Abstract: The antimutagenic activity of Lactobacillus bulgaricus and acidophilus to potent mutagen, 2-nitrofluorene was examined by the modified Ames test using Salmonella typhimurium TA100. The results showed that the supernatants, heat killed cells and viable cells of Lactobacilli, preincubated with mutagenic factor such as 2-nitrofluorene, displayed characteristic antimutagenic activities. L. bulgaricus and L. acidophilus cells showed 98 and 64% antimutagenic activity against 2-nitrofluorene, respectively. Antimutagenic activities of Lactobacilli were higher than their supernatants. All the strains, heated for 15 min at 100°C, antimutagenic activities were reduced by 36-47%.
INTRODUCTION
The presence of mutagen contamination in food and environment is causative of several diseases including tumor (Wollowski et al., 1999). It is important to control mutagenesis and carcinogenesis by identifying the hazardous agents and removing them from the environment. There are many reports that fermented milk products and Lactic acid bacteria inhibit the mutagenicity of a variety of chemical components (Asahara et al., 2006; Hsieh and Chou, 2006). Epidemiological evidences indicate a negative correlation between the incidence of certain cancers and consumption of fermented milk products (Rachid et al., 2002; Abdel-Gawad et al., 2004; Femia et al., 2002). Yoghurt and the lactic acid producing bacteria that it contains, have received much attention as potential cancer-preventing agents in the diet. Yoghurt has been reported to inhibit tumor progression and modulate the immune response and stimulate cellular apoptosis (Leblung and Perdigon, 2004; Rachid et al., 2002). Yoghurt and extracts thereof have been shown to be antimutagenic against a range of mutagens and pro-mutagens in microbial and mammalian cell systems (Nadathur et al., 1995; deMoreno de Le Blance and Perdigon, 2005). Certain epidemiological studies have suggested that consumption of yoghurt and other fermented milk products may reduce the incidence of colon or breast cancer (Parvez et al., 2006; Norat and Riboli, 2003; Perdigon et al., 2002; Rachid et al., 2002). 2-nitroflourene, widespread in the atomosphere, is a direct-acting mutagen and carcinogen (Paul et al., 1994).
In this study, we used a preincubation method, a modification of Ames test (Lo et al., 2002) to investigate the microbiological and antimutagenic characteristics of Lactobacilli, isolated from Iranian yoghurt against 2-nitrofluorene as a potent mutagen.
MATERIALS AND METHODS
Bacterial Strain
Samples of Iranian commercial yoghurts were homogenized with Ringer solution
by shaking for several minutes. 0.1 mL of this suspension was plated on the
surface of MRS agar (Merk, Germany). After anaerobic incubation for 48 h at
37°C, the colonies were tested for catalase reaction. All Lactobacilli
were rod shaped. The isolated strains were maintained as frozen cultures
in MRS broth with 15% glycerol at 80°C. Growth at 15 and 45°C was tested
in MRS broth after 2 and 5 days of incubation.
Assay for Antimutagenicity
The antimutagenicity of Lactobacilli was determined by measuring
the extent of decrease in mutation induced by 2-nitrofluorene (Sigma LTD). A
preincubation method reported by Maron and Ames (1983), which is a modification
of the Ames test (Lo et al., 2002) was used throughout this study. The
following components were added to the sterile glass tubes: Overnight culture
of Salmonella typhimurium TA100 (0.1 mL), MRS culture of Lactobacilli
(0.1 mL) and 2-nitroflorene, (2 μg/plate). The entire mixture was incubated
at 37°C with gentle shaking for 20 min. Then 2 mL of molten top agar (45°
C) containing 0.05 mM histidine (Sigma LTD), 0.05 mM biotin (Sigma LTD) and
0.09 M NaCl was added to this mixture. This combined solution was then poured
on a minimal glucose agar plate. The plate was then incubated at 37°C for
48 h to calculate revertants. Positive control consisted of S. typhimurium
TA100 and 2-nitrofluorene without MRS culture of Lactobacilli and
spontaneous control consisted of S. typhimurium TA100 and MRS medium.
