Denaturing gradient gel electrophoresis (DGGE) of DNA fragments obtained by PCR amplification of the V2-V3 region of the 16S rRNA gene was used to detect the presence of Lactobacillus species in the stomach contents of mice. Lactobacillus isolates cultured from human and porcine gastrointestinal samples were identified to the species level by using a combination of DGGE and species-specific PCR primers that targeted 16S-23S rRNA intergenic spacer region or 16S rRNA gene sequences. The identifications obtained by this approach were confirmed by sequencing the V2-V3 region of the 16S rRNA gene and by a BLAST search of the GenBank database.
A means to accelerate fish sauce fermentation without adversely affecting fish sauce quality was investigated. Starter cultures prepared from Virgibacillus sp. SK33, Virgibacillus sp. SK37, and Staphylococcus sp. SK1-1-5 were added separately to anchovy that was hydrolyzed by 0.25% Alcalase at 60 degrees C for 2 h followed by 0.5% Flavourzyme at 50 degrees C for 4 h. The mixtures were then adjusted to contain 25% solar salt and incubated at 35 degrees C for 4 mo. alpha-Amino contents of all inoculated samples were higher than the control (without the addition of starter culture) during the course of fermentation. After 4-mo fermentation, the samples inoculated with Staphylococcus sp. SK1-1-5 contained the highest alpha-amino content of 733.37 +/- 13.89 mM while that of the control was 682.67 +/- 3.33 mM. Amino acid profiles of inoculated samples showed similar patterns to that of commercial product fermented for 12 mo, with glutamic, aspartic, and lysine being predominant amino acids. Virgibacillus sp. SK33 appeared to decrease histamine content of fish sauce by 50% when compared to the control. Volatile compounds analyzed by GC-MS of all inoculated samples fermented for 4 mo exhibited a similar pattern to those of the 12-mo-old commercial product. Samples inoculated with Staphylococcus sp. SK1-1-5 produced higher levels of volatile fatty acids and showed similar sensory characteristics to the commercial fish sauce fermented for 12 mo. Staphylococcus sp. SK1-1-5 is a potential strain that can be applied to produce fish sauce with overall sensory characteristics of traditional fish sauce in shorter time.
The potential of Tetragenococcus halophilus as a starter culture for flavor improvement in fish sauce fermentation was elucidated. Four strains of T. halophilus isolated from fish sauce mashes were inoculated to anchovy mixed with 25% NaCl with an approximate cell count of 10(6) CFU/mL. The α-amino content of 6-month-old fish sauce samples inoculated with T. halophilus was 780-784 mM. The addition of T. halophilus MRC10-1-3 and T. halophilus MCD10-5-10 resulted in a reduction of histamine (P < 0.05). Fish sauce inoculated with T. halophilus showed high contents of total amino acids with predominantly high glutamic acid. Major volatile compounds in fish sauce were 2-methylpropanal, 2-methylbutanal, 3-methylbutanal, and benzaldehyde. T. halophilus-inoculated fish sauce samples demonstrated the ability to reduce dimethyl disulfide, a compound contributing to a fecal note. The use of T. halophilus for fish sauce fermentation improves amino acid profiles and volatile compounds as well as reduces biogenic amine content of a fish sauce product.
Lactobacillus isolates were identified by PCR amplification and sequencing of the region between the 16S and 23S rRNA genes (spacer region). The sequences obtained from the isolates were compared to those of reference strains held in GenBank. A similarity of 97.5% or greater was considered to provide identification. To check the reliability of the method, the V2-V3 region of the 16S rRNA gene was amplified and sequenced in the case of isolates whose spacer region sequences were less than 99% similar to that of a reference strain. Confirmation of identity was obtained in all instances. Spacer region sequencing provided rapid and accurate identification ofLactobacillus isolates obtained from gastrointestinal, yoghurt, and silage samples. It had an advantage over 16S V2-V3 sequence comparisons because it distinguished between isolates ofLactobacillus casei and Lactobacillus rhamnosus.
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