WO2011004129A1

WO2011004129A1 - Use of a co-product from a method for extracting lysozyme from egg whites, in order to obtain at least one basic egg white protein

Info

Publication number: WO2011004129A1
Application number: PCT/FR2010/051450
Authority: WO
Inventors: Françoise NAU; Catherine Guerin-Dubiard; Thomas Croguennec; Maryvonne Pasco
Original assignee: Institut Supérieur Des Sciences Agronomiques, Agroalimentaires, Horticoles Et Du Paysage (Agro Campus Ouest)
Priority date: 2009-07-09
Filing date: 2010-07-08
Publication date: 2011-01-13
Also published as: EP2451825A1; FR2947818A1; FR2947818B1

Abstract

The present invention relates to the use of a co-product from a method for extracting lysozyme from egg whites, in order to obtain at least one basic egg white protein. Said co-product is a solution obtained from a step of extracting pre-crystallized lysozyme having a basic pH from an eluate containing the egg white protein fraction kept on a cation exchange chromatography substrate. The present invention also relates to a method for purifying at least one basic egg white protein, including an operation of extracting and purifying said basic protein using said co-product from a method for extracting lysozyme from egg whites.

Description

USE OF A COPRODUCT FROM A METHOD OF EXTRACTING LYSOZYME A

FROM EGG WHITE, FOR OBTAINING AT LEAST

A BASIC PROTEIN OF EGG WHITE

FIELD OF THE INVENTION

The present invention relates to the field of obtaining proteins, and in particular the extraction and purification of proteins.

The invention relates more specifically to the use of a product derived from egg white, for obtaining, advantageously by extraction and purification, basic proteins of egg white, in particular avidin.

PRIOR ART

Egg white, or albumen, is a naturally occurring blend that typically comprises about 88% by weight water and a dry matter content of about 12% by weight. The dry matter consists mainly of proteins (more than 90% by weight, relative to the total weight of dry matter).

Some egg white proteins are commonly used because of their biological activity.

This is particularly the case of so-called "basic" egg white proteins, having an isoelectric point (or "pi") greater than 7.

In particular, lysozyme (whose pi is of the order of 10.7) and avidin (whose pi is of the order of 10) can be cited, which are the subject of numerous studies and a exploitation on an industrial scale.

As an indication, the egg white has an avidin content close to 0.05 g per liter, and a lysozyme content close to 3.5 and 4 g per liter. The mass ratio of the "lysozyme plus avidin" group, relative to the total egg-white proteins, is about 0.04.

Lysozyme is a natural enzyme that was discovered and characterized in the 1920s.

Lysozyme is used in the pharmaceutical industry for its antibacterial activity against gram-positive bacteria. It alters the cell wall of gram-positive bacteria, resulting in cell lysis and death. Lysozyme is effective against a number of bacteria that cause infections in the human body.

The anti-bacterial activities of lysozyme are also used to reduce or block the deterioration of certain foods. One of the most important commercial applications of lysozyme is the ripening of certain cheeses, for example cooked pressed dough cheeses. Lysozyme inhibits the growth of Clostήdium tyrobutyricum, a bacterium that can contaminate milk. The presence of this bacteria in the cheese in formation in particular, the production of butyric acid, which causes an abnormal flavor, and the generation of an excessive concentration of hydrogen and gas in the mass of the cheese paste causing cracks and cracks in said mass.

Lysozyme is also used today to replace sulphites in winemaking processes. Lysozyme inhibits lactic acid bacteria and controls malolactic fermentation. It improves the organoleptic properties (color and taste) of the final wine. In addition, the final wine that is free of sulfite is better tolerated by consumers.

Avidin, which is also found in egg white, is known primarily for its ability to form a complex with biotin. This avidin-biotin complex has been widely used in biotechnology applications, especially for medical diagnosis.

It follows from the foregoing that pharmaceutical and food industry manufacturers have growing needs for basic egg white protein, especially lysozyme and avidin.

The accessibility and the composition of the egg white, and in particular that derived from chicken egg, make it a raw material of choice for the preparation and obtaining of such basic proteins.

However, the extraction of such basic proteins directly from the egg white has certain limitations and disadvantages.

This is particularly the case for processes for the extraction and purification of avidin from egg white. Indeed, this approach does not allow today production of this protein in quantity, quality or cost price sufficient to meet the demand of manufacturers.

More generally, starting from egg white as raw material, avidin purification processes are often complex and expensive to implement, and generate large volumes of waste.

By way of example, avidin purification methods based on cation exchange chromatography techniques are known for the extraction and purification of avidin from egg white.

The Canadian patent document No. ⁰ CA- 1283072 discloses a process for the isolation and separation by chromatography, lysozyme and avidin from egg white. This method comprises repeating a cycle comprising up to twenty times the steps of (i) contacting the egg white with a weakly acidic cation exchange resin so that lysozyme and avidin are adsorbed on said resin then (ii) contacting the resin with a low ionic strength elution buffer, which elutes the lysozyme from the resin while the avidin remains adsorbed. This process ends with the contacting of the resin containing avidin, with a high ionic strength elution buffer eluting this accumulated avidin by adsorption.

One of the disadvantages of the above method is that it requires the treatment of a large volume of egg white, and that it involves the implementation of a large number of steps (corresponding to the repetition cycles of loading the egg white on the resin), to obtain a significant amount of protein of interest.

In addition, the degree of purity of the avidin thus produced is not very high.

Patent Document No. CA-2 431 773 also discloses a process for the preparation of avidin from egg white. This process comprises (i) a step of lyophilizing an egg white homogenized with a buffer followed by (ii) a step of solubilizing the lyophilized egg white in a buffer followed by (iii) a step incubating the solubilized lyophilized product with a cation exchange resin material, then (iv) a filtration step to separate the resin material from the incubation medium, and (v) a step of selectively eluting the avidin using a colored molecule for which avidin has a high affinity.

