Classifications

• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
  • A23J1/00—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites
  • A23J1/009—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from unicellular algae
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
  • A23J3/00—Working-up of proteins for foodstuffs
  • A23J3/30—Working-up of proteins for foodstuffs by hydrolysis
  • A23J3/32—Working-up of proteins for foodstuffs by hydrolysis using chemical agents
  • A23J3/34—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes
  • A23J3/347—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes of proteins from microorganisms or unicellular algae
• • 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
  • A23L27/00—Spices; Flavouring agents or condiments; Artificial sweetening agents; Table salts; Dietetic salt substitutes; Preparation or treatment thereof
  • A23L27/20—Synthetic spices, flavouring agents or condiments
  • A23L27/21—Synthetic spices, flavouring agents or condiments containing amino acids
  • A23L27/22—Synthetic spices, flavouring agents or condiments containing amino acids containing glutamic acids
• • 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
  • A23L27/00—Spices; Flavouring agents or condiments; Artificial sweetening agents; Table salts; Dietetic salt substitutes; Preparation or treatment thereof
  • A23L27/20—Synthetic spices, flavouring agents or condiments
  • A23L27/23—Synthetic spices, flavouring agents or condiments containing nucleotides
  • A23L27/235—Synthetic spices, flavouring agents or condiments containing nucleotides containing also amino acids
• • 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
  • A23L27/00—Spices; Flavouring agents or condiments; Artificial sweetening agents; Table salts; Dietetic salt substitutes; Preparation or treatment thereof
  • A23L27/88—Taste or flavour enhancing agents
• • 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
  • A23L33/18—Peptides; Protein hydrolysates
• • 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
• • 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
• • 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
  • C12P21/00—Preparation of peptides or proteins
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
  • C07K1/14—Extraction; Separation; Purification
  • C07K1/36—Extraction; Separation; Purification by a combination of two or more processes of different types

Definitions

• the present invention relates to a peptide isolate derived from a biomass of microalgae rich in proteins, microalgae of the genus Chlorella, more particularly of the species Chlorella protothecoides.
• the algae, macro and micro have a richness largely unexplored. Their exploitation for food, chemical or bioenergetic purposes is still very marginal. However, they contain components of great value, wealth and abundance.
• Microalgae are indeed sources of vitamins, lipids, proteins, sugars, pigments and antioxidants.
• Algae and microalgae are thus of interest to the industrial sector that uses them for the manufacture of food supplements, functional foods, cosmetics, medicines or for aquaculture.
• Microalgae are primarily photosynthetic microorganisms that colonize all biotopes exposed to light.
• microalgae Some species of microalgae are indeed able to grow in the absence of light: Chlorella, Nitzschia, Cyclotella, Tetraselmis, Crypthecodinium, Schizochytrium.
• the pigments (chlorophyll a, b and c, ⁇ -carotene, astaxanthin, lutein, phycocyanin, xanthophylls, phycoerythrin, etc.) whose demand is increasing, both for their remarkable antioxidant properties and for their contribution of natural colors in the 'food, lipids, in order to optimize their fatty acid content (up to 60% or even 80% by weight of their dry matter), in particular for:
• PUFAs called "essential” (i.e. brought by the diet because are not naturally produced by the man or the animal), or
• proteins in order to optimize their nutritional qualities or, for example, to promote the supply of amino acids of interest by means of the preparation of peptide-rich fractions.
• Peptide fractions can be promoted as functional agents or dietary supplements in many fields.
• Arginine is an amino acid that has many functions in the animal kingdom.
• Arginine can be degraded and thus serve as a source of energy, carbon and nitrogen to the cell that assimilates it.
• arginine is decomposed into ornithine and urea.
• the latter is a nitrogen molecule that can be eliminated (by excretion in the urine) so as to regulate the amount of nitrogen compounds present in the cells of animal organisms.
• Arginine is used to synthesize nitric oxide (NO) with NO synthase, thereby mediating arterial vasodilatation, which reduces the rigidity of blood vessels, increases blood flow, and improves the functioning of blood vessels.
• NO nitric oxide
• Dietary supplements that contain arginine are recommended to promote heart health, vascular function, to prevent "platelet aggregation" (risk of blood clots) and to lower blood pressure.
• arginine in wound healing is related to its role in the formation of proline, another important amino acid for collagen synthesis.
• Arginine is also a component frequently used, especially by athletes, in energy drinks.
