GALDIERIA's method for breaking down glycogen present in biomass

Abstract

The present invention relates to a method for processing glycogen present in the biomass of unicellular red algae (URA), particularly those of the genus Galdieria, and to a product obtained in this manner.

Description

Technical Field

[0001] The present invention relates to a treatment method for the degradation of glycogen present in the biomass of unicellular red algae (URA), particularly of the genus Galdieria, and to an aqueous product thus obtained.

Background Art

[0002] Unicellular red algae (URA) or red algae are characterized by the presence of pigments within their cells. In addition to chlorophyll and carotenoids, unicellular red algae produce phycobiliproteins. These natural pigments derived from photosynthesis are divided into four types: allophycocyanin, C-phycocyanin, phycoerythrin, and phycoerythrosin.

[0003] Phycocyanin has beneficial properties for human and animal health and is currently used in a wide range of fields including the pharmaceutical, cosmetic, and food industries.

[0004] Microalgae belonging to the class Cyanidiophyceae, more specifically, the genera Cyanidioschyzon, Cyanidium, and Galdieria, are of particular interest for phycocyanin production. The production of biomass from unicellular red algae is well known to those skilled in the art, particularly for the production of target molecules, especially proteins such as phycocyanin. Methods for producing and extracting said phycocyanin are described in the literature (International Publication No. 2017 / 093345, International Publication No. 2019 / 228947, International Publication No. 2018 / 178334, International Publication No. 02020 / 161280).

[0005] However, this class, particularly the microalgae of the genus Galdieria, is also known to possess glycogen as its primary storage sugar. This glycogen is a polymer of α(1→4) glucose branched by α-(1→6) bonds. Glycogen from the genus Galdieria has a particular characteristic: it possesses a high proportion of these branchings, i.e., approximately 7-18% of branched glucose randomly distributed along the molecule. This molecular structure gives the glycogen a spherical form and makes it water-soluble (Martinez-Garcia et al., Int J Biol Macromol. (2016) 89:12-8). Furthermore, if the genus Galdieria microalgae possess enzymes for the synthesis of highly branched glycogen, they also possess enzymes for its degradation (Martinez-Garcia et al., Int J Biol Macromol. (2016) 89:12-18). These enzymes are intracellular enzymes. The intracellular culture medium of microalgae in this class has a pH of 6.3–7.1 (Miyagishima et al., Plant Cell Physiol. 62(6):926-941(2021)). Therefore, these enzymes are known to be active within this precise pH range.

[0006] A filtration step may be necessary for the preparation of aqueous extracts from microalgae, such as phycocyanin extract from Galdieria sulphuraria. Since glycogen is readily soluble in cold water, it is found in the aqueous fraction along with the hydrophilic compound of interest, such as phycocyanin. Filters used to purify phycocyanin retain all or part of the glycogen, thus increasing the viscosity of the retaining solution. This creates technical constraints on methods, particularly with tangential filtration membranes, such as increased pressure, reduced flow rate, and clogging. On the other hand, if glycogen is not removed, the prepared extract will be high in glycogen, viscous, and low in phycocyanin.

[0007] Glycogen is known to be resistant to certain enzymes. However, methods involving the addition of suitable exogenous enzymes for its degradation have already been developed (International Publication No. 2020 / 144330).

[0008] If it is not possible to completely eliminate and / or inactivate it, enzymes added for the preparation of food must be listed in the composition. Such unavoidable substances correspond to additives or processing aids that may cause difficulties in marketing and / or formulation.

[0009] Other methods for preparing aqueous extracts from unicellular red algae, particularly Galdieria biomass, are described in the literature (Moon et al., Korean J. Chem. Engl. 2016, 31, 3, pages 490-495). However, these methods are difficult to apply on an industrial scale because they require either the addition of large amounts of ammonium sulfate, resulting in pollutant-rich effluents undesirable for ecological and economic reasons, or the use of very expensive equipment such as chromatographs, or a series of steps that are difficult to carry out on a scale larger than that of a laboratory. Furthermore, none of these methods can selectively remove glycogen from biomass or aqueous microalgae extracts.

[0010] Therefore, there is a need to provide a method for degrading glycogen present in the biomass of unicellular red algae, particularly in the biomass of microalgae of the genus Galdieria, whose main storage sugar is glycogen, while overcoming the problems of conventional technology, especially those detailed above. [Overview of the Initiative]

[0011] According to a first aspect of the present invention, the inventors have developed a method for processing the biomass of uncelled red algae (URA) of the genus Galdieria, and the URA biomass processing method is a) A step of biomass collection by separating the culture medium to obtain the live biomass of URA, b) Step (a) to obtain a lysate, the step of cellular lysis of crude biomass, c) A step of optional dilution of the solubilized product from step (b) to obtain a solubilized product, d) a step of separating insoluble matter suspended in the solubilized material from step (b) or from step (c) for obtaining a clarified fraction, This method includes a glycogenolysis step in which the raw biomass and / or dissolved and / or solubilized material is kept standing for at least 3 hours, during which time the liquid medium containing glycogen is at an acidic pH.

[0012] Surprisingly, glycogenolysis by endogenous microbial enzymes is effective over a wide pH range, not limited to the intracellular pH of the microbial organism, with a more acidic pH being optimal, particularly between 2 and 5. Even more surprisingly, enzymatic degradation functions inside cells not only in processed biomass such as raw biomass, thawed raw biomass, or dried raw biomass, but also in solubles and solubilized products. The present invention further relates to products obtainable by the method according to the present invention.

[0013] Advantageously, the method according to the present invention makes it possible to reduce, or even eliminate, the addition of exogenous enzymes adapted for the degradation of ARU glycogen, and thus reduce the addition of substances that must be qualified as additives and / or processing aids.

[0014] The method according to the present invention can also be applied under time, temperature, and pH conditions ranging from the mildest to the most stringent, and thus can be adapted to the stability of the molecule of interest to be extracted. [Brief explanation of the drawing]

[0015] [Figure 1]This shows the free glucose concentration in Galdieria sulphuraria biomass lysates over time at pH 3.75 and pH 6 at 37°C, with and without the addition of exogenous enzymes (SE: no enzyme), and with 1% exogenous enzymes in the lysate (E: enzyme). [Figure 2] The free glucose concentrations in Galdieria sulphuraria biomass lysates at room temperature ("ambient," i.e., 20°C) at different pH levels in the absence of exogenous enzymes are shown as a function of time. [Figure 3] The free glucose concentration in Galdieria sulphuraria biomass lysate at pH 3.75 at different temperatures, with and without the exogenous enzyme, is shown as a function of time (WE: without enzyme, E: with enzyme). [Figure 4] The free glucose concentration in Galdieria sulphuraria biomass lysate at pH 3.75 at different temperatures in the absence of exogenous enzymes is shown as a function of time. [Figure 5] The free glucose concentration as a function of time is shown in Galdieria sulphuraria biomass lysates and solubilizes at pH 3.75 and 20°C in the absence of the exogenous enzyme, and in Galdieria sulphuraria biomass lysates at pH 3.75 in the presence of the exogenous enzyme (WE: without enzyme, E: with enzyme). [Figure 6] This shows the percentage of glycogen digestion as a function of time in Galdieria sulphuraria biomass clarified at pH 4 and 20°C, either in the absence or presence of exogenous enzymes. [Figure 7] This shows the percentage of glycogen digestion as a function of time in Galdieria sulphuraria biomass lysates, solubilized fractions, and clarified fractions at pH 4 and 20°C in the absence of exogenous enzymes (WE: no enzyme). [Modes for carrying out the invention]

[0016] definition In the context of the present invention, the term "biomass" refers to a collection of microalgae cells that are preferentially produced by fermentation in a bioreactor. The biomass can be regarded as a mass of single-celled organisms.

