EP2576608A1 - Fraction de protéines et peptides issus du blanc d'oeuf et protéine issue du blanc d'oeuf et leur utilisation comme agent anti-listeria - Google Patents
Fraction de protéines et peptides issus du blanc d'oeuf et protéine issue du blanc d'oeuf et leur utilisation comme agent anti-listeriaInfo
- Publication number
- EP2576608A1 EP2576608A1 EP11723948.3A EP11723948A EP2576608A1 EP 2576608 A1 EP2576608 A1 EP 2576608A1 EP 11723948 A EP11723948 A EP 11723948A EP 2576608 A1 EP2576608 A1 EP 2576608A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- fraction
- protein
- egg white
- heparin
- molecule
- 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.)
- Withdrawn
Links
Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—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
- C07K14/465—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from birds
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/76—Albumins
- C07K14/77—Ovalbumin
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
- A01N37/18—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing the group —CO—N<, e.g. carboxylic acid amides or imides; Thio analogues thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Definitions
- the invention relates to a fraction of proteins and / or peptides derived from egg white and having anti Listeria monocytogenes activity.
- the invention also relates to a specific molecule, derived from the egg white, namely the OVAX protein, for its antimicrobial action and more particularly for its targeted action against Listeria monocytogenes.
- the invention finds applications particularly in the field of agri-food, for example during the development of food products for everyday consumption, such as cold cuts and cheeses, in order to eliminate all traces of Listeria monocytogenes in these products. products.
- the invention also has applications in the pharmaceutical field, for example for the preparation of medicaments for treating and / or preventing conditions related to Listeria monocytogenes, such as Listeriosis.
- Listeria monocytogenes is a bacterium found in soil, vegetation, water, wastewater, silage products and human and animal feces. Some animals may carry the bacteria without their knowledge, without being sick. These healthy carriers can be the source of food contamination, such as milk and meat, and by-products. In particular, this bacterium is found repeatedly in cold cuts and raw milk cheeses. Listeria monocytogenes can then cause a condition called Listeriosis, related to food poisoning. Listeriosis can have serious consequences, especially in pregnant women and the elderly or whose immune system is weakened.
- Listeria monocytogenes is known to be sensitive to certain antibiotics, including ampicillin and arnoxicillin. Since persons receiving listeriosis treatment in case of illness are fragile individuals, it is known to treat them via a pool of antibiotics, and in particular penicillin, streptomycin and sulphonamides. This treatment can be prolonged with lactams.
- the invention proposes to use a molecule having antimicrobial activity against Listeria monocytogenes.
- the molecule according to the invention is derived from a protein present in the egg, in particular of hen, and more particularly in egg white, known to contain numerous proteins and peptides having effects against bacteria, and in particular enzymes, such as lysozyme.
- the protein of interest hereafter called OVAX, for "ovalbumin-related protein X" has also been identified, in smaller amounts, in egg yolk, eggshell, yolk sac, etc.
- OVAX The corresponding amino acid sequence of OVAX has been identified, so that it is possible to use the native protein, that is to say isolated from the chicken egg, but also the protein recombinant.
- the invention has also made it possible to demonstrate that a fraction of peptides and / or proteins derived from chicken egg white and able to bind heparin-sepharose (or heparin coupled to any other resin), has an activity anti Listeria monocytogenes.
- the invention made it possible to identify the peptides and proteins of this fraction, fifteen in number, and which comprises in particular OVAX which advantageously represents at least 50%, more precisely at least 56%, in proportion to this fraction.
- the anti Listeria monocytogenes activity of the OVAX protein and of the fraction according to the invention is preserved after digestion by certain digestive enzymes and in particular by trypsin or chymotrypsin.
- trypsin is a digestive enzyme present in gastric juice in humans and in mammals
- chymotrypsin is a digestive enzyme present in the pancreas of men and mammals. Both participate in the digestion of proteins contained in the diet ingested by the animal, human or non-human.
- the invention therefore relates to a molecule of amino acid sequence SEQ ID No. 1, for its antimicrobial activity, and more particularly against Listeria monocytogenes.
