METHOD FOR PRODUCING MICROBIAL PROBIOTIC BIOFILMS AND USES THEREOF

Abstract

The present invention relates to a method for producing microbial probiotic biofilms and their uses in the biomedical, industrial, food and environmental field.

Claims

1. A method for producing a probiotic biofilm, comprising the following steps: a) co-inoculation of a combination of at least two probiotic bacterial strains belonging to the genus Bifidobacterium and Lactobacillus at a concentration comprised between 10.sup.6-10.sup.9 CFU/ml on the surface of a solid support pretreated with a culture medium or into a liquid medium; b) co-culturing the two probiotic bacterial strains for a time period comprised between 24-72 hours in the absence or in the lack of nutrients at a temperature comprised between 12-18 C.

2. The method for producing a probiotic biofilm according to claim 1, wherein said at least two probiotic bacterial strains are Bifidobacterium infantis (DSM20088) and Lactobacillus reuteri (DSM20016).

3. The method for producing a probiotic biofilm according to claim 1, wherein step a) of co-inoculation is carried out on the surface of a solid support made of a material selected from the group consisting of glass, steel, ceramic, polymers, paper and resin.

4. The method for producing a probiotic biofilm according to claim 1, wherein step a) of co-inoculation is carried out at a concentration of 10.sup.8 CFU/ml and step b) of co-culture is carried out for a period of 48 hours at a temperature of 15 C.

5. The method for producing a probiotic biofilm according to claim 3, wherein said polymers are selected among polyethylene, polyvinyl chloride and polypropylene.

6. The method for producing a probiotic biofilm according to claim 3, wherein said resin is made of polycarbonate, preferably of Lexan.

7. The method for producing a probiotic biofilm according to claim 1, wherein said co-culture of step b) in lack of nutrients is carried out in the presence of bacteriological peptone 1%.

8. A probiotic film obtainable by the method according to any one of the claims 1-7.

9. A combination of probiotic bacteria comprising Bifidobacterium infantis (DSM20088) and Lactobacillus reuteri (DSM20016).

10. Use of the combination of probiotic bacteria according to claim 9, for the preparation of a probiotic biofilm.

11. A probiotic biofilm characterized in that it comprises the combination of probiotic bacteria according to claim 9, adhered on a solid support or inoculated in a liquid medium.

12. Use of the probiotic biofilm according to claim 8, for sanification or cleaning of the surface of a structure.

13. Use of the probiotic biofilm according to claim 12, wherein said structure is a device selected from the group consisting of catheter, implant, brace, bite or condom.

14. Use of the probiotic biofilm according to claim 12, wherein said structure is selected from the group consisting of sink, bidet, water closet or piece of furniture.

15. Use of the probiotic biofilm according to claim 8, for the application on a packaging material for use in the food industry.

16. Use of the probiotic biofilm according to claim 15, wherein said packaging material is selected from the group consisting of film, pellicle, combinations for packaging or oven paper.

17. A detergent solution comprising the probiotic biofilm according to claim 8 as active ingredient, optionally together with one or more further detergent agent and/or surfactant.

18. A packaging material for use in the food industry comprising the probiotic biofilm according to claim 8.

19. Use of the probiotic biofilm according to claim 11, for sanification or cleaning of the surface of a structure.

20. Use of the probiotic biofilm according to claim 19, wherein said structure is a device selected from the group consisting of catheter, implant, brace, bite or condom.

21. Use of the probiotic biofilm according to claim 19 wherein said structure is selected from the group consisting of sink, bidet, water closet or piece of furniture.

22. Use of the probiotic biofilm according to claim 11, for the application on a packaging material for use in the food industry.

23. Use of the probiotic biofilm according to claim 22, wherein said packaging material is selected from the group consisting of film, pellicle, combinations for packaging or oven paper.

24. A detergent solution comprising the probiotic biofilm according to claim 11 as active ingredient, optionally together with one or more further detergent agent and/or surfactant.

