EP3847237A1 - Method for enriching a biomass with proteins - Google Patents

Method for enriching a biomass with proteins

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Publication number
EP3847237A1
EP3847237A1 EP19762172.5A EP19762172A EP3847237A1 EP 3847237 A1 EP3847237 A1 EP 3847237A1 EP 19762172 A EP19762172 A EP 19762172A EP 3847237 A1 EP3847237 A1 EP 3847237A1
Authority
EP
European Patent Office
Prior art keywords
biomass
lysed
proteins
microorganisms
less
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP19762172.5A
Other languages
German (de)
French (fr)
Inventor
Olivier CAGNAC
Axel ATHANE
Julien DEMOL
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Fermentalg SA
Original Assignee
Fermentalg SA
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Fermentalg SA filed Critical Fermentalg SA
Publication of EP3847237A1 publication Critical patent/EP3847237A1/en
Pending legal-status Critical Current

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Classifications

    • 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
    • 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
    • C12N1/125—Unicellular algae isolates
    • A—HUMAN NECESSITIES
    • A21—BAKING; EDIBLE DOUGHS
    • A21D—TREATMENT, e.g. PRESERVATION, OF FLOUR OR DOUGH, e.g. BY ADDITION OF MATERIALS; BAKING; BAKERY PRODUCTS; PRESERVATION THEREOF
    • A21D2/00—Treatment of flour or dough by adding materials thereto before or during baking
    • A21D2/08—Treatment of flour or dough by adding materials thereto before or during baking by adding organic substances
    • A21D2/24—Organic nitrogen compounds
    • A21D2/26—Proteins
    • A21D2/264—Vegetable proteins
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
    • A23C19/00—Cheese; Cheese preparations; Making thereof
    • A23C19/06—Treating cheese curd after whey separation; Products obtained thereby
    • A23C19/09—Other cheese preparations; Mixtures of cheese with other foodstuffs
    • A23C19/093—Addition of non-milk fats or non-milk proteins
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
    • A23C9/00—Milk preparations; Milk powder or milk powder preparations
    • A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
    • A23C9/13—Fermented milk preparations; Treatment using microorganisms or enzymes using additives
    • A23C9/1315—Non-milk proteins or fats; Seeds, pulses, cereals or soja; Fatty acids, phospholipids, mono- or diglycerides or derivatives therefrom; Egg products
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
    • A23C9/00—Milk preparations; Milk powder or milk powder preparations
    • A23C9/152—Milk preparations; Milk powder or milk powder preparations containing additives
    • A23C9/1526—Amino acids; Peptides; Protein hydrolysates; Nucleic acids; Derivatives thereof
    • 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
    • A23J1/008—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from microorganisms
    • 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
    • A23J3/20—Proteins from microorganisms or 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
    • A23J3/22—Working-up of proteins for foodstuffs by texturising
    • A23J3/225—Texturised simulated foods with high protein content
    • A23J3/227—Meat-like textured foods
    • 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
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/12—Animal feeding-stuffs obtained by microbiological or biochemical processes by fermentation of natural products, e.g. of vegetable material, animal waste material or biomass
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/30—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms
    • 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
    • A23L11/00—Pulses, i.e. fruits of leguminous plants, for production of food; Products from legumes; Preparation or treatment thereof
    • A23L11/05—Mashed or comminuted pulses or legumes; Products made therefrom
    • 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
    • A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
    • A23L2/385—Concentrates of non-alcoholic beverages
    • A23L2/39—Dry compositions
    • 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
    • A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
    • A23L2/52—Adding ingredients
    • A23L2/66—Proteins
    • 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
    • A23L29/275—Foods or foodstuffs containing additives; Preparation or treatment thereof containing gelling or thickening agents of animal origin, e.g. chitin
    • A23L29/281—Proteins, e.g. gelatin or collagen
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/02—Algae
    • A61K36/04—Rhodophycota or rhodophyta (red algae), e.g. Porphyra
    • 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
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/89—Algae ; Processes using algae

Definitions

  • the present invention relates to a process for enriching proteins in a biomass of red single-celled algae such as Galdieria and the biomass thus obtained.
  • soybeans generally used in the form of oil cakes, a secondary solid residue obtained from the extraction of oil.
  • oil cakes are generally imported from countries which cultivate an intensive soybean culture to the detriment of other plants source of biodiversity.
  • GM genetically modified soybean varieties, which are found mixed with non-GMO soybeans in oil cakes, which does not meet growing demand for food of origin non-GMO vegetable.
  • Spirulina like chlorella, however has the disadvantage of low productivity which does not allow the transition to a culture in high yield fermenter. If their culture meets local and limited demand for traditional food supplements, it does not meet a broader industrial production objective, economically viable, of a source of food protein whose qualities will allow it to replace tomorrow usual sources such as soya in animal and human food.
  • Microalgae such as Galdieria are also studied with regard to their nutritional characteristics (Graziani & al., 2013). Protein extracts from microalgae such as Auranthiochitrium known to produce oils rich in polyunsaturated fatty acids are also described (WO 2017/132407, WO 2017/019125, WO 2016/015013). They are however implemented on a defatted biomass, that is to say after having extracted and eliminated the fatty fraction of the cultivated biomass. This is also what is used for the recovery of chlorella proteins (WO 2016/120548, WO 2016/00194).
  • GMOs genetically modified microorganisms
  • the present invention relates to a method for producing a protein-rich biomass, the method comprising the steps of
  • the fraction of biomass recovered in c. can then be dried.
  • the invention also relates to the biomass thus obtained, its use for human or animal consumption, as well as the foods and food compositions which contain it.
  • Figure 1 represents the protein content (% of acids animated in the Dry Matter) for a spirulina biomass and for a biomass of Galdieria sulphuraria strain UTEX 2919 before and after treatment according to the invention for 1 cycle (PL1) or 4 cycles (PL4) of treatment.
  • FIG. 2 represents the amino acid composition (in g / 100g of DM) for a spirulina biomass and for a biomass of Galdieria sulphuraria strain UTEX 2919 before and after treatment according to the invention for 1 cycle (PL1) or 4 cycles ( PL4) of processing.
  • the present invention relates to a method for producing a biomass of lysed microorganisms rich in proteins, the method comprising the steps of b.1. suspension of a biomass of lysed microorganisms,
  • microorganisms essentially microorganisms which accumulate glycogen soluble in water, such as yeasts or protists which accumulate glycogen soluble in water during their growth, in particular under industrial conditions of culture by fermentation with biomass accumulation
  • the method is particularly suitable for a biomass of microorganisms producing phycobiliproteins.
  • the process advantageously makes it possible to make use of the biomass remaining after extraction of the phycobiliproteins, a by-product of the production of these molecules of interest.
  • ARU unicellular red algae
  • Rhodophytes in particular of the sub-division of Cyanidiophytina, preferably of the class of Cyanidiophyceae, in particular of the order of Cyanidiales, more particularly of the families of Cyanidiaceae or Galdieriaceae, even more particularly of the genera Cyanidioschyzon, Cyanidium or Galdieria.
  • the ARUs are chosen from the Cyanidioschyzon species merolae 10D, Cyanidioschyzon merolae DBV201, Cyanidium caldarium, Cyanidium daedalum, Cyanidium maximum, Cyanidium partitum, Cyanidium rumpens, Galdieria daedala, Galdieria maxima, Galdieria partita or Galdieria sulphuraria, more preferentially of the species Galdieria sulphuraria.
  • the method is implemented with a lysed biomass of which at least 70% of the cells are lysed, more preferably at least 80% of lysed cells, even more preferably 90%, even more preferably 95%.
  • the method comprises a preliminary step a. for lysis of a biomass of microorganisms previously cultivated.
  • Step a. lysis can be done by any means of cell lysis known to those skilled in the art. Mention will be made in particular of grinding or enzymatic lysis. Preferably, cell lysis is carried out by grinding, this method being particularly preferred for the lysis of ARUs, in particular of the species Galdieria sulphuraria.
  • the lysis by grinding is carried out on a biomass of “dry” cells, in particular on a biomass of fresh cells separated from the fermentation medium after culture by any separation method well known to those skilled in the art, in particular by filtration or centrifugation.
  • lysis by grinding is carried out on a biomass of cells previously lyophilized or dried for their conservation.
  • Step b.1. of suspension makes it possible to obtain a suspension of the lysed biomass.
  • the suspension obtained in step b1 generally has between 1% and 40% of dry matter, or more. Preferably it will have between 20% and 30% of dry matter.
  • Step b.2. is implemented by adding an acid to obtain a suspension whose aqueous phase has a pH less than or equal to 5.
  • a pH less than or equal to 4 The pH is adjusted by adding a strong or weak acid, mineral or organic, in the form of a solid or solution, the amount of acid added being determined by the pH of the suspension to be treated and the pH value that a person skilled in the art will seek to obtain.
  • mineral acids well known to those skilled in the art, there will be mentioned more particularly hydrochloric acid, sulfuric acid and phosphoric acid.
  • organic acids well known to those skilled in the art, there will be mentioned in particular acetic acid, citric acid, tartaric acid, lactic acid, preferably citric acid.
  • acids used are acids authorized for food use, in particular phosphoric acid, sulfuric acid, citric acid or tannic acid.
  • the acid suspension obtained by step b.2. preferably has a dry matter content greater than or equal to 1%, advantageously more than 2% of dry matter, preferably from 3 to 30% of dry matter, more preferably from 4 to 15% of dry matter, even more preferably from 5 to 12%, even more preferably from 6 to 10%.
  • steps b.1. and b.2. are consecutive, step b.2 being carried out directly after step b.1 without any other biomass treatment step such as a defatting step, for example.
  • the suspension, or dilution of the suspension is followed by the addition of acid, in solid or liquid form.
  • steps b.1. and b.2. are carried out simultaneously by adding an appropriate amount of an acidic solution to obtain both the desired pH less than or equal to 5 and the desired dry matter content.
  • step a Depending on the lysed biomass recovered from step a., In particular whether the lysis was carried out on a cell suspension in water or on a so-called “dry” biomass, the skilled person will be able to add the amounts of water and acid necessary for obtaining a suspension with the desired dry matter content and at the desired pH, whether these additions are made in two stages or in one single step.
  • Step c. is implemented by any separation means known to a person skilled in the art for separating the solid cell lysis residues from the aqueous solution from the acid suspension comprising the lysed cells, in particular by filtration or by centrifugation.
