1Biomedical Technology and Cell Therapy Research Laboratory-Departments of Biomedical Engineering, Physiology, and Artificial Cells and Organs Research Center, Faculty of Medicine, McGill University, Canada
Received Date: May 28, 2015 Accepted Date: July 06, 2015 Published Date: July 09, 2015
Citation: Kahouli I, Malhotra M, Tomaro-Duchesneau C, Rodes LS, Aloui-Jamali MA, et al. (2015) Identification of Lactobacillus Fermentum Strains with Potential against Colorectal Cancer by Characterizing Short Chain Fatty Acids Production, Anti-Proliferative Activity and Survival in an Intestinal Fluid: In Vitro Analysis. J Bioanal Biomed 7:104-115. doi:10.4172/1948-593X.1000132
Copyright: © 2015 Kahouli I, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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The use of probiotics as preventive agents in colorectal cancer (CRC), as widely suggested in many clinical and pre-clinical studies, was often linked to the potency of short chain fatty acids (SCFAs) in the gut. However, there remains an incomplete understanding of the fatty-acid-producing activity of certain probiotics and their cancer preventive potential. In the current study, L. fermentum strains were investigated for their potential use with CRC treatments. Using cell-free extracts, L. fermentum NCIMB -5221, - 2797, and -8829 were first compared based on their SCFAs production and anti-proliferative activity against Caco-2 colon cancer cells. The corresponding SCFAs synthetic formulations, similar to the ones produced by the bacteria, were prepared and compared with the latter to determine the role and efficacy of naturally produced SCFAs in inhibiting the proliferation of colon cancer cells. Subsequently, the bioactivity and stability of L. fermentum bacterial strains in a simulated intestinal fluid (SIF) was determined. Results showed that L. fermentum NCIMB -5221 and -8829 were the most potent in producing SCFAs, in particular, acetic (192.3 ± 4 mg/L minimum), propionic (69.2 ± 1.6 mg/L minimum), and butyric (35.4 ± 2.9 mg/L minimum) acids. They were also found to inhibit the growth of Caco-2 cells (53.4 ± 1.6%, 72 h, p = 0.021) in comparison with L. acidophilus ATCC 314. Additionally, they showed resistance to SIF (16.3 ± 1.9% minimum, 72 h, p = 0.006) and produced SCFAs in SIF at concentrations high enough to significantly inhibit Caco-2 proliferation (74.73 ± 2.1%, 72 h). Based on characteristics related to bacterial cell survival, SCFA production, and anti-proliferative activity, L. fermentum NCIMB -5221 and - 2797 could potentially be considered as biotherapeutic agents against CRC.
L. fermentum; Probiotics; Colorectal cancer; Short chain fatty acids; Cell proliferation; Intestinal fluid
CRC: Colorectal cancer; SCFA: Short chain fatty acid; GI: Gastrointestinal; L.: Lactobacillus; CM: Conditioned cell culture medium; LAB: Lactic acid bacteria; SIF: Simulated intestinal fluid; LA: Lactic acid; AC: Acetic acid; PA: Propionic acid; BA: Butyric acid; L. a ATCC 314: L. acidophilus ATCC 314; L. f NCIMB 2797: L. fermentum NCIMB 2797; L. f NCIMB 8829: L. fermentum NCIMB 8829; L. f NCIMB 5221: L. fermentum NCIMB 5221
The diagnosis and primary prevention strategies employed for colorectal cancer (CRC) have shown this disease to be an emerging and one of the major common public health problems in developing countries [1,2]. It accounts for 8-9.7% of all cancer cases and cancerrelated deaths  and it is considered, not only a common cancer, but also a complex and multifactorial disease [4,5]. Despite the appreciable understanding of the disease’s pathogenesis, the environment is considered to play a vital role in progression of the disease and the identification of reliable markers used as a primary preventive measure for CRC is still deficient . However, reports have shown that CRC incidence was reduced to a large extent (up to 80%) by healthy lifestyle and environmental factors with diet being a major controlling factor . Dietary interventions have recently attracted increased attention from researchers and clinicians for the prevention and the management of CRC . Within this domain, the probiotics have emerged as potential therapeutic agents and are also regarded as healthy dietary supplements with nutritional and health benefits. Probiotics, the live microbial food supplements with the ability to beneficially affect the gut micro biome, have long been known to augment a variety of immunological and metabolic parameters through diverse mechanisms . One of the main classes of probiotics found to confer multiple health-promoting attributes to the host are lactic-acid-producing microorganisms, the Lactobacillus spp. that are present in fermented foods as well as the gastrointestinal (GI) ecosystem. Several probiotic formulations containing L. fermentum have shown survival in the gut than without it in both GI [9,10] and genital environments . They were found to reduce infection  and over growth of harmful bacteria . In addition, they retained their beneficial metabolic activities when exposed to intestinal conditions, suggesting their potential for targeted colon delivery and for increasing colon bioproduction of anticarcinogenic compounds . L. fermentum have shown potential beneficial GI health attributes including anti-inflammatory [15,16] and anti-tumorigenic [17,18] activities. A number of L. fermentum strains were have shown greater and comaprable effect compared with other probiotic bacteria, such as L. reuteri , Bifidobacterium longum  or L. plantrum .
