CH619731A5 - Process for combatting the autooxidation of substances capable of being degraded by an oxidation involving superoxide ions - Google Patents

Abstract

The process consists in using, in combination with the oxidisable substances, a superoxide dismutase enzyme extracted from marine bacterial strains, from Escherichia coli, from fungi, or from blood. This process applies especially to the protection of foodstuffs and of the antioxidants employed in the food industry.

Description

The subject of the present invention is a method for combating the auto-oxidation of substances liable to be degraded by an oxidation involving superoxide ions, characterized in that said substances are associated with at least one superoxide dismutase enzyme.

This process applies in particular to the protection against auto-oxidation of lipids and of the compounds used as antioxidants in the food industry.

Superoxide dismutases have already been described, extracted from beef erythrocyte (Markovitz, J. Biol. Chem. 234, p. 40.1959) and from Escherichia coli (Keele and Fridovich, J. Biol. Chem. 245 , p. 6176, 1970).

Superoxide dismutases are enzymes capable of inducing the dismutation of superoxide ions, depending on the reaction:

202- + 2H + ^ H202 + O2

They therefore contribute to achieving a self-protection system for the articles or organisms in which they are found, since the O2- ions which are produced during the oxidation reactions under the effect of molecular oxygen are very active and attack between other proteins, by oxidation of tryp-tophane among other amino acids, and nucleic acids.

It has now been found that the superoxide dismutase enzymes of all origins act remarkably as inhibitors of oxidation of oxidizable substances, in particular as inhibitors of the oxidation of lipid, protein or lipoprotein foods, as well as other oxidizable substances and media.

For the purposes of the present invention, the term "oxidizable substance" means any substance capable of being degraded by an oxidation involving the intervention of superoxide ions.

The superoxide dismutases which have proved effective are for example prepared from marine bacterial strains, such as for example strains of Photobacterium phosphoreum, Photobacterium leiognathi or Photobacterium sepia, among others; but these are also all other superoxide dismutases, such as, by way of nonlimiting examples, those extracted from Escherichia coli, from fungi such as Pleurotus olearius or those extracted from blood, in particular erythrocupréins. Among these various strains, there may be mentioned the strains of Photobacterium phosphoreum no ATCC 11,040, Photobacterium leiognathi no ATCC 25,521, Photobacterium sepia no ATCC 15,709, Escherichia coli no ATCC 15,224 and Pleurotus olearius Gillet (Laboratory of Cryptogamy of Paris).

All of these microorganisms are known. In particular, Photobacterium leiognathi has been described by Boisvert, Châtelain and Bassot (Annals of the Institut Pasteur (1967), 112, p. 520-524). Photobacterium sepia has been described by Nicholas and Me El-roy in J. Gen. Microbiology (1964), 35, p. 401-410.

We have been able to determine the remarkable antioxidant effect obtained by these superoxide dismutases on fungi, apples and potatoes, among other foods. These tests are reported in some of the examples which will be found later.

However, techniques for assessing food quality that are versatile and truly objective cannot be developed at present; therefore, we have chosen to have tests also carried out on particular models, which have the advantage of constituting standard models of a large number of foods and food compositions and of being able to be analyzed objectively as to their integrity. Thus, as a model of inhibition of the auto-oxidation of lipo-proteins, the activity of self-oxidation inhibitor provided according to the invention by superoxide dismutases on lysine was tested. Yeast t-RNA ligase, model representing all classes of lipoproteins. Likewise, as a model of nucleo-proteins in their generality, the bacteriophage R 17, which is a ribonucleprotein, was used. In addition, the auto-oxidation of the pancreatic ribonu-clase enzyme was tested as a strictly protein character food model.

On the other hand, the efficiency of superoxide dismutases has also been established according to the invention for the protection against oxidation of bacteria exposed to ultraviolet radiation as well as for the protection of a medium called "Jef medium". fries ”, containing sodium tetrathionate and active for the enrichment of Salmonella.

As specified above, the enzyme superoxide dismutase used according to the invention can be extracted from any suitable source, such as: erythococcin, Escherichia coli, fungi such as Pleurotus olearius, marine bacterial strains such as Photobacterium phosphoreum, Photobacterium leiognathi and Photobacterium sepia and others. Whatever the source of the enzyme, the assessment of the effectiveness of the latter in the application considered is the same, since it is measured in absolute terms: the activity of the enzyme is measured by inhibition of the lumol chemoluminescence reaction, reaction produced by the hypoxanthine / xanthine oxidase / oxy-

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uncomfortable; to carry out this reaction, each time an injection was made of 1 ml of 0.33 × 1 CHM hypoxanthine in a mixture of 0.3 ml of luminol 10 ~ 3M, 0.3 ml of gly-cine-NaOH buffer 1 M pH 9, 0.3 ml of 10'3M EDTA, 1.8 ml of water and 0.005 ml of xanthine oxidase solution at 1 mg / ml. The experimental device used for measuring the light intensities 1) and 2) after inhibition by means of superoxide dismutase included a silver vessel intended to receive the mixture to be tested and placed in front of a photomultiplier. The reaction was initiated by injecting the substrate into the tank and there was then an emission of a flow of photons which gave rise, under the action of the photomultiplier, to a current which was measured using of a picoampeter, and recorded the intensity. If an appropriate quantity of the superoxide dismutase to be evaluated was introduced into the reaction mixture, before the initiation of the reaction, this light emission was inhibited. The chosen activity unit arbitrarily corresponds to the quantity of enzyme causing a reduction of 50% in the maximum light intensity in the absence of enzyme inhibition. This unit is therefore independent of the source of enzyme and of the enzyme superoxide dismutase used.

In certain cases, it may be advantageous, in order to assess over a reasonable period of time the effectiveness of the superoxide dismutases in the application considered, to activate the oxidation of the materials. To do this, one operates in practice either using flavins reduced by irradiation to 365 mu, or by means of an enzymatic system, for example the xanthine oxidase / hypoxanthine / oxygen system.

When the oxidation by the reduced flavins is accelerated, a solution of flavin monocucleotide (FMN) 10_4M, EDTA 10_3M and phosphate buffer 10_2M pH 7.0 is irradiated in a quartz tank placed in a Zeiss spectrofluorimeter '' an M 365 filter; one follows the photoreduction, which occurs quickly, by following the decrease in the intensity of fluorescence emitted at 530 m | X.

At the end of the reduction, the solutions are taken up to shake them vigorously in the presence of air, thus obtaining their complete reoxidation, which produces superoxide O 2 -r ions. It should be noted that this flavin reduction-reoxidation cycle can be repeated several times, without the structure of the mononucleotide flavin being altered. Alternatively, irradiation can be carried out at 365 m (x, using a B 100 A lamp supplied by Ultraviolet Products Inc., on solutions as described above, continuously stirred.

When it is desired to accelerate the oxidation by the hypoxanthine / xanthine oxidase / oxygenic enzyme system, it is advantageous to use a solution containing 0.3 ml of hypoxanthine 10 ~ 3M, 0.3 ml of 1 M phosphate buffer pH 7.8, 0.3 ml of EDTA 10 ~ 3M, 2.1 ml of water and 0.05 ml of a xanthine oxidase solution at 1 mg / ml.

