KJM056 02 09 Korean Journal DD

Korean Journal of Microbiology (2020) Vol. 56, No. 2, pp. 160-169 DOI https://doi.org/10.7845/kjm.2020.0038 Copyright ⓒ 2020, The Microbiological Society of Korea pISSN 0440-2413 eISSN 2383-9902 Isolation of Lactobacillus from donkey dung and its probiotic characterization Suyash Arunrao Kathade1 , Mayur Arjun Aswani2 Niricharan Kunchiraman Bipinraj1* , Pashmin Kaur Anand1 , Suresh Jagtap2 , and 1 Bharati Vidyapeeth (Deemed to be University), Rajiv Gandhi Institute of I.T. and Biotechnology, Katraj 411046 and Pune, Maharashtra, India 2 IRSHA, Bharati Vidyapeeth Deemed to be University, Katraj 411046 and Pune, Maharashtra, India (Received April 22, 2020; Revised June 8, 2020; Accepted June 9, 2020) 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. of antimicrobial compounds, modulation of the immune response, Keywords: Lactobacillus, donkey dung, probiotics researchers due to its chemical composition which is similar to 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 acidbase 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 that of human milk and hence is suitable for consumption for infants (Cariminati et al., 2014). Nutritional components reveal Micro-organisms that reside in digestive tracts of hosts have that donkey milk poses a high amount of lactose and low levels a large impact on host health (Kobierecka et al., 2017). Probiotics of casein and fat; essential for the survival of lactic acid bacteria. are defined as live micro-organisms when ingested in adequate There are many reports of bacteria especially, Lactobacillus amounts confer a health benefit to host such as the production group that had been isolated from various animal sources such *For correspondence. E-mail: bipinrajnk@gmail.com, k.bipinraj@bharatividyapeeth.edu; Tel.: +020-2437-9013 as milk and dung of cow and buffalo. Infant animal dung is considered as the best source of probiotic bacteria since they Lactobacillus from donkey dung ∙ 161 are dependent on mother’s milk which is the rich source of was performed as per the method of (Reynolds et al., 2009) for nutrient for the gut bacteria to establish themselves in a gastro- confirmation of acid-fast bacilli, (Goyal et al., 2012) method intestinal environment. So far there is no report on probiotic was used for performing catalase test and to detect the presence bacteria enumerated from donkey dung hence objective of the or absence of enzyme catalase. present research is to isolate, identify and characterize probiotic microorganisms from donkey dung. Carbohydrate fermentation and gas production Bromothymol blue broth base medium containing different 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 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 Gastroenterology Organization. Accordingly following assays were performed. Man Rogosa and Sharpe (MRS, Himedia) medium. The plates were incubated at 37°C for 48 h under anaerobic conditions in Toxicity assay an anaerobic jar. To maintain anaerobic conditions 0.1% sodium For toxicity assay, isolates were spot inoculated on sheep thioglycolate was added as a reducing agent in all experiments. blood agar plates and incubated at 37°C for 24 h in anaerobic After incubation, morphologically distinct colonies were isolated jars. Toxicity was determined by the pattern of haemolysis on and observed microscopically. Cells that showed Lactobacillus blood agar plates (Papadimitriou et al., 2015; Pino et al., 2019). morphology were purified and stored in MRS agar slants. These cultures were further inoculated in MRS broth for 48 h, Bile, pH, NaCl, and temperature tolerance centrifuged and suspended in saline to get cell concentration For pH tolerance assay, cultures were inoculated in MRS of 107 CFU/ml. This suspension (1%) was used for further with pH ranging from 1.5 to 10 adjusted using 1 N HCl or 1 experiments. Each experiment was performed in triplicate and N NaOH. Similarly, for bile tolerance assay cultures were mean value were calculated. 