WO2020161280A1 - Procédé optimisé d'exploitation industrielle d'algues rouges unicellulaires - Google Patents
Procédé optimisé d'exploitation industrielle d'algues rouges unicellulaires Download PDFInfo
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- WO2020161280A1 WO2020161280A1 PCT/EP2020/053081 EP2020053081W WO2020161280A1 WO 2020161280 A1 WO2020161280 A1 WO 2020161280A1 EP 2020053081 W EP2020053081 W EP 2020053081W WO 2020161280 A1 WO2020161280 A1 WO 2020161280A1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/12—Unicellular algae; Culture media therefor
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L17/00—Food-from-the-sea products; Fish products; Fish meal; Fish-egg substitutes; Preparation or treatment thereof
- A23L17/60—Edible seaweed
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
- A23L29/20—Foods or foodstuffs containing additives; Preparation or treatment thereof containing gelling or thickening agents
- A23L29/206—Foods or foodstuffs containing additives; Preparation or treatment thereof containing gelling or thickening agents of vegetable origin
- A23L29/256—Foods or foodstuffs containing additives; Preparation or treatment thereof containing gelling or thickening agents of vegetable origin from seaweeds, e.g. alginates, agar or carrageenan
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/17—Amino acids, peptides or proteins
- A23L33/195—Proteins from microorganisms
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/405—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from algae
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/795—Porphyrin- or corrin-ring-containing peptides
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/06—Lysis of microorganisms
- C12N1/066—Lysis of microorganisms by physical methods
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
- C12P17/182—Heterocyclic compounds containing nitrogen atoms as the only ring heteroatoms in the condensed system
Definitions
- the present invention relates to a method for cultivating unicellular red algae (ARUs) optimized for the valorization of culture products, both the biomass obtained, and the phycocyanins which are extracted therefrom or other culture products such as porphyrins or protein extracts. .
- ARUs unicellular red algae
- ARUs unicellular red algae
- the invention relates to an optimized process for the culture and valuation of ARUs, in particular of Galdieria sulphuraria, comprising steps (a) of culture by fermentation of the ARUs, (b) of separation of the biomass from the fermentation juice, where appropriate. (c) cell lysis and, where appropriate, a step (d) for extracting valuable products from the lysed biomass, which comprises at least one of the following steps of: (c1) lysis by grinding with maintenance of the biomass during grinding at a temperature below 50 ° C and / or
- the invention also relates to the products obtained by the process, in particular the porphyrins extracted from the fermentation juice, the biomass, the lysed biomass, the proteins and / or the phycocyanins isolated.
- Figure 1 shows a simplified diagram of the manufacturing process of different products from the culture of Galdieria sulphuraria.
- FIG. 2 represents the growth of the strain of Galdieria sulphuraria in “FedBatch” mode on glycerol with a maturation phase.
- FIG. 3 represents the monitoring of the composition of the biomass during culture in “FedBatch” mode on glycerol with a maturation phase.
- FIG. 4 represents the growth of the strain of Galdieria sulphuraria in “Fed-Batch” mode on milk permeate with a maturation phase.
- FIG. 5 represents the monitoring of the composition of the biomass during the culture on milk permeate in “FedBatch” mode with a maturation phase.
- Figure 6 represents the growth monitoring of a strain of Galdieria sulphuraria cultivated continuously on glycerol without production of porphyrins.
- FIG. 7 represents the monitoring of the composition of the biomass during the culture of Galdieria sulphuraria cultivated continuously on glycerol.
- Figure 8 shows the growth monitoring of a strain of Galdieria sulphuraria in continuous mode on glucose.
- FIG. 9 represents the monitoring of the composition of the biomass during the culture of Galdieria sulphuraria cultivated continuously on glucose.
- Figure 10 represents the growth monitoring of a strain of Galdieria sulphuraria in continuous mode on milk permeate.
- FIG. 11 represents the monitoring of the composition of the biomass during the culture of Galdieria sulphuraria cultivated continuously on milk permeate.
- Figure 12 shows HHP Bertoli grinding data (1200bars) without cooling.
- Figure 13 represents HHP Bertoli grinding data (1200bars) with cooling.
- Figure 14 shows the resistance of Phycocyanin at 50 ° C on lysates adjusted to different pH.
- Figure 15 shows the effect of bead diameter on the rate of cell lysis by bead mill.
