Today some strains of lactobacilli are extensively used in the food and pharmaceutical industries as commercial probiotics. Probiotics refer to viable microorganisms that promote or support a beneficial balance of the microbial population of the gut (Holzapfel and Schillinnger, 2002). It is most likely that the influence of probiotics is depending at least partly on the indigenous lactobacilli that are present in the gastrointestinal (GI) tract of the host (Waard et al., 2002).The diversity of these lactoflora varies between individuals depending on their genetic background, physiological and environmental factors (Arici et al., 2004). In addition, because of the strain dependency of health promoting properties of probiotics (Waard et al., 2004), it is necessary to find new probiotics among native strains. (Delgado et al., 2007; Khalil et al., 2007; Xanthopoulos et al., 1999).
According to the guidelines of the evaluation of probiotic organisms, reported by a joint FAO/WHO working group, two of the currently most widely used in vitro tests are resistance to gastric acidity and bile compounds based on both survival and growth studies (Vizoso Pinto et al., 2006). During the evaluation of bile tolerance by growth studies, the growth abilities of the examined strains in their culture media, containing different concentration of bile components can be assessed. These evaluations are obtained through the application of either of the following methods; a) by assessing the strain’s ability in bringing about changes in optical density (Liong and Shah, 2005; Suskovic et al., 2000; Gilliand and Walker, 1990; Gilliland et al., 1984) and pH characteristics (Succi et al., 2005) of the broth culture media; b) by assessing growth ability on solid culture media (Morelli, 2007; Chou and Weimer, 1999; Prasad et al., 1999). The former, differentiates the bile resistant strains, growth rates of which are less affected by the inhibitory effects of the bile. A few growth studies have also addressed the evaluation of the acid tolerance of the examined strains (Nguyen et al., 2006). Instead, in survival studies, cell enumeration of the tested strains are carried out before and after keeping cells under stressful conditions resembling the stressful environment of the stomach or the small intestine transit. Such conditions could be introduced either separately or sequentially in which the latter could display possible interaction between theses stresses (Martin et al., 2006; Vizoso pinto et al., 2006; Succi et al., 2005; Alender et al., 1999; Charteris et al., 1998; Mustapha et al., 1997). Growth studies seem to be easier, more reproducible and cost effective than survival studies and the normal practice in screening for probiotic potentials usually begins with such examinations. Although all the above mentioned in vitro tests have been extensively used as the primary steps to select probiotic potentials, but rarely their correspondence in relation to one another has been disputed in the literature.
The objective of the present research was to apply growth and survival studies to evaluate the potential probiotic properties of three native strains of lactobacilli, isolated from infant’s fecal flora with respect to acid and bile tolerance. There were two aspects in focus here (1) Comparison of native strains with commercial probiotic strains; (2) Compatibility of the growth and survival methods in discriminating amongst acid and bile resistant strains.
To date, this is the first trial involving comparison of acid and bile tolerance of commercial lactobacilli with Lactobacillus isolates originating from Iranian infants’ fecal flora.
MATERIALS AND METHODS
Bacterial strains and chemicals: Three native Lactobacillus stains used in this study were selected from 33 isolates of lactobacilli originating from the fecal samples of 6 infants less than 2 years of age. They were isolated on de Man Rogosa Sharpe (MRS) (Sharlou-Spain) medium containing 1 mg/l Vancomycin, adjusted to pH 5.4 by lactic acid (Mirlohi et al., 2008a) and identified as Lactobacillus acidophilus H26, Lactobacillus rhamnosus L51 and Lactobacillus plantarum A7 based on biochemical tests (Mirlohi et al., 2008b). Identification of Lactobacillus plantarum A7 was also confirmed by specific Polymerase Chain Reaction (PCR), using a reqA gene derived primer, plantF (5´-CGTTTATGCGGAACACCTA-3´) and a general primer pREV (Torriani et al., 2001). Electrophoretic observation of the amplification products with the expected length of 318#bp showed that the given strain was a L. plantarum strain (unpublished data). L. rhamnosus GG was isolated from a pharmaceutical product (Culturelle, USA) and identified based on biochemical and molecular methods (Mirlohi, et al., 2008b). L. acidophilus was purchased from a dairy company (Lactina, Bulgaria) as a commercial probiotic dairy culture and its identity was confirmed based on biochemical and physiological criteria. As the strain of the given commercial L. acidophilus was unknown, in this study, it was arbitrarily named as L. acidophilus Lac. The cultures were stored at -80ºC in 15% (v/v) glycerol. During the experiments, stock cultures were maintained at 4ºC on MRS agar slants, and were subcultured monthly. Working cultures were activated by two successive transfers in sterile MRS broth using 1% (v/v) inoculums at 37ºC before each experiment. Oxgall (Sigma Chemical Co., St. Louis, MO, USA, cat. no. B-3883) was used as bile component; 8N hydrochloric acid and sterilized 8% (w/v) saturated sodium bicarbonate solution were used for adjusting pH to the desired level during the experiments.
