PL232885B1 - Method for protection of materials intended for use in contact with water and application of glycolic acid for protection of such materials - Google Patents

Abstract

The subject of the application is a method for protecting materials intended for use in contact with water against bacterial colonization and/or the formation of bacterial biofilms on their surface, wherein said materials are treated with a glycolic acid solution at a concentration in the range of approximately 0.5 - 15 mg/l in sterile water, preferably distilled water. The application also concerns the use of glycolic acid to protect such materials.

Description

Description of the invention

Technical field

The present invention relates to a method of protecting materials intended for use in contact with water against bacterial colonization and / or the formation of bacterial biofilms on their surface. The invention also relates to the use of glycolic acid to protect such materials.

State of the art

Heterotrophic bacteria living in aquatic environments tend to settle on available surfaces and create spatial structures with a thickness of several - several hundred gm, called biofilms. The surface colonization by bacteria occurs in stages. After contact with the surface and initial attachment to it, the bacteria synthesize the extracellular polysaccharide matrix in which they remain embedded. Such a matrix (biofilm) contains numerous microorganisms that often belong to different species. Living in a biofilm brings many benefits to microorganisms, including: protection against physical factors such as UV radiation; higher temperature in the biofilm - increased metabolic rate; the richness of organic compounds, both those building the colonized particle and those adsorbed on the surface; horizontal gene transfer, more frequent conjugation of bacteria; protection against bactericidal compounds (e.g. detergents or antibiotics); protection against the pressure of protozoa (mainly nano-flagellates), on the other hand, the risk of being eaten by larger protozoa or ciliates or crustaceans increases; immobilization of phages in the biofilm matrix; the produced hydrolases remain within the reach of their producers.

From an industrial and medical point of view, the formation of biofilms by bacteria is often an undesirable phenomenon. Biofilms may destroy the structure of the colonized surface (e.g. bridge spans, ship bottoms) or pose a health risk in the event of the colonization of specialized medical devices (e.g. implants) or clothing, e.g. colonization of diving clothing or neoprene clothing by potentially pathogenic bacteria. The implementation of an effective method of combating biofilms is a significant problem and still unsolved to a satisfactory degree.

Currently, numerous bactericides and detergents are used to remove the resulting biofilms. They include, among others cationic ammonium salts, anionic soaps and fatty acids, organic solvents, e.g. alcohols, heavy metals, oxidizing agents, e.g. hydrogen peroxide, alkylating or denaturing proteins.

In addition to combating the already formed biofilms, it is equally important, if not more important, to prevent their formation. A special case is the protection against the production of biofilms by bacteria living in aquatic environments.

Currently, there are no preparations that use natural mechanisms to inhibit the formation of biofilms in the structures of foam clothing. The available solutions generally require soaking and washing this type of clothing in a variety of preparations. An example would be the preparation of the trade name MiraZyme ™ offered by M Essentials, based on enzyme preparations and detergents. Its action is similar to that of washing powders containing active enzymes and does not allow for quick application in field conditions without the possibility of washing clothes.

There are also industrial bactericidal preparations containing hydroxycarboxylic acids in the form of metal salts or esters, e.g. as described in the application US20080317800A1 or JP58067761A (Composition for forming coating film of hydrolysable resin). However, they do not contain pure glycolic acid in minimal concentrations (only high concentrations of its derivatives) and are not based on the idea of using a small gradient of glycolic acid. Moreover, they do not protect neoprene clothing, but only industrial equipment.

Under natural conditions, aquatic bacteria strive to colonize not only various inanimate surfaces, but also algae and cyanobacterial cells. These microorganisms defend themselves against colonization. They modify, for example, the structure of the cell surface and produce cytotoxic compounds, such as elatol or phlorotannins (Eom et al. 2012) or disrupt the interaction between bacterial cells, for example by influencing the "quorum sensing" mechanism (Punyasl and Wright 2004).