A process for the production of superoxide dismutase by extraction from marine bacterial strains consists in dispersing in water and maintaining a marine bacterial culture at approximately 4 ° C., then adjusting the pH of the medium 6.5-8, and preferably at about 7, to bring the mixture to 50-60 ° C for a few minutes, to cool it to about 4 ° C and, at this temperature, to centrifuge, to carry out on the supernatant fraction a first fractional precipitation by means of neutral salts, centrifuge again and perform a second fractional precipitation with neutral salts on the supernatant and centrifuge.

As a variant, the process can be completed by a purification step, advantageously consisting in dissolving in a phosphate buffer pH 7.8 of the precipitate collected in the last centrifugation and in dialysis of the solution thus obtained, against the same phosphate buffer pH 7.8.

According to another variant of this process, the enzyme extracted from the marine bacterial culture is purified, after taking up the product of the last centrifugation with phosphate buffer pH 7.8, and dialysis against this same buffer, by chromatography which is preferably column chromatography and in particular chromatography on three successive columns which are respectively: a first column of Sephadex G 200 gel, a column of diethylaminoethyl Sephadex (DEAE-Sephadex A-50) and a second column of gel gel Sephadex G 200.

The bacterial culture serving as an enzyme source in the process according to the invention is, for example, a culture of strains of Photobacterium phosphoreum, Photobacterium leiognathi, or Photobacterium sepia. To adjust the pH of the dispersion of bacteria in water to 6.5-8, which constitutes the first step of said process, for example 2N ammonia is used and then potassium chloride, preferably potassium KCl 3M.

The mixture thus constituted is then brought to a temperature of 50 to 60 ° C, for only a few minutes and preferably for 3 to 4 minutes. The mixture is then cooled to approximately 4 ° C., the temperature at which all the following stages of the process according to the invention are carried out.

Each of the fractional precipitations constituting stages of this process is carried out, as indicated above, by means of neutral salts in aqueous solution, and preferably by means of ammonium sulphate (NH4) 2SO4.

In an advantageous embodiment, the first fractional precipitation is carried out by means of ammonium sulfate (NH4) 2SO4 added in an amount such that the mixture or enzymatic extract treated is thus brought to a final concentration of 30 to 35%. approximately saturation at 4 ° C. It should however be noted that it is possible to replace, for all or part, the ammonium sulphate by one or more other suitable salts and the total amount of said salts and then an equivalent and appropriate amount for qu '' it brings the mixture to a final concentration of around 30 to 35% of saturation at 40 C.

Alternatively, one can use an ultrafiltration system, such as for example a system implementing a porous device such as porous tubes, and in particular a system using a first porous tube retaining, by concentrating them, molecules having a molecular weight greater than 40,000, and a second porous tube letting through the desired enzyme molecules but retaining the remaining bacteria, thereby providing a sterile enzyme extract.

Likewise, the second fractional precipitation is advantageously carried out by means of ammonium sulphate (NH4) 2SO4 added in an amount such that the mixture or enzymatic extract treated is thus brought to a final concentration of 70 to 75% approximately of the saturation at 4 ° C.

The superoxide dismutases obtained from different marine bacterial strains are all superoxide dismutases comprising non-hematinic iron, while the enzymes exhibiting superoxide dismutase activity which have been described previously and which are erythrocuprein and the enzyme extracted from Escherichia coli contain divalent cations which are copper and zinc respectively for the first, and manganese for the second.

The search for the presence of a metal in the superoxides dismutases considered can be carried out by an analysis with an atomic absorption spectrograph.

We can also perform a colorimetric test which,

in the present case, consists in coloring an electrophoretic migration gel of an enzyme preparation using a specific dye of the ferrous iron FeFe2 +, bathophé-nanthroline, optionally in the presence of a reducing agent such as hydrazine . For this test, the gel is cut in half in the

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lengthwise and one of the two parts is placed in coomassie blue, and the other part in bathophenan-throline. The test is positive if a pink ring is obtained in the latter case, at the level of the protein band. However, as is known, such an appearance of a pink ring can be considered as an indication of the presence of ferrous iron, here in the protein superoxide dismutase.

Radioactive iron can also be used to label the protein and determine its stoichiometry with respect to the divalent metal cation present.

A superoxide dismutase extracted from marine bacterial cultures therefore comprises non-hematinic iron, it has a molecular weight of approximately 40,000 ± 2,500 and a pHi or isoelectric point of approximately 4 to 7 and exhibits a maximum of enzymatic activity for a pH of 8 , 5 to 10 approximately, with an optimum at pH 9.5 approximately.

The active enzyme can be maintained over a long period of time by storing it in a 70-80% solution of neutral ammonium sulfate (NH <t) 2SO4 at 4 ° C.

To measure the activity of superoxide dismutases, it is possible to evaluate the inhibition by the latter of the chemoluminescence reaction caused by the oxygen-hypoxanthine-xanthine-oxidase-luminol enzyme system, as will be shown below. This reactive enzyme system causes the release of Oz-r ions which are capable of giving a chemiluminescence reaction with luminol. The addition of superoxide dismutase to this system in fact entertains O2- ions and thus causes a reduction in the intensity of the light emitted in this reaction.

Superoxide dismutases catalyze the reaction

2O2- + 2H + - * H202 + O2

If the superoxide ions produced by the enzymatic reaction involving xanthine oxidase and xanthine or hypanthanthine are used as substrates in this reaction, the superoxide ions thus produced are very unstable and spontaneously emit light. The latter is however too weak and the measurements could not have sufficient reproducibility.

This is why the analytical device is completed in practice by using, to demonstrate the quantity of superoxide ions formed, a chemiluminescent substance, luminol or 5-amino-2,3-dihydro-1,4-phthalazinedione.

Luminol + O2-I1V + oxidation product

We have also developed systems producing superoxide ions, catalytic systems capable of promoting the oxidation of luminol consisting of Fe2 +, Ni2 + or Co2 + ions in aqueous solutions of molecular oxygen in the presence of certain ligands.

The superoxide dismutase introduced into the system decreases the amount of Ol- ions and therefore the production of light.

The assay is carried out as follows:

The following reaction mixture is used:

Luminol 10 ~ 3M 0.3 ml

Phosphate buffer 10T3M pH 7.8 0.3 ml

EDTA 10 ~ 3M 0.3 ml

Water qs 2 ml

+ 50 μl of xanthine oxidase (1.05 ml of a 1 mg / ml solution of xanthine oxidase).

This mixture is placed in a silver tank, in front of a photomultiplier. The reaction is initiated by injecting the substrate into the tank: 1 ml of a solution containing 0.3 μmol of hypoxanthine.

There is then an emission of a photon flux, which gives rise, under the action of the photomultiplier, to a current whose intensity is measured by a picoampmeter and recorded. If 5 μl of superoxide dismutase to be evaluated is introduced into the reaction mixture, before the initiation of the reaction, this light emission is inhibited.

The unit of superoxide dismutase enzyme is then arbitrarily defined as being the quantity of this enzyme which causes a 50% inhibition of the emission of light.

Note, however, that one can also evaluate the activity of superoxide dismutases by causing the inhibition of the same reaction as that mentioned above, directly by injecting 1 ml of a solution of 02-r ions, prepared by reduction electrochemical (JM Cord, I. Fridovitch, JBC vol. 244, 25 (1969), pp. 6049-6055), in 2 ml of a solution containing 0.5 [miole of luminol and 0.17 millimole of phosphate buffer pH 7.8.