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 Cultural and colony characteristics under anaerobic condition. To observe temperature tolerance Cultural and colony characterization of all isolates was per- of cultures, cultures were incubated at different temperatures formed based on Bergey’s Manual of Systemic Microbiology, such as 28°C, 37°C, and 42°C anaerobically and growth was Gram staining was performed as per the method of (Coico et observed after 24 h of incubation (Lohith and Anu Appaiah, al., 2005) to observe Gram character of culture, endospore 2014). Tolerance to NaCl was determined by growing the staining was performed as described by (Reynolds et al., 2009) cultures in MRS medium containing different concentration of method for observation of spores in cultures, acid-fast staining NaCl, 1~10% (Islam et al., 2016). Korean Journal of Microbiology, Vol. 56, No. 2 162 ∙ Kathade et al. Auto-aggregation and co-aggregation assay Hydrophobicity assay Cultures were grown in MRS broth for 48 h at 37°C. Cell Hydrophobicity assay indicates the ability of probiotic to pellets were obtained by centrifuging at 5000 rpm for 5 min and adhere to human epithelial cells. For hydrophobicity, the cultures were suspended in 1 ml of PBS (pH 7.4). The cell suspension (1 OD at A600) were suspended in phosphate buffer (pH 6.5) was then diluted to 10 times in PBS (pH 7.4) and vortexed for and treated with xylene in 5:1 ratio. The suspension was 10 sec. The suspension was then incubated at 37°C. After vortexed for 2 min and incubated at 37°C for phase separation. incubation 1 ml of the upper phase was carefully aliquoted at The decrease in absorbance of the aqueous phase was measured different time intervals (0, 2, 4, and 24 h) and the optical density as percent hydrophobicity (H%) and calculated as H% = was determined at 600 nm. For co-aggregation isolate and [(A0-A)/A0] × 100, where A0 and A are the absorbances of the the pathogen in equal amounts were suspended in PBS and culture in the aqueous phase before and after extraction res- incubated for 24 h at 37°C. The OD (600 nm) of the suspension pectively (Vinderola and Reinheimer, 2003; Honey Chandran was measured at 2, 4, and 24 h and compared with pathogen and Keerthi, 2018). 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 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 taurodeoxycholic 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. albicans NCIM 3557. The pathogens were spread on Muller Hinton agar plates and incubated at 37°C for 30 min. Wells (6 Antibiotic susceptibility test mm dia.) were punctured on agar using punch borer and supernatant 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). 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 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 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. 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 Molecular identification 8) containing 0.5% w/v of sodium chloride (Charteris et al., Total genomic DNA was isolated using a genomic DNA 1998). Viable count of the cultures was measured at 1, 90, and isolation kit (Sigma) as per the manufacturer’s instructions and 180 min for gastric and 1, and 240 min for pancreatin by spread used as the template for PCR. Each reaction mixture containing plating on MRS agar plates after serial dilution. The gastric and approximately 10 ng of DNA; 2.5 mM MgCl2; 1× PCR buffer intestinal transit tolerance was evaluated by determining the (Genei) 200 µM each dCTP, dGTP, dATP, and dTTP; 2 pmol viable count of cells after the incubation period (Sourabh et al., of each, forward and reverse primers ABI Prism BigDye 2012) and percentage survival was calculated by the formula: Terminator Cycle Sequencing reaction kit was used for Survival (%) = CFU (Final) × 100/ CFU (Initial) sequencing the PCR product. Combination of universal primers 미생물학회지 제56권 제2호 Lactobacillus from donkey dung ∙ 163 FDD2–RPP2 (universal primers for 1.5 kb fragment amplification can ferment hexoses and pentoses to produce lactic acid and for eubacteria) was used to sequence the nearly completed gene. acetic acid. In this study we are targeted to isolate Lactobacillus The sequencing reaction and template were purified as per the cultures are generally recognized as safe (GRAS) (Khemariya manufacturer’s instructions (Applied Biosystems). Samples et al., 2016; Behera et al., 2018). Out of three samples, a total were run on ABI prism 3100 Genetic Analyzer and sequencing of 16 colonies