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Original Article

Isolation and Characterization of Probiotic Properties of Lactobacilli

Isolated from Rat Fecal Microbiota

Prasant Kumar Jena1, Disha Trivedi1, Kirati Thakore1, Harshita Chaudhary1, Sib Sankar

Giri2, Sriram Seshadri1*

1 Institute of Science, Nirma University, Chharodi, Ahmedabad-382481, Gujarat, India.

2 Department of Biotechnology, Periyar Maniammai University, Thanjavur-613403,

Tamilnadu, India.

Running Head: Probiotic Lactobacilli from Rat Gutflora

*Corresponding author:

Dr. Sriram Seshadri Institute of Science, Nirma University

Sarkhej-Gandhinagar Highway Chharodi, Ahmedabad-382481, Gujarat, India Phone: +91 2717 241901-04 ext752, Fax: +91 2717 241916 E-mail: sriram.seshadri@nirmauni.ac.in, sriramsjpr@gmail.com

This article has been accepted for publication and undergone full peer review but has not been through the copyediting,

typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of

Record. Please cite this article as doi: 10.1111/1348-0421.12054.

This article is protected by copyright. All rights reserved.

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ABSTRACT

The objective of the present study was to characterize the lactobacilli isolates from the

feces of male wistar rat. Some physiological features of the candidate probiotic isolates

were preliminarily investigated such as tolerance to simulated gastric juice and bile salts,

antimicrobial activity, antibiotic susceptibility, and in vitro aggregations assay. Four

potential probiotic isolates (CS2, CS3, CS4, and CS7) were screened based on their

morphological and biochemical characteristics. The isolates showed good tolerance against

stimulated gastric juice and bile salts. CS4 and CS7 exhibited strong antibacterial activities

against the pathogens as measured in neutralized culture supernatants. All the lactobacilli

isolates were susceptible to all the tested antibiotics, except vancomycin. Moreover, the

isolate CS4 and CS7 were found to posses higher cell surface traits such as hydrophobicity,

auto-aggregation, and co-aggregation capacity. In addition, CS4 and CS7 had the greater β-

galactosidase activities. Biochemical tests and 16S rRNA gene sequencing confirmed that

CS2, CS3, CS4, and CS7 were Lactobacillus intestinalis PJ2, L. sakei PJ3, L. helveticus

PJ4, and L. plantarum PJ7, respectively. Based on the obtained results, L. helveticus PJ4

and L. plantarum PJ7 are ideal in vitro probiotic candidates and required for further in vivo

evaluation.

Key words Probiotics, Lactobacillus spp., Rat feces, Antimicrobial activity.

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List of abbreviations

BCP: Bromocresol Purple, BHI: Brain Heart Infusion, BLAST: Basic Local Assignment

Search Tool, CFU: Colony Forming Unit, EFSA: European Food Safety Authority, GIT:

Gastrointestinal Tract, GRAS: Generally regarded as safe, IAEC: Institutional Animal

Ethical Committee, LAB: Lactic Acid Bacteria, LB: Luria Bertani, MIC: Minimum

Inhibitory Concentration, MRS: deMan Rogosa Sharpe, MTCC: Microbial Type Culture

Collection, NCBI: National Centre of Biotechnology Information, OD: Optical Density,

PBS: Phosphate Buffer Saline, PCR: Polymerase Chain Reaction, U: Unit.

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INTRODUCTION

The growing health awareness in the consumption of microorganisms as probiotics has

encouraged consumers’ worldwide (1). Probiotics are defined as ‘live microorganisms

when administered in adequate amounts; confer a health benefit on the host’ (2). The genus

lactobacillus is an important group belonging to the normal mucosal microbiota of humans

and animals (3). They have been considered to be an important group of bacteria in

maintaining the stability of the gastrointestinal tract (GIT), in preventing intestinal

infections, and generally, in supporting intestinal health (4). Several species of lactobacilli

have generally regarded as safe (GRAS) status and some of them have the ability to interact

with intestinal epithelial cells. Their possible applications as mucosal vaccine vector (5),

probiotics (3) and ability to influence metabolic activities (e.g. cholesterol assimilation,

lactase activity) and vitamin production) (6) have generated interest.

