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Combination of culture-independent and culture-dependent molecular methods for the determination of bacterial community of iru, a fermented Parkia biglobosa seeds.

Adewumi GA, Oguntoyinbo FA, Keisam S, Romi W, Jeyaram K - Front Microbiol (2013)

Bottom Line: DNA sequencing of the highly variable V3 region of the 16S rRNA genes obtained from PCR-DGGE identified species related to Bacillus subtilis as consistent bacterial species in the fermented samples, while other major bands were identified as close relatives of Staphylococcus vitulinus, Morganella morganii, B. thuringiensis, S. saprophyticus, Tetragenococcus halophilus, Ureibacillus thermosphaericus, Brevibacillus parabrevis, Salinicoccus jeotgali, Brevibacterium sp. and uncultured bacteria clones.Bacillus species were cultured as potential starter cultures and clonal relationship of different isolates determined using amplified ribosomal DNA restriction analysis (ARDRA) combined with 16S-23S rRNA gene internal transcribed spacer (ITS) PCR amplification, restriction analysis (ITS-PCR-RFLP), and randomly amplified polymorphic DNA (RAPD-PCR).This further discriminated B. subtilis and its variants from food-borne pathogens such as B. cereus and suggested the need for development of controlled fermentation processes and good manufacturing practices (GMP) for iru production to achieve product consistency, safety quality, and improved shelf life.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Faculty of Science, University of Lagos Akoka, Lagos, Nigeria ; Microbial Resources Division, Institute of Bioresources and Sustainable Development, Takyelpat Institutional Area Imphal, Manipur, India ; Department of Food Science and Technology, College of Food Sciences, Bells University of Technology Ota, Nigeria.

ABSTRACT
In this study, bacterial composition of iru produced by natural, uncontrolled fermentation of Parkia biglobosa seeds was assessed using culture-independent method in combination with culture-based genotypic typing techniques. PCR-denaturing gradient gel electrophoresis (DGGE) revealed similarity in DNA fragments with the two DNA extraction methods used and confirmed bacterial diversity in the 16 iru samples from different production regions. DNA sequencing of the highly variable V3 region of the 16S rRNA genes obtained from PCR-DGGE identified species related to Bacillus subtilis as consistent bacterial species in the fermented samples, while other major bands were identified as close relatives of Staphylococcus vitulinus, Morganella morganii, B. thuringiensis, S. saprophyticus, Tetragenococcus halophilus, Ureibacillus thermosphaericus, Brevibacillus parabrevis, Salinicoccus jeotgali, Brevibacterium sp. and uncultured bacteria clones. Bacillus species were cultured as potential starter cultures and clonal relationship of different isolates determined using amplified ribosomal DNA restriction analysis (ARDRA) combined with 16S-23S rRNA gene internal transcribed spacer (ITS) PCR amplification, restriction analysis (ITS-PCR-RFLP), and randomly amplified polymorphic DNA (RAPD-PCR). This further discriminated B. subtilis and its variants from food-borne pathogens such as B. cereus and suggested the need for development of controlled fermentation processes and good manufacturing practices (GMP) for iru production to achieve product consistency, safety quality, and improved shelf life.

No MeSH data available.


Related in: MedlinePlus

Non-metric Multidimensional Scaling analysis of DGGE data Group (a): iru Abeokuta 2 southwest Nigeria (); iru Abeokuta 3 southwest Nigeria (); iru Lagos 1 southwest Nigeria (); iru Lagos 2 southwest Nigeria (•); iru Ilorin 1 northcentral Nigeria (); iru Ilori n 2 northcentral Nigeria (); iru Ibadan 1 southwest Nigeria (). Group (b): iru Abeokuta 4 southwest Nigeria (). Group (c): iru Abeokuta 1 southwest Nigeria (); iru Abeokuta 5 southwest Nigeria (); iru Abeokuta 8 southwest Nigeria (); iru Oyo southwest Nigeria (); iru Ado-Ekiti southwest Nigeria (). Group (d): iru Abeokuta 6 southwest Nigeria (); iru lbadan 2 southwest Nigeria (). Group (e): iru Abeokuta 7 southwest Nigeria ().
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Figure 1: Non-metric Multidimensional Scaling analysis of DGGE data Group (a): iru Abeokuta 2 southwest Nigeria (); iru Abeokuta 3 southwest Nigeria (); iru Lagos 1 southwest Nigeria (); iru Lagos 2 southwest Nigeria (•); iru Ilorin 1 northcentral Nigeria (); iru Ilori n 2 northcentral Nigeria (); iru Ibadan 1 southwest Nigeria (). Group (b): iru Abeokuta 4 southwest Nigeria (). Group (c): iru Abeokuta 1 southwest Nigeria (); iru Abeokuta 5 southwest Nigeria (); iru Abeokuta 8 southwest Nigeria (); iru Oyo southwest Nigeria (); iru Ado-Ekiti southwest Nigeria (). Group (d): iru Abeokuta 6 southwest Nigeria (); iru lbadan 2 southwest Nigeria (). Group (e): iru Abeokuta 7 southwest Nigeria ().