showed colony characteristics similar to the output was analysed using DNA sequence analyser computer Lactobacillus genus and those colonies were purified (Table software. The sequence was compared with the National Centre 1). They were further screened for biochemical properties. Out for Biotechnology Information GenBank entries by using the of sixteen isolates, when screened further twelve cultures were BLAST algorithm. found to be Gram-positive rod, endospore negative, acid-fast and catalase-negative as well which could ferment glucose, Statistical analysis lactose and sucrose (Table 1). All 12 cultures were further screened for the probiotic property. Each experiment was performed in triplicate and data were subjected to a one-way analysis of variance (ANOVA) and results Toxicity assay are expressed as Mean ± SD. Statistical analysis was done by PRISM software. Differences were considered statistically Haemolysis is a test to determine the ability of microorganisms significant when p < 0.05 (p > 0.05 = ns, p < 0.05 = *, p < 0.01 to bind mammalian cells such as platelets, which fibronectin, = **, p < 0.001 = ***). 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 Results around the colony, this is considered as beta haemolysis. The Isolation and characterization bacteria which can reduce haemoglobin to methaemoglobin show Lactobacillus species, a dominant group of bacterial species greenish zone around the colonies called alpha haemolysis found in the human gastrointestinal tract, is a member of (Pelczar et al., 1977) Gamma haemolysis is types of haemolysis Firmicutes phyla, a group of Gram-positive, nonpathogenic, where no change is observed in the medium and is reported to catalase-negative, non-spore forming, anaerobic bacteria, that 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 Catalase Carbohydrate fermentation Lactose 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 - - - + + + - - Gas production + (lactose) +, positive; –, negative. Korean Journal of Microbiology, Vol. 56, No. 2 164 ∙ Kathade et al. 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 Fig. 1. Auto aggregation percentage of different isolates (p > 0.05 = ns, p < 0.05 = *, p < 0.01 = **, p <0.001 = ***). al., 2016; Gupta and Sharma, 2017). In the different stages of gastrointestinal tract with different pH and enzymatic condition more than 40% aggregation and maximum 100% after 24 h in mammalian gastric transits pH 1.5~2.0 along with proteolytic with significant results at incubation after 2 h P = 0.009, 4 h P enzymes such as pepsin and in intestinal condition pH 4.5~7.8 = 0.004, 24 h P = 0.001 (Fig. 1). with bile acid with 0.3% concentration w/v (Chou and Weimer, Co-aggregation is a process where different strain or species 1999; Jacobsen et al., 1999; Çakir, 2003). In this study, out of of microorganisms bind together which used to eliminate 12 cultures screened, nine cultures survived the presence of bile pathogenic microorganisms (Ochiai et al., 1993; Malik et al., acid. The normal concentration of bile in the intestine is around 2003; Corno et al., 2014). Isolates showed good co-aggregation 0.3% to 2% (Gotcheva et al., 2013). The nine cultures could percentage with tested pathogenic organisms. They showed tolerate up to 1.2% of bile salt. Further 6 of them showed 100% co-aggregation with pathogens E. coli, E. faecalis, and S. growth in the range of pH from 1.5 to 10. Similarly, all 6 aureus after 24 h The significance of co-aggregation for culture cultures could tolerate temperature up to 42°C with best results designated as LB-VII was found to be at 2 h incubation period. was observed with isolate LB-VII with growth at 37°C with E. coli p = 0.003, LB-VII + E. coli p = 0.006, E. faecalis p = significance at 28°C P = 0.08, 37°C P = 0.006, and 42°C P = 0.023, LB-VII + E. faecalis p = 0.0048, S. aureus p = 0.001, 0.064, respectively. After passing through the acidic stomach LB-VII + S. aureus p = 0.008, at 4 h E. coli p = 0.0031, LB-VII conditions, probiotic strains must be able to tolerate the bile salt + E. coli p = 0.0042, E. faecalis p = 0.053, LB-VII + E. faecalis in the intestine. Six isolates of the present study could survive p = 0.0078, S. aureus p = 0.004, LB-VII + S. aureus p = 0.008, bile acid as well as a wide range of pH and temperature. 