Emerging Synbiotics Consisting of Catechin and Probiotic Bacteria: Exploring Aggregation, Adhesion, Antioxidant, and Anticancer Effects
Sena Davran Bulut, Buse Nur Derebası, Sevilay Günay, Yavuz Erden, Hasan Ufuk Celebioglu, Mustafa Ümit Ünal
TL;DR
This study explores how catechin, a natural compound, interacts with probiotic bacteria to enhance their health benefits, including antioxidant and anticancer effects.
Contribution
The study introduces a novel synbiotic combination of catechin and probiotics and demonstrates its enhanced physiological and anticancer properties.
Findings
Catechin improved bacterial growth, auto-aggregation, and adhesion of probiotic strains LA-5 and LGG.
The synbiotic combination showed increased antioxidant activity and induced higher cell death in colon cancer cells.
Catechin enhanced probiotic properties without affecting genotoxicity of LA-5.
Abstract
Probiotic bacteria are microorganisms that confer health benefits on the host, when administered in adequate amounts. Catechin, naturally found in nature and human diet, has strong antioxidant, antibacterial, and anticancer potentials. Phenolic compounds and probiotic bacteria are found together in gastrointestinal tract (GIT), and their potential synbiotic interactions are not fully understood. The present study aims to investigate the probiotic properties effects of catechin on (bacterial growth, auto-aggregation, co-aggregation) on well-known probiotic bacteria Lactobacillus acidophilus LA-5 (LA-5) and Lacticaseibacillus rhamnosus GG (LGG), and in vitro cytotoxic effects of the synbiotic combination of catechin and probiotic bacteria on colon cancer cells Caco-2. Results show that catechin contributed to bacterial growth of LA-5, auto-aggregation of LA-5, co-aggregation of LA-5 with…
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Taxonomy
TopicsTea Polyphenols and Effects · Microbial Metabolites in Food Biotechnology · Gut microbiota and health
Introduction
The human microbiome, primarily composed of commensal bacteria, is distributed throughout different areas of the body, including the gut and airways. These bacteria play a crucial role in regulating immunophysiological functions such as metabolism, ontogeny, and defense against pathogens. The colon, in particular, hosts a substantial quantity of commensal bacteria, with an estimated several hundred bacterial species residing in the intestines [1]. Probiotic is defined as “live microorganism that, when administered in adequate amounts, confer a health benefit on the host.” by Joint FAO/WHO [2]. Certain criteria need to be met for bacteria to qualify as probiotics: Probiotic bacteria must (1) be orally safe, meaning they should not produce or metabolize substances that have adverse effects on human health [3] (2) withstand the presence of pepsin and the acidic pH of the stomach [4] (3) be able to survive in the gastrointestinal tract (GIT) by tolerating bile salts, enabling them to adhere to the intestinal mucosa [5] (4) adhere to the intestinal mucosa to establish colonization and exert beneficial effects [6]. Bifidobacteria and Lactobacilli are among the most well-known and extensively studied genera of probiotic bacteria. Within the Lactobacilli genus, various species and strains, such as Lacticaseibacillus rhamnosus, Lactobacillus acidophilus, Lactobacillus casei, and Lactobacillus helveticus, have been thoroughly examined for their positive effects on human health [7].
Phenolic compounds are widely distributed in the plant kingdom, serving as secondary plant metabolites with more than 8000 known structures [8]. Constituting an essential component of the human diet, these compounds are present in all plant organs [9]. Phenolics play a crucial role in plant defense against factors such as ultraviolet (UV) radiation, pathogens, and other predators. Additionally, they contribute to the bitterness of fruits and the vibrant coloring of many fruits and vegetables [10]. Research suggests that the consumption of phenolics significantly impacts the prevention and treatment of various diseases, including cardiovascular diseases, inflammatory cardiovascular risks, hypertension, and diabetes [11]. Catechin, notable phenolic compound abundant in green tea, exhibits numerous beneficial features for human health, such as antioxidant, anticancer, and anti-inflammatory activities [12]. Beyond its positive effects, recent studies suggest its potential therapeutic applications in preventing and treating various diseases, including cardiovascular diseases, digestive system dysfunctions, microbial diseases, oxidative damage, and immune system disorders [13].
