Deficiency in Opu Systems Imparts Salt-Sensitivity to Weizmannia coagulans.

Weizmannia coagulans can be used as a starter strain in fermented foods or as a probiotic. However, it is salt-sensitive. Here, W. coagulans genomes were compared with the genomes of strains of Bacillus species (B. licheniformis, B. siamensis, B. subtilis, and B. velezensis) that were isolated from fermented foods and show salt tolerance, to identify the basis for the salt-sensitivity of W. coagulans. Osmoprotectant uptake (Opu) systems transport compatible solutes into cells to help them tolerate osmotic stress. B. siamensis, B. subtilis, and B. velezensis each possess five Opu systems (OpuA, OpuB, OpuC, OpuD, and OpuE); B. licheniformis has all except OpuB. However, W. coagulans only has the OpuC system. Based on these findings, the opuA and opuB operons, and the opuD and opuE genes, were amplified from B. velezensis. Expression of each of these systems, respectively, in W. coagulans increased salt-tolerance. W. coagulans expressing B. velezensis opuA, opuD, or opuE grew in 10.5% NaCl (w/v), whereas wild-type W. coagulans could not grow in 3.5% NaCl. The salt resistance of B. subtilis was also increased by overexpression of B. velezensis opuA, opuB, opuD, or opuE. These results indicate that the salt-susceptibility of W. coagulans arises because it is deficient in Opu systems.

The addition of jogi, Micropogonias undulates, affects amino acid content in kimchi fermentation.

The effects of jogi (the fish Atlantic croaker, Micropogonias undulatus) on the production of physicochemical components, such as color, organic acids, and amino acids, in kimchi, a traditional fermented vegetable food of Korea, were determined. As fermentation progressed, the color change of jogi-added kimchi increased, but in comparison with that of the control group without jogi-added kimchi, was difficult to distinguish with the naked eye. Reducing sugar decreased in all experimental groups, and as fermentation progressed, kimchi with jogi showed a lower value. Acetic acid, citric acid, lactic acid, and ethanol, were highly produced in both types of kimchi, and above all, the jogi-baechu-kimchi group showed higher acetic acid and lactic acid contents than the control group. The increase and decrease of amino acids were similar in both types of kimchi. However, significantly, immediately after manufacture, the savory components aspartic acid and glutamic acid were detected higher than the control group. Subsequently, the fermentation tended to decrease as it progressed, but the content was higher than that of the control group. The above results show that jogi addition has a greater effect on the contents of amino acid, especially the savory component, than on the physicochemical components.

Current status of the novel food ingredient safety evaluation system.

Increasing demand for new foods, technological development, and vegan market growth have led to an increase in new food ingredients, so the need for safety assessment of these ingredients is important. Representative safety assessment systems are the Generally Recognized as Safe (GRAS) notification of the Food and Drug Administration in the USA and the novel food system of the European Food Safety Authority in the European Union. GRAS is a notification system for information on food ingredients, food additives and functional foods under the responsibility of the applicant, while the novel food system assesses the safety of food ingredients excluding food additives. In Korea, a safety evaluation system is established for temporary food ingredients, which includes food ingredients without a domestic intake history. However, safety assessment systems for novel foods from other countries and food ingredients produced by the application of new technology need to be improved.

Comparison of four multilocus sequence typing schemes and amino acid biosynthesis based on genomic analysis of Bacillus subtilis.

Bacillus subtilis, a valuable industrial microorganism used in starter cultures in soybean fermentation, is a species of bacteria with interspecies diversity. Here, four multilocus sequence typing (MLST) schemes developed to assess the diversity of B. subtilis or Bacillus spp. were applied and compared to confirm the interspecies diversity of B. subtilis. In addition, we analyzed correlations between amino acid biosynthesis genes and sequence types (STs); this is important because amino acids are key taste components in fermented foods. On applying the four MLST methods to 38 strains and the type strain of B. subtilis, 30 to 32 STs were identified. The discriminatory power was 0.362-0.964 for the genes used in the MLST methods; the larger the gene, the greater the number of alleles and polymorphic sites. All four MLST methods showed a correlation between STs and strains that do not possess the hutHUIG operon (which contains genes required for the production of glutamate from histidine). This correlation was verified using 168 further genome-sequence strains.