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, Auto-aggregation and co-aggregation LB-VII + S. aureus p = 0.0067 (Fig. 2A, B, and C). Auto-aggregation is a microscopic observation of formation of clusters and binding to the inorganic or extracellular metrics Gastric and intestinal tolerance of cells. Auto aggregation is important step to check ability to An ideal probiotic culture should survive at least 90 min of colonise and formation of biofilm in the colon (Sorroche et al., exposure to gastric and 240 min exposure to intestinal con- 2012; Kragh et al., 2016; Trunk et al., 2018). Auto-aggregation ditions of pH 2 and pH 8, respectively. In the present study, percentage of isolates was measured by comparing the initial isolates DD-IVA, DD-IVC, and LB-VII showed varying degrees absorbance at 600 nm and auto-aggregation percentage at of resistance when exposed to pepsin (gastric condition) and different time intervals. For good probiotic isolates, it has been only LB-VII showed good resistance to pancreatin (intestinal recommended that auto-aggregation property should be more condition). LB-VII culture survived in both conditions with 64% than 40% and all 6 cultures tested for auto-aggregation showed at 90 min and 38% at 180 min in gastric simulated conditions 미생물학회지 제56권 제2호 Lactobacillus from donkey dung ∙ (A) Co-aggregation with S. aureus (A) Tolerance to gastric environment (B) Co-aggregation with E. coli (B) Tolerance to intestinal environment (C) 165 Co-aggregation with E. faecalis 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 = ***). 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. 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 = ***). Bile salt hydrolysis Bile is a yellow green aqueous solution synthesised by liver and stored in gall bladder, majorly it present bile acid, cholesterol, with significance at 0 min P = 0.001, 1 min P = 0.001, 90 min phospholipids and biliverdin (De Smet et al., 1995). It plays an P = 0.008, 180 min P = 0.044, whereas the isolate LB-VII important role in fat digestion and dissolve lipids, conjugated exhibited 74% survival rate at 240 min in intestinal environment, bile is important to convert into deconjugated bile for passive with significance at 0 min P = 0.001, 1 min P = 0.007 and 240 reabsorption to liver. Probiotic microorganisms could produce min P = 0.009 however, the viability of cultures decreased after BSH enzyme to convert it into absorbable form (Boyer, 2013). incubation period of 120~240 min. Studies from Charteris et al. Bile salt hydrolase enzyme hydrolyses the amide bond liberating (1998), showed that extreme pH conditions down-regulated glycine and taurine resulting decannulated form of bile salt the growth of reported probiotics such as Lactobacillus and (Dawson and Karpen, 2014). The BSH activity was determined Bifidobacterium, after the treatment of such harsh enzymes by precipitation around colonies, after 72 h of incubation. At similar to the present study (Fig. 3A and B). 37°C incubation temperature, precipitation was observed around LB-VII colonies, indicating bile salt hydrolase activity of the NaCl tolerance culture (Fig. 4) (Zheng et al., 2013). High NaCl is an inhibitory substance that may inhibit growth Korean Journal of Microbiology, Vol. 56, No. 2 166 ∙ Kathade et al. 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 Fig. 4. Bile salt hydrolase activity. (10 μg), suphatried (300 μg) and tetracycline (25 μg), whereas intermediate to penicillin-G (1 unit) as per the interpretation Antimicrobial activity of zones of inhibition (in mm) for Kirby-Bauer antibiotic 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 susceptibility test as reported in Fall (2011) (Fig. 6). Probiotic characterization of isolate LB-VII is given in Table 2. antimicrobial substances and bacteriocins (Klaenhammer and Kullen, 1999; Çakir, 2003; Quwehand et al., 2004). Bioactive Bacterial identification compounds present in supernatant were tested antimicrobial Bacterial isolate LB-VII was genotypically sequenced and activity against Gram-positive and Gram-negative bacteria. The analysed by 16S rRNA region. After comparing 16S rRNA results exhibited that LB-VII culture was able to inhibit