GIT serves as the initial site where probiotics engage with dietary substances [14]. The interaction between prebiotics and probiotics is known as synbiotics, denoting the combination of these compounds. Synbiotics enhance the viability of probiotics and exert more beneficial effects on human health. This synergistic combination has the potential to create a more favorable microbial environment in the colon [15]. Recent studies have shown promising results, indicating that the synbiotic relationship between prebiotics and probiotics can reduce the risks of certain diseases and help manage the symptoms of intestinal disorders [16]. Thus, the primary objective of the present study is to elucidate the impact of catechin on probiotic bacteria (LGG and LA-5), focusing on probiotic properties (growth, auto-aggregation, co-aggregation, adhesion, and antioxidant properties). Additionally, the present study aims to investigate the in vitro cytotoxic, genotoxic, and metabolic effects resulting from the potential synbiotic interaction of these components on colon cancer cells, using Caco-2 cell lines.
Material and Methods
Growth of Probiotic Bacteria in the Presence of Catechin
Lactobacillus acidophilus LA-5 (LA-5) and Lacticaseibacillus rhamnosus GG (LGG), which were kind gifts of Chr. Hansen, Turkey, were grown in semisynthetic medium for lactic acid bacteria (LABSEM) without shaking at 37 °C [17]. The bacterial cultures were divided into groups and treated with varying concentrations of catechin (0–1000 µg/mL), one group as control without catechin (LABSEM only). The growth of LA-5 and LGG treated with catechin and without catechin was monitored every 4 h by measuring their absorbance at a wavelength of 600 nm.
Auto-aggregation and Co-aggregation Assays
For the auto-aggregation assay, LA-5 and LGG were divided into groups and treated with varying concentrations of catechin (0–1000 µg/mL), one group as control without catechin (LABSEM only). LA-5 and LGG treated with catechin and without catechin were grown in LABSEM without shaking at 37 °C. Probiotics were collected in the late logarithmic phase and centrifuged at 3200 × g at 15 min. After centrifugation, pellets were washed with phosphate-buffered saline (PBS) and re-suspended in PBS to OD600 0.5 [18]. Auto-aggregation was determined by adding 4 mL of bacterial suspensions to the test tubes after vortex for 10 s. After incubation, 100 μL from the upper portion of the suspensions was taken, added to the tube containing 900 μL of PBS, and the absorbance was measured at 600 nm. The percentage of auto-aggregation was calculated using the following formula:
\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\%\;\mathrm{Auto}-\mathrm{aggregation}=(1-\frac{A_t}{A_0})\times100$$\end{document}where A_t_ is the absorbance measured after incubation (every hour for 4 h), and A_0_ is the absorbance measured before incubation [19].
Escherichia coli and Staphylococcus aureus were used to determine co-aggregation of LA-5 and LGG. LA-5 and LGG treated with varying concentrations of catechin (0–1000 µg/mL), and without catechin were grown in LABSEM without shaking at 37 °C. Probiotics were collected in the late logarithmic phase and centrifuged at 3200 × g at 15 min. After centrifugation, pellets were washed with phosphate-buffered saline (PBS) and re-suspended in PBS to OD600 0.5. E. coli and S. aureus were grown in LB Broth, collected in the late logarithmic phase, and centrifuged at 3200 × g at 15 min. After centrifugation, pellets (E. coli and S. aureus) were washed with phosphate-buffered saline (PBS) and re-suspended in PBS to OD600 0.5. 2 mL of LA-5 and LGG suspension was mixed with 2 mL of either E. coli or S. aureus suspension. In every hour, 100 µL of upper part of the suspension was taken and mixed with 900 µL of PBS and the absorbance was read using a spectrophotometer (600 nm). The percentage of co-aggregation is calculated according to the formula given above for the auto-aggregation [19].
