Adhesion Properties of Indigenous Dadih Lactic Acid Bacteria on Human Intestinal Mucosal Surface

Dadih is Indonesian traditional fermented buffalo milk believed by the natives to have beneficial effects on human health. This may be due to the probiotic properties possessed by the lactic acid bacteria (LAB) involved in its fermentation process. It was discovered that ten strains of dadih lactic isolates possessed some probiotic properties in vitro. In this study, the adhesion properties of dadih LAB, in comparison with documented probiotic strains, were investigated in vitro by using mucin extracted from human faeces and Caco-2 cells as the models for human intestinal mucosal surface and intestinal cells respectively. The adhesion results showed the distinction of Lactobacillus reuteri IS-27560 in adhering to both mucus layer and Caco-2 cells. The competition assay for adhesion to the mucus layer between dadih LAB and selected pathogens indicated the competence of Lactococcus lactis IS-16183 and Lactobacillus rhamnosus IS-7257 in significantly inhibiting the adhesion of Escherichia coli O157:H7. Accordingly, these two strains may be potential candidates for use as probiotic strains. Overall, the adhesion properties of all dadih LAB strains were relatively comparable to that of Lactobacillus casei Shirota and Lactobacillus rhamnosus GG, the documented probiotic strains. (Asian-Aust. J. Anim. Sci. 2006. Vol 19, No. 5 : 751-755)


INTRODUCTION
The emerging of probiotics these days is predominantly driven by the demands of consumers for foods that are able to provide other benefits for health beyond their basic nutritional value.Salminen et al. (1999) defined probiotics as microbial cell preparations or components of microbial cells that have a beneficial effect on the health and wellbeing of the host.Many lactic acid bacteria (LAB) strains have been classified as probiotics due to their beneficial effects towards human health; in particular, the gastrointestinal (GI) tract.Those reported include prevention of colon cancer, reduction of cholesterol levels, inhibition of pathogenic microflora, stimulation of immune response, reduction of constipation, enhancement of lactose digestion in lactase deficient subjects as well as alleviation of food allergy (Salminen et al., 1998;Kaur et al., 2002;Ouwehand et al., 2002;Saarela et al., 2002).
There are a few criteria in selecting potential probiotic strains as illustrated by Saarela et al. (2000), and one of them is the ability of the bacteria to adhere to epithelial cell surfaces.Adhesion of probiotic strains on mucosal surface is one of the most important probiotic properties because it is often recognized as the prerequisite for colonization of the human GI tract (Beachey, 1981).Besides, adhesion is also a means of competitive exclusion of the pathogenic bacteria from the intestinal epithelium; and the adherent bacteria strains induce immune effects more effectively and stabilize the intestinal mucosal barrier from invasion by pathogens (Salminen et al., 1996).
Dadih is Indonesian traditional fermented buffalo milk and is believed by the natives to be beneficial for consumers' health.This might be contributed by the probiotic properties exerted by the indigenous lactic acid bacteria present in dadih.Some studies on probiotic properties of indigenous lactic acid bacteria isolated from dadih have shown to exhibit antimutagenic, hypocholesterolemic, acid and bile tolerance as well as antipathogenic properties (Hosono et al., 1990;Surono and Hosono, 1996;Surono, 2003;Pato et al., 2004).
In this study, the adhesion properties of 10 LAB strains isolated from dadih on human intestinal mucosal surface were investigated.Two probiotic strains, Lactobacillus casei strain Shirota (LCS) and Lactobacillus rhamnosus strain GG (LGG), were used for comparison study.The adhesion assays were carried out in vitro by using mucin extracted from human faeces and Caco-2 cells as the models for intestinal surface and intestinal cells respectively.Furthermore, the ability of dadih LAB to compete with the intestinal pathogens, namely Escherichia coli O157:H7, Salmonella typhimurium E10 and Helicobacter pylori, in adhesion on mucin was also observed.

