Isolation of a Potential Probiotic Levilactobacillus brevis and Evaluation of Its Exopolysaccharide for Antioxidant and α-Glucosidase Inhibitory Activities.

The probiotic properties of ten lactic acid bacteria and antioxidant and α-glucosidase inhibitory activities of the exopolysaccharide (EPS) of the selected strain were investigated in this study. Levilactobacillus brevis L010 was one of the most active strains across all the in vitro tests. The cell-free supernatant (50 g/l) of L. brevis L010 showed high levels of both α-glucosidase inhibitory activity (98.73 ± 1.32%) and 2-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging activity (32.29 ± 3.86%). The EPS isolated from cell-free supernatant of L. brevis L010 showed 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) radical-scavenging activity (80.27 ± 2.51%) at 80 g/l, DPPH radical-scavenging activity (38.19 ± 9.61%) at 40 g/l, and ferric reducing antioxidant power (17.35 ± 0.20 mg/l) at 80 g/l. Further, EPS exhibited inhibitory activities against α-glucosidase at different substrate concentrations. Kinetic analysis suggests that the mode of inhibition was competitive, with a kinetic constant of Km = 2.87 ± 0.88 mM and Vmax = 0.39 ± 0.06 µmole/min. It was concluded that the EPS might be one of the plausible candidates for possible antioxidant and α-glucosidase activities of the L. brevis L010 strain.

Draft genome sequence data of multi-stress tolerant yeast Millerozyma farinosa KCTC27753 isolated from nuruk.

The strain Millerozyma farinosa KCTC27753, isolated from nuruk, is a multi-stress tolerant yeast which grows at 46 °C temperature and pH 3.0. This strain can withstand fermentation inhibitors, such as furfural and phenolic compounds released from biomass. Hence, this strain could be used for bioethanol production. The draft genome sequence of M. farinosa KCTC27753 was analyzed by PacBio RSII. The genome length is 21,255,474 bp and it consists of 17 contigs. The GC content of the genome is 41.1%. The genome analysis identified a total of 10,910 plausible gene-coding regions in this strain.

Draft genome sequence data of the starch-utilizing yeast Saccharomycopsis fibuligera MBY1320 isolated from Nuruk.

Saccharomycopsis fibuligera MBY1320 was isolated from Nuruk, a traditional Korean starter for makgeolli (rice wine) production. This isolate has previously been reported to exhibit thermotolerance and starch-degrading activities. In this data description, we present the draft genome sequence of S. fibuligera MBY1320. The genome contained 19,138,941 bp in 16 contigs, and the internal transcribed spacer region of S. fibuligera MBY1320 rRNA gene showed the highest similarity with that of S. fibuligera NRRL Y-2388. The BioProject sequence has been deposited at DDBJ/EMBL/GenBank and Mendeley database. The GenBank accession numbers are PRJNA598085 for the BioProject data, SAMN13698230 for the BioSample data, and GCA_012062855 for the GenBank data.

Isolation of acid tolerant lactic acid bacteria and evaluation of α-glucosidase inhibitory activity.

In this study, lactic acid bacteria strains (LABs) were isolated from Korean traditional fermented food and examined as potential probiotics using in vitro methods. Ten LAB strains survived in de Man, Rogosa and Sharpe broth adjusted to pH 2.5 were tested for resistance to acidic conditions and bile, antimicrobial activity, and α-glucosidase inhibitory activity. Among them, strain MBEL1397 showed antimicrobial activity against Bacillus cereus and exhibited survival rates of over 97% in acidic and bile conditions. The α-glucosidase inhibitory activity was 3.91 ± 0.25%, corresponding to approximately 2.3 times higher than that of acarbose. MBEL1397 was susceptible to ampicillin, erythromycin, and penicillin G and identified as Lactobacillus sakei. It was deposited to Korean Collection for Type Culture (KCTC) as KCTC14037BP. In conclusion, these results demonstrate that L. sakei MBEL1397 might be prominent probiotics with potential hypoglycemic effects.

Complete genome sequence data of Lactobacillus sakei MBEL1397 isolated from kimchi.

