Deficiency in Opu Systems Imparts SaltSensitivity 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.

Bacterial diversity of baechu-kimchi with seafood based on culture-independent investigations.

Baechu-kimchi is a traditional Korean dish of fermented vegetables, in which kimchi cabbage is the major ingredient. Seafood is added to baechu-kimchi in coastal areas, giving this dish regional diversity. However, little is known about how the addition of seafood affects the bacterial diversity of kimchi. Therefore, in this study, the bacterial diversity of five varieties of baechu-kimchi with seafood and one variety of baechu-kimchi without seafood was analyzed using culture-independent techniques. In 81.7% of all kimchi analyzed, the predominant species were members of the phylum Firmicutes and the lactic acid bacteria, Latilactobacillus sakei, Leuconostoc mesenteroides, Pediococcus inopinatus, and Weissella koreensis. These organisms were similar to those identified in baechu-kimchi without the addition of seafood, which was used as a control group, and bacterial community of previously reported kimchi. Therefore, the results of this study confirmed that the addition of seafood did not significantly affect the bacterial community in baechu-kimchi.

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.

Palmitoylation-driven PHF2 ubiquitination remodels lipid metabolism through the SREBP1c axis in hepatocellular carcinoma.

Palmitic acid (PA) is the most common fatty acid in humans and mediates palmitoylation through its conversion into palmitoyl coenzyme A. Although palmitoylation affects many proteins, its pathophysiological functions are only partially understood. Here we demonstrate that PA acts as a molecular checkpoint of lipid reprogramming in HepG2 and Hep3B cells. The zinc finger DHHC-type palmitoyltransferase 23 (ZDHHC23) mediates the palmitoylation of plant homeodomain finger protein 2 (PHF2), subsequently enhancing ubiquitin-dependent degradation of PHF2. This study also reveals that PHF2 functions as a tumor suppressor by acting as an E3 ubiquitin ligase of sterol regulatory element-binding protein 1c (SREBP1c), a master transcription factor of lipogenesis. PHF2 directly destabilizes SREBP1c and reduces SREBP1c-dependent lipogenesis. Notably, SREBP1c increases free fatty acids in hepatocellular carcinoma (HCC) cells, and the consequent PA induction triggers the PHF2/SREBP1c axis. Since PA seems central to activating this axis, we suggest that levels of dietary PA should be carefully monitored in patients with HCC.

Genomic insight into the salt tolerance and proteolytic activity of Bacillus subtilis.

We assessed the salt tolerance and proteolytic activity of 40 genome-published Bacillus subtilis strains isolated from fermented Korean foods to illuminate the genomic background behind the functionality of B. subtilis in high-salt fermentation. On the basis of the salt tolerance and phenotypic proteolytic activity of the 40 strains, we selected five strains exhibiting different phenotypic characteristics. Comparative genomic analyses of these five strains provided genomic insight into the salt tolerance and proteolytic activity of B. subtilis. Two-component system (TCS) genes annotated as ybdGJK and laterally acquired authentic ATP-binding cassette (ABC) transporter system genes of tandem repeat structure might contribute to increase salt tolerance. The additional possession of gene homologs for CAAX protease family proteins and components of Clp (caseinolytic protease) complex, ATP-dependent Clp proteolytic subunit ClpP and AAA+ (ATPases associated with diverse cellular Activities) family ATPases, might determine the proteolytic activity of B. subtilis. This study established the scientific foundation for the viability and functionality of B. subtilis in high-salt fermentation.

Novel Strain Leuconostoc lactis DMLL10 from Traditional Korean Fermented Kimchi as a Starter Candidate for Fermented Foods.