As a variant, the activity of the superoxide dismutases can be evaluated as follows:

0.3 ml of IM glycine-NaOH buffer, pH 9, 0.3 ml of neutralized EDTA 10 ~ 3M, 1.1 ml of flavin-mononucleotide 10 ~ 5M and 0.3 ml are introduced into the tank mentioned above. luminol (20 mg / 70 ml). 1 ml of 10 ~ 2M NaBKU is injected. Under these conditions, a signal is obtained evaluated at IO-7 at 10 ~ 8A for a voltage of 1500 V. It will be noted that the Na BH4 solution must be prepared extemporaneously, while the luminol must be kept away from light. at 0 ° C and must be added separately to the reaction mixture.

The addition of superoxide dismutase to this system causes a certain inhibition of the light signal, the latter varying linearly with the amount of enzyme up to the rate of 75%.

According to yet another variant, the activity of the superoxide dismutases can be evaluated using a reaction mixture composed of 0.3 ml of glycine-NaOH IM buffer, pH 9, 0.3 ml of ED3 10_3M, 1.1 ml of and 0.3 ml of luminol 10-4M. 5 µl of xanthine oxidase (1 mg / ml) and an aliquot of superoxide dismutase are added to this mixture. 1 ml of hypoxanthine is injected (0.3 ml of 10_3M hypoxanthine, diluted at the last moment with water up to 10 ml). It should be noted that the luminol, which must be stored protected from light and at 0 ° C., is to be added separately to the reaction mixture.

The signal obtained for the assay of the control (without superoxide dismutase) is approximately 10_8A for a voltage of 1500 V and the inhibition of the light signal is linear is proportional to the amount of superoxide dismutase up to 50%.

Having admitted that, under appropriate conditions, the metal ions Fe2 +, Ni2 + and Co2 + can give rise to superoxide radicals, the licensee has addressed the problem of lipid oxidation, in particular in foods containing it, and has brought the reaction mechanism closer to that of the formation of superoxide ions by a system comprising the aforementioned metal ions and an appropriate ligand. It was then observed that the antioxidants commonly used in the food industry, such as pyrogallol-type free radical chain switches, for example propyl gallate, or inhibitors of free radical production, such as EDTA and l ascorbic acid, can effectively promote against all expectations certain catalyzed oxidations either enzymatically or under the effect of metal ions, instead of acting as antioxidants as expected.

It was then that it was established that, under appropriate pH conditions, the superoxide dismutases according to the present invention inhibit oxidizing systems such as those consisting of 02-r electrochemically produced ions, of FMNHb /

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Oz, or Fe2 +, Ni2 + or Co2 + ions in aqueous molecular oxygen solutions in the presence of suitable ligands. Thus, 7 units of superoxide dismutase extracted from Photobacterium leiognathi were found to inhibit at 16.5% the emission of light due to the action of the Co2 + / 02 / tetraglycine system on the luminol at pH 9.7 and at around 40% that due to the action of the Ni2 + / 02 / cyanide system on the luminol at pH 9.

It has been shown that superoxide dismutases effectively protect lipids and antioxidants and other preservatives commonly used in the food industry, by very strongly inhibiting reactions linked to the production of the superoxide ion O2. In particular, it has been established that the auto-oxidation of unsaturated lipids from Anchoveta is very strongly inhibited by superoxide dismutases. In addition, other tests have shown that superoxide dismutases exert a protective action against the auto-oxidation of certain antioxidants, in particular of antioxidants used for the preservation of food, such as for example pyrogallol or ascorbic acid.

The method according to the invention therefore provides food compositions, compositions obtained from strains of bacteria or viruses or culture media, comprising, in association with said foods, bacteria or viruses or other principles of the environment, an effective amount of a superoxide dismutase enzyme.

It is clear that the amounts of superoxide dismutase enzymes to be combined with the substances to be protected are not critical and the determination of the most appropriate amounts for each case is within the reach of those skilled in the art. Routine tests make it possible to determine the effectiveness of the protection conferred by the enzyme and possibly adapt or modify the quantity of the latter accordingly, using for this purpose activity tests similar to those described above. -above.

By way of examples, it can however be specified that amounts of superoxide dismutase enzyme from 1 to 100 units per milliliter or per gram are particularly suitable for the protection of auto-oxidizable substances as defined above.

As shown above, superoxide dismutases effectively protect lipids and antioxidants and other preservatives commonly used in the food industry, by very strongly inhibiting reactions linked to the production of the superoxide ion O2- . In particular, it has been established that the auto-oxidation of unsaturated lipids from fish is very strongly inhibited by superoxide dismutases. In addition, other tests have shown that the superoxide dismutases exert a protective action against the auto-oxidation of certain antioxidants, in particular of antioxidants used for the preservation of food, such as for example pyrogallol or ascorbic acid.

The invention is described in more detail in the examples below, which do not limit it in any way.

Example 1

Photobacterium leiognathi bacteria of strain ATCC 25 521 were cultivated on a synthetic medium containing, in grams per liter:

NaCl

Na2HP04,12H2O KH2PO4 MgS04,7H2O (NH4) 2HP04 Glucose Glycerol Trypticase Yeast extract

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This medium was brought to pH 7.2 with sodium hydroxide and sterilized for 1 hour at 110 ° C.

A 1 liter preculture was used, grown overnight and distributed between 4 Erlenmeyer flasks containing 250 ml of medium each, to inoculate a fermenter of 12 liters of medium.

The culture was continued for 12 hours at 20 ° C. with strong aeration and thus 100 g of bacteria were obtained, expressed by weight of wet product.

135 g by wet weight of Photobacterium leiognathi bacteria, from several cultures, were dispersed in 650 ml of water and allowed to stand at 4 ° C overnight. 2N ammonia was added until the pH of the medium was brought to 7-8 and then 18 ml of 3M KCl was added.

The mixture was brought to 58 ° C and kept at this temperature for 4 minutes, after which it was cooled to 4 and centrifuged for 10 minutes at the speed of 10,000 rpm. Still operating at 4 ° C., the supernatants were adjusted to 35% saturation with solid ammonium sulfate at pH 8; it was centrifuged and the supernatant was brought to 75% ammonium sulphate saturation and allowed to stand overnight at 4 ° C. The precipitated protein was harvested by centrifugation and stored in a 75% ammonium sulfate solution.

The activity of a solution of 9 mg of protein per milliliter (Biuret) was 40 units / mg, while it was, for comparison, 0 unit / mg for catalase in solution of 9 mg of protein / ml.

After a further fractional precipitation using ammonium sulfate with a concentration gradient of 35 to 75% of saturation, a superoxide dismutase was obtained which, in solution at 14.8 mg protein / ml (Biuret) exhibited a activity from 134 units / mg to 500 units / mg.

The protein precipitate was dissolved in pH 7.8 phosphate buffer and dialyzed for 48 hours at 4 ° C. The enzyme superoxide dismutase was stored at -20 ° C.

To determine the activity of the latter, a tank was used placed in front of a photomultiplier and containing the reaction mixture below:

40 Luminol

Phosphate buffer

EDTA

Water

Xanthine oxidase

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10 ~ 3MpH 7.8 10-3M

(1 mg / ml)

0.3 ml 0.3 ml 0.3 ml 1.0 ml 0.050 ml

The reaction was initiated by injecting 1 ml of 3 × 10 ~ 4M hypoxanthine into the tank.

A stream of photons was then emitted, which gave rise, under the action of the photomultiplier, to a current whose intensity was measured at the pico-ammeter and the variations in this intensity were also recorded.

By introducing into the reaction mixture, before initiation of the reaction, 5 μl of a solution of superoxide dismutase prepared as indicated above, an inhibition of light emission was obtained and it was arbitrarily considered that 1 unit of enzyme superoxide dismutase would be the amount of enzyme that causes a 50% inhibition of light emission.

