An optimized de Man Rogosa Sharpe- McCoy’s 5A medium

Vol. 13 | No. 02 | 2020 Philippine Science Letters

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An optimized de Man Rogosa Sharpe-McCoy’s 5A medium formulation for the in vitro screening of cytotoxic effects by probiotic candidates against colorectal cancer cells Ranelle Janine L. Asi1, Christian Luke D. Badua2, Jason Bryan D. Atun2, Joshua Ismael L. Lapus2, Sheena May O. Lopez2, Sonia D. Jacinto1, Ahmad Reza F. Mazahery1, and Marilen P. Balolong*2 1Institute of Biology, College of Science, University of the Philippines Diliman, Quezon City, Metro

Manila 1101 Philippines 2Department of Biology, College of Arts and Sciences, University of the Philippines Manila, Ermita,

Metro Manila 1000 Philippines

s attention for the therapeutic potential of postbiotics increases, the efficiency of in vitro screening methods for cell-free supernatants (CFS) should also be improved. DeMan Rogosa and Sharpe (MRS) broth has been reliable in bacterial

cultivation but presents considerable limitations during cytotoxicity screening. Here we show that MRS broth alone is detrimental to HCT-116 colorectal cancer cells at 12.5%v/v and above and is unlikely the optimal vehicle during screening. Hence, we compared different media for cultivating locally-isolated lactic acid bacteria (LAB) Lactobacillus plantarum BS25 (LPBS25) and Pediococcus acidilactici S3 (PAS3) and determined the effects of their CFS on HCT-116 cell viability. Through time-course spectrophotometry and agar colony counts, we show that a combination medium (CM) consisting of 10:90 MRS broth-McCoy’s 5A medium was able to appreciably promote LAB growth. During cytotoxicity screening through MTT assay, CM as a vehicle exerted no significant effects on

HCT-116 cells across all concentrations tested (50% to 1.5625%v/v) while CFS from CM-grown LPBS5 and PAS3 exerted up to 54.29% and 63.23% cytotoxicity, respectively. Based on IC50 values, CFS from MRS-grown LAB exhibited higher cytotoxicity compared to CFS from CM-grown LAB, suggesting some compounding effects from the MRS vehicle that may lead to overestimation. Overall, we provide an efficient media formulation that stably supports LAB growth while minimizing vehicle-mediated effects during cytotoxicity screening against colorectal cancer cells. Additionally, this study is the first to report cytotoxic activity from the two LAB strains, LPBS25 and PAS3.

KEYWORDS lactic acid bacteria, postbiotics, colorectal cancer, MTT assay INTRODUCTION Colorectal cancer (CRC) is one of the leading causes of malignancy-driven mortalities globally and in the Philippines, CRC mortality rate ranks second in male populations and third in females (Bray et al. 2018). Research in the last few decades

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*Corresponding author Email Address: [email protected] Date received: July 6, 2020 Date revised: September 15, 2020 Date accepted: October 5, 2020

