PROBIOTIC BIOFILM ON CARRIER SURFACE: A NOVEL PROMISING APPLICATION FOR FOOD INDUSTRY

In this essay work, the ability of probiotic bio ﬁ lm formation on carrier surface was demonstrated. Probiotic bio ﬁ lms exhibit the same properties as pathogen microbial bio ﬁ lms but with higher resistance to low pH values and bile salts. The ability of different probiotic strains ( Lactobacillus acidophilus, Biﬁ dobacterium breve, Biﬁ dobacterium longum ) to interact with pre-selected carriers divided into 3 categories (polymers, complex food matrices, and inorganic compounds) was tested. Lactobacillus acidophilus and Biﬁ dobacterium longum combined with inorganic silica carrier exhibited the interaction leading to bio ﬁ lm formation only. Prepared bio ﬁ lm ( Lactobacillus acidophilus ) was then subjected to comparative study with planktonic bacterial culture. The ability to survive in the presence of low pH value (pH 1–3) and bile salts (0.3% solution) was evaluated. Low pH value (pH 1) had a harsh effect on free cell culture causing decreased cell viability (71.9±3.2% of viable cells). Bio ﬁ lm culture exhibited higher resistance to low pH value, the viability exceeded 90%. The exposure of free cell probiotic culture to porcine bile resulted in an almost constant decrease in viability during the study period (68.2±1.1% of viable cells, after 240 min incubation). Viability of bio ﬁ lm after the exposition to bile was almost constant with a slight decrease of no more than 5% during the study.

The fi rst mention of probiotics appeared at the beginning of the 20 th century in the writings of Elie Metchnikoff. He suggested that the longevity and healthy life of Bulgarian people is hidden in their consumption of fermented milk products (TRIPATHI & GIRI, 2014). In 2001, probiotics as "live microorganisms, which when administered in adequate amounts confer a health benefi t on the host" (FAO/WHO, 2001) was defi ned.
During the last 15-20 years, it was recognized that the developmental process of biofi lm formation is the natural way of life for microorganisms, without differences between harmful and commensal microorganisms. Therefore, on one hand, biofi lm formation is an important clinical pathogenic mechanism, a menace to the aging population, immunocompromised and poly-traumatic patients, on the other hand, a modern medical instrumental intervention (RÖMLING et al., 2014). In the common line, biofi lm cells have a higher resistance to antimicrobial agents and agents harmful to microbes in biofi lm than planktonic bacteria. The formation of a barrier or biofi lm prohibits the direct contact with harmful agents (SREY et al., 2013).
Acta Alimentaria 46, 2017 Probiotics, including Lactobacillus and Bifi dobacterium species, have been found also to naturally exist in the gut in the complex biological conglomerate called biofi lm, which tightly adheres to the gut lining (CAGGIANIELLO et al., 2016). Biofi lm is a sessile high-density community of bacterial cells, which is shielded by a self-secreted protective layer formed by extracellular polymeric substance (EPS) (CHEOW & HADINOTO, 2013). Although the exact mechanism of acting of EPS is still unknown, cells involved in biofi lm exhibit a specifi c rate of resistance presumably (CAGGIANIELLO et al., 2016) due to (i) the specifi c dormant metabolic state of biofi lm cells and their specifi c communication called quorum sensing and/or (ii) the protective function of the EPS (VENTOLINI, 2015). Moreover, the protection of bacterial cells against harsh biotic and abiotic conditions including temperature, pH, and osmotic stress during passage through intestinal tract, EPS can also be involved in adhesion to surfaces and biofi lm formation and in cell adhesion/recognition mechanisms (DONOT et al., 2012;SALAZAR et al., 2016).
The protective function of biofi lm on cells starts considerations on the application of this knowledge in pharmaceutical and food industry, where probiotic cells are stressed by handling, storage, and digestion and, therefore, they lose their potential healthy activity.
The aims of the present study were to evaluate the abilities of probiotic strains to form biofi lm on a wide range of carriers, and to compare biofi lm formation on the selected surfaces. The viability of biofi lm at low pH and in the presence of bile was evaluated under model conditions. Thus, harsh conditions of the gastrointestinal tract were induced to assess differences in the survival of planktonic and biofi lm forms of probiotic bacteria.

