Materials treating method

The invention relates to a method of treating materials, in particular, packaging, using UV laser UV radiation.

It is known that the sterilization of the packages, for example, the inner surface of the cardboard boxes, is done in several ways, used together or separately. One method is to use heat, for example, in the form of hot air or steam, at a temperature of more than 1 ° C for several seconds. Another treatment is the use of bactericidal wavelength UV radiation (for example, 254nm) giving an average density of irradiation power in the surface treatment of several mW / cm², for example 8 mW / cm², for several seconds. Subsequent treatment consists of using a relatively concentrated solution of H₂0₂for a few seconds. The combination of two or more treatments is common and justifies reducing treatment parameters, such as temperature, duration of treatment and / or H concentration.₂0₂. For example, W0-A-80/01457 discloses a method of sterilization applicable to liquids, for example, wastewater and cooling water from canning plants, but especially to surface sterilization, for example, wall surface and hospital furniture or surface of food containers. The latter are treated with a solution of H₂0₂ with a concentration not exceeding 10% by weight, for example, by passing the container or material from which the container will be manufactured through a reservoir containing the H solution₂0₂ or by spraying the respective solution on the surfaces of the container or material. The irradiation is carried out with a UV lamp placed in such a way that the containers or materials that have come out of the tank or from the sprayer are subjected to UV radiation throughout the spectrum or predominantly below 325 nm, so that the microorganisms are destroyed by the synergy between UV and H₂0₂.

The use of UV radiation as a physical agent to reduce the amount of microorganisms is well established. Cellular DNA absorbs radiation energy between 250 and 260 nm, which leads to the formation of chemical bonds between adjacent thymine nucleotide bases. This distorts the structure of DNA, interfering with the replication and reproduction and thus the notion of genes. The event is inevitable if the essential genes are blocked or if DNA replication is stalled. The number of vegetative cells destroyed by UV radiation depends on the exposure and the dose in mW / cm²and mJ / cm², respectively, and UV radiation is often ineffective in destroying bacterial endospores, mainly because of the low penetration power of UV radiation. The combined use of UV radiation, hydrogen peroxide (the peroxide radical reacts with most chemical bonds) and heat, performs sterilization, but at a high price and time (for example, at least 10 seconds treatment time per carton) for packaging. Also, the use of a chemical, such as, for example, H₂0₂, for the treatment of food packaging has ethical implications and is regarded as a less desirable alternative. It is therefore of interest to look for an alternative sterilization system.

WD-A-88/03369 makes known a system for the sterilization of environments such as air, water, various foods and food packaging through intermittent pulses, very intense, with very short duration of light of frequencies in the visible and in the near field, such as would be the light produced by flash lamps. The components of each pulse cover a wide spectral range and may include far and near UV radiation for deactivation of microorganisms by photochemical effects. Such pulses abundant in UV radiation have at least 15% and preferably at least 30% of their energy at wavelengths below 300nm. Such pulses rich in UV radiation can usually have a relatively low total energy density, from

RO 112247 Bl cca.0.01 at 15J / cm² and typically from 0.1 to 3J / cm² However, for treating the surface of food products, it may be desirable to filter portions of the spectrum so that at least approx. 90% of the radiation energy is distributed between 300 and 2500nm. In such methods where the UV component of the flash pulses is suppressed or substantially eliminated, the intensity of the pulsating light must be sufficient to heat a superficial layer in the food or packaging with a thickness of less than 10 μ from the at least 50 ° C, up to a temperature of at least 75 ° C or, preferably, at least 100 ° C.

EP-A-0201650 describes a system for laser disinfection of fluids, in which a laser beam that radiates UV light is directed on a fluid flow to be treated. The system is especially applicable to water treatment and uses a pulsed gas laser, the speed of emitting pulses and therefore the average intensity of UV light can be adjusted. The UV beam covers the entire cross-section of the water stream. The speed (frequency) of the pulse emission can vary from slow to very fast, so that the beam appears continuously and is adjusted according to the changes of the water flow that flows perpendicular to the laser beam and can also be adjusted according to Alternatively, the fluid flow may be varied and the UV beam intensity maintained constant. One or more surfaces of the fluid-guiding pipe may be made relatively strongly reflective of UV radiation, so as to limit the scattering of the beam due to suspended particles in water and to decrease the cross-sectional area of ​​the beam to compensate for beam alteration, so that a roughly uniform intensity is maintained throughout the region where the beam and fluid interact. The penetration angle of the fluid beam can be adjusted according to the changes in water turbidity.

Moreover, the geometry of the beam, and in particular its length, can be changed in response to changing water characteristics, such as content in organic substances. An excimer laser with krypton fluoride producing a wavelength of 249 nm is preferred.

US-A-3817703 refers to a method for destroying suspended living matter in a light transmitting material. It reveals the possibility of simultaneous sterilization of containers and materials stored in them by directing a laser beam to the container, so that the beam comes into contact with the entire inner surface of the container during the sterilization process and thereby subject the entire material from the container to rays of light. Living matter suspended in irradiated material or deposited on the inside walls of the container is destroyed by it. One or more variable position mirrors are used to determine a container leakage through one or more laser beams.

US-A-3941670 discloses the use of an IR laser whose target can be scanned by the laser beam by the oscillation of a mirror. Light changes the biological activity of macromolecular species by excitating the vibrational and rotational states of irradiated species. Different targets can be sterilized, for example air or other fluids, plastics or metals, such as an aluminum foil tape or ribbon.

