Biochemical identification of lactobacilli from chicken intestine and their potential probiotic activity

Probiotic Lactobacilli are associated with normal microbial ecosystem of gastrointestinal tract (GT) of humans and chickens and plays an essential role in in-vivo interactions occurring in GT and hence exert health benefits beyond inherent basic nutrition. In recent study, some potential probiotic Lactobacillus bacteria were screened from chicken intestine. Twenty strains have been selected keeping in notice their growth in Lactobacillus selective media i.e. De Man, Rogosa and Sharpe agar (MRS). They were subjected to gram staining, catalase activity and oxidase test. Out of these, 5 strains have been selected for detailed studies on the basis of best antimicrobial activity and Lactobacillus specific biochemical tests. These five strains were named as (LS1, LS2, LS3, LS4 and LS5) and their probiotic potential has been evaluated. Out of these five strains, LS4 has shown maximum antimicrobial activity (25.10 ± 2.008) by producing antimicrobial bacteriocins like compounds, which have inhibitory property against potential pathogenic strains. All the strains were stable in both acidic and alkaline pH. These strains were also tolerant to different concentrations of bile salts and sodium chloride (NaCl). Their temperature tolerance was also evaluated and observed that is an important selection criteria for probiotics. This study is extremely promising that underscores the important role of Lactobacillus strains, having probiotic effects, which may play an important role in food industry as starter-culture, co-culture and bio-protective cultures to standardize and enhance quality and safety of preserved food and beverages.


Introduction
Lactobacilli are non-pathogenic bacteria widely distributed in nature.These probiotic bacteria have a remarkable role in the preservation of foods and fermented products and therefore generally regarded as safe.
Probiotics are living micro-organisms, upon intake in specific numbers, exerts health advantages beyond innate basic immunity [1,2].Conventionally, the genus Lactobacillus, has the ability of producing lactic acid as the main end-product from carbohydrate metabolism.They are coupled with normal microbial population of GT of humans and chickens.The genus Lactobacillus comprised of a wide group of non-spore producing gram-positive bacteria.They are rod shaped and are generally deficient in catalase, however pseudocatalase can be found but they are in rare cases.They are chemoorganotrophic and grow only in complex media.Fermentable carbohydrates are utilized as energy source.Hexoses are broken down mainly to lactate (homofermentatives) or supplementary products such as acetate, ethanol, carbon dioxide (CO2), formate or succinate (heterofermentatives).Lactobacilli are found in foods, sewage, plants as well as within the intestinal, genital and respiratory tracts of humans and other animals [3,4].A number of low molecular mass compounds together with acids, alcohols, CO2, diacetyl, hydrogen peroxide and various metabolites are generated by these bacteria.Many of those metabolites has a wide range of activities against different species.A mixture of antagonistic substances produced by Lactobacilli consist of end products which are resulted from metabolism of antibacterial peptides and substances similar to antibiotics which are called bacteriocins [5].The potential of bacteriocins produced by Lactobacilli, to inhabit pathogenic microorganisms, may be either limited that is to inhabit only those bacteria that are closely associated to them or they may inhabit a wide range of gram-positive bacteria.The antimicrobial peptides of Lactobacilli are regarded safe for normal bio-preservation as they are considered to be digested by the enzymes present in the gastrointestinal tract [6].Resistance of bacteria to antibiotics is one of the major health issues nowadays.In order to come up with some solution to this health issue, research is mainly focused on finding such drugs that are more affective against these resistant bacteria.In such situations Lactobacilli and their products can be a better choice to be used as a substitute to antibiotics.Presently, most people including customers and researchers are attracted by these bacteria to be used as health promoters because more knowledge is available on their beneficial effects in food, health and diet [7,8].

Bacterial isolation
The intestinal samples were obtained in sterile containers and were immediately transported to the laboratory.A total of 25 intestinal samples were processed for the isolation of Lactobacilli.After 24 hours of incubation in CO2 incubator with 10% CO2, the tubes were checked for turbidity of the medium in order to confirm the cell growth.MRS medium was used for bacterial culture.After incubation, the broth culture was transferred to sterile plates containing MRS agar medium and incubated at 37°C in CO2 incubator for 24 hours.For colony purification, streak plate method was used.After culturing, the morphological characteristics including colony morphology, gram staining and catalase activity of the isolates were observed for initial identification.Gram positive and catalase negative bacilli were selected as Lactobacilli and preserved in glycerol stocks for long term storage