Determination of Probiotic Adhesion
Adhesion was assayed in 96-well microplates according to the technique described by Leccese Terraf et. al (2014) with some modifications [20]. Plates were covered with 200 µL of 0.1 mg/mL mucin in PBS and incubated at 4 °C overnight, followed by washing with PBS. For the adhesion assay, LA-5 and LGG treated with varying concentrations of catechin (0–1000 µg/mL), and without catechin were grown in LABSEM without shaking at 37 °C. Probiotics were collected in the late logarithmic phase and centrifuged at 3200 × g at 15 min. After centrifugation, pellets were washed with phosphate-buffered saline (PBS) and re-suspended in PBS to OD_600_ 1.5. 200 µL of LA-5 and LGG suspension were added to each well and plates were incubated for 2 h at 37 °C. After removing nonadhered cells by washing with 200 µL of PBS, adhered cells were detected by staining with crystal violet. Crystal violet was added to each well and incubated for 30 min. Then, crystal violet was removed and 200 µL of iodine solution was added to each well. After 2–3 min, the plate was washed with PBS twice and the stain was released with 30% acetic acid (200 µL per well). Absorbance was measured at 570 nm in a plate reader [20]. Relative absorbance percentages were determined with resect to the initial OD of the bacterial suspensions.
Determination of Antioxidant Activity
Determination of the antioxidant activity of catechin and probiotic bacteria together was performed by investigating by measuring DPPH scavenging effect [21]. For this, 25 mg/L DPPH as a free radical was prepared in methanol. LA-5 and LGG treated with varying concentrations of catechin (0–1000 µg/mL), and without catechin were grown in LABSEM without shaking at 37 °C. Probiotics were collected in the late logarithmic phase and centrifuged at 3200 × g at 15 min. One hundred microliters of cell free supernatants of probiotics and 100 µL of DPPH solution were added in microplate with a total volume of 200 µL. The mixture was incubated at room temperature for 30 min in the dark, and then the absorbance was read at 517 nm in a microplate reader. Decrease in absorbance, thus remaining amount of DPPH is determined as free radical scavenging activity. Results were calculated according to the formula:
\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\%=\frac{\text{Control ABS}-\text{Sample ABS}}{\text{Control ABS}}\times 100$$\end{document}In Vitro Cytotoxicity Assay of Probiotic Bacteria and Catechin for Caco-2 Cells
Cell viabilities of Caco-2, and thus the cytotoxic effects of LGG grown in the presence of catechin were determined by the Thiazolyl Blue Te-trazolium Bromide (MTT) assay [22]. For Caco-2 cells, the feeding medium high glucose Dulbecco’s Modified Eagle Medium (DMEM) was used. 10% FBS, 1% penicillin streptomycin and 1% non-essential amino acid solutions were added in crude medium. In cytotoxicity assay, cell-free supernatant (CFS) of probiotic bacteria grown either with or without catechin were used. CFSs were prepared by centrifugation (3200 × g, 10 min), filtration (0.22-µm syringe filters). CFSs were diluted with DMEM medium as 1/2, 1/5, or 1/10 (v/v). Catechin in LABSEM (without bacteria) was used to determine the effects of only catechin on the cells. CFS of probiotic bacteria grown in LABSEM without cinnamic acid were used as probiotic control. Then, cancer cells seeded in 96-well plates with a density of 15 × 10^3^ cells per well (final volume 200 µL) were treated with CFSs of bacterial culture for 24 h [23]. After treatments, cells were incubated in 50 µL 0.5 mg/mL MTT. The optical densities of the cells in the plates were measured in the microplate reader at 570 nm [22]. Cell viability was calculated as percentage of absorbance measured for treated groups relative to absorbance of the control group.
In Vitro Genotoxicity Assay (Comet Analysis) of Probiotic Bacteria and Catechin for Caco-2 Cells
“Comet Analysis”, also known as single-cell gel electrophoresis, is a widely used method to determine DNA damage (Genotoxicity) in mammals [24]. Approximately 5 × 10^5^ cells were grown in 6-well plates and treated with five-time diluted CFS medium (final volume 3 mL; this dilution was determined after MTT analyses) for 24 h. Collected cells were mixed with 1% low melting agarose and transferred onto slides coated with 1% normal melting agarose. The preparations were carried out in horizontal electrophoresis after the lysis process. After neutralization, the slides were stained with ethidium bromide and viewed under a fluorescence micro-scope (Zeiss Axioscope, Germany). Tail DNA (%), tail length (TL), tail moment (TM), and tail intensity (TI) parameters of 250 cells randomly selected from each group were evaluated and analyzed in TriTek Comet Score Images using the TriTek Comet Software [25].
Statistical Analyses
Statistical analyses were performed using GraphPad Prism 8 package program. One-way ANOVA was used to determine the differences between the groups and Tukey’s test was used for multiple comparisons. Quantitative data were expressed as mean with standard deviation (mean ± SD) of at least three different biological and at least three technical replicates and p < 0.05 was considered as statistically significant for each experimental analysis.
