Korean Fermented Food Research Articles

Prevalence of Bacillus cereus bacteriophages in fermented foods and characterization of phage JBP901

Bacillus cereus is widely distributed in the environment and is responsible for foodborne illnesses, causing diarrhea and vomiting. In this study, the prevalence of B. cereus bacteriophages in Korean fermented foods was studied using 6 reference strains, and JBP901 was further characterized. In a total of 47 samples, 19 (40.4%) contained more than one kind of B. cereus phage. Fourteen phages were purified and determined for host lysis profile. Among them, JBP901 was specific to B. cereus group strains (forming plaques in 13 of 14 isolates) and not able to lyse other tested Gram-positive and Gram-negative strains including Bacillus subtilis. Transmission electron microscopy analysis suggested that JBP901 is a member of the family Myoviridae. Restriction enzyme digestion pattern analysis and one-step growth study indicated a genome size of approximately 150 kbp and a burst size of 100. Furthermore, bacterial lysis studies revealed that JBP901 could be used to control the growth of B. cereus in liquid culture and in food. This is the first report to examine the prevalence of phages in fermented foods in Korea, and suggests that fermented foods are useful sources of B. cereus phage isolation; moreover, B. cereus phages could provide an additional tool to tackle problems associated with B. cereus contamination.

Multiplex PCR assay for the detection of aflatoxigenic and non-aflatoxigenic fungi in meju, a Korean fermented soybean food starter

Aflatoxins are toxic secondary metabolites produced commonly by Aspergillus flavus and Aspergillus parasiticus. In this study, the possibility of using multiplex PCR was investigated to speed up and specify the detection of aflatoxigenic Aspergillus species in meju, a traditional Korean fermented soybean food starter. Two different sets of three primers were designed specifically for the omtB, ver-1, aflR, and omtA genes present in the aflatoxin biosynthesis cluster. The optimized multiplex PCR showed that only aflatoxigenic Aspergillus species gave three band patterns in both primer sets. The detection limits were determined as 125 pg/μl for genomic DNA from aflatoxigenic A. parasiticus KCCM 35078, and 105 spores/g of meju sample for DNA extracted directly from meju. A total of 65 Aspergillus isolates from meju were tested for the presence of aflatoxigenic fungi by the application of multiplex PCR, and were analyzed by TLC and HPLC for the aflatoxin production in the culture filtrates. Results showed a good correlation between the presence of the aflatoxin biosynthesis genes analyzed by multiplex PCR and aflatoxin production by TLC and HPLC. This suggests that this multiplex PCR method may provide an accurate and specific detection of aflatoxigenic Aspergillus species in fermented soybean foods.

Immunomodulatory activity ofLactobacillusstrains isolated from fermented vegetables and infant stool

Four Lactobacillus strains- Lactobacillus plantarum CJLP133, L. plantarum CJLP243, L. plantarum CJNR26, and Lactobacillus gasseri CJMF3- were isolated from Korean fermented food or healthy infant feces, and their capacity to modulate cellular and humoral immune responses was studied. Feeding of the tested lactobacilli for 8weeks did not alter the weight of and cell numbers in the spleen of mice. However, CJLP133 and CJLP243 strains increased the T lymphocyte population in the spleen of mice, while CJNR26 and CJMF3 increased the B lymphocyte population. In splenocytes treated with concanavalin A, ingestion of CJLP133 and CJLP243 promoted T lymphocyte proliferation and secretion of T cell cytokines, whereas feeding of the CJNR26 and CJMF3 strains enhanced B lymphocyte proliferation in splenocytes treated with lipopolysaccharide and plaque formation. These results suggest that CJLP133 and CJLP243 have immunostimulating activity through the enhancement of T cell activation, while CJNR26 and CJMF3 exhibit immunopotentiation through the increment of B cell activation.