It is an object of the present invention to provide a means for effectively protecting materials intended for use in water from colonization by bacteria. The inventor of the present invention has surprisingly found that one of the methods of defending photosynthetic algae against bacterial colonization can be used to protect materials submerged in natural waters by using very low concentrations of glycolic acid to protect them.

PL 232 885 B1

The essence of the invention

The subject of the invention is a method of protecting materials intended for use in contact with water against bacterial colonization and / or the formation of bacterial biofilms on their surface, in which the said materials are treated with a glycolic acid solution with a concentration in the range of 0.5-15 mg / l in sterile water, preferably distilled water.

The range of 0.5-15 mg / L corresponds to 0.00005-0.0015% by weight.

In a preferred embodiment of the process according to the invention, the glycolic acid concentration is 10 mg / l.

The value of 10 mg / l corresponds to 0.001% by weight.

Other concentrations of glycolic acid can also be used in the process according to the invention, in particular 0.5, 1.2 mg / l (respectively 0.00005, 0.0001, 0.0002% by weight, and also e.g. about 3, 4). , 5, 6, 7, 8, 9 mg / L (about 0.0003, 0.0004, 0.0005, 0.0006, 0.0007, 0.008, 0.0009 wt.%, Respectively) or most preferably 10 mg / L l (i.e. 0.001 wt.%).

Higher values of glycolic acid, i.e. values exceeding 15 mg / l, cease to imitate the natural mechanisms and are no longer completely neutral for the natural environment.

In an embodiment of the process according to the invention, the glycolic acid solution additionally comprises auxiliary ingredients and / or fragrances.

Preferably, the glycolic acid solution is packaged in an atomizer and the materials are treated by spraying them.

In an embodiment of the method according to the invention, the glycolic acid solution is applied to the surface of the material in an amount of 0.5-2 ml / cm ^{2 of the} material, preferably 1 ml / 10 cm ^{2 of the} material.

In an embodiment of the method according to the invention, the material, after contact with water, is pre-dried for 15-20 minutes before being treated with the glycolic acid solution.

Preferably, the material for use in contact with water is diving, surfing and / or windsurfing clothing, preferably neoprene.

The subject of the invention is also the use of glycolic acid to protect materials intended for use in contact with water, against colonization by bacteria and / or formation of bacterial biofilms on their surface, where glycolic acid is used for materials in a concentration in the range of 0.5-15 mg / l, in the form of a solution in sterile water, preferably distilled water.

The i range of 0.5-15 mg / L corresponds to 0.00005-0.0015-% by weight.

In a preferred embodiment for the use according to the invention, the glycolic acid concentration is 10 mg / L.

The value of 10 mg / l corresponds to 0.001% by weight.

Other concentrations of glycolic acid can also be used in the use according to the invention, in particular 0.5, 1, 2 mg / l (respectively 0.00005, 0.0001, 0.0002 wt.%, And also e.g. about 3, 4). , 5, 6, 7, 8, 9 mg / L (about 0.0003, 0.0004, 0.0005, 0.0006, 0.0007, 0.008, 0.0009 wt.%, Respectively) or most preferably 10 mg / L l (i.e. 0.001 wt.%).

In an embodiment for use according to the invention, the glycolic acid solution further comprises auxiliary ingredients and / or fragrances.

Preferably, the glycolic acid solution is packaged in an atomizer and the materials are treated by spraying them.

In an embodiment for the use according to the invention, the glycolic acid solution is applied to the surface of said material in an amount of about 0.5-2 ml / cm ^{2 of} material, preferably 1 ml / 10 cm ^{2 of} material.

In an embodiment for use according to the invention, the material is pre-dried for about 15-20 minutes prior to treatment with the glycolic acid solution.

Preferably, the material for use in contact with water is diving, surfing and / or windsurfing clothing, preferably neoprene.

