DESCRIPTION

Field of application The present invention relates to the sector of the chemical industry in general and in particular the chemical industry which uses enzymatic reactions.

More particularly, the present invention relates to a process and a system for carrying out enzymatic reactions intended to obtain products of interest for nutrition or animal husbandry.

Prior art

In the continuation of the present description reference will be made to the animal feed industry and to enzymatic processes for obtaining feed from lignocellulosic biomass, but it must be understood that the enzymatic processes according to the present invention may find an application also in other industrial sectors, such as the food, dietary and pharmaceutical sectors.

It is also known that every year, throughout the world, millions of tonnes of cereals are used for the nutrition of livestock animals and that it would be desirable, also from the point of view of environmental sustainability, to have available alternative food sources.

It is also known that lignocellulosic biomass may not constitute a food source for monogastric animals, such as pigs, chickens and turkeys, since these animals, differently from plant-eating animals, are unable to digest and convert into glucose lignocellulosic materials such as agricultural waste, owing to the lack of enzymes needed to convert the cellulose and the hemicellulose into glucose.

The patent application WO 2014/202716 has recently proposed a process for the production of an additive composition for animal feed from lignocellulosic biomass, comprising a physical, chemical or biological biomass pre-treatment step, such as to allow the subsequent incubation for 3- 120 hours of the biomass pretreated with enzymes having an endoglucanase, beta-glucosidase and endoxylanase activity and substantially without a beta-xylosidase and/ or alpha-L- arabinofuranosidase activity, followed by drying and, where necessary, packaging.

The composition thus obtained is suitable also for the feeding of monogastric animals.

The aforementioned pre-treatment step may consist for example of a dry or wet grinding step, a treatment using high-pressure and high- temperature saturated steam ("steam explosion"), hydro thermolysis, wet oxidation, ammonia fibre explosion (AFEX), a treatment using solvents, ammonia percolation, an acid or alkali treatment, a treatment using microorganisms able to degrade the lignin and the cellulose, such as certain fungi which produce lignocellulolytic enzymes or others which produce hydrogen peroxide.

This pre-treatment step, however carried out, involves further costs and longer times, in addition to those of the following enzymatic treatment, as well as the possible permanence in the final product of the chemical substances or microorganisms used for the pre-treatment.

The process described in WO 2014/202716 is not suitable for being carried out continuously.

The problem underlying the present invention is that of providing a process for continuously carrying out enzymatic reactions on organic substrates, without the need for any pre-treatment of these organic substrates.

Summary of the invention

The problem has been solved by providing a process for continuously carrying out enzymatic reactions on an organic substrate, which comprises the steps of: a) providing a turbo-reactor comprising a cylindrical tubular body having at least one opening for the introduction of reagents and at least one discharge opening, an optional heating or cooling jacket for adjusting the temperature of said tubular body to a predetermined temperature, and a rotor, arranged inside the cylindrical tubular body and comprising a shaft provided with elements projecting radially therefrom, and feeding a continuous flow of said organic substrate into said turbo-reactor, in which the rotor is rotated at a speed greater than or equal to 150 rpm, in order to disperse said continuous flow of organic substrate into a flow of particles of said organic substrate, b) feeding into said turbo-reactor, together with said flow of organic substrate, a continuous flow of an aqueous solution of at least one enzyme adapted to convert said organic substrate into a desired product, which is dispersed by said rotor into a flow of minute droplets, c) centrifuging said particles and said droplets against the inner wall of said turbo-reactor, with formation of a highly turbulent, dynamic, thin, tubular fluid layer, in which the particles of organic substrate and said droplets of aqueous solution of at least one enzyme are kept mechanically in intimate contact by the radially projecting elements of said rotor, while advancing in substantial contact with said inner wall of said turbo-reactor towards the discharge opening; d) starting the conversion of the organic substrate into said desired product by means of said at least one enzyme in said thin layer as it advances substantially in contact with said inner wall of the turbo-reactor towards the discharge opening; e) discharging from said discharge opening a continuous flow of a mixture comprising said product, said at least one enzyme as well as organic substrate that has not yet reacted, f) feeding said continuous flow into a maturation reactor, comprising a box-shaped body, having a bottom, a cover and side walls and provided with at least one inlet opening and at least one discharge opening arranged opposite said inlet opening, a screw-type rotor arranged on the bottom of said box-shaped body, and elements in the form of V-blades and/ or beaters attached to said side walls in the region of said bottom; g) subjecting said continuous flow to continuous mixing by means of said screw- type rotor and said elements in the form of V-blades and/ or beaters, with which the screw-type rotor interacts, while said flow gradually advances towards said discharge opening; h) discharging from said discharge opening a continuous flow of said product and said at least one enzyme; i) subjecting said continuous flow to a thermal treatment at a temperature of at least 70°C, in order to inactivate said at least one enzyme.