25. A packaging material for use in the food industry comprising the probiotic biofilm according to claim 11.

Description

EXAMPLE 1: ASSESSMENT OF THE TENDENCY OF PROBIOTIC MICROORGANISMS TO ADHERE TO DIFFERENT SURFACES

[0042] In order to assess the tendency of probiotic-like microorganisms (virtuous, nonpathogenic) to adhere to different surfaces, 5 strains of bifidobacteria (strains 1-5), 5 strains of lactobacilli (strains 6-10) and 5 strains of probiotic-like yeast (strains 11-15) were investigated. Glass tiles of AISI316L stainless steel (25 mm50 mm, 0.5 mm thick) and Lexan (25 mm75 mm, 0.5 mm thick) were used as surface samples for biofilm formation, suitably treated to remove any fingerprints, grease or oily substances present on the material.

[0043] The tiles were immersed in a 3.5% sodium hypochlorite solution for 15 minutes, washed with distilled water and then transferred to a 0.7% phosphoric acid solution. After 15 minutes, the tiles were rinsed with distilled water and allowed to dry for 24 hours.

[0044] For biofilm formation, optimum substrate aliquots (MRS broth, Oxoid) were distributed in containers containing each 5 tiles in a vertical position so as to have the entire surface exposed to the medium. Samples containing glass tiles, stainless steel tiles and tiles made of a particular resin belonging to the polycarbonate family (Lexan) were prepared, all sterilized in autoclave at 121 C. for 15 minutes.

[0045] Each sample was inoculated with the related microorganism to be tested (initial inoculum 10.sup.2 UFC/ml) and incubated, without stirring, for 5 days at the respective optimum temperature (37 C. for bifidobacteria, 30 C. for lactobacilli, 25 C. for yeasts).

[0046] Prior to each test, the inoculum was prepared by revitalizing the strains in the respective culture broths and incubating them at their optimal temperatures for 24 hours. For each microbial target tested, the inoculum was harvested by centrifuging the corresponding pre-culture at 4000 rpm for 10 minutes at 4 C. in an ALC 4239R centrifuge (ALC, Milan, Italy) and re-suspending in sterile physiological solution (9 g/l NaCl) tempered at 4 C.

[0047] During the tests, the trend of the microbial population in sessile form was monitored by sampling at specified time intervals. The adhered cell detachment was induced by sonication treatment for 3 minutes at 20 watts. Viable and culturable cells were counted on appropriate media.

[0048] The following Table 1 shows the maximum cellular load values reached in stationary phase by the 15 microorganisms tested.

TABLE-US-00001 TABLE 1 STRAINS A (log CFU/cm.sup.2) Bifidobacteria STEEL GLASS LEXAN 1. B. animalis 4.660 0.057 .sup.A* 4.557 0.216 .sup.A 3.841 0.416 .sup.A DSM10140 2. B. subtilis 5.093 0.175 .sup.B 4.684 0.096 .sup.A 5.010 0.016 .sup.B DSM20096 3. B. infantis 6.155 0.235 .sup.B 5.145 0.001 .sup.B 4.813 0.421 .sup.B DSM20088 4. B. longum 4.797 0.155 .sup.A 4.371 0.278 .sup.A 5.237 0.170 .sup.B DSM20219 5. B. breve 4.407 0.076 .sup.A 4.831 0.043 .sup.A 4.053 0.023 .sup.A DSM20213 Lactobacilli 6. L. plantarum 6.364 0.152 .sup.A 5.642 0.137 .sup.A 5.729 0.140 .sup.A DSM2601 7. L. casei 4.083 0.166 .sup.B 3.692 0.144 .sup.B 5.108 0.240 .sup.B DSM20011 8. L. delbrueckii 6.075 0.311 .sup.A 5.869 0.400 .sup.A 4.946 0.200 .sup.B DSM20081 9. L. paracasei 6.228 0.288 .sup.A 6.078 0.311 .sup.A 5.583 0.300 .sup.A DSM20207 10. L. reuteri 6.203 0.259 .sup.A 5.885 0.318 .sup.A 6.305 0.210 .sup.C DSM20016 Yeasts 11. Kluyveromyces No adhesion No adhesion No adhesion lactis ATCC8585 12. S. cerevisiae boulardii 3.002 0.100 .sup.A 3.025 0.156 .sup.A 2.895 0.200 .sup.A ATCC MYA-796 13. S. cerevisiae 3.234 0.150 .sup.A 3.309 0.130.sup.A 3.225 0.250 .sup.A WL21** 14. S. cerevisiae 4.364 0.230 .sup.B 4.847 0.250 .sup.B 4.153 0.116 .sup.B WL40** 15. S. cerevisiae 3.062 0.200 .sup.A 3.308 0.300.sup.A 3.890 0.205 .sup.B WL45** *Within each genus, the values of the same column with the same superscripts do not differ significantly (p > 0.05) **Autochthonous yeast probiotic-like strains isolated from wine and belonging to the Department of the Science of Agriculture, Food and Environment - University of Foggia