Leuconostoc lactis strain DMLL10 was isolated from kimchi, a fermented vegetable, as a starter candidate through safety and technological assessments. Strain DMLL10 was susceptible to ampicillin, chloramphenicol, clindamycin, erythromycin, gentamicin, kanamycin, streptomycin, and tetracycline. It did not show any hemolytic activity. Regarding its phenotypic results related to its safety properties, genomic analysis revealed that strain DMLL10 did not encode for any toxin genes such as hemolysin found in the same genus. It did not acquire antibiotic resistance genes either. Strain DMLL10 showed protease activity on agar containing NaCl up to 3%. The genome of DMLL10 encoded for protease genes and possessed genes associated with hetero- and homo-lactic fermentative pathways for lactate production. Finally, strain DMLL10 showed antibacterial activity against seven common foodborne pathogens, although bacteriocin genes were not identified from its genome. These results indicates that strain DMLL10 is a novel starter candidate with safety, enzyme activity, and bacteriocin activity. The complete genomic sequence of DMLL10 will contribute to our understanding of the genetic basis of probiotic properties and allow for assessment of the effectiveness of this strain as a starter or probiotic for use in the food industry.

Correction to: Food-derived coagulase-negative Staphylococcus as starter cultures for fermented foods.

[This corrects the article DOI: 10.1007/s10068-020-00789-5.].

Novel Starter Strain Enterococcus faecium DMEA09 from Traditional Korean Fermented Meju.

The Enterococcus faecium strain DMEA09 was previously isolated from traditional Korean fermented meju. The objective of the current study was to investigate the traits of E. faecium strain DMEA09 as a starter candidate, focusing on its safety and technological properties. Regarding its safety, the DMEA09 strain was found to be sensitive to nine antibiotics (ampicillin, chloramphenicol, erythromycin, gentamicin, kanamycin, streptomycin, tetracycline, tylosin, and vancomycin) by showing lower minimum inhibitory concentrations (MICs) than the cut-off values suggested by the European Union Food Safety Authority for these nine antibiotics. However, its MIC value for clindamycin was twice as high as the cut-off value. A genomic analysis revealed that strain DMEA09 did not encode the acquired antibiotic resistance genes, including those for clindamycin. The DMEA09 strain did not show hemolysis as a result of analyzing α- and ß-hemolysis. It did not form biofilm either. A genomic analysis revealed that strain DMEA09 did not encode for any virulence factors including hemolysin. Most importantly, multilocus sequence typing revealed that the clonal group of strain DMEA09 was distinguished from clinical isolates. Regarding its technological properties, strain DMEA09 could grow in the presence of 6% salt. It showed protease activity when the salt concentration was 3%. It did not exhibit lipase activity. Its genome possessed 37 putative protease genes and salt-tolerance genes for survivability under salt conditions. Consequently, strain DMEA09 shows safe and technological properties as a new starter candidate. This was confirmed by genome analysis.

Staphylococcus equorum plasmid pKS1030-3 encodes auxiliary biofilm formation and trans-acting gene mobilization systems.

The foodborne bacterium Staphylococcus equorum strain KS1030 harbours plasmid pSELNU1, which encodes a lincomycin resistance gene. pSELNU1 undergoes horizontal transfer between bacterial strains, thus spreading antibiotic resistance. However, the genes required for horizontal plasmid transfer are not encoded in pSELNU1. Interestingly, a relaxase gene, a type of gene related to horizontal plasmid transfer, is encoded in another plasmid of S. equorum KS1030, pKS1030-3. The complete genome of pKS1030-3 is 13,583 bp long and encodes genes for plasmid replication, biofilm formation (the ica operon), and horizontal gene transfer. The replication system of pKS1030-3 possesses the replication protein-encoding gene repB, a double-stranded origin of replication, and two single-stranded origins of replication. The ica operon, relaxase gene, and a mobilization protein-encoding gene were detected in pKS1030-3 strain-specifically. When expressed in S. aureus RN4220, the ica operon and relaxase operon of pKS1030-3 conferred biofilm formation ability and horizontal gene transfer ability, respectively. The results of our analyses show that the horizontal transfer of pSELNU1 of S. equorum strain KS1030 depends on the relaxase encoded by pKS1030-3, which is therefore trans-acting. Genes encoded in pKS1030-3 contribute to important strain-specific properties of S. equorum KS1030. These results could contribute to preventing the horizontal transfer of antibiotic resistance genes in food.

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.

Quality characteristics of soybean fermented by Mucor, Rhizopus, and Aspergillus from meju.