Bacterial strains
The indigenous LAB were isolated from Bukit Tinggi

Caco-2 cells
The Caco-2 cells (obtained from American Type Culture Collection, Rockville, USA) were cultured in the Minimal Essential Medium (MEM) with Earle's salts that contained 25 mM glucose, supplemented with 20% (vol/vol) heatinactivated (30 min, 56°C) Fetal Bovine Serum, 2 mM Lglutamine, Penicillin G (100 U/mL), Streptomycin (100 µg/mL), 1 mM sodium pyruvate, 0.1 mM non-essential amino acids (all from GIBCO-BRL, USA) and 0.15% NaHCO 3 (Merck, Germany).The cells were cultured in the 75 cm 2 tissue culture flask (Falcon, England) for 14±2 days at 37°C in 5% (vol/vol) CO 2 in air atmosphere to become a confluent layer.For adhesion assays, monolayer Caco-2 cells were transferred to 24-well tissue culture plates (Nunclon, Denmark) at a concentration of approximately 1×10 5 viable cells per well in 1 mL of culture medium.The cells were cultured for another 14±2 days to obtain their confluence prior to adhesion assay.The culture medium was replaced every 2 to 3 days.

Human intestinal mucus
The mucin was extracted from faeces of healthy adults by extraction and dual ethanol precipitation (Kirjavainen et al., 1998).Prior to adhesion assays, the crude mucin extract was dissolved in Hank's balanced salt solution (GIBCO) containing HEPES (N-2-hydroxyethylpiperazine-N'-2ethanesulfonic acid, Sigma) at pH 7.4-7.6(HH buffer) to remove intestinal contents.A hundred µl of mucin dissolved in HH buffer (0.05% wt/vol) was then passively immobilized to each well of polystyrene Maxisorp microtiter plate wells (Nunc, Denmark) for 18±2 h at 4°C in air atmosphere.

In vitro adhesion assays
The adhesion assays on mucus layer and Caco-2 cells were carried out by incubating the radioactive labelled bacterial cells (labelled with 113 Ci mmol -1 of [methyl, 1', 2'-3 H] Thymidine obtained from Amersham, UK) on immobilized mucin and on Caco-2 cells respectively for 1 h at 37°C.After being washed to remove the free bacterial cells, the adhesions of LAB on mucus layer and Caco-2 cells were determined from their radioactivity measurement by the scintillation counter (Beckman-LS-6500).Similarly, the competition assay in adhesion on mucus layer was performed by labelling either LAB or pathogens with radioactive 3H and incubating them on immobilized mucin for 1 h at 37°C.The degree of adhesion was expressed as colony forming unit per well (cfu/well).The degree of competition between LAB and pathogens was expressed in percentage of change, by determining the changes in adhesion.

Statistical analysis
The data were analyzed by using SPPS program 11.0 for Windows.Student's t-test or the least significant difference (LSD) test after analysis of variance (ANOVA) was used to identify the differences in the competition assay.A p value <0.05 is considered statistically different at 95% confident level.

Adhesion of LAB on Caco-2 cells
The chart in Figure 1 indicates that all dadih LAB strain had the ability to adhere on Caco-2 cells to various extents, when 5×10 7 cfu of LAB were added to a well containing approximately 1×10 5 viable Caco-2 cells.The number of bacteria adhered on Caco-2 cells varied for each LAB strain and it ranged from 2.4×10 5 to 8.4×10 5 cfu/well or around 0.5% to 1.7% of the LAB added.Among dadih LAB strains, Lb. reuteri IS-27560 had the highest number of bacteria adhered on Caco-2 cells.The results also showed that LGG had approximately 1% adhered bacteria on Caco-2 cells while LCS had 0.33%.In general, dadih LABs were comparable with LGG and LCS in adhering on Caco-2 cells.

Adhesion of LAB on mucin
Each dadih LAB strain also showed various degree of adhesion on mucin when 5×10 6 cfu of LAB was added to a well containing immobilized mucin without the presence of pathogens.Figure 2 shows that the number of dadih LAB adhered on mucin ranged from 1.7×10 5 to 6.0×10 5 cfu/well or about 3.5% to 12% of the bacteria added, with Lb. reuteri IS-27560 as the most adherent strain.It is also shown that the adhesion on mucin of all dadih LAB strains, except Lc. lactis IS-11857, were significantly higher compared with that of LCS and LGG, which had about 1% adhesion.