Results
Effects of Catechin on Growth Kinetics of Probiotic Bacteria
Effect of catechin concentration on the growth the LA-5 and LGG was investigated in the range of 0–1000 µg/mL (Fig. 1). The results showed catechin concentrations studied did not show inhibitory effects against LA-5 and LGG. On the contrary, catechin positively modulated the growth of LA-5. There are contradictory results in the literature reporting that catechin had antibacterial properties against Gram-negative and Gram-positive bacteria [26]. Results indicated that catechin can selectivity modulate probiotic bacteria.Fig. 1. Growth kinetics of A Lactobacillus acidophilus LA-5 and B Lacticaseibacillus rhamnosus GG grown in the presence of 0–1000 µg/mL catechin (Control LA-5 and Control LGG: probiotics grown without catechin)
Effects of Catechin on Aggregation Properties of Probiotic Bacteria
In the present study, the effect of catechin concentrations on the auto-aggregation ability of LA-5 and LGG was investigated for 4 h (Fig. 2). A total of 1000 µg/mL of catechin significantly (p < 0.05) contributed to the auto-aggregation ability of LA-5, however catechin concentrations did not changed the auto-aggregation potential of LGG statistically (p < 0.05).Fig. 2. Effects of catechin on auto-aggregation of A Lactobacillus acidophilus LA-5 and B Lacticaseibacillus rhamnosus GG for 4 h. Lower case letters indicate that there is statistically significant difference (p < 0.05) according to one-way ANOVA and quantitative data were expressed as mean with standard deviation (mean ± SD) of at least three different biological replicates (Control LA-5 and Control LGG: probiotics grown without catechin)
Furthermore, effects of catechin on the co-aggregation ability of LA-5 and LGG with E. coli and S. aureus were also evaluated for 4 h (Figs. 3 and 4). All the catechin concentrations studied did not exert a statistically significant impact (p < 0.05) on the co-aggregation ability of LA-5 with E. coli, whereas catechin, especially 125 µg/mL concentration, significantly enhanced the co-aggregation ability of LA-5 with S. aureus. None of the catechin had positive impact statistically (p < 0.05) on the co-aggregation ability of LGG with E. coli and S. aureus.Fig. 3. Effects of catechin on co-aggregation of Lactobacillus acidophilus LA-5 with A E. coli and B S. aureus for 4 h. Lower case letters indicate that there is statistically significant difference (p < 0.05) according to one-way ANOVA and quantitative data were expressed as mean with standard deviation (mean ± SD) of at least three different biological replicates (Control LA-5: probiotics grown without catechin)Fig. 4. Effects of catechin on co-aggregation of Lacticaseibacillus rhamnosus GG with A E. coli and B S. aureus for 4 h. Lower case letters indicate that there is statistically significant difference (p < 0.05) according to one-way ANOVA and quantitative data were expressed as mean with standard deviation (mean ± SD) of at least three different biological replicates (Control LGG: probiotics grown without catechin)
Effects of Catechin on Adhesion of Probiotic Bacteria to Mucin
Figure 5 shows the effects of catechin concentration on the adhesion of LA-5 and LGG. Results indicated that LA-5 grown in the presence of 1000 µg/mL catechin and LGG grown in the presence of 500 µg/mL catechin had statistically significant (p < 0.05) higher adhesion potential to mucin as compared to control groups. Thus, catechin enhanced the adhesion property of LA-5 and LGG.Fig. 5. Effects of catechin on adhesion of A Lactobacillus acidophilus LA-5 and B Lacticaseibacillus rhamnosus GG. Lower case letters indicate that there is statistically significant difference (p < 0.05) according to one-way ANOVA and quantitative data were expressed as mean with standard deviation (mean ± SD) of at least three different biological replicates (Control LA-5 and Control LGG: probiotics grown without catechin)
Effects of Catechin on Antioxidant Property of Probiotic Bacteria