DE-A-2914075 presents a system for sterilizing the interior surfaces of preformed and folded cardboard boxes. UV sources, for example, vertical lamps with mercury vapor can be inserted from top to bottom inside the respective boxes, at different positions relative to the side walls of the boxes, so that each cardboard receiving the UV source, UV radiation of intensity enough fall on the different surfaces available, created by the bottom and the sides of the cardboard. a

EN 112247 With some arrangement, UV sterilization can be combined with heat sterilization so that yeasts and molds are eliminated. Q Such thermal sterilization can be done by steam or by irradiation, namely with infrared radiation sources, which are similarly inserted in the cardboard box, before or after irradiation with UV sources.

The method, according to the invention, presents a way of treating the inner surfaces of certain types of packaging, namely cardboard packaging, protected internally with a polymer film, using UV laser radiation, with bactericidal wavelength.

UV laser radiation can be subsequently or simultaneously accompanied by IR or IR laser. The use of UV and IR laser radiation (or IR laser) has a synergistic bactericidal effect.

In order to be able to fully understand the invention and apply it easily, references will be made to the following figures:

FIG. 1, shows in diagram form the apparatus used for exposing the test strips to UV laser radiation, and

- Figures 2 and 3, schematically show two alternative systems that can be used to obtain a substantially uniform distribution of UV laser energy on the surface of a cardboard box with the lid open.

It is of course well known that the thermoplastic surfaces of the packages, for example the surfaces of thin polymeric layers, are particularly sensitive to heating. However, it was found that using UV laser radiation, it is possible to irradiate these surfaces with an average power density greater than 1 W / cm.², with suitable values ​​in the range of 4 W / cm² and 10 W / cm², without affecting the surface of the thermoplastic material. Convenient values ​​of energy density are greater than 50 mJ / sec / cm², applied for less than 10 s.

It is also possible to use UV radiation, whether they are laser radiation or not, to sterilize a material and at the same time to use IR radiation, whether they are laser radiation or not, to heat that material, heating it. material for the purpose of promoting faster sterilization and / or for the purpose of causing the material to become viscous or molten for sealing and / or for the purpose of removing the foam formed inside the package, during filling. In this way, the application of IR and UV radiation is either simultaneous or overlapping for a limited period of time.

In order to obtain a relatively high power density, UV and / or IR radiation can be focused on a space that subsequently runs a scan, for example by using mirrors. IR and / or UV radiation can be diffracted by a computer-generated hologram to obtain a relatively uniform power density on the three-dimensional surface to be treated. Although an important aspect of the invention is the use of only UV laser radiation to sterilize solid surfaces, oxygenated water can be used in conjunction with UV (and IR) laser radiation to produce sterilization. It will be shown in the embodiments that the use of UV laser radiation combined with H₂0₂ has a bactericidal synergistic effect.

From the embodiments, it is also observed that, at the increasing levels of the density of microorganisms, there is a relatively sharp drop in the viability of the sterilization method, presenting the so-called "bearing" effect.

It has been found that UV radiation in pulses is particularly advantageous, especially due to the increased efficiency in solid surface sterilization, compared to continuous UV radiation.

The wavelengths used in the UV range are preferably in the range of 150 to 320 nm, more advantageously between 240 and 280 nm, for example, approximately 250 nm. The wavelength of the IR radiation used is

RO 112247 Bl preferably in the range of about 1OOO to about 10000 nm.

The use of a laser to deliver UV, as opposed to a UV emitting lamp with germicidal properties, has been shown to have the following advantages:

1. Relatively high absolute energy densities;

2. Low divergence, perfectly directed output beam, which makes it particularly convenient for treating certain surfaces inside cardboard boxes, where contamination can be considerable, for example, at corners or at joints.

To understand the method, the following are some examples of embodiments:

Example Ί. /. 1. Materials and methods

The body chosen to investigate laser treatment was the bacterium that can form endospores: Bacillus subtilis var. alobiaii. This bacterium is classified as Gram positive, aerobic, rod-shaped, mobile and is non-pathogenic. It is ubiquitous as a habitat and can be found in soil, faeces, hay, milk and water. Spore formation allows the bacterium to survive in a dormant state (dormant and variable) for a long time and makes it particularly important because sterilization systems are capable of killing these endospores. Because spores are resistant to temperatures above 100 ° C and other destructive (chemical) agents, they are useful indicators of sterilization efficiency and are regularly used to track chemical sterilization and heat treatments. Standard microbiological tests and microscopic investigations are used in testing the samples for evaluation of the new sterilization procedure. Identification of the spores under the microscope is facilitated by their high refraction and their reduced susceptibility to staining and their specific reaction to certain dyes.

In the study, cardboard material (a thick paper layer laminated with polyethylene layers, with or without an aluminum foil partition) and a spore suspension of Bacillus subtilis var were used as controls. globigii obtained from Elopak A / S from Norway. □ the second bacterial culture (NCIMP 8649) was used for control together with the above-mentioned culture, serving as an independent confirmation of the species. The methodology is divided into three main stages, namely: coating the test strips with bacteria along with all the preliminary procedures for this coating, exposing the strips to UV laser radiation and further analysis of these strips.

1.2.1. Preparation before exposure to laser radiation

A test system was developed to deposit the culture on cardboard in the form of test strips, insisting on the sterility of these strips prior to coating and on the specific placement of the bacterium on the sterile surface, so that the sterility of the strips is ensured.

1.2.1. a. Sterilization of the cardboard

The cardboard was cut with scalpel blades to form 6cm test pieces² and 2cm². These test strips have been exposed to different sterilization treatments:

- 35% hydrogen peroxide, 1 ml / test strip, for 2 minutes;

- 2% hydrogen peroxide, ml / test strip, for 2 s and for min;

- steam sterilization in an autoclave at 121 ° C for 20 minutes.

All the strips were allowed to dry well before applying the bacterial culture layer and a number of sterile strips were set aside as witnesses.