In order to obtain a final preparation of purified avidin, the above method, which comprises a large number of steps, requires the implementation of a final step of removal of the added dye for the production of the purified avidin. elution stage.

An alternative is the extraction of avidin by affinity chromatography from egg white (Heney and Orr, Anal Biochem, 14: 92-96, 1981. Airenne et al., Gene, 144: 75 80, 1997, Kusnadi et al., Biotechnol Prog, 14: 149-155, 1998, Hood et al., Adv Exp Med Biol, 464: 127-147, 1999).

But large-scale avidin extraction would require the use of large volumes of affinity chromatography media; however, these supports are very expensive, and this technique is rather used at the laboratory scale.

Known methods require the use of a large volume of egg white to obtain the desired amounts of proteins of interest. In addition, the use of egg white generates large volumes of waste derived from egg white, which would be economically important to value.

Thus, with the known methods, the use of egg white as a starting material for obtaining certain basic proteins such as avidin results in the implementation of numerous, complex and / or expensive steps.

There is thus a need for novel processes for obtaining, in particular by extraction and purification, certain egg-white proteins, in particular basic proteins such as avidin, which are alternative or improved over known processes, advantageously with a lower cost compared with known methods. SUMMARY OF THE INVENTION

New methods are provided according to the invention for obtaining purified egg-white proteins, including purified basic egg-white proteins, such as purified avidin, which are advantageous over known methods.

The subject of the present invention is in particular the use of a co-product for extracting lysozyme from egg white, for obtaining (more preferably for the extraction and purification) basic proteins of egg white, which coproduct is a solution (advantageously in the form of a brine) obtained at the end of a step of extraction of the lysozyme previously crystallized at basic pH from an eluate containing the egg white protein fraction retained on a cation exchange chromatography support.

Preferably, this coproduct consists of a filtrate obtained at the end of a filtration step of the eluate containing the egg white protein fraction retained on cation exchange chromatography support, said filtration step being adapted to purify in the filtration retentate, the lysozyme previously crystallized at basic pH.

The present invention also relates to the use of the co-product in concentrated and desalted / demineralized form, which verifies the following characteristics:

a conductivity less than 50 mS / cm,

an avidin concentration greater than 1 g / l,

a mass ratio (lysozyme + avidin) / total proteins greater than 0.1 and

an avidine / lysozyme mass ratio greater than 0.1.

Another subject of the present invention relates to the process for purifying at least one basic egg-white protein, comprising an operation for extracting and purifying said basic protein from a coproduct resulting from a process extraction of lysozyme from egg white, which coproduct is a solution obtained at the end of a step of extraction of lysozyme previously crystallized at basic pH from an eluate containing the protein fraction of white d egg retained on a cation exchange chromatography support.

The coproduct is advantageously a filtrate obtained at the end of a filtration step of an eluate containing the egg white protein fraction retained on a cation exchange chromatography support, said filtration step being adapted to purify the lysozyme previously crystallized at basic pH in the filtration retentate.

This co-product, used in the process, is advantageously obtained by the following succession of steps:

(a) passage of the egg white on a cation exchange chromatography support for the retention of an egg white protein fraction containing the lysozyme,

(b) eluting the lysozyme-enriched protein fraction retained on said chromatographic support during step (a), using a brine solution,

(c) recovering the lysozyme enriched eluate from step (b), (d) crystallization at basic pH of the lysozyme contained in said eluate obtained in step (c),

(e) filtering said eluate to purify the crystallized lysozyme in the retentate, and

(f) recovering the filtrate from step (e), forming the co-product of interest.

More preferably, this co-product has the following characteristics:

a conductivity of between 60 and 100 mS / cm,

an avidin concentration of between 0.05 and 0.8 g / l,

a mass ratio (lysozyme + avidin) / total proteins, greater than 0.1 and

an avidine / lysozyme mass ratio, greater than 0.1.

More preferably, the coproduct used in the process is in a concentrated and desalted / demineralized form.

In this case, the concentrated and demineralized coproduct advantageously satisfies the following characteristics:

a conductivity less than 50 mS / cm,

an avidin concentration greater than 1 g / l,

a mass ratio (lysozyme + avidin) / total proteins, greater than 0.1 and

an avidine / lysozyme mass ratio, greater than 0.1.

The present invention also provides a composition for the extraction and purification of at least one basic egg-white protein, consisting of a co-product from a lysozyme extraction process in a concentrated and desalted form, which co-product is a filtrate obtained at the end of a process for extracting lysozyme, in a concentrated and desalted form, which co-product is a solution obtained at the end of a step of extraction of the lysozyme previously crystallized at pH base from an eluate containing the egg white protein fraction retained on a cation exchange chromatography support. This coproduct advantageously consists of a filtrate obtained at the end of a filtration step of an eluate containing the egg white protein fraction retained on a cation exchange chromatography support, said filtration step being adapted to purify the lysozyme previously crystallized at basic pH in the filtration retentate.

The present invention also relates to a process for the purification of avidin, comprising an operation for extracting and purifying avidin from a composition containing, in particular, avidin and lysozyme (advantageously egg white or a product derived from egg white), which operation for extracting and purifying avidin comprising the following steps:

(a) a first pass on a cation exchange chromatography support with said composition, the loading of said support being effected beyond its fixing capacities so, on the one hand, to retain the lysozyme (advantageously all or at least minus approximately all lysozyme), and on the other hand, to recover the excluded fraction enriched in avidin and devoid of lysozyme, (b) a second passage on a cation exchange chromatography support with said excluded fraction obtained at the end of step (a), the loading of said support taking place in such a way that its attachment sites are occupied by the avidin (advantageously all these sites),

(c) eluting and collecting the purified avidin-containing eluate fraction retained on said support in step (b).

DESCRIPTION OF THE FIGURES

FIG. 1 shows the gel filtration HPLC profile (chromatogram with value of the optical density at 280 nm depending on the time in minutes), to evaluate the degree of purity of the avidin extracted by the process according to the invention.