  • separation means known to a person skilled in the art for separating the solid cell lysis residues from the aqueous solution from the acid suspension comprising the lysed cells, in particular by filtration or by centrifugation.
  • tangential filtration for example tangential filtration on ceramic membranes or organic membranes such as hollow polyethersulfone fibers.
  • Front filtration can also be used (plate filter or filter press).
  • the suspension / acidification / recovery cycle (b.1./b.2./c.) Can be repeated several times on the fraction of lysed biomass insoluble at pH less than or equal to 5 recovered in c. so as to exhaust it in soluble elements at acidic pH.
  • the suspension / acidification / recovery cycle is repeated 1, 2 or 3 times on the lysed biomass recovered in c.
  • the fraction of protein-enriched biomass recovered in c. can then be dried using any known method of drying.
  • the mother liquors of the suspension are generally recovered in order to be treated so as to extract therefrom soluble components at acid pH which can be valued on their side.
  • This is particularly the case for phycocyanins when the microorganisms are microorganisms producing phycobiliproteins as defined above.
  • Such a process is described in particular in the patent application PCT / EP2018 / 058294 filed on March 30, 2018.
  • the biomass obtained generally has at least 60% of proteins (evaluated by the determination of total nitrogen, N * 6.25) relative to the dry matter, preferably more than 65%, even more preferably more than 68%, more than 70% or even more than 80%.
  • the biomass also preferably has a total sugar content of less than 20% relative to the dry matter, more preferably less than 10%, less than 5%.
  • the biomass obtained also preferably has a glycogen content of less than 10% relative to the dry matter, more preferably less than 5%, or even less than 1%, even more preferably less than 0.1%.
  • the lysed biomass insoluble at pH less than or equal to 5 may have a fat content, in particular lipids, which may be at least 5%, or even at least 10% relative to the material. dry, or at least 15%, at least 16%, up to 20%.
  • the fat composition will also depend on the microorganisms.
  • the lysed biomass according to the invention generally comprises polyunsaturated fatty acids of omega 3, 6 and 9 type, in particular oleic, palmitic and alpha-linolenic acids which represent at least 30% of the fatty matter, preferably 40% , even more preferably 50% of the fat.
  • proteins can be expressed both with respect to the proteins themselves and with respect to the amino acids contained in said proteins.
  • proteins is meant, according to the present invention, any protein, peptide or amino acid insoluble at an acid pH less than or equal to 5.
  • the present invention also relates to the biomass as defined above, capable of being obtained by the method according to the invention.
  • glycogen preferably less than 10% of glycogen, preferably less than 5%, more preferably less than 1% or even more preferably less than 0.1%, and
  • Another subject of the invention is the use of a biomass as described above in the cosmetic, pharmaceutical, human or animal food fields.
  • a protein-enriched biomass according to the invention for improving the performance of animals.
  • This improvement in performance can be assessed, in particular, by measuring consumption, weight gain or even the "Feed Conversion Ratio".
  • farmed animal in particular grazing animals (especially cattle raised for meat, milk, cheese and leather; sheep raised for meat, wool and cheese; goats), pigs, rabbits, poultry (chickens, hens, turkeys, ducks, geese and others), equines (ponies, horses, foals), intended to support human activities (transport, leisure) or their food , aquatic animals (eg fish, shrimp, shellfish (especially oysters and mussels)).
  • grazing animals especially cattle raised for meat, milk, cheese and leather; sheep raised for meat, wool and cheese; goats
  • poultry chickens, hens, turkeys, ducks, geese and others
  • equines ponies, horses, foals
  • aquatic animals eg fish, shrimp, shellfish (especially oysters and mussels)
  • the invention also relates to a food, or food composition, for humans or animals, comprising a biomass according to the invention as described above.
  • Food means any composition which can be used for food for humans or animals.
  • the food may comprise only biomass, dried or not, transformed or not, or biomass, dried or not, transformed or not, mixed with any other additive, vehicle or support, used in the field.
  • food or feed such as food preservatives, colorings, flavor enhancers, pH regulators, or pharmaceutical additives such as growth hormones, antibiotics.
  • the present invention relates in particular to animal feed and more particularly to farm animals.
  • These foods are usually in the form of flour, granules or soup in which the biomass according to the invention is incorporated.
  • “Animal food” means anything that can be used to feed animals.
  • feed may include, in addition to algal biomass, a nutritional base and nutritional additives.
  • Most of the animal's food ration then consists of the "nutritional base” and the algal biomass. This base is constituted, for example, by a mixture of cereals, proteins and fats of animal and / or vegetable origin.
  • the nutritional bases for animals are suitable for feeding these animals and are well known to those skilled in the art.
  • these nutritional bases include, for example, corn, wheat, peas and soy. These nutritional bases are adapted to the needs of the different animal species for which they are intended. These nutritional bases may already contain nutritional additives such as vitamins, minerals and amino acids.
  • Additives used in animal feed can be added to improve certain characteristics of food, for example to enhance the taste, to make the raw materials of animal feed more digestible or to protect animals. They are frequently used in large scale intensive farming.
  • the additives used in animal feed are available, in particular, in the following subcategories (source EFSA):
  • additives for example, preservatives, antioxidants, emulsifiers, stabilizers, acidity regulators and additives for silage;
  • - nutritional additives for example, vitamins, amino acids and trace elements
  • zootechnical additives for example, digestibility enhancers, intestinal flora stabilizers;
  • the invention relates to feed for farm animals comprising between 1 and 60%, preferably between 1 and 20%, quite preferably between 3 and 8% of a dried biomass obtained by the method according to the invention.
  • the invention relates to feed for farm animals comprising between 1 and 40%, preferably between 5 and 10% of an undried biomass obtained by the process of the invention.
  • the food is intended for farm animals, especially cattle, sheep, pigs, rabbits, poultry and horses.
  • the food is intended for aquatic animals, in particular for fish, at least up to the fry stage, including farmed and ornamental fish.
  • the food is intended for domestic animals, pets and / or leisure animals and NACs.
  • the food composition is intended for humans.
  • the subject of the invention is also a cosmetic, nutraceutical or pharmaceutical composition for humans or animals comprising a biomass according to the invention as described above.
  • the cosmetic, nutraceutical or pharmaceutical composition may only comprise biomass, dried or not, transformed or not, or biomass, dried or not, transformed or not, mixed with any other additive, vehicle or support, used in the field of cosmetics or pharmacy such as, for example, preservatives, dyes, pH regulators.
  • the invention also relates to the use of biomass as described above in therapy, as well as in the prevention and treatment of malnutrition.
  • the biomasses treated by the process according to the invention are biomasses of spirulina (Arthrospira platensis) and of Galdieria sulphuraria strain UTEX 2919 (UTEX Culture Collection of Algae, 205 W. 24th St, Biological Labs 218, The University of Texas at Austin ( A6700), Austin, TX 78712 USA) cultivated according to the usual methods for cultivating these strains.
  • the lysed biomass has a lysed cell content of 95%
  • the dry matter content is 10%
  • the enrichment process is carried out by repeating a rinsing of the pellet of the crushed biomass under an acid pH condition.
  • the operation involves the use of a centrifuge, here described by the use of a SORVALL RC5B plus and a Sorvall SLA 3000 fixed angle rotor.
  • the crushed biomass is centrifuged at 12,000 xg for 15 min, allowing the fraction to be pelleted as much as possible insoluble.
  • the “soluble fraction” supernatant is removed and replaced with water.
  • the pellet is resuspended before repeating the centrifugation operation, and the pH is checked and adjusted if necessary to a value below pH 4.8. This process, thus repeated 3 to 4 times, maximizes the protein and lipid enrichment of the insoluble fraction.
  • Total proteins are estimated using the Kjeldahl method known to those skilled in the art.
  • the amino acid profile by acid hydrolysis is carried out according to the ISO 13903: 2005 method; EU 152/2009.
  • the analysis of the lipid content and of the composition is done by GC / FID and internal calibration known to those skilled in the art.
  • the sugar profile is determined by ion chromatography - pulsed amperometric.
  • the protein content of the different biomasses is shown in Figure 1. If we look in detail at the amino acid composition in the different products shown in Figure 2 we can see an overall increase in all amino acids on the phases heavy, and amounts greater than or equal to those of Spirulina for most of them except for alanine.
  • C-PC and APC phycocyanin
  • Phycocyanin is known to be a molecule with strong antioxidant power.
  • the fatty acid composition shows significant amounts of omega 3, 6 and 9 type unsaturated fatty acids, in the form of polar lipids, representing more than 50% of the fatty mass which represents approximately 5% to 20% of the mass the heavy phase (Table 3).
  • the starting biomass comprises more than 70% of proteins compared to the DM. After three treatments according to the invention, a biomass is obtained with more than 80% of proteins, an increase of more than 14%.
  • the heavy phase enriched in proteins thus obtained can be subsequently enzymatically treated with proteases in order to obtain a protein hydrolyzate and to increase the solubility and digestibility of the product.
  • ANC Nutritional Intakes
  • the protein-enriched fraction produced by the process described in this invention can be consumed directly in the form of a cold pressed tablet, in powder form in the form of a capsule, or directly in powder form to be mixed with other ingredients.
  • This fraction can also be integrated into other food preparations which can be used for the manufacture of cake bread, biscuit or other pastry, or bars of cereals, or any preparation in replacement of the traditional flour in order to enrich in proteins the food thus prepared.
  • This protein fraction can also be incorporated into preparations intended to serve as a substitute for meat such as vegetable burgers, or in protein sticks in surimi manner, these proteins being able to be textured or used as such.
  • Another example of use would be in the form of powder to be added to drinks such as Smoothie or Shake.
  • chickpea flour (26%), seaweed powder (25%), onion, carrot, wheat gluten (3%), lemon juice, yeast extract, potato starch, vegetable oil (sunflower) ), turmeric, garlic, acidity regulator (potassium chloride), antioxidant, potato fibers, thickener (carrageenan), starch (corn, wheat), emulsifier (guar gum).
  • Dairy product enriched with algae proteins Dairy product enriched with algae proteins.
  • the protein-enriched alga fraction is added to the coagulated milk proteins before pressing the dough.
  • the percentage of algae fraction enriched in protein can vary from 1% to 50%. In this case the limiting factor will be linked to the appearance of the final product and in particular to its texture and the resistance to cutting.
  • Powder for beverages enriched with algae proteins Powder for beverages enriched with algae proteins.