Several bacterial compounds were found responsible for the mechanisms associated to these effects such as SCFAs that are produced by the gut microflora and known by their ability to induce cancer cells death and present a source of energy to colonocytes . SCFAs, result of the microbial metabolism of nondigestible carbohydrates in the gut and play a central role in the host homeostasis . SCFAs have anticancer actions through apoptosis, promotion of cancer cell cycle arrest, and inhibition of cancer cell invasion and inflammation in the colon . Recently, an in vitro study showed the adherence property of L. fermentum to cancer cells and the resulting anti-proliferative effect through the bioproduction of SCFAs . However, comparative studies investigating the anti-proliferative effect of these bacteria, in vitro, against CRC cells as well as their activity in intestinal conditions were rare or non-conclusive [14,26,27]. To address this gap, the current study screen a number of L. fermentum bacteria (NCIMB -5221, -2797 and -8829) according to different criteria to evaluate a potential for use as biotherapeutics against CRC . Those strains were previously investigated for the production of certain anti-inflammatory acids , cholesterol assimilation , in relation to targeted colon oral delivery  and for use in metabolic syndrome (MS) . In this study, the aim is to provide details on SCFA production and anti-proliferative effect against colon cancer cells as well bacterial stability in intestinal conditions.
Cell culture media including Dulbecco’s modified Eagle’s medium (DMEM) and Eagle’s Minimum Essential Medium (EMEM) were purchased from, as well as fetal bovine serum (FBS) and phosphatebuffered saline (PBS). Bacterial culture broth De Man Rogosa Sharpe (MRS) and agar used for plating were obtained from Fisher Scientific (Ottawa, ON, Canada). Water was purified with two systems from Barnstead (Dubuque, IA, USA): an Easy Pure reverse osmosis system then a Nano Pure Diamond Life Science (UV/UF) ultrapure water system from Barnstead, Dubuque, IA, USA). Reagent and acids used such as propionate, acetate, and butyrate and sodium L-Lactate were obtained from Sigma (St. Louis, MO, USA).
L. fermentumNCIMB -5221, -8829, and -2797 were obtained from the National Collection of Industrial and Marine Bacteria (NCIMB, Aberdeen, Scotland, UK). L. acidophilus ATCC 314 was purchased from Cederlane Laboratories (Burlington, ON, Canada). To maintain bacterial cultures, they were daily inoculated in new MRS broth at 1% (v/v). Growth and viability of bacterial cells were determined with OD620 nm (Perkin Elmer 1420 Multilabel Counter, USA) and colony counting using agar plates.
Caco-2 human epithelial colorectal cancer adenocarcinoma cell line was purchased from American Type Culture Collection (ATCC, Manassas, VA). Cells were maintained in EMEM +20% FBS and incubated in a CO2 incubator (37°C, 5% CO2) for about two weeks to full differentiation. Caco-2 colon cancer cells were left to attach for u to 28 h and reach 50-60 % confluence in 96-well plates in DMEM. +10% FBS (37°C, 5% CO2), before experiments. During assays, cell culture medium was substituted by probiotic conditioned medium (CM) and serum and antibiotic-free media (DMEM. + 10% FBS).
Preparation of probiotic treatments
For the probiotic treatment used on cancer cells, a conditioned cell culture medium (CM) was prepared according to Grabig et al.  and Kim et al. , with slight modifications. Bacterial culture of L. fermentumand L. acidophilus were passaged for 72 h (37°C, 5% CO2) to reach late exponential phase (~16 h). The bacterial cell were collected from the culture broth by centrifugation (1000 × g, 15 min, 4°C) and washed with PBS. This bacterial pellet (107-109 cfu/ mL) was incubated in DMEM for 2 h (37°C, 5% CO2). The medium was centrifuged (1000 × g, 15 min, 4°C) to remove the bacteria, then sterile-filtered (0.2 μM-pore-size filter, Millipore). The pH was adjusted to 7 using 2M NaOH and 2M HCl. Before use, the CM of each bacterium was diluted twice with DMEM.
Preparation of simulated intestinal fluid (SIF)
To determine the ability of L. fermentum bacteria in surviving intestinal conditions, a simulated intestinal fluid (SIF) was prepared as described previously by Qian Zhao et al.  with some modifications. In brief, the solution of SIF contained glucose (5.5 g/L), yeast extract (3.5 g/L), pancreatin (2 g/L), oxgall (1.5 g/L), pectin (2 g/L), inulin (0.54 g/L), fructooligosaccharides (0.85 g/L), starch (3 g/L), and monobasic potassium phosphate (KH2PO4, 3.3 g/L) dissolved in deionized water. The pH was adjusted to 6.8 using 2M NaOH and 2M HCL, the solution was autoclaved at 120°C for 15 min and cooled at RT before use.