Identification of the genes involved in 1-deoxynojirimycin synthesis in Bacillus subtilis MORI 3K-85

1-Deoxynojirimycin (DNJ), a D-glucose analogue with a nitrogen atom substituting for the ring oxygen, is a strong inhibitor of intestinal α-glucosidase. DNJ has several promising biological activities, including its antidiabetic, antitumor, and antiviral activities. Nevertheless, only limited amounts of DNJ are available because it can only be extracted from some higher plants, including the mulberry tree, or purified from the culture broth of several types of soil bacteria, such as Streptomyces sp. and Bacillus sp. In our previous study, a DNJ-producing bacterium, Bacillus subtilis MORI, was isolated from the traditional Korean fermented food Chungkookjang. In the present study, we report the identification of the DNJ biosynthetic genes in B. subtilis MORI 3K-85 strain, a DNJ-overproducing derivate of the B. subtilis MORI strain generated by γ-irradiation, xhe genomic DNA library of B. subtilis MORI 3K-85 was constructed in Escherichia coli, and clones showing α-glucosidase inhibition activity were selected. After DNA sequencing and a series of subcloning, we were able to identify a putative Operon which consists of gabT1, yktc1, and gutB1 genes predicted to encode putative transaminase, phosphatase, and oxidoreductase, respectively. When a recombinant plasmid containing this Operon sequence was transformed into an E. coli strain, the resulting transformant was able to produce DNJ into the culture medium. Our results indicate that the gabT1, yktc1, and gutB1 genes are involved in the DNJ biosynthetic pathway in B. subtilis MORI, suggesting the possibility of employing these genes to establish a large-scale microbial DNJ overproduction system through genetic engineering and process optimization.

유산균 발효에 의한 톳(Hizikia fusiforme) 추출액의 이화학적 및 관능적 특성 변화

This study was conducted to improve the food functional and sensory food quality of Hizikia fusiforme by the fermentation of lactic acid bacteria. Seven strains of lactic acid bacteria isolated from traditional Korean fermented food were inoculated and cultivated in H. fusiforme water extract. Among them, Lactobacillus brevis LB-20, isolated from Kimchi, was selected for further study by considering the results of bacterial growth, DPPH radical scavenging activity, and sensory evaluation. No significant differences in proximate compositions (moisture, crude protein, crude fat, and crude ash) were observed by the fermentation of L. brevis LB-20. The most dramatical change was the conversion from glutamate to <TEX>${\gamma}$</TEX>-aminobutyric acid (GABA) in H. fusiforme water extract fermented by L. brevis LB-20. The GABA content increased approximately 60-fold after 48 hr of fermentation. The bacterial fermentation also resulted in low-molecularization of the extract. The particle size of the fermented extract became approximately 4-fold smaller than that of the law extract. In addition, the analysis of volatile flavor compounds using GC/MS revealed that the bacterial fermentation dramatically removed off-flavors such as acetaldehyde, haxanal, diallyl disulphide and 1-penten-2-ol in the H. fusiforme extract.

Ubiquinone contents in Korean fermented foods and average daily intakes

Coenzyme Q (CoQs, ubiquinones) in food may have antioxidative and other beneficial effects on human health. Data on the ubiquinones (CoQ 10 and CoQ 9) content of Korean fermented foods were determined, then the Korean daily intake of ubiquinones from those foods was estimated. The analytical method employed saponification before solvent extraction and quantification HPLC/MS equipped with diode array detection. Contents of CoQs in the fermented foods varied from 315.9 ± 12.6 μg/g ( Jeotgal, a fermented fish) to below the detection limit ( Soy sauce) on a dry basis. Doenjang (a fermented soy paste) was the richest sources of CoQ 9 (110.1 μg/g), while CoQ 10 was found highest contents in Jeotgal (291.0 μg/g). However, the major fermented food source of CoQs intake in the Korean population was Kimchi, representing 88.2% of the total CoQs (Q 9 + Q 10) intake. The estimated daily intake of total CoQs from the analyzed fermented foods was 11.6 mg/day/person, which was significantly higher than those of other populations reported (3–6 mg). It could be suggested that Korean fermented foods may have higher ubiquinone than unfermented foods due to their diverse microflora during the process of fermentation.