[0049] The data are presented as an average of two repetitions and are accompanied by the standard deviation.

[0050] The 5 bifidobacteria tested all guaranteed a good adhesion, with some differences between the tested surfaces.

[0051] On steel surfaces, all the strains showed similar behavior in terms of lag phase, adhesion rates and maximum load of adhered cells in stationary phase, with the exception of B. infantis DSM20088 (strain 3), characterized by increased adhesion rate and capable of achieving a higher load of adhering cells in less time (few hours), greater than 6 log CFU/cm.sup.2. Unlike steel, the analysis of glass-adhesion tests did not reveal any significant differences between the tested microorganisms, as all strains investigated exhibited good adhesion capacity even after a few hours from the inoculum, reaching a maximum load of adhering cells equal to about 4.40-5.14 log CFU/cm.sup.2. Similar results were recorded for adhesion on Lexan.

[0052] As for the lactobacilli tested, the 5 strains guaranteed good in vitro adhesion to the three surfaces tested. The strains showed similar lag phase and adhesion rates, starting to form biofilm after a few hours from the inoculum and reaching the maximum load of adhering cells on the second incubation day. In particular, higher cellular loads were observed than those observed for bifidobacteria. Yeast strains showed a lower tendency to adhesion than the other microbial targets tested.

[0053] In this way, it was possible to identify, among the probiotic microorganisms tested, the strains of B. infantis DSM20088 (strain 3) and L. reuteri DSM20016 (strain 10) as two bacterial strains with good ability to adhere to the tested surfaces.

EXAMPLE 2: OPTIMIZATION OF CONDITIONS ABLE TO PROMOTE A BETTER FORMATION OF FUNCTIONAL BIOFILMS IN TERMS OF SPEED AND AMOUNT OF ADHERING CELLS

[0054] In order to identify the optimal conditions to ensure the maximum adhesion of probiotic microorganisms in terms of the rate of formation and the amount of adhering cells, the optimal conditions (inoculation level and culture conditions) adapted to stimulate greater biofilm formation were identified for the first time.

[0055] B. infantis DSM20088 and L. reuteri DSM20016 were used in this step. Lexan tiles (25 mm75 mm, 0.5 mm thick) were used as surfaces for the biofilm formation.

[0056] In order to assess the ability to form biofilm under suboptimal conditions (nutrient deficiency and low temperature), a first test was conducted in which peptone water aliquots (1% of bacteriological peptone, Oxoid) were distributed in 50 ml plastic tubes containing each 1 tile in vertical position so as to have the entire surface exposed to the medium. Samples were inoculated with the relevant microorganism to be tested (initial inoculum 10.sup.8 CFU/ml) and incubated at 15 C. for 48 hours. The inoculum was prepared as described above. Every hour, the samples were stirred for 10 min at 70 rpm. The trend of the microbial population in sessile form was monitored at specified time intervals, as described above. The tests were conducted in double. Microbial counts were transformed into logarithms before standard averages and deviations were calculated and reported in log CFU/cm.sup.2.

[0057] Both microorganisms tested were able to adhere to the Lexan surface in the lack of nutrients at 15 C. and under dynamic conditions, reaching a cellular load of about 4.8-5.3 log CFU/cm.sup.2, already after an hour of contact.