- sequence SEQ ID No. 1 corresponds to the amino acid sequence of the OVAX protein.
- the inventors have discovered that the molecule according to SEQ ID No. 1 has a particular affinity for heparin, a negatively charged glycosaminoglycan, and could therefore bind to negatively charged surfaces such as the lipopolysaccharide of certain Gram-negative bacteria. , and the peptidoglycan of certain Gram-positive bacteria (due to the presence of teichoic acid).
- the bond between at least one of these positively charged sequences and the surface of a negatively charged microorganism results in destabilization of the wall of said microorganism, leading to its lysis.
- the molecule according to the invention is therefore able to inhibit the growth of such microorganisms.
- the invention is particularly directed to the molecule of sequence SEQ ID No. 1 for its anti-Listeria monocytogenes action, as is more specifically set out below.
- the invention also relates to a composition comprising a molecule of sequence SEQ ID No. 1, as an antimicrobial active principle and especially as an anti-Listeria monocytogenes active ingredient.
- composition according to the invention comprising the antimicrobial molecule of amino acid sequence SEQ ID No. 1, may especially be intended for the manufacture of an anti-Listeria monocytogenes drug.
- a composition may in particular be a solution intended to be ingested, and comprising the required excipients. Otherwise, the composition may be a powder to be suspended later, or to be packaged such as, in capsule form.
- composition according to the invention can also be used for the manufacture of food, intended for animal feed, human or non-human, as an anti-Listeria monocytogenes food additive.
- the composition is then used during the method of manufacturing the food product, said composition being added to the preparation intended to form the food product, during its preparation.
- the composition can be used as a food additive during the preparation of sausages such as pâtés, rillettes, sausages, etc.
- the composition may also be added to milk for use in making cheese.
- the composition is added to the preparation after any cooking step and / or any step in which said preparation is subjected to temperatures above 60 ° C.
- SEQ ID No. 1 in the composition may be between 15 g / mL and 400 g / mL, and preferably between 20 g / mL and 100 g / mL. Even more preferably, the concentration of the molecule of sequence SEQ ID No. 1 in the composition is between 25 g / ml and 60 g / ml.
- the invention also relates to a fraction of peptides and / or proteins, derived from egg white and able to bind heparin, for its action against Listeria monocytogenes.
- egg white it is meant that the peptides and proteins present in the fraction according to the invention are all in the egg white, and can be obtained from the egg white.
- these peptides and proteins may have been obtained otherwise than by purification from egg white, and in particular by chemical synthesis or produced in recombinant form.
- the fraction according to the invention is likely to comprise all or part of the peptides and / or proteins derived from egg white that can bind to heparin.
- This fraction may contain some contaminating molecules of ovalbumin, a major protein of egg white, which represents more than 50% of the total protein present in the egg white, and which does not have any affinity for the egg white. heparin.
- peptide and protein molecules were isolated directly from chicken egg white, by affinity chromatography using heparin-sepharose beads, then identified by mass spectrometry.
- the fraction according to the invention thus comprises at least one of the peptides and proteins below (the references G1 below are extracted from the NCBI database, updated in April 2011):
- the OVAX with a molecular weight of approximately 45 kDa, more precisely approximately 44 kDa, of sequence SEQ ID No. 1, and which has 1 HBS binding site heparin;
- Lysozyme C (Gl: 229157), with a molecular weight of approximately
- beta-defensin 11 (Gl: 49169808), with a molecular weight of approximately 12 kDa; this molecule seems to have no binding HBS consensus site heparin, which also suggests that it has one or more conformational binding sites to heparin;
- the TENP protein (Gl: 46048814) with a molecular weight of approximately 47 kDa; this molecule seems to have no heparin-binding HBS consensus site, which suggests that it also has one or more conformational binding sites to heparin;
- cyclophilin B (peptidylprolyl isomerase B) (Gl: 45382027) with a molecular weight of about 22 kDa and having a heparin-binding HBS consensus site;
- Vmo-I protein for "vitelline membrane Outer Layer Protein I"
- ovotransferrin Conalbumin
- Gl 71274075
- ovocleidine 17 (Gl: 31615312), with a molecular weight of approximately 15 kDa and having no heparin-binding HBS consensus site, which suggests that it also has one or more conformational binding sites at heparin;
- ovalbumin (Gl: 28566340), with a molecular weight of approximately 43 kDa and having a heparin binding HBS consensus site;
- Clusterin (Gl: 45382467), with a molecular weight of about 51 kDa and having 2 heparin binding HBS sites;
- heparin conformational binding site is meant a specific exposure of positively charged amino acids, when the molecule is shaped, and forming a site capable of binding to heparin.