In UV spectroscopy, the superoxide dismutase extracted gave the classical spectrum of non-hematinic proteins, with the shoulder of tryptophan at 290 m [x.

To determine the molecular weight, two known techniques were used, one consisting in determining a sucrose centrifugation gradient and the other using a Sephadex G 200 gel.

To do this, we used the following markers, whose molecular weight was known (P.M.):

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P.M.

Yeast ADH 150,000

Bovine albumin 66,000

Peroxidase 40,200

It was concluded that a molecular weight of 40,000 ± 2,500.

To determine the subunits of the protein structure of the enzyme, an electrophoresis was carried out in polyacrylamide gel supplemented with SDS (sodium dodecyl sulfate) at 10%.

Only one type of subunit was observed, with a molecular weight of approximately 21,000. The molecular weight of the superoxide dismutase obtained can therefore be estimated at 21,000 × 2, or 42,000.

Still on the polyacrylamide gel, a red band was obtained in the presence of bathophenanthroline and hydrazine, precisely at the level of the superoxide dismutase band revealed by coomassie blue.

A colorimetric determination of a protein solution was carried out and a value for iron was obtained which, by estimating the molecular weight of the enzyme at 42,000, gave approximately 2 iron atoms per mole.

An atomic absorption spectrometry of a 0.2 mg / ml protein solution confirmed the figure.

We can therefore reasonably estimate the number of iron atoms per mole of superoxide dismutase at 2.

Furthermore, the enzyme extracted according to this example has been found not to suffer an appreciable loss of activity after 5 minutes at a temperature of 70 ° C.

Electrofocusing of the superoxide dismutase made it possible to evaluate the pHi or isoelectric point of this enzyme at 4.4.

The enzyme exhibited maximum activity at a pH of around 9.5.

An identical enzyme with similar characteristics was obtained from a bacterial strain of Photobacterium leiognathi no ATCC 25 587.

Example 2

A culture of bacteria of Photobacterium sepia strain ATCC 15709 was subjected to lysis by stirring in cold water, at the rate of 1 g of wet weight of bacteria per 4 ml of water. It was then centrifuged at 16,000 rpm for 20 minutes at 4 ° C. 3M KCl was added to the light yellow supernatant to a final concentration of 0.1 M.

The solution was heated for 3 to 4 minutes in a water bath brought to 60 ° C. It was then cooled to 4 ° C and clarified by centrifugation.

The supernatant was subjected to fractional precipitation by addition of solid ammonium sulfate. The active fraction precipitated between 45 and 75% saturation with ammonium sulfate and was separated by centrifugation; it was redissolved in the minimum volume of K2HPO4 5 × 10 ~ 3M, pH 7.8, and dialyzed overnight against the same phosphate buffer. The product of this dialysis was then directed onto a column of Sephadex G 100 or G 200 gel, equilibrated with phosphate buffer 5 × 10 ~ 3M, pH 7.8. The active fraction eluted was concentrated using a Diaflo PM-10 ultracentrifugation membrane, and then dialyzed overnight against 3M 2x5 K2HPO4, pH 7.8.

The enzyme superoxide dismutase was adsorbed on a column of DEAE-Sephadex A-50 calibrated with K2HPO4 5 X 10 "3M, pH 7.8. The protein was eluted from the column with a linear gradient of K2HPO4, pH 7 , 8 (from 5 X 10_3M to 3 X 10 ^ M). Superoxide dismutase was thus eluted at a phosphate concentration of 1.4 X 10_1M and it was then concentrated. A new filtration was carried out under the same conditions than the previous one on a column of

DEAE-Sephadex A-50. The enzyme was thus eluted at a phosphate concentration of 1.6x10 IM and concentrated.

The protein thus extracted and purified gave a unique band to acrylamide gel electrophoresis (100 (xg of protein for one gel).

Thus, 3 mg of pure superoxide dismutase was obtained from 20 g of frozen cells of Photobacterium sepia (with a superoxide dismutase activity of 250 to 5000 units / mg).

The active enzyme was maintained over a long period of time by storing in a 70-80% solution of (NH4) zSO4 at 4 ° C.

The molecular weight of the purified enzyme was determined by ultracentrifugation at 45,000 rpm and for 16 hours at 4 ° C. The sedimentation rate was measured by the method of Martin and Ames, with a linear sucrose gradient of 5 to 20 (in weight / volume) and using a Beckman Spinco model L2-65B apparatus, with a SW 65 K rotor. A sedimentation coefficient of 3.2 was found for this dismutase, compared with a coefficient of 4.82 and 7.4 respectively for alcohol dehydrogenases from horse liver and yeast. From this sedimentation constant, the molecular weight of the extracted superoxide dismutase was therefore calculated to be approximately 42,500.

It has also been established that the molecule of this protein contains 2 iron atoms.

Electrophoresis in acrylamide gels gave a unique band for dismutase corresponding to a molecular weight of 20,000 to 20,500, thus showing that the proteinaceous molecule was composed of two identical subunits.

Superoxide dismutase has also been shown to be very resistant to the proteolytic action of trypsin, since a treatment for 60 minutes of 150 μg of this superoxide dismutase with 10 μg of trypsin at 20 ° C. did not cause any change in the enzymatic activity, no more besides than in the electrophoretic mobility of the undissociated protein.

The enzyme obtained was very stable with respect to heat: no loss of enzymatic activity appeared after 30 minutes at 20 ° C, 30 ° C and even 40 ° C; after 15 minutes at 50 ° C, a decrease in activity of 28% appeared; after 30 minutes at 50 ° C, the loss of activity was still only 50% and it was only 10% and 50% after 3 minutes and 10 minutes respectively at 60 ° C.

Electrofocusing of the superoxide dismutase made it possible to evaluate the pHi or isoelectric point of this enzyme at 4.1.

It was determined by means of an 02T ion solution prepared electrolytically, that the enzyme exhibited a maximum of activity for a pH of 8.5 to 10, with an optimum at pH 9.5.

Example 3

We treated a strain no ATCC 11 040 of Photobacterium phosphoreum bacteria which are marine bacteria and therefore have a high internal salt concentration.

The lysis of the bacteria was spontaneous in a solution of EDTA 10_3M, pH 7.8, at 4 ° C.

Cellular debris was removed by centrifugation at 16,000 rpm for 20 minutes at 0 ° C.

Preliminary studies had shown that the enzyme superoxide dismutase was stable at 50 ° C and we therefore eliminated the other proteins, themselves heat-labile, by carrying the lysate for 3 minutes at 50 ° C, after adding KCl up to at a molarity of 0.1, then the lysate was cooled to 4 ° C. and the denatured proteins were separated by conventional centrifugation.

Still at 4 ° C., fractional precipitation was carried out using ammonium sulphate (NH4) 2SO4 added in one s

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quantity such that the mixture or enzymatic extract treated is brought to a concentration gradient from 0 to 30% approximately of the saturation at 4 ° C. The precipitated fraction was removed by centrifugation for 45 minutes at 16,000 rpm and at OC.

The amount of neutral ammonium sulfate required to bring it to 75% saturation at 4 ° C was added to the supernatant.

The precipitated fraction was combined by centrifugation similar to the previous one.

In order to purify the superoxide dismutase thus extracted, the precipitate collected was dissolved in a little phosphate buffer 5 × 10 ~ 3M, pH 7.8 and dialyzed against this same buffer for 48 hours at 4 ° C., to obtain an extract. (AT).