[0017] The biomass can be subjected to various treatments and can be raw biomass, lysed biomass, thawed raw biomass, and / or dried raw biomass.

[0018] In the context of the present application, the characteristics of the biomass correspond to the average of the characteristics of all the cells constituting the biomass. In other words, lysed biomass is biomass that contains at least 50% lysed cells with respect to the total number of cells, and raw biomass can contain lysed cells resulting from the collection step as long as the number of lysed cells remains small compared to the number of non-lysed cells, that is, less than 50%, and is not regarded as lysed biomass.

[0019] The term "raw biomass" refers to the biomass obtained after collection, that is, after recovery of the fermentation broth and subsequent separation of the cells from at least a part of the culture medium that may have been thawed and / or dried.

[0020] The expression "lysed biomass" or "lysate" refers to microalgae biomass in which at least 50% of the cells are lysed, preferably at least 70% are lysed, more preferably at least 80%, 85%, 90%, 95%, up to 100% of the cells are lysed.

[0021] The expression "thawed raw biomass" refers to raw biomass that has been frozen, possibly for reasons of storage and / or transportation, and then thawed to reach a temperature suitable for the treatment according to the present invention, particularly a temperature suitable for the glycogenolysis step of the biomass according to the present invention.

[0022] According to the present invention, the expression "dry raw biomass" refers to raw microalgae biomass that has been dried using methods known to those skilled in the art and has a water content of less than 10%, preferably less than 7%, more preferably 5% to 1% water relative to the total weight of the biomass. Known drying methods include natural air drying, spray drying, fluidized air bed drying, roller dryer drying, and freeze drying.

[0023] The term "solubilizate" refers to a dissolved biomass or solute that has undergone a step of dilution using an aqueous solution of neutral, acidic, or basic pH.

[0024] The term "clarified fraction" refers to an aqueous extract obtained after separation of insoluble substances suspended in a solute or solubilizate.

[0025] The term "standing" refers to a stage where the chemical properties of preferably dried or frozen raw biomass, solute, and / or solubilizate are not modified by the addition or extraction of one or more components. Standing allows the raw biomass, solute, solubilizate, and / or clarified fraction to be mixed under non-destructive conditions that do not affect the chemical properties of the raw biomass, solute, and / or solubilizate, i.e., it does not prevent stirring.

[0026] The term "liquid medium containing glycogen" describes the intracellular medium of the cells constituting the raw biomass and / or, where applicable, the liquid phase of the solute, solubilizate, and / or clarified fraction.

[0027] The term "biomass treatment" refers to any method applied to biomass, particularly to modify its physical / chemical properties, extract target molecules, and / or purify them.

[0028] It should be noted that all the recited numerical intervals are intended to include all intermediate numbers (e.g., an interval of 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 4.32, and 5).

[0029] It should be noted that all the numbers listed refer to the actual values ​​given, as well as approximations of these values ​​estimated based on the common convention that the last digit indicated corresponds to the precision of the measurement. Where there is no precise error limit, the maximum error of the last digit to be specified should be estimated using rounding conventions.

[0030] Processing method according to the present invention The method according to the present invention is a method for processing the biomass of single-celled red algae (URA) of the genus Galdieria, and the URA biomass processing method is a) A step of biomass collection by separating the culture medium to obtain the live biomass of URA, b) Step (a) to obtain a lysate, the step of cellular lysis of crude biomass, c) A step of optional dilution of the solubilized product from step (b) to obtain a solubilized product, d) a step of separating insoluble matter suspended in the solubilized material from step (b) or from step (c) for obtaining a clarified fraction, This method includes a glycogenolysis step in which the raw biomass and / or dissolved and / or solubilized material is kept standing for at least 3 hours, during which time the liquid medium containing glycogen is at an acidic pH.

[0031] Ideally, throughout this method, the temperature, time, and pH conditions are specifically adapted to promote glycogenolysis during the standing stage while preventing the degradation of the target compound in the aqueous extract, such as phycocyanin.

[0032] In one embodiment, the glycogen degradation step is carried out on raw biomass. Then, in this embodiment, a) A step of collecting biomass by separating the culture medium for obtaining the biomass of URA, The raw biomass is allowed to stand for at least 3 hours, during which time the liquid medium containing glycogen is at an acidic pH. b) Step (a) to obtain a lysate, the step of cellular lysis of crude biomass, c) A step of optional dilution of the solubilized product from step (b) to obtain a solubilized product, d) A step of separating insoluble matter suspended in the dissolved substance from step (b) or the solubilized substance from step (c) for obtaining a clarified fraction, The method optionally includes the step of adjusting the pH of the raw biomass.

[0033] In this embodiment, glycogen is present in the intracellular culture medium of the cells constituting the biomass, the liquid medium is acidic in pH, and the biomass is preferably dried or thawed. In the following two embodiments, after cell lysis, glycogen is found in the liquid phase of the lysate and, if applicable, the solubilized product.

[0034] In one embodiment, the glycogenolysis step is performed on the soluble product. Then, in this embodiment, a) A step of biomass collection by separating the culture medium to obtain the live biomass of URA, b) A step of cellular lysis of crude biomass from step (a) for obtaining a lysate, comprising the step of allowing the lysate to stand for at least 3 hours in a liquid medium containing glycogen at an acidic pH, c) A step of optional dilution of the solubilized product from step (b) to obtain a solubilized product, d) A step of separating insoluble matter suspended in the dissolved substance from step (b) or the solubilized substance from step (c) for obtaining a clarified fraction, The method optionally includes adjusting the pH of the biomass or dissolved material.

[0035] To obtain the acidic pH required for the lysate standing step, it may be necessary to adjust the pH of the liquid medium containing the biomass and / or lysate.

[0036] In another embodiment, the glycogenolysis step is carried out on the solubilized product. Then, in this embodiment, a) A step of biomass collection by separating the culture medium to obtain the live biomass of URA, b) a step of cellular lysis of crude biomass from step (a) to obtain a lysate, and c) a step of optional dilution of the lysate from step (b) to obtain a solubilized product, The solubilized material is allowed to stand for at least 3 hours, during which time the liquid medium containing glycogen is at an acidic pH. d) Steps to obtain a clarified fraction: a step of separating insoluble substances suspended in the solubilized material from step (c), The method optionally includes adjusting the pH of the biomass, dissolved material, or solubilized material.

[0037] To obtain the acidic pH required for the solubilizer standing step, it may be necessary to adjust the pH of the liquid medium containing the biomass, lysate, and / or solubilizer.

[0038] a) Collect biomass The biomass according to the present invention is the biomass of unicellular red algae (URA), more specifically, the biomass of phycocyanin-producing microalgae having a high glycogen content.

[0039] These microalgae belong to the class Cyanidiophyceae, which includes the genera Galdieria, Cyanidium, and Cyanidioschyzon. Preferably, the microalgae belong to the genus Galdieria.

[0040] Microalgae of the genus Galdieria include Galdieria daedala, Galdieria maxima, Galdieria partita, Galdieria sulphuraria, Galdieria phlegrea, Galdieria javensis, Galdieria yellowstonensis, and Galdieria sp. Preferredly, the biomass according to the present invention is the biomass of Galdieria sulphuraria.

[0041] Methods for producing biomass from unicellular red algae (URAs), particularly from the genus Galdieria, are well known to those skilled in the art. In the context of the present invention, microalgae can be cultured using any known culture technique in a container suitable for growing microorganisms, also known as a bioreactor, bioreactor, or fermenter.