• Glutamic acid is not only one of the building blocks used for protein synthesis, but it is also the excitatory neurotransmitter more prevalent in the central nervous system (brain + spinal cord) and is a precursor of GABA in GABAergic neurons.
• glutamate is used as a food flavor enhancer. It is added to food preparations to enhance their taste.
• the Codex Alimentarius has also recognized as flavor enhancers its salts of sodium (E621), potassium (E622), calcium (E623), ammonium (E624) and magnesium (E625).
• Glutamate (or its salts) is often present in ready-made dishes (soups, sauces, chips, ready meals). It is also commonly used in Asian cooking.
• kitchen auxiliaries cubic broths, sauces, sauces, etc.
• these peptides must be extracted from the microalgae with the required compositions, in terms of:
• the present invention relates to a peptide isolate derived from a biomass of microalgae rich in proteins, microalgae of the genus Chlorella, more particularly of the species Chlorella protothecoides.
• the present invention relates to a microalgae isolate characterized by its remarkably high content of arginine and glutamic acid.
• the present invention also relates to the biomass of microalgae rich in proteins as such, biomass particularly suitable for the preparation of said peptide isolate.
• the present invention also relates to the process for enriching and depigmenting the biomass of microalgae, more particularly of the genus Chlorella, more particularly of the species Chlorella protothecoides.
• the present invention finally relates to the process for preparing this peptide isolate from the biomass of microalgae rich in proteins and depigmented.
• the term "essentially composed of arginine and glutamic acid” means a richness in arginine and glutamic acid that can be understood as an arginine and glutamic acid content of more than 80, 85, 90 or 95% by weight expressed relative to the total amino acids.
• these two amino acids represent 85 to 99% relative to the total amino acids, preferably between 90 and 98% relative to the total amino acids, and in particular between 95 and 98%.
• the term "essentially composed of arginine and glutamic acid” means a richness in arginine and glutamic acid that can be understood as containing:
• arginine in particular between 40% and 60%, preferably around 50%;
• At least 40% glutamic acid in particular between 40% and 60%, preferably about 50%
• amino acid content other than arginine and glutamic acid is less than 10%, preferably less than 5%, especially less than 3%.
• the content of the isolate is as follows:
• This isolate of peptides can be prepared from a biomass of microalgae of the genus Chlorella, more particularly of the species Chlorella protothecoides, microalgae discolored and having a protein content, expressed in N 6.25 greater than 60%, for example more than 65%.
• the preferred method of microalgae fermentation is a two-step process comprising:
• the residual salt content of the soluble fraction of the fermentation must does not exceed 6 g / l.
• the biomass thus prepared is then washed in order to purify it from its interstitial solubles (in particular soluble salts), brought to a solids content of between 15 and 30%, preferably to a solids content of between 20 and 30%, and then is heat-treated. at a temperature between 50 and 150 ° C, for a period of between 5 seconds and 5 minutes.
• interstitial solubles in particular soluble salts
• the residual biomass is removed, and the recovered soluble fraction is then clarified, precipitated, concentrated and then dried to form the peptide isolate according to the invention.
• the present invention thus relates to an isolate obtained or obtainable from a protein rich microalgae biomass prepared by a fermentative method described herein. It also relates to an isolate obtained or obtainable from the biomass of high protein microalgae by a method of treating biomass as described herein. Detailed description of the invention
• the invention relates to a peptide isolate prepared from a biomass of microalgae grown so as to enrich it with proteins, microalgae of the genus Chlorella, more particularly Chlorella protothecoides.
• the peptide isolate according to the invention obtained from this protein-rich biomass, is characterized in that it comprises:
• the term “comprises” means that the isolate consists essentially of these peptides but may comprise other minority components.
• “essentially constituted” means at least 90, 95 or 99% by dry weight of the isolate.
• the molecular weight of said peptides is measured by chromatography according to the following method:
• the sample is dissolved in 0.5% water in HPLC grade water.
• The% of the different fractions is then calculated on the basis of the retention times of each control.
• the measurement of the protein content is conventionally determined by the measurement of N.6.25, known in general
• amino acid composition is determined according to NF EN ISO 13903 (November 2005).
• arginine and glutamic acid contents of the isolate are as specified hereinabove.
• the high content of arginine and glutamic acid is understood here for example by a content:
• the peptide isolate comprises less than 3% of total sugars (carbohydrates).
