WO2015044257A2

WO2015044257A2 - Process for the inactivation of microorganisms present in an aqueous liquid contained in a closed container and apparatus to carry out said process

Info

Publication number: WO2015044257A2
Application number: PCT/EP2014/070468
Authority: WO
Inventors: Dante ALFIERI; Pavel Koulik
Original assignee: Alfieri Enterprise S.R.L.
Priority date: 2013-09-25
Filing date: 2014-09-25
Publication date: 2015-04-02
Also published as: ITMI20131585A1; WO2015044257A3

Abstract

This invention concerns a process for the inactivation of microorganisms present in an aqueous liquid contained in a closed container comprising multiple phases of heating and temperature maintenance to obtain adequate sterilisation of the treated liquid.

Description

PROCESS FOR THE INACTIVATION OF MICROORGANISMS PRESENT IN AN AQUEOUS LIQUID CONTAINED IN A CLOSED CONTAINER AND APPARATUS TO

CARRY OUT SAID PROCESS

DESCRIPTION

The present invention refers to a process for the inactivation of microorganisms present in an aqueous liquid contained in a closed container and an apparatus to carry out said process. The main purpose of the inactivation of microorganisms present in foodstuffs, commonly designated by the terms pasteurisation or steril isation, is to mini mise the health hazards associated with heat sensitive pathogens, such as bacteria in their vegetative state, fungi and yeast, with a minor alteration in the chemical, physical and organoleptic characteristics of the foodstuff.

Pasteurisation or steril isation of liquids contained in closed containers such as. for instance, sugary drinks, is particularly difficult. In fact, said procedure does not allow treatments to have a uniform effect on the entire contents of containers, causing partial inactivation of microorganisms and. therefore, making it impossible to sell the product.

In most cases the closed containers containing liquids to be sterilised are made of thermosensitive materials that can be deformed and release hazardous substances at lower temperatures than those used for normal steri l isation/pasteurisation processes. Specifically, a material that is widely used to produce food containers for liquids is PET. a thermoplastic polymer, whose heating temperature is l imited to 70-75°C, above which level the material starts deforming and can release hazardous substances.

The most used method by the liquid pasteurisation industry is the thermal method, which consists of raising the temperature of the liquid above the temperature at which the microorganisms contained therein are destroyed. This temperature level varies in the range of 90-130°C, depending on the nature of the microorganisms concerned. Pasteurisation with the autoclave reaches temperatures over 200°C by using "dry" heat. The duration of the pasteurisation process varies from a few minutes to a few hours. This method is generally accompanied by an alteration in the product. Furthermore, it is not appl icable to products in containers that are not resistant to high temperatures. Particularly, said method of inactivation of microorganisms is not appl icable to foodstuffs contained in containers made of plastic material, such as PET.

Many products are preserved from microbiological alteration by means of the chemical method according to which chemical products that inactivate microorganisms and allow long- term conservation are added to the product. Since these chemical products are hazardous for the human body, they are added to foodstuffs in small doses and are strictly controlled. However, nobody precisely knows the long-term effects of chemical additives on human health and. to date, the tendency is to remove any chemical prod uct/ pre servat i ve from foodstuffs and to maintain the product in its natural condition.

Methods based on penetrating ionising radiations (x rays, β and γ radiations, and others) are often used to sterilise foodstuffs, especial ly drinks that are closed in thei containers, but studies report that said radiations could create new molecular formations in the product due to the formation of free radicals, which are hazardous for the human body.

UV radiations have a steril ising action, particularly on packaged products, as long as the packaging and the product are transparent to said radiations. Moreover. UV radiations destroy groups of microorganisms that are concentrated in multi -layers only on the surface; therefore, they do not allow effective sterilisation. Said steri l isation method is only used very selectively as. for example, to steri lise liquids that are not contained in closed containers.

The method that appl ies high pressures in the range of ten kilobars has proven to be effective for the sterilisation of drinks in w hich there is a prevalence of microorganisms in the vegetative state but it has scarce effects on spores, which are. instead, hard to destroy with this procedure. Furthermore, the industrial application of this process is l imited to the treatment of containers filled with a certain product and divided into "lots," strongly penal ising continuous production and high performance.

One of the most w idely used physical processes to steril ise foodstuffs and. especially, drinks, is electroporation. w hich consists of submitting the product to the action of large amplitude pulsating electric fields. This method allows to steri lise the product at a low temperature and. therefore, to preserve its natural properties. However, said procedure is hardly appl icable in practice due to the risk of stretching, which alters the product. This method, applied to sealed drinks, would need the application of high voltages in the range of hundreds of kilovolts. which is hard to implement in the framework of the drinks industry.

Sonoporation consists of acting on the protective membrane of microorganisms through ultrasonic waves that cause local cavitation effects, creating irreversible perforations in the membrane. This method is poorly developed and needs long exposure times to be effective. Due to the long implementation times and high management costs, said method is scarcely used in the industrial framework.

PCT/NZ2006/000069 describes the combination of pressure pasteurisation processes with thermal pasteurisation processes. This combination aims at reducing the pressure level required to pasteurise the product, compared to pasteurisation processes that only use pressure. However, this reduction is not very important, and the results proved that acceptable pasteurisation only takes place at pressure levels in the range of ki lobars.

