FR2613725A1 - Process for the production of active lactoperoxydase and lactoferrin from whey and the substances produced by this process - Google Patents

Abstract

This process consists in: a adjusting the pH of the whey to a value close to neutral pH; b bringing the whey thus treated into contact with an anion exchanger; c separating from the whey the anionic exchanger upon which the lactoperoxydase and the lactoferrin are bound, then washing it in order to remove any other protein and the contaminants derived from the whey; and d releasing the lactoperoxydase by successive elutions with increase in pH, using a volatile base and using, as solution for elution, a solution of a volatile salt of the said base, and then, if desired, releasing the lactoferrin. In particular, there are used as volatile base, ammonium hydroxide and, as volatile salt of the said base, ammonium acetate or formate. The eluted lactoperoxydase can be obtained in a pure state directly by freeze-drying.

Description

 The present invention relates to a simple and advantageous method for obtaining active lactoperoxidase and lactoferrin from whey. This process is part of a program to promote cheese whey and caseinery.

 Indeed, it is known that the fraction having the highest value of the whey consists of the proteins it contains. Most often, these are recovered by ultrafiltration, or by thermocoagulation at acidic pH, to be used for food or feed. However, without rejecting this use, we can think that some of the proteins present in the whey, if they could be isolated from the latter, would have interesting outlets, especially those that have a biological activity and that can be active in concentrations very low.

 Among these proteins of interest are lactoperoxidase, the most abundant enzyme in cow's milk (10-30 mg / liter), which is found entirely in whey and retains its activity after normal pasteurization at 72 ° C. 'C, for 15 seconds, being destroyed more or less completely when the temperature is higher or the time longer.

To illustrate the interest of lactoperoxidase. several of its applications can be mentioned - it can thus be used as an additive for
milk in which the lactoperoxidase has been destroyed,
or for other dietary liquids. She permits
then to ensure a conservation of these liquids by
its bactericidal activity in the presence of hydrogen peroxide and
of thiocyanate, as described in the British patent
No. 1,468,405 - it can also be used in preparations
pharmaceuticals for the treatment of gastro-intestinal infections
intestinal in animals. We put there also
profit its bactericidal activity, following the same principle
only as above. British Patent No. 1,546,747
is concerned with such pharmaceutical compositions
bacterial.We also know food supplements
based on lactoperoxidase and lactoferrin
for the preventive treatment of bacterial diarrhea
in young ruminants - it is commonly used for the marking of
radioactive iodine proteins and for labeling
enzymatic antibody or antigen, in the
ELISA techniques (Enzyme Linked Immunosorbent Assay)
antigen or antibody assay - finally, lactoperoxidase can be used for esti
Boehringer blood glucose measurement
(W. Werner, HG Rey, H. Wielinger.
Congress "Biochemische Analytik 70", Munich, April 29
May 2, 1970), using glucose oxidase and
peroxidase.

Lactoperoxidase has been known for a long time because it has been isolated in the crystallized state by
Theorell, H. et al. Kemi, Mineral, Geol., 17B, 7 (1943) and 18A, 12 (1944), relatively simple techniques for preparation by chromatography on anionic supports have been available since 1957. Thus, can refer to the article by Mr. Morrison,
HB Hamilton and E. Stotz, entitled "The Isolation and Purification of Lactoperoxidase by Ion Exchange Chromatography, The Journal of Biological Chemistry, 28, <1957> 767. These techniques are based on the fact that only a few Milk proteins (lactoferrin, lactoperoxidase and immunoglobulins) bind to such pH-neutral media. Several methods of this type, allowing for laboratory preparation, have been published.

By way of illustration, mention may be made of the techniques reported by M. Morrison and DE Hultquist, The Journal of Biological Chemistry, 238, (1963) 2847; ML Groves, Biochimica and Biophysica Acta 100 (1965); KG Paul, PI Ohlsson and A. Henriksson, FEBS Letters 110, No. 2, (1980) 200; and by C. Dumontet and B. Rousset, The Journal of
Biological Chemistry, 253, <1983) 14116.