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Abstract

The present invention relates to a method for protein enrichment of a biomass of red unicellular algae such as Galdieria and to the biomass thus obtained.

Description

PROCÉDÉ D’ENRICHISSEMENT D’UNE BIOMASSE EN PROTÉINES  PROCESS FOR ENRICHMENT OF PROTEIN BIOMASS
DOMAINE DE L'INVENTION FIELD OF THE INVENTION
La présente invention concerne un procédé d’enrichissement en protéines d’une biomasse d’algues unicellulaires rouges telles que Galdieria et la biomasse ainsi obtenue.  The present invention relates to a process for enriching proteins in a biomass of red single-celled algae such as Galdieria and the biomass thus obtained.
ETAT DE LA TECHNIQUE  STATE OF THE ART
On connaît plusieurs sources de protéines végétales pour leur utilisation dans l’alimentation humaine ou animale, directement ou comme compléments alimentaires, pour apporter aux animaux et humains des acides aminés nécessaires à leur métabolisme. Ces sources de protéines sont entendues comme des sources d’acides aminés disponibles pour les animaux ou humains une fois les aliments ingérés.  Several sources of vegetable proteins are known for their use in human or animal food, directly or as food supplements, to provide animals and humans with amino acids necessary for their metabolism. These sources of protein are understood to be sources of amino acids available to animals or humans once food is ingested.
La plus connue des sources de protéines végétales employée dans l’alimentation animale est le soja, généralement employé sous forme de tourteaux, résidu solide secondaire issu de l’extraction de l’huile. Toutefois, l’emploi de tourteaux de soja présente plusieurs inconvénients associés à leur origine. Les tourteaux sont généralement importés de pays qui pratiquent une culture intensive du soja au détriment d’autres végétaux source de biodiversité. En outre, de nombreux pays favorisent la culture de variétés de soja génétiquement modifiées (OGM), que l’on retrouve mélangées aux sojas non OGM dans les tourteaux, ce qui ne permet pas de répondre à une demande croissante de produits alimentaires d’origine végétale sans OGM.  The best known source of vegetable protein used in animal feed is soybeans, generally used in the form of oil cakes, a secondary solid residue obtained from the extraction of oil. However, the use of soybean meal has several disadvantages associated with their origin. The oil cakes are generally imported from countries which cultivate an intensive soybean culture to the detriment of other plants source of biodiversity. In addition, many countries favor the cultivation of genetically modified (GM) soybean varieties, which are found mixed with non-GMO soybeans in oil cakes, which does not meet growing demand for food of origin non-GMO vegetable.
On connaît d’autres sources de protéines végétales, notamment la spiruline ou la chlorelle, employées comme compléments alimentaires chez l’homme.  Other sources of vegetable proteins are known, in particular spirulina or chlorella, used as food supplements in humans.
La spiruline, comme la chlorelle, présentent toutefois l’inconvénient d’une faible productivité qui ne permet pas le passage à une culture en fermenteur à rendement élevé. Si leur culture permet de répondre à une demande locale et limitée en compléments alimentaires traditionnels, elle ne permet pas de répondre à un objectif de production industrielle plus large, économiquement viable, d’une source de protéines alimentaires dont les qualités lui permettront de remplacer demain les sources usuelles comme le soja dans l’alimentation des animaux et de l’homme.  Spirulina, like chlorella, however has the disadvantage of low productivity which does not allow the transition to a culture in high yield fermenter. If their culture meets local and limited demand for traditional food supplements, it does not meet a broader industrial production objective, economically viable, of a source of food protein whose qualities will allow it to replace tomorrow usual sources such as soya in animal and human food.
Des microalgues comme Galdieria sont également étudiées vis-à-vis de leurs caractéristiques nutritionnelles (Graziani & al., 2013). Des extraits protéiques de microalgues comme Auranthiochitrium connues pour produire des huiles riches en acides gras polyinsaturés sont également décrits (WO 2017/132407, WO 2017/019125, WO 2016/015013). Ils sont toutefois mis en œuvre sur une biomasse délipidée, c’est-à-dire après avoir extrait et éliminé la fraction grasse de la biomasse cultivée. C’est également ce qui est mis en oeyvre pour la récupération de protéines de chlorelles (WO 2016/120548, WO 2016/00194). Microalgae such as Galdieria are also studied with regard to their nutritional characteristics (Graziani & al., 2013). Protein extracts from microalgae such as Auranthiochitrium known to produce oils rich in polyunsaturated fatty acids are also described (WO 2017/132407, WO 2017/019125, WO 2016/015013). They are however implemented on a defatted biomass, that is to say after having extracted and eliminated the fatty fraction of the cultivated biomass. This is also what is used for the recovery of chlorella proteins (WO 2016/120548, WO 2016/00194).
On connaît des méthodes de production de biomasses de microorganismes riches en protéines, qui passent par exemple par la sélection de souches particulièrement adaptées à la surexpression de protéines et/ou à la mise en œuvre de méthodes de culture qui permettent de favoriser cette surexpression et enrichir la biomasse en protéines. Toutefois, les biomasses issues de microorganismes génétiquement modifiés (OGM) ne sont pas appropriées pour certains marchés de l’alimentation humaine et animale, y compris l’aquaculture (notamment en Europe).  Methods are known for producing biomasses of protein-rich microorganisms, which for example involve the selection of strains which are particularly suitable for the overexpression of proteins and / or the implementation of culture methods which make it possible to promote this overexpression and to enrich protein biomass. However, biomass from genetically modified microorganisms (GMOs) is not suitable for certain food and feed markets, including aquaculture (especially in Europe).
Il y a un intérêt à pouvoir proposer des sources de biomasses non-OGM riches en protéines répondant à un objectif de production industrielle large, économiquement viable, comme alternative aux sources usuelles de protéines comme le soja.  There is an interest in being able to offer sources of non-GMO biomass rich in proteins meeting a broad industrial production objective, economically viable, as an alternative to the usual sources of proteins such as soybeans.
EXPOSE DE L'INVENTION  STATEMENT OF THE INVENTION
La présente invention concerne un procédé de production d’une biomasse riche en protéines, le procédé comprenant les étapes de  The present invention relates to a method for producing a protein-rich biomass, the method comprising the steps of
b.1. mise en suspension d’une biomasse de microorganismes lysés, b.1. suspension of a biomass of lysed microorganisms,
b.2. acidification de la suspension jusqu’à un pH inférieur ou égal à 5, b.2. acidification of the suspension to a pH less than or equal to 5,
c. récupération de la fraction de biomasse lysée insoluble à pH inférieur ou égal à 5, et vs. recovery of the fraction of lysed biomass insoluble at pH less than or equal to 5, and
le cas échéant répétition des étapes b.1 ., b.2. et c. sur la fraction de biomasse lysée insoluble à pH inférieur ou égal à 5 récupérée en c. if necessary repeat steps b.1., b.2. and c. on the fraction of lysed biomass insoluble at pH less than or equal to 5 recovered in c.
La fraction de biomasse récupérée en c. peut ensuite être séchée.  The fraction of biomass recovered in c. can then be dried.
L’invention concerne aussi la biomasse ainsi obtenue, son utilisation pour l’alimentation humaine ou animale, ainsi que les aliments et compositions alimentaires qui la contiennent.  The invention also relates to the biomass thus obtained, its use for human or animal consumption, as well as the foods and food compositions which contain it.
DESCRIPTION DES FIGURES  DESCRIPTION OF THE FIGURES
La Figure 1 représente la teneur en protéines (% d’acides animés dans la Matière Sèche) pour une biomasse de spiruline et pour une biomasse de Galdieria sulphuraria souche UTEX 2919 avant et après traitement selon l’invention pour 1 cycle (PL1 ) ou 4 cycles (PL4) de traitement. Figure 1 represents the protein content (% of acids animated in the Dry Matter) for a spirulina biomass and for a biomass of Galdieria sulphuraria strain UTEX 2919 before and after treatment according to the invention for 1 cycle (PL1) or 4 cycles (PL4) of treatment.
La Figure 2 représente la composition en acides aminés (en g/100g de MS) pour une biomasse de spiruline et pour une biomasse de Galdieria sulphuraria souche UTEX 2919 avant et après traitement selon l’invention pour 1 cycle (PL1 ) ou 4 cycles (PL4) de traitement.  FIG. 2 represents the amino acid composition (in g / 100g of DM) for a spirulina biomass and for a biomass of Galdieria sulphuraria strain UTEX 2919 before and after treatment according to the invention for 1 cycle (PL1) or 4 cycles ( PL4) of processing.
DESCRIPTION DETAILLEE DE L'INVENTION DETAILED DESCRIPTION OF THE INVENTION
La présente invention concerne un procédé de production d’une biomasse de microorganismes lysés riche en protéines, le procédé comprenant les étapes de b.1. mise en suspension d’une biomasse de microorganismes lysés,  The present invention relates to a method for producing a biomass of lysed microorganisms rich in proteins, the method comprising the steps of b.1. suspension of a biomass of lysed microorganisms,
b.2. acidification de la suspension jusqu’à un pH inférieur ou égal à 5 b.2. acidification of the suspension to a pH less than or equal to 5
c. récupération de la fraction de biomasse lysée insoluble à pH inférieur ou égal à 5, et vs. recovery of the fraction of lysed biomass insoluble at pH less than or equal to 5, and
le cas échéant répétition des étapes b.1., b.2. et c. sur la fraction de biomasse lysée insoluble à pH inférieur ou égal à 5 récupérée en c. if necessary repeat steps b.1., b.2. and c. on the fraction of lysed biomass insoluble at pH less than or equal to 5 recovered in c.
Par microorganismes on entend selon l’invention essentiellement des microorganismes qui accumulent du glycogène soluble dans l’eau, comme des levures ou des protistes qui accumulent du glycogène soluble dans l’eau pendant leur croissance, en particulier dans des conditions industrielles de culture par fermentation avec accumulation de biomasse  By microorganisms is meant according to the invention essentially microorganisms which accumulate glycogen soluble in water, such as yeasts or protists which accumulate glycogen soluble in water during their growth, in particular under industrial conditions of culture by fermentation with biomass accumulation
De manière avantageuse, le procédé est particulièrement adapté pour une biomasse de microorganismes producteurs de phycobiliprotéines. Le procédé permet avantageusement de valoriser la biomasse restant après extraction des phycobiliprotéines, sous-produit de la production de ces molécules d’intérêt.  Advantageously, the method is particularly suitable for a biomass of microorganisms producing phycobiliproteins. The process advantageously makes it possible to make use of the biomass remaining after extraction of the phycobiliproteins, a by-product of the production of these molecules of interest.
