Yeun Hong

Identification of lactic acid bacteria in salted Chinese cabbage by SDS-PAGE and PCR-DGGE.

BACKGROUND Lactic acid bacteria (LAB) in salted Chinese cabbage, the main ingredient of kimchi, were analyzed by culture-dependent sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by sequencing of the 16S rRNA gene and by culture-independent polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE), followed by sequencing of the V3 region of the 16S rRNA gene. The results were compared to those of LAB that had previously been found in kimchi. RESULTS The two identification methods produced distinct overall LAB profiles. The PCR-DGGE method detected a more diverse microflora, including non-LAB strains. The culture-dependent method uniquely detected Weissella sp. and was able to provide the quantitative distribution of LAB in samples. However, Leuconostoc mesenteroides, Lactobacillus curvatus and Leuconostoc carnosum, which had also been reported as the dominant LAB in kimchi in previous studies, were identified by both methods. CONCLUSION The two identification methods gave different bacterial profiles, while both methods were sufficient to identify the most prevalent LAB in salted Chinese cabbage samples. The quantitative feature of the culture-dependent identification method would make it preferable for studying and monitoring LAB viability in kimchi at each fermentation stage. The availability of the culture-independent identification method to identify a broader bacterial profile, including non-LAB, would make it a more effective tool for controlling contamination of undesirable bacteria during kimchi fermentation.

Quantitative detection of pork in commercial meat products by TaqMan® real-time PCR assay targeting the mitochondrial D-loop region.

The TaqMan® real-time PCR assay using the mitochondrial D-loop region was developed for the quantitative detection of pork in processed meat products. The newly designed primers and probe specifically amplified pork without any cross-reactivity with non-target animal species. The limit of detection of the real-time PCR assay was 0.1pg of heat-treated pork meat and 0.1% (w/w) pork meat in beef and chicken meat mixtures. The quantitative real-time PCR assay was applied to analyze the pork meat content in 22 commercial processed meat products including jerkies, press hams, sausages, hamburger patties and steaks, grilled short rib patties, and nuggets. The developed real-time PCR method was able to detect pork meat in various types of processed meat products that declared the use of pork meat on their label. All processed meat products that declared no use of pork meat showed a negative result in the assay. The method developed in this study showed sensitivity and specificity in the quantification of pork meat in commercial processed meat products.

Aroma characteristics of fermented Korean soybean paste (Doenjang) produced by Bacillus amyloliquefaciens

Four strains of Bacillus amyloliquefaciens with high proteolytic, fibrinolytic, and angiotensin converting enzyme (ACE) inhibitory activities were used to make Bacillus-koji suitable for the production of functional doenjang. Volatile compounds in Bacillus-koji doenjang were analyzed by headspace-solid phase microextraction-GC-MS (HS-SPME-GC-MS) to compare the aroma profile with those of commercially produced doenjang made with traditional meju and with modified koji. Extraction temperatures for SPME were adjusted to 30 and 90°C. Commercial samples contained significantly more esters than Bacillus-koji doenjang and demonstrated a more complex volatile compound profile. Pentanoic acid, butanoic acid, or 2/3-methyl butanoic acid, known as off-flavor compounds in doenjang, were not detected in one of the Bacillus-koji doenjang when extracted at 30°C. From the sensory test results, the cheese character was less intense in Bacillus-koji doenjang than commercial samples, although not all sensory properties projected a significant difference.

Distribution of lactic acid bacteria in garlic (Allium sativum) and green onion (Allium fistulosum) using SDS-PAGE whole cell protein pattern comparison and 16S rRNA gene sequence analysis

Distributions of lactic acid bacteria (LAB) in garlic and green onion samples as kimchi sub-ingredients were analyzed by comparing the SDS-PAGE whole cell protein patterns and 16S rRNA gene sequence analysis. In total, 245 LAB were isolated from 10 garlic samples and differentiated into 7 groups by comparing SDS-PAGE whole cell protein patterns. The groups were identified as Leuconostoc, Weissella, and Lactobacillus through the 16S rRNA gene sequence analysis. A total of 115 LAB were isolated from 7 green onion samples, differentiated into 6 groups, and identified as Weissella, Leuconostoc, and Lactococcus. Leuconostoc was the most dominated LAB in garlic and Weissella was the most dominated LAB in green onion. The LAB identified in this study was found as dominant microorganisms in kimchi. This result suggests the possible contribution of LAB in garlic and green onion to the bacterial microflora of kimchi, especially during early stage of fermentation.