Evaluation of bile and acid tolerance of the strains through growth studies: The bile resistance of the isolates was evaluated by the method of Gilliland et al., (1984). The Lactobacillus strains were grown overnight in MRS broth. One hundred μl of the culture suspension was then inoculated into the tubes containing 20 ml of MRS broth with or without 0.3% (w/v) Oxgall, the latter was considered as control. The inoculated tubes were incubated at 37ºC anaerobically, under 5% CO2 in a CO2-air-jacketed incubator (Memmert, Germany). For each strain, three independent tests, each in duplicate, were carried out. Growth was monitored every 15 min for 10 h by measuring optical density at 620 nm (OD620) using a spectrophotometer (2100, UV-Vis, Unico, USA). The bile tolerance of each strain was defined as the difference in the time required for the absorbance value to increase by 0.3 units between MRS containing Oxgall and the control (Liong and Shah, 2005; Patal et al., 2004; Prasad et al., 1998; Gilliland and Walker, 1990). Bile tolerance was also evaluated through another growth study (Succi et al., 2005). In this procedure, the ability of the examined strains to reduce the pH in the presence of different percentages of bile salts or Oxgall was considered as the bile tolerance. One percent (v/v) of inocula of the activated culture medium of each strain were transferred to MRS broth containing 0, 0.3, 0.5, 1, 1.5 and 2% (w/v) bile component and the changes in pH were monitored (pH/temperature tester, Eutech instrument, Malaysia) at time intervals of 3, 6, 24 and 48 h.
In the evaluation of acid tolerance of the tested strains by the growth study, the method used by (Negugen, et al., 2006) was applied. One percent inocula (w/w) of the activated cultures in MRS broth, acidified to pH 3 with 8N hydrochloric acid were prepared in duplicates. The strains capable of growing to >107 CFU/ml after 24 h (37°C, 5% CO2) were considered as acid resistant strains. Each of the last two experiments is characterized by two independent duplicate tests.
Evaluation of acid and bile tolerance of the strains through survival studies: Survival study was performed based on the method used by (Succi et al., 2005). Activated cultures of each strain were inoculated at 1% (v/v) concentration into 100 ml of MRS broth acidified to pH 2.5 or 3 with 8N hydrochloric acid. The OD620 of all broth cultures was adjusted to 1.5, with a maximum difference of 0.25 between the broth cultures. They were incubated at 37ºC under 5% CO2 for 1 and 2 h. This was followed by increasing the pH of the culture medium with saturated sodium bicarbonate solution to 6.8-7.0 and subsequent addition of 1% (w/v) Oxgall. The incubation was continued for more than 4 h under the same conditions mentioned above. The survival of the tested strain was assessed by sampling of the medium at time intervals of 0, 1, 2 and 4 h during the 6 h of the experiment. Ten fold serial dilutions were made from each 1ml sample using peptone water and then, pour plated on the MRS agar. Approximately 30-250 colonies appeared after 48 h of incubation at 37ºC under a 5% CO2 concentration. The experiment was repeated twice for each strain and the data was presented as mean.
Statistical Analysis: Data analysis was carried out with the Minitab software (version 15). One way analysis of variance (ANOVA) was used to determine significant difference between the means, with significant level at a = 0.05.Tukey’s test was used to perform multiple comparisons between the means. In all growth studies, the mean of two to three times repeated measurements yielded the value for each replicate.
Growth studies: Table 1 presents the results of the mean values of the time required for each strain to increase by 0.3 units. As seen in Table 1, both L. plantarum A7 and L. rhamnosus GG showed superior growth rates when compared to the other strains tested, but after the addition of 0.3% (w/v) Oxgall, only L. plantarum A7 displayed the best growth ability. L. acidophilus H26 and L. rhamnosus L5K1 exhibited nearly the same bile tolerance as that of L. plantarum A7.
