Exhibited antimicrobial activity

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Abstract

Thirteen strains of L. plantarum exhibited antimicrobial activity against Escherichia coli O157:H7 DMST 12743 with zone inhibition ranged between 11.5-12.5 mm. All test strains were evaluated for their ability to survive under gastrointestinal tract conditions with and without cereals; soybean, sesame and Job?s Tears. Regarding gastric tolerance, seven strains of PKWB6-12, PKWB7-1, PKWB7-2, PKWC6-1, PKWC6-9, PKWC7-1 and PKWD7-1 were more tolerant with more than 4.6 log CFU/ml while the viability of other six strains decreased to undetectable level after 180 min exposure. However, all test strains exhibitied high resistance to simulated small intestinal juice pH 8.0 with 0.45% bile salt. The result showed that cereal exerted a protective effect on the survival of all test strains. Interestingly, the addition of cereals especially soybean exhibited a significantly improved the viability of all test strains in simulated gastric juices pH 2.0. Whereas, Job?s Tears showed a greater positive influence on viability of most test strains. These results indicated that soybean, sesame and Job?s Tears could be used as food carriers to delivery probiotic lactic acid bacteria through the gastrointestinal tract system.

Keywords soybean; sesame Job?s Tears gastrointestinal tract conditions

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Introduction

Probiotics are live microbial food supplements which play an important role in promoting and maintaining human health. Probiotics propose many health benefits such as reducing gastrointestinal problems like diarrhea, controlling inflammatory diseases and stimulating the immune system, improving intestinal microbial balance (Prado et al., 2008). In Asian countries, a number of new probiotic strains isolated from fermented vegetable and meat products have been developed. Many researches have been focused on developing new probiotic nondairy foods to meet the increased demand from consumers for alternative to probiotic milk-based product. Probiotics can nowadays be found in non-fermented milk, fruit juices and cereal-based products (Saarela et al., 2006). In order to exert probiotic beneficial effect after consumption, probiotics need to survive passage through the gastrointestinal tract conditions mainly attributed to their acid and bile tolerance which are crucial properties of probiotics. Cereal products have been shown to be suitable substrates for the growth of potential probiotics (Valerio et al., 2006). In previous studies, malt, wheat and barley were found to enhance acid tolerance of L. plantarum NCIMB 8826 (Michida et al., 2006) and bile tolerance of L. reuteri NCIMB 1195 and L. acidophilus NCIMB 8821 and L. plantarum NCIMB 8826 (Patel et al., 2004). The survival of L. plantarum ITM21B and L. paracasei IMPC2.1 was improved during exposure to simulated gastrointestinal digestion when artichokes were used as food carriers (Valerio et al., 2006). Cereals including soybean (Glycine max (L.) Merr.), sesame (Sesamum orientale L.) and Job?s Tears (Coix lacryma-jobi Linn.) are considerable economic crop in eastern Asia used as foods, ingredients and component of drugs. They are high in nutrition value and contain significant amounts of dietary fiber, protein, carbohydrates, energy, minerals and vitamins required for human health. Therefore, the aim of this study was to evaluate antagonistic activity of L. plantarum L. plantarum against Escherichai coli O157:H7 DMST 12743 and the effect of these cereals on the stability of L. plantarum under gastrointestinal tract conditions.

Materials and methods

Microorganisms

Thirteen strains of Lactobacillus plantarum isolated from fermented vegetable with high acid production were used in this study. The test strains were preserved in MRS broth (Merck, Darmstadt, Germany) containing 20% (v/v) glycerol at -20 oC. For routine analysis, the test strains were subcultured twice in MRS broth to maintain freshness. Escherichia coli O157:H7 DMST 12743 was purchased from Department of Medical Science, Ministry of Public Health, Thailand. The culture was grown in Tryptic Soy Broth (TSB) (Difco Laboratories, Detroit, MI, USA) supplemented with 0.6% Yeast extract (TSBYE) at 37 oC.

Determination of antagonistic activity of L. plantarum

L. plantarum were investigated for their antimicrobial activity against E. coli O157:H7 DMST 12743 by agar diffusion method and spot on lawn method. For the inhibitory activity by agar well diffusion method, indicator strains was grown overnight in TSBYE. 20 ml of TSAYE plates (1.5% agar) were inoculated with 50 ?l overnight culture of the indicator microorganism (106 CFU/ml final concentration). The wells (6 mm-diameter) were formed by punching out of the solid agar using sterile cork borer, and 50 ?l volume of L. plantarum culture was added to the holes and incubate at 37 oC for 24 h (Domrongpokkaphan and Wanchaitanawong, 2006). The diameters of the inhibition zones were measured in millimeters (mm).