Oral administration of Lactobacillus strains from Kimchi inhibits atopic dermatitis in NC / Nga mice

Atopic dermatitis (AD) is marked by elevated levels of immunoglobulin E and skin lesions such as oedema and haemorrhage. Kimchi is a Korean fermented food that contains beneficial bacteria for human health. In this study, Lactobacillus plantarum CJLP55, CJLP56, CJLP133 and CJLP136 isolated from Kimchi were investigated for their capacity to inhibit AD. The three strains, CJLP55, CJLP133 and CJLP136, suppressed AD-like skin lesions, high serum IgE levels and epidermal thickening. The three strains diminished the accumulation of eosinophils and mast cells into topical inflammatory sites and the enlargement of axillary lymph nodes, which are responsible for the dorsal dermatitis. CJLP55, CJLP133 and CJLP136 decreased production of type 2 cytokines such as IL-4 and IL-5 in lymph node cell culture. CJLP133 and CJLP136 increased IFN-γ secretion, while CJLP55 enhanced IL-10 production. The three strains isolated from Kimchi suppress house-dust mite-induced dermatitis in NC/Nga mouse, a representative animal model of human AD. These findings suggest that lactobacilli isolated from Kimchi inhibit AD, probably by altering the balance of Th1/Th2 ratio or inducing IL-10 production.

Metagenomic Analysis of Kimchi, a Traditional Korean Fermented Food

Kimchi, a traditional food in the Korean culture, is made from vegetables by fermentation. In this study, metagenomic approaches were used to monitor changes in bacterial populations, metabolic potential, and overall genetic features of the microbial community during the 29-day fermentation process. Metagenomic DNA was extracted from kimchi samples obtained periodically and was sequenced using a 454 GS FLX Titanium system, which yielded a total of 701,556 reads, with an average read length of 438 bp. Phylogenetic analysis based on 16S rRNA genes from the metagenome indicated that the kimchi microbiome was dominated by members of three genera: Leuconostoc, Lactobacillus, and Weissella. Assignment of metagenomic sequences to SEED categories of the Metagenome Rapid Annotation using Subsystem Technology (MG-RAST) server revealed a genetic profile characteristic of heterotrophic lactic acid fermentation of carbohydrates, which was supported by the detection of mannitol, lactate, acetate, and ethanol as fermentation products. When the metagenomic reads were mapped onto the database of completed genomes, the Leuconostoc mesenteroides subsp. mesenteroides ATCC 8293 and Lactobacillus sakei subsp. sakei 23K genomes were highly represented. These same two genera were confirmed to be important in kimchi fermentation when the majority of kimchi metagenomic sequences showed very high identity to Leuconostoc mesenteroides and Lactobacillus genes. Besides microbial genome sequences, a surprisingly large number of phage DNA sequences were identified from the cellular fractions, possibly indicating that a high proportion of cells were infected by bacteriophages during fermentation. Overall, these results provide insights into the kimchi microbial community and also shed light on fermentation processes carried out broadly by complex microbial communities.

Oceanobacillus kimchii sp. nov. Isolated from a traditional Korean fermented food

A moderate halophile, strain X50(T), was isolated from mustard kimchi, a traditional Korean fermented food. The organism grew under conditions ranging from 0-15.0% (w/v) NaCl (optimum: 3.0%), pH 7.0-10.0 (optimum: pH 9.0) and 15-45°C (optimum: 37°C). The morphological, physiological, and biochemical features and the 16S rRNA gene sequences of strain X50(T) were characterized. Colonies of the isolate were creamcolored and the cells were rod-shaped. Phylogenetic analysis based on the 16S rRNA gene sequence indicated that strain X50(T) belongs to the genus Oceanobacillus and is closely related phylogenetically to the type strain O. iheyensis HTE831(T) (98.9%) and O. oncorhynchi subsp. oncorhynchi R-2(T) (97.0%). The cellular fatty acid profiles predominately included anteiso-C(15:0) and iso-C(15:0). The G+C content of the genomic DNA of the isolate was 37.9 mol% and the major isoprenoid quinone was MK-7. Analysis of the 16S rRNA gene sequences, DNA-DNA relatedness and physiological and biochemical tests indicated genotypic and phenotypic differences among strain X50(T) and reference species in the genus Oceanobacillus. Therefore, strain X50(T) was proposed as a novel species and named Oceanobacillus kimchii. The type strain of the new species is X50(T) (=JCM 16803(T) =KACC 14914(T) =DSM 23341(T)).