- the fraction according to the invention comprises at least the molecule of sequence SEQ ID No. 1 which corresponds to the sequence of OVAX as isolated from the egg white.
- the fraction may otherwise or also comprise the OVAX molecule of sequence SEQ ID No. 2, which corresponds to the known sequence which has been predicted from the bioinformatic analysis of the chicken genome, and which is identified in FIG. NCBI database by the reference XP_418984.2, in the name of the similar to Ovalbumin-related protein Y (Gene Y protein), named OVAXdb in Figure 3.
- the fraction according to the invention may advantageously comprise all of the peptides and proteins derived from egg white and able to bind heparin, as listed above.
- the molecule with the sequence SEQ ID No. 1 and / or sequence SEQ ID No. 2 represents at least 50% in proportion to the molecules of the fraction.
- Such a fraction can then be obtained directly by passing the egg white on heparin-sepharose affinity chromatography (or heparin-agarose), and used such that.
- the fraction resulting from this chromatography comprises, in fact, more than 50%, and even more than 56%, of OVAX ( Figure 1, track 5 -HB-EW, task around 45-50 kDa). It is the quantification of the pixels of the 50 kDa band of the gel relative to the total pixel number of the well which made it possible to estimate the quantity of the OVAX molecule in this fraction at least 50%, and even at least 56%, in proportion.
- OVAX in the other bands, but the OVAX being then with other molecules, we can not quantify the part due to the OVAX
- this fraction in the food industry or in the medical field, may be direct, since the egg is a foodstuff commonly used by humans, and whose safety, except for people with allergies, is recognized.
- all or part of the molecules of the fraction according to the invention can also be obtained by chemical synthesis or in the form of a recombinant protein, in particular from the sequence corresponding peptide, since the genome of the hen has already been fully sequenced.
- the subject of the invention is also a composition comprising the fraction of peptides and / or proteins according to the invention as active principle against Listeria monocytogenes.
- the fraction of peptides and / or proteins according to the invention can be used as an active ingredient against Listeria monocytogenes, for the production of a medicinal, food or other composition.
- the total concentration of peptides and / or proteins derived from egg white and able to bind the heparin in the composition may be between 15 g / mL and 400 ⁇ g / mL, and preferably between 20 g / mL and 100 g / mL. Even more preferably, the concentration of these molecules in the composition is between 25 g / ml and 60 g / ml.
- total concentration is meant the cumulative concentration of each of the peptides and / or proteins according to the invention present in the fraction used to produce the composition.
- the fraction present in the composition is digested with at least one digestive enzyme, preferably with trypsin or with chymoptrypsin, prior to its use.
- composition based on the peptide and / or protein fraction according to the invention may in particular be intended for the manufacture of an anti-Listeria monocytogenes drug.
- a composition may be a solution intended to be ingested, and containing the required excipients.
- the composition may be a powder, and in particular an egg white powder optionally enriched in the fraction according to the invention, to be subsequently suspended, or to be packaged in capsule form.
- composition according to the invention can also be used for the manufacture of food, intended for animal feed, human or non-human, as an anti-Listeria monocytogenes food additive.
- the composition is then used during the method of manufacturing the food product, said composition being added to the preparation intended to form the food product, during its preparation.
- the composition can be used as a food additive during the preparation of sausages such as pâtés, rillettes, sausages, etc.
- the composition may also be added to milk for use in making cheese.
- the composition is added to the preparation after any cooking step and / or any step in which said preparation is subjected to temperatures above 60 ° C.