To determination of the inhibitory activity by spot on lawn method, the indicator plates were prepared by overlaying 5 ml of soft TSA (with 0.75 % agar) with 10 ?l of indicator strain at the concentration of about 106 CFU/ml. Cell-free supernatant was obtained from overnight lactobacilli MRS culture. The supernatant were collected by centrifugation at 10,000 rpm for 15 min, adjusted to pH 5.0 and 6.0 with 5 M NaOH and filtered through a 0.2 ?m pore size sterile filter (Minisarts?, satorius). Serial dilutions of cell-free supernatant was spotted (10 ?l) on the surface of the agar. The plates were then incubated at 37 ?C. Inhibition area was revealed by the formation of clear zone in the indicator bacterial lawn. Antimicrobial activity is expressed in arbitrary units (AU) per ml of the original cultures calculated as follows: AU/ml is the highest dilution exhibition inhibition zone per volume of spotting supernatant (Hamsupo, 2005).

Preparation of cereals powder and chemical analysis

Soybean, sesame and Job?s Tears grains were ground and separated with a sieve size of 0.5 mm. Samples of 5 g the resulting powder were mixed with 45 ml of distilled water and sterilize at 121oC for 15 min before used as 10% (w/v) cereals.

Total sugar concentration was determined by the phenol-sulphuric acid method (Dubois et al., 1956). The analysis of the samples was conducted based on a calibration curve (R2=0.993) using an array of glucose standard solution (400 mg/l). Reducing sugar concentration was determined by the 3, 5-dinitrosalicylic acid method (Miller, 1959). The analysis of the samples was conducted on the basis of a calibration curve (R2=0.993) using an array of glucose standard solutions (1 g/l). The buffering capacity of samples was determined by titrating 100 ml of the medium with HCl (1N). The value was expressed as the amount of HCl (mmole) required dropping 1 pH unit per unit volume (l).

Preparation of simulated gastric juices and small intestinal juices

Simulated gastric and small intestinal juices were prepared fresh daily following to method of Michida et al. (2006) and Huang and Adam (2004). Briefly, Simulated gastric juice was prepared by suspending pepsin (1:10,000, ICN) in sterile saline (0.5%, w/v) to a final concentration of 3 g/l and adjusting to pH 2.0 with concentrated HCl using a pH meter (Mettler Toledo, FiveEasy). Simulated small intestinal juice was prepared by suspending pancreatin USP (P-1500, Sigma, Basingstoke, Hampshire, UK) in the sterile saline to a final concentration of 1 g/l with 0.45 % bile salt (Oxoid, Basingstoke, Hampshire, UK) and adjusting to pH 8.0 with sterile 0.1 mol/l NaOH using a pH meter.

Determination of simulated gastrointestinal tract tolerance of L. plantarum

Washed bacterial cell suspension was prepared. Bacterial cell of 20 h culture was centrifuged at 5,000 rpm for 10 min, washed twice with sterile saline (0.5%, w/v) and resuspended in sterile saline using as washed cell suspension. The viable cell count was determined prior to assay of transit tolerance.

The tolerance of a washed cell of L. plantarum to the simulated gastric and small intestinal juices was determined using Michida et al. (2006) method with a little modification. Aliquots (0.2 ml) of the free-cell suspension were transferred to a 2.0 ml Eppendorf tube, mixed with 0.3 ml sterile saline (0.5%, w/v) or cereal (10%, w/w) and finally mix with 1.0 ml of simulated gastric (pH 2.0) or small intestinal juices (pH 8.0). The final concentration of cereals was 2% and then the mixture was incubated at 37 ?C. For simulated gastric juices tolerance determination, viable cell counts were measured after 30, 60, 90, and 180 min. For simulated small intestinal juices tolerance determination, viable cell counts were measured after 240 min. Viable cell counts were determined by the standard plate count method with Lactobacilli MRS medium. The plates were incubated at 37 ?C for 36 h. Viable cell counts were expressed as log10 values. The percentage of cells survival was calculated as follows: % survival = (log N/log N0) ? 100, where N0 number of viable cells (CFU/ml) before exposure to simulated gastric and small intestinal juices and N is number of viable cells (CFU/ml) after exposure to simulated gastric and small intestinal juices.