Preferably the aforementioned thermal inactivation step i) is carried out by means of a turbo-inactivator, comprising a cylindrical tubular body having at least one inlet opening and at least one discharge opening, a heating jacket for adjusting the temperature of said tubular body to a predetermined temperature, and a rotor, arranged inside the cylindrical tubular body and comprising a shaft provided with elements projecting radially therefrom, - feeding said continuous flow of said product and said at least one enzyme exiting said maturation reactor into said turbo-inactivator, through said at least one inlet opening, the inner wall of the turbo- inactivator being kept at a temperature of at least 70°C by means of said heating jacket and the rotor being rotated at a speed of at least 150 rpm; - centrifuging and causing said continuous flow to advance inside the turbo-inactivator through the action of said rotor, and

- discharging from the outlet opening of the turbo-inactivator a continuous flow of said product free from enzymes in active form.

The heating or cooling jacket of the turbo-reactor of step a) is generally intended to be passed through by a heating/ cooling fluid, such as diathermic oil or steam.

The aforementioned elements projecting radially from the shaft of the rotor may be for example rod-like or in the form of blades, V-blades or beaters.

According to one aspect of the present invention, the aforementioned organic substrate is a lignocellulosic biomass, preferably selected from the group comprising corn cobs or husks; corn, wheat, barley, sorghum, soybean and oat straw; grass, beet pulp; rice, corn or wheat bran; rice hulls, citrus peels, palm kernel, sorghum and sugar cane bagasse, miscanthus, switchgrass, cord grass, sawdust, roots, leaves, wood chips, fruits, flowers, corn fiber, cotton, lignin. The aforementioned at least one enzyme is preferably selected from the group comprising endoglucanase, β-glucosidase, endoxylanase, cellulase - in particular cellobiohydrolase I and cellobiohydrolase II -, pectase, polysaccharide monooxygenase and mixtures thereof.

When the aforementioned organic substrates and the aforementioned enzymes are used, the product obtained with the process according to the present invention may be used as a component of feed for animals, including monogastric animals.

In this case the temperature inside the turbo-reactor used in steps a) to e) is kept at a value comprised between 40°C and 70°C, preferably between 45°C and 55°C, by means of the aforementioned heating/cooling jacket.

Preferably, the pH of said solution containing at least one enzyme is adjusted to a value comprised between 5 and 6, preferably between 5.5 and 5.7.

Preferably, the ratio between the flow rate of the organic substrate and the flow rate of said at least one enzyme is comprised between 1 :0.5 and 1 :3.

The concentration of said at least one enzyme in the aforementioned aqueous solution is generally comprised between 0.001% and 0.1% w/v, preferably between 0.005 and 0.03% w/v.

The residence time of the flows of organic substrate and of said at least one enzyme inside the turbo-reactor is generally comprised between 2 and 15 minutes, preferably between 5 and 10 minutes.

The rotational speed of the rotor of the turbo-reactor is generally comprised between 150 and 1000 rpm.

The residence time of the flow of said mixture in said steps f) and g) inside the maturation reactor is generally comprised between 0.5 and 10 hours, preferably 0.8-5 hours, conveniently 1-2 hours. The temperature inside the maturation reactor is preferably kept in a range of 40°-70°C, conveniently between 45°C and 55°C, by means of suitable heat exchange means inside or outside the reactor.

The thermal treatment of the aforementioned step i) is preferably carried out at a temperature comprised between 70°C and 120°C, conveniently between 90°C and 100°C.

The rotational speed of the rotor of the turbo-inactivator is generally between 150 and 1000 rpm.

According to a further aspect thereof, the present invention relates to a system for carrying out the process described above for the conversion of an organic substrate into a product by means of at least one enzyme, comprising the following apparatuses:

- a turbo-reactor comprising a cylindrical tubular body with horizontal or vertical axis, having at least one opening for the introduction of respective continuous flows of said organic substrate and an aqueous solution of said at least one enzyme and at least one discharge opening, an optional heating or cooling jacket for adjusting the temperature of said tubular body to a predetermined temperature, and a rotor, arranged inside the cylindrical tubular body and comprising a shaft which is provided with elements projecting radially therefrom and is able to be rotated at a speed greater than or equal to 150 rpm, and

- a maturation reactor, comprising a box-shaped body, having a bottom, a cover and side walls and provided with at least one inlet opening and at least one discharge opening arranged opposite said inlet opening, a screw- type rotor arranged on the bottom of said box- shaped body, and elements in the form of V-blades and/ or beaters attached to said side walls in the region of said bottom.