[0042] According to the present invention, the biomass of unicellular red algae is obtained from microalgae industrially cultured in a large-capacity reactor to obtain a fermentation mast containing high-density microorganisms. For the purposes of this application, “high density” means an amount corresponding to more than 50 g of dry material per liter of fermentation broth, preferably more than 100 g per liter. Examples of unicellular red algae cultures are described in the patent applications (International Publication Nos. 2017 / 050917, 2017 / 050918, 2017 / 093345, and 2019 / 228947). Those skilled in the art will understand that they can determine the optimal parameters and conditions for culturing microorganisms, such as temperature, lighting, culture time, or the nature and amount of nutrients supplied. The method according to the present invention includes step a) of collecting biomass.

[0043] Once the microorganisms are cultured and biomass is obtained, it is harvested and produced as biomass. Single-celled red algae can be harvested using any technique known to those skilled in the art, possibly including gravimetric or reduced-pressure filtration, decantation, precipitation, followed by gravimetric filtration or centrifugation.

[0044] The method according to the present invention comprises the steps of: a) collecting biomass corresponding to the recovery of fermentation broth; and subsequently separating biomass cells from at least a portion of the culture medium.

[0045] This step produces raw biomass. The raw biomass thus collected can also be preferably washed with water to remove certain soluble impurities.

[0046] After collection, the raw biomass obtained after one or more optional washes contains at least 70% water, up to a maximum of 90%, and preferably 75-88% water.

[0047] Preferably, the raw biomass according to the present invention has a dry material content of 5 to 30% by weight, generally, still preferentially 10 to 25% by weight, and more preferably 10 to 20% by weight, relative to the total weight of the raw biomass.

[0048] Furthermore, preferably, the crude biomass according to the present invention has a C-phycocyanin concentration of 3-12%.

[0049] b) Cell lysis A method for processing single-celled red algae biomass according to the present invention comprises step b) cellular lysis of the biomass from step a) to obtain a lysate.

[0050] Preferably, step b) of cell lysis is performed on live biomass having a dry matter content of 5–30% by weight, preferably 10–25% by weight, and more preferably 10–20% by weight, relative to the total weight of live biomass.

[0051] Furthermore, step b) of cell lysis is preferably performed on crude biomass according to the present invention having a C-phycocyanin concentration of 3-12%.

[0052] Cell lysis can be carried out by any lysis method known to those skilled in the art, in particular by enzymatic, mechanical, and / or chemical means.

[0053] In the context of the present invention, prior to step b) of cell lysis, the raw biomass may have undergone washing, freezing, thawing, drying, and / or rehydration steps. In other words, in the context of the present invention, the raw biomass may be thawed and / or dried biomass.

[0054] In the preferred case of mechanical dissolution, mechanical means that can be used according to the present invention include ball mills, high-shear mixers, high-pressure homogenizers, pin mills, impact mills, ultrasonics, or pulsed electric fields. For apparatus to carry out these methods, see: Ball mill: Discus-100 from Netzsch or ECM-AP60 from WAB; High-pressure homogenizer: Ariete from GEA; High-shear mixer: 700-X from Silverson; Pin mill: Contraplex from Hosokawa; and Impact mill: Condux from Netzsch.

[0055] Cell lysis is preferably carried out by mechanical lysis, preferably by grinding, and even more preferably by using a ball mill.

[0056] Preferably, the resulting solution has a dry material content of 5-30% by weight, preferably 10-25% by weight, and more preferably 10-20% by weight, relative to the total weight of the solution.

[0057] Furthermore, the resulting solution preferably has a C-phycocyanin concentration of 3% to 12% relative to the total weight of the dry material.

[0058] This dissolution step b) can be performed before or after the glycogenolysis step, i.e., before or after the standing step, preferably before.

[0059] c) Optional dilution of the solution According to the present invention, the dissolved biomass or dissolved material can optionally undergo a dilution step.

[0060] In the context of the present invention, the dilution step refers to the addition of a solution to dissolved biomass or dissolved material to reduce its dry substance concentration.

[0061] Next, the diluted dissolved biomass is defined as "solubilized material."

[0062] Advantageously, the dilution step is carried out by adding an aqueous solution.

[0063] In one embodiment, the aqueous solution is water.

[0064] Preferably, the dilution step c) is performed on a solution having a dry substance content of 5–30% by weight, preferably 12–25% by weight, relative to the total weight of the solution.

[0065] Furthermore, preferentially, step c) of dilution is performed on a solution having a C-phycocyanin concentration of 3% to 12% relative to the total weight of the dry material.

[0066] An aqueous solution may additionally contain one or more pH-adjusting compounds. The term "pH-adjusting compound" refers to any organic or inorganic compound (acidity corrector, acid, base, neutralizing agent, or buffer) used to modify pH. Examples of such compounds include sulfuric acid, acetic acid, citric acid, phosphoric acid, sodium citrate, potassium lactate, potassium malate, sodium chloride, disodium phosphate, and potassium phosphate. An aqueous solution will have an acidic or basic pH depending on the pH-adjusting compounds present in the solution.

[0067] Preferably, step c) diluting the dissolved biomass or dissolved material is carried out using an aqueous solution having a pH of 8 or less, particularly 0-6, preferably 1-6, and more preferably 2-5. The pH of the aqueous solution added to the dissolved biomass or dissolved material may be about 2, about 3, about 4, or about 5. Examples of acidic solutions that can be added to dissolved biomass are acid-containing solutions such as those described in the previous paragraph.

[0068] Depending on the circumstances, the solubilized substance may have a pH close to neutral, with a pH of 6-8, 1-6, or 8-14.

[0069] Preferably, the solubilized substance has a pH below 7, especially between 1 and 6, more preferably between 2 and 5, and even more preferably between 3 and 4.

[0070] Preferably, the resulting solubilized product has a dry material content of 1-15% by weight, preferably 3-12% by weight, and more preferably 4-8% by weight, relative to the total weight of the solubilized product.

[0071] Furthermore, the solubilized product obtained according to the present invention preferably has a C-phycocyanin concentration of 0.1-12%, preferably 0.5-8%, and more preferably 1-7%.

[0072] This dilution step c) can be performed before or after the glycogenolysis step, i.e., before or after the standing step.

[0073] Preferably, the dilution step c) is performed after the standing step.

[0074] Therefore, according to this preferred embodiment, the method according to the present invention is a series of steps, a) A step of biomass collection by separating the culture medium to obtain the live biomass of URA, b) A step of cellular lysis of crude biomass from step (a) to obtain a lysate, comprising the step of allowing the lysate to stand at an acidic pH for at least 3 hours, c) A step of diluting the dissolved substance to obtain a solubilized substance, d) a step of separating insoluble matter suspended in the solubilizer from step (c) for obtaining a clarified fraction.

[0075] d) Separate insoluble substances. As part of the method according to the present invention, the dissolved or solubilized substance undergoes step d) to separate the suspended insoluble matter and obtain a clarified fraction.

[0076] This step (d), which separates insoluble substances, is performed after the glycogenolysis step, i.e., after the standing step.

[0077] Step d) separating insoluble substances from the dissolved or solubilized substance can be carried out by any method known to those skilled in the art. These include front filtration and centrifugation.

[0078] According to the present invention, step d) for separating insoluble matter is performed on a solubilized product having a dry matter content of 5 to 30% by weight, preferably 10 to 25% by weight, and more preferably 15 to 20% by weight, relative to the total weight of the solubilized product, or more preferably on a solubilized product having a dry matter content of 1 to 15% by weight, preferably 3 to 12% by weight, and more preferably 4 to 8% by weight, relative to the total weight of the solubilized product.

[0079] Preferably, according to the present invention, step d) for separating insoluble substances is performed on a dissolved or solubilized substance having a pH of less than 7, preferably 1 to 6, more preferably 2 to 5, and still more preferably 3 to 4.