FIG. 2 shows electrophoretic profiles distributed over five tracks, to evaluate the degree of purity of the avidin extracted by the process according to the invention: (1) co-product resulting from a process for extracting lysozyme in a form concentrated and desalted, (2) lysozyme standard, (3) avidin purified by the method according to the invention, (4) avidin standard, (5) lysozyme standard. The bands designated by marks A to D correspond respectively to: (A) ovotransferrin, (B) ovalbumin, (C) avidin and (D) lysozyme.

DESCRIPTION OF THE INVENTION

The Applicant has endeavored to find new processes for obtaining basic proteins of egg white, advantageously purified, the cost of which is moderate.

In this context, the Applicant has shown that a product derived from egg white, which is likely to be obtained as a co-product devoid of market value and which is generated during protein purification processes from white d egg, can be used in industrial processes for obtaining, and especially the extraction and purification, basic protein egg white.

The Applicant has in fact shown that one of the co-products or by-products resulting from a process for extracting lysozyme from egg white can constitute a starting product that can be used for obtaining (advantageously by extraction and purification ) basic egg white proteins, mainly avidin or an avidin / lysozyme mixture.

Thus, it has been identified according to the invention a starting material having a great interest for obtaining (advantageously obtaining by extraction and purification) at least one basic protein of egg white, mainly avidin or a mixture avidin / lysozyme.

The procedure for obtaining the lysozyme extraction coproduct which is used as a starting material for the production of egg white proteins, and (ii) the process for obtaining the egg white protein, will be described in detail below. egg white proteins, in particular avidin, in which the above co-product is used as starting material. Process for obtaining the co-product used as starting material

This starting material of interest consists of a by-product or co-product that is generated during the implementation of a process for extracting lysozyme from liquid egg white, said process for extracting lysozyme comprising:

(i) using egg white which is subjected to a cation exchange chromatography step in which a lysozyme-enriched protein fraction is retained on the chromatographic medium,

(ii) an elution step (advantageously by means of a brine solution) and recovery of the lysozyme-enriched protein fraction, and

(iii) a step of extraction of lysozyme by crystallization at basic pH, resulting in parallel with the production of a lysozyme-depleted by-product or co-product,

said by-product or coproduct corresponding to the product of interest which has been identified according to the invention as a starting product for obtaining a basic protein, advantageously purified, of egg white.

The co-product of interest, which is used according to the invention as a starting product in a process for obtaining a basic protein derived from egg white, is in the form of a solution obtained at the end of step (iii) above extraction of lysozyme crystallized at basic pH from an eluate containing the egg white protein fraction retained on a cation exchange chromatography support.

The co-product of interest advantageously consists of a solution of brine (an aqueous solution of a salt) having a basic pH, whose protein composition is different from that of egg white: it is a solution enriched in basic proteins, other than lysozyme, retained on chromatographic support cation exchanger.

The Applicant has found that this coproduct, nowadays not recovered and considered as waste, is particularly rich in certain basic proteins of egg white, and especially in avidin. It has been shown according to the invention that this co-product is a very attractive starting material for the extraction and purification of these proteins of interest.

More specifically, the co-product of interest, which is used according to the invention as a starting product for obtaining (and in particular purification) basic proteins of egg white, can be obtained by a process of lysozyme extraction comprising the following succession of steps:

(a) passage of liquid egg white on a cation exchange chromatography support, for the retention of an egg white protein fraction containing lysozyme (and also avidin),

(b) eluting the lysozyme-enriched protein fraction retained on said chromatographic support during step (a), using a brine solution, (c) recovering the lysozyme enriched eluate from step (b),

(d) crystallization at basic pH of the lysozyme contained in said eluate obtained in step (c),

(e) extracting crystallized lysozyme from said eluate, and

(f) recovering the solution from step (e), forming the co-product of interest.

It has been found according to the invention that the protein fraction enriched in lysozyme which is recovered in the eluate in step (c) also comprises other egg white proteins, and in particular avidin. It has also been shown that the co-product of interest recovered in step (f), which is depleted of lysozyme, has not been simultaneously depleted of other egg-white proteins. In particular, said co-product of interest was not simultaneously depleted in avidin.

The starting material of the lysozyme extraction process described above is egg white, still commonly referred to as albumen.

The egg white preferably comes from a bird's egg, preferably from a bird's egg selected from the chicken, the turkey, the guinea fowl, the goose, the duck, the quail, the pheasant, the pigeon and ostrich.

The egg white used as starting material of the above process is preferably from a chicken egg.

For carrying out the above lysozyme extraction process, the cation exchange chromatography support is selected from a weak acid type cation exchange support and a strong acid cation exchange support.

According to a preferred embodiment, the cation exchange step is carried out on a chromatographic support consisting of a rigid or semi-rigid gel based on hydrophilic polymers, such as a crosslinked or non-crosslinked agarose gel. crosslinked, a hydrophilic vinyl polymer gel, or a silica gel coated with a hydrophilic polymer, or a polystyrene support divinyl benzene, on which (s) hydrophilic polymer (s) are grafted groups thereof ( their) conferring properties of cation exchanger (s).

Among the functional groups that may be used for the grafting of the polymer (s), mention may be made of (i) the carboxymethyl groups of the CM-polysaccharide type for a weak cation exchange coating, and (ii) the sulfonate groups. or SP-polysaccharide methyl sulfonate (S) or sulfopropyl (SP) for a strong cation exchange coating, which is capable of adsorbing weakly ionized molecules.

The cation exchange chromatography support used in the above lysozyme extraction process is advantageously packaged in a container equipped with at least one starting egg white feed means, at least one means for discharging liquids and at least one means for agitating the chromatographic support particles in the liquid to be treated. This is a "batch" packaging of the chromatographic medium. The container in which the cation exchange chromatography support is arranged may also be equipped with one or more means for supplying buffer solution, in washing solution, or else in solution to regenerate the chromatographic support.