Bioactivity of L. fermentum bacteria
In order to determine if L. fermentum bacteria are metabolically active in CM or SIF, the concentrations of lactic acid, potentially produced by bacterial cells, were separated and measured by HPLC method, which was adapted from Dubey and Mistry, 1996 with modifications [32,33] (described below in details).
Analysis of lactic acid and SCFAs
Lactic acid and SCFAs were separated using a slightly modified HPLC method [26,27]. The HPLC system used (Model 1050 UV, Hewlett- Packard HP1050 series, Agilent Technologies, USA) was equipped with a UV-vis detector and diode array detector (DAD, 210 ± 5 nm). The column used was a prepacked Rezex ROA-organic acid H+ (8%) (150 × 7.80 mm, Phenomenex, Torrance, CA, USA) column attached to an ion-exclusion microguard refill cartridge and heated to 35°C. Data were obtained using ChemStation equipped with LC3D software (Rev A.03.02, Agilent Technologies, CO, USA). The mobile phases (0.05 % of M H2SO4 and 2 % of acetonitrile) were pumped at an isocratic gradient with 0.7-0.8 mL/min flow rate. 100 μl of sample was injected through the autosampler. Lactic, acetic, propionic, and butyric acids were used to prepare standard solutions at concentrations of 1, 10, 100, 500, and 1000 ppm. The concentrations of samples were calculated using the linear regression equations (R2 ≥ 0.99) from each standard curve.
Cancer cell proliferation
The growth of colon cancer cells determined using ATP bioluminescence assay (CellTi- ter-Glo Luminescent Cell Viability Assay; Promega). Caco-2 cells were seeded at 5 × 103 cells/ well onto 96-well culture plates and left to attach for 24-48 h or up to few days for the formation of an epithelium-like monolayer (37°C, 5% CO2).
Caco-2 cells were incubated with the probiotic treatments for 24 h, 48 h and 72 h, (37°C, 5% CO2, pH = 7). Cell growth inhibition and viability were determined according to the manufacturer’s protocol . After incubation, the plate was equilibrated at room temperature (RT, 30 min) and the media was replaced with 100 μL of luminescent reagent and 100 μL of DMEM. The plate was kept on an orbital shaker (200 rpm, 3 min), followed by incubation at RT for 10 min. Signals were recorded using a multi-label microplate reader (Perkin Elmer, Victor 3, Massachusetts, USA).
Determination of bacterial stability in SIF
Each bacterial culture in MRS broth, passaged for 72 h, was used to inoculate 15 ml of SIF at 3%, sealed and incubated micro-anaerobically. At 0 h, 4 h, 8 h, 12 h, 16 h, and 24 h, samples were used to determine the density (OD620 nm) and viable bacterial cell count in SIF. The bacterial supernatant was collected by centrifugation (1000 × g, 30 min, 4°C), using 5 ml of bacterial culture, filtered (0.22 μm sterile filters), then stored at -80°C until use.
Relevance of SCFAs produced by L. fermentum strains
To determine whether the concentrations of SCFAs present within the bacterial free extract were the active compounds behind suppressing CRC cell growth, the anti-proliferative effect of the SCFAs alone was determined. First, lactic, acetic, propionic, and butyric acids produced by the each L. fermentum strain were quantified in CM. Mixtures containing the same composition were formulated in DMEM, then added to the colon cancer cells (37°C, 5% CO2, pH=7, 72 h). Cell viability was determined using an ATP bioluminescence assay, as described above.
Results were presented as means ± standard error of the mean (SEM). Statistical significance was calculated using one-way analysis of variances (ANOVA) with the Tukey’s comparison test and student’s t-test. Pearson correlation method was followed to determine correlation between variables. SPSS statistics software package (version 20.0, IBM corporation, New York, NY, US) was used. P-value of p<0.05 were considered significant.
L. fermentumbacteria do produce lactate in the conditionned medium (CM)
Before using the CM of L. fermentumbacteria as probiotic treatments in vitro, the activity of bacterial cells incubated in the CM was established by quantifying the levels of lactic acid produced. All bacteria were active in CM and produced variable amount of lactic acid (Figure 1). Data showed that L. fermentum NCIMB 5221 (455,3 ± 9,3 mg/L, p<0.001) produced significantly the highest amounts of lactic acid compared with L. fermentum NCIMB -2979 and -8829. But all L. fermentum strains produced significantly less lactic acid than L. acidophilus ATCC 314 (1947.7 ± 23.3, p<0.0001).
Figure 1: Determination control for of the ability of L. fermentum strains to produce lactic acid in conditioned cell culture medium (CM). L. fermentum NCIMB -2797, -5221 and, -8829 were active enough to produce different concentrations of lactic acid when incubated in DMEM (2 h, 37°C, 5% CO2). L. acidophilus ATCC 314 is used as a control/for comparative purpose. Data are presented as mean ± SEM (n = 3). ***p < 0.005.