[0080] The clarified fraction according to the present invention preferably has a dry substance content of 0.1% to 5% by weight, preferably 0.5% to 4% by weight, and more preferably 1% to 3% by weight, based on the total weight of the clarified fraction.

[0081] The clarified fraction according to the present invention preferably has a C-phycocyanin concentration of 0.1 to 20 g / L. In the specific case where the method according to the present invention does not include step c), the clarified fraction according to the present invention has a C-phycocyanin concentration of 7 to 19 g / L, and more preferably 12 to 18 g / L. In the reverse case where the method according to the present invention includes step c), the clarified fraction according to the present invention has a C-phycocyanin concentration of 0.1 to 12 g / L, preferably 0.5 to 8 g / L, and more preferably 1 to 7 g / L.

[0082] Glycogen breakdown The method according to the present invention is characterized by a glycogenolysis step using endogenous enzymes of microorganisms, which involves keeping the raw biomass and / or dissolved product and / or solubilized product in a standing state for at least 3 hours (standing step) (during which time the liquid medium containing glycogen is at an acidic pH).

[0083] For glycogenolysis by endogenous enzymes of microorganisms to be effective during the standing step, these endogenous enzymes must be active and therefore, for example, must not be inactivated by a heating step prior to the standing step.

[0084] The period of the standing-off step A standing period of at least 3 hours is performed on the biomass, lysate, and solubilizer at an acidic pH. In particular, according to the present invention, the standing period can be performed before or after step b) of the cell lysis, i.e., on the biomass, lysate, and / or solubilizer.

[0085] According to the present invention, the settling step can be advantageously carried out under stirring of the raw biomass and / or dissolved and / or solubilized material.

[0086] In principle, the settling step is performed only on dried or thawed raw biomass, dissolved materials, or solubilized materials. In the case of dissolved materials and / or solubilized materials, the settling step is performed after adjusting the pH, if necessary, to obtain the acidic pH required for the settling step.

[0087] Preferably, according to the present invention, the standing is performed after the cell lysis step b), in other words, preferably on the lysate and / or solubilized product, and more preferably on the lysate.

[0088] Preferably, the standing step is performed in a culture medium containing lysed cells, i.e., the lysate from step b) and / or the solubilized product from step c) if applicable.

[0089] Conditions for the standing step According to the present invention, the settling step can be performed on live biomass, dissolved materials, and / or solubilized materials that are immersed in the dark or exposed to natural or artificial light. Preferably, the settling step is performed on live biomass, dissolved materials, or solubilized materials that are immersed in the dark.

[0090] According to the present invention, the settling step is performed on the raw biomass, dissolved material, or solubilized material with or without aeration.

[0091] The "acidic pH" of the glycogen-containing medium during the standing stage is defined as a pH below 7, particularly 1 to 6, preferably 2 to 5, and even more preferably 3 to 4.

[0092] Regarding biomass, glycogen is found in the intracellular culture medium of the cells that make up the biomass, which is a liquid culture medium with an acidic pH.

[0093] Optionally, the method according to the present invention further comprises the step of adjusting the pH of the biomass, dissolved product, and / or solubilized product to the acidic pH of the standing step.

[0094] In this case, the pH of the biomass, dissolved matter, and / or solubilized matter is adjusted to a pH below 7, particularly 1 to 6, more preferably 2 to 5, and even more preferably 3 to 4.

[0095] Reagents for adjusting the pH, i.e., for acidifying or basicizing the biomass, lysate, or solubilizer, can be added in solid or solution form. Advantageously, the pH of the biomass, lysate, or solubilizer is adjusted by adding an acidic or basic solution, preferably in aqueous solution form.

[0096] Examples of pH-adjusting compounds are listed above in the explanation of step c), and include sulfuric acid, hydrochloric acid, nitric acid, phosphoric acid, acetic acid, sodium hydroxide, sodium carbonate, or sodium bicarbonate.

[0097] Those skilled in the art will understand that it is possible to determine whether an acidic or basic compound or solution needs to be added to a biomass, soluble, and / or solubilized product in order to adjust the pH to a desired value.

[0098] If present, this step of adjusting the pH is performed upstream of the standing step, preferably on thawed and / or dried raw biomass, dissolved or solubilized material.

[0099] Advantageously, if present, this step of adjusting the pH is performed on the dissolved product before the standing step, as well as before the dilution and separation steps c) and d).

[0100] Alternatively, the step of adjusting the pH, if present, can be carried out simultaneously with step c) of dilution. In particular, if step c) is present, the step of adjusting the pH can be carried out on the solubilizer simultaneously with step c) of dilution, before the standing step. In this case, after dilution of the solubilizer, the resulting solubilizer will have a pH below 7, particularly 1 to 6, preferably 2 to 5, and even more preferably 3 to 4.

[0101] The standing step can last up to one week or seven days. Preferably, the standing step lasts from 3 hours to one week, more preferably from 3 hours to 48 hours, even more preferably from 6 to 36 hours, and still more preferably from 10 to 24 hours.

[0102] According to the embodiment, the standing period lasts from several hours to several days, particularly approximately 3 hours, 6 hours, 8 hours, 10 hours, 12 hours, 24 hours, 36 hours, 48-72 hours, 2-7 days, 3-7 days, 4 days, 5 days, or 6 days.

[0103] According to one embodiment, the temperature of the live biomass and / or dissolved and / or solubilized material during the standing period is maintained at a temperature of 15°C to 70°C, particularly 15°C to 50°C, preferably 15°C to 40°C, and even more preferably 15°C to 30°C.

[0104] Advantageously, the temperature of the biomass and / or dissolved and / or solubilized material during this standing period is maintained at a constant temperature, particularly 15°C to 70°C, especially 15°C to 50°C, preferably 15°C to 40°C, and even more preferably 15°C to 30°C.

[0105] In another embodiment, the temperature of the biomass and / or dissolved and / or solubilized material during the standing period is maintained at a temperature below 15°C, preferably between 4°C and 15°C.

[0106] Advantageously, according to this other embodiment, the temperature of the biomass and / or dissolved and / or solubilized material during this standing period is maintained at a constant temperature, particularly 4°C to 15°C. It should be noted that the closer the standing step is to the optimal temperature and pH combination, the less time is required for glycogen decomposition. For example, a standing step with a pH of 3 to 4 and a temperature of 15 to 30°C may last 2 to 10 hours, while a standing step with a pH of about 6 and a temperature of about 10°C should last at least 6 to 7 days.

[0107] In the context of the present invention, the standing step is carried out using a liquid medium containing glycogen that is free from significant microbial contamination. Such contamination can be avoided by adding a preservative to the liquid medium containing glycogen. These preservatives are well known to those skilled in the art and are selected, in particular, from sodium benzoate, potassium benzoate, calcium benzoate, benzoic acid, sodium diacetate, calcium propionate, sodium propionate, sodium nitrate, potassium sorbate, sodium sorbate, methyl gallate, propyl gallate, sodium ethylenediaminetetraacetate, methylparaben, natamycin, propylparaben, and mixtures thereof.

[0108] Selective addition of exogenous enzymes This method may further include a step of adding an exogenous enzyme to complement the standing step. This addition of the exogenous enzyme allows glycogenolysis to be completed as needed, particularly if the desired level of degradation is not achieved. Here, "complement" is understood to mean that the majority of the glycogenolysis, i.e., at least 50%, is a result of performing the standing step independently of this possible addition.

[0109] The term "enzyme" refers to a protein that activates or accelerates a chemical or biological reaction. In the context of this application, the term "exogenous enzyme" refers to an enzyme that is not naturally produced by cells or microorganisms of processed biomass of unicellular red algae (URA). In particular, such exogenous enzymes are selected from enzymes extracted from Aspergillus, Bacillus, or Trichoderma.

