WO2001018188A2

WO2001018188A2 - Apparatus generating oxygenated chemical radicals and industrial applications thereof

Info

Publication number: WO2001018188A2
Application number: PCT/FR2000/002438
Authority: WO
Inventors: Marguerite Gabrielle Calone-Bonneau
Original assignee: Bordeau, Philippe
Priority date: 1999-09-07
Filing date: 2000-09-05
Publication date: 2001-03-15
Also published as: WO2001018188A3; FR2798137A1; AU7297800A

Abstract

The invention concerns a novel apparatus for enzymatic catalysis, formatted, structured and designed to produce oxygenated free chemical radicals in continuous concentrated liquid or gas flux specifically adapted to various industrial purposes. The invention concerns a device comprising a sealed chamber provided with at least an orifice wherein are immobilised by adsorption, or by being bound on a support, enzymes of plant, microbial or animal origin, belonging to the oxidoreductase group. The inventive device, after various chemical compositions rich in oxygenated molecules and in specific enzymatic substrates have been introduced in the chamber, enables to generate, by contact with the immobilised enzymes, and in specific pH and temperature conditions, a concentrated and continuous flux of oxygenated free chemical radicals and oxidised substrates provided with biocidal activity. Said biocidal products are applied, in liquid or gas form and in sufficient concentration levels, for decontaminating food products (milk and milk products, meat, fruits and vegetables, beverages), for cleaning and disinfecting equipment (containers, tools, machines, fabrics, packages) and industrial premises, for detoxication and sanitizing water and air, and for destructive treatment of organic waste.

Description

APPARATUS FOR GENERATING OXYGEN CHEMICAL RADICALS AND ITS INDUSTRIAL APPLICATIONS

The present invention relates to the arrangement and the implementation of a new apparatus for enzymatic catalysis comprising immobilized oxidoreductases in order to produce a continuous flow of radicals and oxygenated chemical substrates with compositions suitable for specific industrial applications.

Oxidoreductases are a group of enzymes widely distributed in living organisms where they participate, in a regulated manner, in the various reactions of metabolism. Among these enzymes, peroxidases - and in particular lactoperoxidase (EC N ° 1.11,1.7) which appears abundant in the natural state in the milk of bovine - have been widely studied during the last decades because of their biological properties particularly interesting. Lactoperoxidase (LP), industrially extracted from cheese whey, has proven to be the essential element of a powerful natural antimicrobial system which plays a decisive role in the protection of young new born mammals against neonatal digestive microbial infections .

This natural antimicrobial system has 3 components:

- an enzyme: lactoperoxidase (LP) present in milk at an average rate of 30μgr per milliliter, - an oxidizable substrate: the thiocyanate ion (SCN ^" ) food metabolite secreted naturally in milk at a concentration of 0.02 to 0.26 millimeter,

- an oxygen supplier: hydrogen peroxide (H ₂ O ₂ ) produced both exogenously by certain intestinal bacteria and endogenously during the metabolism of circulating glucose under the action of cellular glucose oxidases of l 'animal.

In this system, in a liquid medium, lactoperoxidase catalyzes, at the expense of hydrogen peroxide, the production of oxygen radicals and oxidized substrates with biocidal activity according to the following reaction:

SCN ^" + H ₂ O ₂ LP OSCN ^" + H ₂ O Thiocyanate Oxythiocyanate

OSCN ^" + H ₂ O ₂ LP SCN ^" + H ₂ O ₂ + 0 ₂ ^"

The ubiquitous O ₂ - superoxide anion is a powerful oxidizing agent which leads to the generation of new free radicals such as hydroxyl radicals. (OH-), hydroxyperoxyle (0 ₂ H) trioxide (.O ₃ ) which are themselves converted into H ₂ O ₂ . The OSCN-oxythiocyanate product is in equilibrium with its acid form HOSCN. In the presence of sufficient quantities of hydrogen peroxide, the oxidation reaction of the thiocyanate ion continues towards even more oxidized oxyacid derivatives according to the following reactions:

OSCN ^" + H ₂ O ₂ LP O ₂ - SCN ^" + H ₂ O Oxythiocyanate + peroxyl (superoxythiocyanate)

O ₂ SCN- + H ₂ O ₂ _LE__ ^ O ₃ - SCN ^' + HO Superoxythiocyanate (trioxythiocyanate)