Antimicrobial activity
Agar well diffusion method was used to determine antimicrobial activity.To evaluate the antimicrobial activity of the isolated strains, three indicator strains were used i.e.
Escherichia coli, Salmonella thyphimurium and Stephylococcus aureus.These strains were grown in nutrient broth at 37°C for 24 hours.For antimicrobial activity, all the isolated strains were grown in MRS broth at 37°C in 10% CO2 for 24 hours.To get the supernatant 1ml from each culture was taken in eppendorf tubes and centrifuged at 4000 rpm for 10 minutes at 4°C.The supernatant was filtered with a sterile filter paper having 0.45μm pore size.A thick lawn of each indicator strain was prepared through sterile cotton swabs by spreading 100μl culture of each indicator strain on plates having BHI agar.Four wells per plate were made by using a 6mm sterile borer.100μl of the supernatant of each isolated strain was added to the wells and left for 2hrs to diffuse completely into the wells [12].All the plates were incubated at 37°C for 24 hours.After 24 hours the inhibition zones were measured.The experiment was performed in triplicate.

pH tolerance
The isolated strains were grown in MRS broth following the incubation at 37°C for 24 hours for the evaluation of pH tolerance.100μl of the overnight culture was inoculated into tubes each containing 10ml of MRS broth adjusted with different pH (2, 4, 6 and 8) followed by incubation in 10% CO2 at 37°C for 24 hours.Tubes having control MRS adjusted with pH 6.5 were also inoculated with cultures.Survival of the isolates and their growth was determined by monitoring the changes in optical density at OD 600nm using spectrophotometer.

Bile tolerance
As the suggested concentration of bile in the intestine is supposed to be 0.3%, and the time for which the food remains in the intestine is thought to be 4 hours, therefore the same concentration of bile was used in this experiment which was run for 4 hours.0.3% bile was added to MRS broth which was previously inoculated with fresh cultures of the isolated strains.After four hours of incubation, growth of the cultures was observed at OD 600nm.

Statistical analysis
Microsoft excel was used to analyze the data.

Results Identification
The bacterial isolates were characterized morphologically, physiologically and biochemically.
After microscopic examination, the isolates were found gram positive and rod shaped as shown in figure 1.They were non spore forming, catalase negative, non-motile and oxidase negative as shown in table 1.The isolates were also able to tolerate different concentrations of NaCl.

Antimicrobial activity
All the isolates showed antimicrobial activity against the three indicator strains in which the highest activity was shown by the strain LS4 against S. typhi (25.10±2.008)mm.In this experiment, the ampicillin antibiotic disc was used as positive control while un-inoculated MRS broth was used as negative control.No activity was observed for Ampicillin against S. typhi and S. aureus, while a zone of 16±0.81 mm was observed against E. coli.No inhibition zone was observed for MRS broth which shows that the zones were purely made by the supernatant of the isolates of Lactobacilli as shown in table 2 and figure 2. Among the 20 Lactobacillus isolates, 5 isolates were selected for further activities which were showing highest activity against the indicator strains.The 5 selected Lactobacillus isolates were LS1, LS2, LS3, LS4 and LS5 as shown in figure 3.

Temperature tolerance
The selected isolates were further screened for temperature tolerance.For this purpose the isolates were inoculated in MRS broth containing bromecresol purple and were followed by incubation for one week at different temperatures (10°C, 15°C, 25°C and 45°C).After incubation the tubes were checked for color change from purple to yellow.For the tubes which were incubated at 10°C, no color change was observed which indicates that no growth was present there while at 15°C only the isolate LS1 was able to grow.At temperature 25°C and 45°C all the 5 isolates were able to grow as all the tubes were observed with yellow color as shown table 4.

NaCl tolerance
All the selected Lactobacillus isolates were subjected to different concentrations of sodium chloride (NaCl) in order to check their NaCl tolerance ability.The concentrations of NaCl were 2%, 3%, 4% and 6.5%.After incubation all the isolates were checked for growth.As the change of medium color indicates the growth of the cells and no change in color indicates the absence of growth.The growth results of all the isolates at different NaCl concentrations are shown in table 5.According to the table 5 growth was observed at 6.5% NaCl concentration while only three isolates (LS1, LS2 and LS5) were able to grow in 4% NaCl concentration.All the isolates were able to grow at 2% and 3% NaCl concentrations as shown table 5.

Bile salt tolerance activity
The selected strains were subjected to different concentrations of bile salt in order to determine their bile salt tolerance.The concentrations of bile salt used in this experiment were 0.1%, 0.2% and 0.3%.During this experiment, the highest growth was observed for the strain LS4 (1.10±0.06)against the concentration 0.1% followed by the strains LS1 (1.03 ± 0.02) and LS5 (1.03 ± 0.034) while the lowest growth was recorded for the strain LS2 (0.39 ± 0.009) against the concentration 0.2%.The results are the means of three replicates.In (Table 6 & Figure 5) showed the results for bile salt activity.