Preferably, prior to step (a) of passage on a chromatographic support, the liquid egg white is adjusted to a pH value of between 7 and 10, and preferably of the order of 8.5.

The cation exchange chromatography support is advantageously equilibrated at the same or approximately the same pH value as the adjusted pH value of the egg white.

In step (a), the lysozyme contained in the starting liquid egg white is retained on the cation exchange chromatography support, and the excluded fraction (not retained on the support) which is depleted in lysozyme is removed. container or chromatography column.

After step (a), and prior to step (b) of elution, the cation exchange chromatography support is subjected to one or more washing steps, preferably with demineralized water, in order to remove the protein or non-protein components of egg white not specifically retained on the chromatographic medium.

The brine solution used in step (b) advantageously consists of an elution solution containing at least one counter-ion selected from the following monovalent cations Na or NH ₄ , and / or the following bivalent cations Mg or Ca.

The salt concentration of this brine solution is advantageously adjusted so as to obtain a conductivity of between 70 and 80 mS / cm; it is for example adjusted to 5% NaCl.

In step (d), the crystallization of lysozyme is selective. It can be carried out by adding sodium hydroxide (NaOH), with stirring, until adjustment to a pH value of between 8 and 11, and preferably of the order of 10.

This step can be implemented in a reactor or "tank" of crystallization, well known to those skilled in the art.

According to a preferred embodiment of steps (e) and (f), the separation between (i) the crystallized lysozyme and (ii) the co-product of interest, is carried out by filtration of the eluate resulting from step (c). ): the crystallized lysozyme is found in the retentate (also called "lysozyme paste"), and the filtrate from step (e) is the co-product of interest depleted in lysozyme and enriched in avidin (also referred to as "brine out filter "in the present description).

The above filtration operation, for carrying out steps (e) and (f) of the lysozyme extraction process above, is for example carried out by means of a filter press.

Preferably, the filter press consists of a pressure band press filter whose membrane is advantageously a microfiltration membrane with a pore size of between 0.1 and 10 μm. Other methods and techniques known to those skilled in the art can still be envisaged for effecting the separation between the crystallized lysozyme and the other components of the eluate obtained at the end of step (c) of the process described above. above.

For example, a conventional method of separation by centrifugation can be mentioned. In this embodiment of steps (e) and (f) of the above method, the co-product of interest constitutes the supernatant or centrifugate; the crystallized lysozyme is collected in the centrifugation pellet.

In step (f) of the above process, the lysozyme extraction coproduct is obtained, which is useful for the processes for obtaining (advantageously for extraction and purification) basic proteins of egg white according to US Pat. invention, said co-product of interest advantageously having the following characteristics:

a conductivity of between 60 and 100 mS / cm,

an avidin concentration of between 0.05 and 0.8 g / l (this avidin concentration depends mainly on the number of recirculations of the brine solution in the lysozyme elution stage (b)),

a mass ratio (lysozyme + avidin) / total protein greater than 0.1 (generally between 0.1 and 2), and

an avidine / lysozyme mass ratio greater than 0.1 (generally between 2 and 25).

Process for obtaining a basic protein derived from egg white

By using said coproduct, which is enriched in particular with avidin, as starting raw material, it is possible to obtain (and advantageously extract and purify) at least one basic egg-white protein by a suitable method, which can also be designated " method of obtaining (advantageously purification) a basic protein of egg white "is detailed below in the present description.

Basic egg-white proteins, which are obtained by performing said method of obtaining a basic egg-white protein described hereinafter, may be used as such in any suitable industrial applications.

By "obtaining at least one basic egg-white protein" is meant primarily a purification process, and still mainly:

(i) separating a mixture of lysozyme and avidin, and the other protein components of said co-product of interest;

(ii) the separation between avidin and the other protein constituents of said co-product of interest;

(iii) the separation between lysozyme and the other protein constituents of said co-product of interest; and

(iv) separating a basic egg white protein, other than avidin or lysozyme, from the other protein components of said co-product of interest. The purification of at least one basic egg-white protein may include, in addition to the separation between the basic protein of interest and the other protein constituents of said co-product of interest, also the separation between the basic protein of interest and non-protein components of said co-product, such as sugars, mineral salts, amino acids, vitamins, etc.

The purification of at least one basic egg-white protein is said to be "selective" because one advantageously (i) is recovered a purified mixture of one or more basic egg-white proteins of interest, (ii) or a basic egg white protein of interest in purified form. In practice, purified avidin or purified lysozyme or a purified mixture of avidin and lysozyme is preferably recovered "selectively".

By "obtaining at least one basic egg-white protein", is also denoted in particular a so-called "enrichment" method for obtaining at least one basic protein of egg white in an enriched form. In particular, this method aims at modifying the ratio of at least one basic protein of interest contained in said co-product of interest, with respect to its other constituents.

In this case, we designate mainly:

(i) enrichment of lysozyme and avidin, relative to the other protein constituents of said co-product of interest;

(ii) avidin enrichment, relative to the other protein components of said co-product of interest;

(iii) enriching a basic egg-white protein, other than avidin or lysozyme, with respect to the other protein components of said co-product of interest.

The enrichment of at least one basic egg-white protein makes it possible in particular to recover the coproduct enriched in one or more basic proteins, in comparison with said coproduct before enrichment.

Any suitable protocol can be used for this purpose, according to methods used individually or in combination, such as fractionation by precipitation and / or filtration, chromatography methods, immunoaffinity techniques using monoclonal antibodies or specific polyclonal, etc.

In order to obtain basic egg white proteins, and in particular for extraction and purification, the lysozyme extraction coproduct is advantageously used in a concentrated and desalted / demineralized form, to obtain a product enriched in proteins. basics of interest.

