EP3942016A1 - Neuartige milchsäurebakterienstämme zur förderung der absorption von calcium-peptiden und assoziierten produkten - Google Patents

Neuartige milchsäurebakterienstämme zur förderung der absorption von calcium-peptiden und assoziierten produkten

Info

Publication number
EP3942016A1
EP3942016A1 EP20725858.3A EP20725858A EP3942016A1 EP 3942016 A1 EP3942016 A1 EP 3942016A1 EP 20725858 A EP20725858 A EP 20725858A EP 3942016 A1 EP3942016 A1 EP 3942016A1
Authority
EP
European Patent Office
Prior art keywords
peptides
seq
milk
strain
identity
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
EP20725858.3A
Other languages
English (en)
French (fr)
Inventor
Benoit Cudennec
François COUTTE
Pascal Dhulster
Djamel Drider
Christophe FLAHAUT
Marc Fremont
Cyril RAVESCHOT
Maxime VAEREMANS
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.)
Vf Bioscience
Original Assignee
Vf Bioscience
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
Priority claimed from FR1902847A external-priority patent/FR3094013A1/fr
Priority claimed from FR1902854A external-priority patent/FR3094015A1/fr
Priority claimed from FR1902851A external-priority patent/FR3094014A1/fr
Application filed by Vf Bioscience filed Critical Vf Bioscience
Publication of EP3942016A1 publication Critical patent/EP3942016A1/de
Pending legal-status Critical Current

Links

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
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • 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
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/59—Compounds containing 9, 10- seco- cyclopenta[a]hydrophenanthrene ring systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/12—Drugs for disorders of the metabolism for electrolyte homeostasis
    • A61P3/14—Drugs for disorders of the metabolism for electrolyte homeostasis for calcium homeostasis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4732—Casein
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/8103—Exopeptidase (E.C. 3.4.11-19) inhibitors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/15—Peptidyl-dipeptidases (3.4.15)
    • C12Y304/15001—Peptidyl-dipeptidase A (3.4.15.1)
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/17—Amino acids, peptides or proteins
    • A23L33/18—Peptides; Protein hydrolysates
    • 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
    • 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
    • A23V2200/00—Function of food ingredients
    • A23V2200/30—Foods, ingredients or supplements having a functional effect on health
    • A23V2200/306—Foods, ingredients or supplements having a functional effect on health having an effect on bone mass, e.g. osteoporosis prevention
    • 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
    • A23V2400/00—Lactic or propionic acid bacteria
    • A23V2400/11—Lactobacillus
    • A23V2400/147—Helveticus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/225—Lactobacillus

Definitions

  • the present invention relates to novel strains of lactic acid bacteria as well as isolated peptides and mixtures of peptides derived from b-casein and produced by these strains. These peptides are active either as ACE inhibitors or as an agent to promote calcium absorption.
  • the present also relates to a food or pharmaceutical composition as well as associated products.
  • Insufficient absorption of calcium can lead to various symptoms or pathologies such as osteoporosis, high blood pressure or metabolic syndrome. Indeed, calcium is involved whatever the age of the patient, in the formation of bones, the solidification of the skeleton, the rigidity of the teeth, muscle contraction and therefore cardiac contraction, the transmission of nerve impulses at the level synapses, learning and memorization, salivary secretion, cell growth and proliferation. Calcium activates many enzymes, releases several hormones, clots the blood, and helps balance body weight.
  • hypocalcemia is also linked to various rare pathologies such as autoimmune hypoparathyroidism, vitamin-D dependent hypocalcemic rickets, progressive bone heteroplasia, isolated hypoparathyroidism, syndromic hypoparathyroidism, pseudo-hypoparathyroidism and hypo-rachitias. more generally diseases caused by an abnormality in the regulation of calcium levels due to the dysfunction of the parathyroid hormone, the absorption of vitamin D or the functioning of the calcium receptor.
  • An aim of the present invention is to provide a strain of lactic acid bacterium resistant to gastrointestinal digestion and capable of acting in the intestine by acidifying the medium, thus making the calcium soluble, in order to facilitate its absorption and its passage to through the intestinal wall.
  • Another object of the present invention is to provide a strain of lactic acid bacteria resistant to gastrointestinal digestion and capable of stimulating the absorption of calcium by the intestinal cells, by acting directly or indirectly on the latter.
  • Another aim of the present invention is to provide a strain of lactic acid bacteria which has greater growth than certain known lactic acid bacteria and / or which allows greater acidification of the fermentation medium, in particular of milk.
  • Another aim of the present invention is to provide a strain of lactic acid bacteria capable of producing new phosphorylated peptides capable of promoting the absorption of calcium in the intestine.
  • Another aim of the present invention is to provide at least one peptide, or even a mixture of peptides, which exhibits an ACE inhibitory activity which is greater than the activity of the IPP and VPP peptides.
  • Another aim of the present invention is to provide a pharmaceutical or food composition capable of treating hypocalcemia and therefore the pathologies which result therefrom.
  • the invention relates to a strain of Lactobacillus helveticus exhibiting the prtH2 and prtH3 genes and capable of reducing, by anaerobic fermentation, the pH of milk, in particular of cows and in particular of skimmed cow's milk to a value substantially equal to or less than 3.36, especially equal to 3.32 after 48 hours of fermentation at 37 ° C.
  • the strain allows the dissolution of compounds containing calcium, making the latter absorbable through the intestinal wall.
  • the present invention also relates to the strain of Lactobacillus helveticus VF45A deposited at the CNCM under the order number CNCM-I-5300, its mutants and variants having at least 80% identity, preferably at least 90%. % identity, preferably at least 95% identity with the genome of said strain VF45A and capable of producing at least one peptide corresponding to the sequences SEQ ID 1 to SEQ ID NO 43 and / or capable of reducing by anaerobic fermentation the pH of the milk, in particular cow's milk and in particular skimmed cow's milk at a value substantially equal to or less than 3.36, in particular equal to 3.32 after 48 hours of fermentation at 37 ° C.
  • the invention also relates to the aforementioned strains with reference to the first aspect for their use as a medicament and in particular as a medicament for the treatment of hypocalcemia or in the treatment of a pathology caused by a deficiency.
  • the present invention also relates to an isolated peptide which can be obtained by fermentation of milk, in particular of cow's milk, by at least one strain according to the first aspect of the invention and chosen from the peptides corresponding to the sequences SEQ ID 1 to SEQ ID NO 43, peptides exhibiting at least 80% identity, in particular at least 90% identity, in particular at least 95% identity with said peptides corresponding to sequences SEQ ID 1 to SEQ ID NO 19 and capable of inhibiting ACE and peptides exhibiting at least 80% identity, in particular at least 90% identity, in particular at least 95% identity with said peptides corresponding to the sequences SEQ ID 20 to SEQ ID NO 43 and capable of increasing the intestinal absorption of calcium.
  • the Applicant has in fact demonstrated either the ACE inhibitory activity of the aforementioned peptides - which activity is also linked to good bone health - or the activity of improving the absorption of calcium through the intestinal wall. due to some of the above peptides.
  • the peptides of the invention act on bone health via the absorption of calcium through the intestinal wall, and / or via the inhibition of ACE.
  • the present invention also relates to a mixture of peptides chosen from the mixtures comprising or consisting of all peptides of sequence SEQ ID NO 1 to SEQ ID NO 43, mixtures comprising or consisting of said peptides of sequence SEQ ID NO 1 to SEQ ID NO 5, the mixtures comprising or consisting of 5 peptides of sequence SEQ ID NO 1 to SEQ ID NO 5 and at least one peptide chosen from the peptides corresponding to the sequences SEQ ID 20 to SEQ ID NO 43, preferably mixtures containing or consisting of 5 peptides of sequence SEQ ID NO 1 to SEQ ID NO 5 and peptides of sequences SEQ ID 20 to SEQ ID NO 43 and mixtures comprising or consisting of peptides of sequence SEQ ID 20 to SEQ ID 43.
  • the present invention also relates to the aforementioned isolated peptides and the aforementioned mixtures for their use as a medicament and in particular as a medicament in the treatment of hypocalcemia or in the treatment of a pathology caused by a deficiency in calcium absorption.
  • hypocalcaemia induced by a pathology chosen from autoimmune hypoparathyroidism, vitamin-D dependent hypocalcemic rickets, progressive bone heteroplasia, isolated hypoparathyroidism, syndromic hypoparathyroidism, pseudo-hypoparathyroidism and hypo-deficiency rickets, diseases caused by an abnormality in the regulation of calcium levels due to dysfunction of the parathyroid hormone, absorption of the vitamin D or the functioning of the calcium receptor, in a subject, in particular a mammal and particularly a human having such a need.
  • the present invention also relates to a food or pharmaceutical composition which contains at least one pharmaceutically acceptable excipient or a medium suitable for ingestion and as active ingredient at least one peptide chosen from the aforementioned isolated peptides with reference to first aspect and / or a mixture of peptides as mentioned above with reference to the first aspect and / or a strain according to the first aspect of the invention and in particular the VF45A strain.
  • the food or pharmaceutical composition comprises, at least or only as active peptides, the 5 peptides corresponding to the sequences SEQ ID NO 1 to SEQ ID NO 5.
  • active peptides are the most active as ACE inhibitors. They are more active than the tripeptides VPP and IPP of the prior art.
  • the food or pharmaceutical composition according to the first aspect of the invention contains as active ingredient or consists of the fermentate obtained by anaerobic fermentation of possibly skimmed cow's milk by means of 'a strain according to the first aspect of the invention, said fermentate having optionally undergone ultrafiltration at a cut-off threshold of 10 kDa or it contains or consists of the fermentate obtained by anaerobic fermentation and optionally after ultrafiltration at a cut-off threshold of 10 kDa a protein substrate chosen from protein substrates of plant origin originating from at least one cereal and / or at least one pea and / or at least one fungus and / or at least one tree nut , mixtures of at least two protein substrates of plant origin, milks of animal origin optionally thermally sterilized, and / or at least partially skimmed, in particular the cow, goat or sheep milk with the exception of yak milk for the non-ultrafiltered fermentate at a cut-off threshold of 10kDa,
  • the fermentate as mentioned above obtained by anaerobic fermentation and ultrafiltration at a cut-off threshold of 10 kDa is part of the fermentates according to the first aspect of the invention, including the ultrafiltered fermentate obtained from yak milk.
  • the fermentate obtained from cow's milk it is advantageously obtained according to the temperature and duration conditions indicated in the part relating to the experimental results.