The solution according to the invention assumes the use of low concentrations of glycolic acid diffusing from the protected surface. The use of glycolic acid according to the invention is in particular aimed at protecting specialized clothing intended for contact with aqueous environments against the formation of biofilms formed by water bacteria on its surfaces and inside the structure of the material from which it is made. Biofilms created in the porous structure of the material can harbor potentially pathogenic bacteria (e.g. Aeromonas hydrophila), and also adversely affect the hygiene and comfort of using clothing, e.g. by the formation of an unpleasant odor resulting from the bacterial decomposition of organic compounds. Although the described preparation

The PL 232 885 B1 can serve in particular to protect clothing intended to come into contact with water, such as neoprene suits, e.g. for diving, surfing, windsurfing, neoprene caps, neoprene boots, neoprene gloves, etc.), it can also be used for protection other materials that undergo dip-dry cycles. Such materials can be, for example, swimwear, towels, but also various types of sealing materials or surface material intended for use in water or for other reasons subjected to cycles of immersion in water and drying.

An innovative aspect of the solution according to the invention is that it mimics the mechanisms occurring in nature with the use of chemical compounds produced naturally by microorganisms living in aquatic ecosystems. It is worth emphasizing that low concentrations of glycolic acid are also non-toxic to aquatic organisms, including bacteria. It does not kill bacteria, but only inhibits their natural tendency to colonize the surface. As a result, materials intended for use in water (e.g. preferably neoprene foams) will not form durable and difficult to remove bacterial biofilms. This solution is different from most of the anti-biofilm agents used today, which prevent the growth of bacteria based on the inhibition of their metabolic activity. They are often bactericides, not indifferent to the functioning of the natural environment. Agents of this type, by causing the death of microorganisms, can lead to the release of intracellular toxins, such as hepatotoxins, microcystins or dermatotoxins, from cyanobacterial cells. In the case of the solution proposed here, this problem does not occur. Glycolic acid in the proposed concentration is non-toxic to humans, and its effective action does not require rinsing the foam clothes after using the preparation, as is the case with other foam preservatives. The method of protecting materials and the use of glycolic acid according to the invention, in addition in very low concentrations, to protect materials immersed in water, in particular clothing intended for contact with water, enabling effective protection of such materials against colonization and the formation of bioflims by bacteria, while not disturbing the balance ecological water ecosystem and contributes to the protection of the natural environment of water bodies.

The method of using the glycolic acid preparation according to the invention allows its easy application, even in the field, without the need for access to running clean water. It preferably consists in spraying the preparation - with a glycolic acid solution in sterile water at a concentration in the range of about 0.5-15 mg / l, on the material, in particular clothing, e.g. neoprene, preferably by spraying the preparation with a glycolic acid solution in a concentration in the range of about 0 , 5-15 mg / l, in sterile water with an atomizer. In an embodiment of the glycolic acid solution preparation, the material (e.g. clothing) should be slightly dried after use (e.g. after being immersed in water), e.g. for approx. 15-20 minutes, and then the material should be sprayed evenly with a thin layer (preferably approx. 1 ml / 10 cm ^{2 of} material) of a glycolic acid solution at a concentration in the range of about 0.5-15 mg / L in sterile water, preferably distilled water, and then allow the material to dry. The material soaked in this way, after adsorption of the glycolic acid solution, is protected against the formation of biofilms by bacteria present in the water trapped in the structure of the material, until it is completely dry. As low concentrations of glycolic acid do not pose a threat to the natural environment and humans (it is used, among others, in numerous cosmetics and is allowed to come into contact with the skin in concentrations higher than in the solutions according to the present invention), it is not required to rinse the material before re-using it. use.

The formulation comprising a solution of glycolic acid in sterile water used in a method of preserving materials and used for such an application may optionally also contain additional excipients and / or fragrances. The auxiliaries may be, for example, dispersants known in the art, enzymes, for example those used in antibacterial or cleaning preparations, buffering substances, cosmetic ingredients, etc. Preferably, a formulation comprising a glycolic acid solution intended to be used in particular for materials to be used. skin contact, such as clothing exposed to water, may contain additional auxiliary ingredients used in cosmetic formulations known in the art, including in particular ingredients of plant origin. Advantageously, a preparation comprising a solution of glycolic acid in sterile water used in a method of protecting materials and used in such application may be packaged in spray cans, for example in a volume of 0.5L. Preferably the atomiser can have a capacity of about 1 ml. of the fluid for 1 press of the washer button.