- Figure 16 shows the amounts of phycocyanins extracted with serial washes compared to one-time washes for different volumes of water.
- recovery according to the invention is meant the technical steps which make it possible to isolate products useful for use in industry,
- the method according to the invention comprises the following successive steps: (a1) culture by fermentation with a maturation phase which comprises limiting the supply of carbon source in the culture medium,
- the method according to the invention comprises the following successive steps:
- the method comprises at least the following steps:
- the method comprises in minus the following steps:
- ARUs are well known to those skilled in the art, in particular ARUs capable of being cultured industrially for the production of biomass and its derivative products, proteins or phycocyanins. Mention will in particular be made of the algae (or microalgae) of the orders of the Cyanidiales.
- Cyanidiales encompasses the families of Cyanidiaceae or Galdieriaceae, themselves subdivided into the genera Cyanidioschyzon, Cyanidium or Galdieria, to which belong among others the species Cyanidioschyzon merolae 10D, Cyanidioschyzon merolae DBV201, Cyanidium caldanidum maximum, Cyanidium caldanidum, , Cyanidium partitum, Cyanidium rumpens, Galdieria daedala, Galdieria maxima, Galdieria partita or Galdieria sulphuraria. Mention will in particular be made of the strain Galdieria sulphuraria (also called Cyanidium caldarium (UTEX 2919).
- microorganisms which produce phycocyanin with a high glycogen content are more particularly identified among the microorganisms mentioned above, in particular the species of the genera Arthrospira, Spirulina, Synechococcus, Cyanidioschyzon, Cyanidium or Galdieria, more particularly Galdieria sulphuraria.
- the invention also relates to the products obtained by the process, in particular the biomass, the porphyrins isolated from the fermentation juice, the lysed biomass, the proteins and the phycocyanins isolated from the lysed biomass.
- Phycocyanins (PC) produced by microorganisms include c-phycocyanins (C-PC) and allophycocyanins.
- C-PC c-phycocyanins
- allophycocyanins c-phycocyanins
- the term “phycocyanins” is understood to mean C-PCs and allophycocyanins, isolated or their mixtures in all proportions, in particular C-PCs.
- the biomass produced includes not only phycocyanins and proteins, but also reserve sugars such as glycogen.
- the glycogen contents in the biomass are of the order of 20 to 50% by mass relative to the total mass of dry matter. However, the greater the glycogen content in the final biomass, the lower the phycocyanin (PC) and protein concentration.
- the glycogen produced by the ARUs in particular in Galdieria, is soluble in cold water and is therefore found in the aqueous phase during the extraction of the PC, which poses technical problems during filtration such as an increase in the viscosity, clogging of the filtration membranes, pressure rises, an accumulation of glycogen in the fraction containing the phycocyanin and therefore obtaining a less pure phycocyanin.
- the invention makes it possible, by “controlling” the fermentation, to reduce the glycogen levels to values below 20% by weight / DM.
- the invention therefore relates to a process for the production of biomass according to the invention which comprises the culture by fermentation of ARUs as defined above with a maturation phase which comprises the limitation of the supply of carbon source in the medium of culture,
- Cultures by fermentation are carried out on a culture medium comprising various nutrients allowing cell growth.
- These culture media include in particular a carbon source, a nitrogen source, a phosphorus source, macroelements, microelements, in appropriate concentrations to allow cell growth.
- the maturation step is implemented in particular in "FedBatch” or continuous cultivation methods.
- the carbon source can be any carbon source known to those skilled in the art and which can be used for the culture of ARUs and in particular of Galdieria sulphuraria, such as polyols, in particular glycerol, sugars, such as glucose or sugar.
- sucrose or lactose ink or complex media comprising lactose such as milk permeates, such as milk permeate, serum permeate, buttermilk and their mixtures and in particular milk permeate.
- the method of culture by fermentation according to the invention comprises a first phase of cell growth on a culture medium comprising a carbon source as defined above, so as to obtain a cell density in the cell.
- culture medium of at least 30 g / L of DM.
- a “maturation” phase is carried out which consists in weaning the strain into organic carbonaceous substrate.
- the maturation phase is triggered once the culture in the growth phase reaches at least 30 g / L of DM, preferably at least 80 g / L of DM, more preferably at least. less 100 g / L of dry matter.