Discussion
In present study, 20 Lactobacilli strains were isolated on the basis of preliminary identification tests and Lactobacillus specific characteristics using MRS broth and MRS agar medium from chicken intestine.Five isolates LS1, LS2, LS3, LS4 and LS5 have been selected on the basis of maximum antimicrobial activity for further identification.Probiotic properties of isolates have also been evaluated.In the initial identification steps, the isolates were rod shaped, gram positive and none of the isolates were oxidase and catalase producers.These results corelate with the results found by [13, 14].All the selected isolates were showing both aerobic and anaerobic growth as they were able to grow in the incubator with 10% CO2 and without CO2.The same results have also been found by Gibson et al. [15].The initial stages of identification that were relied on cell morphology confirmed that the selected isolates were gram positive rods occurring in chains both in pairs and in single form.According to colony morphology, they were rounded and flattened and slightly convex with intact edges.These results are in accordance with [16, 17].After initial identification, all the five selected isolates were processed for further identification through different Lactobacillus specific biochemical tests.The isolates were tested for gas production from glucose by inoculating them in tubes containing a medium in which three sugars i.e. glucose, lactose and sucrose were present.The tubes were incubated for 24 hours and observed for bubbles production in the tubes but no gas bubbles were present which indicated that none of the isolates have the ability of gas production.The same results were found by [16].They were also observed as endospore negative [17, 18].As the isolates were found negative for endospore formation, they were subjected to motility test by inoculating them in a motility agar medium.All the isolates were identified as none motile as their growth was limited only to the stab line and no growth was present around the sides of the stab line [19].The antimicrobial activity of the selected isolates was assessed through agar well diffusion method using Escherichia coli, Salmonella thyphimurium and Stephylococcus aureus.Four strains were showing maximum inhibitory activity against Stephylococcus aureus while the strain LS2 was showing maximum activity against Salmonella thyphimurium.The highest activity was shown by the strain LS3 against Salmonella thyphimurium while the least activity was shown by the strain LS1 against S. aureus.All the isolates were effective against E.coli [20, 21].Members of Lactobacilli having inhibitory potential against pathogenic microorganisms offer a better choice of probiotics which can be used as alternative to antibiotics in poultry as well as animal houses.
During the experiment all the isolates were able to grow at different range of pH either acidic or basic which is an important selection criteria for probiotics.According to our results, maximum growth was recorded at pH 6 and pH 8 by the isolates LS4 (2.0) and LS5 (2.0) respectively while minimum growth was recorded at pH 2 by the isolate LS3 (0.3).The isolates LS2 and LS4 were more stable at low pH [22].The growth of the isolates was monitored at different temperatures (10°C, 15°C, 25°C and 45°C).The incubation time for the isolates at different temperatures was one week.At 10°C no growth was present in these tubes while at 15°C only the isolate LS1 was able to grow.At 25°C and 45°C temperatures all the isolates were able to grow [23].After temperature tolerance the isolates were further subjected to NaCl tolerance.Different NaCl concentrations used during the experiment were 2%, 3%, 4% and 6.5%.These concentrations were used in MRS broth medium which contained bromecresol purple as cells growth indicator.After incubation the tubes were checked for the change of color of the medium from purple to yellow due to cells growth which results in the change of pH of the medium.MRS broth was used as control.During this experiment, no growth was observed at 6.5% NaCl concentration while at 4% NaCl concentration three isolates (LS1, LS2 and LS5) were able to grow.All the isolates were showing better growth at 2% and 3% NaCl concentrations [19].All the selected isolates were also subjected to bile salt tolerance activity which is considered to be the most important selection criteria for probiotics.As the concentration of bile in the human gastrointestinal tract is different at different times but its mean concentration is believed to be 0.3% (w/v) and their resting time is 4 hours [24].During this experiment three concentrations of bile were used which were 0.1%, 0.2% and 0.3%.MRS broth medium containing these concentrations of bile was inoculated with overnight fresh cultures of the isolates.The incubation time was 16 hours.After incubation growth was checked through spectrophotometer at OD 600nm.During this experiment, the highest growth was observed for the isolate LS5 against the concentration 0.1% followed by the isolate LS1 while the lowest growth was recorded for the strain LS2 against the concentration of 0.2%.

Conclusion
From this study, it is concluded that probiotics of Lactobacilli spp.has antimicrobial activity against three different pathogens.These species are able to grow in different environmental conditions like temperature and pH.Thus, these isolated microbes can be further characterized on the basis of probiotic activity.
[9].Identification Identification of these bacterial isolates were made by studying their morphological characteristics and subjecting them to different biochemical and morphological tests such as Gram staining, Catalase test, Endospore formation, Oxidase test, Motility test and tolerance to different concentrations of NaCl [10, 11].

Figure 4 .
Figure 4. Cell growth against different pH.

Figure 5 .
Figure 5. Cell growth against different bile concentration

Table 3 &
Figure 4).According to the table all the strains were able to grow at different range of pH either acidic or basic.