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Isolation and Preliminary Characterization of Probiotic Isolates from Healthy Thai Adult Feces


Paper Type 
Contributed Paper
Title 
Isolation and Preliminary Characterization of Probiotic Isolates from Healthy Thai Adult Feces
Author 
Naruemon Tunsakul, Krit Pongpirul, Lampet Wongsaroj, Chitrasak Kullapanich, Sarn Settachaimongkon and Naraporn Somboonna
Email 
Naraporn.s@chula.ac.th, doctorkrit@gmail.com
Abstract:

     Probiotics are known for health-promoting microorganisms, for examples, assisting gastrointestinal digestion and short chain fatty acid producers. Several current oral human probiotics were derived from human samples; yet, of various ethnic (genetics) and dietary patterns. As probiotics from similar ethnic (genetics) and dietary pattern might confer certain advantages (e.g., more compatibility with hosts), this study isolated and characterized probiotics from healthy Thai adults. To screen for potential probiotic isolates from healthy Thai adult feces, and preliminary characterize these probiotic isolates by biochemical tests and 16S rRNA gene sequencing, following the established Thailand’s Food and Drug Administration (FDA) and National Center for Genetic Engineering and Biotechnology (BIOTEC) guidelines. Twenty fecal samples with clinically verified healthy Thai males and females aged 22-42 years were cultivated on MRS (or M17) media along the broad probiotic screenings (pH 4.0 and 0.1% oxgall) for bacterial probiotic propagation. All derived colonies of different morphologies were continued colony PCR using universal probiotic primers (specific for genera Bifidobacterium, Enterococcus, Lactobacillus, Pediococcus and Streptococcus thermophilus), and only PCR-positive colonies from any of these probiotic primer pair were further cultivation for isolates. Then, these isolates were screened and characterized general probiotic properties that included acid tolerance (pH 2.0), bile salt tolerance (0.3% bile salt), hydrophobicity, antibiotic (ampicillin, ciprofloxacin, erythromycin and vancomycin) susceptibility, no hemolysis activity, catalase, and potassium hydroxide (KOH). The final passing isolates represented potential probiotic isolates that could perhaps maintain in gastrointestinal tract with safety, and were full-length 16S rRNA gene sequenced to identify the genus and species by BLASTN against non-redundant GenBank database. A total of 295 bacterial isolates were obtained from the cultivated on MRS (or M17) media along the broad probiotic screenings from 20 fecal samples. Then, following the general probiotic property characterizations and the full-length 16S rRNA gene sequence analysis, 7 potential isolates were finally derived: 2 Bifidobacterium animalis subsp. lactis, 1 Lactobacillus (new genus name as Limosilactobacillus) reuteri, 1 L. reuteri or L. vaginalis, and 3 Pediococcus pentosaceus. These isolates exhibited strong acid and bile salt tolerances, and relatively high cell surface hydrophobicity. These isolates were susceptible to ampicillin and erythromycin, similar to a reference probiotic control Lactobacillus casei strain Shirota; and the CU13-450 and CU13-451 were also susceptible to vancomycin, similar to a reference control Bifidobacterium breve. Catalase-negative consistent with these bacteria in intestinal tract as anaerobes or facultative anaerobes, and KOH-negative result inferred the Gram-positive bacteria consistent with the full-length 16S rRNA gene sequence identification (Bifidobacterium, Lactobacillus, and Pediococcus are Gram-positive bacteria). No hemolysis activity was observed for these isolates. These 7 probiotic isolates had general probiotic properties and with confirmed the 16S rRNA gene sequences, that follow the Thailand’s FDA and BIOTEC guidelines. However, additional beneficial function investigations, such as in vitro epithelial cell adhesion and in vivo animal model, and whole genome sequencing, should be performed to better validate their possible human health benefits and safety.

Article ID
e2024068
Received Date 
2024-03-26
Revised Date 
2024-07-26
Accepted Date 
2024-07-28
Full Text 
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Keyword 
probiotic, human feces, isolation, characterization, Thai, 16S rRNA gene
Volume 
Vol.51 No.5 (September 2024)
DOI 
https://doi.org/10.12982/CMJS.2024.068
Citation 
Tunsakul N., Pongpirul K., Wongsaroj L., Kullapanich C., Settachaimongkon S. and Somboonna N., Isolation and Preliminary Characterization of Probiotic Isolates from Healthy Thai Adult Feces, Chiang Mai J. Sci., 2024; 51(5): e2024068. DOI 10.12982/CMJS.2024.068.
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Vol.37 No.2 (MAY 2010) View: 576 Download:294
Nanocrystalline Hydroxyapatite Powders by a Polymerized Complex Method
page: 243 - 251
Author:Jutharatana Klinkaewnarong, and Santi Maensiri
Vol.37 No.2 (MAY 2010) View: 582 Download:189



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