This concentrated and desalted coproduct can be obtained by a process comprising the following steps:

(a) optionally preliminary debacterization of the lysozyme extraction coproduct, advantageously by microfiltration,

(b) adjusting the pH of said co-product with a mineral or organic acid, (c) concentration of the acidified coproduct in step (b), preferably by filtration (for example by ultrafiltration, nanofiltration or reverse osmosis), until a concentrated coproduct advantageously containing at least 1 g / L of avidin is obtained,

(d) demineralization / desalting of the concentrated coproduct from step (c), so as to obtain a concentrated coproduct whose salt content is reduced to a conductivity preferably less than 50 mS / cm.

In step (b), the pH is adjusted to a value less than 7.5, preferably to a value ranging from 7.5 to 2.5.

In some embodiments, the pH value is advantageously adjusted in step (b), so as to reach a value close to neutral (more preferably between 6.5 and 7.5), before being concentrated. by the filtration step (c).

The Applicant has furthermore shown that the acidic pH adjustment of the lysozyme extraction coproduct in step (b) of the process, preferably at a pH value ranging from 2.5 to 4.5 (preferably from the order of 3), makes it possible to significantly optimize the efficiency of the avidin concentration step (c) in the concentrated coproduct, in particular in the case of a filtration technique, and more preferably in the context of an ultrafiltration technique; and this makes it possible to obtain high concentration levels of avidin.

Thus, in certain embodiments, the pH value is advantageously adjusted in step (b), to a pH value ranging from 2.5 to 4.5 (advantageously of the order of 3), before be concentrated by the filtration step (c).

In step (c) when the concentration of the acidified coproduct is carried out by ultrafiltration, the ultrafiltration membrane preferably has a cut-off threshold greater than 10,000 Da.

The Applicant has discovered that a membrane whose cut-off point is 100 000 Da makes it possible to increase the avidin / lysozyme ratio of the retentate fraction by a factor of 10 to 50.

As described below, the adjustment of the avidin / lysozyme ratio advantageously depends on the protein of interest to be obtained subsequently.

The demineralization / desalting step (d) may be carried out using any technique known to those skilled in the art, for example by nanofiltration or ultrafiltration with diafiltration, using nanofiltration or ultrafiltration membranes. The demineralization / desalting step (d) may also be carried out by electrodialysis or by passage over an ion exchange resin.

In order to optimize the following operations of extraction and purification, the concentrated and demineralized / desalinated coproduct obtained in step (d) advantageously satisfies the following characteristics:

a conductivity less than 50 mS / cm (and preferably 2 to 15 mS / cm),

an avidin concentration greater than 1 g / l (and preferably from 3 to 15 g / l), a mass ratio (lysozyme + avidin) / total proteins greater than 0.1 (and preferably greater than 1), and

an avidine / lysozyme mass ratio greater than 0.1 (and preferably greater than 2).

The conductivity value is preferably measured using a conductivity meter.

The concentration of avidin is preferably determined by the colorimetric method with HABA (4-Hydroxyazobenzene-2-Carboxylic Acid) according to Green et al (Biochem J, 94: 23-24, 1965) modified by Durance et al (J Food Sci, 56 (3): 707-709, 1991).

Avidin is advantageously detected either by the HABA test or by polyacrylamide gel electrophoresis in the presence of SDS.

Lysozyme is for example detected either by filtration gel HPLC according to the method of Thapon et al (Sci ANm 2: 251-260, 1982), or by polyacrylamide gel electrophoresis in the presence of SDS.

The concentration of lysozyme is determined, for example, by measuring its lytic activity on Micrococcus lysodeikticus cells according to the Litwack method (Proc Soc Exp Biol & Med, 89: 104, 1955) and Prockop et al (New Eng J Med, 240 : 269, 1964).

The total protein concentration is determined, for example, by the Kjeldhal method.

The mass ratio (lysozyme + avidin) / total protein is calculated from the analytical data described above.

The subsequent use of the concentrated and desalted / demineralized coproduct obtained at the end of step (d) advantageously depends on its avidin / lysozyme ratio.

In practice, the fractions whose mass ratio avidin / lysozyme is high (that is to say preferably greater than 5), will be preferred for the purification and subsequent extraction of avidin.

Conversely, the fractions whose avid / lysozyme mass ratio is lower (that is to say advantageously less than 5), will advantageously be used for the subsequent exploitation of the properties of the mixture enriched in avidin / lysozyme. .

The concentrated and desalted product obtained at the end of step (d) can also be treated in such a way as to allow its conservation over time.

The preservation treatment, known to those skilled in the art, can be of the drying, freeze-drying type or conditioning and stabilization by refrigeration (temperature ranging from -5 ° C. to + ^{40 °} C.) or freezing (temperature below -5 ° C. more preferably below -20 ^° C., advantageously below -80 ^° C.).

The present invention also relates to a method for the purification of at least one basic egg-white protein, which is particularly suitable for the use of the lysozyme extraction coproduct described above as a starting material, said co-product of lysozyme extraction being advantageously in concentrated and demineralized form. The Applicant has in particular developed a method for extracting a basic protein from egg white in which a so-called "front-end" chromatography technique is used.

By "front-end" chromatography is meant a method of separating a compound of interest by chromatography according to which:

(i) providing a conditioned chromatography support in a column comprising (i) at least one means for continuously feeding said starting solution column containing said compound of interest, intended to be brought into contact with said support of chromatography and (ii) at least one means for discharging the liquid having been brought into contact with said chromatography medium,

(ii) said chromatography column is fed with a continuous stream of a solution containing a compound of interest to be extracted or purified, so that said solution is brought into continuous contact with the conditioned chromatography medium in the column; said solution having been in contact with the chromatographic medium being discharged from the column also continuously, and feeding the chromatography column with said solution being continued until the molecules of said compound of interest saturate said support chromatography and that said compound of interest is detected in the output stream of the column.

In other words, when a "front-end" chromatography step is carried out, the feed of the chromatography column is continued with the solution containing the compound of interest to be extracted or purified, to exceed the capacity fixing said support for said compound of interest.

Since the chromatographic medium is saturated and once a molecule begins to elute from the support, it is continuously present in the excluded fraction. Molecules with little or no affinity for the chromatographic support are recovered in the excluded fraction before the molecules having greater ability to be retained by the chromatographic support.