ComQXPA quorum-sensing systems contribute to enhancing the protease activity of Bacillus velezensis DMB05 from fermented soybeans.

Bacillus velezensis DMB05, isolated from traditionally fermented soybean, meju, exhibited no protease activity on a TSA plate containing skim milk. To shed light on the genetic background behind this phenotypic non-protease activity, we analyzed the complete genome sequence of strain DMB05 and compared it with those of two B. velezensis strains which did exhibit protease activity. Comparative genome analyses showed no significant difference in the kind or number of proteases between the genomes of the three strains and that all strains possessed the degSU two-component system involved in the gene regulation of protease. However, strain DMB05 possessed a truncated comP which is part of the comQXPA operon that regulates the expression of degQ involved in the activation of DegSU. When the entire comQXPA operon derived from DMB06 was introduced into DMB05, the recombinant expressed proteolytic activity. The results of this experimental study provide evidence for the presence of regulatory genes involved in protease activity, one of several important factors involved in fermentation.

Increased Production of γ-Aminobutyric Acid from Brewer's Spent Grain Through Bacillus Fermentation.

Brewer's spent grain (BSG) is a waste product of the beer industry, and γ-aminobutyric acid (GABA) is a physiologically active substance important for brain and neuron physiology. In this study, we used the bacterial strains Bacillus velezensis DMB06 and B. licheniformis 0DA23-1, respectively, to ferment BSG and produce GABA. The GABA biosynthesis pathways were identified through genomic analysis of the genomes of both strains. We then inoculated the strains into BSG to determine changes in pH, acidity, reducing sugar content, amino-type nitrogen content, and GABA production, which was approximately doubled in BSG inoculated with Bacillus compared to that in uninoculated BSG; however, no significant difference was observed in GABA production between the two bacterial strains. These results provide the experimental basis for expanding the use of BSG by demonstrating the potential gain in increasing GABA production from a waste resource.

Transcriptomic analysis of Staphylococcus equorum KM1031 from the high-salt fermented seafood jeotgal under chloramphenicol, erythromycin and lincomycin stresses.

Staphylococcus equorum strain KM1031 is resistant to chloramphenicol, erythromycin and lincomycin. To shed light on the genetic factors underlying these antibiotic resistances, we determined the global gene expression profile of S. equorum KM1031 using RNA sequencing. During chloramphenicol, erythromycin and lincomycin treatment, 8.3% (183/2,336), 16.0% (354/2,336), and 2.9% (63/2,336) of S. equorum KM1031 genes exhibited significant differences in expression, respectively. These three antibiotics upregulated genes related to efflux and downregulated genes related to transporters. Antibiotic treatment also upregulated osmoprotectant-related genes involved in salt tolerance. To identify specific genes functionally related to antibiotic resistance, we compared the genome of strain KM1031 with those of three S. equorum strains that are sensitive to these three antibiotics. We identified three genes of particular interest: an antibiotic biosynthesis monooxygenase gene (abm, AWC34_RS01805) related to chloramphenicol resistance, an antibiotic ABC transporter ATP-binding protein gene (msr, AWC34_RS11115) related to erythromycin resistance, and a lincosamide nucleotydyltransferase gene (lnuA, AWC34_RS13300) related to lincomycin resistance. These genes were upregulated in response to the corresponding antibiotic; in particular, msr was upregulated more than fourfold by erythromycin treatment. Finally, the results of RNA sequencing were validated by quantitative real-time PCR. This transcriptomic analysis provides genetic evidence regarding antibiotic stress responses of S. equorum strain KM1031.

Bacterial Community of Galchi-Baechu Kimchi Based on Culture-Dependent and - Independent Investigation and Selection of Starter Candidates.

In this study, the bacterial community of galchi-baechu kimchi was determined using culture-based and culture-independent techniques (next generation sequencing:NGS), and showed discrepancies between results. Weissella koreensis and Pediococcus inopinatus were the dominant species according to the NGS results, while Bacillus species and P. inopinatus were dominant in the culture-dependent analysis. To identify safe starter candidates, sixty-five Bacillus strains isolated from galchi-baechu kimchi using culture-dependent methods were evaluated for their antibiotic resistance, presence of toxin genes, and hemolytic activity. Strains were then assessed for salt tolerance and protease and lipase activity. As a result, four strains-B. safensis GN5_10, B. subtilis GN5_19, B. velezensis GN5_25, and B. velezensis GT8-were selected as safe starter candidates for use in fermented foods.