The proteins still present were then separated according to the size of their molecules, using a Sephadex G 100 gel, the grain crosslinking of which is such that it excludes proteins having a molecular weight greater than 100. 000, which therefore leave very quickly outside the gel. The molecules with a lower molecular weight penetrate into the gel and are eluted more or less quickly depending on their size.

To do this, the Sephadex resin was swollen for 3 hours in water, it was degassed and filtered through Büchner to remove the water; it was placed in the filter pad and poured into a column 50 cm long by 3 cm inside diameter. The column was balanced by passing 2 liters of buffer.

The enzyme extract (A) from dialysis was previously taken up to concentrate it on a Diaflo millipore membrane (P.M.-10) under nitrogen pressure, to a volume of 5 ml. This concentrate was deposited on the column prepared as indicated above and was eluted by passing 500 ml of 5 × 10 -3 M phosphate buffer, pH 7.8. The flow rate was 1 drop every 8 seconds and 2.5 ml fractions were collected; those with significant activity were pooled and concentrated on the Diaflo membrane. A concentrated extract (B) was thus obtained.

A new purification step was carried out by chromatography on an ion exchange resin based on DEAE Sephadex, resin on which the proteins are eluted according to their charge, by buffers of increasing ionic strength.

To prepare the column, it was swollen in water, then it was degassed and washed in the following solutions:

0.5 M NaOH

KH2PO4 0.5 M

taking care to rinse the resin with distilled water between each step until the pH is close to neutral. After filtration through Buchner, the resin was suspended in 0.1 M phosphate buffer pH 7.8 and placed in a column 30 cm long by 3 cm inside diameter. The column was balanced by passing 300 ml of 0.1 M buffer through it.

The concentrated extract (B) was placed on the column thus prepared. Once this extract was completely adsorbed, it was eluted with 500 ml of phosphate buffer pH 7.8, composed of 250 ml of 0.1 M phosphate buffer to which 250 ml of 0.5 M phosphate buffer were gradually added. flow rate was 1 drop every 5 seconds and the volume of the collected fractions 2.5 ml. The active fractions were combined and precipitated with ammonium sulphate, then stored in this form at -18 to -20 ° C.

The purity of the enzyme superoxide dismutase could be checked by electrophoresis on polyacrylamide gel.

A determination of the molecular weight of the enzyme by means of an elution study during filtration on Sephadex G 200 made it possible, using the relationship Log (PM) = f (elution volume) and known markers, to assign to the superoxide dismutase extracted a molecular weight of approximately 40,000.

This molecular weight was also determined by a sucrose gradient centrifugation: sucrose gradients of 5 to 20% were poured into 5 × 10 ~ 3M phosphate buffer, pH 7.8, by gradually mixing 2.60 ml of the solution to 20 = 225 ml of the 5% solution, in an appropriate device.

Various protein markers of known molecular weight and superoxide dismutase have been deposited on these sucrose gradients. Balance was achieved by centrifugation at 45,000 rpm, 5 ° C and for 22 hours. The tubes were then pierced through the bottom and the 10-drop fractions were collected, on which the enzymatic activities presented by the different markers used were measured. After plotting the line representing the variation in molecular weight as a function of the number of the elution fraction, the molecular weight of the superoxide dismutase extracted from Photobacterium phosphoreum was estimated at approximately 40,000, which confirms the previous result.

To determine the molecular weight of the protein subunits, the protein, as well as markers for which the molecular weight of the subunits was known, were subjected to electrophoretic migration on polyacrylamide gel in the presence of sodium dodecyl sulfate. The graphical resolution of the values found made it possible to assign the value 20,000 to the molecular weight of each subunit of the superoxide dismutase molecule.

The latter proved to be very stable still at 50 ° C. and to exhibit a maximum of enzymatic activity for a pH of approximately 9.5.

Electrofocusing of the superoxide dismutase led to evaluating the pHi or isoelectric point of this enzyme at 4.2.

A colorimetric test as described previously made it possible to detect the presence of ferrous iron in the protein, a pink ring appearing at the level of the protein band in an electrophoretic migration gel previously added with bathophenanthroline. The number of ferrous iron atoms per molecule has been estimated to be almost 2.

On the other hand, the inhibition of the autooxidation of certain compounds has been determined under the action of superoxide dismutases (SOD) by operating as follows:

Example 4

A solution of pyrogallol 2.5 × HHM was prepared in phosphate buffer K2HPO4 2.0 × 10 −2M, pH 7.7. This solution (A) had a volume of 3 ml.

The increase in optical density (OD) was measured at 440 μm per minute for different systems and the percentage of inhibition was deduced therefrom, for each of the following systems:

Increase DO inhibition at%

440 mti / minute

(A) 0.031 (A) + 25 units of Photobacterium leiognathi SOD (crude) 0.029 6.5 (A) + 250 units of Photobacterium leiognathi SOD (crude) 0.004 87 (A) + 4 [ig of catalase 0.046 -

We therefore see that this superoxide dismutase has an exceptional effect of inhibiting the auto-oxidation of pyrogallol.

s

10

15

20

25

30

35

40

45

50

55

"0

65

619,731

8

Example 5

The procedure was as in Example 4, with the difference that the pyrogallol which was dissolved in the phosphate buffer was respectively 5.0xl (HM, 2x1 (HM and 1CHM. The solutions obtained were called (B ), (C) and (D).

The following results were obtained:

Increase DO inhibition at 440 mji /%

minute (X 10)

- Pyrogallol 5 X10-4M (in phosphate buffer 2.0 X10-2M, pH 7.7)

(B)

(B) + 5 units of Photobacterium leiognathi SOD (raw)

(B) + 10 units of Photobacterium leiognathi SOD (gross)

(B) + 50 units of Photobacterium leiognathi SOD (gross)

(B) + 200 units of Photobacterium leiognathi SOD (gross)

(B) + 4 [ig of catalase

- Pyrogallol 2 X 10 ~ 4M (id.)

(VS)

(C) + 25 units of Photobacterium leiognathi SOD (raw)

(C) + 5 units of Photobacterium leiognathi SOD (raw)

(C) + 10 units of Photobacterium leiognathi SOD (raw)

- Pyrogallol 10 ~ 4M (in phosphate buffer 2.0 X 10 ~ 2M, pH 7.7)

0.175

0.070

0.043

0.004

0.001 0.135

0.054

0.026

0.012

0.005

60

75

98

99.5

52

78

91

(D) 0.030

(D) + 1 unit of SOD of

Photobacterium leiognathi (crude) 0.013

57

20

35

Example 6

Solutions of pyrogallol 10 ~ 4M were prepared respectively in phosphate buffer K2HPO4 5 X 10 ~ 2M and 2X 10_2M, pH 7.7, saturated with molecular oxygen. These solutions (E) and (F) had a volume of 2.5 ml. The increase in optical density was measured at 440 m [i per minute and the percentage of inhibition was deduced therefrom:

Increase in DO to 440 m | U minute (X 10)

Inhibition

%

- K2HPO4 5 X 10_2M

(E)

0.365

-

(E) + 1 unit of SOD of

Photobacterium leiognathi

0.275

25

(E) + 5 units of SOD of

Photobacterium leiognathi

0.183

50

(E) + 10 SOD units of

Photobacterium leiognathi

0.063

83

Increase Inhibition of DO at 440 mu '^

minute (x 10)

10

(E) + 20 SOD units of

Photobacterium leiognathi 0.018 95

- K2HPO4 2 X 10 ~ 2M

(F) 0.062 99 (F) + 10 SOD units of

Photobacterium leiognathi 0.016 74

Note that 1 unit of enzyme in 2.5 ml corresponds to an enzyme 2 X10-9 M.