- the culture is fed with a feed medium comprising at least 100 g / l of carbonaceous substrate, preferably at least 200 g / l of carbonaceous substrate, more preferably at least 500 g / l of carbonaceous substrate. .
- a feed medium comprising at least 100 g / l of carbonaceous substrate, preferably at least 200 g / l of carbonaceous substrate, more preferably at least 500 g / l of carbonaceous substrate.
- a feed medium comprising at least 100 g / l of carbonaceous substrate, preferably at least 200 g / l of carbonaceous substrate, more preferably at least 500 g / l of carbonaceous substrate.
- the growth phase is followed by a maturation phase.
- This maturation phase a drop in dry mass per liter of must can be observed above all, linked to the consumption of reserve sugars accumulated during growth, in particular glycogen. Concomitantly, an increase in the amount of phycocyanin per gram of dry matter can be observed. The same is true for the protein content.
- This maturation process results in low glycogen biomass.
- the culture is fed with a maturation feed medium does not include a carbon source. It is understood that the absence of a carbon source is also observed in the case where the maturation feed medium comprises detectable traces of carbon source.
- the weaning is total, that is to say that one stops feeding the cell culture in culture medium, the cells initiating their maturation by feeding on the residual elements of the fermentation must and their cell reserves.
- the biomass obtained comprises less than 20% of glycogen by mass relative to the mass of dry matter (% of the DM), preferably less than 15% of the DM, more preferably less than 10% of the dry matter. MS.
- the maturation time can be longer or shorter depending on the culture temperature. The closer the temperature is to that of optimum growth, the shorter the maturation phase will be.
- the growth rate for carrying out this maturation is determined as a function of the maximum growth rate of the strain that a person skilled in the art will be able to determine. This growth rate should be less than 80% of the maximum growth rate of the strain, preferably less than 70% of the maximum growth rate of the strain, more preferably less than 50% of the maximum growth rate of the strain.
- the object of the invention is to carry out a continuous culture in order to increase the productivity in biomass and PC compared to a culture in Fed-Batch. It is possible, by the process according to the invention, to achieve a dry mass of 65 -70 g / l, or even more, and a PC content of between 25 and 90 mg / g of DM, or even more.
- the maturation phase is carried out by transferring part of the must from the culture continuously into a tank without nutrient input.
- the “FedBatch” culture method described above there is concomitantly a decrease in the glycogen content and an increase in the phycocyanin and protein content.
- This maturation time will be at least 12 hours, preferably at least 48 hours, more preferably at least 72 hours.
- the growth and maturation stages can also be implemented simultaneously by imposing a reduced growth rate via the flow rate of the feed medium.
- the growth rate is less than 0.06 h-1, preferably less than 0.03 h-1, more preferably less than 0.015 h-1.
- the growth rate is less than 80% of the maximum growth rate of the strain, preferably less than 60% of the maximum growth rate of the strain, more preferably less than 40% of the maximum growth rate of the strain .
- the growth rate was reduced to a value less than 80% of the maximum growth rate, which made it possible to increase the PC and protein content in the biomass and to reduce the glycogen content, compared to the growth rate initially imposed.
- the carbon source content ensures a dry matter content of at least 65 g / L, or even at least 70 g / L, more particularly at least 80 g / L.
- the biomass thus obtained with separate or simultaneous maturation has a glycogen content of less than 20%, advantageously less than 15% or even less than 10%.
- the resulting biomass has a protein content of at least 45% of the DM, preferably at least 50%.
- the content of phycocyanin, in particular C-PC will be at least 20 mg / g of DM, preferably 25 to 50 mg / g of DM. With certain carbon sources, such as polyols, C-PC contents of greater than 50 mg / g DM can be achieved.
- the implementation of the method according to the invention does not lead to excretion of porphyrin as long as a source of organic carbon, in particular glucose, glycerol, lactose, or sucrose, is present. in the middle. Porphyrins are only detected during the maturation phase (without organic carbon in the medium), whether for Fed-batch or continuous culture. When adding organic substrate to the medium after a maturation phase, one can observe a re-consumption of porphyrins by the cells and a return to undetectable levels of these porphyrins in the fermentation juice.
- a fermentation must is obtained comprising a biomass with a low glycogen content, rich in proteins and in PC, as defined above, and a juice containing porphyrins.