Ces microorganismes et leurs modes de culture sont bien connus de l’homme du métier, notamment décrits dans les demandes WO 2017/050917, WO 2017/050918 et WO 2017/093345.  These microorganisms and their culture methods are well known to the skilled person, in particular described in applications WO 2017/050917, WO 2017/050918 and WO 2017/093345.
On citera en particulier les algues rouges unicellulaires (ARUs). Par "ARU" on entend selon l’invention les Rhodophytes, en particulier de la sous-division des Cyanidiophytina, de préférence de la classe des Cyanidiophyceae, en particulier de l'ordre des Cyanidiales, plus particulièrement des familles des Cyanidiaceae ou des Galdieriaceae, encore plus particulièrement des genres Cyanidioschyzon, Cyanidium ou Galdieria.  Mention will be made in particular of unicellular red algae (ARUs). By "ARU" is meant according to the invention the Rhodophytes, in particular of the sub-division of Cyanidiophytina, preferably of the class of Cyanidiophyceae, in particular of the order of Cyanidiales, more particularly of the families of Cyanidiaceae or Galdieriaceae, even more particularly of the genera Cyanidioschyzon, Cyanidium or Galdieria.
De préférence, les ARUs sont choisies parmi les espèces Cyanidioschyzon merolae 10D, Cyanidioschyzon merolae DBV201 , Cyanidium caldarium, Cyanidium daedalum, Cyanidium maximum, Cyanidium partitum, Cyanidium rumpens, Galdieria daedala, Galdieria maxima, Galdieria partita ou encore Galdieria sulphuraria, plus préférentiellement de l'espèce Galdieria sulphuraria. Preferably, the ARUs are chosen from the Cyanidioschyzon species merolae 10D, Cyanidioschyzon merolae DBV201, Cyanidium caldarium, Cyanidium daedalum, Cyanidium maximum, Cyanidium partitum, Cyanidium rumpens, Galdieria daedala, Galdieria maxima, Galdieria partita or Galdieria sulphuraria, more preferentially of the species Galdieria sulphuraria.
De préférence, le procédé est mis en œuvre avec une biomasse lysée dont au moins 70% des cellules sont lysées, plus préférentiellement d’au moins 80 % de cellules lysées, encore plus préférentiellement 90%, encore plus préférentiellement 95%.  Preferably, the method is implemented with a lysed biomass of which at least 70% of the cells are lysed, more preferably at least 80% of lysed cells, even more preferably 90%, even more preferably 95%.
Selon un mode de réalisation de l’invention, le procédé comprend une étape préalable a. de lyse d’une biomasse de microorganismes préalablement cultivés.  According to one embodiment of the invention, the method comprises a preliminary step a. for lysis of a biomass of microorganisms previously cultivated.
L’étape a. de lyse peut se faire par tout moyen de lyse cellulaire connu de l’homme du métier. On citera en particulier le broyage ou la lyse enzymatique. De manière préférée, la lyse cellulaire est réalisée par broyage, cette méthode étant particulièrement préférée pour la lyse des ARUs, en particulier de l'espèce Galdieria sulphuraria.  Step a. lysis can be done by any means of cell lysis known to those skilled in the art. Mention will be made in particular of grinding or enzymatic lysis. Preferably, cell lysis is carried out by grinding, this method being particularly preferred for the lysis of ARUs, in particular of the species Galdieria sulphuraria.
Elle peut se faire alors que les cellules sont en suspension dans l’eau, moût de fermentation ou suspension reconstituée, ou sur une biomasse dite « sèche », c’est à dire séparée du milieu de fermentation, fraiche ou après séchage et conservation, par exemple après lyophilisation.  It can be done while the cells are in suspension in water, fermentation must or reconstituted suspension, or on a so-called “dry” biomass, that is to say separated from the fermentation medium, fresh or after drying and conservation, for example after lyophilization.
De manière avantageuse, la lyse par broyage est réalisée sur une biomasse de cellules « sèches », en particulier sur une biomasse de cellules fraîches séparées du milieu de fermentation après culture par toute méthode de séparation bien connue de l’homme du métier, notamment par filtration ou par centrifugation.  Advantageously, the lysis by grinding is carried out on a biomass of “dry” cells, in particular on a biomass of fresh cells separated from the fermentation medium after culture by any separation method well known to those skilled in the art, in particular by filtration or centrifugation.
Selon un autre mode de réalisation de l’invention, la lyse par broyage est réalisée sur une biomasse de cellules préalablement lyophilisées ou séchées pour leur conservation.  According to another embodiment of the invention, lysis by grinding is carried out on a biomass of cells previously lyophilized or dried for their conservation.
L’étape b.1. de mise en suspension permet d’obtenir une suspension de la biomasse lysée.  Step b.1. of suspension makes it possible to obtain a suspension of the lysed biomass.
La suspension obtenue à l’étape b1 a généralement entre 1 % et 40% de matière sèche, voire plus. De préférence elle aura entre 20% et 30% de matière sèche.  The suspension obtained in step b1 generally has between 1% and 40% of dry matter, or more. Preferably it will have between 20% and 30% of dry matter.
L’étape b.2. est mise en œuvre par l’ajout d’un acide pour obtenir une suspension dont la phase aqueuse a un pH inférieur ou égal à 5. De préférence, on cherchera à obtenir un pH inférieur ou égal à 4. L’ajustement du pH se fait par l’ajout d’un acide, minéral ou organique, fort ou faible, sous forme de solide ou de solution, la quantité d’acide ajoutée étant déterminée par le pH de la suspension à traiter et la valeur de pH que l’homme du métier cherchera à obtenir. Parmi les acides minéraux bien connus de l’homme du métier, on citera plus particulièrement l’acide chlorhydrique, acide sulfurique et l’acide phosphorique. Parmi les acides organiques bien connus de l’homme du métier, on citera en particulier l’acide acétique, l’acide citrique, l’acide tartrique, l’acide lactique, de préférence l’acide citrique. On peut également citer les polyphénols acides comme l’acide rosmarinique, l’acide tannique, l’acide digallique, l’acide quercitannic, l’acide gallotannique, les tannins acides comme la quercitine, les ellagitannines, castalagine, castaline, casuariticine, grandinine, punicaligine punicaline, roburine A, tellimagrandine II, terflavine B, vescaligine, pendunculagine, casuariine, castline, vescaline, de préférence l’acide tannique. De préférence, les acides employés sont des acides autorisés pour un usage alimentaire, en particulier l’acide phosphorique, l’acide sulfurique, l’acide citrique ou l’acide tannique. Step b.2. is implemented by adding an acid to obtain a suspension whose aqueous phase has a pH less than or equal to 5. Preferably, an attempt will be made to obtain a pH less than or equal to 4. The pH is adjusted by adding a strong or weak acid, mineral or organic, in the form of a solid or solution, the amount of acid added being determined by the pH of the suspension to be treated and the pH value that a person skilled in the art will seek to obtain. Among the mineral acids well known to those skilled in the art, there will be mentioned more particularly hydrochloric acid, sulfuric acid and phosphoric acid. Among the organic acids well known to those skilled in the art, there will be mentioned in particular acetic acid, citric acid, tartaric acid, lactic acid, preferably citric acid. Mention may also be made of acid polyphenols such as rosmarinic acid, tannic acid, digallic acid, quercitannic acid, gallotannic acid, acid tannins such as quercitine, ellagitannins, castalagine, castaline, casuariticine, grandinine , punicaligine punicaline, roburine A, tellimagrandine II, terflavine B, vescaligine, pendunculagine, casuariine, castline, vescaline, preferably tannic acid. Preferably, the acids used are acids authorized for food use, in particular phosphoric acid, sulfuric acid, citric acid or tannic acid.
La suspension acide obtenue par l’étape b.2. a de préférence une teneur en matière sèche supérieure ou égale à 1 %, avantageusement plus de 2% de matière sèche, préférentiellement de 3 à 30 % de matière sèche, plus préférentiellement de 4 à 15 % de matière sèche, encore plus préférentiellement de 5 à 12 %, encore plus préférentiellement de 6 à 10 %.  The acid suspension obtained by step b.2. preferably has a dry matter content greater than or equal to 1%, advantageously more than 2% of dry matter, preferably from 3 to 30% of dry matter, more preferably from 4 to 15% of dry matter, even more preferably from 5 to 12%, even more preferably from 6 to 10%.
Selon un mode particulier de réalisation de l’invention, les étapes b.1. et b.2. sont consécutives, l’étape b.2 s’effectuant directement après l’étape b.1 sans autre étape de traitement de la biomasse comme une étape de délipidation, par exemple. La mise en suspension, ou la dilution de la suspension est suivie de l’ajout d’acide, sous forme solide ou liquide.  According to a particular embodiment of the invention, steps b.1. and b.2. are consecutive, step b.2 being carried out directly after step b.1 without any other biomass treatment step such as a defatting step, for example. The suspension, or dilution of the suspension is followed by the addition of acid, in solid or liquid form.
Selon un autre mode particulier de réalisation de l’invention les étapes b.1. et b.2. sont réalisées de manière simultanée par l’ajout d’une quantité appropriée d’une solution acide pour obtenir à la fois le pH recherché inférieur ou égal à 5 et la teneur en matière sèche recherchée.  According to another particular embodiment of the invention, steps b.1. and b.2. are carried out simultaneously by adding an appropriate amount of an acidic solution to obtain both the desired pH less than or equal to 5 and the desired dry matter content.
En fonction de la biomasse lysée récupérée de l’étape a., notamment que la lyse ait été faite sur une suspension cellulaire dans l’eau ou sur une biomasse dite « sèche », l’homme du métier saura ajouter les quantités d’eau et d’acide nécessaires à l’obtention d’une suspension avec la teneur en matière sèche recherchée et au pH recherché, que ces ajouts soient faits en deux étapes ou en une seule étape. Depending on the lysed biomass recovered from step a., In particular whether the lysis was carried out on a cell suspension in water or on a so-called “dry” biomass, the skilled person will be able to add the amounts of water and acid necessary for obtaining a suspension with the desired dry matter content and at the desired pH, whether these additions are made in two stages or in one single step.