- Figure 1 is a photograph of an SDS-PAGE gel showing protein migration from chicken egg white; thus, trypsin alone (T-lane), chymotrypsin alone (CT-lane), egg-white proteins as a whole (EW-lane), proteins of egg-white fraction not binding heparin (HUB-EW lane), proteins of the heparin-binding egg-white fraction (HB-EW lane), proteins of the heparin-binding egg white fraction after digestion with trypsin ( HB-EW-T lane), and heparin-binding proteins of the egg white fraction after chymotrypsin digestion (HB-EW-CT lane).
- Figure 2 is a photograph of an SDS-PAGE gel of the different steps of a first mode of OVAX purification; the first track (1) represents the egg white; the second track (2) the eluted fraction of heparin-sepharose; the third lane (3), the unbound fraction of biotin-sepharose; and the fourth track (4) the peak corresponding to the OVAX after molecular sieving.
- FIG. 3 is an alignment of amino acid sequences between the OVAX sequence from the NCBI database (OVAXdb, XP_418984.2 Gl: 118086485) and the sequence predicted by the Genemark software of the Institute of Technology. from Georgia (http://exon.gatech.edu/eukhmm.cgi) (OVAXgs) from the nucleotide sequence corresponding to the OVAX gene (reference NW_001471638.1 in the NCBI database).
- FIG. 4 comprises two graphs (FIGS. 4A and 4B) respectively showing the antimicrobial activity in liquid medium of Ovalbumin and of OVAX resulting from the first purification mode with respect to Listeria monocytogenes.
- the growth of Listeria monocytogenes was followed at 600 nm for 20 hours without Ovalbumin / OVAX or with increasing concentrations of Ovalbumin / OVAX.
- Figure 5 shows five graphs ( Figures 5A to 5E) showing the anti Listeria monocytogenes activity, in liquid medium, of different egg white samples at different concentrations.
- Figure 5A shows the anti Listeria monocytogenes activity of pure egg white
- Figure 5B shows the anti Listeria monocytogenes activity of the non-heparin-binding egg white fraction
- Figure 5C shows the anti Listeria monocytogenes activity of the heparin-binding egg white fraction
- Figure 5D shows the anti Listeria monocytogenes activity of the heparin-binding egg white fraction, after digestion with trypsin
- Figure 5E shows the anti Listeria monocytogenes activity of the heparin-binding egg white fraction after digestion with chymotrypsin.
- FIG. 6 is a photograph of an SDS-PAGE gel made at the end of a second purification mode of the OVAX, the only track representing the final fraction obtained.
- FIG. 7 is a photograph of an agar medium showing the anti Listeria monocytogenes activity of the OVAX from the second purification mode, at different concentrations, relative to Beta-defensin 11 (positive control) and Ovalbumin (negative control)
- the egg whites were collected from several eggs from laying hens (Isa-Hendrix, St Brieuc, France) and diluted 1 ⁇ 2 in 50 mM Tris-HCl buffer, 150 mM NaCl, pH 7.4.
- the sample was gently homogenized and centrifuged at 14,000 g for 10 minutes at 4 ° C.
- Heparin-Sepharose chromatography was performed using the "batch” technique according to the supplier's instructions (GE Healthcare brand, distributed by Fischer Scientific, W7346E).
- the proteins without affinity for heparin-sepharose are removed by successive washings in 50 mM Tris-HCl buffer, 150 mM NaCl, pH 7.4 and centrifugation of the beads for 5 minutes at 1500 g. at 4 ° C.
- the beads are then loaded onto two 5-mL polypropylene columns (Qiagen, Courtaboeuf, France, 34964).
- the elution of the affine proteins is carried out in 50 mM Tris-HCl buffer,
- the most concentrated elution fractions are pooled and dialyzed for 24 hours against 50 mM Na Phosphate buffer, 50 mM NaCl, pH 7.4 (PBS) in dialysis membranes (3500 Da exclusion) (Brand Spectrum, distributed by Fischer Scientific, 132720).
- the sample is centrifuged at 10000 g for 10 minutes at 4 ° C.