The free radicals produced as well as the oxidized substrates strongly interact with the biochemical components of the cell membranes of microorganisms, disorganize the transport of metabolites and inhibit protein and nucleic biosynthesis. Thus the lactoperoxidase system has been shown to exhibit antimicrobial activities with respect to various microorganisms such as bacteria (Staphylococci, Listerias, Colibacilli, Salmonella, Pseudomonas, Mycoplasmas), protozoa (Cryptosporidia), viruses (Bacteriophages)

This natural antimicrobial system has been adopted as a model for the preservation and conservation of milk and cheese products. Thus, various application methods, subject to patents, have been described in recent decades: one of the simplest consists in adding, to the food to be protected, the complete LP system, preformed with a substrate providing peroxide, (peroxide magnesium for example) or with a complementary enzymatic producing system (glucose-glucose oxidase for example).

The application of these biocidal formulations takes place in the form of bathing, spraying, spraying or dusting the products to be treated and protected. These methods of implementation require the use of quantities of enzymes proportional to the areas or volumes of the products treated and therefore prove to be expensive and incompatible with the economic constraints of the market. Furthermore, the variability of the various environmental parameters linked to the treated products (pH, temperature, salinity, oxygenation, etc.) greatly limit the practical implementation of the system as well as its local efficiency due to rapid degradation of the useful free peroxidase.

To overcome these difficulties, different formulations, various compositions and new application methods have been proposed in recent years.

US Patent No. 5,389,369 describes a bactericidal system based on the contact of a haloperoxidase with microorganisms in the presence of hydrogen peroxide, chlorine or bromine and amino acids.

The different components are prepared and stored separately until they are mixed in solution at the time of use.

Patent No. W96 / 38548 describes peroxidase compositions and methods for producing disinfecting and sterilizing solutions stored in anaerobic pressurized containers that can be activated by oxygen in the air at the time of use. Patent No. W97 / 15661 describes a microporous substance which selectively traps viruses and pathogenic microorganisms in the blood in order to subject them to close biocidal activity generated by an oxidative system linked to the microporous substance.

Unlike the previous inventions, the present invention, the subject of the patent application, relates to a new apparatus with immobilized enzymes making it possible to produce in a practical and high quantity, from different predetermined chemical compositions, a concentrated and continuous flow of oxygenated free radicals and oxidized substrates, in liquid or vapor phase, usable remotely for washing, decontamination, sanitization of various food products, including water, and industrial materials as well as for detoxification and sanitation polluted fluids in environmental engineering.

According to a first non-exclusive embodiment of the invention, the device (figurel) consists of a closed enclosure, for example a cylindrical tank (2), metallic (stainless steel, aluminum, various alloys, etc.) or made of synthetic material (plastic polymers) or glass, provided with at least one loading orifice (l) and / or an evacuation orifice (4) and the inner walls of which are covered with a membrane (3) nylon carrying enzymes immobilized as a mixture, such as beta-galactosidase, glucose oxidase and lactoperoxidase. The retained enzymatic mixture generates, from lactose, hydrogen peroxide (H ₂ 0 ₂ ) according to the following global reaction:

Lactose β.Galactosidase. Galactose + glucose Glucose Oxidase gluconolactone + H ₂ 0 ₂ »

The preparation of the enzyme-carrying membrane can be carried out by simple adsorption of the enzyme solution as follows:

A hydrophilic membrane, nylon 6,6, from the company PALL-France (St Germain en Laye) of dimensions: 30 x 30 cm is used.

Three different solutions of enzymes are prepared in 50 mM sterile citrate-phosphate buffer at pH 7.5: - A solution at 3 mgr / ml of fungal betagalactosidase (E.C. n ° 3.2.1.23) 10U.I./mg.grade VIII. from SIGMA - St Louis United States: betagalactosidase catalyzes the splitting of the lactose molecule into glucose, oxygen and water. A solution of 3 mgr / ml of fungal glucose oxidase (E.C. n ° 1.1.3.4) 20U.I./mg.grade II. SIGMA: this oxidoreductase, in the presence of oxygen, hydrolyzes glucose to D. glucono-lactone and to hydrogen peroxide. - A solution at 3 mgr / ml of bovine lactoperoxidase (E.C. n ° 1.11.1.7) 40U.I./mg.grade II. SIGMA: lactoperoxidase in the presence of hydrogen peroxide catalyzes the oxidation of thiocyanates, sulfites and nitrites possibly present in solution.