Competition in adhesion between LAB and pathogens on mucin
In the competition assay, the adhesions of most dadih LAB strains were generally not affected by the presence of pathogens substantially at 95% confident level (p>0.05) as shown in Figure 2.There was also no significant change observed in the adhesions of all LAB in the presence of H. pylori (data not shown).This might be due to the different sites of adherence between the pathogens and most LAB strains at the receptors on mucus layer, and they did not affect the adhesion of each other.Nonetheless, there were some LAB strains, whose adhesion changed notably in the presence of E. coli and S. typhimurium (p<0.05).
In the presence of E. coli, the adhesions of Lc. lactis IS-11857 and LCS were reduced significantly (p<0.05) when 5x10 6 cfu of radioactive-labelled LAB and equal number of E. coli were added to a well containing immobilized mucin.The number of Lc. lactis IS-11857 adhered on mucin was reduced from 1.7×10 5 cfu/well to 1.2×10 5 cfu/well in the presence of E. coli while it was from 6.8×10 4 cfu/well to 4.6×10 4 cfu/well for LCS.Both LAB had the reduction in adhesion of about 30% in the presence of E. coli.Moreover, the adhesions of Lc. lactis IS-11857 and Lb.brevis IS-23427 were reduced significantly by 40% and 20% correspondingly in the presence of S. typhimurium when equal amount of radioactive-labelled LAB and pathogen (5×10 6 cfu/well) was added.In this case, it could be hypothesized that the pathogens were competing for adhesion sites directly with those LAB strains whose adhesions on mucin extract were reduced significantly.
The effect of LAB on the adhesion of pathogens on mucin was confirmed by determining the capability of LAB to reduce the number of pathogens adhered on the mucosal surface.The pathogens were labelled with the radioactive 3H, and equal amount of LAB and pathogens was added to the mucin.Figure 3 shows that the adhesion of E. coli on mucus was reduced significantly from 6.7×10 4 cfu/well to 5.2×10 4 cfu/well and 4.1×10 4 cfu/well in the presence Lc. lactis IS-16183 and Lb.rhamnosus IS-7257 respectively.The inhibition of adhesion might eventually prevent the invasion of the E. coli on the intestine since adhesion of pathogens is the primary step of pathogenesis (Ouwehand et al., 1999).However, none of dadih LAB was capable of reducing the adhesion of both S. typhimurium and H. pylori notably.
Unexpectedly, the adhesion of Enterococcus faecium IS-27526 increased significantly (p<0.05) up by 33% from 2.1×10 5 cfu/well to 2.8×10 5 cfu/well in the presence of E. coli (Figure 2).On the other hand, this bacterial strain also enhanced the adhesions of S. typhimurium and H. pylori considerably up by 32% and 44% correspondingly (Figure 3).This might be due to the formation of aggregates between the dadih LAB and pathogens.This was presumed to be another mechanism by which probiotics prevent the attachment of pathogens directly on the intestinal surface (Bibiloni et al., 1999;Ouwehand et al., 1999;Tuomola et al., 1999).Nevertheless, further research needs to be carried in order to confirm the ability of Enterococcus faecium IS-27526 to co-aggregate with pathogens.
The adhesion assay performed on mucus layer and Caco-2 cells depicted the distinction of Lb. reuteri IS-27560 in adhering on both substrata.The competence of both Lc.lactis IS-16183 and Lb.rhamnosus IS-7257 in competing with E. coli was also revealed in this study.Ultimately, the adhesion properties of dadih LAB strains were indeed comparable to that of the documented probiotic strains and hence, are potential novel probiotic strains.

Adhesion Properties of Indigenous Dadih Lactic Acid Bacteria on Human Intestinal Mucosal Surface
carried out at 37°C in air atmosphere for 18±2 h before the adhesion study.H. pylori was cultured in the Brain Heart Infusion (BHI) broth (BBL, USA) supplemented with 0.4% (wt/vol) yeast extract (Oxoid, England) and 10% (vol/vol) horse serum.It was incubated at 37°C in the microaerophilic condition for 3 days prior to adhesion study.