Novel probiotics should be selected based on the knowledge of strain

characteristics. In addition to the ability to inhibit the growth of pathogens, the main

selection criteria of probiotics include many functional properties, such as tolerance to

gastric acidity and bile toxicity, adhesion to intestinal epithelium cells and / or mucus, and

ability to improve immune response of the host (3, 7). Probiotic bacteria selected for

specific applications must retain the characteristics for which they were originally selected,

these includes growth and survival during production and commercialization, as well as

during gastrointestinal transit (8).

Lactobacilli constitute one of the dominant genera in the rat large intestine along

with bacteroides, fusiforms, eubacteria, curved rods, and anaerobic Gram-positive cocci

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(9). It has been reported that certain strain of lactobacilli is beneficial for human as well as

in rats like for anti-inflammatory, allergic and side effects of nonsteroidal anti-

inflammatory drugs (10). The characteristics of lactobacilli of various origins, including

those isolated from rat intestine have been studied previously and many of them possess

properties that make them potentially probiotic (3, 11-14). Mice and rats are often used to

test probiotic lactobacilli and microbiota analysis. Hence, it is better to know the

lactobacilli flora with potential probiotic properties of these laboratory animals and to

understand the ecological habitat of the host. For the selection of highly potent probiotic

strains, safety and functional properties such as antibiotic resistance, adhesion to intestinal

mucosa, antimicrobial activity as well as immunomodulation potential are highly

important and should be studied using reliable in vitro screening methods (15).

The objective of this study was to characterize four lactobacilli isolates derived

from the rat fecal microbiota in order to access their potential as probiotic cultures. In vitro

probiotic properties such as gastric juice and bile tolerances, antibiotic susceptibility

profile, cell surface hydrophobicity, auto- and co-aggregation abilities, and β-galactosidase

production ability of the isolates were investigated.

MATERIALS AND METHODS

Bacterial strains and culture conditions

Five pathogenic bacteria were collected from Microbial Type Culture collections

(Chandigarh, India) and used as target bacteria (Table 1). The probiotic reference strain

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Lactobacillus acidophilus NCDC15 (16, 17) was obtained from National Dairy Culture

Collections (National Dairy Research Institute, Karnal, India) and used for preliminary

comparative probiotic characterization. All bacterial strains were stored in 20% glycerol at

–20°C to provide a stable inoculum throughout the study.

Isolation of LAB strains from the feces of male wistar rat

The male wistar rats weighing 200–250 g used in this study were obtained from ‘Central

Animal Facility of Institute of Pharmacy’, Nirma University, Ahmedabad (India). The

animals were maintained at controlled laboratory conditions and fed with standard diet and

water ad libitum. The experimental protocol was approved by the Institutional Animal

Ethics Committee (IAEC), Nirma University (Protocol No. IS/BT/PHD10-11/001).

After 15 days of acclimatization of rats, fecal samples were collected at day 7 and

used for viable counting and isolation of lactobacilli. Fresh fecal samples were 10-fold

serially diluted in saline and inoculated in acidified de Man, Rogosa and Sharpe (MRS)

broth (pH 2.5) for 2 h in order to select acid-tolerant lactobacilli. After acid treatment,

appropriate dilutions were re-cultured anaerobically (Anaerocult system; Merck) on MRS

agar plates at 37°C for 48 h. A total of 157 colonies were counted and designated as CS1–

CS157. Among them, 60 colonies were randomly selected and streaked onto MRS agar

plates supplemented with bromocresol purple (BCP; 0.17 g/L) (18). Typical colonies from

MRS-BCP plates were subjected to morphological and biochemical characteristics. Only

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the colonies showed Gram-positive and catalase-negative reaction were selected for further

studies. Selected isolates were stored at –20oC in MRS broth containing 15% glycerol.