E. coli gene region in the NCBI database, the isolate LB-VII was and E. faecalis (0.5 cm). While no inhibition zone was showed identified as L. plantarum with 16S rRNA sequence as given against, S. aureus and Pseudomonas (Fig. 5A and B). below. Hydrophobicity test possessing useful properties and is industrially employed in L. plantarum is the most popular and versatile species Hydrophobicity of cell surface gives knowledge about structural fermentation and processing of raw foods which are generally properties of microorganisms responsible for aggregation and recognized as safe (GRAS) (Bauer et al., 1959; Khemariya et adhesion. This test signifies the presence of glycoproteinaceous al., 2016). Probiotics in intestine must be safe for its use and material on the cell surface (Kos et al., 2003; Honey Chandran must be assessed for minimum required parameters set by FAO and Keerthi, 2018). It can be determined by bacterial adhesion to n-hexadecane, xylene and toluene reflects cell surface or (A) (B) Fig. 5. Antimicrobial activity of the isolates against (A) E. coli and (B) E. faecalis. 미생물학회지 제56권 제2호 Fig. 6. Antibiotic susceptibility assay. AMP, Ampicillin; C, Chloramphenicol; P, Penicillin G; S, Streptomycin; S3, Sulphatriad; TE, Tetracycline. 167 Lactobacillus from donkey dung ∙ Table 2. Probiotic characterization of the LB-VII Hemolysis Toxicity 1.5 pH tolerance 2.5 3.5 + + + Bile salt tolerance ++ +++ 8 9 10 ++ ++ + 0.6 0.9 1.2 +++ ++ ++ ++ 28°C 37°C 42°C 62 100 67 0% 2% 4% 6% 8% 100 98 74 69 44 2 (h) Auto-aggregation (% aggregation at) Co-aggregation (% aggregation at) h 7 0.3 Temperature tolerance (% growth at) NaCl tolerance (% growth at) 6 10% 0.09 4 (h) 24 (h) 60 74 100 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 E. coli Antimicrobial activity E. faecalis 0.6 cm Antibiotic susceptibility (Zone of inhibition) 0.5 cm Ampicillin Streptomycin Suphatried 4 mm (R) 3 mm (R) Nil (R) Hydrophobicity Tetracycline Penicillin-G Chloramphenicol 13 mm (R) 16 mm (I) 13 mm (I) 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. and WHO. Many studies have shown isolation and probiotic culture was found to be non-hemolytic and could grow at a characterization of L. plantarum. This is the first report of wide range of pH as well as in the presence of the intestinal isolation L. plantarum from donkey dung and its probiotic environment. This culture could inhibit common pathogenic characterization. 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 Conclusion further probiotic characterization. Lactobacillus is one of the most sorted out microorganisms. Many other bacteria and yeast cultures are identified for their Acknowledgments probiotic use in human and animals. Owing to the microbial diversity and functionality, still, there is scope for isolation of The authors are indebted to BV- Rajiv Gandhi Institute of microorganisms and screen them for probiotic potential. The IT and Biotechnology, Bharati Vidyapeeth (Deemed to be present study reports for the first-time isolation of Lactobacillus University) (BVDU), Pune for allowing them to undertake this from donkey dung and its probiotic characterization. The isolated work. Korean Journal of Microbiology, Vol. 56, No. 2 168 ∙ Kathade et al. References Bauer AW, Perry DM, and Kirby WMM. 1959. Single disk antibiotic sensitivity testing of Staphylococci. AMA Arch. Intern. Med. 104, 208–216. Behera SS, Ray RC, and Zdolec N. 2018. Lactobacillus plantarum with functional properties: An approach to increase safety and shelf-life of fermented foods. Biomed Res. Int. 2, 1–18. Bellon-Fontaine MN, Rault J, and van Oss CJ. 1996. Microbial adhesion to solvents: a novel method to determine the electron donor/electron acceptor or lewis acid-base properties of microbial cells. Colloids Surf. B 7, 47–53. Boris S, Suárez JE, Vásquez F, and Barbés C. 1998. Adherence of human vaginal Lactobacilli to vaginal epithelial cells and its interaction with uropathogens. Infect. Immun. 66, 1985–1989. Boyer, JL. 2013. Bile formation and secretion. Compr. Physiol. 3, 1035–1078. Çakır, I. 2003. Determination of some probiotic properties on Lactobacilli and Bifidobacteria. PhD Thesis, Ankara University, Ankara, Turkey. Cariminati D, Tidona F, Fornasari ME, Rossetti L, Meucci A, and Giraffa G. 2014. Biotyping of cultivable lactic acid bacteria isolated from donkey milk. Lett. Appl. Microbiol. 