The three preceding solutions are mixed, in equal parts, in a final solution in which the nylon membrane is immersed (at a rate of 30 ml of solution per membrane of 30 × 30 cm) which adsorbs approximately 80 μg of enzymatic proteins per square centimeter after contact with 15 min at room temperature.

The membrane is then rinsed with 3 times 100 ml of phosphate buffer pH 7.5 and then saturated by immersion in 100 ml of a sterile 2% bovine casein solution in phosphate buffer, pH 7.5, for 15 min at room temperature . After a further rinsing with 3 times 100 ml of phosphate buffer, the membrane is drained and then dried at -20 ° C under vacuum and stored at 4 ° C, in a dehydrated atmosphere until its final insertion by mechanical fixing or by bonding to the internal walls of the tank (Figure n ° 1). The introduction of a given volume of a mixture of cow's milk, preheated to 37 ° C, activates the enzymatic system by endogenous lactose and leads to the catalytic oxidation of sodium thiocyanate (NaSCN) or potassium thiocyanate (KSCN) naturally present in milk. The production of antimicrobial oxythiocyanate (OSCN ^" ) makes it possible to practice a continuous and decreasing self-sanitization of the milk during its cooling and its storage in the tank.

This embodiment of the invention makes it possible to obtain prolonged storage of the milk for an average of 7 days at 10 ° C. (cf. results in table 1 of the bacteriological experimentation carried out during the application of the apparatus). The apparatus thus constituted can be used repeatedly, on several successive batches of milk, for several weeks. It is easily regenerable by renewing the membranes. This set of characteristics makes it compatible with practical, repetitive and inexpensive use, which makes it an effective device for treating, sanitizing and stabilizing liquid foods such as milks and dairy products, fruit juices and drinks for a prolonged storage compatible with enhanced food safety. Another industrial application, according to the invention, consists in using square fragments of 1 cm x 1 cm of a PALL membrane of pre-activated nylon immunodyne type ABC. The peroxidase type enzyme solution is fixed to the membrane by direct contact between the enzyme solution and the membrane, creating a stable covalent chemical bond. The membrane thus obtained, with immobilized peroxidases, can be used to coat, in part or in whole, by fixing or bonding the internal walls of different food containers and packaging, made of cardboard, metal, glass, plastic or composite materials, such as packaging of heat-treated milk (or pasteurized or sterilized), bottles and bottles of fruit juice and wine, the bags used for fruits and vegetables or 4 ^βme range.

These different foods (milks, fruit juices, wines) are rich in water, in natural chemical compounds providing oxygen (glucose, lactose, xanthine, ...) and in endogenous oxidizable substrates (thiocyanate SCN ^" ), brought into contact with the membrane when filling the container, induce a catalytic activation of oxygenated free radicals such as the superoxide O ₂ and trioxide O ₃ anion, the hydroxyl OH and hydroperoxyl HO ₂ radical, oxythiocyanate OSCN, superoxythiocyanate O ₂ SCN, trioxythiocyanate O ₃ SCN, ... which participate in a continuous self-sanitization which leads to an increased shelf life of food. Thus, this embodiment, according to the invention, makes it possible to develop and manufacture active biocidal packaging consisting either of paper and cardboard (cellulose), or of fibers (nylon, glass, carbon), or of organic films or synthetics (nylon, polyethylene, polypropylene, polysulfones, polyamides, polyvinyl ...), either of natural or synthetic fabrics, or of metallic materials (aluminum) intended to contain and protect various sensitive biological products such as food of animal origins (milks, cheeses, meats and meat products, egg products, fish, ...) and vegetables (fruits and vegetables, leaves, seeds and seeds, ...) against attacks and bacterial surface degradation thus leading to durations of prolonged storage conducive to marketing over time and space.

According to a second non-exclusive embodiment of the invention, the device is arranged according to the format of a plan microfiltration module (Figure 2) comprising an inlet (1) and an outlet (5) and consisting of a stack successive and alternating metal plates (2) membrane carriers (3) and metal separating collecting plates (4), clamped between the two end plates of a press. The faces of the plates have grooves facilitating the turbulence of the fluids, on the membrane-carrying plates (2) and the collection of the filtrate on the collecting plates (4).

