Probiotics are live microorganisms which upon ingestion confer

health benefits to the host such as prevention of infection in the

digestive tract, activate, and modulate the immune response

and increase the number of native bacteria in the gut. The

present study was aimed to isolate bacteria from donkey dung

and characterize for probiotic activity. Bacterial cultures were

isolated from excreta of infant donkey and were characterized

using standard procedures. Cultures were grown anaerobically,

and in total 16 cultures showing Lactobacillus morphology

were further screened for the probiotic property. Isolate LB-VII

was found to be non-hemolytic and has the ability to tolerate

1.2% bile, pH 1.5~10, 8% NaCl as well as showed growth at

42°C. The culture survived gastric and intestinal environment

and showed bile salt hydrolysis activity. LB-VII exhibited 100%

auto-aggregation and hydrophobic reaction. The culture could

also co-aggregate with Escherichia coli, Enterococcus faecalis,

and Staphylococcus aureus, a property, which is required to

control pathogens. Moreover, the isolate resist a wide range of

antibiotic. All these characters make LB-VII a good probiotic

culture and was identified as L. plantarum by molecular methods.

Keywords: Lactobacillus, donkey dung, probiotics

Micro-organisms that reside in digestive tracts of hosts have

a large impact on host health (Kobierecka et al., 2017). Probiotics

are defined as live micro-organisms when ingested in adequate

amounts confer a health benefit to host such as the production

of antimicrobial compounds, modulation of the immune response,

confer resistance to food antigens, assimilate cholesterol, prevent

autoimmunity etc. (Paraschiv et al., 2011). Other than these

benefits, probiotics can also enhance digestion and control acid-

base balance in the gut (Yirga, 2015). They also can produce

precursors of aroma compounds such as free amino acids, free

fatty acids etc. (Chen et al., 2010). Isolation and probiotic

characterization from the various environmental source be the

current area of interest (Khisti et al., 2019).

Lactobacillus species, a dominant group of bacterial species

found in the human gastrointestinal tract, is a member of

Firmicutes phyla, a group of Gram-positive, non-pathogenic,

catalase-negative, non-spore forming, anaerobic bacteria, that

can ferment hexoses and pentoses to produce lactic acid and

acetic acid (Khemariya et al., 2016; Behera et al., 2018).

Lactobacillus and Bifidobacterium are most studied and used

probiotic species due to their diverse health benefits (Huang

et al., 2015). Nowadays, donkey milk has attracted many

researchers due to its chemical composition which is similar to

that of human milk and hence is suitable for consumption for

infants (Cariminati et al., 2014). Nutritional components reveal

that donkey milk poses a high amount of lactose and low levels

of casein and fat; essential for the survival of lactic acid bacteria.

There are many reports of bacteria especially, Lactobacillus

group that had been isolated from various animal sources such

as milk and dung of cow and buffalo. Infant animal dung is

considered as the best source of probiotic bacteria since they

Korean Journal of Microbiology (2020) Vol. 56, No. 2, pp. 160-169 pISSN 0440-2413DOI https://doi.org/10.7845/kjm.2020.0038 eISSN 2383-9902Copyright ⓒ 2020, The Microbiological Society of Korea

Isolation of Lactobacillus from donkey dung and its probiotic

characterization

Suyash Arunrao Kathade1 , Mayur Arjun Aswani2 , Pashmin Kaur Anand1 , Suresh Jagtap2 , and

Niricharan Kunchiraman Bipinraj1*

1Bharati Vidyapeeth (Deemed to be University), Rajiv Gandhi Institute of I.T. and Biotechnology, Katraj 411046 and Pune,

Maharashtra, India2IRSHA, Bharati Vidyapeeth Deemed to be University, Katraj 411046 and Pune, Maharashtra, India

(Received April 22, 2020; Revised June 8, 2020; Accepted June 9, 2020)

*For correspondence. E-mail: bipinrajnk@gmail.com,

k.bipinraj@bharatividyapeeth.edu;

Tel.: +020-2437-9013

Lactobacillus from donkey dung ∙ 161

Korean Journal of Microbiology, Vol. 56, No. 2

are dependent on mother’s milk which is the rich source of

nutrient for the gut bacteria to establish themselves in a gastro-

intestinal environment. So far there is no report on probiotic

bacteria enumerated from donkey dung hence objective of the

present research is to isolate, identify and characterize probiotic

microorganisms from donkey dung.