59, 299–305. Charteris WP, Kelly PM, Morelli L, and Collins JK. 1998. Development and application of in vitro methodology to determine the transit tolerance of potentially probiotic Lactobacillus and Bifidobacterium species in upper human gastrointestinal tract. J. Appl. Microbiol. 84, 759–768. Chelliah R, Ramakrishnan SR, Prabhu PR, and Antony U. 2016. Evaluation of antimicrobial activity and probiotic properties of wild-strain Pichia kudriavzevii isolated from frozen idli batter. Yeast 33, 385–401. Chen LS, Ma Y, Maubois JL, He SH, Chen LJ, and Li HM. 2010. Screening for the potential probiotic yeast strains from raw milk to assimilate cholesterol. Dairy Sci. Technol. 90, 537–548. Chou LS and Weimer B. 1999. Isolation and characterization of acid and bile tolerant isolates from strains of Lactobacillus acidophilus. J. Dairy Sci. 82, 23–31. Coico R. 2005. Gram Staining. Curr. Protoc. Microbiol. 00: A.3C.1A.3C.2. Corno G, Coci M, Giardina M, Plechuk S, Campanile F, and Stefani S. 2014. Antibiotics promote aggregation within aquatic bacterial communities. Front Microbiol. 5, 297. Dawson PA and Karpen SJ. 2014. Intestinal transport and metabolism of bile acids. J. Lipid Res. 56, 1085–1099. De Smet, van Hoorde L, Vande Woestyne M, Christiaens H, and Verstraete W. 1995. Significance of bile salt hydrolytic activities of Lactobacilli. J. Appl. Bacteriol. 79, 292–301. Fall. 2011. Jackie Reynolds, Richland 12. BIOL 2420. Gotcheva V, Hristozova E, Hristozova T, Guo M, Roshkova Z, and Angelov A. 2013. Assessment of potential probiotic properties of lactic acid bacteria and yeast strains. Food Biotechnol. 16, 211– 미생물학회지 제56권 제2호 225. Goyal R, Dhingra H, Bajpail P, and Joshi N. 2012. Characterization of the Lactobacillus isolated from different curd samples. Afr. J. Biotechnol. 11, 14448–14452. Gupta A and Sharma N. 2017. Characterization of potential probiotic lactic acid bacteria- Pediococcus acidilactici Ch-2 isolated from Chuli- A traditional apricot product of Himalayan region for the production of novel bioactive compounds with special therapeutic properties. J. Food Microbiol. Saf. Hyg. 2, 1–11. Harty DW, Oakey HJ, Patrikakis M, Hume EB, and Knox KW. 1994. Pathogenic potential of lactobacilli. Int. J. Food Microbiol. 24, 179–189. Honey Chandran HC and Keerthi TR. 2018. Probiotic potency of Lactobacillus plantarum KX519413 and KX519414 isolated from honey bee gut. FEMS Microbiol. Lett. 365, 1–8. Hoque MZ, Akter F, Hossain KM, Rahman MSM, Billah MM, and Islam KMD. 2010. Isolation, identification and analysis of probiotic properties of Lactobacillus spp. from selective regional yoghurts. World J. Dairy Food Sci. 5, 39–46. Huang R, Tao X, Wan C, Li S, Xu H, Xu F, Shah NP, and Wei H. 2015. In vitro probiotic characteristics of Lactobacillus plantarum ZDY 2013 and its modulatory effect on gut microbiota of mice. J. Dairy Sci. 98, 5850–5861. Islam KN, Akbar T, Akhter F, and Islam NN. 2016. Characterization and confirmation of Lactobacillus spp. from selective regional yoghurts for probiotic interference with pathogenic bacterial growth. Asian J. Biol. Sci. 9, 1–9. Jacobsen CN, Nielsen VR, Hayford AE, Moller PL, Michaelsen KF, Paerregaard A, Sandström B, Tvede M, and Jacobsen M. 1999. Screening of probiotic activities of forty-seven strains of Lactobacillus spp. by in vitro techniques and evaluation of the colonization ability of five selected strains in humans. Appl. Environ. Microbiol. 65, 4949–4956. Joint FAO/WHO Working Group. 2002. Report on Drafting Guidelines for the Evaluation of Probiotics in Food: Guidelines for the Evaluation of Probiotics in Food. London, ON: FAO/WHO. Khemariya P, Singh S, Jaiswal N, and Chaurasia SNS. 2016. Isolation and identification of L. plantarum from vegetable samples. Food Biotechnol. 30, 49–62. Khisti UV, Kathade SA, Aswani MA, Anand PK, and Bipinraj NK. 2019. Isolation and identification of Saccharomyces cerevisiae from caterpillar frass and their probiotic characterization. Biosci. Biotech. Res. Asia 16, 179–186. Klaenhammer TR and Kullen MJ. 1999. Selection and design probiotics. Int. J. Food Microbiol. 