The sterilized strips were kept in autoclave bags - at room temperature up to 4 days before being used. Coated test strips were stored in Petri dishes at 4 ° C, up to 4 days.

1.2.1 b. Culture and identification of bacterial species

Bacillus subtilis var. globules in the form of a spore suspension were colRO 112247 Bleached in a nutrient broth (beef extract, pepton, water, pH 6.8], raised to 37 ° C and investigated 24 h later. Standard method of platelets was was used to isolate the colonies and to carry out viable counters The nutrient broth and the sterile distilled water were used, depending on the method chosen in making the plates (slides). Nutrient agar (beef extract, pepton, agar pH 6.8) served as a solid medium for growth of the organism. The observation was made visually, as well as the counting of colonies after 36 h of incubation at 37 ° C. Pure cultures were used for the preparation. smears and subsequent differential staining procedures The dyes used were the Gram dye and the Ziehl-Nielsen endospore dye, and the colored slides were analyzed with an optical microscope (10x100).

1.2.1. C. Bacterial deposition on test strips

Aliquots of 1OO pl from a progressive dilution of the starting culture having 10³ and 10⁶ ufc / ml in distilled water were aseptically poured onto a sterile cardboard test strip, dispersed and left to dry for several hours. The test strip was kept covered in a sterile box during drying time, at room temperature and then kept at 4 ° C. Great care has been taken to keep the Petri dish in a stable position, to avoid accidental contamination of the non-laminated sides of the carton, when reversing the box.

One test strip from a series of dilutions and two sterile strips were placed separately in 10 ml of nutrient broth and growth was investigated after 36 h, at 37 ° C. The turbidity was visually assessed and followed by the deposition of a 100 µl layer from the growth medium over the nutrient plate with agar, an operation followed by a new incubation period of 36 h, at 37 ° C; the smear was then observed according to the procedure described above.

1.2.2. Exposure of test strips to UV laser radiation

The laser used (whose aperture is designated by Q in the diagram in Fig. 1 of the accompanying diagrams) was a Questek 2000 Series excimer laser that starts with a Krypton fluoride (KrF) transition that produces a radiation of 248 nm in UV. The excimer laser is a pulsator, producing pulses with a duration of 10-20 ns at repetition frequencies of several hundred hertz.

The following operating parameters were used:

Pulse energy 200 mJ

Repetition frequency: 100 Hz

The laser emits a beam with cross-sectional dimensions of approximately 3x 2cm (horizontal x vertical).

The optical system used is shown in the figure. The energy density sent on the sample was varied by changing the lens from molten silica L, and / or the distance d from the lens to the mask M and thus, the distance from the lens to the sample S.

Initially, a cylindrical lens with a focal length of 25 cm was used to extend the beam to a horizontal dimension, producing an irradiation surface of approximately 3x3 cm on the M-mask. The resulting energy density per sample per exposure was 40 mJ / cm², leading to an average power density of 4 W / cm² at 100 Hz.

To produce a higher power density on the sample, the distance to the sample was reduced and the sample size was reduced from 3x3 cm to 1x1 cm. The energy per exposure and the average power density were 130 mJ / cm in this configuration² and 13 W / cm respectively². To achieve lower irradiation densities, the cylindrical lens was replaced by a spherical lens with a focal length of 20 cm.

The area where the beam fell over the sample far exceeded the aperture of the mask, leading to energy per exposure and average power density per sample of 10 mJ / cm² and 1W / cm respectively².

In all cases, the energy density was measured in the plane of the mask. The block containing the sample was placed a few cm behind this plane. However, the difference in exposure introduced by this fact was small, as the beam was small compared to the distance d, whose typical value was in the range 40-70 cm.

Different irradiation times were used, depending on the energy and power densities tested:

mJ / shot / cm²: 1s and 6s mJ / exposure / cm²: 1 s and 6s 130 mJ / exposure / cm²: 15s The test strips were mounted using sterilized needles on a balsa wood plate covered with aluminum foil. Both the block and the foil were steam sterilized for 20 minutes at 121 ° C, before use. Depending on the size of the test strips, twelve to sixteen strips were treated on a single block, the block being mounted on a laboratory rake to ensure “sweep”. The mounting of the samples on the block was done hygienically, using a sterilized forceps for fixing the strips.

Experiences with H₂0₂ were made as follows:

a) 100 µl of soil were added.2% H₂0₂ over the test strip (one for each dilution tested), they were spread over the surface of the carton, dried by heating and then exposed to laser beams.

b] as a) but no heating drying was performed before exposure to the laser beam - the test strips were mounted between two sterile quartz plates, treated with the laser beam and then dried by heating.

The applied heating was about 100 ° C, by contacting the bottom silica plate with a laboratory hob. For the drying process, the silica plate from the top was removed.

1.2.3. Testing the sterility of test strips after laser treatment

All the samples exposed to the laser beam, including the control samples, were immediately placed in separate universal containers with 10 ml sterile nutrient broth and incubated for 36 h at 37 ° C. Sterility was investigated by tracking the turbidity of the solution after the incubation period and followed by a second cultivation on nutrient agar plates with a second incubation of 36 h, at 37 ° C and the growth was monitored under the microscope in the presence of a dye indicator.

1.3. Results

1.3.1. before exposure to the laser beam

Test strip sterilization treatment has shown that the autoclave method is the most reliable and effective procedure.

The method of drying the bacterial colony on the test strips was effective in growing viable colonies on samples prepared no more than 4 days (test strips older than 4 days were not tested).