Preferably the system according to the present invention further comprises: - a turbo-inactivator, comprising a cylindrical tubular body with horizontal or vertical axis, having at least one inlet opening and at least one discharge opening, a heating jacket, for adjusting the temperature of said tubular body to a predetermined temperature, and a rotor, arranged inside the cylindrical tubular body and comprising a shaft which is provided with elements projecting radially therefrom and is able to be rotated at a speed of at least 150 rpm. The aforementioned heating or cooling jacket of the turbo-reactor is intended to be passed through by a heating fluid or cooling fluid, preferably consisting of diathermic oil or steam.

The aforementioned heating or cooling jacket of the turbo-inactivator is intended to be passed through by a heating fluid, preferably consisting of diathermic oil or steam.

The aforementioned elements projecting radially from the shaft of the rotor of the turbo-reactor and of the turbo-inactivator may be rod-like or in the form of blades, V-blades or beaters.

The process for enzymatic conversion of organic substrates according to the present invention, differently from the processes known from the prior art, does not require any preliminary treatment of the organic starting substrate, with a consequent marked reduction in the production times and costs.

Furthermore, the intense mechanical action exerted by the rotor of the turbo-reactor used in steps a)-d) is such that a significant quantity of kinetic energy is transmitted to the substrate and enzyme flows such as to trigger rapidly the enzymatic reaction. This triggering step generally does not require more than ten minutes and the creation of the dynamic, thin, tubular layer is able to avoid the negative effects observed in many processes of the prior art, due to the increase in the viscosity of the organic substrate as the reaction proceeds.

Moreover, again owing to the creation of the dynamic, thin, tubular layer it is possible to reduce, compared to the known processes, the quantity of water required to achieve optimum wetting of the substrate to be treated. Finally, owing to the heating/ cooling jacket of the turbo-reactor used in the aforementioned steps a)-d), it is possible to control precisely the temperature inside the turbo-reactor so that it remains in the region of the optimum temperature value for the reaction of a given enzyme or a given enzyme mixture.

Brief description of the drawings

The advantages and characteristic features of this invention will emerge more clearly from the description below of an example of implementation, provided hereinbelow by way non-limiting illustration, with reference to the apparatuses schematically shown in the attached figure.

Detailed description of a preferred embodiment

With reference to the figure, an apparatus used for the process according to the invention comprises a turbo-reactor T consisting essentially of a cylindrical tubular body 1 , closed at the opposite ends by end plates 2, 3 and coaxially provided with a heating (or cooling) jacket 4 intended to be passed through by a fluid, for example diathermic oil, so as to keep the inner wall of the body 1 at a predefined temperature. The tubular body 1 is provided with inlet openings 5, 6 respectively for the organic substrate and the aqueous solution of at least one enzyme, and with a discharge opening 7.

The tubular body 1 rotatably supports internally a rotor comprising a shaft 8 provided with elements 9 radially projecting therefrom in the form of blades, these blades 9 being arranged helically and oriented so as to centrifuge and at the same time convey towards the discharge outlet 7 the flows of organic substrate and aqueous solution of at least one enzyme and the mixture resulting from mixing and the initial reaction between the aforementioned two flows. A motor M is envisaged for operation of the bladed rotor at variable speeds ranging from 150 to 1500 rpm.

The intense mechanical action exerted by the blades of the rotor results in the creation of a dynamic, tubular, thin layer which advances gradually along the inner wall of the tubular body, producing on the inside thereof optimum wetting of the substrate by the aqueous solution of the at least one enzyme with triggering of the reaction catalyzed by the latter, while the temperature is kept constantly at the operating temperature of the enzyme by means of the heating/ cooling jacket 4.

When it exits from the turbo-reactor, the aforementioned wet mixture is fed via a pipe 1 1 , communicating with the discharge opening 7 of the turbo-reactor, to the maturation reactor M. The latter comprises a box-shaped body 12, provided with a bottom 13, cover 14 and side walls 15 and provided with at least one inlet opening 16 and at least one discharge opening 17 arranged in a position opposite to that of said inlet opening, close to the bottom, and with a screw-type rotor 18 arranged on the bottom of said box- shaped body and with elements 19 in the form of beaters and/ or V-blades attached to said bottom 13 and to said side walls 15 in the region of said bottom.