Bile salt and acid tolerance

Tolerance of the isolates to bile salts and simulated gastric juice was assayed as described

by Kumar et al (19). For bile tolerance test, MRS broth was supplemented with 0.3% (w/v)

bile salts (ox gall, Himedia, India). The composition of simulated gastric juice was (g/L):

1.28 g NaCl, 0.239 g KCl, 6.4 g NaHCO3, 0.3% bile salts (Central Drug House, Mumbai),

0.1% (w/v) pepsin (Himedia, India) and the pH was adjusted to 2.5.

For all the tolerance tests, 5 mL culture of lactobacilli isolates grown overnight in

MRS broth were collected by centrifugation and washed twice with 4 mL of phosphate-

buffered saline (PBS; pH 7.0) and inoculated (at 107-8 cfu/mL) in modified MRS broth

containing bile salts and in simulated gastric juice which was then incubated at 37°C for 48

h. The number of viable cells was determined by serial dilution and plate-count method.

Tolerance to phenol

The survival ability of the isolates in the presence of phenol solution was investigated

according to the method of Xanthopoulos et al. (20). Briefly, bacterial cultures (107cfu/mL)

were incubated in MRS broth containing 0.4% phenol, and incubated for 24 h at 37°C.

Bacterial enumeration was done on MRS agar employing serial dilution after 0 h and after

24 h of incubation, respectively.

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Antimicrobial activity

To measure antimicrobial activity, lactobacilli culture filtrates were prepared by filtration-

sterilization (0.22 µm filter, Millipore) of supernatants obtained via centrifugation from

overnight cultures of the isolates. Filtrates were neutralized (set to pH 6.5) with 5 N NaOH.

MRS agar plates were flooded with 100 µL of pathogenic bacteria, air-dried, and then 6-

mm diameter wells were punctured in each plate. The prepared supernatants were poured

into respective wells (100 µL) and incubated for 24 h at 37°C. An agar-containing well

filled with MRS broth was used to determine the inhibitory activity of the medium.

Antibiotic susceptibility

The minimum inhibitory concentration (MIC) was determined for the following antibiotics:

chloramphenicol, quinuspristin+dalfopristin, clindamycin, erythromycin, vancomycin,

ampicillin, streptomycin, tetracycline, kanamycin and gentamycin. These antibiotics were

chosen to maximize the identification of resistance genotypes to the most commonly used

antimicrobials by assessing the resistance phenotypes (21). Lactobacilli isolates were

grown at 37°C for 18 h in MRS broth supplemented with antibiotics at different

concentrations. The growth was calculated by measuring OD at 600 nm and comparing

with the growth in the same medium without antibiotics. The isolates were categorized as

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susceptible or resistant to antibiotics according to the breakpoints levels described by

European Food safety Authority (EFSA) (21).

The antimicrobials were tested in the concentration ranges (mg/L) given in

parentheses: chloramphenicol (0.125-256), quinupristin/dalfopristin (tested as 30:70 ratio:

0.032-64), clindamycin (0.032-32), erythromycin (0.016-32), vancomycin (0.12-256)

ampicillin (0.032-64), streptomycin (2-4096), kanamycin (1-256) and gentamicin (1-2048).

Cell surface hydrophobicity assay

The degree of hydrophobicity of the isolates was determined by employing the method

described by Ekmekci et al (22), based on affinity of cells (cultured overnight in MRS

broth) to toluene in a two phase system. Hydrophobicity was calculated from three

replicates as the percent decrease in optical density of the original bacterial suspension due

to cells partitioning into the hydrocarbon layer. The cell surface hydrophobicity (%) of

isolate adhering to solvent was calculated as:

Hydrophobicity % = [(OD600 before mixing−OD600 after mixing) / (OD600 before mixing)]

× 100.

Autoaggregation assay

Auto-aggregation abilities were measured according to the method described by Collado et

al (23). Overnight grown lactobacilli culture at 37°C in MRS broth was pelleted and

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washed twice with PBS (pH 7.0) and resuspended in PBS. The OD of bacterial suspension

at 600 nm was adjusted to 0.25±0.05 to standardize bacterial concentration (108cfu/mL).

Bacterial cell suspensions were incubated at 37°C for different time periods (2, 4, 6, 12, and

24 h). Auto-aggregation percentage was determined by A600 as:

Auto-aggregation (%) = 1– [At / A0] × 100

where, At represents the absorbance at different time t = 2, 4, 6, 12, or 24 h) and A0 the

absorbance t = 0.