The serial dilution and the subsequently applied lamella method established that the bacterial culture contains 2x10⁷ ufc (colony-forming units) per ml. (5 agar plates from each dilution series were used to determine the average number of colonies). Sterile strains without bacterial addition, used for control throughout the study and the indicators needed to successfully apply aseptic techniques, have been shown to remain sterile in most experiments. In two cases where the cartons were in contact with the nester surfaces, the test strips were put into culture and Gram-negative organisms were identified.

In all experiments, the use of the Gram indicator and the staining of Bacillus subtilis var. globules have made it possible to clearly identify organisms as species of Bacillus subtilis.

The classification of the results in sterile and non-sterile was done unambiguously, as was the differentiation of the organism tested by other potential contaminants.

RO 112247 Bl

/. 3.2. Summary of the results obtained after exposure to laser radiation

Laser treatment conditions ¹ —

<td>Power density (W / cm²)</td><td>Exposure time</td><td>2% H_of0₂used?</td><td>Test strip size (cm x cm)</td><td>Number of repetitions</td><td>Sterilization rate after 36 h, and initial cell density</td>

<td> 13</td><td> 15</td><td>not</td><td>1x1</td><td>2 times</td><td>1 to 10 ⁶</td>

<td> 4</td><td> 1</td><td>Yes</td><td>1x1</td><td>5 times</td><td>1 to 10 ⁶</td>

<td> 4</td><td> 6</td><td>Yes</td><td>1x1</td><td>5 times</td><td>1 to 10 ³3 to 10 ⁶</td>

<td> 4</td><td> 16</td><td>not</td><td>1x1</td><td>2 times</td><td>1 to 10 ⁶, 1 s 2 to 10 ⁶, 6s</td>

<td> 4</td><td> 16</td><td>not</td><td>2,5x2,5</td><td>2 times</td><td>none</td>

<td> 4</td><td> 16</td><td>Yes</td><td>2,5x2,5</td><td>2 times</td><td>2 to 10 ³, 6s 1 to 10 ⁶, 6s 1 to 10 ³, 1 s</td>

<td> 4</td><td> 16</td><td>yes (quartz)</td><td>2,5x2,5</td><td>2 years</td><td>2 to 10 ³, 1 s 2 to 10 ^B, 6 1 * to 10 ^e, 1 s 2 to 10 ³, 6s</td>

<td> 13</td><td> 5</td><td>yes (quartz)</td><td>1x1</td><td>4 times</td><td>4 to 10 ⁶3 * to 10 ³</td>

<td> 1</td><td> 16</td><td>not</td><td>3x3</td><td>5 times</td><td>none</td>

□ The sample was contaminated with the hand

1.4. Conclusions 30 series of encouraging results were obtained, thus demonstrating that the effective destruction of microorganisms can be achieved by the use of UV laser radiation, using an average power density of 4 / Wcm^of and an exposure time of 6 s, the efficiency of killing the microorganisms (defined as the number of sterile test strips / total number of illuminated test strips x 100) was about 30%. The inclusion of hydrogen peroxide in the strip tests has improved it by almost 100%. It should be mentioned that the excellent result obtained with 45 H₂0₂ it was achieved with an illumination time of only one second.

Example 2.11.1. Introduction

The results of Example I were an indication of the successful action of 50 UV laser in killing micro organisms such as Bacillus subtilis. It was shown that the laser illumination times from one second to 6 s at 40 mJ / exposure / cm² (4W / cm²) kill between 1O³ and 1O⁶ bacterial cells in a few samples.

In this example, the quantitative aspect of the process was further investigated: how many microorganisms are killed by a particular laser treatment.

A large number of samples were tested to track the efficiency of destruction of the various laser lighting procedures, paying particular attention to the power density and the frequency of repetition. The sensitivity of the laser light sterilization procedure was measured by observing the survival proportion of the bacteria after different treatments.

Other experimental aspects were researched at this stage.

RO 112247 Bl

These are:

- how the film beneath the strips affects the heat generated and how this additional heat, if it is generated, affects the killing potential of the laser.

- Does the wood used for mounting the strips generate a substance that enters the strips and can help destroy the bacteria? Is this substance, if present in the system, activated by autoclave treatment and taken up by the bands?

- Does the test strip itself generate substances that can affect the speed of killing bacteria? Is the heat generated by the laser necessary to kill bacteria?

- how it affects the reduction of the power density (W / cm²) and / or reducing the repetition frequency (Hz) killing efficiency of UV laser illumination?

II. 2. Materials and methods at this stage were used as controls a cardboard material (laminated with Al foil) and a STUDLAND mounting plate ”. 0 spore suspension of Bacillus subtilis var. globigii was used to prepare a series of dilutions in distilled water. This series of increasingly diluted solutions has been the source of aliquots poured onto the test strips, which were left to dry and exposed to UV laser radiation.

The procedures were divided into three important stages, namely: coating the sterilized test strips with bacteria, exposing the strips to UV laser radiation and further analyzing the strips.

11.2.1. Preparations Before exposure to laser radiation

Cardboard material and plate were cut with a scalpel to give 1.3 cm x 1.3 cm test strips. 18 test strips per block of balsa wood were used (the wood was covered with Al foil or was left uncovered). The strips were placed on the wood in three rows and six lines in a row.

All the strips were kept in the desired position with an autoclave tape so that only 1 cm² from the area either16 whose cardboard strips remained exposed. Each block was placed separately in an autoclave bag and subjected to autoclaving at 121 ° C for 20 min.

50 µl aliquots of each prepared dilution (1Q¹, 10'² , 10’³, 10^-4] starting from an initial culture of Bacillus subtilis var. The globules were aseptically transferred onto the strips and these were allowed to dry in laminar air for several hours. Two bands per block were used for control, for example, without exposure to UV laser radiation and one band per block was used as a control, for example, no bacterial dilution was added to it. Each balsa wood block with the strips mounted on it was then returned to the sterile bag for autoclaving and sealed until UV laser treatment.