Documents US4. 695, 472, EP 1 328 167. W 02008/ 1 141 6. WO2008/ 1 141 1 6 describe the sterilisation processes that combine the thermal method and the electroporation method. In th is case, electroporation is used as a complementary process to the thermal process. The thermal method is applied to heat the product that is treated at a rate greater than 30°C per second.

Following said treatment, the concentration of microorganisms, such as Saccharomyces cerevisiae, Listeria innocua, Aspergillus niger, Byssochlamys fulva, Penicillium glabrum, Candida parapsilosis, Paecilomyces parapsilosis, can be reduced by 6 log.

However, this procedure does not allow to inactivate the many microorganisms that are of interest to the food industry, especially thermo-resistant microorganisms such as. for instance. Geobacillus stearthermophillus or Alicyclobacillus terrestris and. in a broad sense, thermophi le microorganisms that are in the state of "dormant" spores prior to application of the process.

According to many publications, said method is not effective and. as stated in documents WO2008/ 1. 14136 and WO2008/ 1 141 16. over a temperature of 65°C the electric field required to obtain a lethal effect should not exceed a few hundred volts/cm. This means that voltages over one kiloVolt should be appl ied to the container that contains the product to be pasteurised, and this is actually an important technical issue considering the danger of stretching the treated product and. therefore, altering its properties.

The scarce efficacy of electroporation on the inactivation of microorgan isms is confirmed by tests that have been performed and publ ished by several authors, such as. for instance, the monograph "Food Preservation by Pulsed Electric Fields^" ed. by H.L.M. Lel ieveld. S. Notermans and S.W.H. de Haan. CRS PressBoca Raton. Boston. New York. Washington DC. 2007 "Steril ization of Liquid Foods by Pulsed Electric Fields^" Shesha H. Jayaram. DEIS Feature Articles. Vol 16. N°6, 2000, which confirm that the effects of electroporation are practically none at all.

Hence the need for a process to inactivate microorganisms that are present in foodstuffs, particularly in liquids contained in closed containers, a process that can reduce the load of said microorganisms to a non-hazardous level for human health, maintaining the organoleptic and physical characteristics of the liquid contained in the closed container intact.

Moreover, a process for the inactivation of microorganisms that is easy and safe to carry out. and which can be implemented at a low cost is also required. An apparatus for the implementation of a process of inactivation of microorganisms, which allows a high degree of inactivation. is easy to carry out and maintain, and envisages low management and preparation costs, is also required.

Hence, the purpose of the present invention is to provide a process for the inactivation of microorganisms that are present in foodstuffs, a process that is appl icable to liquids contained in closed containers and which can. therefore, reduce the bacterial load in the entire quantity contained in said container.

A further purpose of the present invention is to provide a process for the inactivation of microorganisms that are present in foodstuffs, a process that does not alter the characteristics of the closed container in which the liquids to be treated are contained, and which, at the same time, does not alter the organoleptic characteristics of the liquid.

Another purpose of this invention is to provide a process for the inactivation of microorganisms that are present in foodstuffs, a process that is easy to carry out. that does not present implementation difficulties dictated by operating conditions that cannot be repeated in the industrial framework, and w hich has a low cost.

Another purpose of this invention is to provide an apparatus for the inactivation of microorganisms present in the aqueous liquid contained in a closed container, an apparatus that allows to implement an effective and lasting process, is easy to install and maintain, and envisages low management costs.

The above objectives are achieved w ith a process for the inactivation of microorganisms present in an aqueous liquid contained in a closed container, comprising:

• a first step of heating said liquid at a rate from 15 to 40°C/minute up to a temperature Ti_, w herein Ti is w ithin a range of +10% of the highest germination temperature T of the microorganisms present in said l iquid;

• a second step of maintaining said temperature T| for a time T i equal to one tenth of the highest reviviscence time / of the microorganisms contained in the aqueous liquid to be treated;

• a third step of heating at a rate φ given by the equation

φ =Κ·[ 10 + (Γ/3)⁷⁷³⁰]

w herein 0.3<K<3 up to a temperature T₂ given by the equation

T2 = 1.2 T + 5φ°^'3 + 25(φ* 13.5)° e^{" (φ/13)}

wherein T is the highest germination temperature of the microorganisms present in said liquid; • a fourth step of maintaining said temperature T₂ for a time greater than the time τ₂, wherein τ₂ is given by the equation

x ₂ = L²/a

wherein L is the greater characteristic dimension of the container, and if the thermal exchanges are laminar, a is equal to λ/pc, wherein λ is the thermal conductiv ity of the environment, p is its density e c is the thermal capacity of the environment;

if the thermal exchanges are convective. a is equal to 1 v, where 1 is the amplitude of convective vibrations and v is thei frequency:

• a fi fth step of cooling at a rate from 0.1-30 °C/minute.

Preferably, the liquid contained in the closed container is subjected to the application of a unipolar or bipolar pulsating electric field, whose amplitude E is greater than 1 .OOOV/cm up to power density expressed in kW/L and greater than 0.41 8φ.

Hence, there is greater inactivation of microorganisms present in the l iquid, ensuring higher quality standards.

Preferably the liquid contained in the closed container is subjected to the appl ication of ultrasonic vibrations at a frequency in the range of 20kHz and 5 Hz up to power density expressed in kW/L. and greater than 0.41 8φ.

It is, therefore, possible to contribute to a greater inactivation of microorganisms by increasing the quality of the products treated.

The container that contains the l iquid is preferably subjected to cooling before and/or after every heating or maintenance step.