  • the protein substrate of plant origin can be chosen from vegetable milks such as oat milk, almond milk, rice milk, soy milk, spelled milk, hazelnut milk and mixtures of these milks. Whatever the milk, it can be thermally sterilized before fermentation by the bacteria according to the invention.
  • the food or pharmaceutical composition contains as an active ingredient the peptide (s) and / or the strain. It can also, advantageously also comprise calcium and / or vitamin D.
  • performing ultrafiltration on the fermentate separates the peptides produced by the strain from the proteins.
  • the ultrafiltered fermentate therefore no longer contains proteins.
  • the filtrate obtained by ultrafiltration of the fermentate is found to be more active when it is ingested and digested due to the absence of interference by the peptides resulting from the digestion of the proteins.
  • the food or pharmaceutical composition according to the first aspect of the invention contains a concentration of at least one of said strains and preferably of one of said strains, in particular the Lactobacillus helveticus VF45A strain equal to or greater at 10 7 CFU per gram and / or a concentration of peptides which are identical or different and chosen from peptides of sequence SEQ ID NO 1 to SEQ ID NO 43, greater than or equal to 18 mg per gram.
  • the peptides are the peptides contained in the aforementioned fermentate with reference to the first aspect and in particular the peptides of the sequences SEQ ID NO 1 to SEQ ID No 43.
  • the present invention also relates to a product chosen in particular from food products, in particular fermented milks, juices of fruit (s) and / or of optionally fermented vegetable (s), vegetables and / or fermented fruits, fermented mushrooms, cold meats, food preparations based on fish (s) and dairy products obtained from possibly skimmed milk, in particular from cow, goat, sheep and with the exception of yak milk, or of a mixture of milks.
  • food products in particular fermented milks, juices of fruit (s) and / or of optionally fermented vegetable (s), vegetables and / or fermented fruits, fermented mushrooms, cold meats, food preparations based on fish (s) and dairy products obtained from possibly skimmed milk, in particular from cow, goat, sheep and with the exception of yak milk, or of a mixture of milks.
  • this product contains at least one strain according to the first aspect and / or at least one peptide according to the first aspect and / or at least one mixture of peptides according to the first aspect of the invention and / or or a food or pharmaceutical composition according to the first aspect of the invention.
  • the invention also relates to a food supplement suitable for a subject, in particular a human or an animal in particular chosen from cats, dogs, poultry, sheep, goats, cattle, reptiles, in particular.
  • a food supplement suitable for a subject, in particular a human or an animal in particular chosen from cats, dogs, poultry, sheep, goats, cattle, reptiles, in particular.
  • iguana which characteristically comprises at least one strain according to the first aspect and / or at least one peptide according to the first aspect and / or at least one mixture of peptides according to the first aspect and / or a food or pharmaceutical composition according to the first aspect.
  • the food / pharmaceutical composition or the food supplement can be in the form of a powder, of a solution in particular aqueous, of a tablet, of a capsule containing the strain under lyophilized form and / or the peptide or mixture of peptides in powder or solution form.
  • the capsule may have an outer shell capable of withstanding gastrointestinal digestion so as to deliver its contents into the patient's intestine.
  • the strain can also be, for example, microencapsulated with a lipid coating and be in the form of an ingestible powder.
  • the tablet, the capsule or any other unit form of daily dosage preferably contains at least 0.6 g of active peptides and / or 10 7 CFU of strain according to the first appearance, possibly in a mixture.
  • the pharmaceutical composition may preferably be adapted for oral administration.
  • the dairy product is not limited according to the invention. It can be a milk drink, fermented milk, kefir, yogurt, a product based on milk coagulated by adding acid or a fresh or yeast cheese, for example.
  • the present invention relates to a strain of Lactobacillus helveticus exhibiting the prtH1 gene and capable of reducing by anaerobic fermentation the pH of milk, in particular of cow's milk, to a value substantially equal to or less than 3.45 and in particular equal to 3, 43 after 48 hours at 37 ° C and exhibiting a parietal hydrophobicity of 40% and resistant to acidity and / or capable of increasing the absorption of calcium by the intestinal cells.
  • the present invention relates to the strain of Lactobacillus helveticus VFH049 deposited at the CNCM under the order number CNCM-l-5403 and its mutants and variants having at least 80% identity, preferably at least 90%. identity, preferably at least 95% identity with the genome of said strain VFH049 and capable of producing at least one peptide corresponding to the sequences SEQ ID 44 to SEQ ID NO 86 and / or capable of reducing the pH by anaerobic fermentation milk, in particular from cow's milk, at a value substantially equal to or less than 3.45 and in particular equal to 3.43 after 48 hours at 37 ° C. and / or capable of increasing the absorption of calcium by the intestinal cells.
  • This strain has been shown to be able to form acids which may make calcium soluble in the gut of a host / patient. It is also able to produce, by proteolysis of b-casein, peptides active as an ACE inhibitor or allowing better intestinal absorption of calcium. The strain itself resists digestion and, when in contact with the intestinal cells, allows better absorption of calcium by the latter.
  • the present invention also relates to the strains cited with reference to the second aspect and in particular the strain VFH049 or its mutants or variants for its use as a medicament and in particular as a medicament in the treatment of hypocalcemia or in the treatment of a pathology caused by a deficiency in calcium absorption and in particular chosen from arterial hypertension, metabolic syndrome, osteoporosis or in the treatment of hypocalcemia induced by a pathology chosen from autoimmune hypoparathyroidism , vitamin-D dependent hypocalcemic rickets, progressive bone heteroplasia, isolated hypoparathyroidism, syndromic hypoparathyroidism, pseudo-hypoparathyroidism and hypo-deficiency rickets, diseases caused by an abnormality in the regulation of calcium levels due to the dysfunction of the parathyroid hormone, absorption of vitamin D or the functioning of the calcium receptor, in a subject, especially a mammal and particularly a human having such a need.
  • the present invention also relates to an isolated peptide which can be obtained by fermentation of milk, in particular cow's milk, by at least one strain according to the second aspect of the invention and chosen from the peptides corresponding to the sequences SEQ ID 44 to SEQ ID NO 86, the peptides having at least 80% identity, in particular at least 90% identity, in particular at least 95% identity with said peptides corresponding to the sequences SEQ ID 44 to SEQ ID NO 74 and capable of inhibiting ACE and peptides exhibiting at least 80% identity, in particular at least 90% identity, in particular at least 95% identity with said peptides corresponding to the sequences SEQ ID 75 to SEQ ID NO 86 and capable of increasing intestinal absorption of calcium.
  • the invention also relates to a mixture of peptides chosen from mixtures of peptides containing or consisting of all the peptides of sequence SEQ ID 44 to SEQ ID 86, the mixtures comprising or consisting of peptides of sequence SEQ ID NO 44 to SEQ ID NO 58, the mixtures consisting of peptides of sequence SEQ ID NO 44 to SEQ ID NO 58 and of at least one of the peptides of sequence SEQ ID NO 75 to SEQ ID NO 86, mixtures comprising or consisting of peptides of sequence SEQ ID NO 44 to SEQ ID NO 58 and peptides of sequence SEQ ID NO 75 to SEQ ID NO 86 and mixtures containing or consisting of peptides of sequence SEQ ID NO 75 to SEQ ID NO 86.
  • the invention also relates to an isolated peptide of the second aspect or the mixture according to the second aspect for its use as a medicament and in particular as a medicament in the treatment of hypocalcemia or of a pathology caused by a calcium absorption deficiency and in particular chosen from arterial hypertension, metabolic syndrome, osteoporosis or for the treatment of hypocalcemia induced by a pathology chosen from autoimmune hypoparathyroidism, hypocalcemic rickets vitamin-D dependent, progressive bone heteroplasia, isolated hypoparathyroidism, syndromic hypoparathyroidism, pseudo-hypoparathyroidism and hypo-deficiency rickets, diseases caused by an abnormality in the regulation of calcium levels due to dysfunction of the parathormone, absorption of vitamin D, or calcium receptor function, in a subject, especially a mammal, particularly a human having such a need.
  • the present invention also relates to a food or pharmaceutical composition which contains at least one pharmaceutically acceptable excipient or at least one medium capable of being ingested and, as active ingredient, at least one peptide corresponding to the peptides isolated according to the second aspect of the invention, and / or a mixture of peptides according to the second aspect of the invention, and / or a strain according to the second aspect of the invention, in particular the strain VFH049.
  • the food or pharmaceutical composition contains a mixture containing all the peptides of sequences SEQ ID NO 44 to SEQ ID NO 86 or a mixture containing at least or only the peptides corresponding to the sequences SEQ ID NO 44 to SEQ ID NO 58 and optionally peptides of sequence SEQ ID NO 75 to SEQ ID NO 86.
  • the food or pharmaceutical composition contains or consists of the fermentate obtained by anaerobic fermentation of possibly skimmed cow's milk by means of a strain according to the second aspect of the invention, said fermentate having optionally undergone ultrafiltration at a cut-off threshold of 10 kDa or contains or consists of the fermentate obtained by anaerobic fermentation and optionally after ultrafiltration at a cut-off threshold of 10 kDa of a protein substrate chosen from protein substrates of plant origin originating from at least one cereal and / or at least one pea and / or at least one fungus and / or at least one tree nut, including the almond, mixtures of at least two protein substrates of plant origin, milks of '' animal origin optionally thermally sterilized and / or at least partially skimmed, in particular cow's, goat's or sheep's milk with the exception of mare's milk for the non-ultrafil fermentate at a cut-off point of 10kDa, mixtures of at least two milks
  • This fermentate contains all the peptides of the second aspect of the invention; it is therefore active in promoting bone health, both as an ACE inhibitor and as an agent promoting the intestinal absorption of calcium.
  • the fermentate itself can be considered as a pharmaceutical or food composition.
  • the aforementioned food or pharmaceutical composition preferably contains a concentration of at least one of said strains and preferably one of said strains, in particular the strain Lactobacillus helveticus VFH049 equal to or greater than 10 7 CFU per gram and / or a concentration of peptides which are identical or different and chosen from peptides of sequence SEQ ID NO 44 to SEQ ID NO 86, greater than or equal to 6 mg per gram.
  • the invention also relates to a product chosen in particular from food products, in particular fermented milk, fruit juice (s) and / or vegetable juice (s) optionally fermented, vegetables and / or fermented fruits, fermented mushrooms, cold meats, food preparations based on fish (s) and dairy products obtained from possibly skimmed milk, with the exception of mare's milk, in particular from cow's milk, goat's milk, sheep or a mixture of milk, which contains at least one strain according to the second aspect of the invention and / or at least one peptide according to the second aspect of the invention and / or at least one mixture of peptides according to second aspect of the invention and / or a food or pharmaceutical composition according to the second aspect of the invention.
