trace elements whose purpose is nutritional or physiological. It helps address nutritional and micro-nutritional deficiencies in order to prevent certain pathologies as part of a balanced diet. These food supplements are found in various dosage forms, for example in the form of a green powder, tablet or even syrup.

Among these products, we find SPIRULINE BIO™ from Laboratoires NUTRIMEA which mentions that the benefits of spirulina make it possible to strengthen natural defenses and vitality, to promote the body's resistance and to maintain energy and tone.

PHYCO-BIOTECH Laboratories also offer the OPHYCURE™ range including various food supplements based on spirulina enriched with synthetic mineral or organic trace elements.

We also find the product for dogs and cats from the FLORALPINA Laboratory which is recommended for tone, vitality, maintaining immune defenses and helps fight anemia.

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 even magnesium stearate.

Cyanobacteria such as spirulina advantageously have the capacity to complex different metals, particularly divalent metals, using proteins such as phycocyanin.

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

As a result, various research projects have been carried out around 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.

Cyanobacteria enrichment is generally performed on live cyanobacteria. This results in long enrichment and extraction processes comprising numerous stages during which a significant quantity of trace elements is not fixed inside the cyanobacteria.

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 possibly being trace elements.

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

The phycocyanin contained in cyanobacteria is extracted using different processes.

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

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

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

- 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;

- c) cold grinding (at 2°C, maximum 4°C) of said culture so as to obtain a ground material thereof; And

- d) mixing said ground material in a separation medium and separation of the insoluble part from the soluble part of said ground material by cold tangential filtration allowing a crude extract to be obtained.

Document W 02004070044 discloses photosynthetic microorganisms such as Arthrospira and Spirulina enriched in biologically active molecules by cold incubation with a NaCI solution comprising biologically active molecules, such as trace elements and divalent metals, such as chromium, copper, iron, magnesium, manganese, molybdenum, selenium, zinc, the proportion of said molecules linked to the wall of the microorganism, or accumulated inside the microorganism, or integrated into one components of the cell of the microorganism, or linked 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.

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

Document CN 1947792 discloses a liquid complex of phycocyanin and nano-selenium by a process which does not a priori involve a grinding step. Finally, we know the document WO2015155224 which describes a microalgae, spirulina, enriched with silicon in a water-soluble form. Enrichment is carried out 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. The harvest of part 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.

Such an enrichment process is therefore also complex and time-consuming and a significant quantity of silicon is not fixed by spirulina.

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

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

Spirulina can be enriched with elements of different origin depending on the culture medium where it is found. Thus, uncontrolled culture conditions can lead to obtaining cyanobacteria containing elements dangerous to health such as toxic metals such as lead and/or trace elements from mineral or synthetic organic sources that are poorly assimilable, particularly in human organism.

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

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

These food supplements thus formulated have the disadvantage of containing poorly bioavailable trace elements, that is to say that the proportion of trace elements effectively absorbed by the body is low, which requires a significant intake for effective supplementation.

These food supplements thus formulated are also the cause of other side effects such as digestive system disorders in their user. Taking iron in addition to vitamin C can promote liver damage. An excess of trace elements and minerals can cause fatigue of detoxification organs such as the liver and kidney.

TECHNICAL PROBLEM

Taking into account the above, a problem that the present invention proposes to solve consists of developing a simple, rapid and ecological process. making it possible to enrich in particular the cyanobacteria preferentially spirulina with different varied and bioavailable trace elements from natural sources of trace elements, not presenting the disadvantages of the prior art.

TECHNICAL SOLUTION

The solution to this problem posed has as its first object a process for preparing a cyanobacteria 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 ambient temperature less than or equal to 40°C and greater than 4°C, of a cyanobacterium, said cyanobacteria being chosen from the species of 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 cyanobacteria and the organic matrix allowing the co-extraction of the trace elements contained in the organic matrix by complexation with the phycocyanin of the cyanobacteria; and after possible filtration, recovery of a liquid extract of cyanobacteria comprising phycocyanin enriched with trace elements.

Its second object is an extract of cyanobacteria, preferably spirulina, comprising phycocyanin enriched in trace elements obtained by the process according to the invention.

Its third object is a composition comprising, in a physiologically acceptable medium, an extract according to the invention.

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

ADVANTAGES PROVIDED

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

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

This cyanobacteria 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 cyanobacteria, preferably spirulina, or phycocyanin enriched with trace elements obtained by the process according to the invention. invention, namely advantageously continuous eco-extraction.

Such food supplements comprising an extract of cyanobacteria, preferably spirulina, comprising phycocyanin enriched with trace elements make it possible to meet the daily needs for trace elements of the human body.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention and the advantages which result from it will be better understood on reading the description and the non-limiting embodiments which follow, with regard to the appended figures in which:

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.

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

Figure 3 is a graph representing the extraction yields of the trace elements iron and magnesium in a liquid medium.