The material of the container can. therefore, resist deformations caused by a rise in the temperature inside the container.

.Said container, which contains the liquid, is preferably subjected, before and/or during and/or after said third step of heating to such a degree of stirring that all the surfaces of the container that can be contaminated are wetted by said aqueous l iquid.

This ensures that the entire container is treated and decontaminated from any pathogen.

Preferably said stirring is obtained w ith a series of rotations of the container at a rotation rate in the range of 150-450 rev o 1 u t i o n s/m i n u te .

This guarantees greater efficacy of the microorganism inactivation process.

The container that contains the l iquid is preferably submitted before and/or during every step to the action of static pressure in the range of 0.1 and 1 MPa abs. This allows to uniform the inactivation process of microorganisms in the enti e volume of the container.

The heating steps are preferably implemented by using high frequency electromagnetic radiations.

Heating can. therefore, be rapidly and uniformly implemented on the entire content of the container.

The heating steps are preferably implemented by using microwaves.

The container is preferably made of dielectric material.

Hence, the electromagnetic radiations used to heat the liquid are neither hindered nor altered. An additional scope of this invention is to provide an apparatus for the inactivation of microorganisms present in an aqueous l iquid contained in a closed container.

Further characteristics and benefits of this invention will be highlighted in the description of the preferred forms of implementation, outlined as a non-limiting example in the enclosed figures, wherein:

Figu e 1 illustrates a graph that expresses the relationship between rapid heating rate φ, temperature T₂ and germination temperature T of microorganisms;

Figu e 2 illustrates a diagram of the temperature variation of the treated container and of its content based on the transition time of the container during the steps of the process, according to this invention:

Figure 3 i l lustrates the experimental result that shows the dependence of the residual quantity of microorganisms (Saccharomyces cerevisiae) treated at temperature T2=55°C based on preheating temperature ΤΊ ;

Figu e 4 illustrates the experimental result showing the dependence of the residual quantity of microorganisms (Byssochlamys fulva) treated at temperature T₂=65°C based on preheating temperature T^

Figure 5 is a general diagram of the apparatus for the inactivation of microorganisms present in an aqueous liquid contained in a closed container;

Figures 6 A. 6B, 6C and 7 illustrate an apparatus for the inactivation of microorganisms present in the aqueous liquid contained in a closed container, in which the containers are placed in a flow of water that ensures cooling and compensates for the pressure of the container during treatment. The basic biophysical event of this invention is generally known by the term thermoporation (Yu.A. Chizmadzhev, Bioelectrochem. Bioenerg. 1979, 6. 63 -70). The thermal movement of polar groups initially due to fluctuations of density on the surface of lipid membranes that envelope the microorganisms ensures that the stochastic pores located in them, wh ich are initial ly hydrophobic, are transformed into stable hydrophi lic pores.

The critical radius of pore rc and its energy barrier Ec are determined by the relationship:

rc =σ/γ ; Ec =πσ²/γ

where σ and γ are respectively the linear suiface tension of the rim of the pore and the surface tension of the lipid membrane.

The diffusion and accumulation of electric charges that accompany the evolution of microorganisms in the environment and the temperature conditions to which they are exposed make pore size and the forces to which they are subjected depend on the temperature level and on the conditions in wh ich the latter varies.

It has been found that these two characteristics of pore stability do not depend only on environmental temperature but on the increase rate of said temperature, indicated below with the letter φ.

In the first approximation, this dependence is inverse and linear. This means that, at a certain temperature, a higher temperature increase rate wi ll correspond to a lower energy level at which the pore loses its stabil ity and is, therefore, destroyed. Said phenomenon, which is highlighted in this invention, is called "i reversible dynamic thermoporation.^" The term "irreversible^" is added to specify that it is a case of irreversible destruction of microorganisms, namely that when the pore has reached its status of instabil ity, no factor can contribute to "heal^" it. The membrane is definitely perforated, the cytoplasm is emptied and the microorganism is deactivated.

The mechanisms of "poration^" o microorganisms depend on several factors, including the properties (acid or basic ) of the envi onment in which the microorganism is placed, its aw hydro activity, the presence of the electric field (electroporation), magnetic field (magnetoporation ). pressure pulse, particularly ultrasonic waves that create local cavitation (sonoporation ). Said mechanisms are poorly understood and are only described in the framework of simplified theoretical models. On the other hand, considering their technologically important aspect, they are used, in practice, based on empirical recommendations.

Several tests that allow to highlight the process according to this invention have enabled to determine the rate of temperature increase φ of the treated product and the temperature level T₂ at which the temperature of the treated product must increase based on the germination temperature T of the microorganisms in a neutral environment (pH=7) and of the time t of reviviscence of the same.

According to this invention, the term germination temperature designates the mini mum temperature at which a microorganism shifts from the state of spore to the vegetative state. According to this invention, the term reviviscence time designates the minimum time at which a microorganism that is at the right germination temperature shifts from, the state of spore to the vegetative state.

Germination or reviviscence occurs when environmental conditions are once again favourable to bacterial life and all functions of the microorganism are restored, namely the recoveiy of repl ication and of the expression of pathogenic factors.

Bibliographic references on germination temperature and reviviscence time can be found in: Jean-Paul Larpent. Monique Larpent - Gourgaud. Memento technique de microbiologic. Ed. Lavoisier TEC DOC Londres. Paris. New York. 1997; Bergey^' s Emmanuel. Systematic Bacteriology. Ed. Springer Vol A 1 4 , 2005; Lausig Prescott. Microbiology. 5^th edition. Ed. Mc Graw Hill. October 2002.

