Survival Of Fermented Vegetable Lactobacillus Plantarum Biology Essay

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Thirteen strains of L. plantarum were evaluated for their ability to survive under gastrointestinal tract conditions with and without cereals; soybean, sesame and Job's Tears. In regard to gastric tolerance, seven strains of PKWB6-12, PKWB7-1, PKWB7-2, PKWC6-1, PKWC6-9, PKWC7-1 and PKWD7-1 showed good tolerlance and maintained viability of more than 4.6 log c.f.u/ml, while other six strains of PKWA6-1, PKWA7-1, PKWB6-3, PKWC8-1, PKWD6-10 and PKWD7-2 were susceptible and their viability decreased to undetectable level after 180 min exposure. Furthermore, all test strains were relatively resistant to simulated small intestinal juice (pH 8.0) with 0.45% bile salt for 240 min. The addition of the cereals greatly improved the survival of all test strains in simulated gastric juice (pH 2.0). The protective effect of the cereals on the strain viability depended on their compositions. Soybean was found to be the most protective compound followed by sesame and Job's Tears. Compared with the control group, the viability of all test strains was considerably improved by approximately 2 log c.f.u/ml for the acid-tolerant strains and 9 log c.f.u/ml for the sensitive strains. Furthermore, survival of L. plantarum after sequential exposure to simulated gastric (180 min) and intestinal juices (240 min) was also investigated. All test strains were quite stable with the survival ranging from 92 to 100% in the presence of cereals and 84 to 100% in the absence of cereals. These results indicate that soybean, sesame and Job's Tears could be used as food carriers to deliver probiotic lactic acid bacteria through the gastrointestinal tract system.

Introduction

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Probiotics are live microbial food supplements which play an important role in promoting and maintaining human health. Probiotics propose many health benefits such as the reduction of gastrointestinal problems like diarrhea, lowering of serum cholesterol in blood, control of inflammatory diseases and stimulation of the immune system, improvement of intestinal microbial balance (Prado et al. 2008; Wang et al. 2009). Nowadays, probiotics are available in food and dietary supplements. In addition to yogurt and cheese, the types of foods delivering probiotic have extended to fruit/vegetable juices and cereal-based products (Yoon et al. 2005; Saarela et al. 2006; Rakin et al. 2007; Tang et al. 2007; Kedia et al. 2008; Champagne et al. 2009).

Probiotics are defined as "live microbial supplement that beneficially affects the host animal by improving its intestinal microbial balance" (Fuller 1989). To provide health benefits, probiotics must be able to survive passage through the gastrointestinal tract and arrive alive at its site of action in high enough number (Mattila-Sandholm et al. 2002; Champagne and Gardner 2008; Rivera-Espinoza and Gallardo-Navarro 2010). Viability loss of probiotics in gastrointestinal tract conditions has challenged researchers to find new efficient method to improve viability. One of strategies is incorporation of food and food ingredient such as metabolizable sugars, protein, polysaccharides and amino acid (Hutkins and Nannen 1993; Kos et al. 2000; Charalmpopoulos et al. 2002; Corcoran et al. 2005; Sheng and Marqus 2006). In addition, cereals have been proven to be suitable carriers for delivery probiotic (Valerio et al. 2006; Kedia et al. 2007; Ranadheera et al. 2010; Rivera-Espinoza and Gallardo-Navarro 2010). For example, 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).

Soybean (Glycine max (L.) Merr.), sesame (Sesamum indicum L.) and Job's Tears (Coix lacryma-jobi L.) are widely used in food/beverage industry because they are high content of dietary fibers, proteins, minerals and vitamins. Additionally, the cereals can be applied as source of non-digestible oligosaccharides that can function as prebiotic, beneficially affect the host by selectively stimulating the growth and/or activity of one or a limited number of bacteria in the colon (Mussatto and Mancilha 2007). Therefore, it would be desirable to investigate how the cereals affected the viability of Lactobacillus plantarum under gastrointestinal tract conditions. In our laboratory, 13 strains of L. plantarum were isolated from fermented vegetable and exhibited high antimicrobial activity against human pathogens including Escherichia coli O157:H7 DMST 12743 and Salmonella Typhimurium ATCC 13311. In order to be used as potential probiotics, the strains need to be screened for their capacity of transit tolerance to gastrointestinal tract conditions. Therefore, in the present study, all test strains were evaluated for their viability in simulated gastric condition (pH 2.0) and small intestinal tract condition (pH 8.0 with 0.45% bile salts). Moreover, the effect of soybean, sesame and Job's Tears on simulated gastrointestinal tract tolerance of all test strains was also investigated.