L. fermentumstrains produced variable amounts of SCFAs
In order to confirm that L. fermentum bacteria may produce anti-carcinogenic active compounds in the cell free extract, three SCFAs were quantified in the conditioned cell culture medium CM: acetic, propionic and butyric acids. Results described the quantities of naturally produced SCFAs by the bacteria. For the bioproduction of acetic acid, L. fermentum NCIMB 2797 (206.3 ± 8.7 mg/L, p<0.01) and L. fermentum NCIMB 5221 (192.3 ± 4 mg/L, p<0.01) produced significantly more than both L. acidophilus ATCC 314 (114.2 ± 11.9 mg/L) and L. fermentum NCIMB 8829 (134.3 ± 5.7 mg/L, (Figure 2a). Again, L. fermentum NCIMB -2797 (69.2 ± 1.6 mg/L, p<0.001) and -5221 (85.7 ± 10.9 mg/L, p<0.001) were the only bacteria to produce propionic acid but not L. acidophilus ATCC 314 or L. fermentum NCIMB 8829 (Figure 2b). Similarly, L. fermentum NCIMB 2797 (35.4 ± 2.9 mg/L) and L. fermentum NCIMB 5221 (38.7 ± 4.2 mg/L, p<0.05) produced significantly more butyric acid than L. fermentum NCIMB 8829 (butyric acid not detected) and L. acidophilus ATCC 314. In terms of total SCFAs production, L. fermentum NCIMB -2797 (35.4 ± 2.9 mg/L) and -5221 (38.7 ± 4.2 mg/L) had significantly higher production compared to L. acidophilus ATCC 314 (14.1 ± 5.9, p<0.01) or L. fermentum NCIMB 8829 (Not detectable, p<0.0001, Figure 2d).
Figure 2: Analysis of the bioproduction of SCFAs by L. fermentum strains in the conditioned cell culture medium (CM). L. fermentum strains produced variable levels of SCFAs in a strain-dependent manner. The levels (a) acetic, (b) propionic, (c) butyric acids, and (d) total SCFAs, produced by L. fermentum NCIMB -2797, -5221, and -8829 were quantified in the conditioned medium cell culture and compared with each other while L. acidophilus ATCC 314 was used as a control. Data are presented as mean ± SEM (n = 3). *p < 0.05, **p < 0.01 and ***p < 0.001, compared with L. acidophilus ATCC 314.
L. fermentum inhibits colon cancer cell proliferation
In this experiment, the ability of L. fermentumbacteria to inhibit colon cancer cell growth was investigated. Caco-2 cancer cells were incubated with bacterial CM for 24 h, 48 h and 72 h. The results showed a time-dependent effect of the probiotic extracts on the viability of Caco-2 cells (Figure 3). At 24 h (Figure 3a), only L. fermentum NCIMB 5221 (6.02 ± 1.04, p<0.05), inhibited cancer cell growth when compared with the rest of untreated and untreated cells. After 48 h of probiotic treatment (Figure 3b), results showed that L. fermentumNCIMB 2797 (39.00 ± 1.56 %) and L. fermentum NCIMB 5221 (45.77 ± 0.37 %) were significantly the best in reducing CRC cell proliferation (p<0.001). Data presented in Figure 3c showed that L. fermentum NCIMB 2797 (53.4 ± 1.6 %), and L. fermentum NCIMB 5221 (57.9 ± 0.7 %), effected significantly greater inhibition of colon cancer proliferation compared to all other treatments tested here (p<0.001, 72 h). Moreover, L. fermentum NCIMB 5221 inhibited significantly more cancer cell proliferation than L. fermentum NCIMB 2797 (p=0.033, 72 h).
Figure 3: Screening of L. fermentum strains for a proliferation inhibitory effect against colorectal cancer cells. To investigate the anti-proliferative effect of the different L. fermentum strains, the cell culture conditioned cell culture media (CM) of L. fermentum NCIMB -2797, -5221, and -8829 were incubated with Caco-2 cancer cells. The viability and growth inhibition rate of Caco-2 cells for (a) 24 h, (b) 48 h and (c) 72 h of incubation showed a time-dependent effect. L. acidophilus ATCC 314 is used as a positive control. *p < 0.05, **p < 0.01 and ***p < 0.005 compared to L. acidophilus ATCC 314. Data are presented as mean ± SEM (n = 4).
The inhibition of colon cancer cells correlates with SCFAs production
To relate the ability of L. fermentun bacteria to suppress colon cancer cell growth to the production of SCFAs, a correlation analysis was conducted (Figure 4). Regression analysis showed that the suppression of colon cancer cell proliferation by L. fermentum-CM significantly correlated with the levels of total SCFAs produced by the bacteria in the CM (r=0.87, p<0.001, Figure 4d). Cancer cell inhibition correlated with the production of butyric (r=0.89, p<0.001) and acetic (r=0.0771, p<0.001) acids (Figure 4c and 4b). The highest correlation was with the propionic acid concentrations (r=0.89, p<0.001) and with different combinations of SCFAs (butyrate and propionate) (r=0.95, p<0.001, Figure 4f).