  • food products in particular fermented milk, fruit juice (s) and / or vegetable juice (s) optionally fermented, vegetables and / or fermented fruits, fermented mushrooms, cold meats, food preparations based on fish (s) and dairy products obtained from possibly skimmed milk
  • the invention also relates to a food supplement suitable for a subject, in particular a human or an animal in particular chosen from cats, dogs, poultry, sheep, goats, cattle, reptiles, in particular.
  • a food supplement suitable for a subject, in particular a human or an animal in particular chosen from cats, dogs, poultry, sheep, goats, cattle, reptiles, in particular.
  • the iguana which characteristically comprises at least one strain according to the second aspect of the invention and / or at least one peptide according to the second aspect of the invention and / or at least one mixture of peptides according to the second aspect of the invention the invention and / or a food or pharmaceutical composition according to the second aspect of the invention.
  • the pharmaceutical or food composition is in the form of a liquid, in particular of the aforementioned cow's milk fermentate.
  • a volume of 30 mL of this solution containing 0.2 g of the mixture of peptides of the invention is administered daily orally.
  • the present invention relates to a strain of Lactobacillus delbrueckii ssp. bulgaricus exhibiting the prtB gene and capable of reducing by anaerobic fermentation the pH of milk, in particular from cows, to a value substantially equal to or less than 4.55 and in particular equal to 4.51 after 48 hours at 37 ° C and exhibiting a wall hydrophobicity of at least 35% and / or capable of increasing calcium absorption.
  • One such strain has been shown to be active in the gut and resistant to gastrointestinal digestion. It is able to increase the passage of calcium through the intestinal wall. The acids it produces also help to dissolve calcium.
  • the invention relates to the strain of Lactobacillus delbrueckii ssp. bulgaricus VF50b deposited with the CNCM under the order number CNCM- 1-5316 and its mutants and variants having at least 80% identity, preferably at least 90% identity, preferably at least 95% identity with the genome of said strain VF50b and capable of producing at least one peptide corresponding to the sequences SEQ ID 87 to SEQ ID NO 199 and / or capable of reducing by anaerobic fermentation the pH of milk, in particular from cows, to a value substantially equal to or lower at 4.55 and in particular equal to 4.51 after 48 hours at 37 ° C and / or capable of increasing the intestinal absorption of calcium.
  • the third aspect also relates to each of the strains as mentioned above for its use as a medicament, and in particular as a medicament in the treatment of hypocalcemia or in the treatment of a pathology caused by a deficiency in calcium absorption. and in particular chosen from arterial hypertension, metabolic syndrome, osteoporosis or in the treatment of hypocalcaemia induced by a pathology chosen from autoimmune hypoparathyroidism, vitamin-D dependent hypocalcemic rickets, progressive bone heteroplasia, isolated hypoparathyroidism, syndromic hypoparathyroidism, pseudo-hypoparathyroidism and hypo-deficiency rickets, diseases caused by an abnormality in the regulation of calcium levels due to the dysfunction of the parathyroid hormone, absorption of vitamin D or the functioning of the calcium receptor, in a subject, especially a mammal and particularly a human having such a need.
  • the present invention relates to an isolated peptide chosen from the peptides corresponding to the sequences SEQ ID 87 to SEQ ID NO 199, the peptides having at least 80% identity, in particular at least 90% identity, in particular at least 95% identity with said peptides corresponding to sequences SEQ ID 87 to SEQ ID NO 161 and capable of inhibiting ACE and peptides exhibiting at least 80% identity, in particular at least 90% identity, in particular at least 95% identity with said peptides corresponding to the sequences SEQ ID 162 to SEQ ID NO 199 and capable of increasing the intestinal absorption of calcium.
  • the invention also relates to a mixture of peptides chosen from mixtures containing or consisting of all peptides of sequence SEQ ID NO 87 to SEQ ID 199, mixtures containing or consisting of peptides of sequence SEQ ID 87 to SEQ ID 1 12, mixtures containing or consisting of peptides of sequence SEQ ID 87 to SEQ ID 1 12 and at least one of peptides of sequence SEQ ID NO 162 to SEQ ID NO 199 and mixtures containing or consisting of peptides of sequence SEQ ID NO 162 to SEQ ID NO 199.
  • the invention also relates to an isolated peptide cited with reference to the third aspect or to a mixture mentioned above with reference to the third aspect for its use as a medicament and in particular in the treatment in the treatment of hypocalcemia or of a pathology caused by a deficiency in calcium absorption and in particular chosen from arterial hypertension, metabolic syndrome, osteoporosis or for the treatment of hypocalcemia induced by a pathology chosen from autoimmune hypoparathyroidism, hypocalcemic rickets vitamin-D dependent, progressive bone heteroplasia, isolated hypoparathyroidism, syndromic hypoparathyroidism, pseudo-hypoparathyroidism and hypo-deficiency rickets, diseases caused by an abnormality in the regulation of calcium levels due to dysfunction of the parathormone, '' absorption of vitamin D or the functioning of the calcium, in a subject, in particular a mammal, in particular a human having such a need.
  • the invention also relates to a food or pharmaceutical composition which contains at least one pharmaceutically acceptable excipient or at least one medium capable of being ingested and, as active ingredient, at least one peptide isolated according to the third aspect of the invention and / or a mixture of peptides according to the third aspect.
  • the composition contains a mixture containing at least or only as peptides, the peptides corresponding to the sequences SEQ ID NO 87 to SEQ ID NO 161, or a mixture preferably containing all the peptides corresponding to the sequences SEQ ID 87 to SEQ ID NO 199 and / or a strain according to the third aspect.
  • the pharmaceutical or food composition according to the third aspect of the invention comprises or is constituted by the fermentate obtained by anaerobic fermentation of possibly skimmed cow's milk by means of a strain according to the third aspect of the invention, said fermentate having optionally undergone ultrafiltration at a cut-off threshold of 10 kDa or it contains or consists of the fermentate obtained by anaerobic fermentation and optionally after ultrafiltration at a cut-off threshold of 10 kDa of a protein substrate chosen from among the following: protein substrates of plant origin originating from at least one cereal and / or at least one pea and / or at least one fungus and / or at least one tree nut, mixtures of at least two protein substrates of plant origin, possibly thermally sterilized and / or at least partially skimmed milk, in particular cow's, goat or sheep's milk, with the exception of milk from e yak for said fermentate not ultrafiltered at a cut-off threshold of 10kDa, mixtures of at
  • the fermentate obtained by anaerobic fermentation of yak milk by a strain according to the first or the third aspect of the invention and ultrafiltered at a cutoff threshold of 10 kDa is respectively part of the fermentates according to the first and the third aspect of the invention.
  • the fermentate obtained by anaerobic fermentation of mare's milk and ultrafiltered at a cut-off threshold of 10 kDa is part of the fermentates according to the second aspect of the invention.
  • said food or pharmaceutical composition according to the third aspect of the invention contains a concentration of at least one of said strains and preferably only one of said strains, in particular the strain Lactobacillus delbrueckii ssp.
  • bulgaricus VF50b equal to or greater than 10 7 CFU per gram and / or a concentration of peptides which are identical or different and chosen from peptides of sequence SEQ ID NO 87 to SEQ ID NO 199, greater than or equal to 4 mg per gram.
  • the present invention also relates to a product chosen in particular from food products, in particular fermented milks, juices of fruit (s) and / or vegetable (s) optionally fermented, vegetables and / or fermented fruits, fermented mushrooms, cold meats, food preparations based on fish (s) and dairy products obtained from possibly skimmed milk, in particular from cow, goat, sheep with the exception of yak milk, or from a mixture of milks, which characteristically contains at least one strain according to the third aspect of the invention and / or at least one peptide according to the third aspect and / or at least one mixture of peptides according to the third aspect and / or one food or pharmaceutical composition according to the third aspect of the invention.
  • food products in particular fermented milks, juices of fruit (s) and / or vegetable (s) optionally fermented, vegetables and / or fermented fruits, fermented mushrooms, cold meats, food preparations based on fish (s) and dairy products obtained from possibly skimmed milk, in particular from
  • the present invention also relates to a food supplement suitable for a subject, in particular a human or an animal in particular chosen from cats, dogs, poultry, sheep, goats, cattle, reptiles, in particular.
  • the iguana which characteristically comprises at least one strain according to the third aspect and / or at least one peptide according to the third aspect and / or at least one mixture of peptides according to the third aspect and / or a food or pharmaceutical composition according to the third aspect of the invention.
  • the food or pharmaceutical composition is in the form of a liquid. A volume of 30 ml of this composition containing 0.2 g of peptides are administered daily, orally.
  • the present invention relates to a mixture of at least two strains chosen from the strains of Lactobacillus helveticus exhibiting the prtH2 and p rtH3 genes and capable of reducing the pH of milk, in particular cow's milk, by anaerobic fermentation.
  • bulgaricus exhibiting the prtB gene in particular the strain of Lactobacillus delbrueckii ssp. bulgaricus VF50b deposited with the CNCM under the order number CNCM-l-5316, capable of reducing by anaerobic fermentation the pH of milk, in particular of cow's milk to a value substantially equal to or less than 4.55 and in particular equal to 4.51 after 48 hours at 37 ° C and exhibiting a parietal hydrophobicity of at least 35% and / or capable of increasing the absorption of calcium by the intestinal cells and in particular the mixtures of the three strains VF45A, VFH049 and VF50b.
  • the invention relates to a mixture of peptides chosen from mixtures comprising peptides of sequences SEQ ID NO 1 to SEQ ID NO 199, mixtures containing peptides of sequence SEQ ID NO 1 to SEQ ID NO 5, peptides of sequence SEQ ID NO 44 to SEQ ID NO 86 and peptides of sequence SEQ ID NO 87 to SEQ ID NO 1 12.
  • the invention also relates to an isolated peptide chosen from the peptides of sequences SEQ ID NO 1 to SEQ ID NO 199 or a mixture according to the fourth aspect of the invention for its use as a medicament and in particular in the treatment of hypocalcemia or in the treatment of a pathology caused by a deficiency in calcium absorption and in particular chosen from arterial hypertension, metabolic syndrome, osteoporosis or in the treatment of hypocalcemia induced by a pathology chosen from autoimmune hypoparathyroidism, vitamin-D hypocalcemic rickets dependent, progressive bone heteroplasia, isolated hypoparathyroidism, syndromic hypoparathyroidism, pseudo-hypoparathyroidism and hypo-deficiency rickets, diseases caused by an abnormality in the regulation of calcium levels due to dysfunction of the parathyroid hormone, absorption of vitamin D or how the calcium receptor works.
