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

Naruemon Tunsakul, Krit Pongpirul, Lampet Wongsaroj, Chitrasak Kullapanich, Sarn Settachaimongkon and Naraporn Somboonna
* Author for corresponding; e-mail address: Naraporn.s@chula.ac.th, doctorkrit@gmail.com
Volume: Vol.51 No.5 (September 2024)
Research Article
DOI: https://doi.org/10.12982/CMJS.2024.068
Received: 26 March 2024, Revised: 26 July 2024, Accepted: 28 July 2024, Published: -

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 Journal of Science, 2024; 51(5): e2024068. DOI 10.12982/CMJS.2024.068.

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.

Keywords: probiotic, human feces, isolation, characterization, Thai, 16S rRNA gene

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page: 363 - 370

Udomluk Sompong* [a], Richard W. Castenholz [b], Somboon Anuntalabhochai [a] and Yuwadee Peerapornp

Vol.33 No.3 (SEPTEMBER 2006)
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Microcystin LR Content in Microcystis aeruginosa Kütz Collected from Sri Sakhett, Thailand
page: 231 - 236

Waya Sengpracha [a], Napattarapong Suvannachai [b] and Weerachai Phutdhawong* [b]

Vol.33 No.2 (MAY 2006)
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Subcritical Water Extraction of Polyphenolic Compounds from Terminalia chebula Fruits
page: 103 - 108

Rangsriwong P., Rangkadilok N., and Shotipruk A

Vol.35 No.1 (JANUARY 2008)
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Fabrication and Characterization of High Strength Dental Porcelain Nanocomposite from Thailandís Raw Materials
page: 549 - 553

Attavit Pisitanusorn*[a], Supon Ananta [a], Rattikorn Yimnirun [a] and Sakdipown Thiansem [b]

Vol.32 No.3 (SEPTEMBER 2005)
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Characterization of Various Proteases from SaltTTTolerant Bacteria Isolated from Thai Fermented Foods Foods
page: 149 - 161

Somkid Deejing* [a,b], Mitsuaki Moriguchi [c], Naiyatat Poosaran [d], Griangsak Chairote [e] and Sa

Vol.32 No.2 (MAY 2005)
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Selective Isolation of Sorbitol Utilizing Acetic Acid Bacteria
page: 189 - 192

Jeerapun Jaiinphon, Kreuawan Thonglem and Wasu Pathom-aree*

Vol.29 No.3 (DECEMBER 2002)
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Cloning and Expression of Protease Gene from Thermophilic Bacterium Bacillus stearothermophilus TLS33 in Escherichia coli DH5α
page: 53 - 59

Thammarat Kaosombat [a], Supachok Sinchaikul [b], Hataichanoke Niamsup [a], Shui – Tein Chen [b] and Suree Phutrakul* [a]

Vol.29 No.2 (AUGUST 2002)
Opinion View: 1,396 Download: 314

Screening of Antioxidants from Some Thai Vegetables and Herbs
page: 1 - 5

Anchana Chanwitheesuk, Nuansri Rakariyatham*, Aphiwat Teerawutgulrag and Teerapol Wongchanapiboon

Vol.29 No.1 (APRIL 2002)
Opinion View: 1,221 Download: 367

Fifty Three New Record Species of Benthic Diatoms from Mekong River and Its Tributaries in Thailand
page: 326 - 343

Sutthawan Suphan, and Yuwadee Peerapornpisal

Vol.37 No.2 (MAY 2010)
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Determination of Local Tobacco Cultivars Using ISSR Molecular Marker
page: 293 - 303

Jessada Denduangboripant, Sornsuda Setaphan, Wilasinee Suwanprasart, and Somsak Panha

Vol.37 No.2 (MAY 2010)
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Nanocrystalline Hydroxyapatite Powders by a Polymerized Complex Method
page: 243 - 251

Jutharatana Klinkaewnarong, and Santi Maensiri

Vol.37 No.2 (MAY 2010)
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Chiang Mai Journal of Science

1,647

Q3 0.80

Q3 1.3

7 days

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