Materials and methods

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Microorganisms

Thirteen strains of L. plantarum were used in this study. The strains were isolated from fermented vegetable with high acid production ability and antimicrobial activity against E. coli O157:H7 DMST 12743 and S. Typhimurium ATCC 13311. All 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.

Preparation of cereals and chemical analysis

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

Total sugar concentration was determined by the phenol-sulphuric acid method (Dubois et al. 1956) and the sample analysis was conducted based on a calibration curve (R2=0.993) by the application of an array of glucose standard solution (400 mg/l). Reducing sugar concentration was determined by the 3, 5-dinitrosalicylic acid method (Miller 1959) and the sample analysis was conducted on the basis of a calibration curve (R2=0.993) employing 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 washed cell suspension

L. plantarum was grown in MRS broth at 37 oC for 20 h. Cell culture (7 ml) of each test strain was centrifuged at 5,000 g for 10 min. After washing twice with sterile saline (0.5% w/v), the cell pellet was resuspended in the same solution. The viable cell count of the washed cell suspension was determined prior to assay of transit tolerance.

Preparation of simulated gastric juice and small intestinal juice

Simulated gastric juice was prepared fresh daily by dissolving pepsin (1:10,000, ICN, Sigma, Basingstoke, Hampshire, UK) in sterile 0.5% ( w/v) NaCl solution to a final concentration of 3 g/l and adjusting the pH to 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 the pH to 8.0 with sterile 0.1 mol/l NaOH.

Survival of L. plantarum in simulated gastric and intestinal juices

The survival of L. plantarum in simulated gastric juice and small intestinal juice was determined following the method of Michida et al. (2006). An aliquots (0.2 ml) of each washed cell suspension was transferred to a sterile tube and then mixed with 0.3 ml sterile 0.5% ( w/v) NaCl solution and 1.0 ml of simulated gastric juice (pH 2.0) or small intestinal juice (pH 8.0). The mixture was then incubated at 37 °C. The changes in total viable count were monitor during exposure to simulated gastric juice (180 min) and simulated small intestinal (240 min) by the standard plate count method using MRS agar. The plates were incubated at 37 °C for 36 h. Viable cell counts were expressed as log10 value. The percentage of cells survival was defined as follows: % survival = (log N/log N0) ´ 100, where N represents the number of viable cells (c.f.u/ml) after exposure and N0 is the initial viable cell count (c.f.u/ml) before exposure.

Effect of cereals on survival of L. plantarum in simulated gastric and intestinal juices

The tolerance of washed cell suspensions of L. plantarum strains to simulated gastric and intestinal juices was determined as described above, with the exception that 0.3 ml of 10% (w/v) cereal suspension replaced the sterile saline addition. Survival of L. plantarum was determined as described above.

Survival of L. plantarum after sequential incubation in simulated gastric and intestinal juices

After exposure to simulated gastric juice (pH 2.0, 180 min), the cell suspension were centrifuged at 5,000 g for 10 min and subsequently resuspended in simulated small intestinal juice (pH 8.0) (Valerio et al. 2006). The suspensions were then incubated at 37 °C for 240 min. The samples for total viable cell counts were taken at 60, 90 and 180 min during exposure to simulated gastric juice and at 60, 120 and 240 min during exposure to simulated small intestinal juice. The same procedure was used with addition of the cereals. Survival of L. plantarum was determined as described above.

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Scanning electron microscopy

After exposure to simulated gastric juice (pH 2.0) with soybean, sesame and Job's Tear, L. plantarum 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 h. After three times washing with phosphate buffer, the samples were fixed with 1% osmium tetroxide for 1 h and washed once more with distilled water three times. The samples were then dehydrated through a graded series of ethanol soaks (30%, 50%, 70%, 90% and three times in 100 % ethanol) and dried with liquid carbondioxide. The dried samples were coated with Au and determined with the application of scanning electron microscope JSM 5600 LV (JEOL Ltd, Tokyo, Japan).

Statistical analysis

Results were expressed as the mean ± standard deviations of three determinations. The data were analyzed by analysis of variance (ANOVA) with significant at P < 0.05. Significant difference among mean values was determined by Duncan s multiple range test. All statistical analyses were performed using SPSS Software Version 12.

