Elsevier

Anaerobe

Volume 33, June 2015, Pages 117-123
Anaerobe

Clinical microbiology
Characterization of Lactobacillus isolated from dairy samples for probiotic properties

https://doi.org/10.1016/j.anaerobe.2015.03.004Get rights and content

Highlights

  • Paper reports screening of 12 dairy isolates for standard probiotic properties.

  • Isolate LBS2 identified as Lactobacillus rhamnosus with 16S rDNA sequencing.

  • L. rhamnosus LBS2 successfully adhered to rat epithelial cells in in vitro conditions.

  • In SEM study high surface roughness observed in LBS2 using in vitro high bile salt.

Abstract

In the present study twelve Lactobacillus isolates (LBS 1-LBS 12) were characterized for probiotic properties. Out of the twelve, eight isolates (LBS 1–6, 8 and 11) were bile resistant (survival > 50% at 0.3% bile salt w/v) and five isolates (LBS 1, 2, 5, 6 and 11) were found acid pH value resistant (survival > 50% at pH 3). All twelve isolates inhibited the growth of Staphylococcus aureus whereas isolate LBS 2 also inhibited the growth of Escherichia coli and Salmonella typhimurium. Antibiotic susceptibility testing of isolates was also performed and isolate LBS 2 was selected for further study based on its broad spectrum effect in clinical pathogen inhibition. LBS 2 was characterized phenotypically at Institute of Microbial Technology (IMTECH), Chandigarh, India and was confirmed as Lactobacillus rhamnosus by 16S rDNA sequencing and subsequent analysis using BLAST. The gene sequence was deposited in GenBank with accession number KJ562858. Scanning electron microscopy (SEM) study was used to study in vitro epithelial cell adherence and bile salt effect on isolate LBS 2. Epithelial cells adherence assay showed positive results and surface roughness of LBS 2 increased with increase in bile salt (0.15–0.45% w/v).

Introduction

Probiotics are live microorganisms which, when administered in adequate amounts, confer a health benefit on the host [1]. Most probiotic microorganism belongs to the lactic acid bacteria (LAB) viz. Lactobacillus spp., Enterococcus spp. and Bifidobacterium [2]. Most of the species of the genus Lactobacillus are part of human and animal commensal intestinal flora [3] and consists of a physiologically and genetically diverse group of rod-shaped, Gram-positive, non-pigmented, non-spore forming [4], catalase negative, microaerophilic to anaerobic LAB [5] with widespread use in fermented food production [6]. These microorganisms are also called friendly bacteria and are generally recognized as safe (GRAS) microorganisms [7]. The lactobacilli isolated from dairy products have long history of safe use [1] and are widely used as starter cultures in the food industry viz. fermented milk, alcoholic beverages, sourdough and silage preparation [8]. Several health benefits associated with the consumption of probiotic bacteria include; controlling intestinal infections, improving lactose utilization, lowering blood ammonia level [9], [10], influencing immune system and low serum cholesterol level [11], [12]. There is an increase in use of probiotic bacteria in variety of food products viz. yoghurt, cheese, drinks and dietary supplements based on their therapeutic benefits [13]. Keeping in view the importance of probiotics, the present study is endeavored to screen twelve previously reported Lactobacillus isolates (8 from household curd and 4 from household milk) [14] for the probiotic properties viz. resistance against bile salt, acid pH values, antibiotics, pathogenic microbe inhibition and select a potential isolate for further investigation.

Section snippets

Bacterial strains

Thirty dairy samples (household milk and curd) were collected from local area of Solan, H.P., India. Serial dilutions of 1 ml dairy samples were prepared in peptone water and then 100 μl sample from different dilutions were spread over the solidified MRS (de Man, Rogosa and Sharpe) medium (HiMedia, India) and incubated at 37 °C for 24–48 h under anaerobic conditions for the isolation of Lactobacillus. Pure cultures of isolated colonies were obtained after re-streaking on MRS agar plate. Each

Results

Thirty bacterial isolates were initially obtained from thirty dairy samples collected from Solan district in Himachal Pradesh, India. Out of thirty, 14 isolates were from milk sample and 16 isolates were from curd samples. Characterization of isolates was performed on the basis of their morphological characteristics viz., type of colony, color, margin, elevation, opacity and presence of pigment and Lactobacillus specific biochemical tests. Based on morphological, cultural and biochemical

Discussion

In the present study twelve Lactobacillus spp. isolated from dairy samples were characterized for probiotic properties and results were compared with the existing literature. In screening study eight, out of twelve isolates were bile resistant and Lactobacillus isolates showing resistance ≥50% at 0.3% (w/v) bile salt were considered as bile resistance strains [16]. Highest resistance (72.36% and 68.62%) was observed with isolate LBS 2 at 0.3% and 0.45% of bile salt respectively. Gilliland [26]

Conclusion

In conclusion one potential probiotic isolate LBS 2 was obtained after screening of twelve Lactobacillus isolates for the probiotic properties. This isolate LBS 2 from the household curd sample of Solan, district of Himachal Pradesh was identified as L. rhamnosus LBS 2 after phenotypic and genotypic characterization. High resistance to bile salt, tolerance to acid pH values, broad spectrum microbial pathogen inhibition and adherence to epithelial cells seems to be a potential advantage of this

Acknowledgments

The authors are thankful to Dr. Rohit Goyal, Faculty of Pharmaceutical Sciences, Shoolini University, Solan, India for his help in epithelial cells adherence assay and Er. Sandeep, Indian Institute of Technology, Mandi, India for SEM analysis in the present study.

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