Coaggregation assay

Bacterial cell (lactobacilli isolates/ pathogenic bacteria) suspensions were prepared as

described above (autoaggregation assay). Absorbance (OD600) of bacterial suspensions was

adjusted to 0.25 ± 0.05 in order to standardize the bacterial concentration (107 -108

cfu/mL). Lactobacilli cell suspension (2 mL each) was mixed at least 10s by a vortex mixer

with 2 mL of the different pathogenic bacteria suspensions (E. coli MTCC 443, S. aureus

MTCC 737, S. flexnerii MTCC 1457, S. Typhimurium MTCC 733 and P. aeruginosa

MTCC 1688) and incubated for 4 h 37oC. The co-aggregation ability was expressed as

follows:

Co-aggregation % = 100 × [(Amix0 – Amixt) / (Amix0)]

where Amix0 represents the absorbance (A600 nm) of a bacterial mixture at t = 0, and Amixt

represents the absorbance (A600 nm) of a bacterial mixture after 4 h of incubation.

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β - Galactosidase activity

β -galactosidase activity of the isolates was assayed according to the method of Hsu et al

(24). Absorbance was measured at 420 nm using a spectrophotometer (Shimadzu, model

2467). A unit of β-galactosidase activity was defined as the amount of enzyme required for

catalyzing the formation of 1 μmoL/min of o-nitrophenol under the assay conditions.

Identification of bacteria and phylogenetic analysis

Selected isolates were identified on the basis of morphological, physiological, and

biochemical characterizations as well as 16S rRNA gene sequencing. For 16S rRNA gene

sequencing, chromosomal DNA was extracted as previously described (25) and 16S rRNA

gene was amplified with universal primers: forward, 5’-GAGTTTGATCCTGGCTCA-3’

and reverse, 5’-CGGCTACCTTGTTACGACTT-3’ (26). The PCR products were purified

using the QIAquick PCR purification kit (Qiagen) and sequencing was carried out using

automatic ABI 310 DNA Sequencer (Big Dye Terminator Cycle Sequencing Ready

Reaction Kit, Perkin Elmer). The Basic Local Alignment Search Tool (BLAST) from the

NCBI (http://www.ncbi.nlm.nih.gov/) was used for nucleotide comparison for percentage

similarity. Sequences of close relatives together with the newly determined sequences were

aligned using the ClustalW software program (NCBI, Bethesda, MD, USA) (27). A

phylogenetic tree was constructed by using the neighbor-joining method, which produced a

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unique final tree under the principle of minimum evolution using the MEGA5 programs

(28).

Statistical analysis

One way analysis of variance (ANOVA) was used to analyse the data. Multiple

comparisons were performed with Tukey’s test. All statistical analyses were performed

using the OriginPro software (version 8; OriginLab Corporation, Northampton, U.S.A).

Statistical significance was accepted at P < 0.05 and the results are expressed as mean±SD.

RESULTS

Isolation and identification of LAB

Biochemical tests revealed that only four isolates (CS2, CS3, CS4, and CS7) showed

Gram-positive and catalase negative reaction. Thus, these four isolates were used in

subsequent experiments.

The morphological and biochemical characters of CS2, CS3, CS4, and CS7

resembled those of Lactobacillus species. The comparative 16S rRNA gene sequencing

revealed that, isolates CS2 (590 bp), CS3 (650 bp), CS4 (950 bp), and CS7 (430 bp) had

98% , 99%, 99%, and 98% nucleotide base homology with L. intestinalis DSM 6629, L.

sakei DSM 20017, L. helveticus DSM 20075, and L. plantarum NRRL B-14768,

respectively. The NCBI GenBank accession numbers of these sequences are JQ068822,

JQ041698, JQ068823, and JN853601, respectively. The phylogenetic tree showed the

species relatedness of the isolates (Fig. 1).

