Characterization and selection of probiotic lactic acid bacteria from different dietary sources for development of functional foods

Frontiers in Microbiology · 3 authors, 2 centres

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This study isolated 60 lactic acid bacteria from dietary sources like curd, pickle, milk, and wheat dough, and identified two strains—Lactobacillus acidophilus CM1 and Lactobacillus delbrueckii OS1—as the most promising probiotics. These strains survived acidic conditions (pH 2–3), high salt (up to 6% NaCl), and bile salts (up to 2%), and showed antimicrobial activity against pathogens such as Staphylococcus aureus and Escherichia coli. The findings support the potential use of these strains in developing millet-based probiotic functional foods.

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**Background:** Probiotics, particularly lactic acid bacteria (LAB), are beneficial microorganisms commonly found in fermented foods and are known to improve digestive health, boost immunity, and reduce disease risk. This study aimed to isolate, characterize, and identify LAB from various dietary sources (curd, pickle, milk, wheat dough) and the human oral cavity, and to evaluate their probiotic properties for potential use in millet-based functional foods.

**Methods:** A total of 50 food samples (cow milk, buffalo milk, curd, pickle, wheat dough) and 10 oral cavity samples were collected from regions in Punjab and Haryana, India. Bacteria were isolated on MRS agar under anaerobic conditions at 37°C for 24–48 h. Sixty isolates were initially screened based on Gram staining, catalase test, motility, and endospore formation. Sixteen Gram-positive, rod-shaped, catalase-negative isolates were selected for further characterization. Biochemical tests included MR-VP, indole, citrate utilization, nitrate reduction, H2S production, and sugar fermentation (11 sugars). Probiotic attributes were assessed via acid tolerance (pH 1–4), NaCl tolerance (2–8%), bile salt tolerance (0.5–2%), and antimicrobial activity against Bacillus cereus, Staphylococcus aureus, Salmonella typhimurium, Escherichia coli, and Enterococcus faecalis using the agar well diffusion method. Hemolytic activity was tested on blood agar. Molecular identification of the two best isolates (CM1 and OS1) was performed via 16S rRNA gene sequencing and phylogenetic analysis. Statistical analysis used SPSS version 20 with one-way ANOVA and Tukey's post-hoc test (p < 0.05).

**Key Results:** Among 60 isolates, 16 were Gram-positive, rod-shaped, and catalase-negative. Two isolates, CM1 (from cow milk) and OS1 (from oral cavity), exhibited the strongest probiotic properties. Both were identified as Lactobacillus acidophilus CM1 and Lactobacillus delbrueckii OS1, with GenBank accession numbers OP811266.1 and OP824643.1, respectively. Acid tolerance: CM1 and OS1 survived at pH 2 and 3 for 3 h, while most other isolates survived less than 90 min. NaCl tolerance: Both strains grew at 4% and 6% NaCl; only six isolates grew at 6%, and one at 8%. Bile tolerance: Eight isolates (including CM1 and OS1) tolerated up to 2% bile salt. Antimicrobial activity: CM1 showed inhibition zones of 11 mm against B. cereus and E. coli, 10 mm against E. faecalis, 12 mm against S. aureus, and 10 mm against S. typhimurium. OS1 also showed activity against all tested pathogens. All 16 isolates were non-hemolytic (γ-hemolysis). Sugar fermentation: Glucose (91.63%), lactose (83.3%), maltose and fructose (66.64%) were most utilized; mannose (58.31%), galactose (49.98%), mannitol, ribose, sucrose (41.65%), starch (16.66%), and arabinose (8.33%) were less utilized.

**Clinical Implications:** The identified LAB strains demonstrate key probiotic traits—survival in gastrointestinal conditions (acid, bile, salt), safety (non-hemolytic), and antimicrobial activity against common pathogens. These characteristics make them suitable candidates for developing millet-based probiotic beverages, which could offer health benefits such as improved digestion and pathogen inhibition. However, further in vivo studies are needed to confirm efficacy and safety in humans. This research provides a foundation for functional food development using locally sourced probiotics.

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other

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gastroenterology

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ORIGINAL PAPER

PMC10196247

Characterization and selection of probiotic lactic acid bacteria from different dietary sources for development of functional foods

Frontiers in Microbiology

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ABSTRACT

Abstract Introduction Dietary sources have an abundance of bacteria, mainly lactic acid bacteria (LABs), which have long been regarded as probiotics in humans and animals. Lactic acid bacteria (LAB) have been used as probiotic agents due to their ability to produce a variety of beneficial compounds for cultivars and their status as safe microorganisms. Methods In this current study, the lactic acid bacteria (LAB) were isolated from several dietary sources such as curd, pickle, milk, and wheat dough. The principal focus of this study was to determine the survivability of these microorganisms in the gastrointestinal tract and to use promising strains to create probiotic drinks with numerous health benefits. The isolates were identified using a combination of morphological, biochemical, molecular and sugar fermentation patterns, like phenotypic characteristics, sugar fermentation, MR-VP reaction, catalase test, urease test, oxidase test, H 2 S production, NH 3 production synthesis from arginine, citrate utilization, indole test, and 16s rRNA sequencing. Results Two (CM1 and OS1) of the 60 isolates obtained showed the best probiotic results and were identified as Lactobacillus acidophilus CM1 and Lactobacillus delbrueckii OS1. These organism sequences were submitted to Gen bank with accession numbers OP811266.1 and OP824643.1 , respectively. The acid tolerance test results indicated that most strains could survive significantly in an acidic environment with pH levels of 2 and 3. Similarly, the salt tolerance test results showed that both Lactobacillus acidophilus CM1 and Lactobacillus delbrueckii OS1 could survive at 4 and 6% NaCl levels significantly. The isolates also showed their ability to ferment sugars such as lactose xylose, glucose, sucrose, and fructose. Discussion In conclusion, the study showed that the bacteria isolated from different food sources were indeed probiotic lactic acid bacteria and had probiotic properties. These isolates hold potential for future research in the formulation of millet-based probiotic beverages. However, further studies are required to confirm their effectiveness and safety in improving human health. This research provides a foundation for developing functional foods and drinks that can positively affect human health by incorporating probiotic microorganism. Keywords: Lactobacillus delbrueckii , Lactobacillus acidophilus , millet, sugar utilization, curd, pickle

CITATION EXPORT

Khushboo, Arun Karnwal, Tabarak Malik. Characterization and selection of probiotic lactic acid bacteria from different dietary sources for development of functional foods. Frontiers in Microbiology. (2023). https://doi.org/10.3389/fmicb.2023.1170725

AUTHORS

3 · 2 CENTRES

Khushboo, Arun Karnwal, Tabarak Malik

1Department of Microbiology, School of Bioengineering and Biosciences, Lovely Professional University, Phagwara, India
2Department of Biomedical Sciences, Jimma University, Jimma, Ethiopia