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produced mounting evidence of the prophylactic and even direct or adjuvant therapeutic action of probiotic lactic acid bacteria (LAB) in colon carcinogenesis (Cruz et al. 2020; Brasiel et al. 2020; Javanmard et al. 2018; Vivarelli et al. 2019; Degnan et al. 2012). However, probiotic research is plagued by inconsistent results in in vivo and clinical studies due to the biological limitations of administering live probiotics such as low gut survival and highly variable host gut microbiome interactions (Kleerebezem et al. 2019; Brüssow et al. 2019; Aarti et al 2017). In vitro, live co-culture systems of bacterial and mammalian cells present challenges in optimizing variables brought about by increased metabolism of both cell types, premature depletion of media and pH destabilization from bacterial fermentation (Papadimitriou et al. 2015; Duell et al. 2011). These are factors that contribute to the notoriously low translational efficiency from pre-clinical models to clinical trials for the therapeutic use of probiotics (Suez et al. 2019; Kleerebezem et al. 2019). Such inconsistencies imply the need for better strategies in conducting in vitro experiments and more accurate screening of probiotic strains. Due to the limitations of live probiotics, attention has recently shifted towards harnessing the potential of postbiotics, which are the extracellular metabolites, cell surface molecules and other components found in non-viable probiotic bacteria (Rad et al 2020; Wang et al. 2014). In the right culture conditions, bacterial cells have the natural ability to rapidly produce metabolites that could later be purified and developed as therapeutic agents (Mora-Villalobos et al. 2020; Kleerebezem et al. 2019). Indeed, whole cell-free supernatants (CFS) and novel purified postbiotics in the form of secreted proteins and exopolysaccharides from probiotic LAB cultures have been shown to exert anticancer activities and other beneficial effects in vitro and in vivo (Chuah et al. 2019; Gao et al. 2019; Izuddin et al. 2019; De Marco et al. 2018; Wang et al. 2014; Escamilla et al. 2012). Focusing on postbiotics provides several advantages in research. Postbiotic samples present less complex abiotic systems to investigate and narrow down the list of potential factors that produce bio-functional effects. Postbiotic metabolites are also relatively easy to gather through centrifugation and filtration. Unlike in screening live probiotics using in vitro cell culture systems, microbial viability or overgrowth will not be an issue. These advantages allow for easier and quicker screening of several to hundreds of strains for drug discovery (Chuah et al. 2019; Higgs et al. 2001). Metabolites can vary depending on the substrates present in the medium (Bhuyar et al 2019; VanderMolen et al., 2013). Secondary metabolites, usually produced during stationary growth phase, are non-essential for growth but provide additional pro-survival benefits which can be harnessed for industrial or medical applications (Mora-Villalobos et al. 2020; de Frias et al. 2018; Higgs et al. 2001; Khammee et al. 2020). Hence the selection of culture medium for growing LAB strains affects the secondary metabolites present in the CFS. Culturing LAB in a wider array of non-essential but metabolizable nutrients may improve the production of postbiotic secondary metabolites that can be identified, isolated and purified for bacterial natural product drug discovery. The de Man Rogossa Sharpe (MRS) medium developed in 1960 by de Man et al. is a standard medium used for growing lactobacilli. Since its conception, it has been used for more than half a century by industries and research laboratories in growing and studying probiotic LAB. From its original formulation, MRS broth has been scarcely modified by manufacturers and remain to be comprised of bacto-peptone, beef extract, yeast extract, dextrose, polysorbate 80, ammonium citrate, sodium acetate, magnesium sulfate, manganese sulfate and dipotassium hydrogen sulfate. On the other hand, the McCoy’s 5A medium

is a cell culture basal medium formulated by Neuman and McCoy (1958) while cultivating carcinosarcoma and hepatoma cells and is now commonly used for the culture of widely-distributed cancer cell lines such as HCT-116, HepG2 and primary cells. Its components contain all necessary nutrients for cellular growth such as non-essential amino acids, B vitamins and carbohydrate sources. Unlike in MRS broth formulation which derives these components from undefined amounts from yeast and beef extracts, McCoy’s 5A medium formulation is highly defined. Moreover, the production of this medium involves rigorous tests for endotoxins by manufacturers (Patterson and Dell’orco 1978). McCoy’s 5A also contains high concentrations of components that have not been defined in MRS broth such as folic acid and ascorbic acid, which are precursors that influence the growth and metabolism of selected LAB strains (Revuelta et al. 2018; Yao et al. 2018; Rossi et al. 2011; Sybesma et al. 2003). Several studies employ the method of extracting cell-free supernatants (CFS) from MRS-grown LAB and directly adding these to cultured cancer cells (Chuah et al. 2019; Shyu et al. 2014; Escamilla et al. 2012). Other studies utilized MRS broth to expand the LAB cultures but later resuspended the live cells in cell culture medium such as DMEM or RPMI and incubated them at various time periods prior to CFS collection (Lee et al. 2019; De Marco et al. 2018; Kahouli et al. 2015). Such variations in culture and CFS collection methods can contribute to the inconsistency of screening probiotic metabolites in vitro against cancer cells. In the Philippines, numerous bacterial strains isolated from local food items have been reported to have medicinal potential (Saguibo et al. 2019; Banaay et al. 2013; Dalmacio et al. 2011). However, very few of these strains have been studied for anticancer properties. In this study, we investigate the growth and cytotoxic properties of two LAB strains Lactobacillus plantarum BS-25 (LPBS25) and Pediococcus acidilactici S3 (PAS3) previously isolated from Philippine local fermented food products, Balao-Balao (shrimp-rice mixture) and longganisa (fermented sausage), respectively (Banaay et al. 2004; Banaay et al. 2013). This study is the first report of cytotoxic activity of these two locally-isolated LAB strains against colorectal cancer cells. Initially, we demonstrate that MRS broth has a significant cytotoxic effect on HCT-116 colorectal cancer cells and may compound with the effects coming from LAB CFS. These results present an issue in overestimating the cytotoxic effects of MRS-extracted LAB CFS and implies reconsideration in using other media formulations that minimize the impact of vehicle media. Hence, in this study we aimed to: compare the growth of LPBS25 and PAS3 in MRS broth, McCoy’s 5A medium and a combination medium (CM) composed of 10% MRS broth and 90% McCoy’s 5A medium, determine whether these growth media can impact the effects of LAB CFS on HCT-116 colorectal cancer cells, and provide a more accurate measure of postbiotic cytotoxicity by minimizing vehicle-mediated effects. Briefly, we report that CM as a vehicle consistently did not impact the viability of HCT-116 cells through various concentrations and that CM-grown LAB CFS exerted significant cytotoxic effects. We show that through decreasing the compounding effects of MRS broth as a vehicle, we can increase the range of detecting cytotoxic activity for more accurate screening of LAB for natural product discovery. Ultimately, this study provides a more accurate method for cytotoxic screening of postbiotics using a combination of two common commercially-available growth media.