Example 7

A 10_4M ascorbic acid solution was prepared in a 2 X 10_2M phosphate buffer, pH 7.7.

The variation in optical density (OD) was measured at 265 m (i per minute for each of the following systems:

Variation of DO at Inhibition 265 mu / minute%

Witness 0.016

Control + 7.5 units of Photobacterium leiognathi SOD (crude) 0.005 Control + 10 units of Photobacterium leiognathi SOD (crude) 0 Control + 15 units of Photobacterium leiognathi SOD (crude) 0 Control + 20 units of Photobacterium leiognathi SOD (gross) 0

69

100

100

100

Example 8

The procedure was as in Example 7, except that the 10-4 M ascorbic acid was dissolved in 40 5 × 10 2 M phosphate buffer, pH 8.8.

Variation of DO at 265 mji / minute

Inhibition

%

"Witness

0.031

Control + 7.5 units of SOD

of Photobacterium

leiognathi (raw)

0.011

65

Control + 10 units of SOD from

s »Photobacterium leio

gnathi (raw)

0.004

87

Example 9

55 The action of superoxide dismutases on the oxidation of luminol catalyzed by metal ions was determined as follows:

Imax. (x 10 'quanta / s / ml)

Inhibition

%

16.2

65

- Oxidizing catalytic system: C02 + / 02 / NH40Ac / di-hydroxyfumaric acid.

- pH 9.0 control

- pH 9.0 control + 140 units of Photobacterium sepia SOD

7.3

55

9

619,731

Imax. (X 10 'Inhibition quanta / s / ml)%

- pH 9.8 control 16.5 -

- pH 9.8 control + 140 units of Photobacterium sepia 11.0 33

- Oxidizing catalytic system: Ni2 + / 02 / NH40Ac / di-hydroxyfumaric acid.

- pH 9.0 control 4.7 -

- pH 9.0 control + 140 SOD of Photobacterium sepia 0.88 81

- pH 9.8 control 240 -

- control pH 9.8 + 140 units of Photobacterium sepia 80 67

Example 10

A suspension was prepared at 10 = by weight per volume of Anchoveta lipid in 0.1 M phosphate buffer, pH 8.

Crude photobacterium leiognathi superoxide dismutase was added thereto in an amount of 0.01% by weight of enzyme relative to the lipid (i.e. 0.1 mg of crude enzyme per g of lipid) .

The oxygen consumption by this system was measured in a Warburg apparatus, by relating it each time to the value obtained with a lipid control of the same composition, but not comprising the addition of superoxide dismutase. The measurements were carried out over successive periods of time and the results obtained were expressed in the form of a percentage of oxidation inhibition relative to the control not comprising the addition of superoxide dismutase.

Time (in hours)

0-24

24-40

40-42

42-44

% inhibition

61

74

72

78

Example 11

5 g of fresh button mushrooms were used, which we cut into thin slices and got rid of their colored strips, because the latter could have disturbed the coloring of the medium and made it difficult to assess the result.

The mushrooms thus prepared were distributed in several 100 ml beakers, in each of which 50 ml of a solution buffered at pH 7.8 (0.01 M phosphate buffer) and containing 0.02% acid were then introduced. ascorbic.

In one of the beakers were added 50 units of superoxide dismutase from Photobacterium leiognathi of strain no ATCC 25 521.

Each beaker was covered with a double sheet of paper and allowed to stand at laboratory temperature.

After 40 hours, the optical density was evaluated at 600 m | i of the supernatant of each beaker: the optical density found made it possible to deduce an increase in optical density of 0.173 on average for the controls, while the increase in density optical was only 0.092 for the supernatant comprising superoxide dismutase. The oxidation in the beaker thus treated was therefore R7% lower than it was in the control beakers.

Example 12

Apple slices were added to a solution of

3 units of superoxide dismutase from Photobacterium leo-ignathi of strain no ATCC 25 521 per ml, in a phosphate buffer pH 7.8. After a few minutes, the apple slices were removed from the solution and placed in petri dishes where they were left; after a few days, the slices thus preserved were compared with slices prepared in the same way, but preserved without treatment with a superoxide dismutase. The latter appeared browner than those treated according to the invention.

. Example 13

Potato rings were placed in beakers each containing three units of photobacterium leiognathi superoxide dismutase strain ATCC 2521 per ml, in phosphate buffer pH 7.8; they were removed a few minutes later and then put in Petri dishes where they were left. After 4 days, the discs thus treated appeared significantly less oxidized than identical discs, subjected to the same treatment, but without any addition of superoxide dismutase.

Example 14

It is known that certain yeast t-RNA ligases are lipoproteins in which the enzymatic activity is a function of the integrity of the lipid part. We therefore have here a material whose degradation can be objectively determined by a simple evaluation of the enzymatic activity and which, because of its essentially lipo-proteinic constitution, can be taken as a model for all lipo-protein foods.

The ligases that were used were extracted from yeast cells (Saccharomycetes cerevisiae). To prepare them, the procedure was as follows: strains of Saccharomycetes cerevisiae no ATCC 9841 were cultivated in a medium containing 30 g of glucose and 5 g of yeast extract, and the culture was allowed to develop until the medium of the logarithmic growth phase. A centrifugation was then carried out, then the cells were washed and crushed in a device known under the name of French Press; 67 g of yeast were thus treated with 67 ml of buffer (consisting of 0.01 M tris pH 8, 0.01 M MgCh, 1 mM EDTA, 10% glycerol and 2 mM phenyl-methyl-sulfonium fluoride). After grinding in the device called French Press, repeated three times, it was centrifuged for 30 minutes at 15,000 rpm. The supernatant was taken up and centrifuged again for 2 hours at 50,000 rpm. The supernatant from this last centrifugation was purified on a DEAE-cellulose DE-52 column of 2 cm × 36 cm comprising 80 g of cellulose, said column being equilibrated with 0.02 M potassium phosphate buffer pH 7.5, 0, 02 M mercaptoethanol, 1 mM MgCk and 10% glycerol. Once the said supernatant was placed on the column, it was washed with 370 ml of the same buffer. The ligases were then eluted with 0.25 M potassium phosphate buffer pH 6.5, 0.02 M mercaptoethanol, 1 mM MgCh and 10% glycerol.

The optical density of the fractions collected at the bottom of the column was determined and their activity was tested with lysine, as well as with the other amino acids (the activity values are reported in Table I below). ). Each extract was then concentrated by ultrafiltration, to a protein concentration of 10 mg / ml and assayed by measuring the load of t-RNA. The incubation medium used was 100 (it and contained 50 mM of N-morpholino-3-propane sulfuric acid (pH 6.5), 10 mM of MgCh, 2 mM of ATP (adenosine-triphosphoric acid), 400 μM (picomoles) of carbon 14-labeled lysine and 10 units of optical density at 260 mfi of total yeast t-RNA.

After incubation for a determined time, 50 μl of the reaction mixture were deposited on Whatmann DE-81 paper.

5

10

15

20

25

30

35

40

45

50

55

60

65

619,731

10

and washed for 1.5 hours with 8.7% acetic acid and 2.5% formic acid. The paper thus washed was then dried and, on the spots formed, a counting was carried out using a scintillator mixture based on toluene.