PL 232 885 B1

The solution according to the invention can be used, for example, for clothing exposed to contact with water inhabited by microorganisms (e.g. diving, windsurfing suits). Such clothes, after use, until they are completely dry, are exposed to colonization of porous surfaces by microorganisms living in the water (including pathogenic). It is at this point, when the clothes are left without water flow, often not fully dried, transported from the place of use to the place of storage, that the colonization processes of the clothes by bacteria can be most effective. This leads to the colonization of the clothes by bacteria and, consequently, lowering the hygiene of its use. The solutions according to the invention counteract this negative phenomenon.

The present invention uses natural mechanisms to inhibit bacterial biofilm formation, and the proposed low concentrations of glycolic acid do not kill microorganisms, so there is no risk of releasing toxic compounds from decaying dead bacteria and cyanobacteria.

Glycolic acid at the proposed low concentrations is non-toxic to the environment and humans.

This is an inexpensive way to protect materials intended for use in contact with water, since the production cost of the formulation used in the method of use according to the invention is relatively low.

The glycolic acid preparation used according to the invention allows to protect the material to be used in contact with water, preferably clothing, at a crucial moment - shortly after use, especially when in the field it is not possible to thoroughly, immediately wash and / or dry it. The use of a glycolic acid preparation can protect the materials, preferably clothing, in transport from being permanently colonized by bacteria derived from the water in which the material was used.

Brief description of the figures

Fig. 1 are microscopic pictures comparing the image of microorganisms colonizing a surface emitting glycolic acid (A) and not emitting glycolic acid (B).

Fig. 2 shows the relative number of bacteria present on the surface of a 10 cm long dialysis bag after their detachment by sonication (in 20 ml volume), 3 ml depositing on a polycarbonate filter and observation in a field of view of 5519.02 gm ² , for the surface from which increasing concentrations of glycolic acid are emitted.

Fig. 3 shows the potential maximal activity of aminopeptidase associated with the presence of bacteria colonizing the surface of filters emitting increasing concentrations of glycolic acid.

Fig. 4 is a comparison of microscopic images of the treated and untreated neoprene surface of neoprene foam coated with polypropylene fibers according to the method of use (over 400x, dyed with DAPI).

Fig. 5 is a comparison of the number of bacteria (average of 15 fields of vision of the area. 5519.02 gm ²⁾ visualized on the foam surface the treated and the untreated product during the experiment.

Fig. 6 shows the number of bacteria present on the surface of filters closing water tanks in variants with the addition of: sodium azide (0.3% - inhibition of active filter colonization), algae extract X5 (Ext. Alga. X5 - five times concentrated in the lake water filtrate by 0.2 gm filter algae extract from J. Mikołajski), control (no enrichment), with LB medium (enrichment with LB medium, 5 mg / l), with glycolic acid (enrichment with glycolic acid, 5 mg / l), with glucose (enriched with glucose, 5 mg / l), after 48 hours incubation.

Fig. 7 shows the changes in the taxonomic structure of bacteria found on the surface of filters closing the vessels with water, for the various variants from Fig. 6.

Examples

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Influence of changes in the concentration of glycolic acid gradient on surface colonization - inhibition of colonization of the surface of dialysis bags containing increasing concentrations of glycolic acid.

Dialysis bags were filled with autoclaved lake water (J. Mikołajskie, summer growing season, July 2014) filtered through a polycarbonate filter (Millipore) with a pore diameter of 0.2 gm, containing the following increasing concentrations of sterile glycolic acid - 0, 0.25, 0 , 5 and 1 mg / l. The bags prepared in this way in 3 replications were suspended in the epilimnion zone of J. Mikołajski and left for 5 days. After this period, the surface (2 cm ² ) of the pouches was fixed in a formalin vapor, stained with DAPI at a concentration of 1 gg / ml and dried. The surface of the pouches was observed under a Nikon Eclipse 400 epifluorescence microscope. The photos were taken at 1000x magnification using a Nikon DXM1200F camera and Nikon NIS Element software (Nikon Corporation).