- the supernatant is then loaded onto a 1 mL polypropylene column, in which 1.5 mL of agarose beads coupled to biotin (Sigma-Aldrich, Saint Quentin Fallavier, France, B0519) were previously equilibrated in buffer PBS.
- the non-fixed column is recovered and concentrated on 3500 Da exclusion cell (Millipore, UFC900324). This step allows the elimination of avidin from the sample, the latter having a high affinity for biotin.
- the concentrated sample is then injected onto a TSK-gel filtration exclusion chromatography G3000SW (Tosoh Bioscience, Hampton, UK) pre-equilibrated in PBS buffer.
- the major peak corresponding to OVAX is collected and concentrated on 3500 Da exclusion cell (Millipore, UFC900324).
- Proteins were separated by SDS-PAGE under reducing conditions and stained with Coomassie blue.
- the 45 kDa major band was extracted from the gel and rinsed with water and acetonitrile.
- Proteins were reduced with dithiothreithiol, alkylated with iodoacetamide and digested with trypsin (12.5 ng / L) (Roche Diagnostics GmbH, Mannheim, Germany) overnight at 37 ° C in 25 mM NH 2 HCO 3 buffer. according to the protocol described by Shevchenko et al.
- the peptide extracts were then dried.
- MALDI-TOF mapping (Matrix Assisted Laser Desorption-lonization-Time of Flight)
- NanoLC-MS / MS analysis was conducted using a CapLC system coupled to a quadrupole time-of-flight hybrid mass spectrometer (Q-TOF Ultima Global, Waters, Manchester Great Britain) equipped with a source of Z-spray ion.
- the device is calibrated with a solution of GluF at 500 fmol / L (50% of an acid solution formic 1% / 50% acetonitrile, v / v).
- the samples are desalted and concentrated in line by a precolumn (Monolithic trap column, 200 ⁇ id X 5mm (PS-DVB), Dionex, ref 163972).
- the peptides were separated on a reverse phase capillary column (Acclaim PepMap 100 C18, 5 ⁇ , 75 ⁇ m I.D., 25 cm long, Dionex) with a flow rate of 200 nL / min.
- the gradient profile is as follows: Equilibration of the columns with 95% solvent A (0.1% formic acid / 2% acetonitrile / 98% H2O, v / v) and 5% solvent B (0.1% formic acid / 20% H2O / 80% acetonitrile, v / v), Gradient from 5 to 55% B in 80 min; Bearing at 95% B for 10 min.
- the acquisition of data is done automatically between the MS and MS / MS (fragmentation) modes: an MS survey scan is followed by three MS / MS scans on the three peaks detected the most intense.
- the collision energy is selected based on the mass and charge of the ion.
- the raw data is processed by ProteinLynx Global Server V 2.2.5 (PLGS) software to create .pkl files with all precursors selected for fragmentation as well as the list of associated fragment ions.
- PLGS ProteinLynx Global Server V 2.2.5
- the sample is sonicated a few minutes before depositing on the target.
- the matrix used is a solution of CHCA ( ⁇ -cyano-4-hydroxycinnamic acid) at 5 mg / ml in 50% ethanol / 50% acetonitrile / 0.1% TFA / 10 mM 18-C-6 crown ether.
- the MALDI-TOF device was calibrated externally with peptides from tryptic digestion of BSA (Bovine Serum Albumin) at 300 fmol / L.
- the MALDI-TOF spectra are obtained using a MALDI L / R P / N mass spectrometer (Waters, Manchester, UK).
- the analyzes were carried out: in positive mode, in reflectron mode, with an acceleration voltage of 15 KV, draws Voltage: 2226, with a mass range (m / z) of 500 Da to 5000 Da.
- the computer processing of the spectra is done using the software MassLynx V4.0 (Waters).
- MS / MS were confronted with a non-redundant database (NCBInr 20071102, then 20110403) and using the MASCOT software (Matrix Science UK, http://www.matrixscience.com).
- Enzymatic specificity was established as trypsin, including two missing cleavages.
- variable modifications were selected: carbamidomethylcysteine and oxidation of methionine.
- the search was performed in all taxonomies. Mass accuracy was set to ⁇ 0.3 Da.