• the peptide isolate according to the invention can be prepared from a biomass of microalgae of the genus Chlorella, more particularly of the species Chlorella protothecoides, microalgae discolored and having a protein content, expressed in N.6, 25 greater than 60%.
• a person skilled in the art chooses to control the growth of microalgae by controlling the fermentation conditions (Tp, pH, etc.), or by the regulated feeding into nutritional components (nitrogen or carbon sources) of the fermentation medium. , under semi-continuous conditions called "fed-batch".
• Chlorella protothecoides is justly recognized as one of the best oil-producing microalgae.
• the C / N ratio is here decisive, and it is accepted that the best results are obtained by acting directly on the nitrogen content, the glucose content not being limiting.
• Chlorella protothecoides can also be used for its ability to produce proteins.
• the heterotrophic culture process of said microalgae developed by the Applicant Company to increase the protein content of the biomass then comprises:
• a so-called "batch" fermentation phase characterized, after the fermenter has been inoculated, with the addition of a quantity of glucose of between 15 and 25 g / l, preferably of the order of 20 g / l, in one go;
• the NH 3 contribution remarkably rapidly induces an increase in the level of proteins synthesized in the cell, which results in an increase in the intracellular N 6,25 level to a value exceeding the 60%.
• a complete analysis of the amino acids present in the biomass was then performed on a sample taken just before the change of pH regulation, and on several other samples taken after said change.
• the content of glutamic acid and arginine on total amino acids is more than 45%
• N.6, 25 is therefore directly correlated with the increase in the synthesis of glutamic acid and arginine.
• This biomass is particularly well suited to the preparation of the peptic isolate according to the invention, by implementing the following method:
• washing the biomass so as to eliminate the interstitial soluble compounds optionally, washing the biomass so as to eliminate the interstitial soluble compounds
• thermal permeabilization of the biomass at a temperature of between 50 and 150 ° C., preferably between approximately 80 and 150 ° C., for a duration of between approximately 10 seconds and approximately 5 minutes, preferably for a duration of between approximately 10 seconds and approximately 1 minute, elimination of the biomass thus permeabilized by a solid-liquid separation technique, preferably chosen from the group consisting of frontal or tangential filtration, flocculation and centrifugation, more particularly multi-stage centrifugation, to obtain a soluble fraction,
• the biomass is collected by solid-liquid separation, by frontal or tangential filtration or by any means known to those skilled in the art.
• the Applicant Company recommends washing the biomass so as to eliminate the interstitial soluble compounds by a succession of concentration (by centrifugation) / dilution of the biomass.
• interstitial soluble compounds means all the soluble organic contaminants of the fermentation medium, for example the water-soluble compounds such as soluble salts, residual glucose, oligosaccharides of degree of polymerization (or DP) 2 or 3 or the peptides.
• This biomass thus purified of its interstitial soluble compounds is then adjusted preferably to a solids content of between 15 and 30% by weight, preferably at a solids content of between 20 and 30%.
• the heat treatment is then carried out at a temperature of between 50 and
• 150 ° C preferably between about 80 and 150 ° C, for a period of time between about
• the heat treatment is carried out at a temperature of about 140 ° C for a period of about 10 seconds.
• the heat treatment is carried out at a temperature of about 85 ° C for a period of about 1 minute.
• This treatment makes it possible to allow the intracellular components to diffuse into the reaction medium.
• the biomass is cooled to a temperature below 40 ° C, preferably cooled to a temperature of the order of 4 ° C.
• the applicant company considers that the heat treatment, carried out under these operating conditions, could act thus as a membrane embrittlement process that allows the spontaneous release of soluble components of the intracellular compartment, or even the extracellular matrix.
• organic substances such as carbohydrates (predominantly DP1 and DP2), peptides and polypeptides are drained out of the cell.
• the process according to the invention therefore does not lead to the formation of an emulsion, but to an aqueous suspension.
• a lag time may be necessary to allow sufficient diffusion after the heat treatment which permeabilizes the membrane.
• reaction time between 5 seconds and 5 minutes.
• the method of the invention advantageously exploits the thermal permeabilization phenomenon to extract the solubilized peptide fraction of the residual biomass.
• the residual biomass is then removed by a solid-liquid separation technique by frontal or tangential filtration, by flocculation, by centrifugation, or by any other means known to those skilled in the art, which makes it easy to recover the fraction. soluble free of microalgae cells.
• the yield and quality of this separation step can be improved by diluting the permeabilized cells (for example by multi-stage dilution / centrifugation).