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Bile salt and acid tolerance

The results of bile salt and acid tolerance are summarized in Table 2. All the isolates

showed relatively high resistance to stimulated gastric juice and bile salts. Among the

isolates, CS4 showed strongest resistance (P < 0.05) to gastric juice at any point of time.

After 6 h exposure to acidic (pH 2.5) condition CS2, CS3, CS4, and CS7 showed

concentrations of 106.13cfu/mL, 105.95 cfu/mL, 107.46cfu/mL, and 106.94cfu/mL, respectively.

In case of bile salts tolerance, all the tested isolates were able to grow in 0.3% bile (Table

3). Isolates CS2 and CS3 showed significantly lower tolerance to bile compared to the

reference strain.

Phenol tolerance

Among the isolates, CS4 (108.05 cfu/mL) showed significantly higher (P < 0.05) growth in

the presence of 0.4% phenol compared to the reference strain and other isolates (Table 4).

The isolate CS7 (106.28 cfu/mL) showed least resistance to phenol.

Antimicrobial activity

All four isolates (in the form of cell-free spent broth) showed inhibitory activities against

the indicator bacteria, although degree of inhibition varied (Fig. 2). In particular, isolate

CS4 showed strongest inhibitory activity (inhibition zone ranged from 15.66 mm to

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23.6mm) against the pathogens; although there was no significant difference with inhibitory

activities of L. acidophilus NCDC15. CS3 and CS7 exhibited a moderate inhibitory activity

towards pathogens (inhibition zone ranged from 12.33 mm to 20.33 mm) and CS2 was least

effective against the pathogens (inhibition zone 6 mm to 16 mm).

Antibiotic susceptibility

According to breakpoints levels established by EFSA (21), the isolates were susceptible to

all the tested antibiotics, except vancomycin (data not shown). It was suggested that

probiotic strains lack acquired antimicrobial resistance properties is an important requisite

when considering them to be safe for animal consumption (29).

Cell surface hydrophobicity assay

Lactobacillus isolates CS4 (78.3±1.8 %) and CS7 (74.2±2.2 %) showed remarkable

hydrophobicity activity (P < 0.05) in the toluene as compared to the reference strain

(57.54±4.7 %). CS2 and CS3 had lower hydrophobicity activity i.e. 44.74±2.6 % and

37.39±2 %, respectively.

Aggregation assay

Autoaggregation abilities of the isolates increased with increase in incubation period (Fig.

3). Among the isolates, CS4 showed the highest autoaggregation ability (47.2%) after 24 h

of incubation (Fig. 3), followed by CS7 (45.5 %) and CS3 (33.2 %). The reference strain

(L. acidophilus NCDC15) showed 47.72% autoaggregation potential after 24 h of

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incubation. This result indicates that the CS4, CS3, and CS7 possess high potential ability

to adhere to epithelial cells and mucosal surfaces.

All the tested isolates showed coaggregation abilities with pathogens (Fig. 4).

Among the isolates, CS4 and CS7 had shown better coaggregation ability with pathogens.

CS4 showed higher coaggregation percentage with S. aureus (38.22 %) and lesser with P.

aeruginosa (25.20 %). Likewise, CS7 showed higher co-aggregation properties with S.

aureus (36.61 %) but lesser with S. Typhimurium (24.99 %). CS3 showed least co-

aggregation abilities with S. flexnerii (11.89%). In the other hand probiotic strain L.

acidophilus NCDC15 showed higher coaggregation percentage with S. aureus (38.05%)

and lower activity against S. Typhimurium (25.16%).

β-galactosidase activity

All the isolates produced β -galactosidase after 24 h of incubation at 37ºC. CS4 was the

highest producer of β-galactosidase (3.38±0.04 U/mL) and CS7 produced second highest

activity (3.1±0.04 U/mL). These results did not vary significantly with the β-galactosidase

of L. acidophilus NDC15 (3.80±0.37 U/mL). The lowest β-galactosidase activities were

found in isolates CS2 (2.17±0.04 U/mL) and CS3 (2.23±0.05 U/mL).

