CHAPTER 8 IN VITRO CHARACTERIZATION OF LACTIC ACID BACTERIA STRAINS FOR PROBIOTIC CHARACTERISTICS

CHAPTER 8 IN VITRO CHARACTERIZATION OF LACTIC ACID BACTERIA STRAINS FOR PROBIOTIC CHARACTERISTICS 8.1. Introduction Probiotic bacteria must overcome physical and chemical barriers such as acid and bile in the gastrointestinal tract in order to provide health benefits (Liong & Shah, 2005). The highly acidic condition of the stomach requires that the organism should have a high tolerance to acid. This is frequently measured by evaluating its ability to survive ph 3 or lower for 3 h, an average passage time through the stomach. Similarly, bacterial strains need sufficient tolerance to bile to enable safe passage through the duodenum to their site of action (O Sullivan et al., 2001). Previous studies have confirmed that some lactobacilli could lower total cholesterol and low-density lipoprotein (LDL) cholesterol (Anderson & Gilliland, 1999; Sanders, 2000). Milk fermented with lactobacilli was first demonstrated to exhibit hypocholestrolemic effects in humans as early as 1963 (Sharp et al., 1963). The precise mechanism of cholesterol reduction by probiotic bacteria remains skeptical. However, certain strains of Lactobacillus acidophilus were found to secrete bile salt hydrolase, which catalyzes the hydrolysis of glycine- or taurine-conjugated bile salts into amino acid residues and free bile acids (Corzo & Gillilan, 1999). Free bile salts are less soluble than conjugated bile salts, resulting in lower absorption in the intestinal lumen. Deconjugation of bile acids can reduce serum cholesterol levels by increasing the formation of new bile acids that are needed to replace those that have escaped the enteropathogenic circulation (Reynier et al., 1981). It was also postulated that certain strains of lactobacilli assimilate or incorporate some of the cholesterol removed from medium into the cellular membranes during 114

growth (Noh et al., 1997). As a result cholesterol incorporated into or adhered to the bacterial cells would be less available for absorption from the intestine into the blood. Adhesion of lactic acid bacterial strains to the intestinal surface and the subsequent colonization of the human gastrointestinal tract have been suggested as an important prerequisite for probiotic action. Adherent strains of probiotic bacteria are likely to persist longer in the intestinal tract and therefore have a better possibility to show metabolic and immunomodulatory effects than nonadhering strains. Adhesion provides an interaction with the mucosal surface facilitating the contact with gut associated lymphoid tissue mediating local and systemic immune effects (Saarala et al., 2000). Adhesion may also facilitate competitive exclusion of pathogenic bacteria from the intestinal epithelium: Exclusion of pathogen by lactic acid bacteria has been shown in vitro using CaCo-2 and HT-29 cell lines (Johnson-Henry et al., 2008; Spurbeck & Arvidson, 2010; Alemka et al., 2010; Satish et al., 2011). 8.2. Materials and methods 8.2.1. Bacterial strains, cell line and chemicals Lactic acid bacterial strains Streptococcus phocae PI80, Enterococcus faecium MC13, and Lactobacillus plantarum AS1 isolated in our lab (Gopalakannan, 2006; Satish et al., 2010) were cultured in deman Ragosa Sharpe (MRS) medium for 16 h at 37 C. Adenocarcinoma cell line HT-29 was purchased from National Center for Cell Science (NCCS), Pune, India. Bacteriological media was purchased from HiMedia, Mumbai, India. Cell culture medium and chemicals were purchased from Sigma, USA. Plasticwares were purchased from Tarsons, Kolkatta, India. 115