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MATERIALS AND METHODS Lactic Acid Bacteria (LAB) Strains Two strains of Lactic Acid Bacteria (LAB), Lactobacillus plantarum BS25 (LPBS25) and Pediococcus acidilactici S3 (PAS3), previously isolated from fermented rice-shrimp mixture “balao-balao” and fermented sausage “longganisa” respectively, were provided by Dr. Francisco B. Elegado, National Institutes of Molecular Biology and Biotechnology (BIOTECH), University of the Philippines Los Banos, Laguna (Banaay et al., 2004; Elegado et al., 2004). These strains were revived in Lactobacilli de Man, Rogosa Sharpe (MRS) broth (Difco Laboratories, Detroit, Michigan) and incubated at 37°C for 24 hours under microaerophilic conditions prior to experiments. Purity was monitored through Gram staining and catalase test. Determination of Growth Curves and Colony-Forming Units Revived LAB strains (LPBS25 and PAS3) were adjusted to approximately 0.1 optical density reading at 620nm (OD620) and inoculated in 10mL MRS broth or in 1mL MRS and 9mL McCoy’c 5A combination medium . To measure growth across time, 100-uL samples were obtained from each of the tubes at various time points (0, 1, 2, 6, 10, 24 and 48h) and spectrophotometrically measured at 620nm. To track LAB viability, 100-uL samples were serially diluted in 900uL PBS up to 10-8 dilution. The three highest dilutions (10-6 to 10-8) were spread plated in duplicates on MRS agar (Difco Laboratories) at a volume of 100uL and incubated at 37°C in microaerophilic conditions. After 48 hours, colonies were counted and CFU/mL was computed using the following formula:

𝐶𝐶𝐶𝐶𝐶𝐶/𝑚𝑚𝑚𝑚 = (𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎 𝑎𝑎𝑎𝑎𝑚𝑚𝑣𝑣𝑣𝑣 𝑝𝑝𝑚𝑚𝑎𝑎𝑝𝑝𝑎𝑎 𝑐𝑐𝑐𝑐𝐶𝐶𝑐𝑐𝑝𝑝𝑐𝑐) ∗ 𝑣𝑣𝑣𝑣𝑚𝑚𝐶𝐶𝑝𝑝𝑣𝑣𝑐𝑐𝑐𝑐 𝐶𝐶𝑎𝑎𝑐𝑐𝑝𝑝𝑐𝑐𝑎𝑎