To provide the baseline for estimating bacterial killing, the number of colony-forming units deposited on tapes from different dilutions was determined by a detergent washing method as follows:

the strips prepared with a certain bacterial dilution were immersed in a 0.1% solution of Tween 20 (dilution made with sterile water).

The efficiency of Tween 2Q solution recovery of the contaminated strips was compared with that of the sterile water, so that a proof of the correctness of the chosen principle can be established.

Other variants of the medulla were used to track their effect on the number of bacteria, including comparisons between bands mounted on balsa wood or left unmounted in sterile Petri dishes prior to detergent recovery.

11.2.2 Exposure of test strips to UV laser radiation

The laser used was the same as in Example I, but had a higher pulse energy of 250 mJ. The only variations introduced were the repetition frequency (Hz), the illumination time and the energy density used. The tested variations were grouped in the column "ParaRO 112247 Bl laser meters" in the following table.

A 25 cm cylindrical lens was used again to reduce the vertical size of the beam in order to achieve an energy density of 40 5 mJ / exposure / cm². A lens with a focal length of 35 cm was used to enlarge the cross-section of the beam so that an energy density lower than 6 mJ / exposure / cm² 10 can be achieved.

Up to 18 tapes were mounted on a single block, the block being mounted on a J-type laboratory rack in order to allow the scanning. 15

The sweeping procedure was performed under hygienic conditions, and the blocks were placed in sterile autoclave bags after treatment. The bags were sealed and kept at room temperature up to 20 at the beginning of subsequent analytical procedures. On most blocks there were three bands that were not illuminated with UV laser radiation (two of these samples were used for control and 25 one, without bacteria, was used as a control).

III. 2.3 Procedures after the treatment with UV laser radiation

All assembled strips were aseptically removed from the blocks, placed in separate bottles containing 5 ml of diluent and stirred for a minimum of 30 s. at 37 ° C, for a minimum of 24 hours.

The plates were visually examined to track the growth of the colonies and to count them. 40

II. 3 Results

II. 3.1 Preliminary results for laser exposure

The stretching procedure on nutrient agar plates of the progressive dilutions led to a repeatable number of colonies per bacterial concentration and allowed the calculation of the recovery factor.

It was found that the best way to recover bacteria from the strips is to stir in the diluent for one minute. Recovery ranged from 50% to 90% of the bacteria in the band. Experimentation with progressive dilutions showed that they existed

1.5 x1O⁷ ufc / ml in the initial culture.

The two types of material tested did not show significant differences in the recovery speeds obtained. The experiment with test strips mounted on balsa wood, in comparison with strips left unmounted in Petri dishes, did not reveal any difference in results, excluding any potential chemical effect exerted by the support used.

II. 3.2 Laser post exposure

The results of experiments performed before exposure to laser radiation, such as the research of potential bactericidal substances from used materials or wood, were further studied with test strips subjected to UV laser illumination, but their effects were not observed. Any potential heating effect on the killing rate of bacteria was therefore eliminated when the calorific intensity of the UV laser illumination was reduced to exclude thermal effects (the exposure time was adjusted to obtain the same total number of photons, for example, 10 pulses per second for 10 s instead of 100 pulses in one second).

//. 3.3 Summary of the results from example II

<td>Laser parameters</td><td>Total energy (J / cm²)</td><td>Number of samples^x</td><td>log_1Q decrease^xx</td>

<td>40mJ / shot / cm²100 Hz, 1 sec</td><td> 4</td><td> 36</td><td> &gt;5,2</td>

RO 112247 Bl

20

Continue the table

<td>40mJ / shot / cm²30 Hz, 1 s</td><td> 1,2</td><td> 1</td><td> &gt;5,2</td>

<td>6mJ / shot / cm2 100Hz, 1 sec</td><td> 0,6</td><td> 3</td><td> 2,7</td>

<td>40mJ / shot / cm²2 Hz, 5 s</td><td> 0,4</td><td> 2</td><td> &lt;1,0</td>

<td>40mJ / shot / cm²3 Hz, 1 s</td><td> 1,2</td><td> 2</td><td> 3,3</td>

<td>6mJ / shot / cm²2 Hz, 5 s</td><td> 0,06</td><td> 3</td><td> 1,8</td>

x Each sample has 1.5 x10⁵ increase by 1 cm^s target surface *^x Defined as log_1o (1.5 x 10⁵/ number of survivors]

Using an energy density 15 of 55 mJ / exposure / cm² for a duration of more than 2 years, the physical deterioration of the bands (formation of blisters and blackening).

11.4 Conclusions 20

The experimental results show that the destructive efficiency of UV laser radiation is dependent on the parameters of the laser used and the number of bacterial organisms present, but 25 is independent of the substrate material. Most of the laser test parameters had a high killing efficiency, with bacteria numbers below 10⁶ cfu. 30

Illumination period of one second at 40 mJ / exposure / cm², 100 Hz, achieved a 100% destruction of a bacterial concentration of 1.5x10⁵cfu. High kill efficiency of 35 columns by 1.5x10⁵ cfu at illumination times of one second and relatively low energy or relatively low repetition frequency of 6 mJ / exposure / cm² at 100 Hz and 40 mJ / exposure / cm² at 3 Hz 40 is of great importance for the sterilization of the carton.

In order to obtain a uniform distribution of energy throughout the entire inner surface of the carton, the 45 two systems illustrated in fig. 2 and 3, offer possible alternative solutions, which can offer energy distributions adapted to the requirements.