8.2.2. Bile tolerance Bile tolerance of S. phocae PI80, E. faecium MC13, and L. plantarum AS1 were determined as described (John & Alicia, 2001). Bacterial strains were cultivated in MRS broth at 37ºC for 16 h. Cells were harvested by centrifugation at 5,000 g for 10 min and washed twice 0.1 M phosphate buffer, ph 7.0. Cells were resuspended to the original volume with the buffer by vortexing, 0.5% from this suspension was used to inoculate sterilized MRS and MRSO broth (MRS broth supplemented with 0.05%, 0.1%, 0.15% and 0.3% of bile oxgall) and incubated at 37ºC. Absorbance was read at 560 nm at every two hours for the 24 h of incubation. 8.2.3. Acid resistance Artificial juice (NaCl 0.2%, Pepsin 3.2 g/l, ph 2.0) was prepared and sterilized by filtration (filter membrane 0.22 µm). As a control artificial gastric juice adjusted at a final ph 6.0 with 1N NaOH was taken. It was inoculated with 2% bacterial cell suspension containing 8.929 logcfu/ml viable cells and both media were incubated at 37ºC in an orbital shaker at 100 rpm. Samples were taken at 0, 1, 2, 3 and 4 h and after 24 h for cell viability by plating in MRS agar from 10-fold serial dilution prepared in 0.1% peptone water as described earlier (John & Alicia, 2001). Plates were incubated at 37ºC for 24 h. Results were expressed as colony forming unit per milliliter (CFU/ml). 8.2.4. Cholesterol reduction assay Freshly prepared MRS broth was supplemented with 0.3% oxgall as a bile salt. Water-soluble cholesterol (Sigma, USA) was filter-sterilized and added to the broth at a final concentration of 100 μg/ml, inoculated with each strain (at 1%), and incubated at 37 C for 20 h. After the 116

incubation period, cells were centrifuged and the remaining cholesterol concentration in the broth was determined using a modified colorimetric method as described (Rudel & Morris, 1973). One milliliter of the aliquot was added with 1 ml of KOH (33% w/v) and 2 ml of absolute ethanol, vortexed for 1 min, and heated at 37 C for 15 min. After cooling, 2 ml of distilled water and 3 ml of hexane were added and vortexed for 1 min. One milliliter of the hexane layer was transferred into a glass tube and evaporated under nitrogen. The residue was immediately dissolved in 2 ml of o-phthalaldehyde reagent. After complete mixing, 0.5 ml of concentrated sulfuric acid was added and the mixture was vortexed for 1 min. Absorbance was read at 550 nm (Hitachi, Japan) after 10 min. All experiments were replicated twice. 8.2.5. Adhesion assay Adhesion of S. phocae PI80, E. faecium MC13, and L. plantarum AS1 were assayed as per the method described by Kaushik et al., (2009). Initially 10 5 HT-29 cells per milliliter were seeded in each well of 24-well tissue culture plates. The Dulbecco s modified Eagle s minimal essential medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS), 100 U/ml penicillin, and 100 mg/ml streptomycin was used for culturing. The medium was changed with fresh medium every alternate day. Adhesion assay was done after 15 days of post confluency. The cells were then washed twice with 3 ml phosphate-buffered saline (ph 7.4). Bacterial cultures at concentration 10 8 CFU/ml were suspended in 1 ml DMEM medium (without serum and antibiotics) and added to different wells. The plates were incubated at 37 C for 1 h in the presence of 5% CO 2 /95% air atmosphere. The monolayers were washed with sterile PBS and the cells were detached by trypsinization. One ml of 0.1% trypsin solution was added to each well and incubated for 10 min at room temperature. The cell suspension was plated on MRS agar by 117