DESCRIPTION OF THE EMBODIMENTS

The invention relates to a process for preparing a cyanobacteria extract comprising phycocyanin enriched in trace elements comprising the following steps: brought into contact in an aqueous liquid solvent chosen from water, an aqueous solution of calcium chloride or a buffer aqueous phosphate having a pH between 7.8 and 8.2 at ambient temperature less than or equal to 40°C and greater than 4°C, of a cyanobacteria, said cyanobacteria 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 cyanobacteria and the organic matrix allowing the co-extraction of the trace elements contained in the organic matrix by complexation with the phycocyanin of the cyanobacteria; and after possible filtration, recovery of a liquid extract of cyanobacteria comprising phycocyanin enriched with trace elements.

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 pH8, 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 phycocyanin to be kept stable, preferably at a pH between 4 and 8 , more preferably 8.

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

The contact with the aqueous liquid solvent is carried out 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. vs.

By cyanobacteria, we mean all photosynthetic prokaryotic organisms also called “blue-green algae” or micro-algae.

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 s antannae, 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 brevia rticu la ta, Spirulina brevis, 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, rulina legitima, 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, ulina sigmoidea, Spirulina socialis , Spirulina spirulinoides, Spirulina stagnicola, Spirulina subsala, Spirulina subtilissima, Spirulina tenerrima, Spirulina tenuior, Spirulina tenuissima, Spirulina thermolis, Spirulina undulans, Spirulina vaginata, Spirulina versicolor and Spirulina weissii.

According to a preferred embodiment, the cyanobacteria used in the process 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.

According to another embodiment of the process 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.

By organic matrix we mean any matrix made up of organic elements such as proteins, sugars or lipids. Generally speaking, according to the invention, an organic matrix constitutes everything which 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.

The organic matrix which can be used in the process according to the invention may belong to the plant kingdom or to the Fungi kingdom.

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 pH8, a cyanobacteria preferably being spirulina with an organic matrix being chosen from terrestrial plants, marine macroalgae or lichens.

Preferably, the terrestrial plants are chosen from spinach, Brazil nut, reishi, turmeric root, camu camu berry, carob bean or cranberry berry and the marine macro-algae 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.

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

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.

Preferably, the cyanobacteria, preferably spirulina, used in the process according to the invention is in dried solid form, more preferably with a humidity level of less than 5% in the form of powder or flakes, or fresh in the form of paste or in the form of a liquid extract, more preferably prepared directly after harvest 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 cyanobacteria, preferably spirulina.

Grinding is carried out 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. The grinding is more preferably carried out in the wet process at a pH between 4 and 8, at high shear at an 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, for a period of between 1 minute and 60 minutes.

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

Advantageously, co-extraction makes it possible to complex the phycocyanin contained in the cyanobacteria, 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.

Preferably, high shear grinding is carried out at rotational speeds of 9000 rpm (rotation 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.

More preferably, the high shear grinding is carried out in the wet process 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 period between 5 and 30 minutes.

High shear grinding in the wet process 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, is transposable in 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 duration 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 duration is adapted and advantageously recirculation or a second high shear mill can be used in series.

Extraction at room temperature allows an increase in extraction yields and is of major benefit in reducing phycocyanin extraction time. The short extraction time helps preserve quality and limits degradation.

This grinding makes it possible to reduce and break the membranes of the cyanobacteria, preferably spirulina, and thus to extract the phycocyanin using the aqueous liquid solvent.

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

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.

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

It is thus possible to implement the process according to the invention in an industrial manner using a high quantity from the start during contacting in a ratio of between 1:8:1 and 1:90:9 for the cyanobacteria. preferably spirulina, the aqueous liquid solvent and the organic matrix in order to obtain a cyanobacteria extract, preferably spirulina, comprising phycocyanin enriched in trace elements in a continuous manner.

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

Preferably, the process is implemented by progressive addition of the cyanobacteria, preferably spirulina, and the organic matrix, in the aqueous liquid solvent. The solvent is stirred by initiating the grinding then the cyanobacteria, 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 process according to the invention, the liquid extract of cyanobacteria, preferably spirulina, comprising phycocyanin enriched in trace elements is filtered. Said filtration step is preferably carried out by centrifugation and/or by tangential filtration on a membrane.

This filtration step eliminates cellular debris and obtains a liquid extract of phycocyanin enriched with pure trace elements.

Tangential filtration makes it possible to eliminate unwanted compounds having 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 chloride of calcium. A low cutoff of 10 kDa allows the purification of small compounds below 10 kDa. Choosing a higher threshold such as 100 kDa allows the elimination of a higher quantity of compounds.

The different filtration modes envisaged can be used in a coupled or individual manner.

Advantageously, the extract of cyanobacteria, preferably spirulina, comprising phycocyanin enriched in filtered trace elements is an extract comprising phycocyanin enriched in pure trace elements devoid of cellular debris.

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

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

Advantageously, in order to reduce energy costs, the dry matter content contained in the cyanobacteria extract, preferably spirulina, must be high before drying to reduce the quantity of water to be eliminated.

Once dried by conventional drying processes such as freeze-drying or atomization, the cyanobacteria 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.