- the candidate proteins were validated when the individual scores of the ions had a level of significance greater than p ⁇ 0.05.
- Proteins were separated by SDS-PAGE under reducing conditions and transferred to polyvinylidene membrane.
- the major band (40 kDa) was excised from the gel and analyzed by Edman sequencing.
- amino-terminal sequence of the protein was determined by automatic degradation of Edman using the LF 3000 sequencer (Beckman / Porton) equipped with an on-line Gold HPLC System HPLC system, Beckman Coulter) for the detection of derivatives amino-acids-Phenylthiohydantoin.
- the prediction of the OVAX sequence was made from the gene sequence available on the site "Ensembl” (http://www.ensembl.org/Gallus_gallus/lnfo/lndex, ENSGALG00000019551), then later from the sequence referenced in the NCBI database under number NW_001471638.1, using the Genemark software (Georgia Institute of Technology (http://exon.biology.gatech.edu/).
- EW Egg white
- HAB-EW non-heparin binding
- HB-EW heparin binding
- the protein concentration was determined using the Protein DC test (Biorad, Saint-Quentin, France) with bovine serum albumin (Interchim, Montlucon, France) as a control.
- HB-EW samples (0.9 mg / mL) were digested with either 5.5 ⁇ trypsin (HB-EW-T) or 1 ⁇ M chymotrypsin (HB-EW-CT).
- the bacterial strain EGD of Listeria monocytogenes was preserved by freezing in liquid nitrogen in the medium Broth Brain Heart (Oxoid, Hampshire, England) containing 15% (v / v) glycerol.
- the bacterial strain was cultured overnight at 37 ° C in Tryptone Soy broth (TSB, Oxoid) without shaking.
- the culture was then incubated at 37 ° C with shaking and the optical density was measured regularly at 600 nm to obtain mid-exponential phase bacteria (about 1. 8 CFU.mL- 1 after about 5 hours). 'incubation).
- bacterial growth was monitored in the presence of different concentrations of Ovalbumine or OVAX (0-400 g / mL) using Bioscreen C plate reader coupled to Biolink (Oy Growth Curves Ab) software. Ltd.) in the TSB culture medium.
- Each egg white fraction (EW, HUB-EW, HB-EW, HB-EW digested with trypsin or chymotrypsin) was added at different concentrations.
- EW, HUB-EW, HB-EW, HB-EW digested with trypsin or chymotrypsin was added at different concentrations.
- increasing concentrations of trypsin were added after two successive dilutions of the heparin-binding egg-white fraction (HB-EW) to obtain concentration of 0.9 g / ml of HB-EW and 5.5 ⁇ of trypsin.
- Figure 1 is a photograph of a gel of egg white proteins, at different levels of purification.
- the third track (EW) represents the migration of egg white proteins as a whole.
- the fifth track represents a fraction according to the invention, namely the fraction of heparin-sepharose-binding proteins and peptides of egg white.
- This first purification makes it possible in particular to eliminate ovalbumin, a major protein in the egg white (the fourth track, HUB-EW, shows the proteins of the egg white that does not bind heparin). It appears that this fraction contains mainly a protein of molecular weight of about 45 kDa.
- the same band at approximately 45 kDa is obtained during the successive steps of purification of the protein fraction and peptides from the egg white and binding the heparin, as can be seen in FIG. 2.
- the purification on heparin-sepharose ( lane 2), followed by purification on biotin-agarose (lane 3) and gel filtration (lane 4) allows the protein of molecular weight 45 kDa to be isolated.
- Lane 4 represents a 92% pure protein, identified as OVAX.
- the majority of contaminants in this fraction represent OVAX multimers (high masses appearing on the gel).
- a western-blot analysis has shown that they react with anti-ovalbumin antibodies and disappear after reduction in the presence of beta-mercaptoethanol.
- the concentration of OVAX is estimated at about 0.5 mg / mL of egg white as a minimum, which makes it possible to envisage numerous applications, particularly in the field of the food industry, directly by purification of the protein from egg white. 1 / 3.2- Identification of the OVAX
- the identification by mass spectrometry and sequencing made it possible to isolate and identify a protein of sequence SEQ ID No. 1, corresponding to the previously isolated OVAX, which has a sequence that is almost identical to the predicted sequence already known (SEQ ID No. 2) from the bioinformatic analysis of the chicken genome, and available in the NCBI databases.