Table 1 shows some values that are adopted in microbiology laboratories for germination temperatures and reviviscence times of certain microorganisms that are more or less thermophil ic in a nourishing aqueous environment (a_w~l).

Table 1

According to this invention the temperature increase rate φ to which the temperature of the treated product must be raised based on germination temperature T of microorganisms is given by the equation [1]:

φ =Κ·[ 10 + (Γ/3 ) ⁷⁷³⁰]

wherein K is a factor that varies from 0.3 to 3 (0.3<K<3), preferably from 0.5 to 2 (0.5<K<2). According to this invention, the temperature T₂ to which the temperature of the treated product must be raised based on germination temperature T of microorganisms is given by the equation [2 j :

T₂ = 1.2 T + 5φ^0·3 + 25(φ* 13.5)° e^{" (φ/13)}

The values T₂ and φ are given in °C and in °C/second (°C/s), respectively.

Table 2 shows some values of T₂ and of φ calculated according to equations [1] and [2] described above.

Table 2

According to this invention, the process for the inactivation of microorganisms present in an aqueous liquid that is contained in a closed container comprises a first step of heating said liquid at a rate below 40°C/minute, particularly at a rate in the range of 15-40°C/minute up to a temperature T| .

Temperature T_t is within a range of +10% of the highest germination temperature T of the microorganisms contained in the aqueous liquid to be treated. Ti = T ± \ (Wc

Particularly, temperature Ti is within a range of 5-70°C in order to include the germination temperatures T of the most common pathogens present in l iquids.

According to this invention, in the second step of the process, the aqueous liquid treated is maintained at a temperature T I for a time Ti that is equal to one tenth of the highest reviviscence time t of microorganisms contained in the aqueous liquid to be treated;

I, = O. l f

Particularly τ_χ is in a range of 1.5-50 minutes to include the rev iviscence times t of the most common pathogens present in liquids.

According to this invention, the microorganisms involved in the inactivation process present a germination temperature T in the range of 20-60°C and a duration of reviviscence t in the range of 20-200 minutes.

Experimental tests conducted with a process based on this invention, as il lustrated in the examples below, have proven that the necessary condition to inactivate, through thermoporation. l iquids containing thermophilic microorganisms in the form of spores, such as Byssochlamys fulva, Alicyclobacillus terrestris or Geobacillus stearothermophillus, is preventive heating to not less than 30°C/min and maintenance at a temperature that is at least 45-50°C for at least 1 2- 1 8 minutes.

Conversely, for inactivation through thermoporation of microorganisms such as Candida parapsilosis or saccharomyces cerevisiae, it suffices to preventively heat them to a temperature of 25-35°C and to maintain them at this temperature for 3-7 minutes. Many microbiological tests, as indicated in the cited example, have been conducted in various experimental conditions. If these results are compared with data in Table 1, we note that the preheating rate must be below 30°C/min, and the duration x of maintenance of the solution at a preheating temperature Ti equal to Γ+10% (wherein T is the germination temperature of microorganisms) must be at least equal to one tenth of the duration of reviviscence τ of the microorganisms at temperature T.

These necessary preheating conditions are explained by the fact that, during this procedure, microorganisms are hydrolysed and adapt to the optimal conditions of germination (reviviscence ) for which they are most vulnerable to thermoporation. as proven by the test results. The event of reviviscence is a slow process of hydrolysis that is subjected to the laws of diffusion; hence the times, measured in tens of minutes, required to conduct this process. The steps of preheating up to temperature Ti and of maintenance at this temperature during the time interval ii are followed by a thi d phase of rapid heating from temperature T| to temperature T₂ at a heating rate of φ. The heating rate φ is calculated according to the equation:

φ =Κ·[10 + (Γ/3)^Γ/3°]

wherein K is a factor that varies from 0.3 to 3 (0.3<K<3), preferably from 0.5 to 2 (0.5< <2), and T is the germination temperature of microorganisms.

Temperature T2 to which the liquid to be treated is rapidly heated starting from temperature T I is calculated based on the equation:

Tj = 1.2 T + 5φ⁰-³ + 25(φ* 13.5)° e^{" (φ/13)}

as indicated above.

The third step of rapid heating from temperature T i to temperature T2 is followed by a fourth step of maintenance at temperature T2 of the treated aqueous liquid.

The duration of the fourth step of maintenance of temperature T2 is greater than duration τ₂ of the propagation of thermal waves in the cell containing the container. This step is necessary to ensure that the liquid is treated uniformly throughout its volume. The duration of τ₂ is calculated according to the equation:

τ 2 = L²/a

wherein L is the greater characteristic dimension (actual diameter or characteristic dimension of the container), and is equal to λ/pc (λ is thermal conductivity of the environment, p is its density, c is the thermal capacity of the environment ) if the thermal exchanges are laminar, and is equal to l^"v (where I is the amplitude of convective vibrations and v is thei frequency ) if the thermal exchanges are convective. The greater characteristic dimension is determined according to the description provided in "Hans Dieter Baehr, Karl Stephan HEAT AND MASS TRANSFER^" Heidlelberg. Dordrecht. London. New York Springer Verlag. Berlin. Heidelberg. 20 1 1 .

