Characterization of antibacterial, antibiofilm and antioxidant properties of  Exopolysaccharides produced by Lactobacillus casei

doi:10.21203/rs.3.rs-4876119/v1

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Characterization of antibacterial, antibiofilm and antioxidant properties of Exopolysaccharides produced by Lactobacillus casei

https://doi.org/10.21203/rs.3.rs-4876119/v1

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Periodontal diseases and tooth decay are the most common oral and dental diseases in the world. Oxidation, habits, diet and bacterial infections are among the effective factors in the mentioned diseases. Streptococcus mutans is the main bacteria that causes tooth decay. New strategies needed in order to prevent these diseases. Bacterial exopolysaccharides (EPSs) could be considered for this purpose. In this study, minimum inhibitory concentration (MIC) and minimum batericidal concentration (MBC) tests were used to determine the antibacterial properties of EPS against S. mutans. Then, using the crystal violet method, the postbiotic effect on the reduction of biofilm formation (adhesion) by S. mutans bacteria was investigated. In addition, the antioxidant properties of these compounds were measured using the DPPH method. The results showed that exopolysaccharide has a significant inhibitory effect against the S. mutans growth. The minimum inhibitory concentration as well as the minimum bactericidal concentration were 18.75 and 37.5 mg/ml, respectively. The concentration of 18.75 mg/ml significantly reduced biofilm adhesion. The results obtained from the evaluation of the antioxidant property showed that EPS indicated good antioxidant properties at much lower concentrations (4 mg/ml).

Exopolysaccharide

Lactobacillus casei

Streptococcus mutans

antibiofilm

The World Health Organization reported that almost half of population suffer from oral diseases [1]. Dental caries and periodontal disease are classified through the most common noncommunicable diseases in the world. Tooth decay starts with the formation of dental plaque, a sticky biofilm of bacteria which constantly produces on the teeth. Over the interaction with sugars and starches derived from dietary intake, the bacteria in the plaque generate acids leading to the tooth enamel demineralization. The repetitive acid attack weaken and destroy the enamel, causing tooth decay [2, 3].

S. mutans make an important role in the formation of dental plaque by making biofilm on dental surfaces. These biofilms are complex communities of microorganisms that adhere together and to the tooth surface, giving a protective environment for the bacteria to resist antimicrobial agents [4, 5]. S. mutans helps bacteria adherence through synthesizing polysaccharides, such as dextran. This matrix is a key component of dental plaque. S. mutans expresses three genetically separate glucosyltransferases (Gtfs) which are responsible for the synthesis of glucans from sucrose [6, 7]. Glucan synthesis is critical for biofilm formation, which provides a protective environment for the bacteria [8]. S. mutans isolates from caries-active individuals synthesize insoluble glucans more than those from caries-free individuals. The regulation of glucan synthesis involves many factors, including environmental conditions, quorum-sensing signaling, and the expression of specific genes [6].

Periodontal disease is a significant health concern that affects not only oral health but also overall health. In periodontal disease, oxidative stress is involved in the pathogenesis of the condition. When bacteria in the mouth infect the gums, they trigger an immune response that leads to the production of free radicals [9]. These free radicals can cause damage to the surrounding tissues, including the gums and bone, leading to inflammation and tissue destruction [10].

In vivo studies have also mentioned the role of probiotics in the cure or prevention of periodontal diseases and tooth decay [11]. Studies showed that their antimicrobial effects is related to the substances they produce. According to the challenges and limitations for using probiotics in food products, metabolites or their non-living components (postbiotics) can be an appropriate substitute for them [12, 13]. Exopolysaccharides(EPS) are biopolymers produced by probiotics, which can be isolated and characterized. EPS produced by lactobacilli have been found to exhibit a range of bioactivities, including antioxidant, antibiofilm, and antibacterial activities [14–16]. These bioactivities are attributed to the unique chemical structure and composition of the EPS, which can be differ depending on the specific lactobacillus strain and fermentation conditions [17]. EPS from lactobacilli have also been found to exhibit other bioactivities, including immunomodulatory, anti-inflammatory, and prebiotic properties [18–20]. These bioactivities can contributed to their potential health benefits and applications in the food and pharmaceutical industries [21].