Materials
The following materials were used in the present work as carriers: poly-(vinyl-pyrrolidoneco-vinyl acetate), nano-cotton, potatoes fi bre, sodium alginate, carrageenan, oat fi bre, calcium phosphate, zinc oxide, silica, and fi nely milled complex food matrices (i.e. fl our, groats of oats, pea, lentils, poppy, and buckwheat). Complex food matrices were bought from local markets and milled in the laboratory. Silica was provided by Pharmaceutical Biotechnology (CZ). All other ingredients used in the cultivation step were of pharmaceutical quality and could be purchased as standard pharmaceutical ingredients for food supplements production or drug production.

Microorganisms and cultivation media
The adherence abilities of probiotic strains to selected carriers were evaluated for three different bacteria: Lactobacillus acidophilus (CCM 4833), Bifi dobacterium longum (CCM 4990), and Bifi dobacterium breve (CCM 3763). All tested bacteria in this study were provided by the Czech Collection of Microorganisms in Brno. Probiotic bacterial cultures were grown in commercial deMan, Rogosa and Sharpe (MRS) broth (Lactobacillus acidophilus) and Bifi dobacterium broth with and without carriers. Both media were delivered by Himedia. The media were supplemented with 1.5% w/v carries. All strains were cultivated at 37 °C for 16-24 h.

Determination of biofi lm formation
The creation of biofi lm was performed by static fermentation. The biofi lm formulation was assessed by Gram-staining and observed under optical microscope (Nikon Eclipse E 400). The biofi lm formation was confi rmed by electron microscope (Mira3 Tescan).

Determination of viability in low pH environment
A solution simulating the stomach environment with a composition of NaCl (2.05 g l -1 ), KH 2 PO 4 (0.60 g l -1 ), CaCl 2 (0.11 g l -1 ), and KCl (0.37 g l -1 ) was prepared according to CORCORAN and co-workers (2005). The pH of the solution was adjusted to values 1, 2, and 3 by 1 M HCl. Fresh biofi lm culture after cultivation, where the high degree of adhesion was confi rmed by electron microscope, and planktonic form culture were incubated in prepared acidic solutions in a shaker at 37 °C for 120 min. At 30, 60, and 120 min, the samples were taken and neutralized by 1 M NaHCO 3 . The number of CFU in the prepared sample was determined according to a modifi ed Miles-Misra method (MILES, 1979;HEDGES, 2002).
Each experiment was performed in three replications. The viability of the biofi lm and planktonic form was expressed as a percentage of viable cells at the sampling time to the viable cell at the start of the experiment.

Determination of probiotic cells viability in bile
The bile tolerance of Lactobacillus acidophilus CCM 4833 strain in planktonic and biofi lm form was evaluated. Samples of probiotic cells' fresh cultures grown in the presence of silica carrier and without carrier were incubated in the 0.3% solution of bile salts (Sigma) for 4 h at 37 °C (RUAS-MADIEDO & DE LOS REYES- GAVILAN, 2005). In the period of 1 hour the samples were taken and the CFU number was determined according to Miles-Misra methods (MILES, 1979;HEDGES, 2002). Each experiment was performed in three replications.

Statistical analysis
Two-sample Student's unpaired t-test for the evaluation has been used. This test can determine whether the two normal distributions with the same variance, coming from two independent samples, have the same mean. The conformity of variances has been verifi ed by F-test. If the resulting P-value>0.05, the variances of both data can be treated as equal, t-test can be used.