Figure 6 shows the effects of varying catechin concentrations on antioxidant potential of LA-5 and LGG. No difference was found between antioxidant capacity of LA-5 and LA-5 grown in the presence of catechin although catechin had a high antioxidant potential against DPPH. In addition, 1000 µg/mL of catechin reduced the antioxidant capacity of LA-5. On the other hand, LGG grown with catechin had higher antioxidant capacities when compared to LGG control group.Fig. 6. Effects of catechin on antioxidant of A Lactobacillus acidophilus LA-5 and B Lacticaseibacillus rhamnosus GG. Lower case letters indicate that there is statistically significant difference (p < 0.05) according to one-way ANOVA and quantitative data were expressed as mean with standard deviation (mean ± SD) of at least three different biological replicates (Control LA-5 and Control LGG: probiotics grown without catechin)
Effects of Catechin on Anticancer Effects of Probiotic Bacteria
The in vitro cytotoxic effects of synbiotic combination of catechin and probiotic bacteria L. acidophilus LA-5 and L. rhamnosus GG on colon cancer cells (Caco-2) were investigated using the MTT Assay at different dilutions (Figs. 7 and 8). 1/2 and 1/3 dilution of catechin significantly reduced cell viability, whereas cell viability increased at 1/5 dilution.Fig. 7. Cytotoxic effects of cell-free extract of Lactobacillus acidophilus LA-5 and catechin on Caco-2 cells. The cell-free extracts were diluted as A 1/2, B 1/3, and C 1/5. Lower case letters indicate that there is statistically significant difference (p < 0.05) according to one-way ANOVA and quantitative data were expressed as mean with standard deviation (mean ± SD) of at least three different biological replicates (Control LA-5: probiotics grown without catechin)Fig. 8. Cytotoxic effects of cell-free extract of Lacticaseibacillus rhamnosus GG and catechin on Caco-2 cells. The cell-free extracts were diluted as A 1/2, B 1/3, and C 1/5. Lower case letters indicate that there is statistically significant difference (p < 0.05) according to one-way ANOVA and quantitative data were expressed as mean with standard deviation (mean ± SD) of at least three different biological replicates (Control LGG: probiotics grown without catechin)
Although all dilutions of LA-5 had no cytotoxic effect on Caco-2 cells, LA-5 grown with added catechin reduced the viability of the cells. Although 1/3 diluted LA-5 and 1000 µg/mL catechin did not affect the cell viability of Caco-2 cells, LA-5 grown with 1000 µg/mL catechin had a statistically (p < 0.05) significant cytotoxic effect on Caco-2 cells (Fig. 7). Thus, catechin contributed to the cytotoxicity of LA-5 against Caco-2 cells and synbiotic interaction of catechin and LA-5 may have the potential to be a cytotoxic agent against Caco-2 cancer cells.
1/2 and 1/3 dilutions of LGG and catechin had a cytotoxic effect as they reduced the viability of Caco-2 cells, but LGG reduced cell viability more than all catechin concentrations studied. Otherwise, there is no difference in cell viability between LGG grown with catechin and LGG (Fig. 8). The results indicated that catechin did not enhance the cytotoxic effects of LGG.
Additionally, genotoxic effects of catechin and probiotic bacteria grown with added catechin on DNA damage levels in the Caco-2 cells were evaluated by single cell gel electrophoresis (Comet) analyses. Administration of LA-5, LA-5 grown with catechin, and catechin did not changed the TL, TI, and TM parameters levels of Caco-2 cells compared with control in Comet analyses (Table 1). Parameters are similar in LA-5 and LA-5 grown with catechin. Thus, catechin did not improve the genotoxicity of LA-5. Table 1. Comet assay results of Caco-2 cells after LA-5, catechin and synbiotic of LA-5 and catechin treatmentCaco-2 (24 h)Medium controlLA-5LA-5 + 250 µg/mL catechin250 µg/mL catechinTail length (px)52 (1–161)69 (1–209)63 (1–169)46 (1–136)Tail intensity328,462 (11,113–2,458,730)190,248 (10,552–1,674,397)168,945.78 (11,880–812,922)152,358.3 (11,344–1,086,216)Tail moment30.9 (0.1–135.9)36.4 (0.10–170.9)38.75 (0.52–113.3)22.7 (0.11–109.96)