Results and Discussion

Effects of soybean, sesame and Job's Tears on viability of L. plantarum under simulated gastric juice (pH 2.0)

The acid tolerance of thirteen strains of L. plantarum was tested at pH 2.0 (Fig. 1). It was observed that all test strains exhibited certain resistance abilities to gastric juice for 180 min. Among the thirteen strains tested, seven strains (PKWB6-12, PKWB7-1, PKWB7-2, PKWC6-1, PKWC6-9, PKWC7-1 and PKWD7-1) appeared to be more capable to survive and their viable cell populations were higher than 4.5 log c.f.u/ml. After incubation for 180 min, PKWB6-12 was found to be the most tolerant with a reduction rate of 28.80 ± 8.43 % (from 9.73 ± 0.28 to 6.93 ± 0.82 log c.f.u/ml). Meanwhile, the 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 levels after exposure for 180 min.

Fig. 1 Viability of L. plantarum during exposure to simulated gastric juice for 180 min

Furthermore, the effect of cereals on the survival of all test strains during an exposure to acid juice was shown in Table 1. The addition of soybean, sesame and Job's Tears exhibited a significantly improved the gastric tolerance (P<0.05). All test strains were able to maintain viable cell levels of 4.93 ± 0.09 to 9.64 ± 0.06 log c.f.u/ml for 180 min exposure depending on the cereal type. Remarkably, sensitive strains (PKWA6-1, PKWA7-1, PKWB6-3, PKWC8-1, PKWD6-10 and PKWD7-2) displayed high survival rates after 180 min exposure compared with the control group. Results also showed that acid tolerance was dependant on the type of strain and cereal. Soybean was found to be the most protective compound followed by sesame and Job's Tears. Compared with control group, 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 c.f.u/ml in the presence of soybean, sesame and Job's Tears, 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. Furthermore, the addition of Job's Tears resulted in the highest viability of 95.72 % for PKWC7-1 (Fig 2). The acid tolerlance of all test strains suspended in the cereals could be explained by the protective action of component such as protein, fat, polysaccharide, prebiotic carbohydrates and free amino acids (Charalampopoulos et al. 2002; Charalampopoulos et al. 2003; Michida et al. 2006; Valerio et al. 2006, Wang et al. 2007). In the presence of metabolizable sugar, it provides ATP to F1F0-ATPase via glycolysis, enabling proton exclusion and thereby enhancing survival during gastric transit (McDonald et al. 1990, Cotter and Hill 2003, Corcoran et al. 2005). In addition, sugar content and buffering capacity of the cereals (Table 2) may have a synergistic effect on the gastrointestinal tolerance (Michida et al. 2006).

From SEM results, it was clear that a high cell density of L. plantarum PKWB6-12 was entrapped on the fiber structure of soybean, sesame and Job's Tears (Fig. 3). The roughness of the structure may offer protection to the cells in the acid condition (Valerio et al. 2006). It was obvious that chemical composition and physical structures of cereals appeared to play a major role in the protective effects (Ranadheera et al. 2010). Therefore, the cereals could be used as means for probiotics to enhance their stability during exposure to gastric digestion.

Table 1 Viability of thirteen strains of L. plantarum during exposure to simulated gastric juices in the presence of soybean, sesame and Job's tears

L. plantarumStrain

Cereal

Viablie cell count in therm of log c.f.u/ml ± SD)