In this study, we assessed the inhibitory effect of lactobacillus casei-derived EPS (C-EPS) against S. mutans growth and biofilm formation, and also its antioxidant activity.

2.1. Bacterial strains and culture condition

A potential probiotic bacterial strain Lactobacillus casei L431 was used in this study. Pathogenic strain was Gram positive, “Streptococcus mutans”. Lactobacillus strain was cultivated in DeMan-Rogosa-Sharpe MRS broth at 37 ◦C and pathogenic bacteria was grown at 37 ◦C in 95% air/5% CO2 in brain heart infusion broth (BHI).

2.2. Extraction of Exopolysaccharide (EPS) from L. casei l431

The L. casei l431 suspension was centrifuged at 12000g for 20 min. After centrifugation, the solubilized EPSs were present in the supernatant. EPS were precipitated by addition of 2 volumes of chilled ethanol (99.9%) to the supernatant. After precipitation at 4°C for 24 hours, the sample was centrifuged at 8 000g for 20 min and trichloroacetic acid (TCA)14% was added to the white sediment and kept at 4°C for 24 h, in order to remove soluble proteins. The suspension was centrifuged at 8000g for 20 min at 4°C and dialysed against distilled water, to obtain EPS.

2.3. Assessment of antimicrobial activity of EPS

2.3.1. Preparation of bacterial suspension with a concentration of 0.5 McFarland

To prepare the bacterial suspension of S. mutans with a concentration of 0.5 McFarland in this study, turbidity test was used. Some of an overnight culture S. mutans bacteria transferred to a test tube by sampler, which containing fresh culture medium, until the absorption of suspension reaches 0.08–0.13 at the wavelength of 625 nm. In this range of absorption, the bacterial suspension has 0.5 McFarland concentration.

2.3.2. Minimum Inhibitory Concentration (MIC)

The EPS minimum inhibitory concentration (MIC) was measured in sterile 96-well plates with a final volume of 100 µL Mueller–Hinton broth. 50 microliters of Muller Hinton broth was added to the wells of columns 1 to 10 of 96-well plates. Then, 50 microliters of EPS solution with a concentration of 300 mg/ml was added into the wells of the first column of all three rows. Next, 50 microliters of the mixture of culture medium and post-biotic was removed from each well of the first column and transferred to the second column to halve the concentration of the EPS. Again, 50 microliters were removed from each well of the second column Added to the third column. Therefore, 50 microliters of the mixture of culture medium and exopolysaccharide with serial concentrations were provided in Column wells 1 to 10. Then a bacterial suspension of S. mutans with a concentration of 0.5 McFarland was prepared and diluted up to 100 times to reach the concentration of bacterial suspension from 108 CFU/ml to 106 CFU/ml. In the next step, 50 microliters of this suspension were added to each of cell until the concentration of bacteria in each cell reached 5.105 CFU/ml. The two end columns of the 96-cell plates are also considered as negative control wells (containing only culture medium without bacterial cells) and positive control wells (containing culture medium and bacterial suspension) were considered. Finally, the 96-well plate was incubated for 24 hours at 37°C under Microaerophilic condition. Growth of bacteria was determined visually and based on cloudiness the culture medium and observing the mass accumulation of bacteria in the shape of a button at the bottom of the well.

2.3.3. Minimum bactericidal concentration (MBC)

In order to determine MBC, samples was taken from the wells which no bacterial growth was observed in the MIC test and cultured on Mueller Hinton agar culture medium enriched with 5% sheep blood. Then the plates were incubated for 24 hours under microaerophilic conditions. The lowest concentration that reduced growth by at least 99.9% which almost no growth of bacteria was observed was determined as the minimum bactericidal concentration.