The membranes used in the device can be, preferably but not exclusively, microfiltration membranes, made of hydrophilic nylon, of porosity 5 μ, previously activated of the Immunodyne ABC-Pall France type.

/ -

Table 1

Evolution of the bacterial flora in milk stored at 10 ° c.

ιαp. _iji u _ϋ uψc w ^il ^ ^d ^ ^tll tr

Time (game

C.F.U./ l C.F.U./ml treated milk control milk treated milk

AT

10. ^•

10. ⁴ 10 ² ' ⁵ 10, ⁵

10- ⁵ ' ⁵ 10. ² ' ⁵

10. ⁵ ₁₀ . ²

10. ⁵ ' ⁵ 10-' 10- ⁶ 10. ¹

10. ⁶ 0- 10- ⁶ ' ³ ta ¹

10. ⁶ ' ⁸ 0- 10 ⁶ ' ⁹ o.

10. ⁷ 0- 10. ⁷ 0,

10. ⁷ 10. ¹ 10. ⁷ o. The fixation of an oxidoreductase of plant origin such as black radish peroxidase (Horse Radish Peroxidase) (EC n ° 1.11.1.7) grade VI.300U.I./mg - SIGMA - or soy peroxidase (EC n ° 1.11.1.7) is carried out as described above, by depositing 30 ml of a sterile buffered solution of enzyme at 1 mg / ml on an Immunodyne membrane of section 30 x 30 cm, a contact time of 15 min at ambient temperature. After washing, saturation with casein and rinsing, the membranes carrying peroxidase are interposed between a membrane-carrying plate and a collecting plate (FIG. 1).

The membranes are definitively fixed on the plates by bringing together and pressing the end plates of the filter press.

The planar module is supplied by the continuous introduction, under pressure at 0.5 bar, of a cold aqueous solution (10 ° C) of the following formulation:

1) Sodium Meta Bisulfite (Na ₂ S ₂ Os): 1 gr or Sodium Bisulfite (NaHSOa) or Sodium Hydrosulfite (Na ₂ S_0)

2) Peracetic acid (CH ₃ CO ₃ H): 2 gr

3) Water qs (H ₂ O), 1000 ml

Sulfites (bisulfite NaHSO ₃ , metabisulfite a ₂ S ₂ θ5) have long been known as antimicrobial agents used in the production of wines and the preservation of fresh fruits and vegetables. Oxidation by immobilized peroxidase (HRP) of metabisulfite in the presence of peracetic acid which supplies hydrogen peroxide, generates free radicals such as sulfur trioxide SO ₃ ^' , and its acid derivative HSO3 ^' , as well as hydrogen peroxide H ₂ O ₂ . The titration of the hydrogen peroxide produced is carried out with ceric sulphate in the presence of feroin. The titration of sulfur-oxidized radicals is carried out according to the method described by Mottley and Coll. 1982 Mol.Pharmaco! .22: 732-737.

The filtrate stream from the module contains hydrogen peroxide and oxidized sulfite ions with marked biocidal activities in vitro. Indeed, a volume of 1 ml of a bacterial suspension of Staphylococcus at 10 ^B CFU / ml mixed with 1 ml of the aqueous flow leaving the module, for 5 min at 10 ° C, is completely inactivated. An identical result is obtained according to the same experimental methods on a culture of a strain of Colibacille at 5 × 10 ⁸ CFU / ml. The apparatus which is the subject of the invention, of simple use, makes it possible to generate flows of oxygenated radicals in solution with synergistic and enhanced biocidal activities, at doses lower than those usually necessary for the sulfites used alone. The biocidal effects observed, after microbiological experimentation according to the AFNOR standard, are bactericidal (Staphylococcus, Lactobacillus, Listeria, Colibacillus, Pseudomonas, ...) and virucidal (Bacteriophage) at low concentrations of oxygenated radicals: 400 ppm for sulfites and 200 ppm for H ₂ O ₂

As a preference, non-exclusive, flow oxygen radical obtained by this second embodiment are applied to washing and disinfection of fresh fruits and vegetables (which range 4 ^ee), herbal teas, grains, mushrooms as well as decontamination and health protection of carcass surfaces and meat products from slaughterhouses.