[0110] The exogenous enzymes according to the present invention have glycogenolytic activity. These enzymes are well known to those skilled in the art and are selected from, in particular, enzymes having α1-4 glucuronidase activity, α1-4 glucosidase activity, α1-6 glucosidase activity, and amylase activity. It should be noted that any one of these enzymes or a mixture thereof can be used. These enzymes have been found to reduce the size of the glycoside chains of glycogen present in the culture medium, and then eliminate them along with their degradation byproducts in subsequent stages.

[0111] Enzymes having α1-4 glucuronidase and / or α1-4 glucosidase activity include pectinases known to decompose pectin, particularly pectinases extracted from filamentous fungi such as Aspergillus, and more specifically, pectinases extracted from Aspergillus aculeatus, such as the enzyme sold by Novozymes under the name Pectinex®.

[0112] Examples of enzymes possessing α1-6 glucosidase activity include, in particular, pullulanases known to hydrolyze the α1-6 glucosidic bond of pullulan, which are also known to debranch starch. These are generally enzymes extracted from bacteria of the genus Bacillus, among others. U.S. Patents 6,074,854 and 5,817,498, and International Publication No. 2009 / 075682 describe such pullulanases extracted from Bacillus deramificans or Bacillus acidopullulyticus. Commercially available pullulanases include, in particular, Promozyme D2 and Novozym 26062 from Novozymes, or Optimax L1000 from DuPont-Genencor.

[0113] Many enzymes possessing amylase activity known to break down starch are known from the prior art and are described in the literature, particularly in patent applications such as International Publication No. 2019 / 036721. Commercially available amylases are known in particular by the names "Amylase AG XXL" (from Novozyme) or "Panzym® AG XXL" (from Eaton).

[0114] Enzymes can be used in their pure or concentrated form, or optionally, as a mixture with one or more excipients. The enzymes used in the method of the present invention are in powder or solution form. In the latter case, the enzymes are preferentially soluble in water.

[0115] Preferred conditions for using exogenous enzymes are a pH below 7 and a reaction temperature below 60°C, preferably below 50°C, and even more preferably below 30°C. In particular, the temperature of the solution to which the exogenous enzyme is added and the enzymatic reaction occurs is 4 to 60°C, preferably 20 to 42°C, and the pH of the solution is 5 or less, preferably about 4.5. The exogenous enzyme can be added to the culture medium either in free form or immobilized on a support.

[0116] Exogenous enzymes can be added to the lysate, in which case the enzymatic reaction with the exogenous enzyme is carried out in the lysate, while the standing step is carried out in the raw biomass beforehand and / or in the lysate beforehand and / or simultaneously in the lysate.

[0117] In a preferred embodiment, the exogenous enzyme is added after the standing step. In this case, If the settling step is performed on live biomass, exogenous enzymes can be added to the lysate and / or solubilized and / or clarified fractions to complete the glycogenolysis resulting from the settling of live biomass. - If the standing step is performed on the lysate, exogenous enzymes can be added to the lysate after the standing step, or to the solubilizer and / or clarification fraction, to complete the glycogenolysis resulting from the standing of the lysate. - If the standing step is performed on the solubilized product, exogenous enzymes can be added to the solubilized product after the standing step and / or to the clarified fraction to complete the glycogenolysis resulting from the standing of the solubilized product.

[0118] In a more preferred embodiment, the standing step is performed on the lysate, and preferably, the optional addition of exogenous enzymes is performed on the clarified fraction or the solubilized product.

[0119] This combination of static incubation and exogenous enzyme addition reduces the amount of exogenous enzyme used compared to conventional methods.

[0120] The exogenous enzyme is an enzyme having enzymatic activity equivalent to that of the Pectinex Ultra SP-L enzyme, with an activity declared by the manufacturer at 3300 PGNU / g, and is added in a proportion of less than 0.01% by weight relative to the total weight of the raw biomass, lysate, solubilized product, or clarified fraction being processed.

[0121] In one embodiment, the added exogenous enzyme content is 0.005% by weight or less, preferably 0.0025% by weight or less, and more preferably 0.0001% by weight or less. Note that, if necessary, the concentration of the exogenous enzyme is adjusted according to the activity of an exogenous enzyme selected to have activity in the reaction medium equivalent to that of Pectinex Ultra SP-L at the concentration according to the present invention at 3300 PGNU / g.

[0122] Those skilled in the art will also be able to determine how to adjust the amount of enzyme added during the process to increase the breakdown of glycogen present in the lysate, solubilized, and / or clarified fraction.

[0123] The glycogenolysis step, with or without the addition of exogenous enzymes, is considered sufficient, particularly to improve the subsequent filtration step, when at least 10%, preferably at least 50%, and more preferably 50% to 80% of the initial glycogen content in the biomass is degraded.

[0124] e) Concentration The method according to the present invention may further include step e) concentrating the clarified fraction using a conventional water removal method to obtain a concentrated aqueous extract.

[0125] Conventional water removal methods are known to those skilled in the art and include filtration, evaporation under atmospheric pressure or vacuum, spraying, infrared drying, refracting window drying, and freeze-drying.

[0126] In particular, step e) of the method according to the present invention allows for the concentration of the target molecule while preserving essential components of the clarified fraction.

[0127] Depending on the concentration method selected, step e) of concentration, in particular in concentration by filtration, removes all or some of the impurities present in the clarified fraction, such as solid residues resulting from glycogenolysis, residual glycogen, oligomers, and sugars.

[0128] More preferably, the biomass treatment method according to the present invention includes step e) concentrating the clarified fraction by filtration, particularly by tangential filtration such as ultrafiltration.

[0129] Preferably, the biomass treatment method according to the present invention includes a concentration step e) in which the clarified fraction is concentrated 2 to 1000 times, and more preferably 20 to 60 times.

[0130] Preferably, step e) is performed at a pH of less than 7, preferably between 1 and 6, even more preferably between 2 and 5, and still more preferably between 3 and 4.

[0131] In addition to further degradation by the addition of enzymes and / or concentration of the clarified fraction, the method according to the present invention may include subsequent steps for purifying the clarified fraction and / or concentrated aqueous extract, particularly for purifying proteins in solution.

[0132] Products according to the present invention The products according to the present invention include a variety of organic substances, depending on the processing method applied, including water-soluble proteins containing phycocyanin, sugars containing glycogenolysis by-products (glucose oligomers), and possibly residual undigested glycogen and insoluble substances.

[0133] If present, phycocyanins may include phycocyanins that are resistant to acidic pH. Acidic pH-resistant phycocyanins are those that are stable at acidic pH; that is, they do not precipitate or lose their color at acidic pH. Tolerance or stability to acidic pH can be measured as a loss of color of less than 10% after exposure to an acidic pH, i.e., a pH below 7, particularly a pH between 2 and 5, for at least 10 minutes. Stability at acidic pH can also be measured by other methods, such as protein structure monitoring.

[0134] The presence of phycocyanin that exhibits tolerance to acidic pH results from performing steps d) and optionally e) at a pH below 7, preferably 1–6, more preferably 2–5, and even more preferably 3–4, according to the extraction method described in International Publication No. 2018 / 178334.

[0135] In a preferred form, the product according to the present invention contains phycocyanin that exhibits resistance to acidic pH.

[0136] A concentrated aqueous extract is obtained by a method according to the present invention, which includes step e) of concentrating the clarified fraction as described above. Conversely, the clarified fraction is obtained by a method according to the present invention, which does not include step e) of concentrating the clarified fraction as described above.

[0137] Preferably, the product according to the present invention contains, in particular, a concentration of exogenous enzymes that is either free of exogenous enzymes or undetectable by conventional assay methods, after protein precipitation with acetonitrile, digestion with trypsin, and subsequent analysis by mass spectrometry (LC-MS-MS).