Materials and Methods

Sample collection

Donkey dung of 1-month young domesticated Indian donkey

foal was collected in sterile containers from a farm near Pune

District, Maharashtra, India. The samples were transferred to

the lab in ice bucket filled with ice and stored at 4°C before

processed for culture isolation.

Culture isolation

Sample (100 g) was properly homogenized to get a uniform

consistency and 1,000 mg of dung was aseptically transferred

into 100 ml sterile saline (0.8%) and vortexed to make a

suspension. It was then serially diluted and spread plated on De

Man Rogosa and Sharpe (MRS, Himedia) medium. The plates

were incubated at 37°C for 48 h under anaerobic conditions in

an anaerobic jar. To maintain anaerobic conditions 0.1% sodium

thioglycolate was added as a reducing agent in all experiments.

After incubation, morphologically distinct colonies were isolated

and observed microscopically. Cells that showed Lactobacillus

morphology were purified and stored in MRS agar slants.

These cultures were further inoculated in MRS broth for 48 h,

centrifuged and suspended in saline to get cell concentration

of 107 CFU/ml. This suspension (1%) was used for further

experiments. Each experiment was performed in triplicate and

mean value were calculated.

Cultural and colony characteristics

Cultural and colony characterization of all isolates was per-

formed based on Bergey’s Manual of Systemic Microbiology,

Gram staining was performed as per the method of (Coico et

al., 2005) to observe Gram character of culture, endospore

staining was performed as described by (Reynolds et al., 2009)

method for observation of spores in cultures, acid-fast staining

was performed as per the method of (Reynolds et al., 2009) for

confirmation of acid-fast bacilli, (Goyal et al., 2012) method

was used for performing catalase test and to detect the presence

or absence of enzyme catalase.

Carbohydrate fermentation and gas production

Bromothymol blue broth base medium containing different

carbohydrates (1000 mg, 1% w/v) namely lactose, glucose,

sucrose, xylose and starch were used with and without Durham

tube for gas production and carbohydrate fermentation assay

respectively. After the inoculation, the media were incubated

for 37°C for 24 h anaerobically. The positive reaction was

indicated by colour change for carbohydrate fermentation and

gas formation in Durham’s tube for gas production assay

(Thakur et al., 2017).

Probiotic characterization

Probiotic characterization assays were performed as per the

guidelines given by ICMR-DBT, WHO and the World Gastro-

enterology Organization. Accordingly following assays were

performed.

Toxicity assay

For toxicity assay, isolates were spot inoculated on sheep

blood agar plates and incubated at 37°C for 24 h in anaerobic

jars. Toxicity was determined by the pattern of haemolysis on

blood agar plates (Papadimitriou et al., 2015; Pino et al., 2019).

Bile, pH, NaCl, and temperature tolerance

For pH tolerance assay, cultures were inoculated in MRS

with pH ranging from 1.5 to 10 adjusted using 1 N HCl or 1

N NaOH. Similarly, for bile tolerance assay cultures were

inoculated in media with different bile concentrations of 0.3,

0.6, 0.9, and 1.2%. The media were incubated at 37°C for 24 h

under anaerobic condition. To observe temperature tolerance

of cultures, cultures were incubated at different temperatures

such as 28°C, 37°C, and 42°C anaerobically and growth was

observed after 24 h of incubation (Lohith and Anu Appaiah,

2014). Tolerance to NaCl was determined by growing the

cultures in MRS medium containing different concentration of

NaCl, 1~10% (Islam et al., 2016).

162 ∙ Kathade et al.