By way of non-limiting example, the washing and decontamination of 4 ^th range salads are performed at 2 showers, successive, plants for 5 min at 10 ° C with 5 volumes of an aqueous solution at 2 % of oxide radicals per 1 volume of plants which make it possible to obtain a reduction of 4 to 5 log on average of resident (Pseudomonas 10 ⁵ CFU / ml) or experimental (Listeria 10 ⁵ CFU / ml) contaminants compared to control salads not processed.

A variant of this second embodiment of the device is also indicated in a nonlimiting manner within the scope of the invention. It consists of fixing on equal square surfaces of 30cm side of Immunodyne ABC membrane. Pall

(5μ):

- A first solution in citrate-phosphate buffer 50 mM, pH 7.5 to 1 mg / ml of vegetable horseradish peroxidase (Horse Radish Peroxidase) (E.C. n ° 1.11.1.7) - grade VI-300 U.l./mg SIGMA

- A second solution at 2 mg / ml of bacterial nitrate reductase (E.C. n ° 1.9.6.1) -50 U.I./mg-SIGMA either

- A third solution of nitric oxide syπthetase animal (ECn ° 1.14.13.39) - SIGMA at 2 mg / l. The procedures for immobilizing enzymes on the membrane are identical to those described above. The first part of the plan module consists of a first series of plates carrying bacterial nitrate reductase membranes, the second part consists of plates carrying nitric oxide synthetase membranes of animal origin, the third terminal part consists of plates carriers of vegetable peroxidase membranes. The planar module is continuously supplied by a pump injecting, at the inlet of the device, at a pressure of 0.2 bar, a specific solution of substrates, of the following formulation:

Sodium metabisulfite (Na_.S_.O5) 1gr Sodium nitrite (NaN0 ₂ ) 1gr or Potassium nitrite (KN0 ₂ )

Arginine 1 gr

Peracetic acid (CH ₄ CO ₃ ) 1gr

Water q.s.p. 1000 ml

The filtrate leaving the module consists of water, hydrogen peroxide H ₂ O ₂ and various nitrogen and sulfur radicals oxidized by the peroxidases such as

Nitrite ion (NO ₂ ^" ), nitric oxide NO ^" , titrated trioxide (N ² O ³ ), nitric dioxide

(NO ² ), peroxynitrite (ONOO), hydroperoxynitrite (ONHO ₂ )

Sulfite ion (SO ₂ ^" ), sulfur oxide (SO ^" ), sulfur dioxide (SO ₂ ), sulfur trioxide (SO3) and its acid (HSO3)

The mixture of these different compounds exhibits instant and high antimicrobial activities against populations of microorganisms such as viruses (herpes, pox, influenza, retrovirus, enterovirus, hepatitis, etc.), bacteria (staphylococci, streptococci, listeria) , colibacilli, pseudomonas, legionella, bacillus, ...), fungi (fungi and molds), protozoa (amoebae, cryptosporidia, ...).

The industrial applications of the device according to the invention relate to the production of large quantities of antiseptic solutions which can be used for cleaning, washing and disinfecting materials, machines, tools, textiles, containers, pipes, equipment and premises (floors and walls) used in particular and not exclusively in the agrifood production chains and in hospital services It should be emphasized that different possible configurations of filtration modules such as tubular module, hollow fiber module, spiral module and cartridge can advantageously be retained, within the framework of the invention, to receive the immobilized enzyme membranes, and thus constitute devices according to the invention, specially adapted to different specific flow constraints of the various industrial applications mentioned.

Similarly, the membranes which can be used in the planar module can be made of different materials, the essential characteristics of which are their specific aptitudes for fixing, by electrostatic adsorption, or by affinity, or by chemical bond, various proteins with biological activity such as enzymes by example. Thus, according to the specific methods of the industrial applications concerned, the membranes with fixed enzymes used in the composition and the production of the apparatus according to the invention may advantageously be made up of cellulose acetate, polysulfones, polyamides, acrylic copolymers , of polypropylene or polypropylene polymers, or of polyolefin, of aromatic polymers, of ceramic, of silica, of carbon, of natural or synthetic fabrics.

Likewise, the various enzymes fixed in the apparatus according to the invention are advantageously chosen, according to specific industrial applications, from: - enzymes of plant origin such as for example black radish peroxidase (EC n ° 1.11.1.7) , or soy, nitrate oxidoreductase-NADPH (EC n ° 1.6.6.1) of cereals, ...