Three candidate starter strains-Aspergillus oryzae SNU-G, Mucor sp. KACC 46077, and Rhizopus oryzae KACC 40256-were inoculated into soybean, as individual strains or in combination, to assess their roles in fermentation. All the strains increased the pH, amino-type nitrogen, and moisture content of the soybean during fermentation, and decreased the lightness, redness, and yellowness. The inoculated strains increased to an average density of 1.37 × 108 spores/g (from the initial 5.0 × 107 spores/g) after 20 days of fermentation. Forty-two volatile compounds, including an acid, alcohols, carbonyls, furans, and a pyrazine, were more abundant in soybean fermented with starters than in controls. A. oryzae SNU-G increased the pH more than the other strains and produced more volatile alcohol compounds. R. oryzae KACC 40256 resulted in the lowest reduction of redness and yellowness during the fermentation and produced large amounts of carbonyl compounds, including two specific volatile compounds, 2-hydroxy-3-methylcyclopent-2-en-1-one and (3E)-3-ethyl-2-methylhexa-1,3-diene. Mucor sp. KACC 46077 contributed the least to pH change and volatile compound production, and did not produce specific volatile compounds. Although no significant synergy in the production of volatile compounds was found when using mixtures of strains compared with application of single strains, the quality of fermented soybeans was confirmed to be different depending on the strain(s) applied.

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.

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.

Prevalence of Indigenous Antibiotic-Resistant Salmonella Isolates and Their Application to Explore a Lytic Phage vB_SalS_KFSSM with an Intra-Broad Specificity.

The consumption of fresh produce has led to increase in antibiotic-resistant (AR) Salmonella outbreaks. In this study, indigenous Salmonella was isolated from a total of two hundred-two samples including fresh produce and agricultural environmental samples in Korea. After biochemical confirmation using the Indole, Methyl Red, Voges-Proskauer, Citrate tests, presumable Salmonella isolates were identified by 16S rRNA sequencing. Identified Salmonella isolates were evaluated for antibiotic susceptibility against twenty-two antibiotics. The specificity and the efficiency of plating (EOP) of vB_SalS_KFSSM were evaluated against fifty-three bacterial strains. Twenty-five suspected Salmonella were isolated and confirmed by the positive result for methyl red and citrate, of which ten were identified as Salmonella spp. through 16S rRNA gene sequencing. Eight Salmonella isolates (4.0%, n = 8/202) were resistant to at least one antibiotic, among which five were multi-drug resistant. As a lytic phage against Salmonella spp. CMGS-1, vB_SalS_KFSSM was isolated from cow manure. The phage was observed as a tailed phage belonging to the class Caudoviricetes. It exhibited an intra-broad specificity against four indigenous AR Salmonella isolates, two indigenous Salmonella isolates, and five other Salmonella serotypes with great efficiencies (EOP ≥ 0.75). Thus, this study suggested the potential of vB_SalS_KFSSM to combat indigenous AR Salmonella.

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.

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.

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.

Antibiotic susceptibility of Bacillus velezensis.

We evaluated the antibiotic minimum inhibitory concentrations (MICs) of 123 Bacillus velezensis strains predominantly isolated from fermented soybean foods from Korea. When the 2018 European Food Safety Authority breakpoint values for Bacillus spp. were applied, all the strains were sensitive to chloramphenicol, clindamycin, erythromycin, gentamicin, kanamycin, tetracycline and vancomycin, and eight strains (6.5%) were resistant to streptomycin. The population distribution in MIC tests with streptomycin was continuous and the profile was clearly different from that expected for acquired antibiotic resistance. As of 25 October 2021, there were 181 complete published genomes of B. velezensis strains; 175 (96.7%) and 136 (75.2%) of these strains, respectively, possess potential tetracycline and streptomycin resistance genes tetL and ant(6) in the chromosome. In Bacillus licheniformis, SpeG confers resistance to clindamycin and there is an 'speG' gene annotated in the genomes of 180 B. velezensis strains; however, the gene products exhibit ≤26.6% amino acid identity with that from B. licheniformis DSM 13T. All the potential antibiotic resistance genes in the 181 B. velezensis strains were intrinsic, and traits of lateral gene transfer were not found. In this context, B. velezensis may not present a high risk in terms of antibiotic resistance in food fermentation or human use.