[0058] Having ascertained that capacity, a second test was conducted to evaluate the effect of the inoculum level on the tendency to adhesion and the possible presence of interactions.

[0059] To this aim, in addition to L. reuteri DSM20016 (L) and B. infantis DSM20088 (B), the yeast strain Saccharomyces cerevisiae boulardii ATCC MYA-796 (SB) was tested.

[0060] The inoculum levels were modulated in accordance with a simple centroid; this type of experimental design involves three different variables, each of which can take 3 different levels, identified with code 0 (minimum), 1 (maximum), 0.5 (half of the range considered). As independent variables, the inoculum levels of the three target probiotics investigated were selected.

[0061] Table 2 shows both encoded values and those actually tested of the independent variables of the experimental design. To the above centroid combinations, control samples were added in which each microorganism was inoculated individually at the three levels of inoculation tested.

TABLE-US-00002 TABLE 2 Encoded values Actual values (log CFU/ml) L B SB L B SB 1 1 0 0 9 5 3 2 0 1 0 5 9 3 3 0 0 1 5 5 7 4 0.50 0.50 0 7 7 3 5 0.50 0 0.50 7 5 5 6 0 0.50 0.50 5 7 5

[0062] Biofilm formation was favored as described above (suboptimal nutrient deficiency and low temperature conditions).

[0063] Samples were inoculated with their microorganisms at the levels indicated in the experimental design. All samples were incubated at 15 C. for 72 hours; every hour, the samples were stirred for 10 min at 70 rpm (with a night stop of about 12 hours).

[0064] The trend of the microbial population in sessile form was monitored by sampling at specified time intervals, as already described. Viable and culturable cells were counted on appropriate selective media. The data are presented as an average of two repetitions and accompanied by the standard deviation.

[0065] Cell counts were used as inputs for the construction of a polynomial equation in order to evaluate the effects of each variable on the adhesion of each target probiotic tested.

[0066] Yeast was not able to adhere to the surfaces under any of the tested conditions. The highest levels of adhesion were observed when at least one of the other two strains (L. reuteri DSM 20016 or B. infantis DSM 20088) was inoculated at the highest level.

[0067] As the level of inoculation of each target increased, its tendency to adhesion increased. In addition, the presence of L. reuteri stimulated the adhesion of B. infantis and vice versa. No effect was recorded for yeast.

[0068] These results are shown in the following Tables 3 and 4.

TABLE-US-00003 TABLE 3 Cellular load in sessile form (log CFU/cm.sup.2) observed for Lactobacillus reuteri DSM 20016 and Bifidobacterium infantis DSM 20088 after 6 hours of contact with Lexan L. reuteri B. infantis DSM20016 DSM20088 L + B 5.689 0.200 6.129 0.132 (combination 1) L 5.469 0.108 (single inoculum) B 2.462 0.230 (single inoculum) L + B 5.543 0.023 5.402 0.205 (combination 2) L 2.800 0.145 (single inoculum) B 4.947 0.015 (single inoculum)

TABLE-US-00004 TABLE 4 Standardized effects of the inoculum levels of each of the probiotic targets tested on the adhesion tendency of the other. The bold data indicates significant effects. Bifidobacterium Lactobacillus S. cerevisiae infantis reuteri boulardii DSM 20088 DSM 20016 ATCC MYA-796 Effect of the 4.814 0.915 4.914 0.915 1.103 0.915 inoculum level of L. reuteri DSM 20016 Effect of the 4.652 0.814 5.382 0.814 2.083 0.814 inoculum level of B. infantis DSM 20088 Effect of the 0.322 0.519 0.033 0.519 1.966 0.519 inoculum level of S. cerevisiae boulardii ATCC MYA-796

EXAMPLE 3: MEASUREMENT OF THE EFFECT OF PROBIOTIC BIOFILMS ON THE DEVELOPMENT OF PATHOGENIC MICROORGANISMS

[0069] The activities carried out in Examples 1 and 2 allowed the identification for the first time of a method for the formation of a functional biofilm in which the two probiotic strains to be used (B. infantis DSM20088 and L. reuteri DSM20016) are indicated, but also the optimum conditions to ensure maximum adhesion, in terms of adhesion rate and amount of adhering cells.