- enzymes of fungal origin such as for example glucose oxidase (ECnM.1.3.4), catalase (ECn 1.11.1.6), beta-galactosidase (ECn 3.2.2.23) and nitrate oxidoreductase -NADPH. (E.C. n ° 1.6.6.2.) Of aspergillus ...

- enzymes of bacterial origin such as for example NADH- Peroxidase (EC n ° 11.1.1) from Enterococcus, NADPH oxidoreductase (EC n ° 1.6.99.3.) from Vibrio, nitrate reductase (EC n ° 1.9.6.1 ) of colibacillus, Lactic oxidutase dismutase (EC n ° 1.1.3.2) of pediococcus, superoxide dismutase ((EC n ° 1.15.1.1.) of colibacille, Arthromyces peroxidase, (EC n ° 1.11.1.1.), beta-galactosidase (ECn ° 3.2.1.23.) from colibacillus - enzymes of animal origin such as, for example, xanthine oxidase

(E.C. n ° 1.1.3.22) of milk, lactoperoxidase (E.C. n ° 1.11.1.7.) Of milk, leukocyte myeioperoxidase (E.C. n ° 1.11.1.7.), Nitric oxide synthetase

(ECn ° 1.14.13.39) of nervous tissues, superoxide dismutase (ECn 1.15.1.1.) Of erythrocytes, sulfite oxidase (ECn ⁰ 1.8.3.1) of hepatocytes ...

A third embodiment (Figure 3) of non-exclusive embodiment of the invention relates to a cylindrical enclosure (3), metallic or synthetic, provided with an orifice at each end (1) and (4), and the internal volume of 5 liters, for example, is filled with microspheres (2) of latex or acrylic-oxirane or silica or polystyrene beads or of composite materials on which are fixed, by covalent bond, the oxidoreductases alone or associated with other enzymes of interest chosen for a predetermined industrial application. The preparation and implementation of glass beads carrying enzymes are described below, by way of nonlimiting example and leading to an apparatus having the largest possible reactive surface for a given volume.

A volume of 5 liters of non-porous glass beads with a mean diameter of 30 μm (Corning-USA) is washed in distilled water brought to 80 ° C., at the rate of 10 volumes of water for 1 volume of beads. The beads are drained and then immersed in 5 liters of a 10% solution of sodium aluminate, for 2 hours at 65 ° C. The beads are then washed again in distilled water, drained then suspended and stirred for 30 min at 25 ° C in 5 liters of an aqueous solution of polyethyleneimine (solution at 100 mgr - pH 10).

After another washing in phosphate buffer pH 7.4, the beads are resuspended by gentle stirring in 5 liters of a 5% solution of glutaraldehyde for 3 hours. The beads are then washed and incubated again for 24 hours in an aqueous solution of polyethyleneimine at 50 mgr / l - pH 7.0 added with 2 gr of NaBHsCN. After a last washing in phosphate buffer (3 volumes of buffer for 1 volume of beads) and draining, the beads are immersed in 5 liters of a buffered solution (50mM citrate buffer pH 7.5) of a mixture with equal parts of bacterial nitrate reductase (ECn01.9.6.1) .50U.I. / mg-SIGMA in solution at 1 mg / l and vegetable acid phosphatase (ECn ° 3.1.3.2) type IV 10U.I./mg-SIGMA- in solution at 5 mg / ml. The immobilization of the enzymes is obtained by gentle agitation of the beads in the solution (60 min at room temperature) followed by addition to the glutaraldehyde solution at the final concentration of 1% and a further additional incubation of a hour. The beads, thus activated, are washed with buffered water (citrate buffer 0.50 mM pH 7.5) then introduced and locked in the cylindrical enclosure. The orifices of the enclosure are connected respectively to a supply pipe and to a discharge pipe.

The industrial application of this type of apparatus, according to the invention, is involved in environmental engineering, in the treatment of detoxification of water polluted by nitrates and phosphates. The introduction of polluted water into the device, causes, at a temperature of 10 ° C, an enzymatic degradation and a continuous hydrolysis of nitrates and phosphates., In solution according to the reactions:

NO ₃ Nitrate reductase NO ₂ Nitrate reductase NO

PO ₄ Phosphatase PO ₃ Phosphatase PO ₂ Phosphatase PO

A first variation of this third embodiment of the device consists in loading the interior volume of the enclosure with glass beads, prepared and activated as indicated above and carrying, on their surface, vegetable peroxidase of black radish (EC n ° 1.11.1.7) grade VI.300U.I./mg - SIGMA -

The apparatus thus obtained is intended for the treatment of raw water or for the regeneration and decontamination of waste water. To do this, the apparatus, object of the invention, is used in a circuit connected to preexisting installations of a water treatment chain, downstream for example, devices with ultraviolet radiation or following devices for ozonation.