serial dilution for determining the adherent bacterial cells. The plates were incubated for 24 h at 37 C and colonies were counted. Bacterial cells initially added were also counted. The results of the adhesion assay were expressed as adhesion percentage, the ratio between adherent bacteria and added bacteria per well. Three independent experiments (n = 3) with two replicates in each experiment with HT-29 cells of same passage were carried out. Concurrently, we performed the cover slip assay to visualize the bacterial cells attached to HT-29 cell line. Sterile cover slips were inserted into 12 well plates. Adhesion assay was followed as mentioned above. Cover slips were fixed with 100% methanol and air dried completely. Further they were cut into 10 10 mm size, mounted on a specimen holder and coated with gold particles. Adhered bacterial cells were visualized using Hitachi S-3400N scanning electron microscope (SEM) (Hitachi, Japan) at 15,000 resolution. 8.3. Results 8.3.1. Bile tolerance by LAB strains S. phocae PI80 didn t show any inhibition to bile salt and growth pattern was normal in all the concentration of bile salt (Fig. 11A). E. faecium MC13 was found to be tolerant of increased bile salt concentration, i.e. 0.05%, 0.1%, 0.15%, and 0.3% as shown by the growth curve of bacteria (Fig. 11B). Initially, the growth of bacteria in MRSO was delayed up to 6 h when compared with MRS broth but thereafter growth rate was similar. So, this showed that E. faecium MC13 could grow normally in the presence of bile salt. Similar, growth pattern was observed in the case of L. plantarum AS1 as well (Fig. 11C). Hence, all the tested strains of lactic acid bacteria showed normal growth in the presence of bile salt concentrations that are found in normal human gut. 118

Fig. 11A. Bile salt tolerance by Streptococcus phocae PI80 Fig. 11B. Bile salt tolerance by Enterococcus faecium MC13 119

Fig. 11C. Bile salt tolerance by Lactobacillus plantarum AS1 8.3.2. Artificial gastric juice tolerance S. phocae PI80 was able to survive artificial gastric juice up to 3 h with retention of 2.6 ± 0.032 logcfu/ml viable cells that was 32.21% of initial population added to acidic medium. E. faecium MC13 was viable for 2 h at ph 2.0 but bacterial count of was decreased compared to control (ph 6.0). Initial count of E. faecium MC13 was 7.049 ± 0.108 logcfu/ml at ph 2.0 and 7.107 ± 0.112 logcfu/ml at ph 6.0 which got reduced to 2.477 ± 0.084 logcfu/ml and 6.982 ± 0.096 logcfu/ml respectively after 2 h of incubation. L. plantarum AS1 was viable at ph 2.0 even after 24 h of incubation however the bacterial count of L. plantarum AS1 was lower compared to control (ph 6.0). Initially when L. plantarum AS1 was inoculated its count was 6.97 ± 0.2 logcfu/ml at ph 2.0 and 8.146 ± 0.16 logcfu/ml at ph 6.0 which was reduced to 6.079 ±.08 logcfu/ml and 7.732 ± 0.26 logcfu/ml respectively after 24 h (Table 13). In other sense 120

there was 12.82% reduction in bacterial count at ph 2.0 compared to 5.08% at ph 6.0. Hence, majority of cells were viable at ph 2.0 even after 24 h of incubation. Table 13. Artificial gastric juice tolerance of S. phocae PI80, E. faecium MC13, and L. plantarum AS1 Incubati- ph 2.0 (logcfu/ml) ph 6.0 (logcfu/ml) on time S. phocae E. faecium L. plantarum S. phocae E. faecium L. plantarum (h) PI80 MC13 AS1 PI80 MC13 AS1 0 8.07 ± 0.04 7.05 ± 0.11 6.97 ± 0.05 8.33 ± 0.1 7.11 ± 0.1 8.15 ± 0.14 1 4.5 ± 0.05 3.51 ± 0.05 6.34 ± 0.016 7.11 ± 0.07 6.97 ± 0.03 7.11 ± 0.09 2 3.45 ± 0.08 2.48 ± 0.08 6.48 ± 0.18 7.05 ± 0.09 6.98 ± 0.1 7.73 ± 0.08 3 2.6 ± 0.032 Nil 6.15 ± 0.08 7.3 ± 0.11 7.25 ± 0.04 7.81 ± 0.12 4 Nil Nil 6.48 ± 0.05 7.97 ± 0.05 7.33 ± 0.06 7.81 ± 0.14 24 Nil Nil 6.08 ± 0.04 --- --- 7.73 ± 0.08 8.3.3. Cholesterol reduction In case of E. faecium MC13 absorbance at 550 nm was 0.298 ± 5 for standard 100 µg/ml cholesterol (uninoculated MRSCHO broth). Absorbance of test sample (inoculated MRSCHO broth) was 0.253±4. Residual cholesterol in the inoculated MRSCHO broth (MRS broth with 0.3% bile salt and 100 µg/ml cholesterol) was determined to be 84.8 µg/ml i.e. total cholesterol reduced or assimilated was 15.2 µg/ml. Hence, E. faecium MC13 reduced cholesterol by 15.2%. S. phocae PI80 reduced or assimilated 20.81% of water soluble lipid present in the MRSCHO broth. However, in the case of L. plantarum AS1 residual cholesterol in the inoculated 121