LGG increased the TL, TI, and TM parameter levels in the Caco-2 cells as compared to the control group significantly (p < 0.05). These results indicate that LGG has a cytotoxic effect but also a genotoxic effect on Caco-2 cells. However, there is no difference in DNA damage parameter levels between LGG and catechin-grown LGG. Since LGG already had a highly genotoxic effect against Caco-2 cells, catechin did not affect the genotoxicity of LGG (Table 2). Table 2. Comet assay results of Caco-2 cells after LGG, catechin and synbiotic of LGG and catechin treatmentCaco-2 (24 h)Medium controlLGGLGG + 250 µg/mL catechin250 µg/mL catechinTail length (px)52 (1–161)92 (3–244)*47 (1–172)46 (1–136)Tail intensity328,462 (11,113–2,458,730)264,917.5 (12,330–2,032,618)*141,121 (838,151–11,892)152,358.3 (11,344–1,086,216)Tail moment30.9 (0.11–135.9)71.2 (0.45–191.2)27.9 (0.37–103.84)22.7 (0.11–109.96)
Discussion
Catechin is a natural phenolic compound that has GRAS (generally recognized as safe) status [27] and has emerged as a potent alternative in the treatment of both gram-positive and gram-negative bacterial strains, including antibiotic-resistant ones [28]. There are two leading hypotheses explaining the antibacterial mechanism of catechins. First one is that catechins enter the lipid bilayer, leading to lateral expansion and membrane disruption. Another hypothesis suggests that catechins are oxidized in the cell culture medium and produce hydrogen peroxide, which causes DNA damage and protein oxidation [29]. A previous research indicated that catechin disrupt the bacterial cell wall by binding to the peptidoglycan layer and damaging cross-linking peptides [30]. Another study done by Jeon et al. showed antibacterial potential of epigallocatechin gallate (EGCG) and green tea extracts (GTE) against Gram-positive bacteria, Pseudomonas aeruginosa and Escherichia coli [31]. Furthermore, they noted that EGCG and GTE may be alternative antimicrobial agents against infections resistant to conventional antibiotic therapy [31]. Bai et al. stated that EGCG showed antimicrobial activity against Gram-negative bacterium Streptococcus mutans by inhibiting the growth and biofilm formation [32]. In the present study, catechin did not affect the microbial growth of L. acidophilus LA-5 (LA-5) and L. rhamnosus GG (LGG), which are Gram-positive bacteria.
Probiotics found in the healthy microbiota in the intestines prevent the growth of harmful or unwanted microorganisms by adhering to the host’s epithelial surfaces and establishing colonies in the gastrointestinal tract [33]. Therefore, probiotic bacteria need to reach sufficient biomass as aggregates to exert their benefit the host. Auto-aggregation is the property of cells to self-adhere belonging the same species by adhering to each other and colonize the environment [34].
Probiotics, thanks to their auto-aggregation properties, form a barrier on intestinal epithelial cells and prevent pathogens from adhering. Auto-aggregation is the first step in the adhesion and colonization of probiotic bacteria in the GIT [35]. Studies demonstrated that L. acidophilus ADH and L. rhamnosus GG have high auto-aggregation property around 35.0%, and around 50.0%, respectively [36, 37]. Our results showed that LA-5 and LGG had high auto-aggregation rate, and 1000 µg/mL catechin increased the auto-aggregation property of LA-5 significantly (p < 0.05). However, not all of the catechin concentration changed the auto-aggregation property of LGG. Catechin’s improvement in the auto-aggregation property of LA-5 may be contributed to a further increase in its biomass in the gastrointestinal tract, thus enhancing its adhesion properties and exerting more benefits to host [38].
Co-aggregation is defined as the highly specific recognition and adhesion of different types of microorganisms. It is one of the crucial properties for probiotic bacteria to prevent attachment and colonization of pathogens to the mucosal layer [39]. Probiotics can inhibit the adhesion of the pathogen to mucosal epithelial cells through different mechanisms such as exclusion, competition, or displacement. In the exclusion, probiotics can colonize on the epithelial cells and do not leave a free surface for pathogens to adhere to their binding sites. During migration process, probiotics have potential mechanism to remove pathogens from epithelial cells. In the other mechanism, probiotics and pathogens compete for nutrients in the environment. Thus, a microbiota composition, especially probiotic-enriched, plays a crucial role in maintaining intestinal balance and host health [40].