The association, consecutively, of the apparatus, according to the invention, with these installations leads to an accelerated production of oxygenated free radicals and to a catalytic oxidation of organic and metallic micropollutants. The water flow leaving the device can be connected to an activated carbon device which fixes and retains residual organic compounds and metal oxides. A ^'TNSI, the apparatus according to the invention, used for treatment of polluted waters by agricultural pesticides such as atrazine and simazine provides an effective radical oxidation of these pollutants and their notable decrease in water. The results obtained indicate that a single usual treatment of water with ozone (3 minutes to 4 mgr / l) makes it possible to reduce the initial content of atrazine by 50%. The consecutive associated use of the device, according to the invention leads to a 90% reduction in atrazine for an ozone dose equal to 1 mgr / l and an H ₂ O ₂ / Os ratio = 0.4 g / g.

Likewise, the above device can be used independently and in a unitary manner for the sanitization of drinking water. To do this, the device is supplied with water to be treated, previously added with potassium permanganate (Mn0 ₄ K) in 1% solution, or sodium hypochlorite NaOCI in aqueous solution at 0.5 ppm The radicals generated (oxypermanganate, hypochlorous acid HOCI, ...) induce an oxidative reaction and bleaching of the organic substrates contained in the liquid effluents and in the decantation sludge of wastewater.

Furthermore, another possible application includes the use of potassium permanganate (Mn0 ₄ K), entering the apparatus, in aqueous solution at a concentration of between 5 and 10%. The outgoing flow, rich in oxypermanganate, is applied by spraying on greasy solid organic waste from treatment plants and causes its degradation by accelerated oxidation.

Likewise, the above apparatus is advantageously used for oxidizing the volatile aromatic compounds and the mineral compounds present in industrial fumes and gases. This type of application consists in injecting the fumes and polluting gases into the enclosure and in making them diffuse and bubbled through the aqueous solution of oxygenated radicals. The oxidation obtained is a function of the flow rate adopted and leads to the degradation and the partial or total reduction of the aromatic and mineral compounds present.

This third embodiment of the device, object of the invention, can also make use, depending on the industrial applications chosen, of solid enzymatic supports such as gels of activated polysaccharides (agarose, sepharose, dextran, cellulose, starch , ...) or silica gels.

Claims

EVEND1CATIONS

1) An enzymatic catalysis apparatus characterized by a closed enclosure comprising at least one loading orifice and the internal, intrinsic and contained surfaces of which are covered with redox enzymes immobilized by adsorption, or by stable covalent chemical bonding. 2) Apparatus according to claim 1 characterized in that the intrinsic and contained internal surfaces, carrying immobilized redoxases, are made of different materials, alone or associated, in different formats, such as for example and without limitation :

- membranes (in cellulose acetate, in polysulfones, in polyamides, in polyvinyl, in acrylic copolymers, in nylon, in natural or synthetic fabrics, in polyethylene, in polyolefin, in polypropylene, in aromatic polymers, in silica, in carbon, ceramic, ...) arranged in a flat module, in a tubular module, in a hollow fiber module, in a spiral module, in a filter cartridge - beads or particles of agarose, cellulose, acrylamide, polystyrene , latex, glass, acrylic oxirane, metal (stainless steel)

- nylon, glass, metal fibers (stainless steel)

- specific walls of the enclosure, made of glass, plastic polymers or metal (stainless steel, aluminum, ...).

3) Apparatus according to one of claims 1 or 2, characterized in that the immobilized enzymes belong to one or more different molecular species associated with the group of oxidoreductases, cited as an example below without limitation : - enzymes of plant origin such as for example the peroxidase of black radish (EC n ° 1.11.1.7), or of soya, the nitrate oxydo-reductase-NADPH

(E.C. π ° 1.6.6.1) of cereals, ...