Each assay was performed in triplicate and the antimutagenicity was expressed
as percentage of inhibition:
Inhibition (%) = [(A-B)/(A-C)]x100 |
A= No. of his. revertants in the absence of Lactobacilli (positive control),
B = No. of his. revertants in the presence of Lactobacilli,
C = Spontaneous revertants (negative control).
Preincubation of Lactobacilli Cultures with 2-Nitrofluorene
To evaluate the effect of preincubation of Lactobacilli cultures
with 2-nitrofluorene, MRS culture of Lactobacilli (0.1 mL) and 2-nitroflorene
2 μg/plate were initially mixed and incubated at 37°C for 20 min. Then
0.1 mL of S. typhimurium TA100 was added to the mixture and the incubation
was carried out at 37°C for another 20 min. Antimutagenicity of Lactobacilli
was determined as mentioned above. Positive control consisted of S. typhimurium
TA100 and 2-nitrofluorene without Lactobacilli cultures and spontaneous
control consisted of S. typhimurium TA100 and MRS medium.
Preincubation of Lactobacilli Cultures with S. Typhimurium TA100
To determine the effect of preincubation of Lactobacilli cultures
with S. typhimurium TA100, MRS culture of Lactobacilli (0.1 mL)
and S. typhimuriumTA100 (0.1 mL) were mixed and incubated at 37°C
for 20 min with gentle shaking. Then 2 μg/plate of 2-nitrofluorene was
added to this mixture and the incubation was carried out at 37°C for another
20 min. Antimutagenicity was assayed as mentioned above.
Antimutagenicity of Lactobacilli Cells on 2-Nitrofluorene
Cell suspension in PBS (0.1 mL) and 2-nitrofluorene were mixed and preincubated
for 20 min with gentle shaking, then S. typhimurium TA100 (01 mL) was
added. The solution was then incubated at 37°C for 20 min with gentle shaking.
Antimutagenicity assay was performed as mentioned above. Positive control consisted
of S. typhimurium TA100 and 2-nitrofluorene without Lactobacilli
and spontaneous control consisted of S. typhimurium TA100 and PBS.
Heat Treated Cells on 2-Nitroflourene
Cell suspensions of Lactobacilli in PBS were heated in boiling water
bath for 20 min. After heat treatment, the cells were vortexed for 5 min to
break coagulum and plated in MRS culture to confirm the lethal effect of the
heat treatment.
Statistical Analysis
The results are expressed as mean±standard deviation (n. as indicated).
T-test was used to assess the statistical significance between the test groups
and control group. The data were subjected to the Analysis of Variance (ANOVA).
A significant difference (p<0.05) was determined between the means.
RESULTS
Identification of Lactobacillus Isolated from Yoghurt
Fifteen Lactobacillus strains were isolated from 17 mild yoghurts
and 1 bio-yoghurt. All the isolates were catalase negative rod, producing no
gas from glucose. They were presumptively identified by determining the growth
behavior at 15 and 45°C. Three strains were identified as L. casei,
6 strains as L. acidophilus and 6 strains as L. bulgaricus.
Inhibitory Effect of Lactobacillus acidophilus Cells on 2-Nitroflourene
Antimutagenic activities of viable cells of L. acidophilus were detected
by using modified Ames test. Inhibitory effect of viable cells of L. acidophilus
was 64.45±9.50 on 2-nitroflourene (2 μg/plate) (Table
1) while it was only 30.11±0.59 and 36.66±5.12 for the supernatant
and heat killed cells of L.acidophilus, respectively (p<0.05).
Inhibitory Effect of Lactobacillus bulgaricus Cells on 2-Nitroflourene
The antimutagenic activities of the viable cells of L. bulgaricus were
detected by modified Ames test. Viable cells of L. bulgaricus showed
more than 97.99±0.88 inhibitory effect on 2-nitroflourene (2 μg/plate)
(Table 2) but supernatant and heat killed cells of L. bulgaricus
only showed 34.11±6.9 and 47.75±5.85 antimutagenicity, respectively,
which were significantly lower than those of the viable cells of L. bulgaricus
(p<0.05).