PL 232 885 B1

In order to accurately quantify the number of bacteria on the surface of the bag, 10 cm fragments of dialysis bags were placed in 5 ml of sodium pyrophosphate solution in deionized water (final concentration of sodium pyrophosphate in the samples: 50 mmol I ^-1 ) in order to reduce the surface tension on the surface of the bag. The samples prepared in this way were placed on ice and after 20 minutes they were gently sonicated at the lowest power range of the UDM-10 type sonicator. (6 five-second pulses, power 300 W, 22 μm, pause between pulses 40 s). It allowed the detachment of sedentary bacteria, without disintegrating the bacterial cells. The number of bacteria after sonication was determined according to Porter and Feig (1980) by direct counting under an epifluorescence microscope (Nikon E400) after depositing detached bacteria on polycarbonate filters (0.2 μm Nuclepore, black) and staining with DAPI (4 ', 6'-diamidino- 2-phenylindole, final sample concentration 1 μg / ml).

On the basis of the conducted experiment, the effective concentration of glycolic acid was determined to prevent intensive colonization of the surface by bacteria.

Results

The conducted research has shown that the presence of glycolic acid (in concentrations from a few to several mg per liter) on the experimental model surfaces significantly reduces the colonization of these surfaces by water bacteria. Figures 1A and 1B show micrographs comparing the colonization density of the surface of the dialysis bag submerged in lake waters (for a period of 5 days) from which glycolic acid was secreted (Fig. 1A, concentration gradient from 1 mg / l) to the surface where it was secreted (Fig. 1B). Detailed analysis of the surface colonization showed that the number of bacteria colonizing it decreased with increasing concentration of glycolic acid emitted by the surface (Fig. 2).

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Influence of the presence of a glycolic acid gradient on the proteolytic activity of bacteria colonizing surfaces.

Autoclaved lake water filtered through a 0.2 μm filter (August 2015, J. Mikołajskie) containing increasing concentrations of sterile glycolic acid (0, 0.5, 1 and 2 mg / l) was filled with a series of 50 ml Falcon vessels (in 3 replications) ) and closed with a polycarbonate filter with a pore diameter of 0.02 µm. The vessels were placed in a 10 l mesocosm containing water taken from the surface layer of J. Mikołajski (August 2015). The systems were incubated at 20-24 ° C for 72 hours. After this period, the filters were cut and placed in 3.9 ml of sterile, 0.2 µm filtered lake water. Then 0.1 ml of the substrate (L-leucine-4-methyl-coumarinylamide hydrochloride, LMCA) was added to the sample prepared in this way to obtain the final substrate concentration of 15 μM. Immediately after the addition of the LMCA, the fluorescence of the samples was measured at 380 nm excitation and 440 nm emission (Kiersztyn et al. 2007). The results obtained during t = 0 constituted a blank for the tested samples. The samples were incubated at 20-24 ° C for 60 minutes depending on the expected rate of LMCA hydrolysis, after which the fluorescence was measured again. The rate of increase in the concentration of the product, AMC (aminomethylcumarine) in the tested samples was calculated on the basis of the difference in fluorescence of the sample measured at time t = 0 and after incubation, on the basis of the previously prepared standard curve. A standard curve was prepared by measuring the fluorescence of 8 calibration solutions containing AMC, which were prepared by adding to 3.9 ml of distilled water 0.1 ml of appropriately concentrated AMC solutions in ethanol to give a final concentration of 0.1; 0.5; 1.0; 2.5; 5; 10; 20 and 50 nmol I ^-1 . Measurements of the fluorescence of the samples were performed on a Shimadzu RF 1501 spectrofluorimeter. The substrate degradation rate by bacterial leucine aminopeptidases was obtained, expressed in nMol / h.

Results

It was shown that the enzymatic activity of bacteria found on the surfaces of polycarbonate filters decreased with increasing concentration of glycolic acid emitted by these surfaces (Fig. 3).