[0070] Specifically, these optimal conditions were identified in: [0071] absence/lack of nutrients (only water or 1% bacteriological peptone) [0072] low temperature (i.e. 15 C.) [0073] co-inoculation of high concentration strains (10.sup.8 CFU/ml).

[0074] Once the method had been developed, in order to evaluate the effect of probiotic biofilms on the development of pathogenic microorganisms, evidence was provided on the growth in sessile form of Listeria monocytogenes, Escherichia coli O157:H7, Staphylococcus aureus and Salmonella enteritidis.

[0075] Biofilm formation was favored by simultaneously inoculating the cocktail of identified probiotics (B. infantis DSM20088 and L. reuteri DSM20016, about 10.sup.8 CFU/ml) and the pathogenic target (10.sup.7 CFU/ml) on Lexan tiles (25 mm75 mm, 0.5 mm thick), left at room temperature (20 C.) for two hours. After this time interval, the tiles were transferred to aliquots of peptone water (1% bacteriological peptone) and incubated at 15 C. for 48 hours. For each pathogenic target, two samples were set up: [0076] ACTIVE (ATT, sample with adhering probiotics) [0077] CONTROL (CNT, sample without probiotics).

[0078] The trend of the microbial population in sessile form was monitored by sampling on selective media at specified time intervals. The data are presented as an average of two repetitions and are accompanied by the standard deviation.

[0079] Microbial counts were transformed into logarithms before standard averages and deviations were calculated and reported in log CFU/cm.sup.2.

[0080] The following Table 5 shows the cellular loads in sessile form relating to the targets studied; the data analysis shows how the pathogens studied were able to develop in all samples.

TABLE-US-00005 TABLE 5 Cellular loads in sessile form (log CFU/cm.sup.2) relating to Listeria monocytogenes, Escherichia coli O157: H7, Staphylococcus aureus and Salmonella enteritidis inoculated in samples ATT and CNT Hours CNT ATT Listeria monocytogenes 0 4.209 0.007 .sup.A* 3.457 0.115 .sup.B 4 4.823 0.125 .sup.A 3.388 0.200 .sup.B 24 4.832 0.132 .sup.A 4.090 0.102 .sup.B 30 5.181 0.250 .sup.A 4.306 0.108 .sup.B 48 4.907 0.005 .sup.A 4.230 0.102 .sup.B Escherichia coli O157: H7 0 5.486 0.005 .sup.A 5.204 0.254 .sup.A 4 5.430 0.140 .sup.A 4.190 0.224 .sup.B 24 5.557 0.410 .sup.A 4.101 0.005 .sup.B 30 6.129 0.300 .sup.A 3.820 0.012 .sup.B 48 6.004 0.250 .sup.A 3.800 0.200 .sup.B Staphylococcus aureus 0 5.069 0.200 .sup.A 4.879 0.005 .sup.A 4 5.016 0.033 .sup.A 4.705 0.325 .sup.A 24 5.570 0.140 .sup.A 4.889 0.187 .sup.B 30 5.156 0.010 .sup.A 3.845 0.220 .sup.B 48 5.164 0.300 .sup.A 3.705 0.050 .sup.B Salmonella enteritidis 0 5.942 0.100 .sup.A 4.373 0.100 .sup.B 4 5.380 0.100 .sup.A 4.469 0.156 .sup.B 24 5.526 0.150 .sup.A 4.536 0.130 .sup.B 30 5.350 0.230 .sup.A 4.877 0.050 .sup.B 48 4.984 0.300 .sup.A 4.773 0.003 .sup.B *Values of the same row with the same superscript do not differ significantly (p > 0.05)