L’étape c. est mise en œuvre par tout moyen de séparation connu de l’homme du métier pour séparer les résidus solides de lyse cellulaire de la solution aqueuse à partir de la suspension acide comprenant les cellules lysées, notamment par filtration ou par centrifugation. Pour la filtration, on citera en particulier la filtration tangentielle, par exemple la filtration tangentielle sur membranes céramiques ou membranes organiques tel que les fibres creuses en polyéthersulfone. La filtration frontale peut également être utilisée (filtre-plaque ou filtre-presse).  Step c. is implemented by any separation means known to a person skilled in the art for separating the solid cell lysis residues from the aqueous solution from the acid suspension comprising the lysed cells, in particular by filtration or by centrifugation. For filtration, mention will be made in particular of tangential filtration, for example tangential filtration on ceramic membranes or organic membranes such as hollow polyethersulfone fibers. Front filtration can also be used (plate filter or filter press).
Le cas échéant, le cycle de suspension/acidification/récupération (b.1./b.2./c.) peut être répété plusieurs fois sur la fraction de biomasse lysée insoluble à pH inférieur ou égal à 5 récupérée en c. de manière à épuiser celle-ci en éléments solubles à pH acide. Avantageusement, le cycle suspension/acidification/récupération est répété 1 , 2 ou 3 fois sur la biomasse lysée récupérée en c.  If necessary, the suspension / acidification / recovery cycle (b.1./b.2./c.) Can be repeated several times on the fraction of lysed biomass insoluble at pH less than or equal to 5 recovered in c. so as to exhaust it in soluble elements at acidic pH. Advantageously, the suspension / acidification / recovery cycle is repeated 1, 2 or 3 times on the lysed biomass recovered in c.
La fraction de biomasse enrichie en protéines récupérée en c. peut ensuite être séchée selon toute méthode connue de séchage.  The fraction of protein-enriched biomass recovered in c. can then be dried using any known method of drying.
Les eaux-mères de la suspension sont généralement récupérées pour être traitées de manière à en extraire des composants solubles à pH acides susceptibles d’être valorisés de leur côté. C’est le cas notamment des phycocyanines lorsque les microorganismes sont des microorganismes producteurs de phycobiliprotéines tels que définis ci-dessus. Un tel procédé est notamment décrit dans la demande de brevet PCT/EP2018/058294 déposée le 30 mars 2018.  The mother liquors of the suspension are generally recovered in order to be treated so as to extract therefrom soluble components at acid pH which can be valued on their side. This is particularly the case for phycocyanins when the microorganisms are microorganisms producing phycobiliproteins as defined above. Such a process is described in particular in the patent application PCT / EP2018 / 058294 filed on March 30, 2018.
La biomasse obtenue a généralement au moins 60% de protéines (évaluées par le dosage de l’azote total, N*6,25) par rapport à la matière sèche, de préférence plus de 65%, encore plus préférentiellement plus de 68%, plus de 70% voire plus de 80%. The biomass obtained generally has at least 60% of proteins (evaluated by the determination of total nitrogen, N * 6.25) relative to the dry matter, preferably more than 65%, even more preferably more than 68%, more than 70% or even more than 80%.
La biomasse a également de préférence une teneur en sucres totaux inférieure à 20% par rapport à la matière sèche, plus préférentiellement moins de 10%, moins de 5%.  The biomass also preferably has a total sugar content of less than 20% relative to the dry matter, more preferably less than 10%, less than 5%.
La biomasse obtenue a également de préférence une teneur en glycogène inférieure à 10% par rapport à la matière sèche, plus préférentiellement inférieure à 5%, voire inférieure à 1 %, encore plus préférentiellement inférieure à 0,1 %.  The biomass obtained also preferably has a glycogen content of less than 10% relative to the dry matter, more preferably less than 5%, or even less than 1%, even more preferably less than 0.1%.
Selon les microorganismes employés, la biomasse lysée insoluble à pH inférieur ou égal à 5, peut avoir une teneur en matières grasses, notamment en lipides, pouvant être d’au moins 5%, voire d’au moins 10% par rapport à la matière sèche, voire d’au moins 15%, d’au moins 16%, jusqu’à 20%. La composition en matières grasse dépendra également des microorganismes. Pour les ARU, la biomasse lysée selon l’invention comprend généralement des acides gras polyinsaturés de type oméga 3, 6 et 9, notamment des acides oléique, palmitique et alpha-linolénique qui représentent au moins 30% de la matière grasse, préférentiellement 40%, encore plus préférentiellement 50% de la matière grasse. Depending on the microorganisms used, the lysed biomass insoluble at pH less than or equal to 5, may have a fat content, in particular lipids, which may be at least 5%, or even at least 10% relative to the material. dry, or at least 15%, at least 16%, up to 20%. The fat composition will also depend on the microorganisms. For ARUs, the lysed biomass according to the invention generally comprises polyunsaturated fatty acids of omega 3, 6 and 9 type, in particular oleic, palmitic and alpha-linolenic acids which represent at least 30% of the fatty matter, preferably 40% , even more preferably 50% of the fat.
L’homme du métier saura adapter les conditions de mises en œuvre du procédé selon l’invention, notamment le pH de la suspension et le nombre de cycles de suspension/acidification/récupération à répéter sur la biomasse lysée récupérée pour obtenir une biomasse avec les quantités de protéines, de sucres, de glycogène et de lipides recherchées et notamment telles que définies ci-dessus.  Those skilled in the art will be able to adapt the conditions for implementing the method according to the invention, in particular the pH of the suspension and the number of suspension / acidification / recovery cycles to be repeated on the lysed biomass recovered to obtain a biomass with the quantities of proteins, sugars, glycogen and lipids sought and in particular as defined above.
Les pourcentages en poids en protéines peuvent être exprimés tant par rapport aux protéines elles-mêmes que par rapport aux acides aminés contenus dans lesdites protéines. Par protéines on entend, selon la présente invention, toute protéine, peptide ou acide aminé insolubles à pH acide inférieur ou égal à 5.  The percentages by weight of proteins can be expressed both with respect to the proteins themselves and with respect to the amino acids contained in said proteins. By proteins is meant, according to the present invention, any protein, peptide or amino acid insoluble at an acid pH less than or equal to 5.
La présente invention concerne également la biomasse telle que définie précédemment, susceptible d’être obtenue par le procédé selon l’invention.  The present invention also relates to the biomass as defined above, capable of being obtained by the method according to the invention.
Cette biomasse a avantageusement :  This biomass advantageously has:
- au moins 60% de protéines, de préférence plus de 65%, encore plus préférentiellement plus de 68%, plus de 70% voire plus de 80%,  - at least 60% protein, preferably more than 65%, even more preferably more than 68%, more than 70% or even more than 80%,
- moins de 20 % de sucres totaux, préférentiellement moins de 10%, plus préférentiellement moins de 5%,  - less than 20% of total sugars, preferably less than 10%, more preferably less than 5%,
- moins de 10% de glycogène, de préférence moins de 5%, plus préférentiellement moins de 1 % voire encore plus préférentiellement moins de 0,1 %, et  less than 10% of glycogen, preferably less than 5%, more preferably less than 1% or even more preferably less than 0.1%, and
- jusqu’à 20% de matières grasses,  - up to 20% fat,
les pourcentages étant exprimés par rapport à la matière sèche de la biomasse. the percentages being expressed relative to the dry matter of the biomass.
L'invention a encore pour objet l'utilisation d'une biomasse telle que décrite précédemment dans les domaines cosmétiques, pharmaceutiques, alimentaires humain ou animal.  Another subject of the invention is the use of a biomass as described above in the cosmetic, pharmaceutical, human or animal food fields.
Elle concerne en particulier l’utilisation d’une biomasse enrichie en protéines selon l’invention, telle que décrite ci-dessus et ci-après, pour l’amélioration des performances des animaux. Cette amélioration de la performance peut être évaluée, en particulier, par la mesure de la consommation, du gain de poids ou encore du « Feed Conversion Ratio » (rapport de conversion de l’aliment). On pourra distinguer dans l’alimentation animale, l’alimentation des animaux d’élevage, en particulier en élevage industriel et celle des animaux domestiques ou encore des animaux de compagnie ou des animaux dits de « loisir », comme les poissons d’aquarium ou les oiseaux de volière ou en cage ou encore les Nouveaux Animaux de Compagnie (NAC). It relates in particular to the use of a protein-enriched biomass according to the invention, as described above and below, for improving the performance of animals. This improvement in performance can be assessed, in particular, by measuring consumption, weight gain or even the "Feed Conversion Ratio". We can distinguish in animal feed, the feeding of farm animals, in particular in industrial farming and that of domestic animals or even pets or so-called "leisure" animals, such as aquarium fish or aviary or cage birds or New Pets (NAC).
Par « animal d'élevage » on entend notamment les animaux de pacage (notamment les bovins élevés pour la viande, le lait, le fromage et le cuir ; les ovins élevés pour la viande, la laine et le fromage ; les caprins), les porcins, les lapins, les volailles (les poulets, les poules, les dindes, les canards, les oies et autres), les équidés (poneys, chevaux, poulains), destinés à supporter des activités humaines (transport, loisirs) ou leur alimentation, les animaux aquatiques (par exemple les poissons, les crevettes, les coquillages (notamment les huîtres et les moules)). On pourra toutefois distinguer l’alimentation des poissons jusqu’au stade d’alevins, et celles des poissons élevés, y compris les aliments et compositions alimentaires qui leur sont destinées.  By "farm animal" is meant in particular grazing animals (especially cattle raised for meat, milk, cheese and leather; sheep raised for meat, wool and cheese; goats), pigs, rabbits, poultry (chickens, hens, turkeys, ducks, geese and others), equines (ponies, horses, foals), intended to support human activities (transport, leisure) or their food , aquatic animals (eg fish, shrimp, shellfish (especially oysters and mussels)). However, a distinction can be made between feeding fish up to the fry stage and that of farmed fish, including feed and food compositions intended for them.
On les distinguera des animaux domestiques, animaux de compagnie ou animaux de loisir qui comprennent également des mammifères, ruminants ou non, des oiseaux ou des poissons. Ils comprennent en particulier les chiens et les chats.  They will be distinguished from domestic animals, pets or leisure animals which also include mammals, ruminants or not, birds or fish. They include in particular dogs and cats.