Initial

30 min

60 min

90 min

180 min

PKWA6-1

Control

9.87 ± 0.52

6.90 ± 0.27

6.28 ± 0.07

4.95 ± 0.99

0.00 ± 0.00 d

Soybean

9.81 ± 0.13

9.64 ± 0.03

9.56 ± 0.13

9.45 ± 0.02 a

Sesame

9.23 ± 0.07

9.12 ± 0.06

8.91 ± 0.21

8.27 ± 0.01 b

Job's tear

9.29 ± 0.15

7.07 ± 0.11

6.03 ± 0.13

5.69 ± 0.11 c

PKWA7-1

Control

9.98 ± 0.11

7.49 ± 0.54

5.66 ± 0.84

0.00 ± 0.00

0.00 ± 0.00 d

Soybean

9.78 ± 0.08

9.62 ± 0.12

9.63 ± 0.04

9.41 ± 0.01 a

Sesame

9.64 ± 0.06

9.70 ± 0.02

9.59 ± 0.05

8.12 ± 0.10 b

Job's tear

9.21 ± 0.11

8.85 ± 0.04

8.05 ± 0.08

7.14 ± 0.03 c

PKWB6-3

Control

9.98 ± 0.65

8.26 ± 0.79

7.30 ± 0.56

6.58 ± 0.83

0.00 ± 0.00 b

Soybean

9.82 ± 0.15

9.65 ± 0.03

9.44 ± 0.11

9.17 ± 0.01 a

Sesame

9.79 ± 0.13

9.51 ± 0.11

9.24 ± 0.09

9.16 ± 0.02 a

Job's tear

9.71 ± 0.01

9.41 ± 0.06

9.45 ± 0.11

9.17 ± 0.01 a

PKWB6-12

Control

9.73 ± 0.28

9.01 ± 0.69

8.68 ± 0.68

8.23 ± 0.70

6.93 ± 0.82 b

Soybean

9.67 ± 0.02

9.69 ± 0.12

9.19 ± 0.04

9.06 ± 0.15 a

Sesame

9.38 ± 0.11

9.43 ± 0.03

9.13 ± 0.05

9.01 ± 0.18 a

Job's tear

9.52 ± 0.02

9.36 ± 0.11

9.37 ± 0.01

9.03 ± 0.07 a

KUBB7-1

Control

10.10 ± 0.55

9.07 ± 0.35

7.20 ± 0.36

6.92 ± 0.53

6.44 ± 0.82 b

Soybean

10.08 ± 0.22

9.91 ± 0.21

9.66 ± 0.02

9.48 ± 0.04 a

Sesame

9.56 ± 0.05

9.31 ± 0.01

8.88 ± 0.22

8.57 ± 0.11 a

Job's tear

9.60 ± 0.05

8.74 ± 0.06

8.77 ± 0.07

8.55 ± 0.15 a

PKWB7-2

Control

9.75 ± 0.36

9.08 ± 0.94

8.18 ± 0.93

7.32 ± 0.67

4.66 ± 0.12 d

Soybean

9.80 ± 0.12

9.86 ± 0.02

9.55 ± 0.05

9.06 ± 0.03 a

Sesame

9.33 ± 0.09

9.40 ± 0.06

8.58 ± 0.02

7.96 ± 0.09 b

Job's tear

9.98 ± 0.04

9.35 ± 0.06

8.83 ± 0.13

8.23 ± 0.15 c

PKWC6-1

Control

9.97 ± 0.52

9.23 ± 0.26

7.75 ± 0.75

7.65 ± 0.58

5.39 ± 0.84 b

Soybean

9.99 ± 0.02

9.82 ± 0.06

9.66 ± 0.09

9.49 ± 0.04 a

Sesame

9.25 ± 0.04

8.69 ± 0.10

8.68 ± 0.37

8.76 ± 0.08 a

Job's tear

9.55 ± 0.11

9.52 ± 0.04

9.36 ± 0.09

9.27 ± 0.01 a

PKWC6-9

Control

9.84 ± 0.38

8.73 ± 0.54

8.00 ± 0.24

7.26 ± 0.53

6.39 ± 0.60 b

Soybean

9.87 ± 0.02

9.66 ± 0.16

9.33 ± 0.07

9.03 ± 0.02 a

Sesame

9.89 ± 0.02

9.66 ± 0.06

9.47 ± 0.07

9.09 ± 0.01 a

Job's tear

9.66 ± 0.02

9.63 ± 0.03

9.32 ± 0.04

9.16 ± 0.03 a

KUBC7-1

Control

10.03 ± 0.40

8.44 ± 0.83

6.42 ± 0.13

5.84 ± 0.07

5.47 ± 0.18 c

Soybean

9.82 ± 0.06

9.77 ± 0.10

9.50 ± 0.02

9.37 ± 0.01 ab

Sesame

9.69 ± 0.06

9.56 ± 0.07

9.34 ± 0.04

9.06 ± 0.37 b

Job's tear

9.73 ± 0.07

9.78 ± 0.02

9.69 ± 0.12

9.61 ± 0.06 a

PKWC8-1

Control

9.59 ± 0.52

4.42 ± 0.51

3.59 ± 0.63

0.00 ± 0.00

0.00 ± 0.00 c

Soybean

9.47 ± 0.02

9.38 ± 0.05

9.08 ± 0.02

8.09 ± 0.02 a

Sesame

9.78 ± 0.08

8.66 ± 0.07

8.73 ± 0.05

8.04 ± 0.09 a

Job's tear

8.57 ± 0.03

7.09 ± 0.03

6.75 ± 0.03

4.93 ± 0.09 b

PKWD6-10

Control

9.85 ± 0.54

4.37 ± 0.07

0.00 ± 0.00

0.00 ± 0.00

0.00 ± 0.00 c

Soybean

9.70 ± 0.07

9.64 ± 0.10

9.44 ± 0.06

9.23 ± 0.33 a

Sesame

9.51 ± 0.04

9.41 ± 0.05

9.49 ± 0.00

9.27 ± 0.02 a

Job's tear

9.68 ± 0.03

8.36 ± 0.02

7.17 ± 0.01

6.30 ± 0.20 b

PKWD7-1

Control

9.87 ± 0.28

9.39 ± 0.06

8.24 ± 0.97

7.40 ± 0.92

5.46 ± 0.33 c

Soybean

9.84 ± 0.03

9.78 ± 0.01