Probiotic biofi lm formation on carrier surface
Materials investigated in this work were chosen according to the following criteria: (i) carrier has to be commonly used in the pharmaceutical industry, (ii) according to the legislation, carrier has to be used in food supplements, and (iii) carrier has to be insoluble in boiling water. The screening of the selected materials studied for biofi lm formation for three different probiotic strains is given in Table 1. Surprisingly, the best biofi lm formation was observed in the case of Lactobacillus acidophilus and Bifi dobacterium longum on silica. On the other hand, silica was not an appropriate carrier for Bifi dobacterium breve. Figure 1 shows the rate of adhesion of Lactobacillus acidophilus and Bifi dobacterium longum on the silica carrier. Sodium alginate, potatoes fi bre, carrageenan, oat fi bre, pea, and buckwheat showed partial adhesion of Lactobacillus strain only, which was later on rejected. Thanks to a complex and polymeric structure of studied materials, it was later found that probiotic cells are detained in the complex structure of carriers and biofi lm formation was not confi rmed. Sodium alginate, potatoes fi bre, oat fi bre, and buckwheat show the same properties in the presence of Bifi dobacterium longum; and sodium alginate, oat fi bre, pea, and buckwheat in the presence of Bifi dobacterium breve. Nano-cotton, poly(vinylpyrrolidone-co-vinyl acetate), corn starch, calcium phosphate, kaolin, fl our, groats of oats, lentils, and poppy were not suitable materials for adhesion of Lactobacillus and Bifi dobacterium strains. In Figure 2 biofi lm formation in the presence of silica observed by optical microscopy is introduced. Furthermore, Bifi dobacterium longum did not exhibit the ability to form biofi lm on the surface of carrageenan and fi nely milled pea, and Bifi dobacterium breve did not form biofi lm in the presence of potatoes fi bre and carrageenan.  The data presented in Table 1 show that probiotic strains are able to adhere to some carrier surface and form biofi lm. Furthermore, the present results suggest that the adherence is driven by properties of both selected carrier and probiotic strain. The properties of surfaces, carrier top layer and probiotic cell, are responsible for adhesion, and thus, this ability is ensured by the sum of specifi c (mediated by different pililike structures managed by genetic assembly system (LONGO et al., 2014)) and nonspecifi c (driven by electrostatic and hydrophobic forces, steric hindrance, van der Waals forces, temperature, pH, and hydrodynamic forces (DUNNE, 2002)) interactions between these two surfaces. Although biofi lm formation on different carriers is well known in the case of pathogenic or commensal microorganisms, probiotic strains have not yet been subjected to study for biofi lm formation, especially on free carriers. There are some studies focusing on biofi lm formation on polystyrene microplates (AOUDIA et al., 2016), but silica and other here presented materials have not been tested yet.
The tests performed have not confi rmed adherence of probiotic strain to more than one carrier. Elementary/single carrier particles are proposed as the preferable material suitable for adhesion of probiotic strains. On the other hand, food matrices contain a wide range of components and they may not offer a coherent surface for probiotic adhesion.
The properties of cell surface are connected to the type of microorganism. This study confi rmed that the ability of adhesion and subsequent biofi lm formation on the carrier is dependent on the type of microorganism. Similarities among studied strains and carrier suitable for all studied probiotic strains were not found. Therefore, we expect that strain dependent properties play unmistakable roles also in biofi lm formation. This concept was proposed also by other authors who studied biofi lm formation on polystyrene microplate (AOUDIA et al., 2016).

