Screening of Bacteriocinogenic Lactic Acid Bacteria and Their Characterization as Potential Probiotics
Abstract
:1. Introduction
2. Materials and Methods
2.1. Microorganisms and Growth Conditions
2.2. Screening of Antimicrobial Activity of each LAB Isolate
2.3. LAB Identification
2.3.1. 16S rRNA Sequencing
2.3.2. Whole-Genome Sequencing
2.4. Safety Criteria of Potential Probiotics
2.4.1. Presence of Virulence Factors
Screening-Test of Biogenic Amines Production
Production of Hydrolytic Enzymes
Presence of Virulence Genes
2.4.2. Antibiotic Resistance
2.5. Functional Criteria of Potential Probiotics
2.5.1. Inoculum
2.5.2. Ability to Resist pH 2.5, pH 2.5 with Pepsin and Bile Salts
2.5.3. Resistance to Simulated Gastrointestinal Tract Conditions
2.5.4. Ability to Adhere to Human Colon Adenocarcinoma Cell Lines Caco-2
Preparation of Cell Lines Caco-2
Preparation of LAB
In Vitro Adherence Assays
2.6. Physiological Criteria of Potential Probiotics
Screening of the Isolates for Bile-Salt Hydrolase (BSH) Activity
2.7. Statistical Analysis
3. Results and Discussion
3.1. Screening of Antimicrobial Activity of each LAB Isolate and Its Identification by 16S rRNA Sequencing
3.2. In Vitro Screening of Probiotic Properties of Selected LAB
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Conflicts of Interest
References
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Microorganisms | Species | Source |
---|---|---|
Gram-positive | Bacillus cereus Bacillus subtilis Bacillus stearothermophilus Listeria innocua 2030c Staphylococcus aureus 18N (methicillin-resistant > Staphylococcus aureus-MRSA) Staphylococcus aureus 2037 M1 (methicillin- sensitive Staphylococcus aureus-MSSA) | ESB culture collection |
Enterococcus faecalis ATCC 29212 Staphylococcus aureus ATCC 29213 | ATCC | |
Enterococcus faecalis DSMZ 12956 Enterococcus faecium DSMZ 13590 Enterococcus flavescens DSMZ 7370 Enterococcus casseliflavus DSMZ 20680 Enterococcus gallinarum DSMZ 20628 | DSMZ | |
Listeria monocytogenes L7946 Listeria monocytogenes L7947 | McLauchlin et al. [16] | |
Gram-negative | Acinetobacter baumannii R Acinetobacter baumannii S-1 Acinetobacter baumannii S-2 Acinetobacter calcoaceticus R Acinetobacter calcoaceticus S Klebsiella pneumoniae Proteus mirabilis Proteus vulgaris Pseudomonas aeruginosa Salmonella Braenderup Salmonella Enteritidis Salmonella Typhimurium Yersinia enterocolitica | ESB culture collection |
Escherichia coli ATCC 25922 | ATCC | |
Yersinia enterocolitica NCTC 10406 | NCTC | |
Yeasts | Candida albicans Saccharomyces cerevisiae | ESB |
Amp | Gen | Kan | Str | Ery | Chl | Tet | |
---|---|---|---|---|---|---|---|
R23 | 0.5 | ≤4 | ≤16 | n.r. | ≤0,5 | ≤2 | ≤4 |
Q42 | 1 | ≤4 | ≤16 | ≤32 | ≤0,5 | ≤2 | ≤4 |
Q43 | 2 | ≤4 | ≤16 | ≤32 | ≤0,5 | ≤2 | ≤4 |
CFF4 | 2 | ≤4 | ≤16 | ≤32 | ≤0,5 | ≤2 | ≤4 |
CFF5 | 2 | ≤4 | ≤16 | ≤32 | ≤0,5 | ≤2 | ≤4 |
CFF51 | 1 | ≤4 | ≤16 | ≤32 | ≤0,5 | ≤2 | ≤4 |
CFF202 | 1 | ≤4 | ≤16 | ≤32 | ≤0,5 | ≤2 | ≤4 |
Log CFU/mL (*) | |||
---|---|---|---|
Isolate | 0 min | 60 min (#) | 120 min (†) |
L. plantarum R23 | 6.82 ± 0.06Aa | 4.49 ± 0.04Ba | 4.56 ± 0.05Ba |
P. pentosaceus Q42 | 7.16 ± 0.02Aa | 6.82 ± 0.06Ab | 6.64 ± 0.09Ab |
P. pentosaceus Q43 | 6.87 ± 0.20Aa | 7.18 ± 0.20Ab | 6.65 ± 0.09Ab |
P. pentosaceus CFF4 | 7.25 ± 0.12Aa | 7.12 ± 0.03Ab | 6.90 ± 0.22Abc |
P. pentosaceus CFF5 | 7.25 ± 0.03Aa | 7.02 ± 0.17Ab | 7.17 ± 0.13Ac |
P. pentosaceus CFF51 | 7.06 ± 0.27Aa | 7.30 ± 0.19Ab | 7.04 ± 0.05Abc |
P. pentosaceus CFF202 | 7.12 ± 0.07Aa | 7.07 ± 0.16Ab | 6.87 ± 0.06Abc |
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Pinto, A.; Barbosa, J.; Albano, H.; Isidro, J.; Teixeira, P. Screening of Bacteriocinogenic Lactic Acid Bacteria and Their Characterization as Potential Probiotics. Microorganisms 2020, 8, 393. https://doi.org/10.3390/microorganisms8030393
Pinto A, Barbosa J, Albano H, Isidro J, Teixeira P. Screening of Bacteriocinogenic Lactic Acid Bacteria and Their Characterization as Potential Probiotics. Microorganisms. 2020; 8(3):393. https://doi.org/10.3390/microorganisms8030393
Chicago/Turabian StylePinto, Ana, Joana Barbosa, Helena Albano, Joana Isidro, and Paula Teixeira. 2020. "Screening of Bacteriocinogenic Lactic Acid Bacteria and Their Characterization as Potential Probiotics" Microorganisms 8, no. 3: 393. https://doi.org/10.3390/microorganisms8030393
APA StylePinto, A., Barbosa, J., Albano, H., Isidro, J., & Teixeira, P. (2020). Screening of Bacteriocinogenic Lactic Acid Bacteria and Their Characterization as Potential Probiotics. Microorganisms, 8(3), 393. https://doi.org/10.3390/microorganisms8030393