Safety Assessment Systems for Microbial Starters Derived from Fermented Foods.

Microorganisms involved in food fermentation not only improve the aroma and taste of the food, but also enhance its preservation. Thus, they are added as starter cultures to boost the final product quality of commercial fermented foods. Although these microorganisms originate from fermented foods and have a long history of consumption, the European Union recently applied the concept of Qualified presumption of Safety (QPS), which is a safety evaluation system for microorganisms used in food or feed in Europe. The QPS system is a species-level safety system and shares results with the European Novel Food System, a strain-level safety evaluation system. In the United States, microorganisms added to fermented foods are considered as food additives or Generally Recognized as Safe substance. In Korea, food microbe lists are presented at the species level. Moreover, the nation has established a strain-oriented evaluation system that applies temporary safety evaluation methods for food raw materials as well as new raw materials. However, when it comes to microorganisms isolated from traditional fermented foods and other fermented food products, there is no definition of the term "species," and there is a lack of an evaluation system at the species level. Therefore, such an evaluation system for microbial species used in Korean fermented foods is necessary.

Discrimination of Bacillus subtilis from Other Bacillus Species Using Specific Oligonucleotide Primers for the Pyruvate Carboxylase and Shikimate Dehydrogenase Genes.

Bacillus subtilis is a useful bacterium in the food industry with applications as a starter strain for fermented food and as a probiotic. However, it is difficult to discriminate B. subtilis from other Bacillus species because of high phenotypic and genetic similarity. In this study, we employed five previously constructed multilocus sequence typing (MLST) methods for the discrimination of B. subtilis from other Bacillus species and all five MLST assays clearly distinguished B. subtilis. Additionally, the 17 housekeeping genes used in the five MLST assays also clearly distinguished B. subtilis. The pyruvate carboxylase (pyrA) and shikimate dehydrogenase (aroE) genes were selected for the discrimination of B. subtilis because of their high number of polymorphic sites and the fact that they displayed the lowest homology among the 17 housekeeping genes. Specific primer sets for the pyrA and aroE genes were designed and PCR products were specifically amplified from B. subtilis, demonstrating the high specificity of the two housekeeping genes for B. subtilis. This species-specific PCR method provides a quick, simple, powerful, and reliable alternative to conventional methods in the detection and identification of B. subtilis.

Risk factors and the role of bedside colonoscopy for lower gastrointestinal hemorrhage in critically ill patients.

BACKGROUND/AIMS: We designed this study to determine the clinical characteristics of critically ill patients with lower gastrointestinal hemorrhage (LGIH) and to evaluate the effectiveness of bedside colonoscopy in diagnosing and managing LGIH in the intensive care unit (ICU) setting. METHODOLOGY: We retrospectively reviewed the medical records of patients who underwent bedside colonoscopy for LGIH that developed while in the ICU at Severance Hospital from January 1999 to June 2006. The control group was a random sampling of patients hospitalized in the ICU without GIH. RESULTS: A total of 28,101 patients were hospitalized in ICU during the study period. Of these, 43 patients were included in each group. There were no significant differences in age, sex, and primary and underlying diseases between the two groups. Hypoalbuminemia and high BUN levels were found to be risk factors for LGIH in ICU patients. Bleeding focus was recognized in 28 patients (65.1%) through bedside colonoscopy. Major endoscopic diagnoses were ischemic colitis (13, 30.2%), rectal ulcer (7, 20.9%), and hemorrhoids (6, 13.9%). Primary endoscopic hemostasis was achieved in 11 of 28 diagnosed patients (39.3%). CONCLUSIONS: Our data suggest that patients' underlying conditions may affect LGIH in the ICU setting. Moreover, urgent bedside colonoscopy may be effective in diagnosing and guiding appropriate treatments for LGIH in critically ill patients.