MRSCHO broth was determined to be 42.7 µg/ml i.e. total cholesterol reduced or assimilated was 57.3 µg/ml (Table 14). Thus, L. plantarum AS1 reduced cholesterol by 57.3%. Table 14. Cholesterol reduction by S. phocae PI80, E. faecium MC13, and L. plantarum AS1 S. No. Bacterial Strain Cholesterol Reduction (%) 1 Streptococcus phocae PI80 20.81 2 Enterococcus faecium MC13 15.20 3 Lactobacillus plantarum AS1 57.30 8.3.4. Intestinal epithelial cell line (HT-29) adhesion assay E. faecium MC13 viable cells adhered to intestinal cell line (HT-29) were enumerated by plating on selective medium (MRS). Post-adhesion E. faecium MC13 count was 4.26 ± 0.24 logcfu/ml which was 51.95% of initially added bacterial population. So, even after washing the cells with PBS thrice there was significant retention in bacterial population. To visualize E. faecium MC13 adhered to HT-29 cell surfaces scanning electron microscope (SEM) analysis was performed. Bacterial cells were observed to be adhering profoundly to the HT-29 cells, thereby providing evidence for significant post-adhesion E. faecium MC13 population. Similarly, post-adhesion count of S. phocae PI80 was enumerated to be 3.98 ± 0.16 logcfu/ml which was 49.75% of initial bacteria added. In case of L. plantarum AS1 adhesion assay with 8 logcfu/ml bacterial concentrations, 4.9 ± 0.08 logcfu/ml i.e. almost 60% of initial population inoculated to HT-29 cell line were retained. This result was further substantiated by scanning electron microscope 122

analysis where significant populations of L. plantarum AS1 were adhered. L. plantarum AS1 was observed to be strongly bound in a diffuse pattern to HT-29 cell line. 8.4. Disussion Earlier in vivo studies on S. phocae PI80, E. faecium MC13 and L. plantarum AS1 showed them as non-pathogenic and potential probiotics strains. For instance, Swain et al., (2009) reported remarkable inhibitory activity of S. phocae PI80 and E. faecium MC13 against vibriosis caused by V. parahaemolyticus and V. harveyi when applied in the shrimp culture of Penaeus monodon. Similarly, Gopalakannan & Arul, (2011) demonstrated significant control in Aeromonas hydrophila infection spread in Cyprinus carpio using E. faecium MC13. Both S. phocae PI80 and E. faecium MC13 were safe on administration to albino rats as well as acted as efficient biopreservatives (Paari et al., 2011a,b). L. plantarum AS1 isolated from Kallappam showed tolerance to wide ph, safe on administration to albino rats at 8 logcfu/ml/day for 30 days. It also decreased the count of pathogenic coliforms, yeast and molds in treated rats compared to untreated control rats (Satish et al., 2010). Nevertheless, its important to follow in vitro experiments to substantiate in vivo results as in vitro assays are better quantification tools. To be a successful probiont, a bacterium must resist harsh conditions persisting in stomach and gut regions, it must be able to colonize intestinal epithelium for its probiotic action. Also, as mentioned in introduction section cholesterol reduction is a special property of probiotic bacteria which is indirectly related to bile salt hydrolase activity of the bacterium. In this study, S. phocae PI80 and E. faecium MC13 tolerated varying concentration of bile salt provided in the growth medium. This effect could be due to the production of bile hydrolase enzyme by the bacterium. They also survived artificial gastric juice up to 3 h and 2 h 123