9.67 ± 0.01

9.64 ± 0.06 a

Sesame

9.78 ± 0.06

9.63 ± 0.21

9.56 ± 0.07

9.33 ± 0.16 ab

Job's tear

9.60 ± 0.01

9.42 ± 0.04

9.28 ± 0.04

9.08 ± 0.10 b

PKWD7-2

Control

9.87 ± 0.17

5.57 ± 0.59

5.18 ± 0.26

4.91 ± 0.10

0.00 ± 0.00 d

Soybean

9.71 ± 0.17

9.50 ± 0.01

9.59 ± 0.21

9.40 ± 0.05 a

Sesame

9.11 ± 0.02

8.88 ± 0.11

8.56 ± 0.03

7.96 ± 0.02 b

Job's tear

9.05 ± 0.07

8.57 ± 0.02

8.04 ± 0.01

7.12 ± 0.01 c

Values in the same column of each strain test with different lowercase letters are significant by Duncan's multiple range test (P<0.05).

Table 2 Chemical composition of sterile cereal suspensions

Soybean

Sesame

Job's Tears

pH

6.40 ± 0.02 a

6.04 ± 0.02 b

6.37 ± 0.01 a

Total sugars (g/l)

18.41 ± 0.62 a

6.74 ± 1.12 b

8.83 ± 0.67 b

Reducing sugars (g/l)

0.66 ± 0.02 a

0.16 ± 0.01 b

0.15 ± 0.01 b

Buffering capacity (mmol/pH.l)

20.05 ± 0.07 a

5.25 ± 0.07 b

4.00 ± 0.14 c

Values in the same row with different lowercase letter are significant different by Duncan's multiple range test (P<0.05)

Fig. 2 Viability of thirteen strains of L.plantarum under exposure to simulated gastric juice pH 2.0 for 180 min

Different lowercase letters in each strain are significant different by Duncan's multiple range test (P<0.05)

Fig. 3 Scanning electron micrographs of L. plantarum PKWB6-12 in the presence of (a) soybean (4000x), (b) sesame (5000x) and (c) Job's Tears (5000x)

Effects of soybean, sesame and Job's Tears on viability of L. plantarum under simulated intestinal juice

Bile tolerance of L. plantarum was carried out for 240 min exposure to simulated small intestinal juice (pH 8.0) with 0.45% bile salt (Huang and Adam 2004; Michida et al. 2006). As shown in Table 3, all test strains appeared to be capable to survive during exposure to simulated small intestinal juice with 0.45% bile salt for 240 min and the reduction of the viable cells count ranged from 0.26 to 2.64 log c.f.u/ml. Of thirteen strains, a slightly decreased viability was observed in PKWD6-10 and PKWA6-1 and their cell population slightly decreased from 9.95 to 9.69 log c.f.u/ml (97.28 ± 0.11% survival) and 9.27 to 8.86 log c.f.u/ml (95.42 ± 2.20% survival), respectively. Survival of other eleven strains ranged from 73.14 to 89.86% (Fig. 4).

In the present of soybean, sesame and Job's Tears, although these cereals may function as buffering agents, only PKWD7-1 and PKWB7-1 were significantly improved (P<0.05) in viability with cell reduction of approximately 1 log c.f.u/ml as compared to the control group after exposure to simulated small intestinal juice with 0.45% bile salt for 240 min. The survival rates of PKWD7-1 and PKWB7-1 were 94.80 to 99.65% (~109 c.f.u/ml) and 83.11 to 87.39% (~108 c.f.u/ml), respectively (Table 2). Moreover, it was observed that Job's Tears appeared to be more effective in protecting cells from harsh environmental conditions than soybean and sesame and most of test strains could survive better in the presence of Job's Tears.

Table 3 Viability of L. plantarum during exposure to simulated small intestinal juice (pH 8.0) with 0.45% bile salt for 240 min in the presence of cereals

Strains

Viable cell count (log c.f.u/ml) ± SD.