2.4. Assessment of Antibiofilm activity of EPS

The biofilm reduction test was performed by crystal violet method. First, BHI culture medium. The broth was enriched with sterilized glucose1%. Then the next steps are to enter the culture medium, different EPS concentrations and bacterial suspension of S. mutans in a flat bottom 96-well plate, exactly according to the mentioned method done in the MIC test (2-3-2). After 24 hours incubation, invert the plate and tapped on it gently to remove the solution inside the wells. Each well was washed twice with 100 microliter of phosphate buffered saline and inverted again. Then 100 microliters of methanol was added to each well of the plate at room temperature and after 20 minutes its content were poured out. 100 microliters of crystal violet solution was added into the wells and after 30 minutes, the wells were washed twice with distilled water. Finally, 150 microliters of acetic acid 33% was added in order to releasing the crystal violet color absorbed by the biofilm inside the wells. The optical density (OD) of the wells was measured at a wavelength of 570 nm using a microplate reader.

2.5. Assessment of Antioxidant activity of EPS (Scavenging activity of DPPH radical)

The ability of EPS samples to scavenge the 2,2-diphenyl-1- (2,4,6-trinitrophenyl) hydrazyl (DPPH) radical was assessed following the methodology outlined by [22]with some modification. Different concentrations (0.5-6 mg/mL) of EPS samples in aqueous solution (1 mL) were combined with an ethanolic solution of the DPPH radical (0.05 mmol/L, 2 mL). This mixture was homogenized and left to incubate in darkness at room temperature for 30 minutes. Subsequently, the absorbance of the solution was measured at 517 nm in three times after centrifugation at 5000 g for 5 minutes. The scavenging capacity of the DPPH radical was defined as following equation:

Scavenging activity (%) = [1- (A_sample – A_blank) / A_control] × 100

A_sample is the absorbance of the solution with DPPH and EPS; A_blank is the absorbance of the EPS ethanolic solution (ethanol instead of DPPH) and; A_control refers to the absorbance of the solution with DPPH and water.

2.6. Statistical analysis

Results were statically evaluated by analysis of variance (ANOVA) and Tukey test was used to determine significant differences by using GraphPad Prism 7.0 software.

3.1. Minimum Inhibitory Concentration (MIC)

Some turbidity occurred after adding the EPS into the culture media of culture medium, which made the visual observation analysis rather inaccurate. The MIC value for postbiotic was 18.75 µg/mL. Based on the cloudiness and button shape accumulation seen at the bottom of the well (Fig. 1).

3.2. Minimum bactericidal concentration (MBC)

After culturing the wells without growth related to the MIC test in Mueller Hinton agar plates enriched with 5% sheep blood, it was found that the concentration of 18.75 mg/ml greatly reduced the growth of S. mutans compared to the control and at the concentration of 37.5 ml showed almost no bacterial growth, so it was selected as MBC.

3.3. Assessment of antibiofilm activity of EPS

In the present study, the effect of exopolysaccharide on adhesion (biofilm formation) was investigated. The released crystal violet color can be seen in each well, has a direct relationship with the amount of biofilm. As illustrated in Fig. 3, which represents the impact of different concentrations of exopolysaccharide on its anti-biofilm efficacy, increasing the concentration enhances the anti-biofilm activity. According to results, the concentration of 37.5 mg/ml(2MIC) and higher concentrations significantly reduced biofilm adhesion (P ≤ 0.01).

3.4. Assessment of antioxidant activity of EPS

The antioxidant activity of EPS evaluated by determining their ability to scavenging 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical. The scavenging capacity of DPPH radicals of the EPS increased by dose-dependent mode. Figure 3 shows that 6 mg/ml concentration of EPS displays the highest antioxidant activity (87%), however it is similar to antioxidant capacity of EPS at a concentration of 4mg/ml (no significant difference, p > 0.05).

The prevention and treatment of oral diseases have significantly enhanced since the 20th century, reducing the occurrence of tooth caries and oral inflammation. Dental caries is initiated by the formation of biofilm by cariogenic bacteria, such as S. mutans. The overuse of antibiotics for treating infections has resulted in the development of antibiotic-resistant pathogens [23]. Many of oral problems caused by imbalance between antioxidants and reactive oxygen species (ROS), which is related to occurrence of dental diseases [9]. Antioxidants are able to inactivate free radicals prior to attack the body's cells and make role in the treatment of oral disease like periodontitis and also prevent the growth and reproduction of oral cancer cells.