P r z k ł a d 3

Assessment of the effectiveness of the application of the preparation in the form of an aerosol in order to protect neoprene material cyclically exposed to lake water against the formation of bacterial biofilms on it.

The experiment involved fragments of windsurfing clothing, 10 cm ² fragments of 1.5 mm thick neoprene foam covered with polyethylene fibers. The foam fragments were placed in 5-liter microcosms containing water from the surface layer (depth 1.5 m) of the eutrophic J. Mikołajski (August 2016). The water was changed once a day at 10:00 every day during the experiment from 03/08/2016 to 09/08/2016. The treated foam fragments (P) and the control foam (K) were placed in the lake water 3 times

Every day for a period of 2-3 hours. After the time of presence in the water, the pieces were taken out of the water and left at room temperature to dry slightly for 15 minutes at approx. 20 ° C. After this time, fragment P was sprayed with an aerosol (1 spray per 10 cm ² - 0.5 ml of liquid) containing glycolic acid at a concentration of 10 mg / L in sterile distilled water. The control fragment K was not sprayed. The fragments were allowed to dry for subsequent placement in the lake water. After 3, 5 and 6 days of the experimental procedure, 1 cm sections of the foam were cut, dried and stained by applying to a dried fragment of the DAPI preparation (2 µg / ml). After 10 min, the foam pieces were observed and photographed under a Nicon Eclipse 400 epifluorescence microscope equipped with a digital camera. Pictures were taken at 400x magnification. The microscopic image was analyzed using Nikon NIS-Elements AR software. The analysis of the number of microorganisms present on the surface protected with the preparation and the surface of the control fragments was performed.

Results

Fig. 4 shows a series of photographs of the surface of the Control foam (K) and the pieces treated with the formulation (P) during the test period. In the untreated variant, after 4 days of cyclic exposure to microorganisms living in lake water, the formation of numerous bacterial biofilm structures was observed. Such a phenomenon did not occur in the variant with the material protected with the preparation. After 6 days, numerous, often large bacteria (cell length> 4 μm) were observed on the surface of the control material. The fibers of the secured material contained only a few small microorganisms. The analysis of the number of bacteria observed on the one-sided surface of the fiber showed clearly many times higher (over 17 times on the fourth day of exposure to water bacteria) the number of bacteria on the fibers not protected with the preparation than in the fragments protected with the preparation (Fig. 5).

The experiment proved that the application of the preparation according to the invention effectively protected the neoprene foam material against colonization by bacteria during its simulated intensive use.

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Comparison of the influence of the emission of glycolic acid with the emission of other chemical compounds on the colonization of the surface by bacteria.

The samples were taken in July 2015 from the photo zone of J. Mikołajski. 50 ml sterile Falcon vessels were prepared, containing 0.2 μm filtered water from the surface zone of J. Mikołajski. The water in the vessels was supplemented with the additives listed below or left untreated and then sealed with a polycarbonate filter (Millipore, 0.2 µm). The following variants were prepared: Sodium azide (0.3% - inhibition of active filter colonization), Algae extract X5 (Ext. Alga. X5 - five times concentrated in the lake water filtrate through a 0.2 μm filter algae extract from J. Mikołajski), Control ( no enrichment whatsoever). LB medium (enriched with LB medium, 5 mg / l), glycolic acid (enriched with glycolic acid, 5 mg / l), glucose (enriched with glucose, 5 mg / l). The containers prepared in this way were placed in J. Mikołajski's water, previously devoid of biofilms, by filtering through a polycarbonate filter with a pore diameter of 3.0 μm. Chemical compounds with which individual variants were enriched were emitted from the filter surfaces. After 48 hours incubation, the bacteria on the surface of the filters closing the tanks were counted. The filters were stained with DAPI at a concentration of 1 µg / ml and dried. The surface of the filters was observed under a Nikon Eclipse 400 epifluorescence microscope. The photos were taken at 1000x magnification using a Nikon DXM1200F camera and Nikon NIS Element software (Nikon Corporation).