DISCUSSION

In the present investigation, four potential probiotic isolates (CS2, CS3, CS4, and CS7)

were identified as Lactobacillus intestinalis PJ2, Lactobacillus sakei PJ3, Lactobacillus

helveticus PJ4, and Lactobacillus plantarum PJ7, respectively. Various lactic acid bacteria

(LAB) such as L. intestinalis (10), L. plantarum (10, 30), L. reuteri (3), L. helveticus, and

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L. sakei (13) isolated from fish (30), canine (31), pig (3, 32) or veal calves (14) have been

reported to have probiotic activity. Escherichia coli isolated from rat gut have also been

reported to have probiotic activity (33).

Probiotic bacteria should have the ability to survive in gastric acidic environments

in order to be effective. In the present study, we found that stimulated gastric juice did not

cause any significant decrease (table 2) in viability of L. intestinalis PJ2, L. sakei PJ3, L.

helveticus PJ4, and L. plantarum PJ7. From this result, we suggest that these strains may

survive in the acidic environment of the stomach. Our results are in agreement with those

obtained from previous studies where lactobacilli strains showed viability at low pH values

(3, 11, 12, 14, 34).

Bile tolerance is one of the essential properties required for LAB to survive in the

small intestine and to be functionally effective in intestine. Bile plays a fundamental role in

specific and non-specific defence mechanisms in the gut; the magnitude of its inhibitory

effects is determined primarily by the concentrations of bile salts (34). In the present study,

bile acid and pH 2.5 had no effect on most of the isolates. Bacteria get stressed by low-pH

conditions in the stomach, and also by bile acid. For this reason, probiotic bacteria should

survive at 0.15%–0.30% bile acid (35). The excellent ability to remain viable in stimulated

gastric juice and good bile tolerance of L. helveticus PJ4 and L. plantarum PJ7 are in

accordance with previous results (11, 12, 36). This observation suggests that these isolates

have the potential to survive in the human GI tract and can likely to survive the passage

through the stomach and the small intestine and remain viable in the large intestine.

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In the present study, L. helveticus PJ4 and L. plantarum PJ7 exhibited higher

survival after 24 h of incubation and remained completely unaffected with 0.4% phenol.

Phenols may be formed in the gut by bacterial deamination of some aromatic amino acids

derived from dietary or endogenously produced proteins (37). Products of colonic protein

degradation and metabolism include ammonia, phenols, indoles and amines which have

been shown to exert toxic effects in vitro and in animal models. These compounds are

present in faecal samples suggesting that they may exert gut mucosal effects (38) and can

exert a bacteriostatic effect against some lactobacilli strains. Hence, tolerance to phenol is a

characteristic probiotic property. Thus, PJ4 and PJ7 may able to survive in the harsh gut

environment and provide health benefits.

Neutralized supernatant from PJ4 exhibited strong inhibitory activity against

pathogens tested, except S. flexnerii; while PJ3 and PJ7 showed moderate inhibition zone

(Fig. 2). The inhibitory activity observed cannot be due to the acidity of the culture, since a

neutralized supernatant was utilized (pH 6.5). The ability to inhibit the growth of harmful

bacteria is also considered as a desirable feature for probiotic bacteria (3). Previous studies

have demonstrated diverse growth inhibition of different pathogens by many LAB strains

originating from various foods (39, 40) or humans (41, 42) and animals (11, 43), including

porcine (3), which is in accordance with our findings.

All bacterial products intended for use as feed additives must be examined to

establish the susceptibility of the component strain(s) to a relevant range of antimicrobials

of human or veterinary importance (21, 29). In order to discard the presence of transferable

antibiotic resistance genes in any of the candidate probiotic strains, antibiotic resistance

profile was assessed. According to breakpoints levels established by EFSA, we found that

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all the strains were susceptible to all the antibiotics tested, except vancomycin,

corroborating their safety as probiotic strains. Bacterial species used in the present study

has already been recognized as intrinsically resistant to vancomycin (21). Vulnerability

against antibiotics is considered to be the most important probiotic characteristic. All the

strains of lactobacilli tested in this study did not demonstrate antimicrobial resistance when

tested according to EFSA guidelines (21).

