Molecular identification, probiotic characterization, anticancer assessment and encapsulation of lactic acid bacteria isolated from vaginas of Iranian fertile women
Probiotics are non-pathogenic microorganisms that positively influence their hosts when ingested in adequate amounts. Research on probiotics has increased over the past years. This trend has resulted in the commercialization of probiotic supplements and functional food. Numerous investigations ha...
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| Format: | Thesis |
| Language: | English |
| Published: |
2015
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| Subjects: | |
| Online Access: | http://psasir.upm.edu.my/id/eprint/67804/1/IB%202015%2043%20IR.pdf |
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| Summary: | Probiotics are non-pathogenic microorganisms that positively influence their hosts
when ingested in adequate amounts. Research on probiotics has increased over the past
years. This trend has resulted in the commercialization of probiotic supplements and
functional food. Numerous investigations have evaluated the potential of probiotic
bacteria for cancer prevention or treatment. Most previous works isolated probiotic
bacteria from dairy products or other fermented foods. This study isolated and
characterized lactic acid bacteria (LAB) from a healthy vaginal ecosystem. LAB that
exhibited probiotic properties were screened for their antimicrobial activity, antibiotic
resistance, and anticancer activity. Isolation was performed using preliminary
phenotypic screening based on Gram-staining and catalase reaction, followed by 16S
rRNA gene sequencing, amplified ribosomal DNA restriction analysis (ARDRA), and
repetitive sequence–based PCR fingerprinting. The bacteria isolated from 40 samples
were classified into three genera with 13 species, three subspecies, and 45 strains. The
combined use of molecular techniques was proven effective for the identification and
classification of vaginal LAB, particularly Lactobacillus, Lactococcus, and
Enterococcus.
The 45 vaginal LAB strains were characterized for their probiotic and therapeutic
potentials. Probiotic characterization included in vitro assays of acid and bile salt
tolerance, antimicrobial activity, and antibiotic resistance. Results showed that the
survival rate of the isolates ranged from 18 to 88% and from 31 to 93% under acidic
(pH 3.0) and 0.3% bile salt conditions in a simulated gastrointestinal environment,
respectively. The strains from each of the 13 species that demonstrated the highest acid
and bile salt tolerance were further evaluated for their antibiotic susceptibility and
antimicrobial activity. Results showed that the survival rates of these 13 isolates under
acidic and bile salt conditions were higher than 58 and 78%, respectively, qualifying
them to be probiotic candidates. All 13 isolates exhibited antimicrobial activity against
different pathogenic bacteria. Among the LAB, Lactococcus lactis 2BL showed the
strongest antimicrobial activity against 15 pathogenic bacteria. The 13 isolates showed different levels of antibiotic resistance depending on antibiotic used. Among the LAB,
Enterococcus avium 7BL and Enterococcus durans 6HL were resistant and sensitive to
all nine antibiotics used, respectively.
The anticancer effects of the 13 isolates on some human cancer cell lines, such as
HeLa, AGS, HT-29, and MCF-7, were also assessed. The human normal cell line
HUVEC was used as a control. The cytotoxicity of these strains and the occurrence of
apoptotic cells were evaluated using DNA fragmentation, fluorescent microscopy, and
flow cytometry. The metabolites produced by Lactobacillus plantarum 5BL,
Lactobacillus acidophilus 36YL, Enterococcus faecalis 16H, and Enterococcus lactis
2BL exhibited remarkable anticancer activity against the tested human carcinoma cell
lines with no significant cytotoxicity on the HUVEC normal cells. Apoptotic cells were
also observed in the cancer cell lines.
Finally, the isolates were subjected to encapsulation to improve their bioavailability
and survival rate under harsh gastrointestinal conditions. Herbal-based biopolymer
matrices, such as alginate, gum arabic, and psyllium, were prepared through extrusion.
Three gel formulations, namely, 1, 1.5 and 2% (w/v) alginate, 2% (w/v) alginate + 0.1
and 0.3% (w/v) gum arabic + psyllium, 1.5% (w/v) alginate + 0.3 and 0.5% (w/v) gum
arabic + psyllium, and 1% (w/v) alginate + 0.4 and 0.6% (w/v) gum arabic/psyllium
were prepared to improve the encapsulation efficiency, gastrointestinal survival, and
colonic release rates of the isolates. All encapsulated bacteria exhibited significantly
improved (P <0.05) encapsulation efficiency (>98%) and survival rate (>75%)
compared with the un-encapsulated bacteria (>45%) under harsh gastrointestinal
conditions. The incorporation of gum arabic and psyllium into the alginate gel
enhanced encapsulation properties. The use of combination gels for encapsulation
improved the survival rate and storage stability of the probiotic strains under
gastrointestinal conditions.
In conclusion, probiotic bacteria were successfully isolated and characterized from a
healthy vaginal ecosystem. These probiotics exhibited antimicrobial activity, antibiotic
resistance, and anticancer activity, making these bacteria potential candidates for the
development of nutraceutical products. |
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