To evaluate the effectiveness of the protection against oxidation of yeast lysine t-RNA ligase provided by a superoxide dismutase, a solution of 0.37 mg of lipoproteins in 1 ml of phosphate buffer (0.5 M) was produced. pH 7.5, and either 0.1% ascorbic acid or 90 units of superoxide dismutase from Photobacterium leiognathi of strain ATCC 25 521 was added thereto; in each case, the solutions were allowed to stand at 4 ° C, aliquots were removed at different times (see Table I below) and the enzyme activity was assayed as indicated above).

Table I

Days

% activity

without protector with acid with 90 units

superoxide ascorbic

0.1%

dismutase from

Photobacterium

leiognathi

0

100

100

100

2

81

31

97

4

62

19

96

7

35

2.8

72

9

27

1.2

67

Similar results were obtained using the same number of superoxide dismutase units from Pleurotus olearius Gillet, Eschirichia coli strain ATCC 15 224 or Ferithrocuprein.

Example 15

For the determination of the protection of bacteria against oxidation by a superoxide dismutase, luminescent bacteria of Photobacterium leiognathi of strain ATCC 25 521 were used, the oxidation of which was artificially accelerated by photo-reduction of FMN, in order to to make the protection phenomenon perceptible over a shorter period of time. The bacteria were cultivated at 28 ° C. on a medium comprising 8 g of nutrient broth (Nutrient Broth), 10 g of NaCl, 14 g of Na2HP04.2 g of KH2PO4 and made up to 1000 ml with water, and adjusted at pH 7.

The cells were counted by spreading 0.1 ml of a bacterial solution diluted with physiological saline on Petri dishes maintained at 25 ° C. and containing a medium consisting of 8 g of nutritive broth (Nutrient Broth ), 10 g of NaCl, 15 g of agar-agar, and water to a complement of 1000 ml, the pH of this medium being adjusted to 7.

A culture of 100 ml of Photobacterium leiognathi growing exponentially (their optical density at 600 m [i was 0.3) was centrifuged at a speed of 10,000 rpm for 10 minutes at 3 ° C. The centrifugation pellet was dispersed in 100 ml of physiological saline. This bacterial suspension was diluted 10 times in FMN 5xlO ~ 5M and 3% NaCl (respective proportions) to a final volume of 10 ml.

The bacteria were irradiated at 25 ° C. at 365 m (i under a B 100 A lamp and with constant stirring. After determined periods of exposure to the lamp, aliquots were taken and the cells were counted, after having previously diluted them further in physiological saline. While 1 × 10 7 cells / ml were had before irradiation, the numbers of cells / ml reported in Table II below were counted after determined irradiation times.

Table II

Duration of exposure

Witness

13.5 bacteria added

(in minutes)

units / ml superoxide dismutase

(in 10 ~ 2M phosphate buffer pH 7)

30

4X106

7 X106

60

5xl0s

3.9 X106

120

8X103

6 X104

Example 16

The procedure was as in Example 15, by putting bacteria of Photobacterium leiognathi of strain no ATCC 25 521 in suspension in a physiological saline solution to a final volume of 10 ml and this suspension was irradiated for 16 hours at 365 mjx using a B 100 A lamp (incident energy 0.55 mW / cm2). The suspension was then placed in petri dishes to determine the survival rate of the bacteria.

Although very prolonged irradiation, since 16 hours, had been used, it was found that there were 3.3 times more surviving bacteria, compared to a control test without superoxide dismutase, in the petri dishes where there was a suspension to which, before irradiation, 53 units per ml of superoxide dismutase (SOD) from Pleurotus olearius Gillet were added, the results obtained being as follows:

- Non-irradiated bacteria 9.7 X104 cells / ml

- Irradiated bacteria (control) 1.5 X102 cells / ml

- Irradiated bacteria (added 53 units /

ml SOD of Pleurotus olearius) 5.0 X102 cells / ml

Example 17

A preservation test was carried out on a bacteriophage, which is a nucleoprotein and will be referred to hereinafter as bacteriophage R 17. This bacteriophage was stored and diluted in a solution comprising 6 g of Na2HPO4, 3 g of KH2PO4, 0, 5 g of NaCl, 1 g of NH4Cl, 100 ml of water and 10 ml of 0.01 M CaCl2. 0.1 ml of this bacteriophage solution was incubated for ten minutes at 0.2 ° C. with 0.2 ml. of a solution of Escherichia coli strain ATCC 15 224, 10 g of trypticase, 5 g of yeast extract, 10 g of NaCl and 1000 ml of water, and this at an optical density of 0.5 to 650 ml and with 3 ml of soft agar suspension (consisting of 8 g of agar, 10 g of trypticase, 1 g of yeast extract, 8 g of NaCl, 1000 ml of water, 2 ml of 1 M CaCk and 5 ml of 20% glucose) which was kept at 45 ° C. The whole was poured into petri dishes containing 20 ml of hard agar (consisting of 12 g of agar, 10 g of trypticase, 1 g of yeast extract, 8 g of NaCl, 1000 ml of water, 2 ml of 1 M CaCk and 5 ml of 20% glucose); the petri dishes were placed at a temperature of 37 ° C.

In order to accelerate the natural oxidation of bacteriophage R 17, we resorted to photoreduction of flavins: we diluted the bacteriophage solution by 10 times its volume of a solution of FMN 10 ~ 4M, EDTA 10 ~ 3M and phosphate buffer pH 7 10 ~ 2M; photoreduction was carried out with the fluorimeter, the final volume of solution being 0.8 ml.

With a solution initially containing 4.1 × 10 8 particles / ml at time t = 0, the results given in Table III below were obtained:

5

10

15

20

25

30

35

40

45

50

55

60

65

Table III

Particles / ml

Number of opera

Control test

With 13.5 units /

Successive protection

(containing ml of dismutase

(B)

photoreduction

0.05 mg dismutase

(report ... )

inactivated at pH 3)

(AT)

(B)

0

4.1 X108

4.1 X108

-

3

4.9 X107

7.5 X107

1.53

6

5 X106

1.5 X107

3.00

9

5 X105

2 XlO6

4.00

Example 18

The procedure was as in the previous example, except that, in order to accelerate the oxidation of bacteriophage R 17, 0.3 ml of the latter was incubated with an enzyme system hypoxanthine / xanthine oxidase / oxygen, the volume of the incubation system being 3 ml.

After 15 minutes, the system was treated again by adding 0.05 ml of xanthine oxidase and 0.3 ml of 10 "3M hypoxanthine.

With, at time t, 0.5 × 10 8 particles / ml, the results of Table IV below were obtained in the absence and in the presence of 200 units / ml of superoxide dismutase originating from Photobacterium sepia of strain no ATCC 15 709. Very similar results have been obtained with the superoxide dismutases of Pleurotus olearius Gillet or erythrocuprein.