L'invention a aussi pour objet un aliment, ou composition alimentaire, pour l’homme ou les animaux, comprenant une biomasse selon l'invention telle que décrite précédemment. On entend par « aliment » toute composition qui peut servir à la nourriture de l’homme ou des animaux.  The invention also relates to a food, or food composition, for humans or animals, comprising a biomass according to the invention as described above. "Food" means any composition which can be used for food for humans or animals.
Selon l'invention l’aliment peut ne comprendre que de la biomasse, séchée ou non, transformée ou non, ou de la biomasse, séchée ou non, transformée ou non, mélangée à tout autre additif, véhicule ou support, utilisé dans le domaine de l'alimentation humaine ou animale, comme par exemple les conservateurs alimentaires, les colorants, les exhausteurs de goût, les régulateurs du pH, ou encore des additifs pharmaceutiques comme par exemple des hormones de croissance, des antibiotiques.  According to the invention, the food may comprise only biomass, dried or not, transformed or not, or biomass, dried or not, transformed or not, mixed with any other additive, vehicle or support, used in the field. food or feed, such as food preservatives, colorings, flavor enhancers, pH regulators, or pharmaceutical additives such as growth hormones, antibiotics.
La présente invention concerne en particulier des aliments pour animaux et plus particulièrement pour les animaux d’élevage. Ces aliments se présentent habituellement sous la forme de farines, de granulés ou de soupe dans lesquels est incorporée la biomasse selon l'invention. On entend par "aliment pour animaux" tout ce qui peut servir à la nourriture des animaux. Pour l'élevage intensif des animaux, les aliments peuvent comprendre, en plus de la biomasse algale, une base nutritionnelle et des additifs nutritionnels. L'essentiel de la ration alimentaire de l'animal est alors constitué par la "base nutritionnelle" et la biomasse algale. Cette base est constituée à titre d'exemple par un mélange de céréales, de protéines et de matières grasses d'origine animale et/ou végétale. The present invention relates in particular to animal feed and more particularly to farm animals. These foods are usually in the form of flour, granules or soup in which the biomass according to the invention is incorporated. "Animal food" means anything that can be used to feed animals. For intensive animal husbandry, feed may include, in addition to algal biomass, a nutritional base and nutritional additives. Most of the animal's food ration then consists of the "nutritional base" and the algal biomass. This base is constituted, for example, by a mixture of cereals, proteins and fats of animal and / or vegetable origin.
Les bases nutritionnelles pour animaux sont adaptées à l'alimentation de ces animaux et sont bien connues de l'homme du métier. Dans le cadre de la présente invention, ces bases nutritionnelles comprennent par exemple du maïs, du blé, du pois et du soja. Ces bases nutritionnelles sont adaptées aux besoins des différentes espèces animales auxquelles elles sont destinées. Ces bases nutritionnelles peuvent déjà contenir des additifs nutritionnels comme des vitamines, des sels minéraux et des acides aminés.  The nutritional bases for animals are suitable for feeding these animals and are well known to those skilled in the art. In the context of the present invention, these nutritional bases include, for example, corn, wheat, peas and soy. These nutritional bases are adapted to the needs of the different animal species for which they are intended. These nutritional bases may already contain nutritional additives such as vitamins, minerals and amino acids.
Les additifs utilisés en alimentation animale peuvent être ajoutés pour améliorer certaines caractéristiques des aliments, par exemple pour en relever le goût, pour rendre plus digestes les matières premières des aliments pour animaux ou protéger les animaux. Ils sont fréquemment utilisés dans les élevages intensifs de grande envergure.  Additives used in animal feed can be added to improve certain characteristics of food, for example to enhance the taste, to make the raw materials of animal feed more digestible or to protect animals. They are frequently used in large scale intensive farming.
Les additifs utilisés en alimentation animale se déclinent, notamment, dans les sous-catégories suivantes (source EFSA):  The additives used in animal feed are available, in particular, in the following subcategories (source EFSA):
- additifs technologiques: par exemple, conservateurs, antioxydants, émulsifiants, stabilisateurs, régulateurs d’acidité et additifs pour l'ensilage;  - technological additives: for example, preservatives, antioxidants, emulsifiers, stabilizers, acidity regulators and additives for silage;
- additifs sensoriels: par exemple, arômes, colorants,  - sensory additives: for example, flavors, colors,
- additifs nutritionnels: par exemple, vitamines, aminoacides et oligo-éléments; - nutritional additives: for example, vitamins, amino acids and trace elements;
- additifs zootechniques: par exemple, améliorateurs de digestibilité, stabilisateurs de flore intestinale; - zootechnical additives: for example, digestibility enhancers, intestinal flora stabilizers;
- coccidiostatiques et histomonostatiques (antiparasitaires).  - coccidiostats and histomonostats (antiparasitic).
Dans un mode de réalisation, l'invention concerne des aliments pour animaux d’élevage comprenant entre 1 et 60 %, de préférence entre 1 et 20%, de façon tout à fait préférentielle entre 3 et 8 % d’une biomasse séchée obtenue par le procédé selon l’invention.  In one embodiment, the invention relates to feed for farm animals comprising between 1 and 60%, preferably between 1 and 20%, quite preferably between 3 and 8% of a dried biomass obtained by the method according to the invention.
Dans un autre mode de réalisation, l'invention concerne des aliments pour animaux d’élevage comprenant entre 1 et 40 %, de préférence entre 5 et 10% d’une biomasse non séchée obtenue par le procédé de l’invention.  In another embodiment, the invention relates to feed for farm animals comprising between 1 and 40%, preferably between 5 and 10% of an undried biomass obtained by the process of the invention.
Selon un mode particulier de réalisation de l’invention, l’aliment est destiné aux animaux d’élevage, en particulier les bovins, les ovins, les porcins, les lapins, les volailles et les équidés. According to a particular embodiment of the invention, the food is intended for farm animals, especially cattle, sheep, pigs, rabbits, poultry and horses.
Selon un autre mode particulier de l’invention, l’aliment est destiné aux animaux aquatiques, en particulier aux poissons, au moins jusqu’au stade alevins, y compris les poissons d’élevage et d’ornement.  According to another particular mode of the invention, the food is intended for aquatic animals, in particular for fish, at least up to the fry stage, including farmed and ornamental fish.
Selon un autre mode particulier de réalisation de l’invention, l’aliment est destiné aux animaux domestiques, animaux de compagnie et/ou animaux de loisirs et les NACs.  According to another particular embodiment of the invention, the food is intended for domestic animals, pets and / or leisure animals and NACs.
Enfin, selon un autre mode de réalisation de l’invention, la composition alimentaire est destinée à l’Homme.  Finally, according to another embodiment of the invention, the food composition is intended for humans.
L'invention à également pour objet une composition cosmétique, nutraceutique ou pharmaceutique pour l’homme ou les animaux comprenant une biomasse selon l'invention telle que décrite précédemment.  The subject of the invention is also a cosmetic, nutraceutical or pharmaceutical composition for humans or animals comprising a biomass according to the invention as described above.
Selon l'invention la composition cosmétique, nutraceutique ou pharmaceutique peut ne comprendre que de la biomasse, séchée ou non, transformée ou non, ou de la biomasse, séchée ou non, transformée ou non, mélangée à tout autre additif, véhicule ou support, utilisé dans le domaine de la cosmétique ou de la pharmacie comme par exemple les conservateurs, les colorants, les régulateurs du pH.  According to the invention, the cosmetic, nutraceutical or pharmaceutical composition may only comprise biomass, dried or not, transformed or not, or biomass, dried or not, transformed or not, mixed with any other additive, vehicle or support, used in the field of cosmetics or pharmacy such as, for example, preservatives, dyes, pH regulators.
L'invention a aussi pour objet l'utilisation de la biomasse telle que décrite précédemment en thérapie, ainsi que dans la prévention et le traitement de la malnutrition.  The invention also relates to the use of biomass as described above in therapy, as well as in the prevention and treatment of malnutrition.
EXEMPLES  EXAMPLES
Matériel et Méthodes  Material and methods
Biomasse lysée  Lysed biomass
Les biomasses traitées par le procédé selon l’invention sont des biomasses de spiruline ( Arthrospira platensis) et de Galdieria sulphuraria souche UTEX 2919 (UTEX Culture Collection of Algae, 205 W. 24th St, Biological Labs 218, The University of Texas at Austin (A6700), Austin, TX 78712 USA) cultivées selon les méthodes usuelles de culture de ces souches.  The biomasses treated by the process according to the invention are biomasses of spirulina (Arthrospira platensis) and of Galdieria sulphuraria strain UTEX 2919 (UTEX Culture Collection of Algae, 205 W. 24th St, Biological Labs 218, The University of Texas at Austin ( A6700), Austin, TX 78712 USA) cultivated according to the usual methods for cultivating these strains.
La biomasse lysée a une teneur en cellules lysées de 95%  The lysed biomass has a lysed cell content of 95%
La teneur en matière sèche est de 10%  The dry matter content is 10%
Mode opératoire  Procedure
Le procédé d’enrichissement s’opère par la répétition d’un rinçage du culot de la biomasse broyée en condition de pH acide. Pour ce faire l’opération implique l’utilisation d’une centrifugeuse, ici décrite par l’utilisation d’une SORVALL RC5B plus et d’un rotor à angle fixe Sorvall SLA 3000. La biomasse broyée est centrifugée à 12000 x g pendant 15min, permettant de culoter au maximum la fraction insoluble. Le surnageant « fraction soluble » est retiré et remplacé par de l’eau. Le culot est resuspendu avant de réitérer l’opération de centrifugation, et le pH est contrôlé et ajusté si nécessaire à une valeur inférieure à pH 4,8. Ce procédé ainsi répété 3 à 4 fois maximise l’enrichissement en protéine et lipides de la fraction insoluble. The enrichment process is carried out by repeating a rinsing of the pellet of the crushed biomass under an acid pH condition. To do this the operation involves the use of a centrifuge, here described by the use of a SORVALL RC5B plus and a Sorvall SLA 3000 fixed angle rotor. The crushed biomass is centrifuged at 12,000 xg for 15 min, allowing the fraction to be pelleted as much as possible insoluble. The “soluble fraction” supernatant is removed and replaced with water. The pellet is resuspended before repeating the centrifugation operation, and the pH is checked and adjusted if necessary to a value below pH 4.8. This process, thus repeated 3 to 4 times, maximizes the protein and lipid enrichment of the insoluble fraction.