Mentions: The two genomic DNA extraction protocols- enzymatic and chemical proved to be efficient in terms of quantity, purity, and amplifiabiltiy, and thus produced DNA suitable for PCR amplification. The results of the PCR-DGGE gel fingerprints of the V3 region of amplified 16S rRNA gene showed identical DGGE patterns and high degree of similarity in DNA fragments with the two DNA extraction methods. The PCR-DGGE gel profiles were also used to assess the bacterial community profile; richness, biodiversity, and dominance indexes of naturally fermented iru samples obtained from different geographical locations in Nigeria. The species richness index (R) determined was highest for DGGE profiles of iru samples from Ilorin (R 19), thus exhibiting the highest number of bands/bacterial species than those of Lagos (16 < R < 18); Ado-Ekiti (R 16); Ibadan (11 < R < 14); Oyo (R 10) and Abeokuta (9 < R < 17) (Table 2). Bacterial diversity index (H) calculated on the basis of number of bands on a gel track was also highest for iru samples from Ilorin (H 2.92) with the lowest being Abeokuta (2.19 < H < 2.80) (Table 2). Similarity and variation among DGGE gel patterns of the various fermented condiments were established based on combined analysis of Dice similarity coefficient and nMDS. The results obtained showed that the 16 iru samples analysed clustered into five groups—a, b, c, d, and e (Figure 1). Noticeable similarity can be said to exist in the bacterial community structure of the different samples of iru obtained in the two geographical zones under study (southwest and north central Nigeria). Apart from two iru samples from Lagos south western Nigeria, that clustered into the same group “a” variation was observed among samples within the same geographical location especially southwest Nigerian samples.


Combination of culture-independent and culture-dependent molecular methods for the determination of bacterial community of iru, a fermented Parkia biglobosa seeds.

Adewumi GA, Oguntoyinbo FA, Keisam S, Romi W, Jeyaram K - Front Microbiol (2013)

Non-metric Multidimensional Scaling analysis of DGGE data Group (a): iru Abeokuta 2 southwest Nigeria (); iru Abeokuta 3 southwest Nigeria (); iru Lagos 1 southwest Nigeria (); iru Lagos 2 southwest Nigeria (•); iru Ilorin 1 northcentral Nigeria (); iru Ilori n 2 northcentral Nigeria (); iru Ibadan 1 southwest Nigeria (). Group (b): iru Abeokuta 4 southwest Nigeria (). Group (c): iru Abeokuta 1 southwest Nigeria (); iru Abeokuta 5 southwest Nigeria (); iru Abeokuta 8 southwest Nigeria (); iru Oyo southwest Nigeria (); iru Ado-Ekiti southwest Nigeria (). Group (d): iru Abeokuta 6 southwest Nigeria (); iru lbadan 2 southwest Nigeria (). Group (e): iru Abeokuta 7 southwest Nigeria ().
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3539807&req=5