𝑉𝑉𝑐𝑐𝑚𝑚𝐶𝐶𝑚𝑚𝑎𝑎 𝑝𝑝𝑚𝑚𝑎𝑎𝑝𝑝𝑎𝑎𝑣𝑣

Interpolation of CFU/mLvalues at the highest OD620nm readings were done using linear correlation according to Beer-Lambert’s law (Lin et al., 2010). Collection of Cell-Free Supernatants (CFS) Revived LAB strains (LPBS25 and PAS3) were loop-inoculated (10uL) in 10-mL pure MRS broth and 10-mL pure McCoy’s Medium 5A (duplicate) and 10-mL of MRS-MC5A 10:90 combination and incubated at 37°C in microaerophilic conditions. After incubation, cultures were centrifuged at 13,000 RPM for 3 minutes and syringe-filtered using 0.22 μm-pore membranes. CFS collection was done 72 hours after inoculation, corresponding to death phases of LPBS25 and PAS3 (determined previously through plate counts). Prior to use, all CFS were adjusted through dilution in vehicle medium to the corresponding value of OD620 = 0.11 reading (corresponding to the lowest read among all cultures) Cell Culture All protocols involving the use of human cell lines were conduc- -ted following the approval of the UP Manila Research Ethics Board. Colorectal cancer cell line HCT 116 (ATCC CCL-247, American Type Culture Collection, Manassas, Virginia) was maintained in McCoy’s 5A medium supplemented with 10% heat-inactivated Fetal Bovine Serum (Gibco, Tokyo, Japan), 1.5% sodium bicarbonate (Cat#25080-094, Gibco, Tokyo, Japan), and 0.1 mL Gentamicin (Cat#15710-064, Gibco, Tokyo, Japan) in T-25 cell culture-treated flasks at 37° C, 5% CO2 and 95% relative humidity. Cells were routinely passaged or seeded in 96-well plates for MTT assays upon reaching 70-80% confluence.

MTT Viability Assay Based on the protocol by Mosmann (1983) with slight modifications, 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay was performed on the HCT-116 cells treated with LAB CFS. This colorimetric assay is based on the reduction of the yellow MTT compound to purple formazan crystals catalyzed by NAD(P)H-dependent oxidoreductase enzymes produced by metabolically active cells (Vistica et al., 1991). Following this reaction, formazan concentration is measured spectrophotometrically and linearly correlated to cellular mitochondrial activity which presents a reliable estimate for overall viable cell count (Stockert et al., 2018). Prior to treatment, HCT-116 cells were seeded at 8 x 103 cells per well and incubated overnight to allow for attachment. Cells were then treated with 2-fold dilutions of vehicle (MRS/McCoy’s) and CFS ranging from 50% to 1.5625% v/v and incubated at 37°C, 5% CO2 and 95% relative humidity. After 72 hours, all spent media were discarded and replaced with 20uL of 5mg/ml MTT dye solution (VWR, Lutterworth, United Kingdom) and incubated at 37°C. After 4 hours, 150uL dimethyl sulfoxide (DMSO) was added to each well to solubilize the formazan crystals. Absorbances were then read at a wavelength of 570nm using Varioskan Flash microplate reader (Thermo Fisher Scientific, US). Inhibitory concentrations at 50% (IC50) for each treatment were computed using GraphPad Prism 8. Based on the methods of Ahmad et al. (2018), percent viability was obtained using the formula:

% 𝑉𝑉𝑣𝑣𝑎𝑎𝑉𝑉𝑣𝑣𝑚𝑚𝑣𝑣𝑝𝑝𝑉𝑉 = (𝑂𝑂𝑂𝑂570 𝑝𝑝𝑎𝑎𝑎𝑎𝑎𝑎𝑝𝑝𝑚𝑚𝑎𝑎𝑐𝑐𝑝𝑝 / 𝑂𝑂𝑂𝑂570 𝑐𝑐𝑐𝑐𝑐𝑐𝑝𝑝𝑎𝑎𝑐𝑐𝑚𝑚) ∗ 100 While percent cytotoxicity was obtained using the formula:

% 𝐶𝐶𝑉𝑉𝑝𝑝𝑐𝑐𝑝𝑝𝑐𝑐𝐶𝐶𝑣𝑣𝑐𝑐𝑣𝑣𝑝𝑝𝑉𝑉 = 100− % 𝑉𝑉𝑣𝑣𝑎𝑎𝑉𝑉𝑣𝑣𝑚𝑚𝑣𝑣𝑝𝑝𝑉𝑉 Statistical Analysis All experiments were performed at least in three independent trials. Statistical significance between two means was computed using nonparametric T-test (Kruskal-Wallis) at alpha = 0.05, while significant differences among three or more means were determined using one-way ANOVA at alpha = 0.05. Curve-fitting to obtain inhibitory concentrations at 50% (IC50) was done using asymmetric sigmoidal 5-parameter (5PL) function set at 99% confidence interval. All analyses were performed using GraphPad Prism 8 statistical software. P < 0.05 was considered significant. RESULTS AND DISCUSSION MRS broth is significantly cytotoxic to HCT-116 cancer cells at concentrations above 12.5% volume/volume During initial screening of cytotoxic properties of cell-free supernatants (CFS) from Lactobacillus plantarum BS25 (LPBS25) and Pediococcus acidilactici S3 (PAS3), we found that MRS broth on its own exerted significant cytotoxic properties on HCT-116 colon cancer cells at high concentrations (Figure 1). At 12.5% v/v and above, MRS broth (through previously neutralized to pH 7.0 with NaOH) caused a decrease in the viability of HCT-116 cells after 72 hours incubation measured through MTT colorimetric assay. Although concentrations may be adjusted to levels with no significant effect from MRS vehicle, it may compound with and lead to the overestimation of the cytotoxic potential of LAB CFS. Furthermore, the formulation of MRS broth itself is highly variable, containing complex undefined components such as yeast and beef extracts. Yeast extract, particularly, has been previously shown to exhibit cytotoxic potential on its own against various cell lines (Shamekhi et al. 2019; Moon et al. 2019). Following this result, we formulated a possible culture


medium combination that would support ample growth of LAB Figure 1: MRS broth on its own is cytotoxic to HCT-116 cancer cells at concentrations above 6.25 % v/v. MTT assay was performed to assess the influence of pure MRS broth on HCT-116 colon cancer cells after 72 hours incubation at 37°C, 5% CO2 and 95% relative humidity. A: Computed percent viabilities of MTT absorbance readings (570nm) of MRS-treated HCT-116 cells relative to those of untreated controls show that cell viability significantly decreases at 12.5% v/v and above. B: Documentation through phase-contrast microscopy shows that HCT-116 cells exhibited clear morphological signs of cell death such as rounding up and detachment from culture wells after 72 hours of incubation. C: Untreated HCT-116 cells exhibit typical epithelial morphology after 72 hours of incubation. Bars show means of three independent trials. Error bars are standard deviation of means. Bars with different letters are significantly different (P < 0.05).

Figure 2: Comparison of the growth of two LAB strains Lactobacillus plantarum BS-25 (LPBS25) and Pediococcus acidilactici S3 (PAS3) in pure MRS broth and MRS combined with McCoy’s 5A (MC5A) medium at 10:90 v/v ratio. LAB strains were inoculated at 1 mL in 9mL MRS or MC5A and sampled at various time points (0, 1, 2, 4, 10, 24 and 48 hours). To determine LAB growth and total biomass in each medium across time, turbidity was measured through spectrophotometric reading (optical density, OD) at a wavelength of 620nm. To determine viability of the LAB strains at each time point, samples were spread-plated in MRS agar and incubated for 48 hours prior to counting of colony-forming units and calculation of cfu/ml. A: Growth and total biomass of LPBS25 in 10:90 MRS-MC5A is evident but lower than MRS broth. B: Growth and total biomass of PAS3 in 10:90 MRS-MC5A is evident but lower than MRS broth. C: Growth of LPBS25 in 10:90 MRS-MC5A exhibited an earlier logarithmic phase and earlier decline of viable population than LPBS25 grown in pure MRS broth. D: Growth of PAS3 in 10:90 MRS-MC5A also exhibited an earlier logarithmic phase and decline of viable population is similar to PAS3 grown in pure MRS broth. Data points are means of three independent trials. Error bars are standard deviation of means.