[0081] However, cellular loads were always lower in ATT samples (presence of probiotic biofilm, about 6.5-7 log CFU/cm.sup.2) compared to the CNT samples (absence of probiotic biofilm), highlighting that the functional biofilm was able to control the growth of all inoculated pathogenic targets. Biofilms were odorless and invisible to the naked eye. For E. coli 0157: H7, there was a significant decrease in cell load compared to control, of more than 1 and 2 logarithmic cycles after 4 and 48 hours of incubation, respectively. For L. monocytogenes, St. aureus and S. enteritidis, cell load reductions ranged from 0.5 to 1.5 logarithmic cycles. Therefore, if left to form ad hoc on surfaces, such as water closets or materials generally used to package foods, it is confirmed that the biofilm proposed can reduce or eliminate the pathogenic species that may be present (present in concentrations far below those tested in the present study) with the possibility of reducing the onset of nosocomial infections and/or ensuring the hygienic-health safety of food products.

EXAMPLE 4: ASSESSMENT OF PERSISTENCE AND SURVIVAL OF BIOFILMS TO SANITIZING AGENTS AND THEIR FORMATION ON CERAMICS AND PACKAGING MATERIALS

[0082] Tests were conducted to evaluate the persistence and survival of functional biofilms to sanitizing agents (step a) and tests to evaluate their formation on the most common packaging materials used in the food packaging industries (polypropylene, PVC, oven paper, paraffin paper and polyethylene films) and on ceramics (step b). In particular, two sanitizing agents (SAN1 and SAN2) were tested on functional biofilms formed on surfaces of Lexan, as proposed in the present invention (cell load of about 6.5-7 log CFU/cm.sup.2). The agents tested were two common disinfectants based on dibenzylketone/ammonium acetate and chlorhexidine, allowed to act for 5 minutes at room temperature.

[0083] After contact, the tiles were rinsed to remove the disinfectant residues and placed under suboptimal conditions (nutrition deficiency and 15 C.) for 24 hours to evaluate the ability to reform the biofilm.

[0084] The results are shown in the following Table 6.

[0085] The two tested sanitizers were able to reduce the cellular load in sessile form but not to completely remove the biofilm, leaving a residual load of about 2.5-3 log CFU/cm.sup.2. This load allowed the biofilm to restore almost completely after 24 hours (over 5 log CFU/cm.sup.2). The results obtained in step b are shown in the following Table 7. The data are presented as an average of two repetitions and are accompanied by the standard deviation.

TABLE-US-00006 TABLE 6 Cellular load in sessile form (log CFU/cm.sup.2) on the tile after 5 minutes treatment with sanitizing agents and after subsequent placement under suboptimal conditions for 24 hours SAN1 SAN2 Starting cell load 6.576 0.313 7.134 0.010 Load after treatment with 2.752 0.230 3.080 0.004 sanitizer (5 minutes) Load after placement under 5.559 0.071 5.388 0.129 suboptimal conditions for 24 hours

TABLE-US-00007 TABLE 7 Cellular load in sessile form (log CFU/cm.sup.2) observed on ceramics and common packaging materials used in the food industry Cellular load in sessile form (log CFU/cm.sup.2) MATERIALS 2 hours 24 hours 96 hours Polypropylene 6.644 0.004.sup.A* 6.144 0.483.sup.A 5.883 0.233.sup.A PVC 6.540 0.142.sup.A 5.646 0.103.sup.A 5.866 0.296.sup.A Oven paper 5.774 0.227.sup.B 5.238 0.148.sup.B 5.248 0.060.sup.B Paraffin paper No adhesion 4.607 0.218.sup.C 4.528 0.134.sup.C Polyethylene film 6.938 0.002.sup.C 6.033 0.376.sup.A 6.544 0.135.sup.D Ceramic 6.855 0.198.sup.C 6.237 0.226.sup.A 6.542 0.143.sup.D *Values of the same column with the same superscript do not differ significantly (p > 0.05)

[0086] Table 7 shows that the functional biofilm according to the invention was successfully formed on all the materials tested, recording a higher cellular load in sessile form on polyethylene and on ceramics.

BIBLIOGRAPHY

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