50, 45–57. Kobierecka PA, Wyszyńska AK, Aleksandrzak-piekarczyk T, Kuczkowski M, Tuzimek T, Piotrowska W, Górecki A, Adamska I, Wieliczk A, Bardowski J, et al. 2017. In vitro characterisitics of Lactobacillus spp. isolated from chicken digestive tract and their role in inhibition of Campylobacter colonization. Microbiologyopen 6, e00512. Koneman EW, Allen SD, Janda WM, Schreckenberger PC, and Winn WC Jr. 1992. Colour Atlas and Textbook of Diagnostic Lactobacillus from donkey dung ∙ Microbiology, 4th ed., J. B. Lippinccott Company. Kos B, Susković J, Vuković S, Simpraga M, Frece J, and Matosić S. 2003. Adhesion and aggregation ability of probiotic strain Lactobacillus acidophilus M92. J. Appl. Microbiol. 94, 981– 987. Kragh KN, Hutchison JB, Melaugh G, Rodesney C, Roberts AEL, Irie Y, Jensen PØ, Diggle SP, Allen RJ, Gordon V, et al. 2016. Role of multicellular aggregates in biofilm formation. MBio 7, e00237–16. Lohith K and Anu Appaiah KA. 2014. In vitro probiotic characterization of yeasts of food and environmental origin. Int. J. Probiotics Prebiotics 9, 87–92. Malik A, Sakamoto M, Hanazaki S, Osawa M, Suzuki T, Tochigi M, and Kakii K. 2003. Coaggregation among nonflocculating bacteria isolated from activated sludge. Appl. Environ. Microbiol. 69, 6056–6063. Ochiai K, Kurita-Ochiai T, Kamino Y, and Ikedo T. 1993. Effect of co-aggregation on the pathogenicity of oral bacteria. J. Med. Microbiol. 39, 183–190. Ogunremi OR, Sanni AI, and Agrawal R. 2015. Probiotic potentials of yeasts isolated from some cereal-based Nigerian traditional fermented food products. J. Appl. Microbiol. 119, 797–808. Papadimitriou K, Zoumpopoulou G, Foligné B, Alexandraki V, Kazou M, Pot B, and Tsakalidou E. 2015. Discovering probiotic microorganisms: in vitro, in vivo, genetic and omics approaches. Front. Microbiol. 6, 58. Paraschiv D, Vasile A, Constantin M, Ciobanu A, and Bahrim G. 2011. Study of physiological properties of some probiotics in multiple cultures with mesophilic lactic acid bacteria by flora Danica Ch. Hansen commercial starter. Food Technol. 35, 56–65. Pelczar MJ Jr., Reid RD, and Chan ECS. 1977. Microbiology, 4th ed., Tata McGraw-Hill Publishing Company Ltd, New Delhi. Pino A, Bartolo E, Caggia C, Cianci A, and Randazzo CL. 2019. Detection of vaginal lactobacilli as probiotic candidates. Sci. Rep. 9, 3355. Quwehand AC and Vesterlund S. eds. 2004. Antimicrobial components from lactic acid bacteria. Lactic Acid Bacteria Microbiological 169 and Functional Aspects. Marcel Dekker Inc., New York, USA. Reynolds J, Moyes R, and Breakwell D. 2009. Differential staining of bacteria endospore stain. Curr. Protoc. Microbiol. 15, A.3J.1– A.3J.5. Shehata MG, El Sohaimy SA, El-Sahn MA, and Youssef MM. 2016. Screening of isolated potential probiotic lactic acid bacteria for cholesterol lowering property and bile salt hydrolase activity. Ann. Agric. Sci. 61, 65–75. Sorroche FG, Spesia MB, Zorreguieta A, and Giordano W. 2012. A positive correlation between bacterial autoaggregation and biofilm formation in native Sinorhizobium meliloti isolates from Argentina. Appl. Environ. Microbiol. 78, 4092–4101. Sourabh A, Kanwar SS, and Sharma OP. 2012. In vitro characterization of Saccharomyces cerevisiae HM535662 obtained from an indigenous fermented food “Bhaturu” of Western Himalayas. Afr. J. Biotechnol. 11, 11447–11454. Tan Q, Xu H, Aguilar ZP, Peng S, Dong S, Wang B, Li P, Chen T, Xu F, and Wei H. 2013. Safety assessment and probiotic evaluation of Enterococcus faecium YF5 isolated from sourdough. J. Food Sci. 78, M587–M593. Thakur M, Deshpande HW, and Bhate MA. 2017. Isolation and identification of lactic acid bacteria and their exploration in non-dairy probiotic drink. Int. J. Curr. Microbiol. Appl. Sci. 6, 1023–1030. Trunk T, Khalil HS, and Leo JC. 2018. Bacterial autoaggregation. AIMS Microbiol. 4, 140–164. Vinderola CG and Reinheimer JA. 2003. Lactic acid starter and probiotic bacteria: a comparative “in vitro” study of probiotic characteristics and biological barrier resistance. Food Res. Int. 36, 895–904. Yirga H. 2015. The use of probiotics in animal nutrition. J. Prob. Health 3, 2. Zheng Y, Yingli LU, Wang J, Yang L, Pan C, and Huang Y. 2013. Probiotic properties of Lactobacillus strains isolated from Tibetian kefir grains. PLoS One 8, 1–8. Korean Journal of Microbiology, Vol. 56, No. 2