- porphyrins produced by ARUs in particular by Galdieria sulphuraria are natural chelating agents which can be used, for example, for treatments against nematodes (US 2006/0206946).
- Certain molecules such as Protoporphyrin IX could also be of interest in the medical field for cancer treatments by phototherapy (Huang et al., 2015)
- the invention therefore relates to a process which comprises a step of recovering the fermentation juice and extracting the porphyrins from this juice.
- the fermentation juice is recovered by all usual methods of separating the biomass, in particular by centrifugation (plate centrifuge or sedicanter), well known to those skilled in the art, or by filtration (plate filter, filter press, ceramic tangential filtration or organic).
- centrifugation plate centrifuge or sedicanter
- filtration plate filter, filter press, ceramic tangential filtration or organic.
- Porphyrins can be extracted by usual methods, such as chromatography (affinity or size exclusion chromatography).
- the extracted porphyrins can be purified and then packaged for their subsequent use, in particular in therapy.
- ARUs and in particular Galdieria sulphuraria have a very resistant cell wall which makes it difficult to lysis by usual methods unless they are put into operating conditions which will degrade the phycocyanins sought.
- the yield of product recovered, phycocyanins and / or proteins does not depend only on the content of product in the biomass, but also on the ability to extract the maximum from this biomass. This extraction capacity will depend on the one hand on the efficiency of cell lysis, but also on its implementation under conditions which do not lead to substantial degradation of the phycocyanins.
- the invention therefore relates to a process for lysis of ARUs cells, in particular of Galdieria sulphuraria, characterized in that the lysis is carried out by grinding with a ball mill while maintaining the ARUs biomass during grinding at a temperature below 50 ° C.
- the invention consists in regulating the temperature of the biomass during grinding, inside the grinding chamber, so that it does not exceed 50 ° C, preferably 47 ° C, more preferably 40 ° C and less.
- This temperature regulation can be done by a water cooling system for the double jacket of the mill or by injection into the mill of biomass previously cooled to temperatures below 20 ° C.
- the grinding process according to the invention is applicable for a biomass regardless of its method of production (mode of fermentation and isolation). It is particularly suitable and preferably for a biomass obtained by the culture process according to the invention described above with a reduced glycogen content.
- the invention also relates to the lysed biomass thus obtained.
- the inventors were able to observe that the crushed biomass according to the invention allowed better digestibility of the proteins than the unground biomass. This improvement in digestibility is shown by in vitro digestibility tests (Boisen and Fernandez, 1995).
- the invention therefore relates to a crushed ARUs biomass and in particular a Galdieria sulphuraria biomass capable of being obtained by the crushing process according to the invention.
- the invention relates in particular to a ground biomass of Galdieria sulphuraria of the composition described below.
- the amino acid composition is given in the following Table.
- the Lipid composition is as follows:
- the invention also relates to the use of this crushed biomass as a food supplement or as food for human or animal consumption.
- the invention also relates to a process for extracting phycocyanin from a biomass of lysed cells of ARUs, in particular of Galdieria sulphuraria, characterized in that it comprises successive washes in quantities of water representing at least total less than 4 times, preferably 2 to 3 times, more preferably about 3 times the total volume of lysed biomass.
- This lysed biomass comprises a suspension of insoluble cellular residues in an aqueous solution comprising various cellular extracts solubilized following cell lysis, including phycocyanins.
- the lysed biomass advantageously comprises a dry matter of at least 2%, preferably at least 5%, more preferably at least 7%.
- the total volume of water (Ve) required for the extraction is calculated based on the volume of the lysed biomass to be treated (Vb) and will represent up to 4 times this volume (Ve / Vb is less than or equal to 4).
- the invention can of course be implemented with a larger total volume of water, but the economics of the process then remain less attractive because of the volumes of water to be treated subsequently to recover the phycocyanin.
- This total volume of water is then split into several fractions which will be used to extract the phycocyanin by successive passes over the biomass, the number of fractions (n) being at least 2, preferably at least 3.
- the person from the field profession can plan to perform extraction with more than 3 water fractions, while having to take into account all the economic parameters of the implementation of the process, such as the cost price of the immobilization of the equipment and the repetition of the biomass.
- the number of fractions is 3.
