Probiotic Properties of Bacillus Strains Isolated from Stingless Bee (Heterotrigona itama) Honey Collected across Malaysia
Abstract
:1. Introduction
2. Materials and Methods
2.1. Honey Samples
2.2. Isolation of Bacterial Strains from Stingless Bee Honey
2.3. Bacterial Strains and Growth Conditions
2.4. Identification of Bacteria Using Molecular Technique
Genomic DNA Extraction, 16S rRNA Amplification, and Gene Analysis
2.5. Antimicrobial Activity Assessment
2.6. Screening for Probiotic Properties
2.6.1. Acid and Bile Tolerance
2.6.2. Hydrophobicity
2.6.3. Autoaggregation
2.7. Safety Assessment
2.7.1. Antibiotic Susceptibility
2.7.2. Blood Hemolysis
3. Results
3.1. Isolation and Preliminary Detection of Bacillus Isolates
3.2. Molecular Identification through 16S rRNA Gene Sequence Analysis
3.3. Distribution of the Bacillus Species Isolated from Stingless Bee Honey from Different Geographical Locations
3.4. Antimicrobial Test against Pathogenic Bacteria
3.5. Tolerance to Acidic Conditions and Bile Salts
3.6. Cell Adhesion Activity of Bacillus Species
3.7. Antibiotic Susceptibility and Hemolytic Activity
4. Discussion
5. Conclusions
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Geographical Location | CFU/g | No. of Selected Isolates | Gram-Staining | Catalase Test | Tolerance to 7% NaCl | ||||
---|---|---|---|---|---|---|---|---|---|
Gram + ve | Gram − ve | Bacilli | Cocci | +ve | −ve | ||||
Batang Benar, Negeri Sembilan | 2.2 × 102 | 17 | 17 | - | 17 | - | All isolates exhibited positive results for catalase test | 6 | |
Segamat, Johor | 2.4 × 102 | 23 | 21 | 2 | 23 | - | 9 | ||
Seri Kembangan, Selangor | 1.1 × 101 | 3 | 3 | - | 3 | - | 2 | ||
Sematan, Sarawak | 9.7 × 100 | 4 | 3 | 1 | 3 | 1 | 3 | 2 | 2 |
Serdang, Selangor | 3.7 × 102 | 11 | 11 | - | 11 | - | 6 | 5 | 4 |
TOTAL | 23 |
Bacterial Isolates | Inhibition Zones against Pathogenic Bacteria, mm | |||||
---|---|---|---|---|---|---|
Staphlococcus aureus | Bacillus cereus | Salmonella thyphimurium | Escheria coli | Klebsiella pneumonia | Pseudomonas aeruginosa | |
B. pumilus HTI-1 | +++ | NI | NI | NI | NI | NI |
B. pumilus HTI-2 | +++ | NI | NI | NI | NI | NI |
B. pumilus HTI-3 | NI | NI | NI | NI | NI | NI |
B. pumilus HTI-4 | ++ | NI | NI | NI | + | NI |
B. pumilus HTI-5 | ++ | NI | NI | NI | NI | NI |
B. pumilus HTI-6 | +++ | NI | NI | NI | NI | NI |
B. pumilus HTI-7 | +++ | ++ | NI | NI | ++ | NI |
B. pumilus HTI-8 | +++ | NI | NI | NI | NI | NI |
B. altitudinis HTI-11 | NI | NI | NI | NI | NI | ++ |
B. altitudinis HTI-14 | +++ | NI | NI | NI | NI | NI |
B. altitudinis HTI-15 | +++ | NI | NI | NI | NI | NI |
B. megaterium HTI-16 | NI | NI | NI | NI | NI | NI |
B. megaterium HTI-17 | NI | NI | NI | NI | NI | NI |
B. megaterium HTI-18 | NI | NI | NI | NI | NI | NI |
B. amyloliquefaciens HTI-19 | +++ | ++ | ++ | ++ | NI | + |
B. amyloliquefaciens HTI-20 | +++ | NI | NI | +++ | NI | NI |
B. aryabhattai HTI-21 | NI | NI | NI | NI | NI | NI |
B. aryabhattai HTI-22 | NI | NI | NI | NI | NI | NI |
B. subtilis HTI-23 | + | NI | +++ | NI | ++ | ++ |
L. rhamnosus GG | +++ | +++ | +++ | +++ | ++ | +++ |
Tetracycline(20ug/µl) | +++ | +++ | +++ | +++ | +++ | +++ |
Isolates | Survival Rates, % | |
---|---|---|
Acid Tolerance | Bile Tolerance | |
pH 2.0 | 0.3% | |
B. amyloliquefaciens HTI-19 | 86.56 a | 129.10 a |
B. subtilis HTI-23 | 86.72 a | 140.50 b |
L. rhamnosus GG | 97.46 b | 106.76 c |
Isolates | Autoaggregation (%) | Hydrophobicity (%) |
---|---|---|
B. amyloliquefaciens HTI-19 | 84.13 a | 53.64 a |
B. subtilis HTI-23 | 57.51 b | 60.82 a |
L. rhamnosus GG | 69.99 ab | 61.04 a |
Isolates | Susceptibility to Antibiotics | Hemolytic Activity | ||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
AMP10 | C30 | CIP5 | E15 | CN10 | K30 | TE30 | TEC30 | VA30 | RD30 | S10 | ||
B. amyloliquefaciens HTI-19 | S | S | S | S | S | S | S | S | S | S | S | α-hemolytic |
B. subtilis HTI-23 | S | S | S | S | S | S | S | S | S | S | S | γ-hemolytic |
L. rhamnosus GG | S | S | S | S | S | S | S | R | R | S | S | γ-hemolytic |
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Zulkhairi Amin, F.A.; Sabri, S.; Ismail, M.; Chan, K.W.; Ismail, N.; Mohd Esa, N.; Mohd Lila, M.A.; Zawawi, N. Probiotic Properties of Bacillus Strains Isolated from Stingless Bee (Heterotrigona itama) Honey Collected across Malaysia. Int. J. Environ. Res. Public Health 2020, 17, 278. https://doi.org/10.3390/ijerph17010278
Zulkhairi Amin FA, Sabri S, Ismail M, Chan KW, Ismail N, Mohd Esa N, Mohd Lila MA, Zawawi N. Probiotic Properties of Bacillus Strains Isolated from Stingless Bee (Heterotrigona itama) Honey Collected across Malaysia. International Journal of Environmental Research and Public Health. 2020; 17(1):278. https://doi.org/10.3390/ijerph17010278
Chicago/Turabian StyleZulkhairi Amin, Fatin Aina, Suriana Sabri, Maznah Ismail, Kim Wei Chan, Norsharina Ismail, Norhaizan Mohd Esa, Mohd Azmi Mohd Lila, and Norhasnida Zawawi. 2020. "Probiotic Properties of Bacillus Strains Isolated from Stingless Bee (Heterotrigona itama) Honey Collected across Malaysia" International Journal of Environmental Research and Public Health 17, no. 1: 278. https://doi.org/10.3390/ijerph17010278