Table IV

Particles / ml

Number

Without dismutase

With dismutase

Mortality

Mortality

of operations

(witness)

(200 units)

%

%

enzymatic

(witness)

(with successive

dismutase)

0

5.0 XlO8

5.0 XlO8

-

-

î

3.5 XlO8

5 XlO8

30

0

2

2 XlO8

3 XlO8

60

40

Example 19

Protection by superoxide dismutases against oxidation of the pancreatic ribonuclease was tested, by carrying out an oxidation using photoreduction of FMN to make the phenomenon perceptible over a more suitable period. To this end, a solution of pancreatic ribonuclease (0.1 mg / ml in 10 ~ 2M of phosphate buffer pH 7 and 10-3M of EDTA) was diluted in 10 times its volume of a solution made up of FMN 10 ~ 4M. , EDTA 10 ~ 3M, and phosphate buffer 10 "2M pH 7, the final volume being 0.8 ml, the pancreatic ribonuclease protein was thus subjected to several photoreduction cycles, in the absence and in the presence of 67.5 units / ml of superoxide dismutase from Pleurotus olearius Gillet or in the presence of the same denatured and inactive dismutase at pH 3.

The activity of the ribonuclease enzyme was determined using a spectrophotometer by monitoring the increase in optical density at 280 m [i of a solution of ribonucleic acid as described above. The results obtained are shown in Table V below:

Table V

% of residual activity

Number of Control (without Control with dis- Dismutase cycles dismutase) mutase (0.05 mg) 67.5 units / ml inactivated at pH 3

0 100 100 100

3 67 56 89

619,731

Table V

This is residual activity

Number of

Witness (without

Witness with dis

Dismutase cycles dismutase)

mutase (0.05 mg)

67.5 ml units

inactivated at pH 3

6

45

45

89

9

22

22

67

Example 20

An enzyme superoxide dismutase of the strain Photobacterium leiognathi no ATCC 25 521 was used in an attempt to improve the conservation of the medium known as Jeffries medium, containing sodium tetrathionate.

This medium is a Salmonella enrichment medium and it is conventionally used as follows: a sample containing a small quantity of Salmonella and a large quantity of Escherichia coli bacteria is introduced into such a medium with sodium tetrathionate. After a 24 hour stay in an oven at 37 ° C., a drop of this culture was seeded on an agar culture medium in a petri dish. After a 24 hour incubation at 37 ° C., a very large number of Salmonella colonies were observed in this culture, but a total absence of Escherichia coli was noted.

It has thus been confirmed that said sodium tetrathionate medium inhibits Escherichia coli, but promotes the development of Salmonella. However, it has also been confirmed that this medium is only really usable for a maximum of three weeks, from its preparation, and even practically for a maximum of two weeks. By this very fact, one cannot envisage until now that a manufacture in small batches of such a medium, which makes the management of a stock difficult and in strike the cost price.

To carry out a test for protection against oxidation of the Jeffries medium, 100 tubes of the same batch of this medium with sodium tetrathionate were taken; in 11 of these tubes which each contained 20 ml of medium, 2 ml of a superoxide dismutase solution of Photobacterium leiognathi was added at a rate of 15 units / ml, all the tubes being considered as controls. All 100 tubes were stored at + 40 ° C.

Analyzes were carried out to determine the conservation of the media, one analysis per week for 7 weeks and then one analysis every two weeks only. For each of these analyzes, a tube containing the enzyme was compared with:

- 1 control tube for the first three tests,

- 2 control tubes for the 4th and 5th tests,

- 3 control tubes for the last five tests.

For the analysis, the procedure described above was followed at the beginning of this example.

The results are collated in Table VI below:

Table VI

Control Tetrathionate Weeks (sodium tetrathionate)

sodium + enzyme

1st

2nd

3rd

1

+

+

///////////

//////////////

2

+

+

//////////////

///////////

3

+

+

//////////////

///////////

4

+

+

-

///////////

5

+

+

-

///////////

6

+

+

+

-

7

+

-

-

-

9

+

+

+

+

11

+

+

0

-

13

+

+

+

0

(poor)

11

5

10

15

20

25

30

35

40

45

50

55

60

65

619,731

12

From this table, it immediately emerges that the contents of the tubes into which superoxide dismutase enzymes were introduced have retained all their properties. As for the control tube, it was only satisfactory during the first three tests, which corresponds to the period of validity considered hitherto normal for commercial batches of sodium tetrathionate media. During the following tests it was found that the results for the control tubes were very irregular, which prevents the product from being considered as marketable. It is therefore found that this addition of 2 ml of a solution of enzyme superoxide dismutase of the Photobacterium leiognathi strain no ATCC 25 521 per 20 ml tube of sodium tetrathionate prolonged the oxidation resistance of the latter by at least 10 weeks and even beyond.

Example 21 Storage of potatoes A. 250 g of peeled potatoes are cooked in water at 100 ° C for 30 minutes. 60 ml of water are added to them and the mixture is passed through until a homogeneous mass is obtained. Four batches of 50 g are placed in beakers and to each beaker 20 ml of water are added and mixed well with the puree.

Spade PI Witness

P2 500 units of superoxide dismutase (10 units / g)

(P. leiognathi) are added and mixed P3 Control plus 0.5 ml 2.0% ascorbic acid

(final concentration 0.02% ascorbic acid) P4 Like P3 but 500 units of superoxide dismutase are also added.

All the samples are then lyophilized and left at room temperature.

B. Potato flakes containing 25 mg / kg of BHT (ditertiobutyl paracresol) and BHA (butyl-s hydroxy-anisole) and glycerol monostearate (1%) are used.

In 250 ml of boiling water, 45 g of the above flakes are added, the mixture is left for 2 minutes and stirred with a glass rod. Two batches of 50 g of this puree are taken and mixed with 50 ml of water.

10

Miss Witness

M2 500 units of superoxide dismutase

(10 units / g of puree) are added and mixed well

15

The two samples are then lyophilized and the powder left at room temperature.

After two months, the odor of samples A and B was compared by six people. In the presence of superoxide dis-20 mutase the odor is different from that of the controls, less acidic and more pleasant.

Example 22

25 Storage of carrots

We used exactly the same process as that described for potatoes, but in the case of boiled carrots, with the same amount of superoxide dismutase / g. After two months, the odor of the samples was compared by six people. In the presence of superoxide dismutase, the odor is different from that of the controls, less acidic and more pleasant.

B

Claims (12)

619731 1. Method for fighting against the auto-oxidation of substances liable to be degraded by an oxidation involving superoxide ions, characterized in that the said substances are associated with at least one enzyme superoxide dismutase. 2. Method according to claim 1, characterized in that said substances are foods, in particular lipid, protein or lipo-protein foods. 2 3. Method according to claim 1, characterized in that said substances are bacteria or viruses. 4. Method according to claim 1, characterized in that said substances are culture media used for cell growth. 5. Method according to claim 1, characterized in that said substances are lipids. 6. Method according to claim X, characterized in that said substances are antioxidants used in the food industry. 7. Method according to claim 1, characterized in that said substances are lipids and antioxidants. 8. Method according to one of claims 6 and 7, characterized in that said antioxidants are chosen from pyrogallol and ascorbic acid. 9. Method according to claim 1, characterized in that the superoxide dismutase is incorporated into said substance in an amount of 1 to 200 units of enzyme superoxide dismutase per milliliter or per gram of substance. 10. Application of the method according to claim 1 to the fight against auto-oxidation by means of a superoxide dismutase enzyme chosen from those extracted from marine bacterial strains, from Escherichia coli, from fungi, and those extracted from blood. 11. Application according to claim 10, characterized in that said marine strains are strains of Photobacte-rium leiognathi, Photobacterium phosphoreum or Photo-bacterium sepia. 12. Application according to claim 10, characterized in that said marine strains are chosen from a strain of Photobacterium leiognathi No ATCC 25 521, a strain of Photobacterium phosphoreium No ATCC 11 040 and a strain of Photobacterium sepia No ATCC 15 709.