  • the invention relates to a food or pharmaceutical composition which contains a pharmaceutically acceptable excipient or a medium suitable for ingestion and at least one peptide as mentioned above or a mixture of peptides according to the fourth aspect of the invention and / or a mixture of strains according to the fourth aspect of the invention.
  • the invention also relates to a product chosen from food products, in particular fermented milks, fruit juice (s) and / or vegetable juice (s) optionally fermented, vegetables and / or fermented fruits, fermented mushrooms, cold meats, food preparations based on fish (s) and dairy products obtained from possibly skimmed milk, cow, goat, sheep or a mixture of milk which contains a mixture of strains according to the fourth aspect of the invention and / or at least one mixture of peptides according to the fourth aspect of the invention and / or a food or pharmaceutical composition according to the fourth aspect of the invention.
  • a product chosen from food products in particular fermented milks, fruit juice (s) and / or vegetable juice (s) optionally fermented, vegetables and / or fermented fruits, fermented mushrooms, cold meats, food preparations based on fish (s) and dairy products obtained from possibly skimmed milk, cow, goat, sheep or a mixture of milk which contains a mixture of strains according to the fourth aspect of the invention and
  • the food or pharmaceutical composition contains a concentration of said strains equal to or greater than 10 7 CFU per gram of product and / or a concentration of peptides which are identical or different greater than or equal to 4 mg per gram.
  • milk in the absence of precision, refers to cow's milk.
  • yogurt refers to a dairy product produced by fermentation by adding lactic acid bacteria Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus.
  • milk refers to a dairy product obtained by coagulation of caseins following the addition of a lactic acid ferment to milk.
  • fertilizate designates the supernatant obtained at the end of the fermentation.
  • increase calcium absorption refers to the ability of the strain or peptides to increase the overall passage of calcium through the intestinal wall of a mammal (between cells of the latter due to increased expression of the cld-2 gene, for example), or to increase fluorescence in Caco-2 cells as indicated in the experimental part (greater passage of calcium through the cells of the intestinal wall) or to increase the expression of at least one gene chosen from trpv6, and vdr in intestinal cells as indicated in the experimental part of the present application (better absorption by the cells themselves).
  • the terms “acid resistant” refer to class 3 indicated in the experimental part of this application with reference to the acidity resistance.
  • the term “probiotic” refers to a microorganism capable of surviving in the intestine of a host, especially a human, and of interacting with the cells of the intestinal wall and / or of interacting with other microorganisms living in it. intestine of the host and exert a beneficial effect on the health of the host.
  • hypocalcaemia refers to a concentration of calcium in the blood, corrected with the level of albumin less than 2.20 mmol / L.
  • active ingredient means that the ingredient considered (peptide, mixture of peptides and / or strain) is contained in a quantity / concentration making it possible to obtain a technical effect at least in vitro on the inhibition of ACE and / or on calcium absorption.
  • subject refers to a human or an animal, especially a mammal (in particular, a dog, a cat, a poultry, a sheep, a bovine, a goat or a reptile).
  • treatment encompasses both preventive (prophylactic) treatment and treatment allowing improvement of at least one symptom of the pathology in question.
  • Percent identity refers to the percentage of identical amino acid residues between the sequence to be compared and the reference sequence, aligning the entire sequence compared to the reference sequence.
  • the protein sequence which has a% identity with the reference sequence can thus comprise insertions or deletions.
  • the percent identity is calculated by determining the number of positions at which an identical amino acid residue is observed for the two sequences compared, then dividing the number of positions for which there is identity between the two nucleic bases, or between the two amino acid residues, by the total number of positions in the comparison window, then by multiplying the result by one hundred in order to obtain the percentage identity in nucleotides or in amino acids of the two sequences between them.
  • the peptides defined by their% identity can thus comprise one or more deletions and / or one or more substitutions, the insertion or the substitution with a dextrorotatory amino acid, the replacement of the terminal carboxylic acid group and / or of the terminal amine group.
  • a protective group for example, the introduction of a retro-type bond (that is to say a peptide bond -NH-CO- resulting from the reaction between the amine function of the first amino acid in the sequence with the carboxylic acid function of the next amino acid in the same sequence) or the introduction of a retro-inverso type bond (that is to say the introduction of a peptide bond of the aforementioned retro type with a amino acid in the reverse configuration than that of the amino acid in the reference sequence) or have a longer chain of amino acids.
  • a retro-type bond that is to say a peptide bond -NH-CO- resulting from the reaction between the amine function of the first amino acid in the sequence with the carboxylic acid function of the next amino acid in the same sequence
  • a retro-inverso type bond that is to say the introduction of a peptide bond of the aforementioned retro type with a amino acid in the reverse configuration than that of the amino acid in the reference sequence
  • peptides may or may not be phosphorylated.
  • a pharmaceutical composition is defined as being a composition containing an active ingredient and a pharmaceutically acceptable excipient and in particular chosen from water, oils, alcohols, in particular ethyl alcohol, vegetable oils, cyclodextrins, starch, maltodextrin, lactose, sucrose, propylene glycol and physiological saline.
  • peptide when it is used with reference to one or more peptides indicates that the mixture or the composition contains as peptide (s) only the peptides mentioned with the exception of other peptides but other compounds or constituents. such as solvents, vitamins, proteins or the like may be present.
  • a "suitable for ingestion medium” means any mixture or substance regardless of its physical phase which can be ingested and digested without causing any problem to the digestive tract.
  • the pharmaceutically acceptable excipients and in particular the examples cited in the present application are media capable of being ingested. Throughout the present application, the sequences are conventionally indicated in the direction of their N terminal end towards their C-terminal end.
  • strain VFH049 can also be called VF49d.
  • strain refers without distinction to the living strain, that is to say capable of developing in a suitable nutrient medium and to the inactivated strain, that is to say dead. and therefore unable to develop in an environment suitable for its development.
  • FIG. 1 shows electron microscopic photographs of each of the three strains of the invention; the leftmost photograph represents the VF45A strain, the middle one the VFH049 strain and the right one the VF50b strain.
  • FIG. 2 represents the map obtained by PCR which evaluates the correlations between the proteolytic activity on agar-milk, the growth during a fermentation in liquid medium (milk), the pH at the end of the fermentation, the quantity of peptides produced and 5 other criteria linked to the analysis of the chromatographic profiles of the peptides produced by the strains (distribution in apparent molecular weights);
  • FIG. 3 represents the IC 50 in mg / mL for the ACE inhibitory activity of the control and of the crude fermentates obtained by fermentations of the strains VF45A and VFH049;
  • FIG. 4 represents the IC 50 in mg / mL for the ACE inhibitory activity of the control and of the fermentates obtained by fermentations of the strains VF45A and VFH049 and having undergone an ultrafiltration;
  • FIG. 5 represents the calcium absorption rate (ratio of fluorescence emission / basal emission) of the control and crude fermentates obtained by fermentations of strains VF45A and VFH049;
  • FIG. 6 represents the calcium absorption rate (ratio of fluorescence emission / basal emission) of the control and of the fermentates obtained by fermentations of strains VF45A and VFH049 and having undergone ultrafiltration;
  • FIG. 7 represents the level of mRNA of the trpv6 gene relative to that of the PBS condition of the crude fermentates obtained by fermentations of the VF45A and VFH049 strains
  • FIG. 8 represents the level of mRNA of the trpv6 gene relative to that of the PBS condition of the fermentates obtained by fermentations of the VF45A and VFH049 strains and having undergone an ultrafiltration
  • FIG. 9 represents the percentages of wall hydrophobia obtained for the strains tested according to their acid tolerance class.
  • FIG. 10 represents the level of mRNA relative to the control of the vdr, trpv6 and cld-2 genes obtained after 24 hours of contact with the VFH049 strain and the VF50b strain with HT-29 MTX cells.
  • the three strains of the invention were extracted from different dairy products.
  • Samples of each dairy product are collected in sterile tubes, kept at 4 ° C for a maximum of two days before analysis.
  • the samples are subjected to a series of dilutions in saline buffer (0.85% NaCl solution), then spread on MRS-agar plates (De Man, Rogosa and Sharpe).
  • the plates are incubated for 48 hours at 37 ° C. in an anaerobic medium.
  • the morphologically distinct colonies are separated, subcultured onto plates of the same medium for purification. In order to ensure the purity of the isolates, the re-plating is repeated at least three times.
  • Table 1 below shows the dairy products from which the strains were extracted. All of these dairy products come from Mongolia and were prepared in the fall. Optical microscope observations are made to determine the shape of bacteria. These observations are visible in Fig. 1.
  • the Gram of each strain is determined in a conventional manner by the Gram staining technique and observation under a microscope.
  • the test for catalase is carried out for each strain in a conventional manner by removing each strain from an agar medium and bringing it into contact with hydrogen peroxide.
  • the Lactobacillus helveticus VF45A strain was deposited by the Applicant on March 29, 2018 with the CNCM (National Collection of Cultures of Microorganisms), at 25 rue du Dondel ROUX 75724 Paris Cedex 15 under the order number CNCM-l- 5300 (deposit according to the Budapest Treaty).
  • the Lactobacillus helveticus VFH049 strain was deposited by the Applicant on February 19, 2019 with the CNCM (National Collection of Cultures of Microorganisms), at 25 rue du Dondel ROUX 75724 Paris Cedex 15 under the order number CNCM-l- 5403 (deposit according to the Budapest Treaty). In the remainder of the application, this strain is also called VF49d.
  • strain Lactobacillus delbrueckii ssp. bulgaricus VF50b was deposited by the Applicant on April 20, 2018 with the CNCM (National Collection of Cultures of Microorganisms), at 25 rue du Dondel ROUX 75724 Paris Cedex 15 under the order number CNCM-l-5316 (deposit according to the Budapest Treaty).
  • VF45A and VFH049 strains are indeed strains of the species Lactobacillus helveticus.
  • VF50b strain is indeed a strain of the species Lactobacillus. delbrueckii ssp. bulgaricus.
  • the identification of the parietal proteases present in the selected strains is carried out by PCR detection of the genes encoding these enzymes.
  • the bacterial cells are recovered from the cultures in skimmed milk, the bacterial DNA is then isolated using a wizard genomic DNA purification kit (Promega, Madison, United States).
  • the CNRZ32 CIRM-BIA 103 strain of Lactobacillus helveticus (supplied by the International Center for Microbial Resources - Bacteria of Food Interest, CIRM-BIA, INRA, Rennes, France) was used as a control for the genes of Lactobacillus helveticus.
  • This strain (CNRZ32) is known to have the 4 prtH genes (prtH1 to prtH4).