LAB have been used for a variety of applications in the pharmaceutical and food industries. Probiotics can release bioactive substances like exopolysaccharide with some health benefits. Using these substances is safer than application of live probiotics [24]. This study was aimed to investigate the antibacterial, antibiofilm and antioxidant effects of EPS produced by L. casei.

The results of our research indicate that L. casei produced EPS, caused 40% reduction in biofilm formation by S. mutans. There are several possible explanations for this result. The antibiofilm activities of LAB species are related to the some metabolites found in the cell-free supernatant like EPS, biosurfactants and bacteriocin components [25]. The mechanisms of EPS antibiofilm action are due to their ability to act like surfactants, their stimulatory effect as signaling molecules and their interactions with other adhesive molecules [26]. It is argued that EPS can easily penetrate the biofilm of S. mutans, as natural metabolite and exert their bioactivities easily. The Gtf genes from certain streptococcus species, such as S. mutans, have important role in development of dental caries. These genes play crucial role in the synthesis of water-insoluble glucans (WIGs), known to be a primary factor in the onset of dental caries. The Gtf genes from S. mutans are particularly significant, as they contribute to the formation of dental plaque and the establishment of S. mutans on tooth surfaces. The Gtf genes in S. mutans are essential for smooth-surface caries formation. S. mutans defective in either or both of the gtfB and gtfC genes needed for insoluble glucan synthesis lead to reduce the levels of smooth-surface carious attack compared to the original organism [27, 28]. Similarly, S. mutans defective in the gtfD gene, that is responsible for encoding the glucosyltransferase-S enzyme involved in the biosynthesis of water-soluble glucans, exhibit a reduction in the formation of smooth-surface carious attack [29]. Targeting Gtf genes has been recognized as a promising therapeutic approach to inhibit cariogenic biofilms. EPS have been identified as agents modulate the expression of gtf genes, affect Gtf enzyme activities, or reduce Gtf secretion, thereby impairing the virulence of S. mutans without affecting the resident oral flora. In addition to antimicrobial activity, EPS from LAB may also have direct antibiofilm activity. The results in this study indicate that EPS released by L. casei, are effective on S. mutans growth. A possible explanation for EPS antibacterial activity might be related to several mechanisms as the cell wall and cytoplasmic membrane disruption, cell division damage, and DNA decomposition [16]. It is suggested that the EPS negative charge related to its sulphate group could better interact with the Gram-positive bacteria [19, 21, 30]. In other words, the positive charge of Gram-positive bacteria cell wall is higher. One of the results that emerges from our investigation is C-EPS antioxidant activity even in concentrations lower than antibiofilm and antibacterial efficient concentration [21]. This bioactivity could be related to its monosaccharide composition and the extent of sulphate group [31].

The EPS compound produced by Lactobacillus reuteri BM53-1 has been reported to inhibit the biofilm formation of S. mutans through decreasing the amount of GtfD enzymes, which are necessary to make insoluble glucans sticky; without inhibiting this pathogen growth. In a similar case in, has been assessed antibiofilm effect of released (r-EPS) and cell-bound exopolysaccharides(b-EPS) extracted by Apilactobacillus kunkeei K1.10 and Latilactobacillus curvatus Kar.9b. Type of EPS is known to be associated with their effect on biofilm[32]. It has been reported that r-EPS showed high reduction in S. mutans biofilm formation by preventing the aggregation between cells or initial adhesion to surfaces. On the other hand b-EPS improved biofilm formation. No evidence of these EPSs antimicrobial activity was detected. According to available data, we can infer that postbiotic from L. casei can inhibit gram positive and negative bacteria growth like S. enterica and L. monocytogenes [33]. L. casei supernatant also has been identified as antioxidant agent according to its high phenolic content. Similar finding also reported about exopolysaccharide bioactivities by Nehal et al. (2019). They reported that exopolysaccharide produced by Lactobacillus pentosus 14FE, Lactobacillus plantarum 47FE have ability to interact with DPPH radicals by the way of producing H ions. Moreover, they showed antimicrobial effect against gram positive bacteria [34].In our study the antimicrobial assays revealed that the EPS from L. casei effectively inhibited the growth and biofilm formation of the cariogenic bacterium S. mutans, a key etiological agent of dental caries.