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Auto-aggregation and co-aggregation assay

Cultures were grown in MRS broth for 48 h at 37°C. Cell

pellets were obtained by centrifuging at 5000 rpm for 5 min and

were suspended in 1 ml of PBS (pH 7.4). The cell suspension

was then diluted to 10 times in PBS (pH 7.4) and vortexed for

10 sec. The suspension was then incubated at 37°C. After

incubation 1 ml of the upper phase was carefully aliquoted at

different time intervals (0, 2, 4, and 24 h) and the optical density

was determined at 600 nm. For co-aggregation isolate and

the pathogen in equal amounts were suspended in PBS and

incubated for 24 h at 37°C. The OD (600 nm) of the suspension

was measured at 2, 4, and 24 h and compared with pathogen

suspension incubated under the same conditions (Ogunremi et

al., 2015). Moreover, after 24 h of incubation, suspensions

were pipetted from the bottom of the tube and observed after

staining by methylene blue (Chelliah et al., 2016).

Antimicrobial assay

Antimicrobial assays were performed against enteropathogens

such as Escherichia coli NCIM 3099, Staphylococcus aureus

NCIM 2408, Enterococcus faecalis NCIM 3040, and Candida

albicans NCIM 3557. The pathogens were spread on Muller

Hinton agar plates and incubated at 37°C for 30 min. Wells (6

mm dia.) were punctured on agar using punch borer and super-

natant of the isolates grown in MRS broth were added in the

agar well. The plates were observed for inhibition zones after

24 h incubation (Chelliah et al., 2016).

Simulated gastric and intestinal juice tolerance assay

To determine the ability of the culture to survive during

transit through the gastrointestinal tract, the cultures were

exposed to gastric juice pepsin and pancreatin in vitro. The

culture suspension in PBS (0.2 ml) were added in mixture of

gastric juice pepsin (3 mg/ml, pH 2) or pancreatin (1 mg/ml, pH

8) containing 0.5% w/v of sodium chloride (Charteris et al.,

1998). Viable count of the cultures was measured at 1, 90, and

180 min for gastric and 1, and 240 min for pancreatin by spread

plating on MRS agar plates after serial dilution. The gastric and

intestinal transit tolerance was evaluated by determining the

viable count of cells after the incubation period (Sourabh et al.,

2012) and percentage survival was calculated by the formula:

Survival (%) = CFU (Final) × 100/ CFU (Initial)

Hydrophobicity assay

Hydrophobicity assay indicates the ability of probiotic to

adhere to human epithelial cells. For hydrophobicity, the cultures

(1 OD at A600) were suspended in phosphate buffer (pH 6.5)

and treated with xylene in 5:1 ratio. The suspension was

vortexed for 2 min and incubated at 37°C for phase separation.

The decrease in absorbance of the aqueous phase was measured

as percent hydrophobicity (H%) and calculated as H% =

[(A0-A)/A0] × 100, where A0 and A are the absorbances of the

culture in the aqueous phase before and after extraction res-

pectively (Vinderola and Reinheimer, 2003; Honey Chandran

and Keerthi, 2018).

Bile salt hydrolase (BSH) assay

BSH assay was performed as per the method described by

(Zheng et al., 2013). Isolates were spot inoculated on MRS agar

plates supplemented with 0.5% (w/v) sodium salt of taurodeoxy-

cholic acid (Himedia) and Calcium chloride 0.37% (w/v).

Plates were incubated anaerobically at 37°C for 72 h and BSH

activity was determined by the presence of precipitation around

colonies.

Antibiotic susceptibility test

Antibiotic susceptibility test was performed according to the

Kirby-Bauer antibiotic testing method as described by (Bauer

et al., 1959). Accordingly, cultures were spread plated on MRS

agar medium and exposed to antibiotic discs (Himedia) containing

ampicillin (10 μg), chloramphenicol (25 μg), penicillin-G (1 unit),

streptomycin (10 μg), sulphatried (300 μg), and tetracycline

(25 μg). The plates were incubated at 37°C for 24 h before

measuring the zone of inhibition around each antibiotic.