The results of the analysis of the mean pH values of the inoculated MRS and MRS supplemented with 0.3 and 0.5% (w/v) Oxgall, are represented in Table 2. In MRS without bile, L. plantarum A7 grew faster than the other strains with growth appearing after 6 h of inoculation. This strain together with L. acidophilus Lac and L. rhamnosus GG exhibited higher abilities in decreasing the pH of the MRS medium after 24 h of inoculation in comparison with L. rhamnosus L51 and L. acidophilus H26. By incorporation of 0.3% (w/v) and 0.5% (w/v) Oxgall in the MRS medium, L. plantarum A7 and L. rhamnosus GG appeared to be more tolerant than other strains. Instead, L. acidophilus Lac was shown to be the most sensitive strain under the same conditions. In the presence of 1-1.5% Oxgall (w/v), L. plantarum A7 and L. rhamnosus GG showed signs of growth after 48 h, but the other three strains were highly inhibited. None of the strains grew in the presence of 2% (w/v) Oxgall (data are not shown).
The results of the mean values of the viable cells present in the inoculated MRS (pH 3) immediately after inoculation and also after 24 h are represented in Table 3. All the tested organisms were able to grow in acidic media, however, L. plantarum A7 and L. rhamnosus GG grew better than the other strains resulting in more than 108 CFU/ml after 24 h of incubation.
Survival studies: The results of the effect of acid and bile stress on the survival of the examined strains are shown in Figure 1. Black and white arrows indicate the first exposure to acidic MRS and MRS containing bile, respectively. Both commercial strains, L. acidophilus Lac and L. rhamnosus GG have been shown to be more resistant to acid stress (pH 3) than the three native ones and exposure to the harsher acidic media (pH 2.5) made this difference even more significant. Damage to the commercial strains (L. acidophilus Lac and L. rhamnosus GG) due to the acidic environment was revealed by decreases of 0.3-0.7 and 0.35 - 0.97 log10 CFU/ml, respectively after 2 h of incubation in acidified MRS (pH 2.5).Whereas, decreases of about 0.46-2.54, 0.45-2.86 and 1.9-2.2 log10 CFU/ml were observed for the 3 native strains of L. plantarum A7, L. rhamnosus L5k1 and L. acidophilus H26, respectively. After being in acidic medium (pH 3), subsequent exposure to the neutralized bile containing environment seemed to have a more adverse effect on the survival of the examined strains with the exception of L. plantarum A7 and L. acidophilus H26. They showed further decreases of 1.33-1.6 and 0.8-1.69 log10 CFU/ml under this condition. L. rhamnosus GG and L. rhamnosus L5k1 were more sensitive and their cell counts was decreased by 1.51-2.153 and 2.27-3.49 log10 CFU/ml, respectively (Fig. 1A). Using a stronger acidic medium (pH 2.5), subsequent exposure to bile, noticeably decreased the cell number of L. rhamnosus L5k1 to less than 4 log10 CFU/ml. While, L. rhamnosus GG and L. plantarum A7 did not show further reductions (Fig. 1B) which compared to what was observed before (Figure 1A). Despite the strong acid tolerance characteristic, L. acidophilus Lac revealed remarkable sensitivity to bile stress, resulting in a lower microbial count of 5-6 log10 CFU/ml. This reduction made it the least survivable microorganism among the tested strains.
The results of the growth studies, which obtained from the two tested methods, did not support each other in evaluating the bile tolerance of L. rhamnosus GG, L. rhamnosus L5K1 and L. acidophilus H26. It could be concluded that a major difference in bile resistance of the strains is needed to yield the same results using the two mentioned methods.
Chateau et al. (1994) suggested that lactobacilli could be classified into four groups according to their delay in growth in the presence of bile (d): resistant strains (d £ 15 min), tolerant stains (15 min < d £ 40 min), weakly tolerant stains (40 min < d < 60 min) and sensitive strains (d > 60 min). According to this classification, all the strains used in this study are bile sensitive. As the commercial probiotic microorganisms used in this study, were also categorized as the sensitive strains, it is most likely that this classification is not valid. In addition, the results of this research are different from those of Succi et al. (2005). In the present study, bile stress led to greater inhibitory effects when compared to those observed by Succi and colleagues. Also, there is a particular difference between the results of the present study and the result of Suskovic et al. (2000) regarding the performance of growth studies in evaluation of the bile tolerance of Lactobacillus strains. The latter study claimed that 8 h following inoculation, the treated and control cells of a L. acidophilus strain entered the stationary phase of growth and the final cell count remained constant. In contrast to this finding, despite the inhibitory effects of bile (Table 2), the examined strains in this study continued growing in bile containing medium, 8 h after inoculation.