 Belgian Patent No. 214464 (French Patent No. 2,576,752) also describes a process for the purification of milk proteins whose isoelectric pH is greater than 7.5 (lactoferrin, lactoperoxidase and certain immunoglobulins), according to which the medium containing the desired protein, consisting of milk or whey, is passed over particles of a gellable acid polysaccharide taken in the form of a gel (alginates or carraghenates), the gel particles are washed on which have been fixed the desired proteins, with a saline solution of CaCl2 if it is an alginate gel, or KCl, NH4Cl or RbCl, if it is a carrageenan gel, the elution is carried out proteins by solutions of the same salt, and the proteins are recovered in solution. The originality of this process lies mainly in the preparation of the polysaccharide carrier and in the possible treatment of milk, which can be marketed later, since this process does not require the modification of the pH of the milk.

 It can thus be seen that the prior art does not provide a method for isolating lactoperoxidase by ion exchange chromatography on a commercially available support, in a simple manner and meeting an industrial need.

 The present invention now provides a solution; For this purpose, it proposes, in a conventional technique consisting of a treatment of whey with an anionic exchanger, followed by elution, original conditions of elution of the desired protein, after its attachment to the exchanger. In fact, unlike all the known processes in the laboratory or in industry in which an increase in the ionic strength <increase of the concentration in a salt at a constant pH is used to achieve elution, it is used, according to the present invention, elution by increasing pH. In addition, an eluting reagent is used which is volatile and, to achieve the pH increase, a volatile base. As a result, it is possible to obtain, after drying of the eluted solution, optionally after concentration by ultrafiltration. , pure lactoperoxidase, having retained all its enzymatic activity, free of salts. This shows the practical interest of the process of the invention in terms of industrial profitability and for the intended applications of lactoperoxidase, especially for pharmaceutical purposes.

The subject of the present invention is therefore a process for obtaining active lactoperoxidase and lactoferrin from whey, characterized in that it comprises the steps of: (a) bringing the pH of the whey to a value close to
neutrality <b) bring the whey thus treated into contact with a
anionic exchanger (c) separating from the whey the anionic exchanger on which
are fixed lactoperoxidase and lactoferrin, then
wash it to remove any other protein and
contaminants from whey; and <d) releasing lactoperoxidase by successive elutions
with pH increase, using a base
volatile and implementing, as a solution
of elution, a solution of a volatile salt of said
basis and, if desired, then release the
lactoferrin.

 The starting material used is cheese whey or casein, as obtained from raw milk or pasteurized milk, or whey concentrated by ultrafiltration, for example ten or more times, this second route. being preferred to the previous one.

 In step (a), the pH of the whey is brought to a value close to neutral, by a solution of ammonia, sodium hydroxide or potassium hydroxide.

The use of sodium or potassium hydroxide, which are not volatile bases, is however without disadvantage to achieve this neutralization; indeed, the lactoperoxidase is fixed in the same manner to the anionic exchanger as if an ammonia solution is used, since the washing, which will precede the elution, will eliminate the non-volatile salts. On the other hand, lactoserum freed from lactoperoxidase (as well as lactoferrin, which is eluted after lactoperoxidase) can be used just as well.

 In step (b), any anionic exchanger can be used; for example, a CM-Sepharose Fast Flow type exchanger (Pharmacia), equilibrated with a solution of pH close to 7, of the volatile salt also used in steps (c) and (d). ).

 In step <c), the whey is separated from the anionic exchanger and washed with a solution of the salt used in steps <b) - and (d)> the pH of the wash solution. being lower than those of solutions that elute, and greater than 7; it can be, for example, of the order of 8.5.

 The whey obtained at the end of the step (c 2), which still contains the main whey proteins, namely α-lactalbumin and 13-lactoglobulin, can be used in a conventional manner, such as untreated whey, in particular to recover the aforementioned proteins.

 As for the washing liquid, as well as the liquids from subsequent washings, it can be either recycled or used as a source of carbon and nitrogen in the industrial preparation of microorganisms, for example yeasts.

 In steps (c) and (d), ammonia is used as a volatile base to raise the pH and, as the volatile salt of said base, ammonium acetate or formate, indifferently.

 By the method of the invention, it is possible to obtain the pure and active lactoperoxidase, directly by freeze-drying or by spray-drying (spray drying) of the first eluate obtained in step (d) .This eluate can, however, before lyophilization, be subjected to a concentration by ultrafiltration, using, for example, mineral membranes.