The DNA of microorganisms colonizing the filters in each of the tested variants was also isolated using EURx bacterial DNA extraction kits according to the manufacturer's procedure.

The coding sequence of the 16sRNA of microorganisms colonizing the filters was analyzed, which enables their phylogenetic classification. The metagenomic analysis of the gene encoding the 16S rRNA was performed on the basis of the hypervariable region V3-V4 of the 16S rRNA gene. Specific sequences of commercially available 341F and 785R primers (Illumina, http://support.illumina.com/documents/documentation/chemistry_documentation/16s/16s-metagenomic-library-prep-guide-15044223- were used to amplify the selected region and prepare the library. b.pdf; Klindworth A, et al. (2013) Evaluation of general 16S ribosomal RNA gene PCR primers for classical and next-generation sequencingbased diversity studies. Nucleic Acids Res 41 (1)). The PCR reaction was performed with a Q5 Hotstart High-FidelityDNA Polymerase (NEBNext), reaction conditions as recommended by the manufacturer.

PL 232 885 B1

Sequencing was done on the MiSeq sequencer, in paired-end (PE) technology, 2x250nt, using the Illumina v2 kit. Data analysis was performed on the MiSeq apparatus using the MiSeq Reporter (MSR) v2.4 software; 16S Metagenomics protocol. The analysis consisted of three stages:

1) automatic sample demultiplexing,

2) generating fastq files containing raw readings,

3) classification of paired-end readings into individual taxonomic categories.

The 16S Metagenomics protocol provides a species level classification of the readings within the obtained 247 families (1333 species analyzed), based on the Greengenes v13_5 reference sequence database, modified by Illumin.

Preparation of the reference database of reference sequences included:

1) filtering out sequences shorter than 1250 bp (bp),

2) filtering out sequences containing more than 50 degenerate bases (M, R, W, S, Y, K, V, H, D, B, N),

3) filtering out incompletely classified sequences (no classification to the level of genus or species).

Agglomeration analyzes and comparisons of means were carried out in the Statistica program (StatSoft corp.). For the agglomeration analysis, the Manhattan distance algorithm and the Ward binding method were used based on the share of individual species classified by the Illumina analysis based on the GreenGenes databases.

Results

It was shown that the glycolic acid emitted from the surface significantly decreased the average number of bacteria colonizing the surfaces as compared to the control and other variants emitting various organic compounds (Fig. 6). These differences were statistically significant (Student's t-test, p <0.05). In the variant with glycolic acid, the number of bacteria was only slightly higher than on the surface emitting a highly toxic, inorganic compound - sodium azide, a bactericidal preservative. Glycolic acid with a concentration of 5 mg / l emitted from the surface not only effectively reduced colonization compared to other tested organic compounds, but also changed the taxonomic structure found on the surface of bacteria (Fig. 7). For example, in the variant with glycolic acid, a smaller proportion of bacteria belonging to the Flavobacteriaceae family, typical for biofilm formation, was found on the colonized surface (2.3% of readings) than in other variants (Algae extract X5 - 31.5% , Control - 7.93%, LB Medium - 4%, Glucose - 7.77%). This share was only slightly higher than in the variant in which sodium azide, toxic for bacteria, was emitted (1.68%) and the process of intensive, active surface colonization did not take place. The situation is similar in the case of the Legionellaceae family, which contains potentially pathogenic bacteria causing Pontiac fever and life-threatening legionellosis. In the variant with the emission of glycolic acid and sodium azide, no representatives of this family were found on the surface (0.0%), while in the other variants they were present (X5 algae extract - 0.17%, Control - 0.15%, LB medium - 0.9%, Glucose - 0.22%). On the other hand, a significant difference was observed in the participation of the Alfapnoteobacteria class, representatives of which were more numerous in the variant with glycolic acid (16.35%) than in the other variants (Algae extract X5 - 11.2%, Control - 6.4%, LB medium - 8 , 43%, Glucose - 7.69%), and similarly for the variant with biofilm inhibiting, highly toxic sodium azide (15.46%). Example 4 proved that the emission of low concentrations of glycolic acid, not only, as demonstrated in all the presented examples, effectively reduces the colonization of the surface by bacteria, but also changes the phylogenetic structure of the few bacteria present on this surface. It seems that the presence of bacteria on the glycolic acid emitting surface may be mainly due to the passive adsorption of floating bacteria, and not the particularly undesirable active colonization by biofilm-forming bacteria.