According to this invention the maintenance time of temperature T2 is in the range of 0.1 seconds to 10 minutes.

According to this invention, at the end of the maintenance step of temperature T2, the process includes a fifth step of cool ing o l iquid treated at a cooling rate in the range of 0. 1 - 30°C/minute. The lower limit is determined to avoid the possible "revival" of some microorganisms for which the thermoporation process has not been perfectly irreversible (in th is case too rapid cool ing can lead to regeneration of the lipid layer and to reactivation of the microorganism ). The upper l imit is influenced by the need to submit the treated product to a thermal regimen only for a minimum duration to prevent the thermal process from influencing the qual ities of the treated product.

According to this invention, in order to implement the thermoporation process of microorganisms present in the aqueous liquid contained in the closed containers, the process can envisage the application of a unipolar or bipolar pulsating electric field on the volume of the container. The ampl itude E of the unipolar or bipolar pulsating electric field is greater than I OOV/cm in order to generate irreversible electroporation of the microorganisms and amplify the effect of thermoporation. The appl ication of the pulsating electric field can take place during and/or after the container is taken to temperature Ί The minimum value E corresponds to an energy density of the electric field, SQ E 12 (i¾ is the electric permeability of vacuum ), that is equal to one tenth of the minimum value of the density of the thermal power. pc(T₂-Ti). No important electroporation effect takes place below this value. According to this invention, pow er density E expressed in Kw/L is greater than 4.41 8 φ.

Moreover, the process can envisage the application on the volume of the container of ultrasonic vibrations that cause irreversible sonoporation of the microorganisms at a frequency in the range of 20 kHz and MHz, ensuring thermal power density of 2 kW/L. This ultrasonic energy amplifies the effect claimed by thermoporation. The application of ultrasonic vibrations can take place during and/or after the container is taken to temperature T₂. According to this invention, the power density expressed in Kw/L is greater than 4.41 8 φ. According to the procedure of this invention, it is preferable for the container and its content to be submitted before and/or during and/or after every heating and maintenance step to external cooling to avoid deformations of the material which said container is made of due to the temperature increase of the liquid contained in it.

According to this invention, the process preferably envisages that the container that contains the l iquid to be treated is submitted before and/or during every step to the action of static pressure in the range of 0.1 and 1 MPa abs. The principal effect of this pressure is to uniform the thermoporation process in the entire volume of the container, since it can be disturbed by the presence of either air bubbles or a gaseous volume in the flow. The increased pressure reduces the volume of said air bubbles and gaseous volumes, and encourages thermal and convective exchanges in the treated product. As a secondary effect, the increase in static pressure compensates for the stress that develops in the container as a result of the pressure difference that develops when the l iquid contained in the hermetically sealed container is heated. The level of static pressure in question cannot reasonably be greater than 1 MPa, which is a pressure that can compensate for the stress in the container caused, for example, by heating of a gaseous drink. However, the level of static pressure must not reach that of pressure pasteurisation (in the range of one ki lobar) in order to avoid the well known effects of alteration of the treated product following its pressure pasteurisation.

According to a preferred form of implementation, the procedure of this invention envisages stirring the container before and/or during and/or after the third step of heating.

Hence, the amplitude of the turbulence due to forced convection thus caused is greater than the characteristic size of the container, and the frequency of said turbulence is greater than the reverse of the duration of the third step.

The process of inactivation of microorganisms is implemented through contact with the aqueous liquid contained in the container with the surfaces on which the microorganisms are located, namely the internal surface of said container.

Hence, stirring must be such that, during treatment, al l surfaces that are sensitive to contamination are wetted by the aqueous liquid treated at temperature Ί This measure must be taken whenever gaseous volumes are present in the treated container. Particularly, effective stirring of the treated container that contains one air bubble can be obtained by transmitting to the container a rotation speed that breaks down and transfers the air bubble to the entire volume of the container. Experience has proven that, for one half litre bottle made of PET, filled with a drink and sealed to contain one air bubble of ~ 5 mL. effective stirring is obtained if the bottle is placed horizontal ly and if it is given a rotation speed in the range of 250-350 revolutions/minute.

Hence, it is general ly advisable to submit the container and its contents before and/or during and/or after said thi d step of heating to a series of rotations of the container at a speed in the range of 1 0-450 revolutions/minute to ensure that the amplitude of the convective turbulence generated by the movement of air bubbles inside the container is greater than the characteristic size of the container, and the frequency of said turbulence is higher than the reverse of the duration of the thi d step.

According to this invention, the heating of the liquid contained in the closed container can be implemented with any heating technique normally used in the thermal inactivation processes of microorganisms.

The thi d step of rapid heating of the l iquid to temperature T2 is preferably achieved by using low frequency electromagnetic radiation, or preferably with microwaves. Hence, heating can be uniform on the entire volume of the treated container. To ensu e adequately uniform treatment, it is advisable to use electromagnetic radiation, whose wavelength is much greater (at least three-fold) than the characteristic dimension of the container.

If heating is implemented with high frequency electromagnetic waves or microwaves, the container must be made of a dielectric material. Hence, the electromagnetic radiations are neither hindered nor altered.

The apparatus for the implementation of the process of inactivation of microorganisms present in an aqueous liquid contained in a closed container is shown schematical ly in Figure 5. It includes loading (1) and unloading (2) mechanisms of containers (3) filled with liquid and sealed, at least 5 thermal treatment stations (4 - 8), non-thermal treatment stations (e.g. (9) and (10)), and a transport system (17) of containers that cross said stations.