Effect of Low Salt Concentrations on Microbial Changes During Kimchi Fermentation Monitored by PCR-DGGE and Their Sensory Acceptance.

Various salt concentrations (1.0%, 1.3%, 1.6%, 1.9%, and 2.1% labeled as sample A, B, C, D, and E, respectively) were investigated for microbial diversity, identification of Lactic Acid Bacteria (LAB) in salted kimchi cabbage, prepared under laboratory conditions. These samples were stored at 4°C for 5 weeks in proper aluminum-metalized pouch packaging with calcium hydroxide gas absorber. A culture-independent method known as polymerase chain reaction - denaturing gradient gel electrophoresis was carried out to identify LAB distributions among various salt concentration samples that had identified 2 Weissella (W. confusa and W. soli), 1 Lactobacillus (Lb. sakei), and 3 Leuconostoc (Lc. mesenteroides, Lc. lactis, and Lc. gelidum) in the overall kimchi samples. The pH, titratable acidity, viable cell counts, and coliform counts were not affected by salt variations. In order to assess sensory acceptance, the conducted sensory evaluation using a 9-point hedonic scale had revealed that samples with 1.3% salt concentration (lower than the manufacturers regular salt concentration) was more preferred, indicating that the use of 1.3% salt concentration was acceptable in normal kimchi fermentation for its quality and safety. Despite similarities in pH, titratable acidity, viable cell counts, coliform counts, and LAB distributions among the various salt concentrations of kimchi samples, the sample with 1.3% salt concentration was shown to be the most preferred, indicating that this salt concentration was suitable in kimchi production in order to reduce salt intake through kimchi consumptions.

Predominant lactic acid bacteria in mukeunji, a long-term-aged kimchi, for different aging periods

The distribution of dominant lactic acid bacteria (LAB) in mukeunji was examined at different stages of long-term aging. A total of 87 isolates from 3 samples were classified into 13 groups based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis whole-cell protein patterns and identified using 16S rRNA gene sequencing. The 13 LAB were also identified using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and API® strips. Weissella cibaria and Leuconostoc mesenteroides were the most abundant LAB in freshly prepared samples, followed by Lactobacillus (Lact.) curvatus and Lact. sakei, based on 16S rRNA gene sequencing. The species diversity of LAB in samples aged for 12 and 24 months was reduced over time, and Pediococcus sp. was the dominant LAB species in mukeunji samples. MALDI-TOF MS enabled rapid identification of Lactobacillus species. Identification results with similarities higher than 90% using API® strips provided the same results as 16S rRNA gene sequencing.

Characterization of macrophage-activating lactic acid bacteria isolated from Mukeunji

Lactic acid bacteria (LAB) were reported to comprise the majority of the bacterial population in mukeunji, a long-fermented kimchi. This current study investigated the probiotic abilities of LAB isolated from mukeunji. Forty bacterial strains from mukeunji were identified by SDS-PAGE gel patterns, which were further tested for acid and bile tolerance. An assessment of the immuneboosting effects of these strains in RAW264.7 cells was also carried out by measurement of secreted proinflammatory cytokines. Heat-killed LAB were also tested for their contribution to cytokine production because certain LAB are not durable in the digestive tract. Immune-boosting strains were further characterized by SDS-PAGE of whole-cell protein and 16S rRNA gene sequencing, resulting in the identification of Lactobacillus plantarum, L. sakei, Weissella cibaria, and Pediococcus parvulus. These data and the fact that mukeunji is highly consumed in Korea, together, highlight the need of detailed epidemiological and animal feeding studies.

Discrimination of doenjang samples using a mass spectrometry-based electronic nose and human sensory preference testing

Doenjang samples were discriminated using a mass spectrometry-based electronic nose (MS-E-nose) and discriminant function analysis (DFA). Human sensory preference testing was performed using the same samples. DFA plots indicated classification of doenjang samples into 3 groups. Samples with high discriminate function (DF) 1 and low DF2 scores contained fewer volatile compounds. Grouping results using the MS-E-nose and human sensory preference testing were compared. Fully mashed doenjang samples with more diverse and intense volatile compounds showed low DF1 and high preference scores. DF2 scores for selected samples showed positive correlations to the amount of sample. The MS-E-nose was a useful tool for discrimination of the aroma of doenjang samples and for confirmation of changes in the aroma intensities of doenjang samples.