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medium combination that would support ample growth of LAB while minimizing MRS broth concentration upon addition to cancer cell cultures. The LAB stains were able to survive and appreciably grow in 10:90 MRS-MC5A combination media (CM) LAB strains inoculated in pure de Man Rogosa Sharpe (MRS) broth and a 10:90 combination of MRS and McCoy’s 5A medium exhibited different growth rates (Figure 2). Based on the highest spectrophotometric readings at 620nm, LPBS25 proliferated up to ~2.1 x 1011 cfu/ml in pure MRS broth and up to ~1.1 x 1011 cfu/ml in CM, while PAS3 proliferated up to ~2.7 x 1011 cfu/ml in pure MRS broth and up to ~1.0 x 1011 cfu/ml in CM. No observable turbidity was observed in pure McCoy’s 5A medium even after 48 hours (data not shown). Expectedly, total biomass was highest in MRS broth as a medium containing nutrients and components optimized for expansion of numerous LAB species (de Man et al., 1960), while CM produced approximately half of this biomass for both LAB strains. Similarly, based on the time-course spectrophotometric readings, CM supported slower growth of the LAB strains compared to growth in pure MRS broth (Figure 2 A,B). However, densities and viable cell counts for CM-grown LAB logarithmically increased across time, showing the capability of the formulation to support primary metabolism of the LAB strains. Interestingly, based on plate counts for colony-forming units, cultures in CM entered the logarithmic phase slightly earlier than those in pure MRS broth (Figure 2 C,D). This head start might be due to the presence of unique components MC5A. Cultures in both media eventually showed similar stationary growth phases, peaking at 12 hours and continuing until 24 hours. Death phase was clearly observed for all cultures at 48 hours (Figure 2 C,D). Altogether this demonstrates that the 10:90 MRS-MC5A combination medium, although less efficient than pure MRS broth, supports the survival and proliferation of LAB strains. Collection of CFS at 24-hour and 72-hour LAB cultures has no significant effect on cytotoxicity in HCT-116 cells To determine whether the growth phase of the LAB cultures would affect the cytotoxic activities, we collected CFS from a

24-hour time point and a 72-hour time point to represent the stationary and death phases of each culture. Generally, in these two growth phases, bacterial secondary metabolites such as cytotoxic and antibiotic byproducts would be present in the culture supernatants. We treated HCT-116 cells with MRS-grown LAB CFS collected from these two time points and performed MTT colorimetric assay to assess cell viability. We found that there were no significant differences in cytotoxicity between LAB CFS from 24-hour and 72-hour collection points (Figure 3). We thus proceeded with the succeeding experiment by collecting supernatants from the 72-hour time point. Supernatants from LAB strains grown in MRS broth and 10:90 MRS-MC5A combination medium (CM) exhibited differential cytotoxicity against HCT-116 colon cancer cells To detect whether there is an effect in anticancer activities of LAB grown in different growth media, cell-free supernatants (CFS) were collected from 72-hour LAB cultures grown in pure MRS broth, pure MC5A and 10:90 MRS-MC5A combination. These CFS were then added to the colon cancer cell line HCT-116 in 2-fold dilutions ranging from 50% down to 1.5625% v/v, incubated for 72 hours and terminated using MTT viability assay to measure cytotoxicity. Although pure MC5A vehicle expectedly did not exert any significant effects on HCT-116 cells, CFS collected from MC5A-grown LAB produced minimal cytotoxic effects at the highest concentration of 50% v/v (18.58% and 13.95% cytotoxicity from LPBS25 and PAS3, respectively) (Figure 4A, D). This is possibly due to a low amount of metabolites stemming from the lack of LAB growth in this medium. MRS vehicle, as shown previously (Figure 1), was significantly cytotoxic to HCT-116 viability at several concentrations (50%, 25% and 12.5% v/v), while MRS-grown LAB CFS were detected to exert significant cytotoxic effects only at 25% v/v (62.41% and 50.67% cytotoxicity from LPBS25 and PAS3, respectively) (Figure 4B, E). At 50% v/v, the MRS vehicle induced global cell death as confirmed through phase-contrast microscopy wherein HCT-116 cells were predominantly rounded and detached from culture wells (Figure 4G). Due to this, any effects from the LAB CFS were not detected as seen from the computed % cytotoxicity of nearly zero for both LAB strains (Figure 4E). On the other hand, even at the highest concentration (50 %v/v), the CM vehicle had no

Figure 3: Harvesting cell-free supernatants (CFS) from either stationary- and death-phase cultures in MRS broth does not affect cytotoxic activities of CFS from LPBS25 and PAS3. LAB were inoculated in 100% MRS broth and cultured for 24 and 72 hours prior to collection of CFS through centrifugation and filtration. CFS were added to HCT-116 cells at varying concentrations. MTT colorimetric assay was performed to measure absorbances at 570nm and compute for cell viability relative to untreated controls. Bars show means of three independent trials. Error bars are standard deviation of means.