Molecular identification

Total genomic DNA was isolated using a genomic DNA

isolation kit (Sigma) as per the manufacturer’s instructions and

used as the template for PCR. Each reaction mixture containing

approximately 10 ng of DNA; 2.5 mM MgCl2; 1× PCR buffer

(Genei) 200 µM each dCTP, dGTP, dATP, and dTTP; 2 pmol

of each, forward and reverse primers ABI Prism BigDye

Terminator Cycle Sequencing reaction kit was used for

sequencing the PCR product. Combination of universal primers

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Korean Journal of Microbiology, Vol. 56, No. 2

FDD2–RPP2 (universal primers for 1.5 kb fragment amplification

for eubacteria) was used to sequence the nearly completed gene.

The sequencing reaction and template were purified as per the

manufacturer’s instructions (Applied Biosystems). Samples

were run on ABI prism 3100 Genetic Analyzer and sequencing

output was analysed using DNA sequence analyser computer

software. The sequence was compared with the National Centre

for Biotechnology Information GenBank entries by using the

BLAST algorithm.

Statistical analysis 

Each experiment was performed in triplicate and data were

subjected to a one-way analysis of variance (ANOVA) and results

are expressed as Mean ± SD. Statistical analysis was done by

PRISM software. Differences were considered statistically

significant when p < 0.05 (p > 0.05 = ns, p < 0.05 = *, p < 0.01

= **, p < 0.001 = ***).

Results

Isolation and characterization

Lactobacillus species, a dominant group of bacterial species

found in the human gastrointestinal tract, is a member of

Firmicutes phyla, a group of Gram-positive, nonpathogenic,

catalase-negative, non-spore forming, anaerobic bacteria, that

can ferment hexoses and pentoses to produce lactic acid and

acetic acid. In this study we are targeted to isolate Lactobacillus

cultures are generally recognized as safe (GRAS) (Khemariya

et al., 2016; Behera et al., 2018). Out of three samples, a total

of 16 colonies showed colony characteristics similar to the

Lactobacillus genus and those colonies were purified (Table

1). They were further screened for biochemical properties. Out

of sixteen isolates, when screened further twelve cultures were

found to be Gram-positive rod, endospore negative, acid-fast

and catalase-negative as well which could ferment glucose,

lactose and sucrose (Table 1). All 12 cultures were further

screened for the probiotic property.

Toxicity assay

Haemolysis is a test to determine the ability of microorganisms

to bind mammalian cells such as platelets, which fibronectin,

fibrinogen and collagen (Harty et al., 1994) which can produce

enzymes such as glycosidases, proteases and gelatinases, hence

this test is an appropriate test to screen toxicity of microorganisms

(Tan et al., 2013). Haemolytic bacteria will show clear zone

around the colony, this is considered as beta haemolysis. The

bacteria which can reduce haemoglobin to methaemoglobin show

greenish zone around the colonies called alpha haemolysis

(Pelczar et al., 1977) Gamma haemolysis is types of haemolysis

where no change is observed in the medium and is reported to

be safe (Koneman et al., 1992). Sheep Blood Agar Base is the

Table 1. Cultural characterization of bacterial isolates

Cultures Gram’s Endospore Acid fast CatalaseCarbohydrate fermentation Gas

productionLactose Glucose Sucrose Xylose Starch

DD-IC + Rod - - - + + + + +

DD-IIA + Rod - - - - + + - -

DD-IVA + Rod - - - - + + - -

DD-IVB + Rod - - - - + + - -

DD-IVC + Rod - - - + + + - -

LB-I + Rod - - - - + + - +

LB-II + Rod - - - + + + - -

LB-III + Rod - - - + + + - -

LB-IV + Rod - - - + + + - -

LB-V + Rod - - - - + + + -

LB-VI +Rod - - - - + + - -

LB-VII + Rod - - - + + + - - + (lactose)

+, positive; –, negative.

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best medium that showed the expected beta lysis pattern with

Streptococcus pyogenes in comparison to other blood based

medium and hemolysis was suggested to test toxicity in probiotic

microorganisms in the Joint FAO/WHO (2002) guideline.

In the present study, all 12 cultures screened were found to

be non-hemolytic, hence safe for any industrial applications.