- the fractions have respective volumes different from each other. According to another embodiment of the invention, all the fractions have the same volume equal to Ve / n.
- washing waters recovered for each successive extraction comprising the phycocyanin can be treated separately to recover the phycocyanin or else assembled before this recovery.
- the extraction process according to the invention is suitable for any biomass of ARUs, in particular of Galdieria sulphuraria, lysed, regardless of the culture method used for the production of the biomass and the method used for cell lysis.
- the extraction method according to the invention is particularly suitable for biomass with low glycogen contents obtained by the process according to the invention described above and / or for the biomass lysed by the grinding method according to the invention defined above.
- the phycocyanin solution obtained is generally treated so as to isolate the phycocyanin therefrom.
- Methods for recovering phycocyanin from an aqueous solution are well known to those skilled in the art. Mention will in particular be made of the acid precipitation described in patent application WO 2018/178334.
- the enzymatic lysis of glycogen is carried out at a pH less than or equal to 5, preferably about 4.5, at room temperature.
- These temperature and pH conditions are particularly suitable for preserving the phycocyanin during the enzymatic reaction.
- the enzymes which are active under acidic pH conditions and at ambient temperature are chosen from enzymes known for glucuronidase a1-4, glucosidase a1-4 (or alpha-glucosidase) activity.
- pectinases known to degrade pectin and in particular pectinases extracted from filamentous fungi such as Aspergillus, more particularly from pectinases extracted from Aspegillus aculeatus, such as the enzymes marketed under the name Pectinex® by the company Novozymes.
- the enzymatic lysis of the glycogen can also be carried out with a glucosidase a1-6 in addition to the glucuronidase a1-4 or glucosidase a1-4.
- Glucosidases a1-6 active under the pH and temperature conditions set out above are also known to those skilled in the art.
- pullulanases known to hydrolyze the a1-6 glucosidic bonds of pullulan, in particular known to suppress the branches of starch. These are generally enzymes extracted from bacteria, in particular the genera Bacillus. US 6,074,854, US 5,817,498 and WO 2009/075682 describe such pullulanases extracted from Bacillus deramificans or Bacillus acidopullulyticus. Commercially available pullulanases are also known, in particular under the names “Promozyme D2” (Novozymes), “Novozym 26062” (Novozymes) and “Optimax L 1000” (DuPont-Genencor).
- pullulanase / alpha-amylase mixtures are described in the state of the art, but in particular for producing glucose syrup from starch (US 2017/159090).
- Those skilled in the art will know how to determine the appropriate reaction conditions to best reduce the amounts of glycogen as a function of the initial glycogen content in the solution to be treated, the amount of enzymes employed and the desired purity for the phycocyanin produced. Such a method is described in particular in patent application FR 1900278 filed on January 11, 2019.
- the recovered phycocyanin can then be purified by methods known to those skilled in the art, such as diafiltration.
- the phycocyanin obtained by the extraction process according to the invention has a purity index of at least 2, preferably at least 3, or even greater than 4. This purity index is measured by measuring the absorbance with the method described by Moon & al. (2014).
- the phycocyanin obtained is a phycocyanin which has a glycogen / phycocyanins ratio (by dry weight) of less than 6, advantageously less than 4, preferably less than 3, more preferably less than 2.5, even more preferably less than 1.
- the invention also relates to the use of the phycocyanins obtained as colorants, in particular as food colorants. It also relates to foods, solid or liquid, in particular drinks which comprise a phycocyanin obtained by the extraction process according to the invention.
- the solid residues remaining after washing are also recovered. It is a residue of biomass enriched in proteins which can also be used for the preparation of food supplements or food for human or animal consumption.
- the washing of the lysed biomass comprises an acidification of the biomass suspension to a pH less than or equal to 5.
- the residual biomass obtained after extraction of the phycocyanin comprises at least 60% of proteins relative to the dry matter, and at least a total sugar content of less than 20% with respect to the dry matter and / or a glycogen content of less than 10% with respect to the dry matter and / or fat content of at least 5 % based on dry matter.
- Galdieria sulphuraria UTEX # 2919 also called Cyanidium caldarium.
- Cultures are carried out in bioreactors of 1 to 2 L of useful volume with dedicated automatons and supervision by computer station.
- the pH of the culture is regulated by adding base (14% NH3 w / w ammonia solution) and / or acid (4N sulfuric acid solution).