| Table 1: | Antimutagenicity of Lactobacilli acidophilus against 2-nitrofluorene |
| The results are presented as mean±SD for three plates., Inhibition (%) = [(A-B)/ (A-C)] x100%, A= revertants TA100 colonies in the presence of 2-nitrofluorene (positive control), B= revertant TA100 colonies in the presence of Lactobacillus acidophilus + 2-nitrofluorene C= Spontaneous revertant colonies of TA100 | |
| Table 2: | Antimutagenicity of Lactobacilli bulgaricus against 2-nitrofluorene |
| The results are presented as mean±SD for three plates, Inhibition % = [(A-B)/ (A-C)] x100%, A= revertants TA100 colonies in the presence of 2-nitrofluorene (positive control), B= revertant TA100 colonies in the presence of Lactobacillus bulgaricus + 2-nitrofluorene, C= spontaneous revertant colonies of TA100 | |
DISCUSSION
The role of diet on the etiology of cancer has been received increasing attention in the last 20 years (Le Blung and Perdigon, 2004; Brady et al., 2000). Studies on the conditions that enhance or decrease activation or inactivation of mutagens are important to control their risk to man.
Yoghurt and the lactic acid producing bacteria that it contains, have received much attention as potential cancer-preventing agents in diet.
In this study the inhibitory effect of L. acidophilus and L. bulgaricus strains of yoghurt origin against the 2-nitrofluorene was evaluated in vitro by modified Ames test (Lo, 2002). 2-nitrofluorene is a direct-acting mutagen and carcinogen and its mutagenicity has been proved on experimental animals. Paul et al. ( 1994) showed the nitroreduction of 2-nitrofluorene (2-NF) by a human microflora in female Wistar rats, while our finding showed the number of revertants was significantly reduced by incubation of L. acidophilus and L. bulgaricus.
We isolated Lactobacillus and tested them directly as viable bacteria in culture, which means that antimutagenic activities were exerted by yoghurt bacteria. It seems that these antimutagenic activities are dependent on the survival of LAB in yoghurt and in the intestinal tract.
Adminstration of LAB can also decrease DNA damage induced carcinogenesis (Pool Zoble et al., 1996). The mechanisms by which LAB exerts protective effects on DNA damage and tumorgenesis have not been yet elucidated.
In present study, viable cells of L. acidophilus showed antimutagenecity more than 64% but L. bulgaricus cells showed significantly (97%) higher antimutagenicity than L. acidophilus. It has been well established that various lactic acid bacterial strains originating from fermened milk possess antimutagenic activities (Rhee and Park, 2001; Haza et al., 2005). In humans, a decrease in fecal mutagenicity has been revealed after the consumption of L. acidophilus fermented milk together with fried meat (Lidbeck et al., 1992). L. acidophilus feeding has been shown to decrease the incidence of colon tumors in the rats challenge with the colon carcinogen DMH (Goldin and Gorbach, 1980). L. acidophilus is expected to be the main Lactobacillus species involved in the production of mild and probiotic yoghurt. Milk products fermented by L. bulgaricus and S. thermophilus were shown to exhibit dose dependent antimutagenic activity against a number of direct acting and indirect acting mutagens. Aceton extracts of yoghurt fermented by L. bulgaricus and S. thermophilus have been shown to be antimutagenic via metabolite fractions such as palmitic acid (Nadathur, 1995, 1996). Wollwoski (1999) has found that, oral application of L. bulgaricus in rats could prevent DMH induced DNA break in colon in vivo.
Inactive milk constituents may be converted into anti-mutagens by fermenting organisms, or milk may lack such precursors but serve as a neutral medium for the organisms that ordinarily produce antimutagens during the fermentative growth.
We also investigated supernatant and heat killed cells of Lactobacilli for their antimutagenic abilities. Our findings showed that the antimutagenic activities of the heat killed cells significantly decreased (Table 1 and 2), but Oata Zhang (1990) has been shown that heat killed cells of lactic acid bacteria have higher binding activities than viable bacterial cells.
In conclusion, present results demonstrated that viable L. acidophilus and L. bulgaricus isolated from yoghurt, exhibited strong antimutagenic activity against 2-nitroflorene in vitro, but heat killed cell bacteria has shown weaker antimutagenic activity, which means that these antimutagenic activities are dependent on the survival of LAB in yoghurt and in the intestinal tract.
ACKNOWLEDGMENTS
This research has been supported by Grant from Tarbiat Modares University (Tehran, Iran).