respectively, but their count decreased drastically as only 32.21% and 35.1% of initial cells were viable after 3 h and 2 h respectively. This finding also point towards the need for protective means or encapsulation for better viability of cells inside a host organism. Adherence of bacteria to the epithelial intestinal cells and the subsequent colonization of the gastrointestinal tract have been suggested as an important property for probiotic action. Adherent strains of probiotic bacteria are likely to persist longer in the intestinal tract (Strompfova & Laukova, 2007). In our study, 49.75% and 51.95% of initial viable cells of S. phocae PI80 and E. faecium MC13 respectively, were found attached to intestinal epithelial cell line (HT-29). Hence, this revealed an efficient adhering capacity of S. phocae PI80 and E. faecium MC13. Similarly, probiotic properties of bacteriocin producing E. faecium strain associated with chicken were reported earlier (Strompfova & Laukova, 2007). This strain survived acid conditions up to 3 h, tolerated bile salt and successfully adhered to different mucous surfaces. Enterococci isolated from dog also showed similar results (Strompfova et al., 2004). E. faecium strains isolated from Chungkukjang, a fermented soy product had shown low ph survival ability and bile tolerance. The tested strains were resistant to low ph condition for 3 h, even though bacterial counts reduced by 2-3 log CFU/ml with a survival rate of 42% to 62%. These strains could tolerate bile up to 30 g/l (Mi et al., 2008). L. plantarum AS1 found to be tolerant to bile but there was initial delay in growth rate. This was probably due to the unfavourable condition conferred by bile in the medium. The bacterium was subsequently able to synthesis bile hydrolase enzyme to breakdown bile in the medium and could survive normally. Kaushik et al., (2009) observed similar results with L. plantarum LP9. Bacterial number decreased by 1 log cycle after 2 h incubation at 37ºC in MRS broth containing 1.5 to 2.0% bile(kaushik et al., 2009). Similarly there was delay in growth rate in 0.3% oxgall containing MRS broth for L. acidophilus strains 124

C28, FR1 and FR2 (Gilliland et al., 1984). L. plantarum AS1 was tolerant to artificial gastric juice at ph 2.0 hence it could be administered orally as food supplement. In other studies LP9 isolate found to be tolerant at ph 2.0 but its initial log (CFU/ml) of 8.9 decreased to 8.4 (Kaushik et al., 2009). Caecal Lactobacilli strain could survive at ph 2.0 for up to 2 h of incubation (Jin et al., 1998). L. plantarum AS1 found to be very effective in cholesterol reduction (up to 57.3%). Cholesterol reduction is a special property of probiotic bacteria and many bacteria have been characterized to possess this property but the range of cholesterol reduction or assimilation varied among the Lactobacilli strains. In one study cholesterol assimilation ranged from 0% for strains ATCC4356 and 14F1 to 50% for strain ATCC43121 (James & Stanley, 1999). L. plantarum AS1 adhered to HT-29 cells in a strong but diffused manner as observed by SEM. The results of above study revealed that generally all three strains are having probiotic potential. Among three strains L. plantarum AS1 was found to be a better candidate as it showed superior probiotic properties. Nevertheless, S. phocae PI80 and E. faecium MC13 which had shown effective anti-vibriosis properties could be encouraged to be used as aquaculture probiotics. L. plantarum AS1 must be further employed in applied probiotic research as it could be a possible human probiont. 125