Determination of microbial diversity in meju, fermented cooked soya beans, using nested PCR-denaturing gradient gel electrophoresis

To identify the microbiota in meju, fermented cooked soya beans, that may directly affect the microbial communities of Korean fermented soya bean foods. Using conventional bacterial 16S rDNA, bacilli-specific 16S rDNA or fungi 18S rDNA-specific primers, PCR products were amplified through a series of PCRs using the DNA extracted from ten meju samples. The amplicons were analysed using denaturing gradient gel electrophoresis (DGGE), which showed that Enterococcus durans was commonly detected in nine of ten meju samples. Bacillus subtilis was shown to be the major strain of bacilli in the samples tested. Based on the DGGE analysis of fungi in meju, we determined that Absidia corymbifera, Aspergillus sp. and Candida rugosa were the main fungi in the tested samples. A variety of bacterial and fungal micro-organisms were identified in meju samples, in addition to the micro-organisms already known to be present. This is the first report showing the differences and similarities in the populations of micro-organisms in meju samples using nested PCR-DGGE, a culture-independent method. The results may be applicable to the development of improved meju, in which the indigenous micro-organisms required for fermentation can be standardized.

Lactobacillus kimchicus sp. nov., a β-glucosidase-producing bacterium isolated from kimchi

A Gram-reaction-positive, non-spore-forming, rod-shaped, non-motile, β-glucosidase-producing bacterium, designated DCY51(T), was isolated from kimchi, a Korean fermented vegetable food. 16S rRNA gene sequence analysis revealed that strain DCY51(T) belonged to the genus Lactobacillus and exhibited highest 16S rRNA gene sequence similarity with Lactobacillus paracollinoides AB 74 (96.9 %), L. similis JCM 2765(T) (96.9 %), L. collinoides JCM 1123(T) (96.7 %) and L. hilgardii DSM 20176(T) (95.5 %). Subsequently, pheS sequence analysis confirmed that strain DCY51(T) formed a distinct lineage within the Lactobacillus plantarum group. The major polar lipid of strain DCY51(T) was phosphatidylethanolamine and minor amounts of phosphatidylglycerol and diphosphatidylglycerol were found. Phenotypic characteristics and DNA-DNA relatedness indicated that strain DCY51(T) was clearly distinguished from other type strains of species of the genus Lactobacillus. A novel species, Lactobacillus kimchicus sp. nov., is proposed, with type strain DCY51(T) ( = KCTC 12976(T) = JCM 15530(T)).

Complete Genome Sequence Analysis of Leuconostoc kimchii IMSNU 11154

Leuconostoc kimchii IMSNU 11154, isolated from kimchi, a traditional Korean fermented food, is known to be an important antimicrobial lactic acid bacterium with probiotic potential. Here we announce the complete genome sequence of L. kimchii IMSNU 11154 consisting of a 2,101,787-bp chromosome and five plasmids. The strain has genes for dextran formation from sucrose and for mannitol formation from fructose. Antimicrobial and antioxidative functions of L. kimchii IMSNU 11154 could be attributed to a leucosin B-like peptide and multiple enzymes to reduce hydrogen peroxide and oxidized thiols, respectively.

다시마 추출액의 항산화 및 항염증 활성에 대한 효모 발효의 영향

To examine the effective use of seaweeds, sea tangle (Laminaria japonica) was extracted with water and the resultant extracts were fermented with Saccharomyces cerevisiae. Four strains of S. cerevisiae were cultured in aqueous extracts from sea tangle. S. cerevisiae SC-2, which was isolated from a traditional Korean fermented food (Meju), was selected for further study based on the results of a sensory evaluation. No significant differences in proximate compositions, such as moisture, crude protein, crude fat, and crude ash, of the sea tangle extracts before and after fermentation were observed. The reducing sugar decreased as the fermentation period increased, and the contents of some free amino acids were also affected by S. cerevisiae SC-2 fermentation. However, the content of glutamic acid, which is a major taste compound in sea tangle extract, was not affected by fermentation for up to 36 hr by the SC-2 strain. To determine the antioxidant activity of fermented sea tangle extract (fermented for 36 hr by SC-2 strain), the radical scavenging activities of 1,1-diphenyl-2-picrylhydrazyl (DPPH), superoxide, and nitric oxide were investigated and xanthin oxidase inhibition assay was performed. The antioxidant activity increased by 8 to 35%. The greatest enhancement of antioxidant activity was seen in the superoxide radical scavenging assay with <TEX>$100\;{\mu}g/mL$</TEX> of raw and fermented sea tangle extract. The anti-inflammatory activity of fermented sea tangle extract was also enhanced. The fermented sea tangle extract showed 34.2% inhibitory activity against nitric oxide synthesis versus 11.9% for raw sea tangle extract at <TEX>$100\;{\mu}g/mL$</TEX> concentration. These results suggest that fermented aqueous extracts from sea tangle are a useful resources.