- sequence SEQ ID No. 1 (OVAXgs) has a difference over five amino acid residues with respect to the sequence SEQ ID No. 2 ( OVAXdb).
- the graphs in FIG. 4 make it possible to draw a parallel between the action over time of Ovalbumin (FIG. 4A) and OVAX (FIG. 4B) against Listeria monocytogenes, at increasing concentrations of these molecules, in a liquid medium. .
- Ovalbumin for concentrations below 400 g / mL.
- the effect observed at 400 g / mL is very weak.
- OVAX is observed to have an anti-Listeria monocytogenes activity of 25 mg / ml, at least during the first 10 hours of the test. With 50 g / mL of OVAX, the anti-Listeria monocytogenes activity persists beyond 15 hours.
- the graphs in FIG. 5 make it possible to draw a parallel between the anti-Listeria monocytogenes activity of the egg white (FIG. 5A-EW) and the fraction of egg white that does not bind heparin (FIG. 5B-HUB).
- -EW of the heparin-binding egg-white fraction
- FIG. 5D-HB-EW of the heparin-binding egg-white fraction
- Figure 5E-HB-EW-CT the fraction of heparin-binding egg white after digestion with chtmotrypsin
- Egg white like the egg-white fraction that does not bind heparin, have similar activity profiles. No significant anti-Listeria monocytogenes activity was observed, even at concentrations above 400 g / mL.
- An intermediate action against Listeria monocytogenes is obtained from 28 pg / ml, this action being near-optimal at 56 g / ml.
- a second mode of purification of the OVAX protein has been implemented, in order to obtain the protein with a higher degree of purity.
- the antimicrobial activity thus obtained was tested in agar medium, as detailed below.
- OVAX was purified from chicken egg white.
- Freshly laid chicken eggs (ISA Brown, Hendrix Genetics, Saint-Brieuc, France) were collected, broken, whites and yellows were independently sampled.
- the egg whites were pooled and the resulting mixture was diluted 1/2 in 50 mM Tris-HCl, 50 mM NaCl, pH 7.4 and homogenized. The mixture was then centrifuged at 14,000 rpm at 4 ° C for 10 minutes to remove the viscous proteins. The supernatant was stored at -20 ° C.
- the first purification step is a heparin-Sepharose type affinity chromatography: 30 ml of the supernatant obtained from egg whites prepared as indicated above were diluted by 1 ⁇ 2 in 50 mM Tris-HCl, 300 mM NaCl, pH 7.4, and incubated overnight at 4 ° C with heparin-coupled sepharose beads (GE Healthcare, Uppsala, Sweden).
- the sepharose beads were washed extensively with a buffer
- the eluted fractions were concentrated on concentration membrane (Millipore, Ireland, UFC800324) and the resulting sample was injected onto a Sephacryl S-100 High Resolution column, Hi-prep 16/60 (GE Healthcare, Uppsala, Sweden) pre-equilibrated in 50 mM Tris-HCl buffer, 150 mM NaCl, pH 7.4.
- the major peak containing OVAX was then passed on a biotin-Sepharose type affinity column pre-equilibrated in 50 mM Tris-HCl buffer, 150 mM NaCl, pH 7.4, in order to remove all traces of avidin in the sample, the OVAX being recovered in the unbound fraction of the biotin beads.
- the results show a heterogeneous band of 40 to 50 kDa apparent mass, the theoretical mass of the OVAX being 44.2 kDa.
- the peptides resulting from this digestion were analyzed by a nanoHPLC CapLC system (Waters, Manchester, UK) coupled to a Q-TOF Ultima Global mass spectrometer (Waters Micromass, Manchester, UK) equipped with a Z-ion source. -Spray.
- the apparatus was calibrated with a solution of GluF at 500 ⁇ mol / ⁇ l (50% of a 1% formic acid / 50% acetonitrile solution, v / v).