The Applicant has in particular developed a method of chromatography, in the frontal mode, making it possible to achieve very high levels of avidin purity (advantageously greater than 99%) in only two stages, and with satisfactory yields (advantageously 30 to 30%). 60% of the avidin present in the concentrated / demineralised coproduct, in particular the function of its avidin / lysozyme mass ratio: the higher the avidin / lysozyme ratio, the higher the extraction efficiency).

The method for extracting or purifying a basic protein from the egg white according to the invention consists of a three-step process in which two successive stages of chromatography are carried out, said extraction method comprising the following steps: (a) in a first step, loading a chromatographic support beyond its binding (or adsorption) capacity with a continuous stream of lysozyme extraction coproduct described above, so that the lysozyme retained on said support prevents the retention of avidin, which allows to obtain an excluded fraction depleted, or even deprived, of lysozyme ("délysozymée") and containing most or all of the avidin initially present in the volume of co-product put into contact with the chromatographic medium, and

(b) in a second step, loading a chromatographic support with the excluded fraction délysozymée obtained in step (a), where appropriate beyond the capacity of fixation (or adsorption) of said chromatographic medium, so that the binding sites (or adsorption) are occupied by avidin,

(c) eluting the avidin retained on the chromatographic support in step (b).

It has been shown in the examples that the above extraction method, which was developed by the applicant, allows to obtain a very pure avidin, and with a high yield.

According to a preferred embodiment of the method for extracting or purifying a basic egg white protein of the invention, the following steps can be implemented:

(a) a first pass on a cation exchange chromatography support with the lysozyme extraction coproduct in concentrated and desalted form, the loading of said support being carried out beyond its fixing capacities so, on the one hand , to retain the lysozyme, and on the other hand, to recover the part of the excluded fraction enriched in avidin and devoid of lysozyme,

(b) a second pass on a cation exchange chromatography support with said excluded fraction portion obtained at the end of step (a), the loading of said support being effected in such a way that its attachment sites (the case all sites) are occupied by avidin,

(c) eluting and collecting the eluate fraction containing the purified avidin retained on said support in step (b).

The cation exchange chromatographic support used in step (a) and in step (b) is chosen from a weak type cation exchange chromatographic support or a strong type cation exchange chromatographic support.

According to a preferred embodiment, the cation exchange step is carried out on a chromatographic support consisting of a rigid gel, or semi-rigid, based on hydrophilic polymers, such as an optionally crosslinked agarose gel, a gel of hydrophilic vinyl polymer, or a silica gel coated with a hydrophilic polymer, or a polystyrene divinyl benzene support, on which hydrophilic polymer are grafted groups conferring on said polymer and said gel cation exchange properties. Among the functional groups that may be used for the grafting of the polymer (s), mention may be made of (i) the carboxymethyl groups of the CM-polysaccharide type for a weak cation exchange coating, and (ii) the sulfonate groups. or SP-polysaccharide methyl sulfonate (S) or sulfopropyl (SP) for a strong cation exchange coating, which is capable of adsorbing weakly ionized molecules.

For example, the cation exchange chromatographic support is selected from the following group: S Hyper DF, S Ceramic Hyper DF, SP Spherodex LS, Streamline SPXL, CM Hyper DF, CM Sepharose, Biorex 70, Duolite C464, Amberlite IRC 50, CG -50, DP-1, IRC-72 and 64, Rexyn 102, Merck IV, Zeocarb 226, Diaion WK 10 and WK 11, IMAC Z-5, CP-3050, and Wofafit CP300.

According to a preferred embodiment, the cation exchange chromatography support used in step (a) is identical to that used in step (b); it suffices, for example, to regenerate the support between steps (a) and (b).

Preferably, prior to step (a), the concentrated and desalted coproduct is adjusted, on the one hand, to a conductivity value of between 5 and 60 mS / cm, preferably between 5 and 10 mS / cm, and on the other hand, at a pH value of between 7 and 10, and more preferably of the order of 8.5. This operation makes it possible to maximize the fixing capacity of the support and to favor the adsorption of the basic proteins, and in particular the lysozyme, and to exclude the other proteins contained in the concentrated and desalted coproduct. Preferably, the chromatography medium is balanced to conductivity and pH values that are identical or at least close to the conductivity and pH values of the product applied.

Following this step of adjusting the conductivity and the pH, the concentrated and desalted coproduct is advantageously subjected to a decantation, centrifugation or filtration step, in order to eliminate any insoluble constituents that may hinder the following chromatography steps.

Avidin and / or lysozyme are detected and / or quantified in the chromatographic output fluid.

This operation can be implemented by any technique known to those skilled in the art, and mentioned above.

During the first pass according to step (a), avidin and lysozyme are detected and / or quantified in the chromatographic output fluid (according to for example a technique described above, directly or in an excluded fraction) , so as to exclusively recover the excluded fraction containing avidin (and possibly other basic egg-white proteins) and lacking lysozyme (taking into account the sensitivity of the detection / quantification test).

Preferably, the excluded fractions recovered are those containing no trace of detectable lysozyme. These excluded recovered fractions are pooled to form the excluded fraction enriched in avidin and devoid of lysozyme.

According to other preferred embodiments of the extraction process, prior to step (b), the fraction excluded and collected at the end of step (a), which is enriched in avidin, is adjusted to a pH value of between 7 and 10, and preferably of the order of 8.5.

This operation makes it possible to maximize the fixing capacity of the support and to favor the adsorption of avidin, and to promote the exclusion of the other proteins contained in the medium.

Preferably, prior to step (b) of said method, the chromatography support is balanced to conductivity and pH values that are identical or at least similar to those of the product applied.

During step (b), the avidin is advantageously detected and / or quantified in the chromatographic output fluid (directly or in an excluded fraction), so as to interrupt or stop the passage as soon as a certain concentration of avidin is detected (corresponding to the saturation of the sites of attachment of the chromatography medium by avidin).