The first one, illustrated in FIG. 2, uses a computer-generated hologram H that diffracts the UV light beam into a well-defined model. It consists of a complex surface structure or a network that is formed on the surface of a piece of glass. This model requires the facilities provided by a computer, which can predict how light will interact with the network structure. The network is made by covering the glass with a photoresist and transcribing the model onto the photoresist with an electron beam. The development of the photoresist removes the surfaces that were exposed to the beam. The glass is then attacked to reproduce the pattern that was present in the photoresist deposited on the glass.

The second system, illustrated in FIG. 3, uses two galvanometers (not shown) to move the respective mirrors l \ l, which can be rotated around the respective axes, thereby deflecting the incident laser beam B. Combining the two sweeping systems allows the beam to be directed after two axis. The addition of a third galvanometer (not shown) controlling the translation stage (movement), or in other words a focusing motion F, can be used to vary the separation distance between the two lenses, thus changing the beam divergence.

RO 112247 Bl

It should be noted that the two systems are characterized by a fundamental difference. Hologram H sends light simultaneously at each point of container C, while galvanometer sends light sequentially to different areas.

in addition, the hologram system has no moving parts.

Example III.

III. 1. Introduction

This example describes the sweep of the entire cardboard box and, in particular, the microbiological methods and tests used to investigate the activity and efficiency of UV laser radiation on a range of laser energies and bacterial concentrations.

III. 2 Method

The basic principle of testing consisted of spraying empty cardboard boxes, with the lid open, having a triangular profile lid, of type "ELOPAK" (registered trademark) with a certain organism, irradiation of the laser radiation cards and researching the number of surviving bacteria. after irradiation.

///. 2.1 The indicator organism

The indicator organism used for the tests was Bacillus Subtilis var. alobiaii. To provide a sufficient reserve of spore solution, 0.5 ml of sample was grown in a nutrient broth and allowed to grow at 37 ° C for 48 h, followed by centrifugation and harvesting. a sterile Ringers solution. This solution was left at 4 ° C for a minimum of seven days before being used in experiments.

III. 2.2 Spray the cartons

The five inner surfaces of the cardboard boxes were sprayed with the indicator body. The spray device used was a hand atomizer that sent the cells evenly over the surfaces of the box. The range of bacterial concentrations was between 10⁷and 10⁸ cells per box. The sprayed cartons were left to dry at room temperature under a hood with laminar airflow for up to 18 hours before use.

III. 2.3 Detection of surviving bacteria

In order to detect the surviving bacteria, an elution (washing) test was used.

III. 2.4 Experimental assembly

The spore suspension was diluted in a Ringers solution providing a series of dilutions between 10 '¹ and 1O⁸ compared to the initial solution.

100 pl of each dilution was pipetted onto a plate with nutrient agar, which was labeled, then inverted and incubated at 37 ° C for 24 h before visual examination of bacterial growth. Each 2 ml of the appropriate bacterial dilutions were sprayed onto the cardboard boxes used in the tests and allowed to dry under the conditions described above. All reagent containers and Petri dishes were sterilized prior to use and handled aseptically under CAL (laminar airflow). The Ringers solution was autoclaved by the addition of Tween 20.

The regularity of the spray and the number of cells deposited on each carton were investigated by water tests and the opening of the sides of the boxes and was confirmed by spraying with bacteria, over which an agar layer was deposited, as well as by the recovery tests. Before use, the spray was cleaned with 70% ethanol solution and allowed to dry in CAL. The lids of the cardboard boxes were also prepared by immersion in 70% ethanol solution, followed by a short treatment with 100% ethanol and subsequent drying. After ethanol treatment, a few caps were tested with nutrient broth to check sterility.

Each washing test experiment included controls on the untreated, for example, non-laser-irradiated parts, and unpolished cardboard boxes with bacterial spores. The latter were used to control the contaminant fund. Most experiments included testing of at least two bacterial dilutions. Carrying out a typical experiment would be as follows: 3 boxes

The cartons were sprayed with a 1CT dilution¹ of the spore suspension; 3 other cartons were sprayed with 1O 'dilution² of the spore suspension.

For 1Q dilution¹: boxes 1, 2 and 3 were used in the washing test, boxes 1 and 2 being the test and box 3 the control part, ie a box that was not irradiated with laser.

The same rule was followed for cartons sprayed with 10 'dilution² from the spore suspension.

The cardboard boxes were placed (in a row) in a special box holder, attached to the laser unit. After irradiation, the box was removed from the support, covered with a sterilized lid, and the entire box transported to CAL. The handling of the carton in a non-sterile environment was reduced to a minimum time, but still involved a potential risk of contamination when the box was lowered from the irradiation support and then during the handling of the box.

ml of the wash test solution was pipetted onto each wash test box. A sterilized lid was placed on each box, and the boxes were vigorously shaken at least 3D times on each side, then left for a short time after which the solution volumes were emptied into separate sterile universal containers. □ series of dilutions were prepared from the solution thus recovered, using a sterile Ringers solution. 250 pl or 500 µl of each dilution and the remaining net volume were placed on nutrient agar plates and incubated at 37 ° C for 24 h.

III. 2.5 Details about laser tests

The range of energies tested ranged from 1, □ Joule to 4.0 Joule per cm² with irradiation times of about 10 min (this duration, which is 60 times the commercially reasonable time, ie 10 s per box, was chosen for practical experimental reasons to provide a total dose equal to the dose commercially desirable applied for 10 s). Each cardboard box was placed in the irradiation support in exactly the same position, with the printed code number of the box towards the back of the support. The cardboard box lid was completely opened, but in some cases it was slightly inclined. The irradiation procedure was automated and controlled by the computer.

III. 3 Results

Initial tests performed to investigate the test methods showed that the spray method led to a uniform distribution of bacteria and that the recovery tests led in most experiments to a level of bacterial concentration identical to the initial one. In the following you will find a description of typical examples from individual test methods followed by a summary of the most significant experimental results.