 In addition, after elution of the lactoperoxidase, the lactoferrin still affixed to the column can be obtained by eluting with a solution in water of the base used above, at a higher pH. Lactoferrin can then be isolated in pure form, in the same way as lactoperoxidase.

 It can therefore be seen that the process of the invention has the advantage of a very limited number of operations, all of which can be carried out on the same column, for example in a fluidized bed. One could, likewise, perform the operations automatically continuously with two columns, passing from one column to the other when the first is saturated with lactoperoxidase, which is eluted, while the first column is under load.

 Finally, the technique according to the invention does not modify the whey, inconveniently, since the only treatment thereof is an adjustment of the pH to 7.0, whereby the treated whey can be reused in a controlled manner. conventional.

EXAMPLE
Ten times, by ultrafiltration, cheese whey is concentrated and its pH is brought to 7.0 with a commercial solution of concentrated ammonia (28), at a rate of approximately 90 ml for 5 l of retentate 10. whey in tank, an anionic exchanger of type CM
oe +
Sepharose Fast Flow, in NH4 form at pH 7.0, equilibrated with 80 mM ammonium acetate at pH 7. It is stirred gently for 10 minutes at room temperature, the filter exchanger is recovered, or otherwise, and washed on a filter, or in any other manner, with 80 mM ammonium acetate, the pH of which was brought to pH 8.5 with ammonia.

 The exchanger is then placed in a column. The lactoperoxidase is eluted alone with a solution of ammonium acetate 80 mM, brought to pH 10.5 with ammonia, and obtained pure directly by lyophilization.

 The exchanger is washed and regenerated in column with a 0.2 m ammonia solution, at pH 11.5, which elutes the lactoferrin still fixed on the column. This is recovered in the same way as lactoperoxidase.

 The exchanger is finally washed with ammonium acetate, 80 mM, pH 7, on a column to reduce its pH to around 7, for reuse.

Claims (11)

RLYSXXICATIONS  1 - Process for obtaining active lactoperoxidase and lactoferrin, from whey, characterized in that it comprises the steps of (a) bringing the pH of the whey to a value close to neutrality. contact whey so treated with a  anionic exchanger (c) separating from the whey the anionic exchanger on which  are fixed lactoperoxidase and lactoferrin, then  wash it to remove any other protein and  contaminants from whey; and (d) releasing lactoperoxidase by successive elutions  with pH increase, using a base  volatile and implementing, as a solution  of elution, a solution of a volatile salt of said  basis and, if desired, then release the  lactoferrin.  2 - Process according to claim 1, characterized in that one uses> as starting material, cheese whey or caseinerie, as obtained from raw milk or pasteurized milk.  3 - Process according to claim 1, characterized in that one uses> as starting material, concentrated whey by ultrafiltration.  4 - Method according to one of claims 1 to 3, characterized in that in step <a), the whey pH is brought to a value close to neutrality, using a solution of ammonia, sodium hydroxide or potassium hydroxide.  5 - Process according to one of claims 1 to 4, characterized in that in step <b), a balanced anion exchanger is used with a pH solution close to 7 volatile salt used in steps <c) and <d).  6 - Process according to one of claims 1 to 5, characterized in that in step <c), the anion exchanger is washed with a solution of the salt used in steps <b) and (d>, the pH of the washing solution being lower than that of the solution which elutes and higher than that of the neutralized whey.  7 - Process according to one of claims 5 and 6, characterized in that in steps <b), <c) and <d), ammonia is used as a volatile base for raising the pH, and, as volatile salt of said base, acetate or ammonium formate.  8 - Process according to one of claims 1 to 7, characterized in that one obtains the pure and active lactoperoxidase by direct lyophilization or by spray drying from the first eluate obtained in step <d).  9 - Process according to one of claims 1 to 7, characterized in that one obtains the pure and active lactoperoxidase by lyophilization of the first eluate obtained in step <d) and concentrated by ultrafiltration.  10 - Process according to one of claims 1 to 9, characterized in that in step <d), lactoferrin is obtained by eluting with a solution in water of the base previously used at a higher pH.  11 - Lactoperoxidase and lactoferrin obtained by the process as defined in one of claims 1 to 10.