Studies indicated that co-aggregation ability of LA-5 with E. coli and S. aureus has around 35–40% rate at 24 h and co-aggregation rate of LGG with S. aureus was around 40% at 2 h [36, 41]. The results of the present study are consistent with the literature and show the high co-aggregation ability of probiotics. Furthermore, the results showed that catechin enhanced the co-aggregation of LA-5 with S. aureus. Improving the co-aggregation property of LA-5 may provide more advantages in forming a barrier against S. aureus and thus eliminating S. aureus from GIT. Thus, LA-5 grown with catechin may contribute more to gut and human health [42].
The GIT, which is the habitat of the intestinal microbiota, is covered with mucus and is a place of attachment and accommodation for both probiotics and pathogens. Different mechanisms have been proposed to investigate the adhesion properties of probiotics: adhesion to (i) mucin adsorbed onto abiotic surfaces (e.g., polystyrene) [43], (ii) confluent intestinal cell layer cultures in cell/tissue culture plates, and (iii) extracellular matrix components [44].
Adhesion, which is an important feature that allows microorganisms to stick to surfaces or other cells, is affected by some factors such as protein and non-protein factors, hydrophobicity, aggregation ability, and pH of the environment [45]. Furthermore, the sufficient amount of microorganism, medium grown of microorganism, and incubation time are other significant factors which play role in the adherence of probiotic bacteria [46]. Lebber et al. found that amount of glucose in the medium affected the biofilm formation of L. rhamnosus GG [47]. Role of catechin added to the culture media on the adhesion ability of LA-5 and LGG was investigated in this study. Catechin increased the adhesion rate of LA-5 and LGG to the mucin layer. Enhanced adhesion ability of LA-5 and LGG may provide competitive advantage and increase the number of probiotic bacteria in the GIT, resulting in more colony formation, and thus probiotic bacteria may exert more beneficial effects on host health [48].
Excessive amount of oxygen concentrations causes formation of reactive oxygen species (ROS) such as superoxide anion (O_2_^–^), hydrogen peroxide (H_2_O_2_), and the highly reactive hydroxyl radical (HO·). Accumulation of ROS creates oxidative stress that causes apoptosis and plays an important role in the development of many diseases such as neurodegenerative diseases, cardiovascular diseases, cancer etc. [49]. Furthermore, oxidative stress affects the amount of viable probiotic bacteria by disrupting of proteins, creating DNA mutations, and oxidation of membrane phospholipids [50]. Many studies have shown that probiotics have excellent antioxidant capacity to prevent or eliminate oxidative stress and adverse states resulting from oxidative stress [51]. The regulation mechanisms of the antioxidant potential of probiotics are not fully understood; however, it has been proposed that various mechanisms have been identified for the antioxidant potential of probiotic bacteria such as scavenging ROS, chelating metals, increasing antioxidant enzyme levels, and regulating the microbiota [51]. Probiotic bacteria have antioxidant enzymatic systems that regulate and scavenge free radical groups. With these enzymes such as superoxide dismutases, which convert O_2_^–•^ to O_2_ or H_2_O_2_, as well as peroxidases, which reduce H_2_O_2_ to H_2_O, they prevent the accumulation of free radical groups [52]. Furthermore, probiotic bacteria secrete antioxidant metabolites (postbiotics) such as NADH, NADPH, glutathione, uric acid which may play an important role in increasing antioxidant activity [53].
Catechin protects cells from oxidative stress that causes apoptosis by scavenging free radicals with its redox properties such as reducing agents, hydrogen donor and oxygen quencher. Moreover, it inhibits lipid peroxidation in low-density lipoprotein as a strong chain-breaking antioxidant [54]. Thus, it is a strong antioxidant flavanol with significant potential in preventing the progression of diseases like Alzheimer caused by oxidative stress [55]. In the present study, effects of catechin on the antioxidant property of LA-5 and LGG and antioxidant potential of their synbiotics combinations (catechin and probiotic bacteria) against DPPH radical were investigated. Catechin increased the antioxidant property of LGG. It may contribute to the antioxidant property of LGG by encouraging the releasing of antioxidant enzymes and metabolites. Thus, synbiotics of catechin and LGG may better prevent damage caused by oxidative stress [56].