- enzymes of fungal origin such as for example glucose oxidase (ECnM .1.3.4), catalase (ECn ° 1.11.1.6), beta-galactosidase (ECn ° 3.2.1.23) and nitrate oxydo-reductase- NADPH. (EC n ° 1.6.6.2.) Of aspergillus ... - enzymes of bacterial origin such as for example NADH- Peroxidase (EC n ° 11.1.1) from Enterococcus, NADPH oxydo-reductase (EC n ° 1.6.99.3.) from Vibrio, nitrate reductase (ECn ° 1.9.6.1) of colibacillus, lactic dismutase oxidase (EC n ° 1.1.3.2) of pediococcus, superoxide dismutase ((EC n ° 1.15.1.1.) Of colibacillus, Arthromyces peroxidase,

(E.C. n ° 1.11.1.1.), Beta-galactosidase (E.C. n ° 3.2.1.23.) From colibacillus

- enzymes of animal origin such as for example xanthine oxidase (ECn 1.1.3.22) from milk, lactoperoxidase (ECn 1.11.1.7.) from milk, leukocytic myeloperoxidase (ECn 1.11.1.7.), nitric oxide synthetase (ECn ° 1.14.13.39) of nervous tissues, superoxide dismutase

(E.C. n ° 1.15.1.1.) Of erythrocytes, sulfite oxidase (E.C. n ° 1.8.3.1) of hepatocytes ...

4) Device characterized in that it comprises the apparatus according to one of claims 1, 2 or 3, the implementation of which comprises the introduction into the enclosure in liquid or gas phase, of one or more suppliers d associated oxygen such as for example and without limitation:

- glucose, lactose, xanthine, hydrogen peroxide, magnesium peroxide, potassium permanganate, peracetic acid, etc.

5) Device according to claim 4 characterized by. the fact that its implementation includes the introduction into the enclosure of one or more associated enzymatic substrates such as for example and without limitation; sodium thiocyanate (NaSCN) or potassium thiocyanate (KSCN), sodium bisulfite (NaHSOa), sodium hydrosulfite (a ₂ S ₂ θ ₄ ) and sodium metabisulfite (Na_.S_.O ₅ ), sodium (NaNO ₂ ) or potassium (KNO ₂ ) nitrites, sodium hypochlorite (NaOCI)

6) Device according to one of claims 4 or 5 characterized in that its implementation produces oxygenated free radicals alone or mixture such as for example and without limitation

- The superoxide O ₂ and trioxide O ₃ anion - The hydroxyl radical OH

- The hydroperoxyl radical HO ₂

- Hydrogen peroxide H ₂ 0 ₂

- Nitric oxide NO, titrated trioxide N Û ₃ , nitric dioxide NO ₂

- ONOO peroxynitrite - ONHO ₂ hydroperoxynitrite - OSCN oxythiocyanate ^"

- Superoxythiocyanate 0 ₂ SCN

- The trioxythiocyanate O ₃ SCN

- Sulfur dioxide S0 ₂ - Sulfur trioxide S0 ₃ and its acid HSO ₃

- HOCI hypochlorous acid

7) Oxygenated free radicals and oxidized substrates obtained according to one of claims 4, 5 or 6 characterized in that they have, alone or as a synergistic mixture "in vitro", in liquid or gaseous flows of strong biocidal activities against viruses, bacteria, spores, fungi and protozoa

8) Use of the device according to one of claims 1, 2 or 3 and / or of the device according to one of claims 4, 5 or 6, characterized in that it is used in various industrial applications such as for example, and without limitation to: - Treat, sanitize and stabilize liquid foods such as milks and dairy products, fruit juices and drinks, for prolonged storage and better food safety

- Produce large quantities of biocidal solutions that can be used for cleaning, washing and disinfecting equipment, machines, tools, textiles, containers, piping and the premises of industrial establishments, particularly agrifood and hospitals,

- Producing antiseptic washing solutions for surface decontamination treatments of food products such as fruits and vegetables, leaves and herbal teas, mushrooms, cereals and seeds, carcasses and slaughterhouse products,

- Perform an intensive oxidative treatment and bleaching of waste water and industrial liquid effluents as well as decantation sludge

- Apply an oxidative treatment for the accelerated degradation of organic and greasy waste from treatment plants and for the deodorization of gaseous discharges and industrial fumes,

- Participate in the detoxification and aseptic treatment of large quantities of drinking water for animal and human consumption

- Develop and manufacture active biocidal packaging intended for the prolonged conservation of fresh food, sensitive organic products, grains and coated seeds.