The cyanobacteria extract, preferably spirulina, comprising phycocyanin in powder form can also be used in different dosage forms in order to formulate food supplements.

The invention also relates to an extract of cyanobacteria, 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 cyanobacteria being carried out after grinding at room temperature, the The cyanobacteria 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.

The cyanobacteria 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.

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

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

By physiologically acceptable medium, we mean a medium proportioned to a reasonable benefit/risk ratio, comprising known excipients commonly used in the field of food supplements such as binders, disintegration agents, bulking agents, dispersing agents, agglomerating agents. , lubricants, wetting agents, surfactants, emulsifiers, thickeners, pouring agents, flavoring agents, sweetening agents, coloring agents, film coating agents, stabilizers and/or preservatives.

Those skilled in the art will take care to choose these possible excipients and their quantity so that they do not harm the interesting properties of the compositions according to the invention.

As an example of excipients, we can cite cellulose, preferably microcrystalline cellulose, and silicon dioxide.

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

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.

By micronutritional deficiencies we mean micronutrient deficiencies such as deficiencies in minerals and trace elements essential for the human body.

These micro-nutritional deficiencies are likely to cause: metabolic pathologies such as fibromyalgia, cardiovascular diseases, diabetes, blood system diseases; degenerative pathologies such as cancers, age-related macular degeneration (AMD), neurodegenerative diseases such as Alzheimer's disease or even Parkinson's disease; and/or autoimmune pathologies such as osteoporosis, rheumatic conditions such as rheumatoid arthritis and spondyloarthritis.

Finally, the final object of the invention is the use of a composition as a food supplement.

EXAMPLES

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

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

Two organic matrices were tested using the process which is the subject 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, source natural terrestrial plant; or ao-nori, a marine macroalgae.

These two natural sources of trace elements were brought into contact with different aqueous liquid solvents such as water, an aqueous phosphate buffer at pH8 or an aqueous solution of calcium chloride and with spirulina.

The following different co-extractions were carried out:

20 g of Spirulina - 20 g of spinach - 400 mL of water;

20 g of Spirulina - 20 g of Ao nori - 400 mL of water;

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

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

20 g of Spirulina -20 g of spinach -400 ml of Calcium chloride solution (CaCI2) at 1.5% m/m;

20 g of Spirulina - 20 g of Ao nori - 400 mL Calcium chloride solution (CaCI2) at 1.5% m/m.

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

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.

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. There initial temperature is 19°C and can reach 34°C at the end of grinding without heat treatment (heating or cooling). As the temperature is likely to vary during grinding during 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 of more than 10 minutes.

The spirulina extract comprising phycocyanin enriched with trace elements obtained at the end of this grinding is a liquid with a viscous consistency.

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.

Secondly and optionally, the liquid extract obtained can be purified by using tangential ultrafiltration.

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

The concentration can be increased to a defined phycocyanin titration ranging from 1 g to 150 g/l. The re-concentration from 24 g/l to 115 g/l makes it possible to eliminate numerous impurities and increase 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), or by more than 110%.

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

Freeze-dried liquids did not show any slowdown in lyophilization for phycocyanin concentrations of 1 to 40 g/l. The atomized liquids did not need 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 a higher final purity.

As illustrated in Figure 1, phycocyanin extraction yields vary depending on the natural source of trace elements used during the co-extractions carried out. Two forms of phycocyanins were measured: C-phycocyanin and allophycocyanin. Total phycocyanin representing the sum of these last two.

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

The phycocyanin extraction yield obtained is greater than 90% when the extraction solvent used is water or phosphate buffer at pH 8.

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

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

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

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

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 enrichment observed 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 100g of dry spirulina.

Conclusion :

The implementation of the process according to the invention makes it possible to demonstrate that co-extraction allows the extraction of trace elements from the organic matrix by phycocyanin from spirulina. This thus results in an enrichment of the phycocyanin in trace elements from high 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 field. prior art.

Claims

1. Process for preparing a cyanobacteria 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 ambient temperature less than or equal to 40°C and greater than 4°C of a cyanobacteria, said cyanobacteria 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 cyanobacteria and the organic matrix allowing the co-extraction of the trace elements contained in the organic matrix by complexation with the phycocyanin of the cyanobacteria; and after possible filtration, recovery of a liquid extract of cyanobacteria comprising phycocyanin enriched with trace elements.

2. Method according to claim 1, characterized in that the cyanobacteria 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 Arthro, spira 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 cal daria, 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 legitima, 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, 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 a paste or in the form of a 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, berry cranberry and marine macroalgae are chosen from aonori, wakame, dulse, kumbu, ulva, chondrus, nostoc, porphyra, preferably ao-nori.

5. Method according to one of the preceding claims, characterized in that the grinding is carried out at ambient 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 in the wet process 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 phycocyanin enriched with recovered trace elements is reduced to solid powder by atomization or lyophilization.

10. Cyanobacteria extract, 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 1 1, 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 1 1 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 1 1 or 12 as a food supplement.