More preferably, the passage of the fraction excluded in step (b) is stopped when avidin is detected at the outlet of the chromatographic column.

Before step (c) of eluting and collecting the eluate fraction comprising the purified avidin, the chromatographic support can be rinsed, for example by means of water or a buffer (for example a buffer acetate).

The step (c) of eluting and collecting the eluate fraction comprising the purified avidin is carried out according to a technique known to those skilled in the art, advantageously by increasing the pH to values close to 10 with a glycine-NaOH or carbonate-bicarbonate buffer for example, or by increasing the ionic strength of the elution buffer with a saline solution (for example 0.5 M NaCl).

When the elution step (c) is carried out by increasing the ionic strength, the solution used advantageously consists of an elution solution containing at least one counterion chosen from the following monovalent Na or NH ₄ cations, and or the following divalent cations Mg or Ca.

For example, the solution used for elution may consist of a 0.5M solution of NaCl or KCl salt.

The use of the lysozyme extraction coproduct identified by the applicant (and described with its method of obtaining previously in the present description), as raw material starting in the present method of extraction in the front-end mode according to the present invention, is in practice of great industrial interest, because it allows to obtain a large amount of avidin, high purity (preferably greater than 99 % purity, according to a gel filtration chromatography technique). The overall yield of the above avidin extraction process and the material loss is not a critical aspect of the process, since the lysozyme-extractive coproduct that is used as the starting raw material was lacking, until present invention, of market value.

More generally, the conduct of step (a) of "front-end" chromatography, and where appropriate also of step (b), also makes it possible to avoid the problems of change of scale which are conventionally encountered for chromatographic processes, since there is no elution gradient here that it would be necessary to optimize. One can thus very simply pass from a chromatographic support of a few milliliters, to a chromatographic support of several liters, according to the quantities of avidin to be purified, without adaptation or substantial modification of the general characteristics of the process.

The realization of step (a), and where appropriate also of step (b), according to a "front-end" chromatography technique, also makes it possible to work with reduced volumes of chromatography medium. As an indication, up to about 25 to 30 g of purified avidin can be obtained using one liter of chromatographic support.

It is added that the two-step front chromatographic method can also be used to extract and purify the residual lysozyme contained in the extraction coproduct of the concentrated and desalted lysozyme.

To purify the residual lysozyme, a method comprising the following steps is used:

(a) loading a cation exchange chromatography medium with a lysozyme extraction coproduct in concentrated and desalted form, the loading of said support being effected beyond its fixing capacities so, on the one hand, to retain the lysozyme, and on the other hand, to recover the excluded fraction enriched in avidin and devoid of lysozyme,

(b) washing the chromatographic support on which the lysozyme is retained with a suitable washing solution, such as a 20 mM sodium phosphate buffer at pH 8.5,

(c) eluting the lysozyme retained on the chromatographic support by means of a suitable elution solution, for example by modifying the pH (for example by setting the pH to a value of between 10 and 11) or modifying the ionic strength (for example by means of a 1M NaCl solution), and

(d) collecting the eluate fraction containing the purified lysozyme, retained on said support during the first pass according to step (a) of the desalted and concentrated lysozyme extraction coproduct.

More generally, this two-stage frontal chromatography method can be implemented from any other solution containing a mixture of avidin and lysozyme, namely in particular egg white or any other product derived from white of egg. This solution advantageously contains an avidin / lysozyme ratio greater than 1.5. The starting material of the avidin extraction process according to the invention is also any recombinant solution originating from a bacterial or eukaryotic system.

EXAMPLES

EXAMPLE 1 Process for Concentrating and Demineralizing an Ivsozvme Extractive Co-product from Egg White

Material of departure

129.8 kg of a lysozyme extraction coproduct supplied by OVONOR (62232 ANNEZIN, France), having the following characteristics:

a conductivity of the order of 67 mS / cm,

an avidin concentration of 0.296 g / L,

a mass ratio (lysozyme + avidin) / total proteins, of the order of 0.1,

an avidine / lysozyme mass ratio of the order of 2,

a pH value of the order of 10.

This co-product was obtained by the following sequence of steps:

- adjustment of a chicken egg white to a pH value of 8.5,

passage of this acidified chicken egg white on a Purolite C106 EP cation exchange chromatography support (acrylic support grafted with divinyl benzene and COOH functional groups), in batch form,

rinsing the chromatographic medium with demineralized water,

elution with a 5% NaCl solution,

crystallization of the lysozyme contained in the elution solution, using a solution of NaOH with a pH adjustment of 10,

filtration of the saline solution of lysozyme crystallized on a filter press, using a microfiltration membrane.

Concentration and demineralisation of the starting material

The coproduct obtained is first acidified with a 5M hydrochloric acid solution, until its pH is adjusted to a value of the order of 3.15.

The acidified coproduct is then concentrated by ultrafiltration, using a membrane with a cutoff of 10 kDa ("Molecular Weight Cut Off" or "MWCO" 10 kDa), and the surface is 2.5 m ² .

This ultrafiltration operation is carried out with a retentate flow rate of 965 L / h, an inlet pressure of 1.85 bar and an average permeate flow rate of 20 L / h.

The acidified and concentrated coproduct is then subjected to an ultrafiltration membrane diafiltration operation, with an average permeate flow rate of the order of 40 L / h. The retentate obtained, enriched in basic proteins, has a weight of 11.84 kg and has the following characteristics:

a conductivity of 7.5 mS / cm,

an avidin concentration of 3.24 g / L,

a mass ratio (lysozyme + avidin) / total proteins, of the order of 0.1, and

an avidine / lysozyme mass ratio, of the order of 2.

EXAMPLE 2 Extraction of Avidin from the Concentrated and Demineralized Co-product of Ivsozyme Extraction

Material of departure

The extraction co-product used has the following characteristics:

an initial pH of 6.55

a conductivity of 2.54 mS / cm,

an avidin concentration of 7.48 g / L,

an avidine / lysozyme ratio of 3,

a mass ratio (lysozyme + avidin) / total proteins, of the order of 1.