The first thermal treatment station (4) includes a slow heating device that ensu es a temperature increase in the containers and their content at a rate that is less than 30 degrees per minute, from its initial temperature to temperature ΊΊ , as defined by the equation given above.

The second thermal treatment station (5) includes a thermostat that ensu es maintenance of container temperature and content at level ΊΊ . as defined above.

The third thermal treatment station (6) includes a heater powered by a source of cu rent (11) configured to heat the volume of the container by crossing it at a speed defined by equations [1] and [2] from temperature Ti to temperature T₂.

The fourth thermal treatment station (7) includes a thermostat that ensures maintenance of the temperature of the container and of its content at level T₂ for a period greater than duration T_2i as defined above.

The fifth thermal station (8) includes a cooling system at a rate in the range of l-30°C/minute. The device contains one or more transportation systems (14, 15, 16) that ensure the transfer of containers from one thermal station to another.

In a preferred implementation form, if the container is made of dielectric material, the heater can be a microwave resonator conceived to operate continuously or intermittently by ensuring a power density of ψ uniformly distributed in the volume of the treated liquid.

Conversely, the heater can also be conceived as a condenser that belongs to an I I F resonance circuit that operates continuously or intermittently by ensuring a power density of ψ uniformly distributed in the volume of the treated liquid.

The power density of the heater, expressed in Kw/L. is determined by the equation:

ψ = 4. 1 8 φ

wherein the heating speed φ is expressed in °C/s. The device can contain non-thermal treatment stations (9) and (10) of the containers and of their content, stations that are intended to strengthen the microorganism destroying effects of the process claimed by irreversible dynamic thermoporation. Devices, for example, of electroporation (9) and sonoporation (10), respectively powered by sources of current (13) and (32) can be used to this end. In such cases the containers are transported through systems in which they are subjected to continuous or pulsating electric fields and to ultrasonic waves, which are equally continuous or intermittent. In practice, for these complementary treatments to be effective, the power density of these systems must be greater than ψ/10.

The containers filled with liquid and sealed are transported and unloaded by transportation devices (14), (15) and (16).

Figures 6 A. 6B. 6C provide a schematic diagram of the apparatus, according to this invention. According to this invention, the device is used to pasteurise drink bottles (3) through the thermoporation process. The containers, for example bottles (3), contain an aqueous l iquid (e.g. a drink or an aqueous pharmaceutical product ). The bottles are transported in a vertical position by a conveyor belt (14) that acts as interface between one production l ine and said apparatus. The bottles are placed in a heat exchanger (4) in which their temperature shifts from environmental temperature to temperature T| . The bottles are then recovered by a loading device (1) that transfers them to the conveyor belt, slipping them in a horizontal position inside the dedicated individual container cells, which are fitted on the conveyor belt. The latter follows a path formed by two horizontal sectors and two semi -circular ones. The haul-off unit is instal led in one of the semi-circular sectors. The speed of this conveyor belt determines the productivity of the device. Every containment cell has a cooling system and a bottle stirring system. Cooling is implemented by a flow of water driven by a pump (26) that is fitted with a capacity and pressure regulation system. The increase in cooling water pressure compensates for the pressure that develops in the bottle after it is heated. This increase in external pressure is not necessary unless the resistance of the bottle wall material, the major part of whose volume has a low temperature thanks to cooling, does not suffice to resist the efforts developed by pressure inside the bottle. The containment cel ls are made of dielectric material. The inferior horizontal sector of the conveyor belt with the containment cells is submerged in water contained in the two tubs (22), which are used as tanks and are connected by pipes (19, 25 ) to the cooling systems of the containment cells. Once submerged in water, the containment cel ls enter the heater (6). In the diagram illustrated in Figure 6B, the heater is a plate capacitor (18) that belongs to the resonance circuit of a high frequency generator (11), which ensures heating of both the bottle and its content from ΊΊ to T₂ following the conditions of this invention.

In other words, the heater is a plate capacitor (6) that belongs to a resonator, which is powered by a high frequency wave generator (11) that ensures power density, expressed in kW/L. greater than 4. 1 8 φ (°C/s), since the space between these plates is filled by a liquid, cells (27) w ith the containers, cooling liquid and the containers (3).

Even the cool ing water is subjected to the action of high frequency radiation but its capacity is determined by the pumping conditions to prevent its temperature from varying except by a quantity in the range of 1-2°C during transition inside the containment cell. Following transition in the heater, the containment cells cross the area (7) in which the temperature of the liquid that fills the bottle remains constant, and the l iquid is stirred to preserve this level of uniform temperature. The bottle leaves its containment cell through the top part of the supporting conveyor belt after performing a complete revolution. It is then picked up by the unloading mechanism (2) that places it in a vertical position once again and introduces it into the cooling station (8). After cooling, the bottles are placed on the conveyor belt once again (15), which reinserts them in the production l ine. The direction of bottle movements is illustrated by the arrows.

Figure 7 represents the section of a containment cell that encloses the bottles and illustrates an example of execution. It contains a cover (23) that allows to introduce the bottles into the containment cell and to close it hermetically, an ejection axis (31) and a rotation mechanism (24. 25) that allows to rotate the bottle at a speed by which the air contained in it is distributed in the entire volume of the bottle, providing a turbulent character to the heat exchanges that take place in it.