• the soluble fraction thus obtained may be clarified by microfiltration so as to rid it of the residual insolubles and according to its dry matter, a concentration by evaporation or by any means known to those skilled in the art may be carried out before purification which follows.
• the resulting soluble fraction is ultimately composed essentially of proteins (50-80% w / w) and carbohydrates (10-25% w / w).
• the conventional processes for recovering soluble proteins generally rely on a step of precipitating said proteins with trichloroacetic acid (10% w / v) or with ammonium sulphate.
• the method of the invention then leads to the isolation of the proteins of interest, by precipitation by modulating the properties of the medium.
• the cooling temperature is below 10 ° C, preferably below 4 ° C.
• the pH in addition to the temperature, the pH must be between 2.5 and 6.5 and preferably be close to pHi, ie between 3 and 5.
• the ionic strength of the medium can be adapted to promote precipitation.
• Salting-in the phenomenon of salts
• solubility of the proteins by reducing the solvation layer.
• a demineralization operation prior to precipitation can be added. This is carried out on cationic and anionic resins, dialysis, filtration or by any means known to those skilled in the art.
• the polarity of the medium can be decreased (with dehydration of the medium) by adding an ethanol-type solvent which will generate a more quantitative precipitation of the protein fraction by greatly reducing its solubility.
• the separation of the precipitated fraction is carried out by simple decantation and recovery of the heavy phase or optionally by centrifugation under optimum temperature conditions.
• the pH may be readjusted before drying.
• the drying is carried out by atomization, lyophilization or by any means known to those skilled in the art.
• a soluble protein isolate greater than 90% by weight, rich in arginine and glutamic acid, is then obtained.
• Example 1 Preparation of a biomass of C. protothecoides rich in proteins with a high content of glutamic acid and arginine
• strain CCAP21 1 / 8D The Culture Collection of Algae and Protozoa, Scotland, UK.
• composition of the medium 40 g / l of glucose + 10 g / l of yeast extract.
• This fermentation line makes it possible to obtain a biomass having more than 65% of proteins expressed in N, 6.25.
• the biomass obtained according to Example 1 is harvested at a dry cell solids content of 105 g / L with a purity of 80% (purity defined by the ratio between the dry matter of the biomass on total dry matter).
• the pH is adjusted to 7 with potassium hydroxide and the biomass is thermally treated with UHT with preheating at 70 ° C. and then direct steam injection on a scale of about ten seconds at 140 ° C. and cooling at 40 ° C. by vacuum flotation.
• the heat treatment is pushed to a high scale in order to maximize the partial solubilization of the biomass which sees its purity decrease to 53%.
• composition of the biomass is as follows:
• a slight dilution [0.5: 1] (VeauVmout) is performed online on the second stage (on a configuration with two Alfa Laval FEUX 510 centrifuges in series) with recycling. from the supernatant of the second stage to the first.
• the supernatant of the first stage is thus recovered and concentrates the clarified solubles.
• the pH of the crude solubles is adjusted to 4.5 with phosphoric acid.
• the heavy phase is then extracted by simple phase separation into a separating funnel with a mass yield of 26% and has a solids content of 36.3%.
• This extract is lyophilized to a dry matter content of 97%.
• composition of this isolate is as follows:
• the isolate is characterized by a richness of the order of 95% in amino acids consisting essentially of arginine and glutamic acid (based on the analysis of the distribution of total amino acids).
• the molecular weight of this fraction is essentially between 1 kDa and 20 kDa.

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• Proteomics, Peptides & Aminoacids (AREA)
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• 2015
  • 2015-01-26 FR FR1550569A patent/FR3031985B1/fr active Active
• 2016
  • 2016-01-25 KR KR1020177017770A patent/KR20170105002A/ko unknown
  • 2016-01-25 US US15/546,206 patent/US20180000116A1/en not_active Abandoned
  • 2016-01-25 EP EP16705228.1A patent/EP3250705A1/de not_active Withdrawn
  • 2016-01-25 CN CN201680007169.7A patent/CN107205430A/zh active Pending
  • 2016-01-25 MX MX2017008934A patent/MX2017008934A/es unknown
  • 2016-01-25 WO PCT/FR2016/050139 patent/WO2016120549A1/fr active Application Filing
  • 2016-01-25 BR BR112017014580A patent/BR112017014580A8/pt not_active Application Discontinuation
  • 2016-01-25 JP JP2017539264A patent/JP2018502895A/ja active Pending

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