- the culture temperature is set at 37 ° C.
- the stirring is carried out using 2 stirring wheels: 1 Rushton turbine with 6 straight blades positioned at the lower end of the stirring shaft above the "sparger” and 1 HTPG2 three-bladed propeller placed on the shaft agitation.
- the dissolved oxygen pressure in the liquid phase is regulated in the medium throughout the culture by the speed of rotation of the stirring shaft (250-1800 rpm), the ventilation rate by air and / or oxygen.
- the regulation parameters, integrated into the supervision automaton make it possible to maintain a partial pressure of dissolved oxygen in the liquid phase of between 5 and 30% of the air saturation value under identical conditions of temperature and pressure and composition of the medium.
- the culture time was between 50 and 300 hours
- Cultures are carried out in reactors with a useful volume of 1 to 2 L with dedicated automatons and supervision by computer station.
- the pH of the culture is regulated by adding base (14% ammonia solution (w NH3 / w) and / or acid (4N sulfuric acid solution).
- the culture temperature is set at 37 ° C.
- the stirring is carried out using 2 stirring wheels: 1 Rushton turbine with 6 straight blades positioned at the lower end of the stirring shaft above the "sparger” and 1 HTPG2 three-bladed propeller placed on the stirring shaft
- the pressure of dissolved oxygen in the liquid phase is regulated in the medium throughout the culture, by the speed of rotation of the stirring shaft (250-1800 rpm), the flow rate ventilation by air and / or oxygen.
- the regulation parameters integrated in the supervision automaton, make it possible to maintain a partial pressure of dissolved oxygen in the liquid phase between 5 and 30% of the saturation value by air under identical conditions of temperature, pressure and composition of the medium.
- the culture time was between 50 and 300 hours.
- the feed rate of the continuous fermenter is adjusted so that at no time is the carbon source detected in the culture medium.
- the quantity of carbon source is adjusted according to the target dry mass of the end of “FedBatch” or of 100 g / L of dry mass for the culture in continued. All the other elements of the medium are added while respecting the proportions used for the bottom of the tank medium defined in the examples.
- Example 1 Fermentation in Fed-batch mode with maturation phase on glycerol.
- Example 2 Fermentation in “Fed-batch” mode on glucose with a maturation phase.
- Example 3 Fermentation in "Fed-Batch” mode on sucrose with a maturation phase
- Example 4 Fermentation in "Fed-Batch” mode on milk permeate with maturation phase.
- Example 5 Continuous culture of a strain of Galdieria on glycerol.
- Example 6 Continuous culture of a strain of Galdieria on milk permeate.
- Base 30 g / L milk permeate, 8 g / L (NH 4 ) 2 S0 4 , 716mg / L MgS0 4 , 0.2843849 g / LK 2 S0 4 , 0.07 g / L FeS0 4 , 7H 2 0, 0.01236 g / L Na 2 EDTA, 0.00657 g / L ZnS0 4 , 7H 2 0, 0.0004385 g / L CoCI 2 , 6H 2 0, 0.00728 g / L MnCI 2 , 4H 2 0, 0.005976 g / L (NH4 ) 6MO70 24, 4H 2 0, 0.005976 g / L CuS0 4 5H 2 0, 0.00016 g / L NAVOS, 0.01144 g / LH 3 BO 3, 0.00068 g / L Na 2 Se0 3.
- Example 7 Grinding by HHP without cooling of biomass.
- a biomass obtained from a culture in a continuous mode is washed by successive centrifugations and then concentrated to a concentration of 1.4 ⁇ 10 10 cells / ml.
- a volume of 1 L of biomass is then cooled to 16 ° C before undergoing 3 successive homogenizations at 1200 bars on a Bertoli Atomo homogenizer, without cooling between each series.
- the temperature of the biomass, cell lysis by counting with Malassez cells and the phycocyanin concentration in the biomass are monitored for each of them.
- the grinding temperatures measured for the 3 successive homogenizations are respectively 46.7 ° C, 57.6 ° C and 67 ° C.
- Example 8 Grinding by HHP with cooling of biomass.
- a biomass obtained from a culture in a continuous mode is washed by successive centrifugations and then concentrated to a concentration of 2.10 10 cells / mL.