  • Each strain is tested for 5 pairs of primers corresponding to 5 different genes, namely, prtB, prtH1, prtH2, prtH3 and prtH4.
  • the primers described by Hou et al., 2015 are used for the detection of prtB in Lactobacillus delbrueckii. Concerning the strains of Lactobacillus helveticus, the primers described by Genay et al., 2009 are used for the detection of the prtH1 and prtH2 genes and finally those described by Broadbent et al., 201 1 for the detection of prtH3 and prtH4.
  • the PCR reactions are carried out in a final volume of 25 mL comprising 12.5 mL of PCR Master Mix (2x) (ThermoFisher Scientific, Waltham, USA), 2 mL of each primer (12.5 pM), 2 mL of Bacterial DNA extracted (approximately 200 ng.mL -1 ) and 6.5 mL of H2O.
  • the PCR cycles are carried out in a thermocycler labcycler (SensoQuest, Gôttingen, Germany).
  • the results are shown in Table 2 below.
  • the prtB gene is detected for the VF50b strain of Lactobacillus delbrueckii but not in the VF45A and VFH049 strains of Lactobacillus helveticus.
  • the prtH1 gene is detected for Lactobacillus helveticus strain VFH049 and in the control.
  • other strains (not shown) were tested and it turns out that all Lactobacillus helveticus strains exhibiting the prtH1 gene were isolated from fermented mare's milk.
  • the prtH2 and prtH3 genes are detected in Lactobacillus helveticus strain VF45A and in the control.
  • the prtH4 gene is detected in the control, but not in the strains tested.
  • the tests used cover both fermentation capacity (speed of growth, acidification) and proteolytic activity (ability to hydrolyze proteins).
  • the two aspects are largely related, the ability of bacteria to grow depends on their ability to hydrolyze proteins (since they need the amino acids released during hydrolysis to ensure their growth). It is also already known that the acidification of the medium and the hydrolysis of proteins are correlated.
  • the strain is cultivated in MRS medium (Man, Rogosa, Sharpe agar) liquid for 48 h at 37 ° C under anaerobic condition.
  • MRS medium Man, Rogosa, Sharpe agar
  • the bacterial cells are then washed twice in MRS medium and then finally re-suspended in a volume of MRS medium making it possible to obtain an ODeoo equal to 1 (Prim spectrophotometer, Secomam, Groupe Aqualabo, Champigny, France).
  • a volume of 20 mL of the bacterial suspension is placed in the milk agar wells.
  • the inoculated plates are then incubated for 72 hours at 37 ° C. under anaerobic conditions.
  • the proteolytic activity of the strains is quantified by measuring the diameter (expressed in mm) of the halos appearing around the wells after incubation.
  • a pre-culture of 5 mL is carried out in a medium composed of 100% UHT skimmed milk (Cora, Paris, France), for 72 hours at 37 ° C, in anaerobic condition .
  • the strain is then inoculated into a final volume of 30 mL of the same medium, with an initial OD 600 of 0.3.
  • the cultures take place for 48 hours at 37 ° C. in anaerobic condition.
  • NI Milk uninoculated milk medium
  • the acidity of the medium is evaluated by measuring the pH with a pH meter (Mettler Toledo, G accommodatesee, Switzerland).
  • the peptides produced during the fermentation of the strains are analyzed by two methods.
  • the peptide concentrations are evaluated first by assaying with the Folin-Ciocalteu reagent (FC); peptides are also analyzed according to their apparent molecular weight distribution by size exclusion chromatography (SEC).
  • FC Folin-Ciocalteu reagent
  • SEC size exclusion chromatography
  • a 4 mL sample is taken and then mixed with a volume of 400 mL of a solution of trichloroacetic acid (TCA) at a concentration of 10% (m / v) to reach a final concentration in TCA of 1%.
  • TCA trichloroacetic acid
  • the addition of such a concentration of TCA allows the precipitation of high molecular weight proteins. After centrifugation at 10,000 rpm for 10 min, the supernatant containing the peptides is recovered.
  • the peptides are then purified from the supernatant in order to remove the TCA, the sugars, and the salts.
  • the purification is carried out by a solid phase extraction (SPE), the principle of this method is a separation of the compounds of a mixture by selective adsorption on a solid phase.
  • Bond Elut C 18 1000 mg
  • micro-columns Align Technologies, Santa Clara, USA
  • Bond Elut C 18 1000 mg
  • micro-columns Alent Technologies, Santa Clara, USA
  • these are columns composed of a solid phase of silica grafted with C 18 groups allowing the retention of hydrophobic compounds.
  • the columns are equilibrated by passing a minimum volume of 10 mL of a 100% acetonitrile (ACN) solution supplemented with 0.1% trifluoroacetic acid (TFA) (v / v). After rinsing the column with an H 2 O solution containing 0.1% TFA (v / v), a volume of 4 ml of supernatant containing the extracted peptides is then loaded onto the column. A new rinse with I ⁇ 2O supplemented with 0.1% TFA makes it possible to elute all compounds not retained by the column.
  • ACN acetonitrile
  • TFA trifluoroacetic acid
  • the peptides retained are finally eluted in a volume of 2 ml of an ACN solution at 80% (v / v), 20% H 2 O (v / v) and containing 0.1% TFA.
  • the samples are then stored at -20 ° C until analysis.
  • the extracted peptides are quantified by an assay with the Folin-Ciocalteu reagent (FC). It is a solution of sodium tungstate and molybdate prepared in phosphoric and hydrochloric acids. The complex of phosphotungstic acid and phosphomolybdic acid of yellow color is reduced by residues of tyrosine, tryptophan, cysteine or by peptide bonds in an alkaline medium to give a blue color. The appearance of the blue color, proportional to the peptide concentration, is followed by spectrophotometry.
  • FC Folin-Ciocalteu reagent
  • the reaction is carried out in a final volume of 800 mL comprising 200 mL of extracted peptides, 500 mL of a 500 mM sodium carbonate solution (NaHCOa) and 100 mL of Folin-Ciocalteu reagent (Sigma- Aldrich, St Louis, USA).
  • the reaction is incubated at 37 ° C. in the dark for 30 min, then the OD at 750 nm of the mixture is measured by a Prim spectrophotometer (Secomam, Groupe Aqualabo, Champigny, France).
  • the blank is produced from the solution used to elute the peptides from the SPE columns (80% ACN, 20% H 2 O, 0.1% TFA).
  • the concentration in the samples is determined by a standard range of a commercial peptide solution (peptide digest assay standard, ThermoFisher Scientific, Waltham, USA).
  • SEC size exclusion chromatography
  • a volume of 25 mL of sample is eluted isocratically in a solvent composed of 30% ACN (v / v), 70% H 2 O (v / v) and 0.1% TFA (v / v), at a flow rate of 0.5 m L / min for 60 min.
  • a UV detector at 214 nm allows the detection of peptide bonds.
  • the amount of peptides in the samples is analyzed by integration profiles obtained by SEC. Two ranges of molecular weights were chosen for the integrations, the first for peptides with an apparent molecular weight greater than 1,700 Da (called PHPM, high molecular weight peptides) and the second for peptides with an apparent molecular weight less than 1.
  • PBPM low molecular weight peptides
  • the quantity of peptides belonging to each size class is expressed either as a percentage of the total area of the chromatogram (% PHPM and% PBPM), or as a percentage of the area of the chromatogram obtained for the uninoculated milk control condition (% PHPM / NI Milk and% PBPM / NI Milk).
  • Lactobacillus helveticus VF45A strain is one of the most effective strains with a final peptide concentration greater than 6 g / L (see Table 3 below).
  • the molecular weight of the peptides generated was analyzed by SEC as mentioned above.
  • the quantity of peptides belonging to each size class (less than or greater than 1 .700 Da) is expressed either as a percentage of the total area of the chromatogram (% PHPM and% PBPM), or as a percentage of the area of the chromatogram obtained for the non-inoculated milk control condition (% PHPM / Milk NI and% PBPM / Milk NI). Inoculation and fermentation lead to an increase in low molecular weight peptides compared to the control (1.5 times on average for strains of Lactobacillus delbrueckii, 4.2 times on average for Lactobacillus helveticus).
  • PCA Principal component analyzes
  • PCR analysis took into account 9 quantitative parameters: the proteolytic activity on agar-milk, the growth during fermentation in a liquid medium (milk), the pH at the end of the fermentation, the quantity of peptides produced, and 5 other criteria related to the analysis of the chromatographic profiles of the peptides (distribution in apparent molecular weights.
  • Fig. 2 shows the map obtained.
  • the principal components Dim1 and Dim2 represent 60.82% and 23.56% of the total variance, respectively.
  • a correlation positive is observed between the pH value and high molecular weight peptides.
  • growth is correlated with a series of variables related to the production of peptides (quantification by FC reagent,% PBPM,% PBPM / NI Milk) and the diameter of the proteolysis halo pH value and high molecular weight peptides are negatively correlated with variables such as growth, proportion of PBPM and halo of proteolysis.
  • Each strain is cultivated in a 500 mL bioreactor (MiniBio 500, my-Control, Applikon Biotechnology, Delft, the Netherlands).
  • the medium consists of skimmed milk (10 g / L (see previous paragraph), autoclaved for 30 min at 110 ° C.
  • the strain is inoculated with an OD 600 equal to 0.3.
  • the fermentation is carried out for 72 hours at a constant temperature of 40 ° C. with stirring at 300 rpm in a final volume of 330 mL.
  • the anaerobic condition is obtained by injecting N2 into the bioreactor at a flow rate of 20 mL / min.
  • the bacterial pellet is resuspended in 1 ml of PBS buffer for measurement of the DOooo in order to evaluate the bacterial growth. After 72 hours of fermentation, all of the fermentate is recovered and then centrifuged at 10,000 rpm for 10 min, the supernatant is then stored at -20 ° C until analysis. The concentration of dry matter in the fermentates is measured in a desiccator (XM60, Précisa, Poissy, France) and is always equal to 100 g / L. In order to be able to confirm the impact of bacterial fermentation on the properties of the final product, a control is carried out (CTLF). This is the fermentation medium incubated under the same conditions but not inoculated. After 72 hours of incubation, this medium is centrifuged and stored like the other fermentations. Identification of peptides produced during fermentation at controlled pH
  • part of the crude fermentate is fractionated by means of an ultrafiltration membrane.
  • the membrane is a Hydrosart® cassette (Sartocon® Slice Hydrosart® Cassette, Sartorius, Gôttingen, Germany) of 0.1 m 2 with a cutoff threshold of 10 kDa connected to a Sartocon® Slice 200 Hôlder system (Sartorius, Göttingen, Germany ).