III. 3.1 Dilution series

For each experimental measurement, a series of dilutions of the initial spore stock was prepared, in order to determine the correct bacterial concentration in each sample under test.

III. 3.2 Washing test

The results obtained with the progressive dilutions were used to quantify the bacterial concentration sprayed in the cardboard boxes and to track the accuracy of the recovery tests. A recovery control test was included in each series of experiments and the recovery percentage was calculated for each bacterial concentration. Once it was established that the recovery method was appropriate, the efficiency of laser irradiation could be measured based on the result recorded at recovery, and the actual number of colonies recovered in the test sample was compared with the expected number at the given bacterial concentration.

Typical recovery results are slightly lower than expected.

RO 112247 Bl

Wash test example:

<td>tests</td><td>10 'sample wash test¹</td><td>Sample wash control 10¹</td><td>Series of dilutions</td>

<td>pure</td><td> &gt;200</td><td>completely</td><td>completely</td>

<td> 10'¹</td><td> 50</td><td>completely</td><td>completely</td>

<td> 10²</td><td> 4</td><td>hard to count</td><td>completely</td>

<td> 10³</td><td> 1</td><td> 278</td><td> 1315</td>

<td> 10^</td><td> 0</td><td> 27</td><td> 220</td>

Calculation example: 15

The initial stock concentration of the spore colony was calculated from the results of progressive dilutions: 220 colonies at a dilution of 1O⁴ coming from 100 pl sample on the plate, lead to an initial concentration of the stock of 2.2x10 '⁸

10 'dilution¹ of this initial concentration should lead to the concentration of 2.2x10⁷, but because, or using 2 ml, the concentration deposited on 25 cartons was 4.4x10⁷. Recovery in 20 ml of Ringers / Tween 20 solution and an aliquot of 100 pl on the plate means that any results from the washing tests must be multiplied by these factors. 30 The control sample had 278 colonies at 10 'dilution³ of the recovered volume, so that the recovered concentration is calculated as 278 x IO³ x 10 x 20 =

5.6 x 10⁷. This differs from the concentration of deposition by 1.2x10⁷ which is an insignificant difference, indicating that there are no special problems with the recovery method. The current laser irradiated test sample gave approximately 40,200 colonies starting from an aliquot of 100 μΙ taken from 20 ml net recovered volume, thus indicating that in this sample 4.0x10 survived⁴colonies, leading to log (decrease) &lt;3.04 ie log (4.4 x10⁷ : 4.0x10⁴].

Starting from the 250 μΙ sample taken from a 10 ml wash volume, the calculation factor is x 40 (that is in x 4 x10], and for the 500 μ proba sample taken from a 10 ml wash volume the the calculation is x 20 (ie x 2 x 10).

The definition of the log size is:

log_1o (number of cells per box: number of surviving cells per box],

III. 3.5 Experimental data

Boxes sprayed with 10 ^ 10⁸ cells were exposed to 1.2 or 4 Joules / cm² The UV radiation and the surviving spores were determined by the washing test using 10 ml of washing solution and taking 500 μΙ or 250 μΙ for the preparation of slides. The destruction rates are given in the following table.

III. 3.6 Summary of results

Wash test volume 1O ml / samples of 500 µl or 250 µl

<td>loading cells / box</td><td>Wash test recovered cells</td><td>Cells calculated in 10 ml</td><td>energy J / cm²</td><td>log it drop</td>

<td>2,4x10⁷</td><td> 80^x1</td><td>1,6x10³</td><td> 1</td><td> 4,18</td>

<td>2,4x10⁷</td><td>2,4x10^3x1</td><td>4,8x10⁴</td><td> 1</td><td> 2,7</td>

RO 112247 Bl

28

Continue the table

<td>2,4x10⁸</td><td>2,2x10^4x1</td><td>4,4x10⁵</td><td> 1</td><td> 2,7</td>

<td>1,3x10⁸</td><td>8,6x10^3x2</td><td>3,4x10⁵</td><td> 1</td><td> 2,58</td>

<td>1,3x10⁸</td><td>8,8x10^3x2</td><td>3,5x10⁵</td><td> 1</td><td> 2,57</td>

<td>1,3x10⁷</td><td>1,12x10^2x2</td><td>4,48x10³</td><td> 1</td><td> 3,46</td>

<td>2,6x10⁸</td><td> 65^x2</td><td>2,6x10³</td><td> 2</td><td> 5,0</td>

<td>2,6x10⁸</td><td>1,44x10^2x2</td><td>5,76x10³</td><td> 2</td><td> 4,7</td>

<td>2,6x10⁸</td><td> 85^x2</td><td>3,4x10³</td><td> 2</td><td> 4,9</td>

<td>2,6x10⁸</td><td> 27^x2</td><td>1,08x10³</td><td> 4</td><td> 5,38</td>

<td>2,6x10^of</td><td> 27,5^x2</td><td>1,10x10³</td><td> 4</td><td> 5,37</td>

^&lt;1(5OO μΙ) ^x2(25O μΙ]

III. 4 Conclusions

Irradiation with 2 Joules / cm² led to a value of 5 of the log size (decrease) of microorganisms, and an even stronger dosage of 4 Joules / cm², at a log value (decrease) even greater than 5. Summing up the experimental results, the following ideas can be presented in conclusion:

- UV laser scanning of the entire interior of the cardboard boxes can lead to their sterilization;

- 2 Joules / cm² lead to the value of log size (decrease) for Bacillus subtilis var. globigii ',

- Internal surface of 750 cm² a cardboard box requires, to be sterilized, an energy of 1500 Joules;

- Using a UV laser with the power of 150 W, it would take 10 s to reach the value 5 of the log size (decrease);

- It is possible to use very low concentrations of H₂0₂ In combination with laser irradiation, either to decrease the energy level required for each carton over a certain period of time, or to reduce the irradiation time per carton to the same energy level.