The viability of probiotic cells at low pH
To apply their effect in the intestine and to confer any health benefi ts to the host, probiotic bacteria should be capable of surviving the passage through the gastrointestinal tract, which presents low pH in the stomach and bile in the small intestine tract (TULUMOGLU et al., 2013). Therefore, high acid level tolerance was evaluated as a critical parameter of a good source of probiotics in the pharmaceutical industry.
In the present study, the viability of probiotic cells in biofi lm and in planktonic state subjected to low pH values was studied. The viability of cells was expressed as CFU units and calculated also as a percentage of viable cells in the sample to viable cells in the control culture, which was prepared under standard cultivation conditions of Lactobacillus acidophilus CCM4833 (Table 2). Biofi lm bacterial cells, as it could be expected, are more resistant than planktonic cells at low pH (pH 1) at all times studied. The drop of viability was observed during the whole experiment, and the highest rate of decrease was observed after 120 min. While planktonic culture showed just 71.9±3.2%, biofi lm sample exhibited 90.5±0.1% of viability. Furthermore, a higher pH value (pH 2) indicates a partially suitable environment for probiotic viability. After 30 min, the free cell culture exhibited some kind of adaptation. At other monitored time points differences between viabilities were found. The greatest difference was measured after 120 min of incubation, where biofi lm culture exhibited 90.7±0.4% and free cells sample achieved 75.5±2.3% of viable cells.
At the last tested pH value (pH 3) bacteria also showed a partial adaptation to the environment. Especially, after 60 min incubation, increased resistance to low pH of free cells was observed. A sample without carrier exhibited 88.4±1.3% of viability and biofi lm sample achieved 94.8±0.4% of cell viability.
Acta Alimentaria 46, 2017 The hypothesis that bacterial cells in biofi lm on the carrier are more stable at low pH was confi rmed. The difference between viability of biofi lm and free cells exceeded 15% at all studied pH values after 120 min cultivation. Furthermore, the viabilities of free cells oscillated around 72% and viabilities of biofi lm cultures exceeded 90%.
Although most sources indicate a high decrease in the viability of free cells upon contact with the environment of the stomach compared to encapsulated or immobilized probiotics (GEBARA et al., 2013;FIJAŁKOWSKI et al., 2016), there is still a lack of comprehensive data in the case of biofi lm formed on free carrier. Some kinds of probiotic biofi lm forms were studied, but these works focused on resistance to stresses of self-forming biofi lm encapsulated in natural polymers. Such biofi lms exhibited a higher resistance to processing and digestion stresses than encapsulated planktonic cells (CHEOW et al., 2014), but the resistance of biofi lm on free carriers was not documented yet.
The obtained results are in agreement with the published data of DOLEYRES and LACROIX (2005). According to these authors, the immobilized population of bifi dobacteria with suffi cient high cell density could induce a quorum-sensing response, leading to the improvement in physical and technological characteristics of lactic acid bacteria, especially adaptation to changing environmental conditions (DOLEYRES & LACROIX, 2005). On the other hand, the viability of both free cell culture and biofi lm is strictly dependent on the pH value, but linear dependence of decrease in cell viability as the function of pH was not confi rmed. Furthermore, the results show that free cell cultures can also exhibit a weak resistance to low pH value.

The viability of probiotic cells in the presence of bile salts
The comparison of resistance of planktonic form and biofi lm form cultures is given in Table  3. The viability of planktonic culture was greatly infl uenced by bile salts, and at study points (60, 120, 180, and 240 min) viability decreased to 78.9±1.8%, 77.3±0.7%, 74.2±1.4%, and 68.2±1.1%, respectively. While the percentage of viable cells of planktonic culture dropped to 68.2±1.1%, the percentage viability of the biofi lm cells remained 98.2±1.0% even after 4-h treatment. Whereas free cell culture exhibited a decreasing linear trend, biofi lm culture subjected to 0.3% bile salts solution showed an almost constant value during the whole studied period. Lactobacillus acidophilus CCM 4833 biofi lm on the appropriate carrier was more resistant to bile than free cell culture at all studied time points. The previously presented results indicate that probiotic cells form biofi lm as pathogenic or commensal organisms. Furthermore, probiotic biofi lms gain also other features, such as bile tolerance, studied here. Although bile tolerance of probiotic biofi lm on free carrier was not previously studied, bile tolerance of probiotic cells incorporated in biofi lm on the free carrier can be expected.
Bile tolerance of probiotic cells greatly varies from strain to strain (RUIZ et al., 2013) and may be dependent on the type of source from which the potential organism has been isolated. It has been reported that probiotic strains of human origin have a higher resistance to bile than of different origins (PANICKER & BEHRE, 2014). Furthermore, some environmental factors, such as pH and temperature, may increase bile tolerance or enhance the survival rate of probiotic cells (LI, 2012).

Conclusions
In this study the biofi lm formation of probiotic microorganisms on the surface of different carriers was tested. The prepared biofi lm (Lactobacillus acidophilus CCM 4833) was subjected to low pH (pH 1, 2, and 3) and 0.3% solution of porcine bile salts. From the wide range of materials, insoluble in hot water and authorized for food supplements in the European Union, only silica was found as possible carrier for biofi lm formation. The biofi lm formation on a carrier provides protection to probiotics against low pH for a certain exposure time. Although the planktonic form of studied strain exhibited a weak adaptation at some points, the formation of biofi lm showed better survival of probiotic cells. Bile tolerance of biofi lm was also confi rmed in comparison to free cell culture.
Silica as a carrier of probiotic cultures represents a new direction to increase stability and ensure higher effi ciency of probiotics administered in food supplements or in food products. * The work was supported by the project "Materials Research Centre -Sustainability and Development" Nr. LO1211 of the Ministry of Education, Youth and Sports of the Czech Republic.