It seems that the use of postbiotics instead of probiotics has a similar effect and can be effective in the prevention and treatment of many oral and dental diseases caused by pathogens such as S. mutans and the results of the this study also confirms this. However, this study is only an in-vitro study which the antibacterial, anti-biofilm, and antioxidant effects of postbiotics (exopolysaccharide) were investigated and confirmed. In fact, in-vivo studies are needed to prove the preventive and therapeutic effects of them. On the other hand, since probiotics are acid-producing like decay-causing bacteria, it seems that exopolysaccharides have solved this problem.

Conflict of Interest

The authors have no conflicts of interest to declare related to this work.

Author Contribution

Bahareh pouragha did the tests about exopolsaccharide and wrote the manuscript. Dr Rasuly checked manuscript from editorial aspect. Dr Homayouni is supervisor of thesis which this article is derivated from it.

Acknowledgments

This article is derived from a master's thesis in the field of food science and technology, with the ethical code (IR.TBZMED.REC.1401.874). The authors express their gratitude to Tabriz University of Medical Sciences for funding the expenses associated with this thesis project.

Dye BA (2017) The global burden of oral disease: research and public health significance. J Dent Res 96(4):361–363
Chhaliyil P et al (2022) Impact of refined and unrefined sugar and starch on the microbiota in dental biofilm. J Int Soc Prev Community Dentistry 12(5):554–563
Baker JL, Edlund A (2019) Exploiting the oral microbiome to prevent tooth decay: has evolution already provided the best tools? Front Microbiol 9:437902
Matsumoto-Nakano M (2018) Role of Streptococcus mutans surface proteins for biofilm formation. Japanese Dent Sci Rev 54(1):22–29
Zhu B et al (2018) A novel regulator modulates glucan production, cell aggregation and biofilm formation in Streptococcus sanguinis SK36. Front Microbiol 9:1154
Zheng T et al (2023) Regulatory mechanisms of exopolysaccharide synthesis and biofilm formation in Streptococcus mutans. J Oral Microbiol 15(1):2225257
Senpuku H et al (2019) Effects of complex DNA and MVs with GTF extracted from Streptococcus mutans on the oral biofilm. Molecules 24(17):3131
Yang Y et al (2019) Regulation of water-soluble glucan synthesis by the Streptococcus mutans dexA gene effects biofilm aggregation and cariogenic pathogenicity. Mol oral Microbiol 34(2):51–63
Sczepanik FSC et al (2020) Periodontitis is an inflammatory disease of oxidative stress: We should treat it that way. Periodontol 2000 84(1):45–68
Adwas AA et al (2019) Oxidative stress and antioxidant mechanisms in human body. J Appl Biotechnol Bioeng 6(1):43–47
Di Pierro F (2021) Probiotics to prevent tooth decay. International Journal on Nutraceuticals, Functional Foods and Novel Foods
de Brandi A (2024) Effect of lacteal products containing probiotic in the progression of tooth decay around orthodontic brackets. Int J interdisciplinary dentistry 17(1):15–18
Homayouni Rad A, Pourjafar H, Mirzakhani E (2023) A comprehensive review of the application of probiotics and postbiotics in oral health. Front Cell Infect Microbiol 13:1120995
Song Y et al (2020) Antibiofilm activity of Lactobacillus plantarum 12 exopolysaccharides against Shigella flexneri. Appl Environ Microbiol 86(15):e00694–e00620
Andrew M, Jayaraman G (2020) Structural features of microbial exopolysaccharides in relation to their antioxidant activity. Carbohydr Res 487:107881
Abdalla AK et al (2021) Exopolysaccharides as antimicrobial agents: Mechanism and spectrum of activity. Front Microbiol 12:664395
Zhou Y, Cui Y, Qu X (2019) Exopolysaccharides of lactic acid bacteria: Structure, bioactivity and associations: A review. Carbohydr Polym 207:317–332
Xu Y et al (2019) Purification, characterization and bioactivity of exopolysaccharides produced by Lactobacillus plantarum KX041. Int J Biol Macromol 128:480–492