Improvement of the Functional Qualities of Sea Tangle Extract through Fermentation by Aspergillus oryzae

This study was conducted to evaluate the potential of a microbial fermentation procedure to improve the functional qualities of seaweeds. Aspergillus oryzae, which has been used in traditional Korean fermented foods, was inoculated and cultivated in an aqueous extract of sea tangle (Laminaria japonica). Fermentation of the sea tangle extract by A. oryzae for 4 days resulted in a 3-fold increase in -aminobutyric acid (GABA) content. GABA is known to be a bioactive compound. Fungal fermentation of the extract also enhanced its antioxidant activity and increased its total content of phenolic compounds. It was assumed that these changes stemmed from the biodegradation of active compounds of the sea tangle packaged within its rigid structural matrix or occurred as result of fungal fermentation. These results suggested that the application of microbial fermentation to the processing of seaweeds will help in the development of processed foods to meet consumer demands.

Antioxidant activity and γ-aminobutyric acid (GABA) content in sea tangle fermented by Lactobacillus brevis BJ20 isolated from traditional fermented foods

GABA-producing lactic acid bacteria were isolated from kimchi and salt-fermented Jot-gal, which are traditional Korean fermented foods. The strain, BJ-20, isolated from salt-fermented Jot-gal (cod gut), possessed the highest GABA-producing ability in MRS broth with 1% monosodium glutamate (MSG), as determined by thin layer chromatography. The BJ-20 strain was identified as Lactobacillus brevis and designated as L. brevis BJ20. A sea tangle solution was fermented over 5 days to produce GABA using L. brevis BJ20. During fermentation, the GABA concentration dramatically increased, while the glutamic acid concentration decreased. This result indicates that the glutamic acid was converted to GABA by L. brevis BJ20 in the fermented sea tangle solution. Furthermore, the fermented solution exhibited strong antioxidant activities, such as DPPH scavenging, superoxide scavenging, and xanthine oxidase inhibition, which were higher than those of BHA as a positive control.

Application of quantitative real-time PCR for enumeration of total bacterial, archaeal, and yeast populations in kimchi

Kimchi is a Korean traditional fermented food made of brined vegetables, with a variety of spices. Various microorganisms are associated with the kimchi fermentation process. This study was undertaken in order to apply quantitative real-time PCR targeting the 16S and 26S rRNA genes for the investigation of dynamics of bacterial, archaeal, and yeast communities during fermentation of various types of kimchi. Although the total bacterial and archaeal rRNA gene copy numbers increased during kimchi fermentation, the number of yeasts was not significantly altered. In 1 ng of bulk DNA, the mean number of rRNA gene copies for all strains of bacteria was 5.45 x 10(6) which was 360 and 50 times greater than those for archaea and yeast, respectively. The total gene copy number for each group of microorganisms differed among the different types of kimchi, although the relative ratios among them were similar. The common dominance of bacteria in the whole microbial communities of various types of kimchi suggests that bacteria play a principal role in the kimchi fermentation process.