Hydrophobicity of bacteria is a phenotype related to their adhesive capacity (44). In

the present study, PJ4 and PJ7 exhibited higher hydrophobicity to hydrocarbon toluene.

Patel et al. (1) reported that bacterial cell surface hydrophobicity and auto-aggregation

ability are directly correlated, and hydrophobicity could be one of the determinants of auto-

aggregation. Del Re et al. (44) have reported that strains can adhere to cell monolayers if

they autoaggregate and manifest a well degree of hydrophobicity as determined by

microbial adhesion to hydrocarbons.

Aggregation between microorganisms of the same strain (autoaggregation) or

between genetically different strains (coaggregation) is of considerable importance in

several ecological niches. Aggregating bacteria may gain an adequate mass to form

biofilms or adhere to the mucosal surfaces of the host and exert their functions (45). In the

present study, L. helveticus PJ4 showed highest auto-aggregation ability (47.2±2.4%),

followed by L. plantarum PJ7 (45.5±1.8%), L sakei PJ2 (33.2±1.7%), and L. intestinalis

PJ2 (23.2±2.5). The mechanism of autoaggregation in lactobacilli showed that proteins

present in the culture supernatant and proteins or lipoproteins located on the cell surface are

involved in the cell aggregation (46).

Coaggregation is related to the ability to interact closely with other bacteria (47). In

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the present study, L. helveticus PJ4, and L. plantarum PJ7 co-aggregated at higher

percentage with the pathogens. Coaggregation ability of PJ4 and PJ7 with pathogens could

contribute to the potential probiotic properties ascribed to specific bacteria (23, 45). Co-

aggregation abilities of probiotic bacteria may enable them to form a barrier that prevents

colonization by pathogenic bacteria (23).

Lactose maldigestion and/or intolerance may be treated using lactobacilli from

fermented products that contain the lactose hydrolyzing enzyme β-galactosidase. β-

galactosidase is an intracellular enzyme that appears to act when being released from

bacterial cells during their transit through the small intestine (48). In this study, L.

helveticus PJ4 (3.38±0.04 U/mL) and L. plantarum PJ7 (3.1±0.04 U/mL) was found to have

the higher β-galactosidase activity as compared to other two isolates. Lactobacilli that are

able to hydrolyze lactose might also be useful for compensating the lactase insufficiency

(6).

In conclusion, four Lactobacillus strains isolated in this study from the feces of

wistar rat posses in vitro properties that make them potential candidates for probiotic

applications. Among the strains, L. helveticus PJ4 and L. plantarum PJ7 exhibited

interesting probiotic properties such as excellent pH and bile tolerance, cell surface traits

like hydrophobicity and aggregations, β-galactosidase activity, and suppressed pathogen

growth under in vitro conditions. Moreover, all the strains were susceptible to a number of

clinically effective antibiotics. These results collectively suggest that PJ4 and PJ7 have

promising properties important for potential probiotics. Hence, more research is needed to

exploit other potential probiotic properties of these strains. Further, in vivo trials have to be

carried out to determine if they also function as probiotics in real-life situations.

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ACKNOWLEDGEMENT

The authors are thankful to Nirma Education and Research Foundation (NERF),

Ahmedabad for financial support.

DISCLOSURE

No authors have any conflicts of interest to disclose.

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Figure legends

Fig. 1. Neighbor-joining phylogenetic tree based on 16S rRNA gene sequences showing

the position of isolates (Lactobacillus) isolated from feces of wistar rats. Numbers of nodes

of levels of bootstrap support (%) from a 1,000-record resample dataset. Bar: 0.1, not

substitution per position.

Fig. 2. Antimicrobial activities of neutralized culture supernatants of lactobacilli isolates

and reference strain against enteric pathogens. L. acidophilus NCDC15 was used as a

reference probiotic strain. Data are expressed as mean ± SD (n = 3). * P < 0.05.

Fig. 3. Auto-aggregation percentages of lactobacilli isolates as measured at 2, 4, 6, 12, and

24 h of incubation in 37˚C. L. acidophilus NCDC15 was used as a reference probiotic

strain. Data are expressed as mean ± SD (n = 3). * P < 0.05.