Process for extracting and purifying avidin

The first chromatography was carried out on 200 ml of a S Ceramic Hyper DF resin column equilibrated at pH 8.43.

A volume of 2009 mL of concentrated and desalinated coproduct (ie 15.02 g of avidin) is loaded onto this chromatography column.

The results of the avidin and lysozyme assays on the excluded fractions, at the column outlet, are shown in Table 1 below.

Table 1: Avidin concentration and detection of lysozyme, in the excluded fraction

On leaving the support, a volume of 1075 ml of the excluded fraction is recovered, containing the proteins of the concentrated-desalted coproduct, in particular avidin, with the exception of lysozyme.

This excluded fraction contains 4.38 g / L of avidin, ie 4.71 g of avidin in total. The avidin extraction yield of this first chromatography is 32%.

This avidin-enriched fraction is then further loaded onto 200 ml of S Ceramic Hyper DF resin, now equilibrated to pH 8.

After loading the support, the resin is rinsed with a 20 mM acetate buffer of pH 5.

The avidin retained on the chromatographic support is then eluted with a 0.5M NaCl solution.

The volume of the eluted fraction enriched with avidin is 473 ml. Its avidin concentration is 9.1 g / L, or 4.31 g of avidin in total. The yield of this second chromatography is 91.6%.

The overall extraction yield of the process is 28.7%.

Evaluation of the degree of purity of avidin The degree of purity of avidin extracted is evaluated by gel filtration HPLC (TSK column

SW XL G3000, 50 mM phosphate buffer + 200 mM NaCl, pH 7, flow rate 0.7 mL / min, 280 nm detection). The degree of purity measured is 100% (Figure 1).

The purity of the avidin thus produced is also estimated by polyacrylamide gel electrophoresis in the presence of SDS. We then observe a single band (Figure 2, lane 3).

Claims

1.- Use of a co-product resulting from a process for extracting lysozyme from egg white, for obtaining at least one basic egg-white protein, which co-product is a solution obtained from the result of a step of extraction of lysozyme previously crystallized at basic pH from an eluate containing the egg white protein fraction retained on a cation exchange chromatography support.

2.- Use according to claim 1, characterized in that said co-product is obtained by the following succession of steps:

(c) recovering the lysozyme enriched eluate from step (b),

(f) recovering the filtrate from step (e), forming the co-product of interest.

3.- Use according to any one of claims 1 or 2, characterized in that said co-product has the following characteristics:

a conductivity of between 60 and 100 mS / cm,

an avidin concentration of between 0.05 and 0.8 g / l,

a mass ratio (lysozyme + avidin) / total proteins, greater than 0.1 and

an avidine / lysozyme mass ratio, greater than 0.1.

4.- Use according to any one of claims 1 to 3, characterized in that the co-product is used in a concentrated form and desalinated / demineralized.

5.- Use according to claim 4, characterized in that the concentrated and demineralized coproduct satisfies the following characteristics:

a conductivity less than 50 mS / cm,

an avidin concentration greater than 1 g / l,

a mass ratio (lysozyme + avidin) / total proteins, greater than 0.1 and

an avidine / lysozyme mass ratio, greater than 0.1.

6.- Use according to any one of claims 1 to 5, characterized in that the co-product is used to obtain purified avidin.

7. Process for purifying at least one basic egg-white protein, comprising an operation for extracting and purifying said basic protein from a co-product derived from a process for extracting lysozyme from from egg white, which co-product is a solution obtained at the end of a lysozyme extraction step crystallized at basic pH from an eluate containing the egg white protein fraction retained on a cation exchange chromatography support.

8. A process according to claim 7, characterized in that, prior to the operation of extraction and purification of the basic protein of interest, said co-product is subjected to a preparation operation comprising a concentration step and a step desalting / demineralization.

9. A process according to claim 8, characterized in that said concentration step is carried out by ultrafiltration, and in that, prior to said concentration step, said co-product is acidified in order to adjust its pH to a value less than 7. , 5, preferably at a value ranging from 7.5 to 2.5.

10. A process according to any one of claims 7 to 9, characterized in that the extraction and purification operation allows the purification of avidin.

1 1.- Method according to claim 10, characterized in that said operation of extraction and purification of avidin comprises the following steps:

(a) a first pass on a cation exchange chromatography support with said coproduct in concentrated and desalted form, the loading of said support being effected beyond its fixing capacities so as, on the one hand, to retain the lysozyme and on the other hand, to recover the excluded fraction enriched in avidin and devoid of lysozyme,

(b) a second passage on a cation exchange chromatography support with said excluded fraction obtained at the end of step (a), the loading of said support taking place in such a way that its attachment sites are occupied by the avidin

12. A process according to claim 1 1, characterized in that the cation exchange chromatography support used in step (a) is identical to that used in step (b).

13. A composition for obtaining at least one basic egg-white protein, characterized in that it consists of a co-product derived from a lysozyme extraction process, in a concentrated and desalted form, which coproduct is a solution obtained at the end of a step of extraction of the lysozyme previously crystallized at basic pH from an eluate containing the egg white protein fraction retained on a cation exchange chromatography support.

14. Composition according to claim 13, characterized in that it satisfies the following characteristics:

a conductivity less than 50 mS / cm,

an avidin concentration greater than 1 g / l,

a mass ratio (lysozyme + avidin) / total proteins, greater than 0.1 and

an avidine / lysozyme mass ratio, greater than 0.1.

15. A process for the purification of avidin, comprising an operation for extracting and purifying avidin from a composition containing avidin and lysozyme, which extraction and purification operation of the avidine consists of the following steps:

(a) a first pass on a cation exchange chromatography support with said composition, the loading of said support being effected beyond its fixing capacities so as, on the one hand, to retain the lysozyme, and on the other hand to recover the excluded fraction enriched in avidin and free of lysozyme,