Figure 4: Investigation of the correlation between cell growth inhibition and the different concentrations of naturally produced SCFAs in probiotic CM. The dependent variables used are the values for: (a) acetic acid, (b) propionic acid, (c) butyric acid, (d) total SCFAs, (e) total SCFAs and BA+AA and (f) SCFA combinations: 7x BA and PA+[7xBA], Plots represent the data of cell growth inhibition at 72 h (Figure 2c). The lines were obtained by linear regression analysis. LA: lactic acid; AA: acetic acid; PA: propionic acid.
The action of probiotic naturally produced SCFAs is straindependent
For L. fermentumbacteria, establishing a correlation between their SCFAs production and their anti-proliferative effect against colon cancer cells is not enough to confirm that the inhibition of colon cancer cell growth is only due to SCFAs. Therefore, an additional approach was taken using synthetic SCFAs.
First, pure SCFAs of the same concentrations as produced by the bacteria were tested separately and the data showed that the concentrations of acetic, propionic and butyric acids separately had significantly less inhibition (maximum of 20.3 ± 2.5 %) than L. fermenum-CM (31.2 ± 1.5% minimum, p<0.05, Figure 5a).
Figure 5: Investigation of the role and effectiveness of SCFAs produced by L. fermentum bacteria. (a) The anti-proliferative effect of pure SCFAs at the same concentrations as what was produced by probiotic bacteria in L. fermentum-CM (as described in Figure 4). The inhibitory effect of SCFAs on Caco-2 cells (72 h) increased with higher doses. (b) Comparison of the anti-proliferative effect of SCFA synthetic formulations (SSFs) with the anti-proliferative effect of L. reuteri-CM. The SCFA synthetic formulations are reconstituted mixtures of acetic, propionic, and butyric acids (Table 1) with or without lactic acid, at concentrations similar to the naturally produced ones by L. fermentum bacteria. These formulations, used to treat Caco-2 cells for 72 h, were compared with their corresponding L. fermentum-CM. *p < 0.05, **p < 0.01 and *** p < 0.001. Data are represented as mean ± SEM (n = 5).
Second, SCFA synthetic formulations corresponding to the concentrations of SCFAs produced by the bacteria and containing acetic, propionic and butyric acid were prepared, as described in Table 1. SCFA synthetic formulations were then tested on Caco-2 cells and were compared with L. fermentum-CM (Figure 5b). The findings showed that those mixtures had variable effects on the alteration of cell viability compared with L. fermentum-CM treated cancer cells. For L. acidophilus ATCC 314, the CM (12.6 ± 1.9%) had significantly less efficacy than its corresponding SCFA synthetic formulation (SSF-a, 22.9 ± 1.0 %, p<0.05). For L. fermnutm NCIMB 5221, there was no significant difference (p=0.094) between the SSF (58.9 ± 1.8 %) and CM (57.9 ± 0.7 %). However, for L. fermentum NCIMB 2797 (53.4 ± 1.6 %) and L. fermentum NCIMB 8829 (31.2 ± 1.5 %), L. fermentum-CM was significantly more effective than SCFA synthetic formulations (SSF-f2, 43.8 ± 2.2 %, p=0.026) and SSF-f8 (19.12 ± 1.6 %, p=0.015, Figure 5b). After addition of lactic acid to each formulation, the inhibitory effect of “SSF+LA” was up to more than 50 % lower than both L. fermentum– CM and SSFs (p<0.001, Figure 5b), showing a loss of SCFA efficacy against cancer cells.
L. fermentum bacteria showed resistance in SIF
The growth and viability of L. fermentum bacteria was depending of the strain. For L. fermentum NCIMB -2797 and -5221 the bacterial culture density (0.38 ± 0.001minimum) was significantly higher compared with L. acidophilus ATCC 314 (0.29 ± 0.003, p<0.001, Figure 6a). Between 4 and 8 h, L. fermentum NCIMB -2797 (16.3 ± 1.9 %) and -5221 (28.4 ± 2.4 %) showed a significant increase in bacterial growth compared with the initial count which was not in the case of L. acidophilus ATCC 314 (Figure 6a).
Figure 6: Characterization of L. fermentum bacterial cell resistance in SIF. (a) Bacterial cell culture characterization for L. fermentum strains in a simulated intestinal fluid (SIF), (pH = 6.8, 24 h). It was determined by bacterial viable cell count and cell culture absorbance of L. fermentum NCIMB -5221, -2797, and -8829, in addition to L. acidophilus ATCC 314 used as a control. (b) Death rate of L. fermentum bacteria in a SIF (pH = 6.8, 24 h). The death rate in all bacteria showed a transition at 8 h. The SIF used contained glucose (5.5 g/L), yeast extract (3.5 g/L), pancreatin (2 g/L), oxgall (1.5 g/L), pectin (2 g/L), inulin (0.54 g/L), fructooligosaccharides (0.85 g/L), starch (3 g/L), and monobasic potassium phosphate (KH2PO4, 3.3 g/L). Data are presented as the mean ± SEM (n = 3).