- the tubes containing the digestion products were brought to dryness and 15 ⁇ l of buffer A (0.1% formic acid / 2% acetonitrile / 98% H 2 O, v / v) were added. Samples were desalinated and concentrated online by a precolumn (Monolithic trap column, 200 ⁇ id X 5mnn (PS-DVB), Dionex, ref 163972).
- the peptides were separated on a reverse phase capillary column (Acclaim PepMap 100 C18, 5 ⁇ , 75 ⁇ I.D., 25 cm long, Dionex) with a flow rate of 200 nL / min.
- the gradient profile is as follows:
- the raw data was processed by ProteinLynx Global Server V 2.2.5 software (PLGS) to create .pkl files containing all precursors selected for fragmentation as well as the list of associated fragment ions.
- PLGS ProteinLynx Global Server V 2.2.5 software
- the experimental data was compared to a database using the MASCOT software available on the local server.
- the anti-L activity / 'ster / a monocytogenes OVAX purified as described above was measured by the radial diffusion test according to the method described by Lehrer et al.
- Beta-defensin 11 was used as a positive control (Hervé et al).
- Bacteria Listeria monocytogenes were incubated overnight with shaking at 37 ° C. The preculture obtained was then diluted in TSB (Tryptone Soy Broth) until the optical density measured at 600 or 620 nm was 0.02 so as to standardize the culture conditions. The culture was incubated for 4 hours at 37 ° C. with stirring to to obtain a culture in exponential growth phase. The bacteria were then centrifuged at 900 g for 10 min at 4 ° C., then washed once with cold 10 mM sodium phosphate buffer (pH 7.4) and resuspended in 10 ml of this same buffer.
- TSB Teryptone Soy Broth
- This bacterial suspension was then measured so as to determine the volume to be taken to obtain 7.5.10 6 cfu of bacteria.
- This latter volume was mixed with 25 ml of "underlay" medium (10 mM sodium phosphate buffer, pH 7.4, containing 0.03% [w / v] BH I, 1% [w / v] agarose weak electro-endosmosis [Sigma-Aldrich, St Quentin Fallavier, France] and 0.02% Tween-20) preheated to 46 ° C.
- underlay medium 10 mM sodium phosphate buffer, pH 7.4, containing 0.03% [w / v] BH I, 1% [w / v] agarose weak electro-endosmosis [Sigma-Aldrich, St Quentin Fallavier, France] and 0.02% Tween-20
- the agarose solution containing 7.5 ⁇ 10 6 cfu of bacteria was cast into a petri dish to form a uniform 1 mm layer. 36 wells (3 mm in diameter) regularly spaced were dug into the agar using a punch.
- the petri dish was incubated at 37 ° C for 3h, to allow the molecules to diffuse into the gelose medium containing the bacteria.
- the first layer of agar medium (underlay) was then covered with 25 ml of "overlay" medium (10 mM sodium phosphate buffer pH 7.4, containing 6% [weight / volume] BHI and 1% [weight / volume] agarose ). After overnight incubation at 37 ° C, the diameter in mm of the inhibition zone around each well was determined by the difference between the diameter of the clear zone around the well and the diameter of the well. The larger the diameter, the more active the molecule is against Listeria monocytogenes.
- ovalbumin gene family Structure of the X gene and evolution of duplicated gene split. Cell 20: 625-37.
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FR1054388A FR2960877B1 (fr) | 2010-06-04 | 2010-06-04 | Fraction de proteines et peptides issus du blanc d'oeuf et proteine issue du blanc d'oeuf et leur utilisation comme agent anti-listeria |
PCT/EP2011/059126 WO2011151407A1 (fr) | 2010-06-04 | 2011-06-01 | Fraction de protéines et peptides issus du blanc d'oeuf et protéine issue du blanc d'oeuf et leur utilisation comme agent anti-listeria |
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US (1) | US20130129709A1 (fr) |
EP (1) | EP2576608A1 (fr) |
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CA2801129A1 (fr) | 2011-12-08 |
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US20130129709A1 (en) | 2013-05-23 |
WO2011151407A1 (fr) | 2011-12-08 |
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