  • the fermentate is fed by a peristaltic pump and the pressure of the retentate is maintained at approximately 1 bar during filtration.
  • the ultrafiltration permeate (UFP) containing molecules less than 10 kDa is separated from the retentate (UFR).
  • the two sub-fractions are then stored at -20 ° C until analysis.
  • the ultrafiltration permeates are dried to 10% of the initial volume by centrifugal evaporation (miVac, Gene Vac, Ipswich, United Kingdom) for 15 h at 40 ° C.
  • the concentration of dry matter in the products is then measured in a desiccator (XM60, Précisa, Poissy, France).
  • the peptides purified by SPE from the culture supernatants are dried by centrifugal evaporation (miVac, Gene Vac, Ipswich, United Kingdom) for 2 h at 40 ° C then resuspended in 100 mL of H 2 O containing 0.1% TFA (v / v) and centrifuged for 10 min at 8,000 rpm.
  • Ten microliters of sample are injected onto a C 18 -Kinetex column (150 x 4.6 mm, 2.6 miti, 100 A, Phenomenex, Torrance, USA), connected to an Ultra Performance Liquid Chromatography chromatography system. (UPLC) Acquity (Waters, Manchester, United Kingdom).
  • a linear gradient of ACN containing 0.1% formic acid (FA) (v / v) is used (from 5 to 15% ACN for 30 min, then from 15 to 30% ACN for 60 min , from 30 to 50% ACN for 10 min and finally from 50 to 95% ACN for 10 min) with a flow rate of 500 mL.min -1 .
  • the UPLC eluents are sprayed directly by electrospray at a voltage of 3 kV, the desolvation is carried out by using dinitrogen (N2) at a flow rate of 600 Lh -1 at a temperature of 300 ° C.
  • N2 dinitrogen
  • the chromatographic system is coupled to a Synapt-G2-Si mass spectrometer (Waters, Manchester, UK).
  • MS measurements are made in data depend mode (data depend analysis, DDA) and the data are recovered in a mass range from 100 to 2000 m / z.
  • a maximum of 15 precursor ions with an intensity threshold of 10,000 are selected for fragmentation by the collision-induced dissociation (CID) method at a power of 8 to 9 V for low masses and a power of 40 to 90 V for the high masses.
  • MS / MS spectra are recovered in a mass range from 100 to 2000 m / z.
  • the spectra are processed by Mass Lynx software (version 4.1.) (Waters, Manchester, United Kingdom). Analysis of the control sample (CTLF) allowed the basic heterogeneity to be defined.
  • CID collision-induced dissociation
  • 596 peptides from 22 milk proteins (as-i-casein, b-casein, as2-casein, GLYCAM1, k-casein, BTN1 A1, b-lactoglobulin, FGFBP, lactoperoxidase, osteopontin, perilipine, collagen a2, TRIP6, NPT2B , acylCoA desaturase, dysferlin, COG8, B4GT1, PSME4, DHX9, RALYL and apolipoprotein A2) were identified in this sample. 32% of them come from b-casein. By comparison, between 150 and 722 peptides were identified in the samples fermented by the different strains tested.
  • ⁇ -casein peptides represent 44-67% of all sequences identified. Due to the preponderance of b-casein peptides among the identified peptides, and the natural abundance of b-casein in milk, only peptides derived from b-casein will be studied. The peptides produced by the strains of the invention are therefore all derived from the proteolysis of b-casein in cow's milk.
  • the database search is carried out using Peaks Studio software (version 7.0.) (Bioinformatics Solutions, Waterloo, Canada) using the UniProt database (May 15, 2017) restricted to the complete proteome of the species Bos taurus.
  • the tolerance thresholds for the masses of precursor ions and fragments are defined at 35 ppm and 0.2 Da.
  • the peptide sequences identified by the software are filtered according to a false discovery rate strictly less than 1%. For all the strains, the peptides identified come from the fermentate which has not undergone ultrafiltration. These same peptides should also be found in view of their mass in the ultrafiltered fermentate.
  • Table 4 groups together the non-phosphorylated peptides considered to be new which are produced by the strain VF45A according to the fermentation method indicated above at pH 6.
  • Table 5 groups together the phosphorylated peptides considered to be new which are produced by the strain VF45A according to the fermentation method indicated above at pH 6.
  • Table 6 below groups together the non-phosphorylated peptides considered to be new which are produced by the strain VFH049 according to the fermentation method indicated above at pH 6.
  • Table 7 groups together the phosphorylated peptides considered to be new which are produced by the strain VFH049 according to the fermentation method indicated above at pH 6.
  • Table 8 groups together the non-phosphorylated peptides considered to be new which are produced by the VF50b strain according to the fermentation method indicated above at pH 6.
  • Table 9 groups together the phosphorylated peptides considered to be new which are produced by the strain VF50b according to the fermentation method indicated above at pH 6.
  • Fermentates (before and after ultrafiltration) were tested for two biological activities, namely ACE inhibition and the ability to modulate calcium absorption. Furthermore, the toxicity of the hydrolysates was evaluated. For the analyzes, the various samples are diluted in I ⁇ 2O at a concentration of 15 g / L of dry matter.
  • the aim of this experiment is to test the possible toxicity of fermentates towards intestinal cells.
  • Cell viability is measured by the use of the CCK-8 reagent (Cell Counting Kit-8), this method is based on the reduction of a tetrazolium salt by cellular dehydrogenases producing formazan, the formation of which is followed at 450 nm .
  • the Caco-2 cells are seeded in a 96-well plate at a density of 8,000 cells per well in a volume of 200 mL of complete DMEM medium, and cultured for 7 days at 37 ° C., 5% CO2.
  • the fermentates are diluted in complete DMEM medium at a concentration of 5 and 10 g / L.
  • PBS buffer also diluted in DMEM medium (same dilution factor as the samples) is used as a control.
  • a volume of 150 mL of sample is placed in each well, followed by incubation for 7 or 24 hours at 37 ° C., 5% CO2. After incubation, the Caco-2 cells are washed twice with PBS buffer then a volume of 150 ml of complete DMEM medium supplemented with 5% (v / v) of CCK-8 reagent (Sigma-Aldrich, St Louis, United States ) is added to each well.
  • the plate is then incubated for 2 h at 37 ° C., 5% CO2 in the dark, then read at 450 nm with a Xenius XC spectrofluorimeter (Safas Monaco, Monaco, France). Cell viability is calculated relative to the absorbance obtained for the control wells (corresponding to 100% viable cells).
  • a concentration of 5 g / L the crude fermentates (unfiltered) of each of the strains of the invention exhibit no toxicity towards the Caco-2 cells even after 24 hours of contact. The same observation is made for a concentration of 10 g / L of dry matter.
  • the ultrafiltered fermentates of each of the strains also do not exhibit cytotoxicity.
  • the aim is to study the inhibitory potential of fermentates towards CEA. It is based on the use of a fluorescent substrate, o-aminobenzoyl-Gly-p-nitro-L-Phe-Pro (Abz-Gly-Phe (NO2) -Pro). Hydrolysis of this substrate by ACE generates the Abz-Gly fluorophore moiety which can be followed by excitation and emission wavelengths of 355-375 nm and 400-430 nm respectively. The samples, the enzyme and the substrate are prepared and diluted in Tris-HCl buffer (150 mM) at pH 8.3 to the desired concentrations.
  • Tris-HCl buffer 150 mM
  • the hydrolysis reaction is carried out in a 96-well plate in a final volume of 300 mL comprising 50 mL of sample or Tris-HCl buffer (for the negative inhibition control), 50 mL of an ACE solution ( 0.05 U / mL) (Sigma-Aldrich, St Louis, USA) and 200 mL of Abz-Gly-Phe (NC> 2) -Pro (0.45 mM) substrate (Bachem, Bubendorf, Switzerland). A control not containing enzyme (replaced by Tris-HCl buffer) is also produced.
  • the plate is then incubated at 37 ° C for 1 h, fluorescence measurement takes place every 2 min with excitation and emission wavelengths of 365 and 415 nm respectively by a Xenius XC spectrofluorimeter (Safas Monaco, Monaco, France) equipped with a water bath at 37 ° C.
  • the percentages of inhibition and the IC50s of the different samples tested are calculated based on the negative inhibition control.
  • the crude fermentates exhibit IC 50 ranging from 12.76 mg / mL for the crude control fermentate to 0.56 mg / mL for the fermentate produced by the VF45A strain.
  • the IC 50 of the crude fermenta is 0.76 mg / mL.
  • the IC 50 are 8 mg / mL for the control, 0.47 mg / mL for the fermentate produced by the strain VF45A and 1.7 mg / mL for the fermenta produced by the strain VFH049.
  • the VF45A and VFH049 strains very significantly improve the capacity of the fermentates to inhibit ACE compared with the control. The same effect is obtained with ultrafiltered fermentates. Molecules of interest are therefore produced by the strains during fermentation and exhibit ACE inhibiting activity.
  • the QSAR approach used to determine the IC 50 of our peptides was used on the VPP and IPP peptides of the prior art. Their IC 50 is 26 mM, while some of the peptides of the invention exhibit an IC 50 value below 20 mM (up to 8.7 mM for the most active), and are therefore a priori more effective.
  • the QSAR approach used to determine the I C50 of the peptides of the invention was used on the VPP and IPP peptides described in the prior art. Their IC50 is 26 mM, while some of our peptides are below 20 mM (up to 8.7 mM for the most active), and are therefore a priori more effective.
  • Transcellular calcium transport is an active transport involving the incorporation of calcium into the intestinal cells by a transporter called TRPV6. Calcium is subsequently transported and excreted at the basal pole of the cell in the intestinal mucosa.
  • TRPV6 transporter
  • the ability of the fermentates to modulate the absorption of calcium was evaluated using Caco-2 cells.
  • the test consists of bringing the fermentates (concentration of 10 g / L) into contact with the Caco-2 cells for 7 hours. After contact, the incorporation of calcium by the cells is evaluated by using an intracellular probe capable of emitting fluorescence in the presence of calcium. After rinsing the Caco-2 cells, the FluoForte® probe is added following the instructions of the FluoForte® calcium assay kit (Enzo Life Sciences, Farmingdale, United States), the plate is then incubated at room temperature for 1 h, time to penetration of the probe.
  • the fluorescence emission is then monitored by a Xenius XC spectrofluorimeter (Safas Monaco, Monaco, France) with an excitation wavelength at 490 nm for emission followed at 525 nm.
  • the emission is measured for 30 sec with an injection of 25 mL of a 250 mM CaCL solution between 9 and 10 sec kinetics by means of an injector module coupled to the spectrofluorimeter.