Oxgall is a natural dried bovine bile component containing both conjugated and unconjugated bile salts. Conjugated bile salts comprise a combination or mixture of bile derivatives in which their ratios vary from one individual to another. Depending on the peptide residues and dissociation constant and also bile salt hydrolase activity, conjugated bile salts result in different toxicity effects when compared to one another (Begley et al., 2006; Patel et al., 2004). Therefore, the differences between the results of this study with those of others, could be attributed to the difference between the bile components used.
The results of the survival studies confirmed the observation of other studies in which the commercially used probiotics were less tolerant to either acid are bile or artificial gastric and duodenum juices, when compared to noncommercial strains (Vizoso pinto et al., 2006; Prasad et al., 1999). Long-term subculturing and several passages of the commercial strains might lead to the development of less tolerant genetic variants in industry. (Tuomola, 2001).
Some studies have shown that the presence of bile salts in the bacterial culture medium is much more detrimental than the effects of low pH (Khalil et al., 2007). This claim supports the results of the present study, in that the bile stress leads to more inhibitory effects than acid stress. However, the results of this study are different from those of other studies in which the main decrease in survival of the examined strains has been shown to occur in acidic medium (Succi et al., 2005).
Correlation between growth and survival studies: In the evaluation of the compatibility of the methods applied, the results of the growth studies carried out for assessing bile tolerance were in accordance with the survival studies of L. plantarum A7 and L. acidophilus Lac, which showed the best and the least characteristics in this property. In the evaluation of acid the results of growth studies were not in agreement with those of the tolerance, survival studies. It is most likely that the mechanisms involved in acid resistance in lactobacilli (Cotter and Hill, 2003) do not support the growth rate, to the same extent that they protect cells’ survival under acidic stressful environments. For instance, among the mechanisms responsible for acid tolerance in L. acidophilus strains, cell wall integrity and biochemical structure are considered as key factors in protecting the cells from acidic conditions (Frece et al., 2005; Conway et al., 1986). It is unlikely that these characteristics compensate the growth delay in an equal manner. Hence, it would be reasonable to observe different responses from a Lactobacillus strain in growth and survival studies as the result of this study revealed for the commercial L. acidophilus strain. Alternatively, despite the strain dependency of probiotic properties, when different species with noticeable differences in metabolic activity are challenged by a stressful factor in growth rate, the intensity of their responses to the stress factor might be different depending on their natural growth abilities. As an example, L. plantarum is known as the most adaptable Lactobacillus species due to its large genome, capability in metabolizing different carbon sources and growth ability. By having such characteristics, it enables such a species to colonize different environments (Morelli et al., 2004; Molin, 2001; Kandler and Weiss, 1986). Therefore, when growth rate is under investigation, the strains belonging to this species may demonstrate higher resistance than others. Hence, L. plantarum and L. acidophilus are very different in biochemical and physiological properties; however, a species-related bile resistance has also been observed amongst the closely related species (Morelli, 2007).
The results of this study, confirm the preposition that acid and bile tolerant strians do exist in the population (Cebeci and Gurakan, 2003). Accordingly, the strain L. plantarum A7 can be identified as a potential probiotic strain with respect to in vitro acid and bile experiments. As one of the commercial probiotics, examined here, was lack of bile resistancy, it is thus recommended that food industries and laboratories examine the imported commercial products regarding their probiotic claims before their purchasing. In this way, introduction and development of native strains with an identified origin and specific probiotic features can be very valuable. The results of this study further suggest that when the delay in growth rate is regarded as the point of comparison, the species specificity of the Lactobacillus strains could be considered as an effective parameter. As in vitro studies can only partially mimic the actual in situ conditions in the gut ecosystem, survival of strain A7 under conditions more similar to the human GI tract could provide more clear information regarding the characterization of native probiotic strain. This study is currently in process.