Fig. 4. Co-aggregation percentages of lactobacilli isolates with pathogens measured at 24 h

of incubation in 37˚C. L. acidophilus NCDC15 was used as a reference probiotic strain.

Data are expressed as mean ± SD (n = 3). * P < 0.05.

28

Table 1. Target bacteria and their culture collections

Target Strains Collection Growth medium Growth

temperature (°C)

Salmonella Typhimurium MTCC733 LB 37

Staphylococcus aureus MTCC737 BHI 37

Shigella flexnerii MTCC1457 BHI 37

Escherichia coli MTCC443 LB 37

Pseudomonas aeruginosa MTCC1688 BHI 37

BHI: Brain heart infusion agar, LB: Luria bertani agar (Himedia).

29

Table 2. Acid Tolerance of lactobacilli isolates at 37o C.

Isolates Log cfu/mL

0 h 1 h 2h 4h 6h

L. acidophilus NCDC15 6.39 ± 0.15 6.25 ± 0.19 6.29 ±0 .13 6.42 ± 0.09 6.98 ±0.25

CS2 6.72 ± 0.05 6.68 ± 0.06* 6.48 ± 0.06* 6.21 ± 0.09* 6.13 ±

0.06*

CS3 6.47 ± 0.1 6.39 ± 0.05 6.18 ± 0.04 5.81 ± 0.06* 5.95 ±

0.6*

CS4 6.86 ± 0.06* 6.89 ± 0.08* 7.12 ± 0.1* 7.33 ± 0.05* 7.46 ±

0.07*

CS7 6.65 ± 0.07* 6.53 ± 0.07* 6.80 ± 0.11* 6.84 ± 0.06* 6.94 ±

0.07

Bacterial counts were determined by plate counts on MRS agar plates. L. acidophilus

NCDC15 was used as a reference probiotic strain. Data are expressed as mean ± SD (n =

3). * P < 0.05.

30

Table 3: Tolerance of lactobacilli isolates to bile salts (0.3% Oxgall) at 37o C for 24h.

Isolates Log cfu/mL

MRS broth without Oxgall MRS broth with 0.3% Oxgall

0 h 24h 0 h 24h

L. acidophilus NCDC15 6.34 ± 0.16 8.79 ±0 .22 6.38 ±0 .19 8.32 ± 0.11

CS2 6.77 ± 0.17* 8.48 ±0 .15* 6.87 ±0 .07* 7.44 ± 0.05* CS3 6.65 ± 0.21* 8.60 ±0 .22* 7.07 ± 0.05* 7.56 ±0 .09* CS4 6.89 ± 0.03* 9.62 ±0 .23* 7.23 ± 0.04* 8.80 ± 0.09 CS7 6.46 ±0.08 9.22 ± 0.21* 6.75 ± 0.07* 7.88 ±0 .06 a Bacterial counts were determined by plate counts on MRS agar plates. L. acidophilus

NCDC15 was used as a reference probiotic strain. Data are expressed as mean ± SD (n =

3). * P < 0.05.

31

Table 4. Tolerance of lactobacilli isolates to 0.4% Phenol at 37o C for 24 h.

Isolates Log cfu/mL

MRS broth without Phenol MRS broth with 0.4% phenol

0 h 24h 0 h 24h

L. acidophilus 6.26 ±.09 8.54±.11 6.35 ±.14 7.33 ±.13 NCDC15

CS2 6.83 ±0.05* 8.54 ±0.07 6.83 ±0.02* 6.58 ±0.05* CS3 7.22 ±0.04* 9.50 ±0.06* 7.28 ±0.04* 8.05 ±0.08* CS4 7.22 ±0.04* 9.50 ±0.06* 7.28 ±0.04* 8.05 ±0.08* CS7 5.73 ±0.05* 7.29 ±0.04* 5.69 ±0.04* 6.28 ±0.03* a Bacterial counts were determined by plate counts on MRS agar plates. L. acidophilus

NCDC15 was used as a reference probiotic strain. Data are expressed as mean ± SD (n =

3). * P < 0.05.

32

Figure 1

33

Figure 2

34

Figure 3

35

Figure 4