[0138] In particular, the product according to the present invention preferentially does not contain exogenous enzymes selected from pectinase, amylase, and pullulanase, or contains them in concentrations undetectable by conventional assay methods, especially mass spectrometry.

[0139] If necessary, the products according to the present invention are prepared to eliminate any impurities that would make them unsuitable for consumption, particularly for human consumption.

[0140] The product according to the present invention may also be formulated by means known to those skilled in the art to avoid the decomposition of its constituent materials during storage or subsequent use.

[0141] Finally, the products of the present invention, as potentially formulated, can be packaged for storage and use in either a large-capacity container or a smaller container having a volume corresponding to a single-use portion, known as a single-dose portion for human consumption, for example. In this case, the container may be rigid, like a glass ampoule, or flexible, like a capsule suitable for consumption.

[0142] Following acid hydrolysis of the sample, the sugar content of the product according to the present invention, particularly glucose, mannose, and galactose (referred to as "hydrolyzed glucose," "hydrolyzed galactose," and "hydrolyzed mannose," respectively), is measured by high-performance liquid chromatography (HPLC-RID determination of hydrolyzed sugars) using a Hi-Plex H+ ion exclusion / ligand exchange column and refractive index detection. To do this, 1.5 mL of supernatant from a vortex-homogenized sample is hydrolyzed with 1.5 mL of 2N sulfuric acid at 110°C for 2 hours. The sample is then filtered (0.22 μm) and analyzed.

[0143] It should be noted that the hydrolyzed glucose content measured in this manner includes both undigested residual glycogen glucose and free glucose present in the product.

[0144] According to the present invention, the free glucose content is determined by biochemical analysis, particularly using a YSI® biochemical analyzer in accordance with the manufacturer's recommendations.

[0145] Therefore, in order to estimate the undigested residual glycogen content in the product according to the present invention, simply subtract the free glucose content from the hydrolyzed glucose content.

[0146] The percentage of glycogenolysis is calculated as follows: (concentration of free glucose in the sample under study / maximum concentration of free glucose in an equivalent sample treated with an exogenous enzyme at 1% of the enzyme solution volume relative to the total volume of the sample) × 100.

[0147] According to the present invention, the protein content of the product is determined by the DUMAS method. The sample is subjected to high-temperature combustion in a pure oxygen stream, and the nitrogen oxides produced are reduced by copper. After separation of the reaction byproducts, nitrogen is measured using a thermal conductivity detector, and the result is expressed as N(%). This percentage of nitrogen is then converted to the amount of protein by applying the following formula: N * 6.25(%)(ISO / TS 16634-2:2009).

[0148] Finally, to determine the C-phycocyanin content of the sample according to the present invention, 500 pl of the sample is mixed with 1.5 ml of 100 mM Tris-Cl buffer at pH 7.5, and the absorbance at 652 and 620 nm is measured using a Metier Toledo spectrophotometer. The C-phycocyanin concentration is then calculated using the following formula. [Phycocyanin] mg / mL unit = (0.162 × A620nm - 0.098 × A652nm) × dilution

[0149] According to the present invention, glycogenolysis of less than 10% by weight of total glycogen is not considered sufficient. Furthermore, glycogenolysis of less than 10% does not improve the filtration of the extract. Therefore, glycogenolysis is considered not to be functioning.

[0150] According to one embodiment, the clarified fraction obtained according to the present invention has an estimated undigested residual glycogen (g / L) / C-phycocyanin (g / L) ratio of less than 6, advantageously less than 4, preferably less than 3, more preferably less than 2.5, or even more preferably less than 1.

[0151] The clarified fraction according to the present invention has a total sugar concentration of 40 g / L or less, particularly 0.1 to 40 g / L, preferably 1 to 20 g / L, and more preferably 3 to 11 g / L, as measured by HPLC-RID determination of hydrolyzed sugars. and / or hydrolyzed glucose concentrations of 20 g / L or less, preferably 0.1 to 20 g / L, preferably 1 to 10 g / L, and even more preferably 2 to 5 g / L. and / or hydrolyzed galactose concentrations of 10 g / L or less, particularly 0.01 to 10 g / L, preferably 0.1 to 5 g / L, and more preferably 0.5 to 3 g / L. and / or hydrolyzed mannose concentrations of 10 g / L or less, preferably 0.01 to 10 g / L, preferably 0.1 to 5 g / L, and more preferably 0.5 to 3 g / L. and / or having a free glucose concentration of 10 g / L or less, particularly 0.01 to 10 g / L, preferably 0.1 to 5 g / L, and even more preferably 0.2 to 2 g / L.

[0152] The C-phycocyanin content of the clarified fraction is favorably in the range of 0.1–12 g / L, more preferably 0.5–8 g / L, and more preferably 1–7 g / L.

[0153] Preferably, the clarified fraction has a ratio of at least 0.001, preferably 0.005 to 120, more preferably 0.05 to 12, and even more preferably 0.1 to 3 (C-phycocyanin (g / L) / total sugars (g / L) as measured by HPLC-RID determination of hydrolyzed sugars).

[0154] According to another preferred embodiment of the present invention, the clarified fraction contains C-phycocyanin and total sugars in a ratio of 0.005 to 120, particularly 0.05 to 12, more preferably 0.1 to 3 (C-phycocyanin (g / L) / total sugars (g / L) as measured by HPLC-RID determination of hydrolyzed sugars).

[0155] In certain embodiments where the concentrated aqueous extract has a high C-phycocyanin concentration, the ratio (C-phycocyanin (g / L) / total sugar (g / L)) is at least 90 and may exceed 120.

[0156] Preferably, the clarified fraction has a ratio of at least 0.01, preferably 0.01 to 1200, more preferably 0.1 to 80, and even more preferably 0.1 to 12 (C-phycocyanin (g / L) / hydrolyzed mannose (g / L)).

[0157] Preferably, the clarified fraction has a ratio of at least 0.01, preferably 0.01 to 1200, more preferably 0.1 to 80, and even more preferably 0.1 to 12 (C-phycocyanin (g / L) / hydrolyzed galactose (g / L)).

[0158] The ratio of the clarified fraction (C-phycocyanin (g / L) / hydrolyzed glucose (g / L)) is preferably less than 120, preferably between 0.005 and 120. Preferably this ratio is between 0.05 and 12, and more preferably between 0.1 and 10.

[0159] Preferably, the concentrated aqueous extract according to the present invention has a dry matter content that is 2 to 1000 times higher, and more preferably 20 to 60 times higher, than that of the clarified fraction from which it is derived.

[0160] The C-phycocyanin content of the concentrated aqueous extract is favorably in the range of 7-120 g / L, preferredly in the range of 20-100 g / L, and more preferably in the range of 40-80 g / L.

[0161] The concentrated aqueous extract according to the present invention has a total sugar concentration of 50 g / L or less, particularly 1 to 50 g / L, preferably 5 to 40 g / L, and more preferably 10 to 30 g / L, as measured by HPLC-RID determination of hydrolyzed sugars. and / or hydrolyzed glucose concentrations of 20 g / L or less, preferably 0.1 to 20 g / L, preferably 1 to 10 g / L, and even more preferably 2 to 5 g / L. and / or hydrolyzed galactose concentration of 15 g / L or less, especially 0.5 to 15 g / L, preferably 1 to 13 g / L, and more preferably 3 to 8 g / L. and / or hydrolyzed mannose concentrations of 15 g / L or less, preferably 0.5 to 15 g / L, preferably 1 to 13 g / L, and more preferably 3 to 8 g / L. and / or having a free glucose concentration of 15 g / L or less, particularly 0.01 to 15 g / L, preferably 0.5 to 8 g / L, and even more preferably 1 to 4 g / L.