Figure 4: Cell-free supernatants (CFS) extracted from two LAB strains cultured in different media show differential cytotoxicity. Lactobacillus plantarum BS-25 (LPBS25) and Pediococcus acidilactici (PAS3) were inoculated in pure MRS broth, pure McCoy’s 5A medium (MC5A) and in a combination medium (CM) of 10:90 MRS-MC5A and incubated at 37°C for 72 hours. CFS were collected through centrifugation and filtration and added to HCT-116 cells (seeded at 80,000 cells/well the day prior) at various concentrations (% v/v). To measure viability of HCT-116 cells after treatment, MTT colorimetric assay was performed and resulting absorbances were read spectrophotometrically at a wavelength of 570nm. A-C: Absorbance readings of HCT-116 cells after treatment with CFS from pure MC5A, pure MRS and CM show varying effects across several concentrations. D-F: Computed percent cytotoxicity of LAB CFS relative to their corresponding vehicles. G: Phase-contrast images of HCT-116 cells in all set-ups at 50% v/v treatment after 72-hour incubation. Bars show means of four independent trials. Error bars are standard deviation of means. *statistically significant (P < 0.05)


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cytotoxic effect on HCT-116 cells (Figure 4C) as observed through typical morphologies of HCT-116 cells. Furthermore, CM-grown LAB CFS exerted significant cytotoxicity at 50% v/v (54.29% and 63.23% cytotoxicity from LPBS25 and PAS3, respectively) and at 25% v/v (23.56% and 14.36% cytotoxicity from LPBS25 and PAS3, respectively). Additionally, effects observed in CFS from MRS-grown and CM-grown LAB vary at 25% v/v, wherein the former has much higher % cytotoxicity (Figure 4 E,F). Computed IC50 values for MRS-grown LPBS25 and PAS3 supernatants are 12.51 and 12.71 %v/v, respectively, while IC50 values for CM-grown LPBS25 and PAS3 supernatants are 25.28 and 54.52 % v/v respectively. Lower IC50 values among the MRS-grown LAB CFS denote that these are more cytotoxic compared to CM-grown LAB CFS. It can be inferred that pure MRS broth as a vehicle adds a compounding factor on the effects of LAB CFS by rendering HCT-116 cells more susceptible to cell death due to its inherent cytotoxic properties. Based on these observations, it is likely that the use of pure MRS broth in growing LAB and collecting CFS may lead to overestimation during cytotoxicity screening. On the other hand, CM as a vehicle showed no significant effects on HCT-116 cells across all the concentrations tested. Even at the highest concentration of CM (50% v/v), the MRS component is minimized to a total of 5% v/v which is below the cytotoxic range of pure MRS broth as shown previously (Figure 1). Parallel to this, significant cytotoxic effects are seen from CM-grown LAB CFS with a clearer correlation between concentration and cytotoxicity. CONCLUSION Overall, our study demonstrates the efficiency of 10:90 MRS broth-McCoy’s 5A as a reliable growth medium in harvesting potential anticancer postbiotics present in lactic acid bacteria supernatants and as a vehicle with minimal compounding effects on colorectal cancer cells. ACKNOWLEDGMENTS This study was funded by National Institutes of Health, University of the Philippines Manila (NIH Grant no. 2018-008) and the Office of the Vice-Chancellor for Research and Development, University of the Philippines Diliman (PhDIA Grant no. 181818). All data presented in this study are authentic. The authors would like to thank Dr. Francisco B. Elegado from the University of the Philippines Los Baños for providing the LAB strains used in this study. CONFLICTS OF INTEREST The authors declare no conflict of interest. CONTRIBUTIONS OF INDIVIDUAL AUTHORS RJL Asi conceptualized and performed most experiments and drafted the manuscript. CLD Badua and JBD Atun performed experiments for the optimization of the proposed growth medium and establishment of cytotoxic profiles of the two LAB strains. JIL Lapus and SMO Lopez performed experiments for establishment of growth curves and comparison of growth phase collection points. SD Jacinto is principal investigator of the Mammalian Cell Culture Laboratory (UP Diliman) and provided overall guidance in cancer cytotoxicity. ARF Mazahery is co-project leader of the Mammalian Cell Culture Laboratory and

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An optimized de Man Rogosa Sharpe- McCoy’s 5A medium