Purification and characterization of a novel glucansucrase from Leuconostoc lactis EG001

A gene encoding glucansucrase was identified in Leuconostoc lactis EG001 isolated from lactic acid bacteria (LAB) in Kimchi, a traditional Korean fermented food. The L. lactis EG001 glucansucrase gene consists of 4503 bp open reading frame (ORF) and encodes an enzyme of 1500 amino acids with an apparent molecular mass of 165 kDa. The deduced amino-acid sequence showed the highest amino-acid sequence identity (75%) to that of dextransucrase of L. mesenteroides. The gene was cloned and over-expressed in Escherichia coli strain. The recombinant enzyme was purified via Ni-NTA affinity chromatography and its enzymatic properties were characterized. The enzyme exhibited optimum activity at 30 degrees C and pH 5.0. In addition, the enzyme was able to catalyze the glycosylation of l-ascorbic acid to l-ascorbic acid 2-glucoside. The glycosylated product via EG001 glucansucrase has the potential as an antioxidant in industrial applications.

Characterization of a 27 kDa Fibrinolytic Enzyme from Bacillus amyloliquefaciens CH51 Isolated from Cheonggukjang

Bacillus amyloliquefancies CH51 isolated from cheonggukjang, a traditional Korean fermented soy food, has strong fibrinolytic activity and produces several fibrinolytic enzymes. Among four different growth media, tryptic soy broth was the best in terms of supporting cell growth and fibrinolytic activity of this strain. A protein with fibrinolytic activity was partially purified from the culture supernatant by CMSephadex and Phenyl Sepharose column chromatographies. Tandem mass spectrometric analysis showed that this protein is a homolog of AprE from B. subtilis and it was accordingly named AprE51. The optimum pH and temperature for partially purified AprE51 activity were 6.0 and 45 degrees , respectively. A gene encoding AprE51, aprE51, was cloned from B. amyloliquefaciens CH51 genomic DNA. The aprE51 gene was overexpressed in heterologous B. subtilis strains deficient in fibrinolytic activity using an E.colo-Bacillus Shuttle vector, pHY300PLK.

Carnobacterium jeotgali sp. nov., isolated from a Korean traditional fermented food

A Gram-positive, facultatively anaerobic bacterium, designated strain MS3(T), was isolated from a traditional Korean fermented food made with freshwater shrimp. Strain MS3(T) was able to grow at 4-37 degrees C, at pH 5.5-9.0 and in the presence of 0-5 % (w/v) NaCl. Optimal growth occurred at 30 degrees C, at pH 8.5 and in the presence of 2 % NaCl. The strain was catalase- and oxidase-negative. It was able to metabolize various carbohydrates as energy sources. 16S rRNA gene sequence analysis showed that strain MS3(T) was most closely related to Carnobacterium pleistocenium FTR1(T) (98.95 % similarity), but the level of DNA-DNA relatedness between the two taxa was less than 16.0 %. The genomic G+C content of strain MS3(T) was 43.9 mol% and the major fatty acid components were C(16 : 0), C(16 : 1)omega9c and C(18 : 1)omega9c. On the basis of its genotypic, physiological and biochemical characteristics, strain MS3(T) is considered to represent a novel species of the genus Carnobacterium, for which the name Carnobacterium jeotgali sp. nov. is proposed. The type strain is MS3(T) (=KCTC 13251(T)=JCM 15539(T)).

Antiobestic and antiinflammatory effect of doenjang (Korean fermented soy paste)

Antiobestic effect of Korean fermented soy foods by using SD‐rats and C57BL/6 mice was studied. Doenjang and chungkukjang(short‐fermented soy paste) showed higher antiobestic activities when fed with high‐fat diet. Traditionally prepared doenjang especially exhibited highest antiobesity in the in vivo studies. The levels of serum cholesterol and triglycerides in the rats treated with doenjang diet significantly decreased to the levels of normal diet‐fed rats. Anthocyanins from the coat of black soybeans showed antiobestic effect. Doenjang prepared with A. oryzae and natural salt or bamboo salt for the fermentation increased antiobestic effect. Black soybean doenjang showed the highest antiobestic effect in C57BL/6 mice, expressing lower proteins of PPARγ and SREBP‐1 in the western blot analysis. Doenjang and chungkukjang significantly increased PPARα that suppressed inflammation in YPEN‐1 endothelial cells. The doenjang treated cells also decreased expressions of NF‐κB and COX‐2, but it increased adiponectin expression that caused lipid lysis. The expressions of PPARα and adiponectin increased, however, the NF‐κB, COX‐2 and iNOS protein levels considerably decreased in doenjang and chungkukjang treated SD‐rats.