Figure 1: Non-metric Multidimensional Scaling analysis of DGGE data Group (a): iru Abeokuta 2 southwest Nigeria (); iru Abeokuta 3 southwest Nigeria (); iru Lagos 1 southwest Nigeria (); iru Lagos 2 southwest Nigeria (•); iru Ilorin 1 northcentral Nigeria (); iru Ilori n 2 northcentral Nigeria (); iru Ibadan 1 southwest Nigeria (). Group (b): iru Abeokuta 4 southwest Nigeria (). Group (c): iru Abeokuta 1 southwest Nigeria (); iru Abeokuta 5 southwest Nigeria (); iru Abeokuta 8 southwest Nigeria (); iru Oyo southwest Nigeria (); iru Ado-Ekiti southwest Nigeria (). Group (d): iru Abeokuta 6 southwest Nigeria (); iru lbadan 2 southwest Nigeria (). Group (e): iru Abeokuta 7 southwest Nigeria ().
Mentions: The two genomic DNA extraction protocols- enzymatic and chemical proved to be efficient in terms of quantity, purity, and amplifiabiltiy, and thus produced DNA suitable for PCR amplification. The results of the PCR-DGGE gel fingerprints of the V3 region of amplified 16S rRNA gene showed identical DGGE patterns and high degree of similarity in DNA fragments with the two DNA extraction methods. The PCR-DGGE gel profiles were also used to assess the bacterial community profile; richness, biodiversity, and dominance indexes of naturally fermented iru samples obtained from different geographical locations in Nigeria. The species richness index (R) determined was highest for DGGE profiles of iru samples from Ilorin (R 19), thus exhibiting the highest number of bands/bacterial species than those of Lagos (16 < R < 18); Ado-Ekiti (R 16); Ibadan (11 < R < 14); Oyo (R 10) and Abeokuta (9 < R < 17) (Table 2). Bacterial diversity index (H) calculated on the basis of number of bands on a gel track was also highest for iru samples from Ilorin (H 2.92) with the lowest being Abeokuta (2.19 < H < 2.80) (Table 2). Similarity and variation among DGGE gel patterns of the various fermented condiments were established based on combined analysis of Dice similarity coefficient and nMDS. The results obtained showed that the 16 iru samples analysed clustered into five groups—a, b, c, d, and e (Figure 1). Noticeable similarity can be said to exist in the bacterial community structure of the different samples of iru obtained in the two geographical zones under study (southwest and north central Nigeria). Apart from two iru samples from Lagos south western Nigeria, that clustered into the same group “a” variation was observed among samples within the same geographical location especially southwest Nigerian samples.

Bottom Line: DNA sequencing of the highly variable V3 region of the 16S rRNA genes obtained from PCR-DGGE identified species related to Bacillus subtilis as consistent bacterial species in the fermented samples, while other major bands were identified as close relatives of Staphylococcus vitulinus, Morganella morganii, B. thuringiensis, S. saprophyticus, Tetragenococcus halophilus, Ureibacillus thermosphaericus, Brevibacillus parabrevis, Salinicoccus jeotgali, Brevibacterium sp. and uncultured bacteria clones.Bacillus species were cultured as potential starter cultures and clonal relationship of different isolates determined using amplified ribosomal DNA restriction analysis (ARDRA) combined with 16S-23S rRNA gene internal transcribed spacer (ITS) PCR amplification, restriction analysis (ITS-PCR-RFLP), and randomly amplified polymorphic DNA (RAPD-PCR).This further discriminated B. subtilis and its variants from food-borne pathogens such as B. cereus and suggested the need for development of controlled fermentation processes and good manufacturing practices (GMP) for iru production to achieve product consistency, safety quality, and improved shelf life.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Faculty of Science, University of Lagos Akoka, Lagos, Nigeria ; Microbial Resources Division, Institute of Bioresources and Sustainable Development, Takyelpat Institutional Area Imphal, Manipur, India ; Department of Food Science and Technology, College of Food Sciences, Bells University of Technology Ota, Nigeria.

ABSTRACT
In this study, bacterial composition of iru produced by natural, uncontrolled fermentation of Parkia biglobosa seeds was assessed using culture-independent method in combination with culture-based genotypic typing techniques. PCR-denaturing gradient gel electrophoresis (DGGE) revealed similarity in DNA fragments with the two DNA extraction methods used and confirmed bacterial diversity in the 16 iru samples from different production regions. DNA sequencing of the highly variable V3 region of the 16S rRNA genes obtained from PCR-DGGE identified species related to Bacillus subtilis as consistent bacterial species in the fermented samples, while other major bands were identified as close relatives of Staphylococcus vitulinus, Morganella morganii, B. thuringiensis, S. saprophyticus, Tetragenococcus halophilus, Ureibacillus thermosphaericus, Brevibacillus parabrevis, Salinicoccus jeotgali, Brevibacterium sp. and uncultured bacteria clones. Bacillus species were cultured as potential starter cultures and clonal relationship of different isolates determined using amplified ribosomal DNA restriction analysis (ARDRA) combined with 16S-23S rRNA gene internal transcribed spacer (ITS) PCR amplification, restriction analysis (ITS-PCR-RFLP), and randomly amplified polymorphic DNA (RAPD-PCR). This further discriminated B. subtilis and its variants from food-borne pathogens such as B. cereus and suggested the need for development of controlled fermentation processes and good manufacturing practices (GMP) for iru production to achieve product consistency, safety quality, and improved shelf life.

No MeSH data available.


Related in: MedlinePlus