Scanning electron microscopy

After exposure to simulated gastric juice (pH 2.0) added with soybean, sesame and Job?s Tear, L.plantalum PKWB6-12 was monitored by scanning electron microscopy. The cells were first fixed with a 2.5 % glutaraldehyde in sodium phosphate buffer pH 7.2 for 12 hours. After three times wash with phosphate buffer , the samples were fixed with 1% osmium tetroxide for 1 hour and washed with distilled water for three times. The samples were then dehydrated through a graded series of ethanol soaks (30, 50, 70, 90 and 100 % ethanol, using 100 % ethanol three times) and drying with liquid carbondioxide. The dried samples were coated with Au and determined in a scanning electron microscope JSM 5600 LV (JEOL Ltd, Tokyo, Japan).

Results and Discussion

Antimicrobial activity of thirteen strains of L. plantarum

Thirteen strains of L. plantarum isolated from fermentated vegetable were evaluated for their ability to inhibit the growth of E. coli O157:H7 DMST 12743 using agar well diffusion method. It was found that all test strains had an antimicrobial property against the indicator strain with the inhibition zone ranging from 11.5 ? 0.10 to 12.5 ? 0.10 mm. The cell-free supernatant (pH 3.5-3.6) from all test strains also exhibited antimicrobial activity of approximately 100 - 300 AU/ml. The inhibitory effect observed for all test strains may be attributed to the lowered pH, the undissociated acids and production of other primary and secondary antimicrobial metabolites produced by lactic acid bacteria (H?tt et al., 2006). Furthermore, it was found that the neutralized pH 6.0 supernatant of PKWA6-1, PKWA7-1, PKWB 6-3 and PKWB6-12 inhibited the growth of indicator strain with the antimicrobial activity of 200 - 300 AU/ml. Based on these results, four strains were capable of synthesizing inhibitory substances including bacteriocin-like compounds (Albano et al., 2007; Con et al., 2001; Con et al., 2000).

3.2 Effect of soybean, sesame and Job?s Tears on viability of L. plantarum under simulated gastric juice pH 2.0

The acid tolerance of 13strains of L. plantarum was tested at pH 2.0 (Figure 1). It was observed that all test strains exhibited certain resistance ability to gastric juice for 180 min. Among the 13 strains tested, 7 strains (PKWB6-12, PKWB7-1, PKWB7-2, PKWC6-1, PKWC6-9, PKWC7-1 and PKWD7-1) appered to be more capable to survive higher than 4.5 log CFU/ml. After incubation for 180 min, PKWB6-12 was found to be the most tolerant with the reduction rate of 28.80 ? 8.43 % (from 9.73 ? 0.28 to 6.93 ? 0.82 log CFU/ml). Meanwhile, other six strains (PKWA6-1, PKWA7-1, PKWB6-3, PKWC8-1, PKWD6-10 and PKWD7-2) were susceptible to gastric condition and the number of viable cell decreased to undetectable level after exposure for 180 min.

Furthermore, the effect of cereals on the survival of all test strains during 180 min exposure to acid juice was shown in Figure 2. The addition of soybean, sesame and Job?s Tears progressively improved the gastric tolerance. All test strains were able to maintain viable cell level of 4.93 ? 0.09 to 9.64 ? 0.06 log CFU/ml for 180 min exposure depending on cereal type. Remarkably, sensitive strains (PKWA6-1, PKWA7-1, PKWB6-3, PKWC8-1, PKWD6-10 and PKWD7-2) displayed high survival after 180 min exposure compared with control group. Results also showed that acid tolerance depended on type of strain and cereal. Soybean was found to be the most protective compound followed by sesame and Job?s Tear. Compared with control, the viability of all test strains was considerable improved by approximately 2.13 - 9.45, 2.08 - 9.27 and 2.10 - 9.17 log CFU/ml in the present of soybean, sesame and Job?s Tear, respectively. Among these lactic acid bacteria, addition of soybean and sesame enhanced acid tolerance of PKWD7-1 with the highest survival rate of 97.67 ? 0.56 % and 94.57 ? 1.61%, respectively. Further, the addition of Job?s Tears resulted in the highest viability of 95.72 % for PKWC7-1.