According to this invention, the apparatus is, hence, characterised by the fact that it includes rotation mechanisms for the containers (24, 25 ). bui lt and designed to ensure that the ampl itude of the stirring movements caused by the movement of air bubbles inside the containers and which create convective heat exchanges inside the latter are greater than the characteristic dimension of the container, and thei frequency is higher than the reverse of the duration of step 3 of the process.

Generally the apparatus to start up the process of inactivation of microorganisms that are present in sealed containers that contain an aqueous l iquid (3) and move in a tank (22) fil led with liquid or gas. and which are taken to the conveyor belt that transports them through a system of thermal treatment stations (4—8) is characterised by the fact that the system is made up in sequence by: a first heat exchanger (4), which ensures a slow increase of the temperature of the containers and their contents at a rate lower than 30 7min., from their initial temperature Ti, as defined in Claim 1 ;

a first thermostat (5) that maintains the temperature of the containers and their contents at the temperature Ti for a period given in Claim 1 ;

a heater (6) that operates continuously or intermittently at a power density, expressed in kVV/L. greater than 0.41 8 φ (°C/s), distributed uniformly upon the volume of the container, and configured to heat the volume of the container at a rate φ, as described in Claim 1, from a temperature ΊΊ to a temperature T₂ , as defined in Claim 1 ;

a second thermostat (7) that maintains the temperature of the containers and their contents at temperature T₂ for a period given in Claim 1 ;

a second heat exchanger (8) which ensures transition of the container and of its contents from temperature T₂ to a temperature of less than 40° at a rate from 0.1 to 30 min.

Thermal exchanges between the liquid and the containers during steps 3 to 5 are ensured by the cooler (28) and by the pump (26).

The devices in Figures 5 and 6 include readers of temperatures Ti and T₂ of the liquid and of containers, of cooling water capacity, of the pressure in the containment cells, readers that are not indicated in the diagrams and which measure process parameters and in order to control the performance of irreversible dynamic thermoporation. Likewise, the control devices are not indicated in Figu es 5 and 6. As a rule of thumb, it is, hence, absolutely necessary for the claimed device to have readers of temperatures Ti and T₂ with rotation speed of containers (3), of cool ing liquid pressure, of cooling water capacity of the containers (3) inside the containment cells (27), and of the transport speed of containers given by the mechanism (16). EXAMPLES

The examples given below were implemented by subjecting a sample of liquid contained in a closed container with microorganisms present in a known quantity to heating from its initial temperature to a temperature T 1 in the range of 5-70°C at a heating rate in the range of 15-

40°C/minute. Temperature TI is maintained for a time in the range of 1.5-50 minutes, then the sample is heated up to a temperature T2 in the range of 40-80°C at a heating rate in the range of 10-70°C/minute. Temperature T2 is maintained for a time in the range of 1 .5-50 minutes. The sample is finally cooled at a cooling rate in the range of 0. 1 -30 °C/minute. The sample thus treated is once again analysed to assess the quantity of active microorganisms that are still present.

The analysis of microorganisms is carried out according to rule NF EN IS04833-2 by incubating the microorganisms on Agarose gel for 5 days at a temperature of 30°C.

The results are expressed in concentration of microorganisms contained in the treated sample. Example 1

The example was conducted by subjecting a sample containing Saccharomyces Cerevisiae to the above process. Saccharomyces Cerevisiae presents a germination temperature (J) of 32°C and a reviviscence time (t) of 30 minutes.

Operating conditions:

• initial concentration of microorganisms: from 1.7 to 3.3 10⁶ L/mL

• quantity of samples treated by the regimen: 3

• initial temperature: 15°C

• duration of maintenance at temperature ΊΊ : 30 min.

• K= l

• T, : 15, 20, 25, 35°C

• T₂: 55°C; 60°C; 65°C

• heating rate from T, to T₂ (φ): 15, 25, 40, 60°C/s

• continuous flow-

Results:

Table 3 T, = 15 °C

Table 4 T, = 20 °C

Table 5 T, = 25 °C

Table 6 T, = 35 °C

From the results illustrated in tables 3, 4, 5 and 6 we can notice that, for a treatment temperature of T2 = 55°C, the residual concentration of microorganisms after thermoporation treatment based on preheating temperature T l tends towards a minimum amount of l > 30°C (T = 32°C). The treatment is more effective as the heating rate φ increases. Total inactivation is reached with this value of T2.

Conversely for temperature T2=65°C, total inactivation of liquid is reached wherein φ >

25°C/s and Tl > 25°C.

Example 2

The example was conducted by subjecting a sample containing Byssochlamys Fulva to the above process. Byssochlamys Fulva presents a germination temperature ( T) of 40°C and a reviviscence time (t) of 120 minutes. Operating conditions:

• initial concentration of microorganisms: from 2.3 a 3.1 10⁴ L/mL.

• quantity of samples treated by the regimen: 3

• initial temperature: 15°C

• duration of maintenanc at temperature T 60 min

• K = l

• T, : 15, 20, 35, 40°C

• T₂: 60; 65°C

• heating rate from T, to T₂ (cp): 15, 25, 40, 60°C/s

• continuous flow

Results:

Table 7 T, = 15 °C

Table 9 T, = 35 °C

Table 10 T, =40 °C

From the results illustrated in tables 7. 8. 9 and 10 we can notice that, for a treatment temperature of T2 = 60°C. the residual concentration of microorganisms after thermoporation treatment based on preheating temperature T 1 tends towards a mini mum amount of ΊΊ > 40°C (T = 45°C). The treatment is more effective as the heating rate φ increases. Total sterilisation of l iquid is reached, wherein φ > 40°C/s and Tl > 40°C.