[0162] The weight ratio of the concentrated aqueous extract (C-phycocyanin (g / L) / hydrolyzed glucose (g / L)) is in the range of 2 to 80. Preferably, this ratio is 10 to 70, and more preferably 15 to 60.

[0163] Preferably, the concentrated aqueous extract has a ratio of at least 0.01, preferably 0.01 to 1200, more preferably 0.1 to 80, and even more preferably 0.3 to 12 (C-phycocyanin (g / L) / hydrolyzed mannose (g / L)).

[0164] Preferably, the concentrated aqueous extract has a ratio of at least 0.01, preferably 0.01 to 1200, more preferably 0.1 to 80, and even more preferably 0.3 to 12 (C-phycocyanin (g / L) / hydrolyzed galactose (g / L)).

[0165] Preferably, the concentrated aqueous extract has a ratio of 0.2 to 6.0, more preferably 0.5 to 5.0, and even more preferably 1.0 to 4.5 (protein (%) / estimated undigested residual glycogen (g / L)).

[0166] Preferably, the concentrated aqueous extract has an estimated undigested residual glycogen content (hydrolyzed glucose content (g / L) - free glucose content (g / L)) of 0.1 to 10 g / L, more preferably 0.2 to 7 g / L, and even more preferably 0.3 to 5 g / L.

[0167] Preferably, the concentrated aqueous extract has a ratio of less than 1%, preferredly 1-0.01%, more preferably 0.8-0.05%, and even more preferably 0.6-0.1% (protein (%) / total sugar (g / L) as measured by HPLC-RID determination of hydrolyzed sugars).

[0168] Finally, the present invention relates to a process for preparing a concentrated aqueous extract of phycocyanin-producing microalgae ARU biomass having a high glycogen content, comprising a processing method comprising a previously defined glycogen degradation step and a concentration step.

[0169] use The present invention also relates to the use of all or part of the products obtained by the method according to the present invention as products for the preparation of pharmaceutical or dietary compositions, including foods, pharmaceuticals, nutritional supplements, meals, cosmetics, or industrial compositions. In particular, it relates to the use of products obtained according to the method of the present invention, which contain phycocyanin, for the preparation of pharmaceutical or dietary compositions.

[0170] According to one embodiment, the composition according to the present invention comprises all or part of the product obtained using the method according to the present invention, and one or more excipients.

[0171] According to a preferred embodiment, the composition according to the present invention is an acidic composition having a pH of 6 or less, preferably 5 or less, more preferably 4 or less, and particularly 2 to 4.

[0172] According to the present invention, an acidic composition means any composition comprising all or part of the product obtained using the method according to the present invention, and a mineral acid or an organic acid.

[0173] Mineral acids or organic acids that can be used in the compositions according to the present invention are known to those skilled in the art. Examples of mineral acids include carbonic acid, phosphoric acid, hydrochloric acid, sulfuric acid, perchloric acid, sulfonic acid, and nitric acid. Examples of organic acids include citric acid, lactic acid, malic acid, tartaric acid, and succinic acid.

[0174] The acidic compositions according to the present invention may also include a vehicle, which may contain structural components associated with active compounds identified in terms of their nutritional contribution or their properties beneficial to human or animal health.

[0175] Compositions other than dietary compositions may be pharmaceutical, veterinary, or cosmetic, and may further contain one or more known additives and / or active ingredients used in this type of application.

[0176] The compositions according to the present invention can be provided in any common form known to those skilled in the art. These include solid, liquid, fluid, paste-like, or viscous forms, particularly creams, gels, foams, and pastes. The compositions according to the present invention can also take the form of dried food for cooking, powder for dilution, or gelatinous composition for food compositions.

[0177] In these solid compositions, all or part of the extract obtained according to the present invention is preferably added in powder form. In this case, the product obtained from the method according to the present invention is first dried to take the form of a powder.

[0178] The liquid composition is preferably an aqueous composition in which all or part of the extract obtained according to the present invention is dissolved.

[0179] According to certain embodiments of the present invention, the liquid composition may be a food composition, more specifically, an acidic, carbonated, or non-carbonated beverage. Examples include soda, juice, sports drink, exercise drink, and energy drink. The compositions of these beverages are well known to those skilled in the art and may contain sugars, inorganic salts, food additives, or dissolved gases. An acidic beverage according to the present invention may be a conventional acidic beverage in which the dyes commonly used are replaced, in whole or in part, by a product containing acid pH-tolerant phycocyanin according to the present invention.

[0180] The phycocyanin content in the composition according to the present invention will be consistent with the concentrations typically applied in the intended field of application. [Examples]

[0181] Example 1: Analysis of glycogenolysis with and without the addition of exogenous enzymes at different pH levels. The amount of free glucose over time in Galdieria sulphuraria biomass lysates at 37°C was measured as a function of time at pH 3.75 without enzyme addition, at pH 6 without enzyme addition, and at pH 6 with enzyme addition (1% volume of enzyme solution relative to the total lysate volume, Novozymes' "Amylase AG XXL") (YSI® 2950).

[0182] These dynamics are shown in Figure 1.

[0183] Acidification of the solution is achieved by adding a sufficient amount of citric acid to change the pH from 6 to 3.75.

[0184] The results indicate that more glucose is released at an acidic pH of 3.75 than at pH 6. After 24 hours of incubation at pH 3.75 without enzyme addition, the free glucose in the sample even reached the same level as in the sample to which the exogenous enzyme was added.

[0185] It should also be noted that glucose release occurs at pH 6 in the absence of exogenous enzymes, and its kinetics are simply slower (a gentler gradient).

[0186] Example 2: Analysis of glycogenolysis without the addition of exogenous enzymes at different pH levels. The amount of free glucose over time in Galdieria sulphuraria biomass lysate (20% by weight of dry matter (DM) of the total lysate weight) at room temperature (20°C) with no enzyme addition at pH 3-7 was measured (YSI® 2950).

[0187] These dynamics are shown in Figure 2.

[0188] Acidification and basicization of the dissolved solution are achieved by adding a sufficient amount of citric acid or sodium hydroxide, respectively.

[0189] The results indicate that, over a given period, lower pH levels lead to less glycogen breakdown. Glycogen digestion without the addition of exogenous enzymes on the lysate is primarily active under the most acidic conditions. Here, we find that this activity is high at pH 3 but slightly lower at pH 4. Activity at pH levels 5–7 is the same but slower than at lower pH levels.

[0190] Example 3: Analysis of glycogenolysis at different temperatures with and without the addition of exogenous enzymes The amount of free glucose over time in Galdieria sulphuraria biomass lysate (20% by weight of the dry material (DM) of the total lysate weight) at pH 3.75 was analyzed at different temperatures (4°C, 20°C, and 37°C) with and without the enzyme (1% "Amylase AG XXL" from Novozymes) at pH 3.75 and 37°C (YSI® 2950).

[0191] The results are shown in Figure 3. They demonstrate the digestion of glycogen to free glucose even in the absence of enzyme addition. In particular, after 24 hours at 37°C, the amount of free glucose is the same regardless of whether enzyme addition was present or not.

[0192] Example 4: Analysis of glycogenolysis at different temperatures without the addition of exogenous enzymes. The amount of free glucose over time in Galdieria sulphuraria biomass lysate (20% by weight of the total lysate weight in dry material (DM)) at pH 4 was analyzed at different temperatures (4°C, 15°C, 20°C, 30°C, 40°C, and 50°C) without the addition of exogenous enzymes (YSI® 2950).

[0193] The results are shown in Figure 4. They indicate that, over a given time, the higher the temperature, the greater the amount of glycogen digested during the standing step according to the present invention.