  • This injection causes a sudden entry of calcium into the intestinal cells which leads to an increase in fluorescence emission.
  • the ability of bacterial strains to modulate the incorporation of calcium into cells is determined by this increase in fluorescence after injection of calcium.
  • the results are thus expressed for each well relative to the mean fluorescence measured between 0 and 9 sec of kinetics, considered as the basal fluorescence of a well. An increase in fluorescence ratio, for the various contact conditions, is therefore obtained.
  • the effect of the fermentates obtained with the VF45A and VFH049 strains is compared with that of the PBS buffer and that of the control fermentate.
  • the results show that the crude fermentates obtained with these two strains are capable of positively modulating the incorporation of calcium with respect to the PBS buffer and to the control (FIG. 5).
  • the ratio of fluorescence emission to basal emission is 1.08 for PBS, 1.23 for the control fermentate, 1.3 for the fermentate produced by the VF45A strain and 1.31 for the fermentate produced by the strain VFH049.
  • the effect is less pronounced for strain VF45a than for VF49d (Fig. 6). Nevertheless, a tendency towards increased absorption is observed for both strains.
  • the objective of this part is to evaluate the capacity of fermentates to modulate the expression of the trpv6 gene by intestinal cells.
  • An RT-qPCR approach is conducted using Caco-2 cells as a template.
  • the Caco-2 cells are seeded in a 24-well plate at a density of 40,000 cells per well in a final volume of 500 mL of complete DMEM medium and incubated for 15 days at 37 ° C, 5% CO2 with a change of medium every. the two days after 7 days of culture.
  • the fermentates are diluted in complete DMEM medium at a concentration of 10 g / L of dry matter. PBS buffer diluted in DMEM medium (same dilution factor as for samples) is used as a control.
  • RNA samples are first treated with DNase to remove any DNA fragments co-extracted and / or remaining following the extraction.
  • a volume of 8 mL containing 1000 ng of RNA is mixed with 1 mL of DNase, and 1 mL of DNase buffer (ThermoScientific, Waltham, United States).
  • the reaction takes place at 37 ° C for 30 min, it is stopped by adding 1 mL of 50 mM EDTA solution (ThermoScientific, Waltham, United States) followed by incubation at 65 ° C for 10 minutes. Subsequently, the samples were back-transcribed into cDNA using the RevertAid H minus first strand cDNA synthesis kit (ThermoScientific, Waltham, USA), according to the instructions provided. For the qPCR reaction, the cDNA samples are diluted (1/16) in H 2 O.
  • one cycle comprises denaturation at 95 ° C for 15 sec, hybridization of 58 or 60 ° C depending on the pair of primers used for 30 sec and an elongation at 72 ° C for 30 sec.
  • the realization of a melting curve ends the reaction.
  • the gene studied is the gene encoding the calcium transporter (transient receptor potential selective for calcium) (trpv6) also called CaT 1 (calcium transporter 1).
  • trpv6 transient receptor potential selective for calcium
  • CaT 1 calcium transporter 1
  • ppiA peptidylprolyl isomerase A
  • the fermentate samples were contacted with Caco-2 cells and changes in expression of the trpv6 gene were then studied by qPCR.
  • the fermentates obtained with the VF45A and VFH049 strains are capable of positively modulating the expression of the gene. More particularly, the fermentate produced by the strain VFH049 causes an overexpression of the gene 20 times greater than the control (Fig. 7). The effect of ultrafiltered fermentations is less pronounced, not significant even if a tendency to induce the gene is clearly visible for the two strains (Fig. 8). The fermentations produced by the two strains are therefore capable of modulating both the incorporation of calcium and the expression of trpv6 relative to unfermented milk. Study of probiotic characteristics
  • the significance of the results is assessed by a one-way analysis of variance (ANOVA) followed by a Tukey's test for multiple comparison of means.
  • ANOVA analysis of variance
  • Tukey's test for multiple comparison of means.
  • the post-ANOVA test is Dunn’s test resulting in a comparison to the mean of the control condition. The difference between the means is considered significant for a p value ⁇ 0.05.
  • the statistical tests were carried out under the R software (R core team, 2016, Vienna, Austria), on the R Commander package.
  • the principle of this test is based on the comparison of the survival of a bacterial strain between an incubation of 2 hours at pH 2 and an incubation at the pH of the MRS medium (pH 6.2).
  • each strain in the collection was cultured in liquid MRS medium for 24 hours at 37 ° C. in anaerobic condition.
  • a volume of 200 mL of culture was then diluted (1/10) either in the same MRS medium at pH 6.2 (called tube A), or in MRS medium adjusted to pH 2 with hydrochloric acid (HCl ) (called tube B).
  • Tubes A and B were incubated at 37 ° C. for 2 h then 10 ml of each tube were plated on MRS agar.
  • the inoculated dishes were incubated for 48 hours at 37 ° C. in anaerobic condition.
  • Acidity tolerance was assessed by comparing the number of colonies between plates A and B.
  • the strains were divided into different acid tolerance classes according to the result of the B / A ratio.
  • Class 0 includes strains that are not tolerant to acidity (no colony on box B).
  • class 3 corresponds to acid-tolerant strains (box B shows more than 80% of the colonies in box A).
  • strains were divided into 4 tolerance classes ranging from class 0 (non-tolerant strains) to class 3 (tolerant strains).
  • the objective of this test is to evaluate the hydrophobic character of the wall of the different strains.
  • a simple test has already been developed to assess this criterion, it is the MATS (Microbial Adhesion To Solvents) test. It consists in measuring the affection of a bacterial strain for an apolar hydrocarbon (most often n-hexadecane) in order to estimate the hydrophobicity of the wall.
  • MATS Microbial Adhesion To Solvents
  • the cells are then washed twice by successively repeating a centrifugation step at 10,000 rpm for 10 min, followed by a step of resuspending the cells in a phosphate buffered saline (PBS) buffer, pH 7.4.
  • PBS phosphate buffered saline
  • Optical Density OD at 630nm ( initial OD 630nm ) was determined with an ELx808 spectrophotometer (BioTek Instruments Inc., Vermont, USA).
  • a volume of 1 mL of suspension was then mixed with 100 mL of n-hexadecane. (Acros Organics, Geel, Belgium) by creating a vortex in the mixture for 1 min.
  • the solution was then left to stand at room temperature for 15 min then 100 mL of the aqueous phase were recovered for a new determination of the OD at 630 nm (final OD 630nm ).
  • the wall hydrophobicity of the strains was calculated according to the following formula:
  • Fig. 9 represents the percentages of wall hydrophobia obtained for the strains tested according to their acidity tolerance class.
  • Fig. 9 we note that the VF50b strain does not tolerate acidity, which is not the case with the VFH049 strain.
  • This test makes it possible to evaluate the capacity of the strains to aggregate together in a liquid medium. It is based on the comparison of the bacterial concentration above a suspension at time 0 with the bacterial concentration of the same suspension incubated for a given time without any agitation. Strains capable of strong self-aggregation will aggregate between them, thus increasing their sedimentation rate.
  • the strains are cultured in MRS for 24 h at 37 ° C. then washed and resuspended in PBS buffer at an OD at 600 nm of 1. A volume of 4 mL of suspension is stirred so as to form a vortex for 10 sec and then incubated at room temperature without stirring.
  • The% aggregation of the VFH049 strain is 5.38% ⁇ 5.24 after 2.5 hours and 67.44% ⁇ 6.99 after 5 hours.
  • the% aggregation is 33.33% ⁇ 14.8 after 2.5 hours and 66.67% ⁇ 17.97 after 5 hours. The strains are therefore able to adhere to intestinal cells.
  • the bacterial cells are washed twice and then resuspended in PBS buffer at a concentration of 10 9 CFU / ml.
  • the buccal phase is simulated by adding 8 mL of PBS, adjusted to pH 6.8, to 1 mL of the bacterial suspension. The mixture is incubated with constant agitation at 200 rpm for 5 min at 37 ° C.
  • the gastric phase is simulated by adding 12 mL of PBS at pH 3 supplemented with bovine pepsin (Sigma-Aldrich, St Louis, USA) at 1.56 mg / mL. The suspension is incubated for 2 hours at 37 ° C. with stirring at 200 rpm.
  • the pH of the solution is maintained between 3 and 3.5 by the addition of hydrochloric acid (HCl) (1 M) or sodium hydroxide (NaOH) (1 M).
  • HCl hydrochloric acid
  • NaOH sodium hydroxide
  • 1 M sodium carbonate 1 M sodium carbonate (NaHCO3) is added to the mixture in order to raise the pH to about 7 and inactivate the pepsin.
  • a sample of 100 mL of the reaction mixture is taken, thus for the same digestion, 4 samples are obtained comprising the sample of the mother tube (TM), of the salivary phase (S), of the gastric phase after 2 hours of incubation (G2), and finally that after 2 hours of intestinal phase (I2).
  • Each sample is then diluted (1/10) successively in PBS buffer.
  • 100 mL of the appropriate dilutions are spread on an MRS agar. The seeded plates are then incubated at 37 ° C. for 48 hours in anaerobic condition. After incubation, the count of CFUs allows the determination of the bacterial concentration in the samples.
  • the results are expressed in CFU / mL.
  • the initial number of viable bacterial cells is 10 9 CFU / mL in the salivary phase.
  • the quantity of bacteria decreases more or less depending on the strain, the gastric phase being the most deleterious for all the strains.
  • the VFH049 strain is present in an amount of 10 7 8 CFU / mL while the VF50b strain is present in an amount equal to 10 7 2 CFU / mL. Strains VFH049 and VF50b therefore tolerate gastrointestinal digestion.
  • the objective of this test is to assess the possible toxicity of the strains with respect to the intestinal barrier.
  • Caco-2 and HT-29 MTX cells are used for this purpose.
  • the cells are seeded in a 96-well plate at a density of 8,000 cells per well in a volume of 150 ml of medium and cultured for 7 days at 37 ° C., 5% CO 2.
  • the Intestinal cells are washed twice with PBS buffer.
  • the bacterial strains, for their part, are cultured as described above and suspended in DMEM medium without addition at a concentration of 10 7 CFU / ml. For each well, 150 mL of bacterial suspension is added.
  • PBS buffer diluted with DMEM without addition as for the strains, is used as negative control.
  • Contact between bacterial strains and intestinal cells takes place for 24 h at 37 ° C, 5% C02.
  • the strains are contacted with both Caco-2 cells and HT-29 MTX cells independently. Determination of intestinal cell mortality is used here to assess the possible toxicity of bacterial strains.