It is observed that total inactivation of l iquid is reached for treatment temperature T2=65°C, wherein φ > 25°C/s and T l > 25°C.

The comparison with results of example 1 shows that the process is more effective when the germination temperature T of microorganisms is low. As observed, inactivation of

Byssochlamys Fulva, which is more thermophilic than Saccharomyces Cerevisiae, takes place at values above parameters φ, T 1 and T2.

Claims

Process for the mactivation of microorganisms present in an aqueous l iquid contained in a closed container, comprising:

• a first step of heating said liquid at a rate from 15 to 40 °C/minute up to a temperature Ti_, wherein ΊΊ is within a range of ±10% of the highest germination temperature T of the microorganisms present in said l iquid;

• a second step of maintaining said temperature ΊΊ for a ti me T i equal to one tenth of the highest reviviscence time / of the microorganisms contained in the aqueous liquid to be treated;

• a third step of heating at a rate φ given by the equation

φ = ·[10 + (Γ/3)^Γ/3°]

wherein 0.3<K<3 up to a temperature T₂ given by the equation

T₂ = 1.2 T + 5φ^α3 + 25(φ· 13.5)¹³ e^{" (φ/13)}

w herein T is the highest germination temperature of the microorganisms present in said liquid;

• a fourth step of maintaining said temperature T₂ for a time greater than the time τ₂, wherein τ₂ is given by the equation

τ ₂ = L²/a

w herein L is the greater characteristic dimension of the container, and if the thermal exchanges are laminar, a is equal to λ/pc, w herein λ is the thermal conductivity of the environment, p is its density e c is the thermal capacity of the environment.

if the thermal exchanges are convective. a is equal to 1 v, wherein I is the amplitude of the convective vibrations and v is their frequency;

• a fifth step of cooling at a rate from 0. 1 to 30 °C/minute.

Process according to claim 1, characterized in that said liquid is treated w ith a unipolar or bipolar pulsating electric field having an amplitude E higher than 1 OOOV/cm up to a power density expressed as kW/l higher th n 0.41 8φ.

Process according to one or more of the preceding claims, characterized in that said liquid is treated with ultrasonic vibrations at a frequency from 20kHz to 5 MHz up to a power density expressed as kW/l greater than 0.41 8φ.

4. Process according to one or more of the preceding claims, characterized in that said container containing said liquid is cooled before and/or during and/or after any step of heating or of maintaining said temperature.

5. Process according to one or more of the preceding claims, characterized in that said container containing said liquid is subjected cooled before and/or during and/or after said third heating step to a stirring whereby al l the surfaces of the container that can be contaminated are wetted by said aqueous liquid.

6. Process according to one or more of the preceding claims, characterized in that said stirring is obtained by means of a series of rotation of the container at a speed of from 150 to 450 rounds/minute.

7. Process according to one or more of the preceding claims, characterized in that said container containing said liquid is subjected before and/or during each step to the effect of a static pressure from 0.1 to I MPa.

8. Process according to one or more of the preceding claims, characterized in that said heating steps are carried out by means of a high-frequency electromagnetic radiation.

9. Process according to one or more of the preceding claims, characterized in that said heating steps are carried out by means of microwaves.

10. Process according to one or more of the preceding claims, characterized in that said container consists of a dielectric material.

1 1 . Apparatus for the inactivation of microorganisms present in an aqueous liquid and contained in a closed container, w herein said containers containing said aqueous liquid (3) are transported in a cel l (22) filled with liquid or gas. moved by a conveyor belt w hich convey them through posts of thermal treatment (4, 5. 6. 7. 8), characterized in that said posts of thermal treatment comprise:

• A first heat exchanger (4) which ensures a slow increase of the temperature of the containers and their contents at a rate lower than 0 %nin. from their initial temperature to a temperature Tl ;

• A first thermostat (5) that maintains the temperature of the containers and their contents at the temperature T l for a given period:

• A heater (6), that operates continuously or intermittently at a power density, expressed as kWVl. greater than 0.41 8 φ, distributed uniformly upon the volume of the container, and configured to heat the volume of the container at a rate φ from a temperature Tl to a temperature T2, w herein φ is the rate of heating expressed as °C/s; • A second thermostat (7) that maintains the temperature of the containers and their contents at the temperature T2 for a given period;

* A second heat exchanger (8) which ensures the transition of the containers and their contents from temperature T2 to a temperature of less than 40°C at a rate from 0. 1 to 30 minute.

12. Apparatus according to claim 11, characterized by comprising a device to generate pulses of a unipolar or bipolar pulsating electric field having an amplitude E greater than 1000 V/cm and/or a device to generate ultrasonic waves with a frequency from 5 kHZ to 20 MHz, continuously or through pulses, which ensures a power density greater than 1 kW/l. both positioned downstream said heater.

13. Apparatus according to one or more of claims 11 and 12 characterized by comprising readers of temperature, readers of the speed of rotation of the containers (3), readers of the pressure of the cooling l iquid of the containers (3) within said cells of transport (27), and readers of the speed of rotation of the containers (24 and 25).