In terms of decrease in viable bacterial cells, compared with initial count, significant difference was determined (12-16 h), where L. fermentum NCIMB 2797 (70.11 ± 3.2 % minimum) and L. fermentum NCIMB 5221 (94.02 ± 0.4 % minimum) had higher death rate than L. acidophilusATCC 314 (64.5 ± 0.7 % maximum, p<0.01, Figure 6b).
L. fermentumstrains produced SCFAs in SIF
In order to confirm that, despite the decrease in their viability in SIF, L. fermentum bacteria are still able to excrete an anti-colon-cancer– proliferative activity in an intestinal environment, the production of lactic acid and SCFAs was determined in SIF after 24 h of incubation (Figure 7). Data showed that Both L. fermentum strains produced significantly higher concentrations of lactic (Figure 6a), acetic (Figure 6b), propionic (Figure 6c) acids than L. acidophilus ATCC 314 in SIF. L. fermentum strains showed, also, higher production of total SCFAs in SIF, as described in Figure 6d. L. acidophilus ATCC 314 produced 1968.5 ± 0.3 mg/L and 413.1 ± 0.1 mg/L, respectively. L. fermentum NCIMB 2797 produced 2491.9 ± 11.4 mg/L of lactate, 689.4 ± 2.1 mg/L of acetate and 686.3 ± 35.7 mg/L of propionate. Also, L. fermentum NCIMB 5221 produced 2407.3 ± 42.3 mg/L of lactate, 637.99 ± 5.7 mg/L of acetate and 648.8 ± 17.8 mg/L of propionate. Yet, when considering the concentration of total SCFAs produced depending on the density of the bacterial culture, both L. fermentum NCIMB -2797 and -5221 were significantly more potent than L. acidophilus ATCC 314 (p<0.0001, Figure 7e)
Figure 7: Quantification of the lactic acid/SCFAs produced by L. fermentum strains in SIF. (a) Lactic, (b) acetic, (c) propionic acids and (d) total SCFAs produced by L. fermentum NCIMB -2797 and -5221 were measured in a simulated intestinal fluid (SIF, 24 h, pH=8.6). (e) Comparison of SCFAs production in SIF depending on the bacterial culture density of L. fermentum NCIMB -2797 and -5221 with L. acidophilus ATCC 314 (mg/L/OD620nm x 102). The SIF was prepared by mixing glucose (5.5 g/L), yeast extract (3.5 g/L), pancreatin (2 g/L), oxgall (1.5 g/L), pectin (2 g/L), inulin (0.54 g/L), fructooligosaccharides (0.85 g/L), starch (3 g/L), and monobasic potassium phosphate (KH2PO4, 3.3 g/L). L. acidophilus ATCC 314 is used as a positive control (n=3). L. acidophilus ATCC 314 is used as a control. Data are presented as mean ± SEM (n = 3). *p < 0.05, **p < 0.01 and ***p < 0.005 compared with L. acidophilus ATCC 314.
Efficacy of the levels of SCFAs produced in SIF
In order to verify that L. fermentum bacteria could excrete an antiproliferative activity against colon cancer in intestinal environment, the same concentrations of bacterial SCFAs produced in the SIF were tested on colorectal cancer cells. sSCFA synthetic formulations corresponding to the levels of SCFAs produced by the L. fermentum (NCIMB -2797 and -5221) in SIF (SSF-SIF-f) weres reconstituted. In addition, separate concentrations of propionic and acetic acids at the same levels of what produced in SIF were tested.
Propionic acid doses used were significantly more efficient in inhibiting colon cancer cell growth than acetic acid (p<0.001, Figure 8a). For SCFA synthetic formulations representing the concentrations of SCFAs naturally produced by L. fermentum bacteria in SIF (SSFSIF- f), two formulations were prepared, as described in Table 2. SSF-SIF-f significantly reduced Caco-2 proliferation by 74.73 ± 2.1 % compared to SSF-SIF-a (38.51 ± 2.46 %, p=0.0012) and untreated cells (p=0.0018, Figure 8a). There is no significant difference observed between the two formulations and control in the case of epitheliumlike Caco-2 monolayer (Figure 8b, p=0.382).
Figure 8: Confirmation of the efficacy of SCFAs produced in SIF, by L. fermentum. (a) The inhibitory effect of propionic and acetic acids produced by L. fermentum in SIF was described. The effect of the SCFA synthetic formulations (SSF-SIF-a and SFF-SIF-f) against CRC cells (b) cell culture, and (c) epithelium-like cell culture. SSF-SIF-a and SFF-SIF-f represented synthetic mixtures of SCFAs that have the same composition as the probiotic SCFAs naturally produced in SIF by L. acidophilus ATCC 314 and L. fermentum NCIMB -5221 and -2797, respectively (Table 2). Data are presented as mean ± SEM (n = 5). *p < 0.05 and ***p < 0.005, compared with control or L. acidophilus ATCC 314. SSF-SIF-f: formulation of SCFAs produced in SIF corresponding to both L. fermentum bacteria (NCIMB -5221 and -2797); SSF-SIF-a: SCFA formulation of SCFAs produced in SIF by L. acidophilus ATCC 314.