  • the mortality of the cells is evaluated by assaying the activity of lactate dehydrogenase (LDH). This activity is assayed by the LDH activity assay kit (Sigma-Aldrich, St Louis, United States).
  • this kit is based on the reduction reaction of oxidized Nicotinamide Adenine Dinucleotide (NAD) in its reduced form (NADH) by lactate dehydrogenase, NADH can be detected at 450 nm. After contact, 50 ml of supernatant are taken from each well and the LDH activity is assayed according to the kit protocol. The absorbance of the plate is read at 450 nm by a Xenius XC spectrofluorimeter (Safas Monaco, Monaco, France). The mortality of the intestinal cells is calculated as a percentage of the mean of the absorbances of the control condition (the proportion of LDH released in this condition is taken as a reference to 100%). For strains VFH049 and VF50b, the results show a lack of significant toxicity towards the two cell lines, which suggests that under these experimental conditions, the selected bacterial strains are not deleterious for the intestinal cells.
  • NAD oxidized Nicotinamide Adenine Dinucleotide
  • the objective of this test is to assess the ability of strains to adhere to intestinal cells. It is based on the comparison between the amount of bacterial cells adhering to the Caco-2 cell monolayer versus the amount of bacteria initially added.
  • the strains are cultivated and prepared as described above, a suspension in DMEM medium without addition is prepared at a concentration of 10 7 CFU / ml.
  • the Caco-2 cells are inoculated in 24-well plates at a concentration of 40,000 cells per well in a volume of 500 ml of complete DMEM. After incubation for 7 days at 37 ° C, 5% CO 2, the cells are washed twice with PBS buffer.
  • a volume of 300 mL of bacterial suspension is added to each well, the plate is then incubated at 37 ° C, 5% of C02, for 2 hours.
  • the bacterial suspension used is stored for determining the CFU concentration.
  • the Caco-2 cells are washed twice with PBS buffer in order to remove the non-adherent bacteria.
  • the intestinal cells are then lysed by adding 100 ml of PBS buffer supplemented with 0.1% (v / v) Triton X-100. After incubation for 15 min at room temperature, the lysate and the stock suspension used are successively diluted (1/10) in PBS, then spread on MRS agar. The inoculated plates are incubated for 48 hours at 37 ° C.
  • the percentage of adherent bacterial cells is calculated relative to the concentration obtained in the stock suspension representing the quantity of bacteria added at the start of the experiment (set at 100%).
  • The% of cells adhering to Caco-2 cells is 1.48 ⁇ 0.41 for the VFH049 strain and 0.18 ⁇ 0.13 for the VF50b strain. Strain VFH049 is much more adherent to intestinal cells which suggests that it is more likely to have a probiotic action.
  • This contact protocol is used for each technique used in this part.
  • the bacterial strains are cultured in MRS for 24 hours at 37 ° C. in anaerobic condition.
  • the cells are recovered by centrifugation at 10,000 rpm for 10 min and then resuspended in PBS buffer, this step is repeated a second time to wash the bacterial cells.
  • a bacterial suspension is finally prepared with DMEM medium without addition at a concentration of 10 7 CFU / mL.
  • the HT-29 MTX cells are inoculated in a 24-well plate at a concentration of 40,000 cells per well in a volume of 500 ml of complete DMEM.
  • the Caco-2 cells are seeded in a 96-well plate at a concentration of 8,000 cells per well in a volume of 200 mL of complete DMEM medium.
  • the Caco-2 cells are seeded on inserts placed in a 12-well plate (polyester membrane, 0.4 ⁇ m, Costar®, Corning, New-York, United States) on the apical side in a volume of 500 mL. of complete DMEM medium, a volume of 1,500 mL of this same medium is added to the basal side of the insert.
  • All the cell cultures are cultivated for 15 days with a renewal of the medium every 2 days during the second week of cultivation.
  • the intestinal cells are washed twice with PBS buffer before contact with the bacterial strains.
  • a volume of 150 ml of bacterial suspension is added to each well for a culture in a 96-well plate, and a volume of 300 ml is added for a contact in a 24-well plate or in inserts on a 12-well plate. In all cases, contact takes place for 24 h at 37 ° C, 5% C02.
  • the negative control used is a PBS buffer diluted in complete DMEM medium.
  • Total calcium transport is defined as the part of calcium passing from the apical pole to the basal pole by crossing the epithelial barrier. This transport is evaluated by the variation of the calcium concentration at the basal pole of the cell barrier over time.
  • Caco-2 cells cultured in inserts in a 12-well plate are used for this experiment.
  • 10 mL of a 250 mM solution of calcium chloride (CaCL) are added on the apical side of the insert.
  • 100 mL samples are taken from the basal side of the insert at 30 min, 7 and 24 h of contact. The samples are stored at -20 ° C until analysis.
  • TEER transepithelial electrical resistance
  • This resistance is measured by a MilliCell Electrical Resistance System volt / ohmmeter (Merck Millipore, Burlington, USA). An empty insert without cells is used as a blank to achieve cell barrier resistance. The resistance (in W) measured for each well is then multiplied by the area of the insert (equal to 1, 12 cm 2 for the insert used) to give a resistance in W.cm -2 . The results are expressed as a percentage of the value obtained at 30 min of contact (set at 100%) for each well.
  • an assay of the calcium concentration is carried out in the samples taken at the basal pole of the membrane of Caco-2 cells during contact.
  • the determination of the calcium concentration is carried out using the calcium colorimetry assay kit (Sigma-Aldrich, St Louis, United States). This kit is based on the colorimetric reaction between calcium ions and ortho-cresolphthalein forming a color complex that can be detected at 575 nm.
  • the assay is carried out using 25 mL of samples diluted (1/2) in I ⁇ 20 Milli-Q®, following the kit protocol.
  • the measurement of the absorbance of the samples at 575 nm is carried out by a Xenius XC spectrofluorimeter (Safas Monaco, Monaco, France).
  • the results show that the TEER increases over the incubation time with the strains as well as under the control condition (PBS buffer).
  • the strains of the invention do not make it possible to significantly modulate the absorption after 7 hours of contact compared to the PBS buffer.
  • the calcium concentration decreases in the basal compartment for the control condition, while a significant increase in the amount of calcium is observed for the VFH049 and VF50b strains.
  • RNA extraction is then carried out using the TRIzol TM reagent (Sigma-Aldrich, St Louis, United States). The RNA samples were first treated with DNase to remove any DNA fragments co-extracted and / or remaining following the extraction. A volume of 8 mL containing 1000 ng of RNA is mixed with 1 mL of DNase, and 1 mL of DNase buffer (ThermoScientific, Waltham, United States).
  • the reaction takes place at 37 ° C for 30 min in a Mastercycler gradient thermocycler (Eppendorf, Hamburg, Germany), it is stopped by adding 1 mL of 50 mM EDTA solution (ThermoScientific, Waltham, USA). United) followed by incubation at 65 ° C for 10 min. Subsequently, the samples were back-transcribed into cDNA using the RevertAid H minus first strand cDNA synthesis kit (ThermoScientific, Waltham, USA), according to the instructions provided. For the qPCR reaction, the cDNA samples are diluted (1/16) in I ⁇ 20.
  • qPCR mixture For a volume of 2 mL of sample, 18 mL of qPCR mixture is added containing 10 mL of Power SYBR® Green PCR Master Mix (2x) (Applied Biosystems, Life Technologies, Foster City, USA), 0.6 mL of each primer (10 mM) and 6.8 mL of H2O. Fluorescence is monitored during the reaction in a CFX Connect Real Time Detection System thermocycler (Bio-Rad, Hercules, USA).
  • the results are shown in Fig. 10.
  • the expression of the gene encoding the vitamin D receptor (vdr) is significantly increased in the presence of the VF50b strain; it is increased by 1.8 times relative to the PBS buffer.
  • the VFH049 strain it causes an increase in the expression of the gene encoding the calcium transporter (trpv6) at least 3 times greater than the control.
  • the VF50b strain is the only strain causing a 2.8-fold greater increase in expression of the cld-2 gene compared to the PBS buffer.
  • the growth and acidification properties of the VF50b strain are superior to those of other strains of the same species, which makes it a particularly interesting strain for the artisanal or industrial production of fermented dairy products (use as a fermentation starter) .
  • Table 15 compares the properties of the VF50b strain with those of other strains of the same species (tests carried out with cow's milk):
  • Strain VF50b can be used for making yogurt, for example.

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EP20725858.3A 2019-03-20 2020-03-09 Neuartige milchsäurebakterienstämme zur förderung der absorption von calcium-peptiden und assoziierten produkten Pending EP3942016A1 (de)

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FR1902847A FR3094013A1 (fr) 2019-03-20 2019-03-20 Nouvelles souches de bacteries lactiques favorisant l’absorption du calcium – peptides et produits associes
FR1902854A FR3094015A1 (fr) 2019-03-20 2019-03-20 Nouvelles souches de bacteries lactiques favorisant l’absorption du calcium – peptides et produits associes
FR1902851A FR3094014A1 (fr) 2019-03-20 2019-03-20 Nouvelles souches de bacteries lactiques favorisant l’absorption du calcium – peptides et produits associes
PCT/FR2020/050477 WO2020188181A1 (fr) 2019-03-20 2020-03-09 Nouvelles souches de bacteries lactiques favorisant l'absorption du calcium - peptides et produits associes

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FR3102774B1 (fr) * 2019-10-31 2021-10-01 Vf Bioscience Nouvelle souche de lactobacillus plantarum utile pour l’absorption intestinale du calcium
CN115624175A (zh) * 2022-11-04 2023-01-20 大连澎立生物科技有限公司 一种富硒灵芝酵素小分子肽钙及其制备方法和富硒小分子钙多元蛋白肽

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JP3488722B2 (ja) 1992-03-04 2004-01-19 カルピス株式会社 カルシウム吸収促進活性剤及びその製造法
PT1820849E (pt) 2006-02-20 2009-04-24 Gervais Danone Sa Novas estirpes de lactobacillus helveticus
WO2008002484A2 (en) 2006-06-23 2008-01-03 Lacpro Industries, Llc Novel lactobacillus bulgaricus strain and compositions
JP5686680B2 (ja) 2011-06-24 2015-03-18 カルピス株式会社 乳酸菌発酵によるカゼイン由来ペプチドの製造方法
US20120328735A1 (en) * 2011-06-24 2012-12-27 Naoto Uchida Process for preparing casein-derived peptides by fermentation of lactic acid bacteria

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KR20210142692A (ko) 2021-11-25
US20230287331A1 (en) 2023-09-14
WO2020188181A8 (fr) 2021-11-04
AU2020243322A1 (en) 2021-10-28

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