[0194] Example 5: Analysis of glycogenolysis at different stages of extraction methods The amount of free glucose at room temperature (20°C) in the lysate (20% by weight of dry material (DM) relative to the total weight of the lysate) and Galdieria sulphuraria biomass solubilize (7.5% by weight of DM relative to the total weight of the solubilize) at pH 3.75 without the addition of exogenous enzymes will be analyzed (YSI® 2950). In this context, the dynamics of glucose release at room temperature (20°C) in the Galdieria sulphuraria biomass lysate at pH 3.75 with the addition of an exogenous enzyme (enzyme solution at 1% volume relative to the total volume of the lysate, "Amylase AG XXL", Novozymes) will be considered as a control.

[0195] These dynamics are shown in Figure 5.

[0196] From the results obtained, extrapolation can be concluded that, even without the addition of enzymes, in the case of lysates, all digestible glycogen would be broken down and released as free glucose after 24 hours. On the other hand, in the case of solubilized substances, the reaction appears slower and is not yet complete after 24 hours.

[0197] It should be noted that all samples studied, whether prepared with or without the addition of exogenous enzymes, exhibited more than 10% glycogenolysis, and therefore good filterability after glycogen digestion.

[0198] Example 6: Analysis of glycogenolysis using conventional techniques In International Patent Publication No. 2020 / 144330, Example 2 demonstrates the degradation of glycogen in a crude solution of phycocyanin in the presence and absence of an exogenous enzyme.

[0199] To achieve this, Galdieria sulphuraria biomass is produced and then harvested / collected. The cells are then lysed and diluted to obtain a solubilized aqueous extract. A clarified fraction is then obtained from this aqueous extract by separating the solids through filtration on a 0.22 μm filter.

[0200] The amount of free glucose at room temperature (20°C) in the Galdieria sulphuraria biomass clarified fraction (MS at 7.5% by weight relative to the total weight of the clarified fraction) at pH 4, with and without the addition of an exogenous enzyme (enzyme solution at 0.1% volume relative to the total volume of the clarified fraction, such as Novozymes' "Amylase AG XXL", Novozymes' "Pectinex Ultra SP-L", or Novozymes' "BAN480L"), is analyzed (using a YSI 2700 Biochemestry Analyzer).

[0201] The results are shown in Figure 6.

[0202] The results suggest that without the addition of exogenous enzymes, if the clarified fraction is left standing for 95 hours, glycogen is not yet fully digested (less than 10% breakdown). However, with the addition of enzymes, all glycogen is digested after 95 hours.

[0203] Example 7: Analysis of glycogenolysis at all stages of the extraction method The amount of free glucose at room temperature (20°C) in the lysate (20% by weight DM relative to the total weight of the lysate), solubilizer, and clarified fraction (7.5% by weight DM relative to the total weight of the solubilizer or clarified fraction) of Galdieria sulphuraria biomass at pH 4, without the addition of exogenous enzymes, is analyzed (YSI® 2950). The clarified fraction is obtained after filtering the solubilizer through a 0.22 μm filter.

[0204] In this context, the glucose release dynamics at room temperature (20°C) in the lysate, solubilized, and clarified fractions of Galdieria sulphuraria biomass at pH 4, accompanied by the addition of an exogenous enzyme (1% enzyme solution volume relative to the total lysate volume, "Amylase AG XXL", Novozymes), are used to determine the maximum free glucose concentration of the sample.

[0205] Using this data, we calculated the percentage of glycogenolysis (digestion) shown in Figure 7.

[0206] The results show that, without the addition of exogenous enzymes, more than 10% of glycogen is digested after 48 hours when the lysate and solubilized components are left standing, but the clarified fraction is not digested.

[0207] Example 8: Characterization of extracts prepared according to the present invention Galdieria sulphuraria biomass dissolved (20% by weight of DM of the total dissolved material) is maintained at an acidic pH of 3.7 for 12 hours at 25°C.

[0208] An extract is prepared from this lysate. To achieve this, the lysate is washed with water in an amount representing less than four times the total volume of the lysate biomass. This volume of water is divided into three fractions to carry out three consecutive washes of the lysate biomass. The wash water is recovered according to the teachings of the International Publication of Patent Application No. 2020 / 161280. The resulting product is then filtered over a hollow fiber membrane with a porosity of 70 kDa using a final dialysis filtration step (clarified fraction), and the filtrate is recovered according to the teachings of the International Publication of Patent Application No. 2020 / 144330 (concentrated aqueous extract).

[0209] The aqueous extract concentrate and clarified fraction are analyzed for total sugars, hydrolyzed mannose, hydrolyzed galactose, and hydrolyzed glucose by HPLC-RID, for free glucose by YSI® 2950, ​​and for phycocyanin and protein.

[0210] The results obtained are shown in Table 1 below.

[0211] [Table 1]

Claims

1. A method for processing the biomass of a single-celled red alga (URA) of the genus Galdieria, wherein the URA biomass processing method is a) A step of biomass collection by separating the culture medium to obtain the live biomass of URA, b) Step (a) to obtain a lysate, and the step of cellular lysis of crude biomass, c) A step of optional dilution of the solubilized product from step (b) to obtain the solubilized product, d) a step of separating the dissolved substance from step (b) or the insoluble substance suspended in the solubilized substance from step (c) for obtaining a clarified fraction, A method characterized by comprising a glycogenolysis step, during which the aforementioned live biomass and / or dissolved product and / or solubilized product, if applicable, is kept standing for at least 3 hours (standing step), during which time the liquid medium containing glycogen is at an acidic pH.

2. The method according to claim 1, characterized in that the settling step lasts for 3 hours to 1 week.

3. The method according to claim 1 or 2, characterized in that the settling step is performed at a temperature of 15°C to 70°C.

4. The method according to any one of claims 1 to 3, characterized in that the settling step is performed on the dissolved substance and / or solubilized substance, if applicable, and preferentially on the dissolved substance.

5. The method according to any one of claims 1 to 4, characterized in that, prior to the settling step, the step of adjusting the pH of the live biomass, dissolved material, and / or solubilized material, if applicable, to a pH below 7, particularly 1 to 6, preferably 2 to 5, and more preferably 3 to 4.

6. The method according to any one of claims 1 to 5, further comprising step e) concentrating the clarified fraction to obtain a concentrated aqueous extract.

7. A product obtainable by the method described in any one of claims 1 to 6.

8. The product according to claim 7, characterized by containing an estimated undigested residual glycogen content of 0.1 to 10 g / L, more preferably 0.2 to 7 g / L, and even more preferably 0.3 to 5 g / L.

9. The product according to claim 7 or 8, characterized by containing a hydrolyzed mannose concentration of 15 g / L or less.

10. The product according to claim 7 or 9, characterized by containing a hydrolyzed galactose concentration of 15 g / L or less.

11. The product according to any one of claims 7 to 10, characterized by containing a protein (%) / undigested residual glycogen ratio estimated by HPLC-RID determination of hydrolyzed sugar (g / L) in the range of 0.2 to 6.0, more preferably 0.5 to 5.0, and even more preferably 1.0 to 4.

5.

12. The product according to any one of claims 7 to 11, characterized by containing a protein (%) / total sugar ratio as measured by HPLC-RID determination of hydrolyzed sugar (g / L) in an amount of less than 1%, preferably 1 to 0.01%, more preferably 0.8 to 0.05%, and even more preferably 0.6 to 0.1%.

13. The product according to any one of claims 7 to 12, characterized by containing a C-phycocyanin content of 0.1 to 20 g / L.

14. A nutritional supplement, meal, or cosmetic composition comprising the product described in any one of claims 7 to 13.

15. The composition according to claim 14, characterized by containing a mineral acid or an organic acid.

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