CRC is a leading cause of death and an economic burden with a therapeutic market worth billions of dollars Worldwide . However, thanks to the preventive potential of this cancer , it was found that a lifestyle and dietary measures alone, supplemented with digestive enzymes and probiotics, can prevent CRC up to 70% . It is proposed that increasing the rate of SCFAs, products of carbohydrate fermentation by the gut micro bio data, is key to a healthy colon and prevents intestinal injuries and abnormal cell growth in the lining of the intestines. However, a limited number of probiotic bacteria were investigated for potential against CRC as novel candidates . This study was interested in three L. fermentum strains that demonstrated antioxidant and anti-inflammatory potential by the production of ferulic acid [39,40]. Here, L. fermentum NCIMB -2797, -8829 and -5221 were investigated for anti-cancer associated features such as production of SCFAs and anti-colon-cancer-cell-proliferative effect in vitro. For this end, the cell culture conditioned medium (CM) of each bacterium was used as probiotic extract treatment for the in vitro study. Thus, the metabolic activity of these LAB when incubated in the CM was verified by the concentrations of lactic acid they produced at significantly high levels such in the case of L. fermentun NCIMB 5221 (Figure 1). Lactic acid is used by lactate-utilizing butyrate-producing bacteria in the gut , is considered an anti-inflammatory component  and is found to increase anti-tumor immunoreactivity . SCFAs secreted by gut bacteria induce apoptosis in CRC cells and may therefore be relevant in CRC prevention and therapy. For example, microbial-derived butyrate was found promote the stabilization of transcription factors related to epithelial barrier protection . Butyrate and propionate affected colonocytes and immune cells by inhibiting the activity of histone deacetylases (HDACs) and induced anti-inflammatory effects via the differentiation of regulatory T cell . Thus, SCFAs found to be secreted by L. fermentum were quantified and were produced at significantly different concentrations (Figure 2). L. fermentum NCIMB -2797, -8829 and -5221 produced were significantly higher amount of total SCFAs in their CM compared with L. acidophilusATCC 314 (p<005, Figure 2d), but significantly less amount of lactate in their respective CM (p<0.001, Figure 1). This suggests that L. fermentum may act as an anti-colon cancer agent by the production of higher quantities of SCFAs distinctively from L. acidophilus ATCC 314 that may excrete anti-tumorigenic and anti-inflammatory activities, as shown in a CRC Apc/min mice model , by a higher production of lactic acid and directly providing more substrate for anti-oncogenic bacteria in the gut. Therefore, L. fermentum bacteria may a more direct role in inhibiting epithelial cell damage and suppressing the growth of Caco-2 cells more directly through SCFAs production, than by modulating the gut microbiota, providing growth support for other beneficial microbiota or inhibition of CRC-associated bacteria by lactic acid production . This study showed that the concentrations of acetic acid and propionic acid measured in this study are about half of the optimal doses used in the literature to induce inhibitory effect on Caco-2 cells , which predict a possible cancer-suppressive effect of the probiotic treatments.
L. fermentum-CM significantly inhibited colon cancer cell proliferation, in a time dependent manner, compared with untreated cell and cells treated with L. acidophilus ATCC 314 (p<0.05, Figure 3).
The role of SCFAs in colon carcinogenesis is debatable and poorly understood. Several reports provided evidence on the effect of probiotic bacterial supernatants or separately tested pure SCFAs in the mechanism of cancer cell inhibition. Many of these studies associated the potential anti-cancer activity of probiotic bacteria to the production of SCFAs but few have validated this theory .