Analyses  Analyzes
Les protéines totales sont estimées en utilisant la méthode Kjeldahl connue de l’homme du métier. Le profil en acides aminés par hydrolyse acide est réalisé selon la méthode ISO 13903:2005; EU 152/2009. L’analyse de la teneur en lipides et de la composition se fait par GC/FID et étalonnage interne connue de l’homme du métier. Le profil des sucres est déterminé par Chromatographie ionique - Ampérométrique pulsée.  Total proteins are estimated using the Kjeldahl method known to those skilled in the art. The amino acid profile by acid hydrolysis is carried out according to the ISO 13903: 2005 method; EU 152/2009. The analysis of the lipid content and of the composition is done by GC / FID and internal calibration known to those skilled in the art. The sugar profile is determined by ion chromatography - pulsed amperometric.
Résultats  Results
Nous avons réalisé un suivi de la composition de la phase lourde (PL ou fraction insoluble) après la 1 ère extraction (PL1 ) et la 4eme (PL4) et l’avons comparé à celle du lysat d’entrée pour une biomasse de Galdieria sulphuraria souche UTEX 2919 (Tableaul ) et pour une biomasse de Spiruline (Tableau 2). Les pourcentages sont exprimés par rapport à la Matière Sèche (MS).  We followed the composition of the heavy phase (PL or insoluble fraction) after the 1st extraction (PL1) and the 4th (PL4) and compared it to that of the input lysate for a biomass of Galdieria sulphuraria UTEX 2919 strain (Tableaul) and for a Spirulina biomass (Table 2). The percentages are expressed relative to the Dry Matter (DM).
Tableau 1  Table 1
On observe une nette augmentation de la teneur en protéines de la biomasse lysée dès le premier cycle suspension/acidification/récupération (plus de 6%). Cette augmentation est encore plus marquée après le 4ème cycle de traitement (plus de 16%). Tableau 2 A clear increase in the protein content of the lysed biomass is observed from the first suspension / acidification / recovery cycle (more than 6%). This increase is even more pronounced after the 4th treatment cycle (more than 16%). Table 2
Contrairement aux ARUs comme Galderia, la mise en œuvre du procédé sur la spiruline entraine une diminution de la teneur en protéines dans la biomasse lysée insoluble à pH inférieur ou égal à 5 (Tableau 2). Pour une extraction à pH 4, pH inférieur au pHi de la phycocyanine (C-PC) de l’allophycocyanine (APC) nous pouvons observer une baisse dans de la quantité de phycocyanine et d’allophycocyanine dans la phase lourde. A pH 4, la C-PC et APC n’étant pas soluble dans l’eau, les pertes observées dans la PL4 par rapport à la biomasse sont de certainement dues à une dégradation d’une partie de ces molécules à ce pH. Unlike ARUs like Galderia, the implementation of the process on spirulina leads to a reduction in the protein content in the lysed biomass insoluble at pH less than or equal to 5 (Table 2). For an extraction at pH 4, pH lower than the pHi of phycocyanin (C-PC) of allophycocyanin (APC) we can observe a drop in the quantity of phycocyanin and allophycocyanin in the heavy phase. At pH 4, since C-PC and APC are not soluble in water, the losses observed in PL4 compared to biomass are certainly due to a degradation of part of these molecules at this pH.
Le contenu en protéines des différentes biomasses est représenté sur la figure 1. Si l’on regarde dans le détail la composition en acides aminés dans les différents produits représentés sur la figure 2 on peut voir une augmentation globale de tous les acides aminés sur les phases lourdes, et des quantités supérieures ou égales à celles de la Spiruline pour la plus-part d’entre eux sauf pour l’alanine.  The protein content of the different biomasses is shown in Figure 1. If we look in detail at the amino acid composition in the different products shown in Figure 2 we can see an overall increase in all amino acids on the phases heavy, and amounts greater than or equal to those of Spirulina for most of them except for alanine.
Si l’on prend en compte les recommandations de la FAO concernant les acides aminés essentiels pour l’alimentation humaine et que nous les comparons à la composition des différentes phases lourdes nous pouvons noter que les quantités sont toutes supérieures ou égales aux valeurs recommandées.  If we take into account the recommendations of the FAO concerning essential amino acids for human consumption and compare them to the composition of the different heavy phases, we can note that the quantities are all greater than or equal to the recommended values.
Après plusieurs extractions il reste encore de la phycocyanine (C-PC et APC) dans la phase lourde ce qui représente environ 3% de la masse sèche. La phycocyanine est connue pour être une molécule à fort pouvoir antioxydant. La composition en acides gras montre des quantités non négligeables d’acides gras insaturés de type oméga 3, 6 et 9, sous forme de lipides polaires, représentant plus de 50% de la masse grasse qui représente environ 5% à 20% de la masse sèche de la phase lourde (Tableau 3). Tableau 3 After several extractions, there is still phycocyanin (C-PC and APC) in the heavy phase, which represents approximately 3% of the dry mass. Phycocyanin is known to be a molecule with strong antioxidant power. The fatty acid composition shows significant amounts of omega 3, 6 and 9 type unsaturated fatty acids, in the form of polar lipids, representing more than 50% of the fatty mass which represents approximately 5% to 20% of the mass the heavy phase (Table 3). Table 3
Les sucres sont en dessous du seuil de détection de la méthode, de même que les fibres (Tableau 4). Tableau 4 The sugars are below the detection threshold of the method, as are the fibers (Table 4). Table 4
Le procédé selon l’invention a été répété sur une biomasse de Galdieria sulphuraria souche UTEX 2919 cultivée selon la méthode décrite dans la demande de brevet WO 2017/050917. The process according to the invention was repeated on a biomass of Galdieria sulphuraria strain UTEX 2919 cultivated according to the method described in patent application WO 2017/050917.
La biomasse de départ comprend plus de 70 % de protéines par rapport à la MS. Après trois traitements selon l’invention, on obtient une biomasse avec plus de 80 % de protéines, soit une augmentation de plus de 14%.  The starting biomass comprises more than 70% of proteins compared to the DM. After three treatments according to the invention, a biomass is obtained with more than 80% of proteins, an increase of more than 14%.
Des extractions en série sont réalisées sur 3 biomasses ayant des contenus en glycogène différents. Nous pouvons noter que la quasi-totalité du glycogène soluble de la biomasse se retrouve en solution après la première extraction.  Serial extractions are carried out on 3 biomasses having different glycogen contents. We can note that almost all of the soluble glycogen from the biomass is found in solution after the first extraction.
Tableau 5. Suivi de la solubilisation du glycogène dans le surnageant lors d’extraction en série.  Table 5. Monitoring of the solubilization of glycogen in the supernatant during serial extraction.
La phase lourde enrichie en protéines ainsi obtenue peut être par la suite traitées enzymatiquement par des protéases afin d’obtenir un hydrolysat de protéines et d’augmenter la solubilité et la digestibilité du produit. The heavy phase enriched in proteins thus obtained can be subsequently enzymatically treated with proteases in order to obtain a protein hydrolyzate and to increase the solubility and digestibility of the product.
Selon les différentes périodes de la vie et la dépense énergétique quotidienne propre à chacun, la quantité de protéine recommandée (ANSES, 2007) va varier. Chez l’adulte en bonne santé, les Apports Nutritionnels Conseillés (ANC) en protéines sont fixés à 0,83 g/kg/jour. Mais ces apports varient et sont accrus chez la femme enceinte, la personne âgée ou encore chez certains sportifs. Depending on the different periods of life and the daily energy expenditure specific to each, the amount of protein recommended (ANSES, 2007) will vary. In healthy adults, the Recommended Nutritional Intakes (ANC) in proteins are set at 0.83 g / kg / day. But these contributions vary and are increased in pregnant women, the elderly or in some athletes.
La fraction enrichie en protéines produite par le procédé décrit dans cette invention peut être consommée directement sous forme de comprimé pressé à froid, en poudre sous forme de capsule, ou directement en poudre pour être mélangée à d’autres ingrédients. Cette fraction peut également être intégrée dans d’autres préparations alimentaires pouvant être utilisées pour la fabrication de pain gâteau, biscuit ou autre pâtisserie, ou bars de céréales, ou toute préparation en remplacement de la farine classique afin d’enrichir en protéines les aliments ainsi préparés.  The protein-enriched fraction produced by the process described in this invention can be consumed directly in the form of a cold pressed tablet, in powder form in the form of a capsule, or directly in powder form to be mixed with other ingredients. This fraction can also be integrated into other food preparations which can be used for the manufacture of cake bread, biscuit or other pastry, or bars of cereals, or any preparation in replacement of the traditional flour in order to enrich in proteins the food thus prepared.
Nous pouvons également envisager une utilisation dans des préparations à base de lait (animal ou végétal), lait brut ou fermenté, comme des crèmes desserts, yaourt, fromage. L’ajout de Cela permet d’obtenir une préparation laitière ou fromagère enrichie en protéines tout en réduisant la quantité de matière grasse. Il est envisageable de réaliser les préparations avec des bases de fromages à pâte cru, à pâte cuite pressée ou non, ou de fromages fondus. Les algues peuvent être intégrées dans la masse ou même simplement à l’extérieur sur la croûte si celle-ci est comestible.  We can also consider using it in preparations based on milk (animal or vegetable), raw or fermented milk, such as dessert creams, yogurt, cheese. The addition of It allows to obtain a milk or cheese preparation enriched in proteins while reducing the quantity of fat. It is possible to carry out the preparations with raw cheese bases, cooked or pressed pressed cheese, or processed cheeses. The algae can be incorporated in the mass or even simply outside on the crust if it is edible.
Cette fraction protéique peut être également intégrée dans des préparations visant à servir de substitut à la viande comme les burgers végétaux, ou dans les bâtonnets de protéines façon surimi, ces protéines pouvant être texturées ou utilisées telles quelles.  This protein fraction can also be incorporated into preparations intended to serve as a substitute for meat such as vegetable burgers, or in protein sticks in surimi manner, these proteins being able to be textured or used as such.
Un autre exemple d’utilisation serait sous forme de poudre à ajouter dans des boissons de type Smoothie ou Shake.  Another example of use would be in the form of powder to be added to drinks such as Smoothie or Shake.