There have been reported that proteins, polysaccharides, and free amino acids have a good protective effect on lactic acid bacteria and enhance their viability in the gastrointestinal conditions (Michida et al, 2006; Charalampopoulos et al., 2003). In this present study, the addition of cereals especially soybean displayed a significantly improved the viability of all test strains in simulated gastric juices pH 2.0. Their viability were higher than 8 log CFU/ml at the end of incubation. This indicated that soybean protein played an important role in the enhancement of survival of lactic acid bacteria (Wang et al., 2008). In addition, higher concentration of total sugar, reducing sugar and buffering capacity in soybean (Table 2) may have a synergistic effect on the gastrointestinal tolerance (Michida et al, 2006). Generally, L. plantarum exhibited high cell population owing to its unique ability to tolerate low pH values by maintaining pH homeostasis (Giraud et al., 1998). Under acidic condition, F1F0-ATPase proton pump is an important mechanism responsible for the survival of some gram-positive microorganisms (Cotter and Hill, 2003). The F0F1-ATPase could increase inner pH by generating a proton motive force, via proton expulsion. This reaction requires energy in form of ATP obtained from sugar utilization as carbon-source of L. plantarum. (Sheng and Marquis, 2006; Corcoran et al., 2005; McDonald et al., 1990).

From SEM results, it was clear that high cell density of L. plantalum PKWB6-12 were entrapped on the fiber structure of soybean, sesame and Job?s Tears. The roughness structure may offer protection to the cell in the acid condition (Valerio et al., 2006). Therefore, the cereals could be used as food vehicles for probiotic so as to enhance their stability during exposure to gastric digestion.

3.3 Effect of soybean, sesame and Job?s Tears on survival of L. plantarum under simulated small intestinal juices pH 8.0

Bile tolerance of thirteen test strains of L. plantarum was presented in Table 3. As a general tendency, all test strains appeared to be most capable to survive during exposure to simulated small intestinal juice (pH 8.0) with 0.45% bile salt for 240 min and reduction of viable cell count ranged 0.26 - 2.64 log CFU/ml. Of thirteen strains, a slightly decreased in viability was observed in PKWD6-10 and PKWA6-1 and their cell population slightly decreased from 9.95 to 9.69 log CFU/ml (97.28 ? 0.11% survival) and 9.27 to 8.86 log CFU/ml (95.42 ? 2.20% survival), respectively. For other ten strains, survival ranged between 73.14 - 89.86% (Figure 4).

Futhermore, the influence of soybean, sesame and Job?s Tears on the survival of all test strains under simulated small intestinal juice with 0.45% bile salt for 240 min was examined. Although these cereals may function as buffering agents (Table 2), only PKWD7-1 and PKWB7-1 were significantly (P<0.01) improved in viability of >1 log CFU/ml as compared to control after 240 min incubation retained at the level of 109 and 108 CFU/ml and their survival rate of 94.80%-99.65% for PKWD7-1and 83.11%-87.39% for PKWB7-1 were obtained, respectively (Figure 4). After 240 min incubation time, significantly (P<0.01) decreased in viability of PKWA6-1, PKW D6-10 and PKWD7-2 with addition of sesame and PKWA7-1, PKWB6-3, PKWB6-12 and PKWC6-9 with addition of soybean was obtained. Moreover, it was observed that most of test strains could survive better in the present of Job?s Tears.

Conclusion

The present study has shown that our potential probiotic L. plantarum exhibited antimicrobial activity against E. coli O157:H7. The results indicated that soybean, sesame and Job?s Tears could improve the tolerance of L. plantarum to simulated gastrointestinal tract conditions depending on the composition of cereals. Specially, soybean enhanced the acid tolerance while Job? s Tears improved the bile tolerance.

Results indicated that the cereals could be used as vehicle for delivery of lactic acid bacteria as probiotic to the human gastrointestinal tract. This finding could further lead to the development of new probiotic functional food with the combination of LAB and cereal which reffered to as symbiotic.

Viable cell counts (log CFU/ml) of each strain in the presence of cereals in system were compared with control (no cereal), *p<0.05, **p<0.01 (student?s t-test, two tailed)

Figure 4 Effect of soybean, sesame and Job?s Tears on viability of L. plantarum during exposure to simulated small intestinal juices pH 8.0 with 0.45% bile salt for 240 min

Acknowledgement

This work was granted by the Commission on Higher Education granting. Miss Wanticha Lapsiri was supported for CHE-PhD-SW from the Commission on Higher Education. Thailand.

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Therefore, these six strain was considered as intrinsically tolerant to gastric transit pH 2.0 for 180 min.

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