PL 232 885 B1

Bibliography

Eom SH1, Kim YM, Kim SK. (2012). Antimicrobial effect of phlorotannins from marine brown algae. 50: 3251-5.

Kiersztyn B, Siuda W, Chróst RJ (2012). Persistence of bacterial proteolytic enzymes in Lake ecosystems. FEMS Micro. Eco. 80: 124-134.

Porter KG, Feig YS. 1980. The use of DAPI for identifying and counting aquatic microflora. Limnol. Oceanogr. 25: 943-948.

Punyasl B., Wright PC. (2004) Exploitation of marine algae: biogenic compounds for potential antifouling applications. Planta 219: 561-578.

Stabenau H. and Winkler U. (2005) Antimicrobial effect of phlorotannins from marine brown algae. Phy. Plant. 123: 235-245. 2005.

Claims (14)

Patent claims A method of protecting materials intended for use in contact with water against colonization by bacteria and / or the formation of bacterial biofilms on their surface, characterized in that the said materials are treated with a glycolic acid solution with a concentration in the range of 0.5-15 mg / l in sterile water, preferably distilled water. 2. Method of protecting materials according to claim The process of claim 1, wherein the glycolic acid is at a concentration of 10 mg / l. 3. Method of protecting materials according to claim The method of claim 1 or 2, characterized in that the glycolic acid solution additionally comprises auxiliary components and / or fragrances. 4. Method of protecting materials according to claim A process as claimed in 1-3, characterized in that the glycolic acid solution is packaged in an atomizer and the materials are treated by spraying them. 5. A method of protecting materials according to claim A method according to any of the claims 1-4, characterized in that the glycolic acid solution is applied to the surface of said material in an amount of 0.5-2 ml / cm ^{2 of} material, preferably 1 ml / 10 cm ^{2 of} material. 6. A method of protecting materials according to claim The process as in 1-5, characterized in that the material, after contact with water, is pre-dried for 15-20 minutes prior to treatment with the glycolic acid solution. 7. A method of protecting materials according to claim The method of any of the claims 1-6, characterized in that the material to be used in contact with water is diving, surfing and / or windsurfing clothing, preferably neoprene. 8. The use of glycolic acid to protect materials intended for use in contact with water, against bacterial colonization and / or the formation of bacterial biofilms on their surface, where glycolic acid is used at a concentration of 0.5-15 mg / l, in the form of solution in sterile water, preferably distilled water. 9. Use of glycolic acid for the protection of materials according to claim 1 8. The process according to claim 8, characterized in that the glycolic acid is at a concentration of 10 mg / l. 10. Use of glycolic acid for the protection of materials according to claim 1 The method according to claim 8 or 9, characterized in that the glycolic acid solution additionally contains auxiliary components and / or fragrances. 11. Use of glycolic acid for the protection of materials according to claim 1 8. The process according to claim 8-10, characterized in that the glycolic acid solution is packaged in an atomizer. 12. Use of glycolic acid for the protection of materials according to claim 1, 8. The method according to claim 8-11, characterized in that the glycolic acid solution is applied to the surface of said material in an amount of 0.5-2 ml / cm ^{2 of} material, preferably 1 ml / 10 cm ^{2 of} material. 13. Use of glycolic acid for the protection of materials according to claim 1. 8. The method according to claim 8-12, characterized in that said material is pre-dried for 15-20 minutes prior to treatment with the glycolic acid solution. 14. Use of glycolic acid for the protection of materials according to claim 1, 8. The apparatus as claimed in claim 8-13, characterized in that the material to be used in contact with water is diving, surfing and / or windsurfing clothing, preferably neoprene.