- a volume of 1 L of biomass is then cooled to 16 ° C before undergoing 3 successive homogenizations at 1200 bar on a Bertoli Atomo homogenizer. Between each homogenization, the temperature of the biomass is reduced to 16 ° C.
- the temperature of the biomass, cell lysis by counting with Malassez cells and the phycocyanin concentration in the biomass are followed in the same way.
- the temperatures of the biomass measured at the start and end of grinding are as follows.
- Example 9 Thermosensitivity of phycocyanin in biomass.
- a biomass resulting from a culture in a continuous mode is washed by successive centrifugations then concentrated to a dry matter of 150 mg / g before being ground by ball mill (WAB, multilab) under conditions allowing the preservation of the pigment and a lysis rate of 90%. Lysate samples are adjusted to pH from 2.4 to 6 and a kinetics of 0 to 120 minutes is carried out on different temperatures ranging from 50 to 70 ° C. For each time, a quantification of the phycocyanin is carried out. The results are shown in Figure 14.
- Example 10 Effect of Bead Size on Grinding and Temperature Control.
- Galdieria sulphuraria cells are centrifuged for 5 min at 20,000 g and then re-suspended in 10 mM Tris-CI buffer, pH 7. An aliquot of cells 1/3 the volume of a 2 ml Safelock Eppendorf tube is filled with this suspension, another 1/3 with ceramic balls of the diameter tested (Netzsch 0.8 mm; 0.6 mm; 0.3 mm; Plus 0.2 mm; Nano 0.2 mm; Plus 0.1 mm; and 0, 05 mm). The tubes are placed in an apparatus of the TissueLyser II type (Qiagen) and stirred for 2 min at 30 Hz. The lysis rate is calculated by counting on the Malassez cell compared to the control tube not containing beads.
- the diameter of the balls greatly affects the efficiency of the grinding.
- Example 11 Effect of the filling rate of the chamber on the grinding rate.
- the cells are ground in a Multilab model ball mill from WAB.
- the grinding chamber is filled with ceramic balls with a diameter of 0.8 mm at 50% and 65%.
- the 65% filling rate being the maximum filling rate.
- the grinding module speed and throughput are the same in both cases.
- the lysis rate at the grinding outlet is calculated by counting in the Malassez cell compared to the unshredded inlet biomass.
- the best rate of lysis is obtained when the chamber is at its maximum filling rate recommended by the manufacturer, ie 65%. When the filling rate is less than 65%, the lysis rate decreases.
- Example 12 Effect of Cell Concentration on Grinding Rate and Temperature Control.
- the cells are ground in a Multilab model ball mill from WAB.
- the grinding chamber is filled with 0.8mm diameter ceramic balls at a rate of 65%.
- the grinding module speed and throughput are the same in all cases.
- the rate of lysis at the outlet of grinding is calculated by counting in the Malassez cell compared to the unground inlet biomass.
- Example 13 Estimation of flow rates on an industrial type ball mill.
- Galdieria sulphuraria also called Cyanidium caldarium
- the cells are ground in a Multilab model ball mill from WAB.
- the grinding chamber (600 ml) is filled with ceramic balls of diameter 0.8 mm at a rate of 65%.
- the grinding module speed and feed rate are the same in both cases.
- the lysis rate at the grinding outlet is calculated by counting in the Malassez cell compared to the unshredded inlet biomass. For a grinding rate of 95 -100%, the flow rate applied under these conditions is between 1 and 2 liters per hour. An extrapolation of these flow rates was made using the results obtained in Example 10.
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CN202080013392.9A CN113423719A (zh) | 2019-02-08 | 2020-02-07 | 优化的用于工业利用单细胞红藻的方法 |
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WO2023026097A1 (fr) | 2021-08-24 | 2023-03-02 | Fermentalg | Stabilisation améliorée de phycocyanines dans des compositions acides |
WO2023167322A1 (fr) * | 2022-03-04 | 2023-09-07 | Eneos株式会社 | Procédé de culture d'algue appartenant aux cyanidiophyceae |
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Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
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WO2023026097A1 (fr) | 2021-08-24 | 2023-03-02 | Fermentalg | Stabilisation améliorée de phycocyanines dans des compositions acides |
WO2023167322A1 (fr) * | 2022-03-04 | 2023-09-07 | Eneos株式会社 | Procédé de culture d'algue appartenant aux cyanidiophyceae |
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