Initial

Control

Soybean

Sesame

Job's tears

PKWA6-1

9.27±0.03

8.86±0.19 a

8.40±0.04 b

7.49±0.04 c

8.95±0.04 a

PKWA7-1

9.92±0.04

8.85±0.04 a

7.84±0.05 c

8.74±0.01 b

8.86±0.04 a

PKWB6-3

9.60±0.01

7.88±0.06 a

7.00±0.02 c

7.57±0.01 b

7.80±0.02 a

PKWB6-12

9.66±0.04

8.19±0.01 b

7.94±0.01 c

8.33±0.00 a

8.29±0.04 a

PKWB7-1

9.78±0.04

7.14±0.05 d

8.13±0.05 c

8.28±0.02 b

8.55±0.08 a

PKWB7-2

9.95±0.03

7.72±0.08 b

7.41±0.02 c

7.44±0.07 c

7.90±0.04 a

PKWC6-1

9.37±0.02

7.30±0.00 ab

7.15±0.07 b

7.31±0.03 ab

7.38±0.08 a

PKWC6-9

9.63±0.17

8.72±0.02 a

8.30±0.04 c

8.50±0.10 b

8.16±0.01 c

PKWC7-1

9.56±0.21

7.29±0.15 b

8.04±0.02 a

7.37±0.00 b

7.42±0.04 b

PKWC8-1

9.59±0.67

8.57±0.02 a

7.81±0.16 b

8.27±0.18 a

7.76±0.14 b

PKWD6-10

9.95±0.04

9.69±0.03 a

9.43±0.11 a

9.07±0.01 b

9.57±0.14 a

PKWD7-1

9.72±0.09

8.65±0.01 c

9.21±0.01 b

9.65±0.05 a

9.56±0.14 a

PKWD7-2

9.30±0.10

8.17±0.05 b

8.27±0.02 b

7.20±0.08 c

8.72±0.05 a

Values in the same row with different lowercase letter are significant different by Duncan's multiple range test (P<0.05)

Fig. 4 Effect of soybean, sesame and Job's Tears on viability of L. plantarum during exposure to simulated small intestinal juice (pH 8.0) with 0.45% bile salt for 240 min

B6-12

B7-1

B7-2

C6-1

C6-9

C7-1

D7-1

Fig. 5 Survival of L. plantarum during sequential exposure to simulated gastric juice pH 2.0 and small intestinal juice pH 8.0 in the presence of soybean (), sesame (¾), Job s Tears (¿) and in the absence of cereal (l)

Furthermore, seven acid-tolerant strains of L. plantarum (PKWB6-12, PKWB7-1, PKWB7-2, PKWC6-1, PKWC6-9, PKWC7-1 and PKWD7-1) were selected to test for their viability after sequential exposure to simulated gastric (180 min) and intestinal juices (240 min). Similarly, survival of all strains was improved in the present of the cereals over that of free cell. Interestingly, during exposure to intestinal juice all test strains were quite stable with survival ranging from 92 to 100% in the present of cereal and 84 to 100% in the absence of cereal (Fig. 5). Results revealed that all test strains have an adaptability in unfavourable conditions (Succi et al. 2005). This was probably because stress responses may be used to improve the survival of microorganisms in stressful condition (Cruz et al. 2009). When microorganism is pre-exposed to one stress such as acid condition, the surviving cells can tolerate better a subsequent unfavorable environment (adverse conditions of the GIT) (Burns et al. 2008, Buriti et al. 2010) . This kind of protection from one stress gained from prior exposure to another stress is called cross-protective stress response (Saarela et al. 2004; Buriti et al. 2010). Moreover, these results showed that the addition of the cereals could greatly enhance tolerance of B6-12, B7-2 and D7-1 to gastric and small intestinal transit.

Conclusion

In the present study, the rerults demonstrated that all test strains of L. plantalum exhibited the ability to tolerate simulated gastrointestinal tract conditions. Their tolerances to acid and bile juices were greatly enhanced by the addition of soybean, sesame and Job's Tears. The protective capacity of the cereals was dependant on their composition. Soybean was found to enhance the acid tolerance of the strains while Job's Tears improved the bile tolerance. These findings suggest that these cereals could be used as vehicle for the delivery of lactic acid bacteria as probiotic through the human gastrointestinal tract. This finding could further lead to the development of new symbiotic food products with the combination of probiotic lactic acid bacteria and cereals. Clinical trials for the potential health benefits to the consumer should be investigated.