Oerlemans MM et al (2021) Benefits of bacteria-derived exopolysaccharides on gastrointestinal microbiota, immunity and health. J Funct Foods 76:104289
Angelin J, Kavitha M (2020) Exopolysaccharides from probiotic bacteria and their health potential. Int J Biol Macromol 162:853–865
Daba GM, Elnahas MO, Elkhateeb WA (2021) Contributions of exopolysaccharides from lactic acid bacteria as biotechnological tools in food, pharmaceutical, and medical applications. Int J Biol Macromol 173:79–89
Kumar R et al (2020) Purification, partial structural characterization and health benefits of exopolysaccharides from potential probiotic Pediococcus acidilactici NCDC 252. Process Biochem 99:79–86
Haque M, Sartelli M, Haque SZ (2019) Dental infection and resistance—global health consequences. Dentistry J 7(1):22
Van Holm W et al (2023) Probiotics for oral health: do they deliver what they promise? Front Microbiol 14:1219692
Moradi M, Molaei R, Guimarães JT (2021) A review on preparation and chemical analysis of postbiotics from lactic acid bacteria. Enzym Microb Technol 143:109722
Xu X et al (2020) Antibacterial potential of a novel Lactobacillus casei strain isolated from Chinese northeast sauerkraut and the antibiofilm activity of its exopolysaccharides. Food Funct 11(5):4697–4706
Hoshino T, Fujiwara T (2022) The findings of glucosyltransferase enzymes derived from oral streptococci. Japanese Dent Sci Rev 58:328–335
Rezaei T et al (2023) Factors associated with Streptococcus mutans pathogenicity in the oral cavity. Biointerface Res Appl Chem 13(4):368
Sakagami H et al (2018) Recent progress of basic studies of natural products and their dental application. Medicines 6(1):4
Saif FAA, Sakr EA (2020) Characterization and bioactivities of exopolysaccharide produced from probiotic Lactobacillus plantarum 47FE and Lactobacillus pentosus 68FE. Bioactive Carbohydr Diet Fibre 24:100231
Silva LA, Lopes Neto JHP, Cardarelli HR (2019) Exopolysaccharides produced by Lactobacillus plantarum: technological properties, biological activity, and potential application in the food industry. Ann Microbiol 69:321–328
Karaca B (2023) Exopolysaccharides of lactic acid bacteria isolated from honeybee gut and effects of their antibiofilm activity against Streptococcus mutans, vol 95. Anais da Academia Brasileira de Ciencias, p e20220979. 10.1590/0001-3765202320220979
Noori SMA et al (2022) Antimicrobial and Antioxidant Properties of Natural Postbiotics Derived from Five Lactic Acid Bacteria. Jundishapur J Nat Pharm Prod 18(1):e130785
Nehal F et al (2019) Characterization, high production and antimicrobial activity of exopolysaccharides from Lactococcus lactis F-mou. Microb Pathog 132:10–19

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Characterization of antibacterial, antibiofilm and antioxidant properties of Exopolysaccharides produced by Lactobacillus casei

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Abstract

Figures

1. Inroduction

2. Materials and Methods

2.1. Bacterial strains and culture condition

2.2. Extraction of Exopolysaccharide (EPS) from L. casei l431

2.3. Assessment of antimicrobial activity of EPS

2.3.1. Preparation of bacterial suspension with a concentration of 0.5 McFarland

2.3.2. Minimum Inhibitory Concentration (MIC)

2.3.3. Minimum bactericidal concentration (MBC)

2.4. Assessment of Antibiofilm activity of EPS

2.5. Assessment of Antioxidant activity of EPS (Scavenging activity of DPPH radical)

2.6. Statistical analysis

3. Results

3.1. Minimum Inhibitory Concentration (MIC)

3.2. Minimum bactericidal concentration (MBC)

3.3. Assessment of antibiofilm activity of EPS

3.4. Assessment of antioxidant activity of EPS

4. Discussion

5. Conclusion

Declarations

Conflict of Interest

Author Contribution

Acknowledgments

References

Additional Declarations

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