Tolerance to pH, bile salt, and temperature

Generally, probiotics are administered through the oral route,

so it is utmost important to survive against harsh acidic and

alkaline as well as bile salts present in the intestine (Shehata et

al., 2016; Gupta and Sharma, 2017). In the different stages of

gastrointestinal tract with different pH and enzymatic condition

in mammalian gastric transits pH 1.5~2.0 along with proteolytic

enzymes such as pepsin and in intestinal condition pH 4.5~7.8

with bile acid with 0.3% concentration w/v (Chou and Weimer,

1999; Jacobsen et al., 1999; Çakir, 2003). In this study, out of

12 cultures screened, nine cultures survived the presence of bile

acid. The normal concentration of bile in the intestine is around

0.3% to 2% (Gotcheva et al., 2013). The nine cultures could

tolerate up to 1.2% of bile salt. Further 6 of them showed

growth in the range of pH from 1.5 to 10. Similarly, all 6

cultures could tolerate temperature up to 42°C with best results

was observed with isolate LB-VII with growth at 37°C with

significance at 28°C P = 0.08, 37°C P = 0.006, and 42°C P =

0.064, respectively. After passing through the acidic stomach

conditions, probiotic strains must be able to tolerate the bile salt

in the intestine. Six isolates of the present study could survive

bile acid as well as a wide range of pH and temperature.

Auto-aggregation and co-aggregation

Auto-aggregation is a microscopic observation of formation

of clusters and binding to the inorganic or extracellular metrics

of cells. Auto aggregation is important step to check ability to

colonise and formation of biofilm in the colon (Sorroche et al.,

2012; Kragh et al., 2016; Trunk et al., 2018). Auto-aggregation

percentage of isolates was measured by comparing the initial

absorbance at 600 nm and auto-aggregation percentage at

different time intervals. For good probiotic isolates, it has been

recommended that auto-aggregation property should be more

than 40% and all 6 cultures tested for auto-aggregation showed

more than 40% aggregation and maximum 100% after 24 h

with significant results at incubation after 2 h P = 0.009, 4 h P

= 0.004, 24 h P = 0.001 (Fig. 1).

Co-aggregation is a process where different strain or species

of microorganisms bind together which used to eliminate

pathogenic microorganisms (Ochiai et al., 1993; Malik et al.,

2003; Corno et al., 2014). Isolates showed good co-aggregation

percentage with tested pathogenic organisms. They showed

100% co-aggregation with pathogens E. coli, E. faecalis, and S.

aureus after 24 h The significance of co-aggregation for culture

designated as LB-VII was found to be at 2 h incubation period.

E. coli p = 0.003, LB-VII + E. coli p = 0.006, E. faecalis p =

0.023, LB-VII + E. faecalis p = 0.0048, S. aureus p = 0.001,

LB-VII + S. aureus p = 0.008, at 4 h E. coli p = 0.0031, LB-VII

+ E. coli p = 0.0042, E. faecalis p = 0.053, LB-VII + E. faecalis

p = 0.0078, S. aureus p = 0.004, LB-VII + S. aureus p = 0.008,

at 24 h E. coli p = 0.001, LB-VII + E. coli p = 0.001, E. faecalis

p = 0.009, LB-VII + E. faecalis p = 0.008, S. aureus p = 0.074,

LB-VII + S. aureus p = 0.0067 (Fig. 2A, B, and C).

Gastric and intestinal tolerance

An ideal probiotic culture should survive at least 90 min of

exposure to gastric and 240 min exposure to intestinal con-

ditions of pH 2 and pH 8, respectively. In the present study,

isolates DD-IVA, DD-IVC, and LB-VII showed varying degrees

of resistance when exposed to pepsin (gastric condition) and

only LB-VII showed good resistance to pancreatin (intestinal

condition). LB-VII culture survived in both conditions with 64%

at 90 min and 38% at 180 min in gastric simulated conditions

Fig. 1. Auto aggregation percentage of different isolates (p > 0.05 = ns, p

< 0.05 = *, p < 0.01 = **, p <0.001 = ***).