Exemples de composition:  Examples of composition:
Farine enrichie en protéines d’algues :  Flour enriched with algae proteins:
Composition pour 1 kg de Farine :  Composition for 1 kg of Flour:
800 gr Farine de type de blé (10-12 % de protéines) 200 gr d’extrait d’algue enrichie en protéines (65 à 80 % de protéines)800 gr Wheat type flour (10-12% protein) 200 gr of algae extract enriched in proteins (65 to 80% of proteins)
Soit 16 à 19% de protéines pour 100 g de farine contre 10 à 12% pour une farine de blé type 55 normale. Or 16 to 19% of protein per 100 g of flour against 10 to 12% for a normal type 55 wheat flour.
Cookies fabriqués à la farine enrichie en protéines d’algues  Cookies made with flour enriched with algae protein
Ingrédients pour 100 g de Cookies (7 g à 9 g de protéines):  Ingredients for 100 g of Cookies (7 g to 9 g of protein):
45g de Farine enrichie en protéines d’algues  45g of flour enriched with algae proteins
23g de Beurre  23g Butter
21 ,7g de sucre  21.7g sugar
5g de poudre de cacao  5g cocoa powder
4g Œufs  4g Eggs
1g de sel  1g of salt
0,3g levure chimique  0.3g baking powder
Pain fabriqué à la farine enrichie en protéines d’algues  Bread made with flour enriched with algae proteins
Ingrédients pour 100 g de pain (10 à 25 g de protéines):  Ingredients for 100 g of bread (10 to 25 g of protein):
63,2 gr de Farines enrichie en protéines d’algues  63.2 gr of Flours enriched with algae proteins
34 g d'eau 34 g of water
1 ,8 g de sel 1.8 g salt
1 grammes de levure boulangère  1 grams of baking yeast
Burger Végétal à l’algue  Seaweed Vegetable Burger
Composition pour un Burger de 100 g.  Composition for a Burger of 100 g.
Eau, farine de pois chiche (26%), poudre d’algues (25%), oignon, carotte, gluten de blé (3%), jus de citron, extrait de levure, fécule de pomme de terre, huile végétale (tournesol), curcuma, ail, régulateur d'acidité (chlorure de potassium), anti-oxydant, fibres de pomme de terre, épaississant (carraghénane), amidon (maïs, blé), émulsifiant (gomme de guar).  Water, chickpea flour (26%), seaweed powder (25%), onion, carrot, wheat gluten (3%), lemon juice, yeast extract, potato starch, vegetable oil (sunflower) ), turmeric, garlic, acidity regulator (potassium chloride), antioxidant, potato fibers, thickener (carrageenan), starch (corn, wheat), emulsifier (guar gum).
Cela représente entre 22 et 33 g de protéines par Burger.  This represents between 22 and 33 g of protein per Burger.
Produit laitier enrichi aux protéines d’algues.  Dairy product enriched with algae proteins.
Préparation à base de fromage de type Gouda.  Preparation based on Gouda type cheese.
La fraction d’algue enrichie en protéines est ajoutée aux protéines de lait coagulées avant pressage de la pâte. Le pourcentage de fraction d’algue enrichie en protéine peut varier de 1 % à 50%. Dans ce cas le facteur limitant sera lié à l’aspect du produit final et en particulier à sa texture et la tenue à la coupe.  The protein-enriched alga fraction is added to the coagulated milk proteins before pressing the dough. The percentage of algae fraction enriched in protein can vary from 1% to 50%. In this case the limiting factor will be linked to the appearance of the final product and in particular to its texture and the resistance to cutting.
Tableau 6. Principaux composants en gramme pour 100 grammes de produit. Table 6. Main components in grams per 100 grams of product.
Poudre pour boissons enrichies en protéines d’algues. Powder for beverages enriched with algae proteins.
Composition pour Smoothie:  Composition for Smoothie:
93,8 g de poudre de la fraction enrichies en protéines d’algues  93.8 g of powdered fraction enriched with algae protein
5 g Arôme naturel de fruit  5 g Natural fruit flavor
0,25 g gomme de xanthane  0.25 g xanthan gum
0,2 g gomme de guar  0.2 g guar gum
0,25 g édulcorant ou arôme naturel au pouvoir sucrant  0.25 g sweetener or natural flavor with sweetening power
Mélanger 25 g de poudre dans 200 ml d’eau froide ou de jus de fruit- Cela représente entre 15 et 18,5 g de protéines par boisson.  Mix 25 g of powder in 200 ml of cold water or fruit juice - this represents between 15 and 18.5 g of protein per drink.
Composition pour Shake:  Composition for Shake:
84,35 g de poudre de la fraction enrichies en protéines d’algues  84.35 g of powdered fraction enriched with algae protein
10 g lait en poudre  10 g milk powder
5 g de Cacao  5 g of cocoa
0,2 g gomme de xanthane  0.2 g xanthan gum
0,2 g gomme de guar  0.2 g guar gum
0,25 g édulcorant  0.25 g sweetener
Mélanger 25 g de poudre dans 200 ml d’eau froide ou chaude. Cela représente entre 15 et 20 g de protéines par boisson. Mix 25 g of powder in 200 ml of cold or hot water. This represents between 15 and 20 g of protein per drink.
REFERENCES REFERENCES
WO 2016/00194  WO 2016/00194
WO 2016/120548 WO 2016/120548
WO 2016/015013 WO 2016/015013
WO 2017/019125 WO 2017/019125
WO 2017/050917 WO 2017/050917
WO 2017/050918 WO 2017/050918
WO 2017/093345 WO 2017/093345
WO 2017/050917 WO 2017/050917
WO 2017/132407 WO 2017/132407
US 2017-020834 US 2017-020834
Graziani & al., Food & Function, vol. 4, n° 1 , 2013, 144-152.  Graziani & al., Food & Function, vol. 4, n ° 1, 2013, 144-152.

Claims

REVENDICATIONS
1. Procédé de production d’une biomasse de microorganismes lysés riche en protéines, le procédé comprenant les étapes de 1. Method for producing a biomass of lysed microorganisms rich in proteins, the method comprising the steps of
b.1. mise en suspension d’une biomasse de microorganismes lysés, b.1. suspension of a biomass of lysed microorganisms,
b.2. acidification de la suspension jusqu’à un pH inférieur ou égal à 5, b.2. acidification of the suspension to a pH less than or equal to 5,
c. récupération de la fraction de biomasse lysée insoluble à pH inférieur ou égal à 5, et vs. recovery of the fraction of lysed biomass insoluble at pH less than or equal to 5, and
le cas échéant répétition des étapes b.1., b.2. et c. sur la fraction de biomasse lysée insoluble à pH inférieur ou égal à 5 récupérée en c. if necessary repeat steps b.1., b.2. and c. on the fraction of lysed biomass insoluble at pH less than or equal to 5 recovered in c.
2. Procédé selon la revendication 1 , caractérisé en ce que la suspension acide obtenue en b.2. a un pH inférieur ou égale à 4,5.  2. Method according to claim 1, characterized in that the acid suspension obtained in b.2. has a pH less than or equal to 4.5.
3. Procédé selon l’une des revendications 1 ou 2, caractérisé en ce que la suspension acide obtenue par l’étape b.2. a une teneur en matière sèche de 1% à 30%.  3. Method according to one of claims 1 or 2, characterized in that the acid suspension obtained by step b.2. has a dry matter content of 1% to 30%.
4. Procédé selon l’une des revendications 1 à 3, caractérisé en ce que les étapes b.1. et b.2. sont consécutives ou simultanées.  4. Method according to one of claims 1 to 3, characterized in that steps b.1. and b.2. are consecutive or simultaneous.
5. Procédé selon l’une des revendications 1 à 4, caractérisé en ce que les étapes b.1. et b.2. sont consécutives, l’étape b.2 s’effectuant directement après l’étape b.1 sans autre étape de traitement de la biomasse, en particulier sans une étape de délipidation.  5. Method according to one of claims 1 to 4, characterized in that steps b.1. and b.2. are consecutive, step b.2 being carried out directly after step b.1 without any other step of processing the biomass, in particular without a delipidation step.
6. Procédé selon l’une des revendications 1 à 5, caractérisé en ce qu’il comprend une étape préalable a. de lyse d’une biomasse de microorganismes préalablement cultivés.  6. Method according to one of claims 1 to 5, characterized in that it comprises a prior step a. for lysis of a biomass of microorganisms previously cultivated.
7. Procédé selon l’une des revendications 1 à 6, caractérisé en ce que la biomasse récupérée en c. est séchée.  7. Method according to one of claims 1 to 6, characterized in that the biomass recovered in c. is dried.
8. Procédé selon l’une des revendications 1 à 7, caractérisé en ce que les microorganismes sont choisis parmi les algues rouges unicellulaires (ARUs) des familles des Cyanidiaceae ou des Galdieriaceae.  8. Method according to one of claims 1 to 7, characterized in that the microorganisms are chosen from unicellular red algae (ARUs) from the families of Cyanidiaceae or Galdieriaceae.
9. Biomasse de microorganismes lysés riche en protéines susceptible d’être obtenue par le procédé selon l’une des revendications 1 à 8.  9. Biomass of lysed microorganisms rich in proteins capable of being obtained by the process according to one of claims 1 to 8.
10. Biomasse selon la revendication 9, caractérisée en ce qu’elle comprend au moins 60% de protéines par rapport à la matière sèche, et au moins une teneur en sucres totaux inférieure à 20% par rapport à la matière sèche et/ou une teneur en glycogène inférieure à 10% par rapport à la matière sèche et/ou teneur en matières grasses d’au moins 5% par rapport à la matière sèche. 10. Biomass according to claim 9, characterized in that it comprises at least 60% of proteins relative to the dry matter, and at least a total sugar content of less than 20% relative to the dry matter and / or a content glycogen lower than 10% compared to the dry matter and / or fat content of at least 5% compared to the dry matter.
11. Aliment ou composition alimentaire pour l’homme ou les animaux caractérisée en ce qu’elle comprend une biomasse de microorganismes lysés telle que définie selon les revendications 9 ou 10.  11. Food or food composition for humans or animals characterized in that it comprises a biomass of lysed microorganisms as defined according to claims 9 or 10.
12. Biomasse de microorganismes lysés telle que définie selon les revendications 9 ou 10 pour son utilisation en thérapie.  12. Biomass of lysed microorganisms as defined according to claims 9 or 10 for use in therapy.
13. Biomasse de microorganismes lysés telle que définie selon les revendications 9 ou 10 pour son utilisation dans la prévention et le traitement de la malnutrition.  13. Biomass of lysed microorganisms as defined according to claims 9 or 10 for its use in the prevention and treatment of malnutrition.
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