Therapeutic potential of probiotics provide to prevent or treat many diseases without causing negative side effects due to their health-promoting properties. In particular, with their anticancer properties, they have the potential to reduce the risk of various types of cancer and to replace treatment methods with side effects such as chemotherapy, radiation therapy or surgery [57]. Apoptosis is important in cancer treatment because it has different biochemical events that cause cancer cell death. It is known that therapeutic agents with anticancer activity play a role in the induction of apoptosis [58]. Therapeutic compounds secreted by probiotics such as short-chain fatty acids, bacteriocins, exopolysaccharides that found in probiotic cell-free supernatant, which have strong antiproliferative activities, hold innovative and personalized potential in cancer treatment [59].
In addition to probiotics, the applying of natural products as therapeutic agents is increasing day by day due to their chemopreventive properties in cancer treatment/prevention. Tea, especially green tea, is one of the natural sources investigated in cancer treatment/prevention due to its significant health benefits and chemotherapeutic effects [60]. In various cell culture and in vivo animal studies, it was stated that catechin, the main bioactive polyphenol compound in green tea, is a potential anticancer agent by showing chemopreventive and chemotherapeutic effects [61, 62]. For example, Ogunlaja et al. showed that 25 µg/mL and 50 µg/mL of catechin exhibited significant cytotoxicity towards Caco-2 cells thereby decreasing cell viability [63].
In this study, the effects of catechin, probiotic bacteria and their synbiotic combination on the viability and DNA damage of colon cancer cell lines were investigated by MTT and Comet methods, respectively. 125 µg/mL and 250 µg/mL of catechin decreased the cell viability of Caco-2 cells statistically (p < 0.05) and 250 µg/mL of catechin changed the TM that is DNA damage parameter. Thus, catechin showed the cytotoxic and genotoxic effects on Caco-2 cells. The cell-free extract of LA-5 did not affect the cell viability and DNA damage parameters of Caco-2 cells. Therefore, it did not show cytotoxic and genotoxic effects on Caco-2 cells. LA-5 grown with 250 µg/mL and 500 µg/mL of catechin reduced the viability of Caco-2 cells, although they did not changed the DNA damage parameters of Caco-2 cells. Thus, catechin contributed to the cytotoxic potential of LA-5, but did not change the genotoxic property of LA-5. LGG decreased the cell viability of Caco-2 cells and changed the all of the DNA damage parameters. However, catechin did not contributed to the cytotoxic and genotoxic properties of LGG.
Conclusıon
The effects of the synbiotic relationship formed by catechin and probiotic bacteria co-existing in the gastrointestinal system are unknown. In the present study, the effects of catechin on the probiotic properties such as aggregation, antioxidant, adhesion of Lactobacillus acidophilus LA-5 and Lacticaseibacillus rhamnosus GG were investigated. Furthermore, the effects of the synbiotics of catechin and Lactobacillus acidophilus LA-5 and Lacticaseibacillus rhamnosus GG on in vitro cytotoxic effects against colon cancer cells (Caco-2) were examined. Catechin had no inhibitory properties towards LGG and LA-5; on the contrary, it enhanced the growth of LA-5. Moreover, it improved the adhesion property of LGG and LA-5 and aggregation potential of LA-5. Thus, the adhesion, aggregation, and co-aggregation results for LA-5 supported each other. Catechin and LGG have strong antioxidant capacity and their synbiotic combination showed stronger antioxidant potential against DPPH radicals. Thus, catechin increased the antioxidant property of LGG. Effects of catechin on cytotoxic and genotoxic effects of LA-5 and LGG to Caco-2 cells were investigated by using MTT and Comet analyses. Synbiotic combination of catechin and LA-5 led to higher cell death of Caco-2 cells. Thus, catechin improved the cytotoxic property of LA-5. LGG showed high level cytotoxicity and genotoxicity on Caco-2 cells. However, synbiotic of catechin and LGG caused lower cell death less than LGG alone and catechin did not affect DNA damaging parameters of LGG.
In conclusion, the emerging synbiotic combination of catechin and probiotic bacteria found together in the gastrointestinal tract has the potential to create more beneficial effects such as antibacterial, antioxidant, anticancer on the host. These analyses provide a partial representation of the complex physiological conditions within the human gastrointestinal tract and do not fully capture its overall complexity. In future studies, it is important to investigate additional relevant factors beyond these analyses, including the effects on gut microbiome composition, immune modulation, and metabolic activity. Moreover, in vivo studies are required to understand better potential of synbiotic formulations such as catechin and probiotic bacteria for the regulation of the gastrointestinal tract and the prevention/treatment of many diseases.