Lactobacillus from donkey dung ∙ 165

Korean Journal of Microbiology, Vol. 56, No. 2

with significance at 0 min P = 0.001, 1 min P = 0.001, 90 min

P = 0.008, 180 min P = 0.044, whereas the isolate LB-VII

exhibited 74% survival rate at 240 min in intestinal environment,

with significance at 0 min P = 0.001, 1 min P = 0.007 and 240

min P = 0.009 however, the viability of cultures decreased after

incubation period of 120~240 min. Studies from Charteris et al.

(1998), showed that extreme pH conditions down-regulated

the growth of reported probiotics such as Lactobacillus and

Bifidobacterium, after the treatment of such harsh enzymes

similar to the present study (Fig. 3A and B).

NaCl tolerance

High NaCl is an inhibitory substance that may inhibit growth

of micro-organisms (Hoque et al., 2010). Present study revealed

LB-VII culture could tolerate salt concentration in the range of

2~8% with upto 44% survival rate at 8% NaCl concertation.

Bile salt hydrolysis

Bile is a yellow green aqueous solution synthesised by liver

and stored in gall bladder, majorly it present bile acid, cholesterol,

phospholipids and biliverdin (De Smet et al., 1995). It plays an

important role in fat digestion and dissolve lipids, conjugated

bile is important to convert into deconjugated bile for passive

reabsorption to liver. Probiotic microorganisms could produce

BSH enzyme to convert it into absorbable form (Boyer, 2013).

Bile salt hydrolase enzyme hydrolyses the amide bond liberating

glycine and taurine resulting decannulated form of bile salt

(Dawson and Karpen, 2014). The BSH activity was determined

by precipitation around colonies, after 72 h of incubation. At

37°C incubation temperature, precipitation was observed around

LB-VII colonies, indicating bile salt hydrolase activity of the

culture (Fig. 4) (Zheng et al., 2013).

(A) Co-aggregation with S. aureus

(B) Co-aggregation with E. coli

(C) Co-aggregation with E. faecalis

Fig. 2. Co-aggregation of isolates with different pathogens. (A) S. aureus,

(B) E. coli, and (C) E. faecalis (p > 0.05 = ns, p < 0.05 = *, p < 0.01 = **,

p < 0.001 = ***).

(A) Tolerance to gastric environment

(B) Tolerance to intestinal environment

Fig. 3. (A) Tolerance to gastric environment, (B) Tolerance to intestinal

environment (p > 0.05 = ns, p < 0.05 = *, p < 0.01 = **, p < 0.001 = ***).

166 ∙ Kathade et al.

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Antimicrobial activity

Many probiotic bacteria are reported for their ability to

produce antimicrobial compounds such as organic acids (Lactic,

acetic, propionic etc.), carbon dioxide, hydrogen peroxide, low

antimicrobial substances and bacteriocins (Klaenhammer and

Kullen, 1999; Çakir, 2003; Quwehand et al., 2004). Bioactive

compounds present in supernatant were tested antimicrobial

activity against Gram-positive and Gram-negative bacteria. The

results exhibited that LB-VII culture was able to inhibit E. coli

and E. faecalis (0.5 cm). While no inhibition zone was showed

against, S. aureus and Pseudomonas (Fig. 5A and B).

Hydrophobicity test

Hydrophobicity of cell surface gives knowledge about structural

properties of microorganisms responsible for aggregation and

adhesion. This test signifies the presence of glycoproteinaceous

material on the cell surface (Kos et al., 2003; Honey Chandran

and Keerthi, 2018). It can be determined by bacterial adhesion

to n-hexadecane, xylene and toluene reflects cell surface or

hydrophobicity (Bellon-Fontaine et al., 1996). Strains with

hydrophobicity more than 40% were considered hydrophobic

(Boris et al., 1998). In the present study, LB-VII culture with

initial 1.876 and final 1.25, percentage of hydrophobicity were

calculated using formula and showed 62.6% hydrophobicity.

Antibiotic susceptibility test

In the present study, the culture when exposed different

antibiotics on MRS agar it was revealed that LB-VII is resistant

to ampicillin (10 μg), chloramphenicol (25 μg), streptomycin

(10 μg), suphatried (300 μg) and tetracycline (25 μg), whereas

intermediate to penicillin-G (1 unit) as per the interpretation

of zones of inhibition (in mm) for Kirby-Bauer antibiotic

susceptibility test as reported in Fall (2011) (Fig. 6).