The screw- type rotor 18, which is rotatably supported close to the bottom 13, ensures, as a result of its rotation and the interaction between the screw and the elements 19 in the form of V-blades and/ or beaters, a continuous slow mixing of the mixture so as to allow continuous renewal of the enzymatic attack surface of the substrate. At the same time, the rotor also causes the mixture to advance slowly towards the discharge opening 17.

During this slow advancing movement, lasting generally at least one hour, the reaction between the at least one enzyme and the organic substrate is completed, resulting in the desired product.

Therefore, a flow of mixture containing the product obtained from the reaction of the at least one enzyme with the organic substrate and the at least one enzyme is continuously discharged from the discharge opening 17 of the maturation reactor M. This flow of mixture is continuously fed into the turbo-inactivator T, through the inlet opening 105.

This turbo-inactivator T', which has a structure entirely similar to that of the aforementioned turbo-reactor T, is not described in detail. The components of the turbo-inactivator T' which are the same as those of the turbo-reactor T are indicated by the same reference numbers increased by 100.

The flow of mixture entering the turbo-inactivator T' is centrifuged by the blades 109 of the rotor against the inner wall of the cylindrical tubular body 101 , heated by means of the heating jacket 104 to a temperature of at least 70°C, preferably between 70°C and 120°C, conveniently between 90°C and 100°C. The rotation of the shaft 108 of the bladed rotor at at least 150 rpm, preferably between 150 and 1000 rpm, has the effect that a dynamic, thin, tubular layer of the aforementioned mixture is created against the heated inner wall of the cylindrical tubular body 101 and an intense heat exchange takes place between the product and enzyme mixture and the aforementioned inner wall. In this way complete thermal inactivation of the enzyme occurs and the product output from the discharge opening 107 of the turbo-inactivator is completely harmless and stable during any following processing stages.

Finally, the continuous flow of product output from the turbo-inactivator may be collected inside a container 20 designed to separate it from the water vapour also output from the turbo-inactivator.

EXAMPLE

A flow of beet pulp (100 kg/h) was continuously fed, through the opening 6, into the turbo-reactor T, inside which the bladed rotor 8 was rotated at a speed of 700 rpm. At the same time, a flow (100 1/h) of an aqueous solution of an enzymatic mixture of cellulase and pectinase in a weight ratio of 10: 1 was continuously fed through the opening 5. The concentration of the aqueous solution of enzymatic mixture was equal to 0.01% w/v. The temperature of the inner wall was kept at about 55°C.

Immediately at the inlet of the turbo-reactor T, the flow of pulp was mechanically dispersed into particles which were immediately centrifuged against the inner wall of the reactor, where a dynamic, tubular, thin, fluid layer was formed. At the same time, the aqueous solution of the aforementioned enzymatic mixture fed through the opening 5 was finely atomized by the blades of the rotor 8 which also performed the immediate centrifuging of the very fine droplets obtained. The latter were thus introduced into the dynamic, tubular, thin, fluid layer of pulp particles, with which they were able to interact intimately.

After an average residence time of about 10 minutes inside the turbo- reactor, a flow of a mixture in the form of puree was continuously discharged from the opening 7. This flow was continuously fed into the maturation reactor M, through the opening 16.

Inside the reactor M the temperature was kept at a controlled value of about 55°C while the rotational speed of the screw-type rotor was adjusted so as to cause discharging of the flow of mixture from the opening 17 after an average residence time of about 1 hour.

This latter flow of mixture was continuously fed into the turbo-inactivator T', through the opening 105.

Inside the turbo-inactivator T' the wall temperature was kept at a controlled value of about 100°C, while the rotational speed of the bladed rotor 108 was kept constantly at 1000 rpm.

Owing to the intense mechanical action of the rotor blades, the mixture was immediately dispersed and centrifuged against the inner wall of the turbo-inactivator T in the form of a dynamic, tubular, thin layer and made to advance towards the discharge opening 107. During the advancing movement along the cylindrical tubular body an intense heat exchange occurs between the thin layer of mixture and the heated wall, this resulting in total inactivation of the enzymes present in the mixture.

After an average residence time of 30 seconds inside the turbo-inactivator T', a flow of a product comprising beet pulp treated enzymatically, together with totally inactivated enzymes and water vapour generated inside the turbo-inactivator following the aforementioned intense heat exchange, was continuously discharged therefrom.

The product output from the turbo-inactivator finally was conveyed into the collection container 20 inside which the water vapour was also removed. The product thus obtained was perfectly safe from a toxicological and microbiological point of view and also perfectly suitable for the nutrition of monogastric animals such as pigs and chickens.