Hence, first, a linear regression was established between the percentage of Caco-2 cells inhibited by L. fermentum–CM and the concentrations of SCFAs produced by L. fermentum bacteria, showing a strong correlation (Figure 4e and Figure f). Second, to verify if there are other factors than SCFAs involved in this activity, concentrations of synthetic SCFAs prepared in mixture were tested on colon cancer cells. Figure 4a describes that the artificially prepared doses of pure SCFAs a significantly showed less effect in comparison with the probiotic bacteria extracts CM (p<0.01), which argues for the ability of a one naturally produced SCFA to excrete inhibitory effects (Figure 4). However, the synthetically prepared mixtures of SCFAs, overall, showed a nearer effect to L. fermentum-CM (Figure 5b). More specifically, L. fermentum NCIMB 5221 had the same effect as its corresponding SCFA formulation. The L. fermentum NCIMB -2797 and -8829 significantly inhibited colon cancer cells growth less than the corresponding SCFAs synthetic formulations (p<0.05) suggesting that the bacteria have produced additional anti-cancer products. Yet, For L. acidophilus ATCC 314 was significantly less effective than its SCFAs synthetic cocktail, suggesting the presence of other bacterial factors, produced in the CM, that hindered the effect of the naturally produced probiotic SCFAs. This data suggests that the anti-proliferative effect of the CM is possibly due, in a minor part, to the concentration of bacterial SCFAs but the effect is not only related to the presence of SCFAs. As described in Table 1, lactic acid was added to each SCFA synthetic formulation. These lactic acid containing SCFA mixtures had significantly less effect than both SCFA synthetic formulation and probiotic CM (p<0.001). This implies that the presence of lactic acid may have reduced the efficacy of SCFAs on the metabolism of cancer cells which is supported by a study where L-lactate significantly inhibited uptake of butyrate in cancer cells , suppressing the anticancer effect of the latter. Hence, the lactic acid, added later to the SSFs, could have suppressed the ability of cancer cell to uptake SCFAs resulting in the decreased action SSF containing lactic acid. Some of the bacterial products released by L. fermentumbacteria were indicated as surface  and adhesive  proteins that bind to the intestinal and gastric mucus, or such as DNA fragments or lipopolysaccharide (LPS) . As explained the anti-proliferative effect of L. fermentum may not only based on the activity SCFAs but also to the release of other bacterial products that probably preserve the effect of SCFA.
Another feature related to probiotic bacteria selection was the loss of viability of L. fermentum bacteria in simulated human intestinal conditions and the SCFA production ability. Interestingly, L. fermentum NCIMB -5221 and -8829, that excreted higher anti-colon cancer potential, showed similar density (Figure 6a), resistance and survived the bile exposure for 4 h which was significantly higher compared with L. acidophilus ATCC 3 (p<0.05, Figure 6b). A number of studies showed that L. fermentumhave resistance to gut conditions but this feature varied according to the glucose and other nutrient availability in the gut. L. fermentumtolerance to intestinal conditions was perceived, mainly, for a maximum of 4 h compared with other probiotic bacteria . Between 12 h and 16 h, L. fermentum NCIMB 2797 had significantly lower death rate than L. fermentumNCIMB 5221. Furthermore, at 24 h, L. fermentum bacteria were still viable at log 6-7, strongly suggested an ability to stay viable in an intestinal environment. Another point is that even though L. fermentum NCIMB -5221 and -8829 displayed, at 24 h, significantly less viability in comparison with L. acidophilus ATCC 314 in SIF (p<0.05), they were both able to produce significantly higher concentrations of lactate (Figure 7a), acetate (Figure 7b), propionate (Figure 7c) and total SCFAs (Figure 7d) than L. acidophilus ATCC 314 (Figure 7, p<0.01)). Moreover, SCFAs (propionate, acetate and total SCFAs) per bacterial density were significantly higher for L. fermentum NCIMB -5221 and -8829 compared to L. acidophilus ATCC 314 (p<0.05, Figure 7e). This observation implies that L. fermentum bacterial cells are more active and have the potential of efficiently in excreting higher concentrations of anti-cancer bioactive compounds than L. acidophilus ATCC 314. This was confirmed by testing those concentrations separately on colon cancer cells (Figure 7) . The levels of SCFAs produced by L. fermentum bacteria in SIF were shown to significantly reduce colon cancer cell proliferation compared to L. acidophilus ATCC 314, which is still in conformity to the superior inhibitory effect of L. fermentum cell free extract described in Figure 3. As noted, the only SCFA L. acidophilus ATCC 314 did not produce was propionate Figure 2b. Nevertheless, the propionic acid concentration produced in the SIF seemed to be behind the decrease in cell viability more significantly than acetic acid SIF concentrations (p<0.001, Figure 8a), suggesting that propionate production as a major mechanism for colon cancer inhibition by L. fermentum in intestinal environment.
This is the first study that explores and compares the potential suitability of L. fermentum NCIMB -5221, -2797 and -8829 as colon cancer bio therapeutic in vitro. Those strains were characterized for their production of active molecules relevant to CRC and their tolerance to intestinal stress, and shown to produce SCFAs in CM or SIF and suppress colon cancer cell growth. We were able to compare the anti-proliferative effect of L. fermentum probiotic bacterial strains in vitro, while evaluating the efficacy of SCFAs bioproduction as a mechanism. Our findings identified a significant effect of L. fermentum strains in inhibiting colon cancer cells and correlate with the ability of these bacteria to produce SCFAs. These strains also showed significant efficiency in producing SCFAs in intestinal conditions suggesting an ability to excrete an anti-carcinogenic effect in the colon.
The authors would like to acknowledge a Canadian Institute of Health Research (CIHR) grant (MPO 64308) and grants from Micropharma Limited to Dr. Satya Prakash, a Fonds de Recherche du Québec–Santé (FRSQ) Doctoral Awards and a Faculty of Medicine George G. Harris Fellowship to Imen Kahouli and Meenakshi Malhotra. We, also, thank the Analytical Laboratory Technicians of the Department of Chemical Engineering (McGill University), Andrew Golsztajn and Ranjan Roy for their help with the analysis.
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