Probiotic characterization of isolate LB-VII is given in

Table 2.

Bacterial identification

Bacterial isolate LB-VII was genotypically sequenced and

analysed by 16S rRNA region. After comparing 16S rRNA

gene region in the NCBI database, the isolate LB-VII was

identified as L. plantarum with 16S rRNA sequence as given

below.

L. plantarum is the most popular and versatile species

possessing useful properties and is industrially employed in

fermentation and processing of raw foods which are generally

recognized as safe (GRAS) (Bauer et al., 1959; Khemariya et

al., 2016). Probiotics in intestine must be safe for its use and

must be assessed for minimum required parameters set by FAO

Fig. 4. Bile salt hydrolase activity.

(A) (B)

Fig. 5. Antimicrobial activity of the isolates against (A) E. coli and (B) E.

faecalis.

Fig. 6. Antibiotic susceptibility assay. AMP, Ampicillin; C, Chloramphenicol;

P, Penicillin G; S, Streptomycin; S3, Sulphatriad; TE, Tetracycline.

Lactobacillus from donkey dung ∙ 167

Korean Journal of Microbiology, Vol. 56, No. 2

and WHO. Many studies have shown isolation and probiotic

characterization of L. plantarum. This is the first report of

isolation L. plantarum from donkey dung and its probiotic

characterization.

Conclusion

Lactobacillus is one of the most sorted out microorganisms.

Many other bacteria and yeast cultures are identified for their

probiotic use in human and animals. Owing to the microbial

diversity and functionality, still, there is scope for isolation of

microorganisms and screen them for probiotic potential. The

present study reports for the first-time isolation of Lactobacillus

from donkey dung and its probiotic characterization. The isolated

culture was found to be non-hemolytic and could grow at a

wide range of pH as well as in the presence of the intestinal

environment. This culture could inhibit common pathogenic

organisms and showed good auto-aggregation and co-aggregation

property against E. faecalis and E. coli, considering these

results, the isolated culture LB-VII is an excellent candidate for

further probiotic characterization.

Acknowledgments

The authors are indebted to BV- Rajiv Gandhi Institute of

IT and Biotechnology, Bharati Vidyapeeth (Deemed to be

University) (BVDU), Pune for allowing them to undertake this

work.

Table 2. Probiotic characterization of the LB-VII

ToxicityHemolysis

-

pH tolerance1.5 2.5 3.5 6 7 8 9 10

+ + + ++ +++ ++ ++ +

Bile salt tolerance0.3 0.6 0.9 1.2

+++ ++ ++ ++

Temperature tolerance (% growth at)28°C 37°C 42°C

62 100 67

NaCl tolerance (% growth at)0% 2% 4% 6% 8% 10%

100 98 74 69 44 0.09

Auto-aggregation (% aggregation at)2 (h) 4 (h) 24 (h)

60 74 100

Co-aggregation (% aggregation at) h

E. coli E. faecalis S. aureus

2 4 24 2 4 24 2 4 24

68 92 100 73 38 100 88 95 100

Bile salt hydrolase +

Pepsin tolerance (% growth at)0 (min) 1 (min) 90 (min) 180 (min)

100 80 72 40

Pancreatin tolerance (% growth at)0 1 240

100 87.2 73.2

Antimicrobial activityE. coli E. faecalis

0.6 cm 0.5 cm

Antibiotic susceptibility (Zone of inhibition)Ampicillin Streptomycin Suphatried Tetracycline Penicillin-G Chloramphenicol

4 mm (R) 3 mm (R) Nil (R) 13 mm (R) 16 mm (I) 13 mm (I)

Hydrophobicity 68%

S, susceptibility; R, resistance; I, intermediate; h, hours; %, percentage; cm, centimetre; -, No growth; +, Fair growth; ++, Good Growth; +++, Excellent Growth.

AMP, Ampicillin; STRP, Streptomycin; SUPH, Suphatriad; TET, Tetracycline; PEN-G, Penicillin-G; CPL, Chloramphenicol.

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