Example IV. IV. 1. Introduction

Quantitative assessment of the speed of destruction when combining UV and IR (simultaneous use) radiation is the subject of research in this example.

The experimental setup was designed in such a way as to obtain additional information on the heat developed during the exposure and to study and separately the effect of each of the treatments.

IV. 2 Materials and methods

A cardboard material (laminated with Al foil) was used as a control. The indicator organism was Bacillus subtilis var. alobiaii. A spore solution of this origin was applied to the cardboard material.

The test involved the existence of three main stages, namely, the coating: the surfaces of cardboard pre-sterilized with bacteria, the exposure of the cardboard strips to radiation and the subsequent analysis of the surviving spores on these strips.

IV. 2.1 Preliminary preparations for exposure to laser radiation

The cardboard was cut with a scalpel to give 2 cm x2 cm test strips.

10 and 100-fold dilutions of the initial spore suspension (containing 4.5 x 10⁷ ufc / ml) were performed in sterile saline (0.85% NaCl containing 0.02% Tween 20 to facilitate dispersal).

These dilutions contained 4.5x10⁶and 4.5x10 respectively⁵ cfu / ml.

Aliquots of 100 µl of each diluted suspension were sprayed on the test strips to give a 4.5 χ 10 strip loading.⁵ and respectively

RO 112247 Bl

4,5x10⁴.

The strips were allowed to dry overnight at room temperature. Some of the tapes were not covered to check their sterility. After drying, each strip was placed in a sterile plastic universal box.

During the irradiation the bands were kept upright with presterilized metal hooks type "buldog".

IV. 2.2 Laser calibration

The infrared laser used in these tests was a C0 laser₂ of 14W. It was controlled by a unit that allowed radiation pulses up to

6.6 s and variable output power between □ and 80% of the total output power.

The system was calibrated by measuring the temperature rise of a cardboard test piece of the same size as used in biological tests. The temperature was measured with a thermocouple attached to the front surface of the cardboard plates.

The IR laser generated two 6.6 seconds pulses with 80% power (approximately 40 J / cm²].

The maximum surface temperature that could be reached in front lighting was 40 C. This was determined by the presence of a thin foil. aluminum placed behind a polyethylene coating of the respective face of the cardboard, reflecting a significant proportion of the IR radiation, preventing their absorption inside the plate.

The excimer laser used was a gas laser using a mixture of Krypton and Fluorine and emitted radiation with a wavelength of 248 nm. The energy of a laser pulse was approximately 300 mJ. The incidence laser excimer incidence on the sample was calibrated using a detector placed behind a 2 cm x 2 cm aperture. The pulse energy at this aperture was 125 mJ. In this way it was determined that an energy density of 1 J / cm² requires 32 pulses.

IV. 2.5 Laser irradiation treatments

1. UV only 1.0 J cm '^2;

2. IR on the front of the cardboard;

3. UV 1.0 J cm² and IR (front face), simultaneously;

4. UV 1.0 J cm '² followed by IR (on the front face), a few seconds later (2-3 s);

5. IR (on the front side) followed by UV 1.0 J cm '² many seconds later (30 s).

Control. The bacteria-coated plates were left unirradiated to determine the maximum degree of recovery. Plates uncoated with bacteria were exposed to the surrounding atmosphere for about 30 s to test for the possibility of accidental contamination.

IV. 2.6 Determination of surviving bacteria

After irradiation, the plates were immediately placed in their bottles and kept overnight at 4 ° C.

To each bottle 10 ml of sterile saline containing 0.1% Tween 20 was added. The bottles were vigorously stirred for 30 s, after which the cardboard plates were removed and removed. Two liquid samples (100 µl or 200 µl) were taken aseptically and scattered on the surface of a plate with nutrient agar, and some samples were subjected to serial dilution to make it easier to count the colonies.

The plates were incubated at 37 C for 24 h and then the colonies were counted. For this, the total number of cfu in 10 ml of washing liquid was calculated first and then, the total number of cfu removed from the carton.

IV. 3 Results

The results are presented in the following table.

(IV. 4) Discussion A clear fact that results from experiences is that only IR radiation does not kill bacteria. It is also clear that all treatments using both IR and UV radiation at 1.0 J cm '²(numbers 3 and 4) lead to higher values ​​of log size (decrease) compared to UV radiation treatments,

RO 112247 Bl with IR radiation or with the sum of the effects of UV and IR treatments separately. We can therefore conclude that a synergistic effect occurs when the treatment with IR and UV radiation is applied simultaneously.

<td>Treatment</td><td>loading CFU / ml</td><td>ucf recover</td><td>log (decrease)</td>

<td>1 (i) UV only (Ii)</td><td rowspan="5">4.7 x1O⁵</td><td>4,4x10³5,6x1O³</td><td> 2,03 -1,98 1,92</td>

<td>2 (i) IR only (Ii)</td><td>4,5x10⁵4,5x1O⁵</td><td> 0,02 -0,02 0,02</td>

<td>3 (i) UV and IR, simultaneously (Ii)</td><td>2,4x10³1,3x10³</td><td> 2,29 -2,43 2,56</td>

<td>4 (i) UV-IR, sequential (ii)</td><td>1,40x10³2,2x1O³</td><td> 2,53 -2,43 2,33</td>

<td>5 (i) IR-UV, sequential (ii)</td><td>7,2x10³contaminated</td><td> 1,81 -1,81</td>

claims