Lactobacillus sakei MBEL1397(=KCTC14037BP) was isolated from kimchi, a traditional Korean fermented food, in Gangwon province, Republic of Korea. MBEL1397 is an acid-tolerant strain with antimicrobial activity and α-glucosidase inhibitory activity, which might be preliminary indications of its probiotic properties. Complete genome sequencing of L. sakei MBEL1397 was performed using the PacBio RSII platform. MBEL1397 has a 1,994,569 bp circular chromosome with 41.04% G+C content. The genome includes 1,946 protein-coding genes, 66 transfer RNA genes, and 21 ribosomal RNA genes. The BioProject has been deposited at DDBJ/EMBL/GenBank. The GenBank accession numbers are PRJNA598112 for the BioProject, SAMN13698554 for the BioSample, and CP048116 for the chromosome.

Complete genome sequence data of a broad-spectrum antipathogen, Bacillus amyloliquefaciens KCTC 18343P, isolated from Makgeolli, Korean traditional rice wine.

Bacillus amyloliquefaciens KCTC18343P(=MBE1283) isolated from Makgeolli, Korean traditional rice wine, strongly inhibits the growth of food and plant pathogens. A complete genome sequence of B. amyloliquefaciens KCTC18343P is presented in this report. The genome is 3,979,925 bp in size and harbors 3856 genes. The BioProject has been deposited at DDBJ/EMBL/GenBank. The GenBank accession numbers are PRJNA301202 for the BioProject, NZ_CP013727 for the chromosome, and NZ_CP013728 for the plasmid.

Changes in antioxidant activities and volatile compounds of mixed berry juice through fermentation by lactic acid bacteria.

The objectives of this study were to analyze antioxidant activities and identify volatile compounds in mixed berry juice after fermentation by lactic acid bacteria (LAB). Antioxidant activity of the mixed berry juice increased significantly from 209.57±2.93 to 268.30±1.75 µmol TE/g after 24 h of fermentation. After LAB fermentation, 34 volatile compounds were identified. Among them, three compounds-benzoic acid, benzaldehyde, and vitispirane-showed significant changes in their concentrations. Peak areas of benzoic acid and benzaldehyde, which are known to possess antioxidant activities, increased by 64 and 188%, respectively, after fermentation. However, the peak area of vitispirane, which is the most abundant terpene compound in berry juices, decreased by 92% after fermentation.

Isolation of thermotolerant yeast Pichia kudriavzevii from nuruk.

Thermotolerant yeast strains were isolated from nuruk, a traditional Korean fermentation starter in which variety of microorganisms are present. Among the isolates, the MBY1358 identified as yeast Pichia kudriavzevii showed significantly higher growth rate (0.59 ± 0.00 1/h) at 44 °C than other strains. Maximum ethanol concentration of 8.35 ± 0.03 g/L was obtained from 20 g/L glucose with yield of 0.44 ± 0.01 g/g at 44 °C, which is 1.14 times ethanol production of the control strain of P. kudriavzevii. The MBY1358, which was significantly more thermotolerant than the control strain and fermented 200 g/L glucose to 107.33 ± 5.03 g/L ethanol at 44 °C, was deposited to Korean Collection for Type Cultures (KCTC) under the accession number 27654.

Screening and Characterization of Potential Bacillus Starter Cultures for Fermenting Low-Salt Soybean Paste (Doenjang).

The bacterial strains were screened as potential starters for fermenting low-salt doenjang (a Korean traditional fermented soybean paste) using Korean doenjang based on proteolytic and antipathogenic activities under 6.5-7.5% NaCl conditions. Phylogenetic analysis based on 16S rRNA gene sequences showed that they all belonged to the genus Bacillus. Proteolytic and antipathogenic activities against Escherichia coli, Bacillus cereus, Staphylococcus aureus, Listeria monocytogenes, and Aspergillus flavus, as well as fibrinolytic, amylase, and cellulase activities of the 10 strains were quantitatively evaluated. Of these, strains D2-2, JJ-D34, and D12-5 were selected, based on their activities. The functional, phenotypic, and safety-related characteristics of these three strains were additionally investigated and strains D2-2 and D12-5, which lacked antibiotic resistance, were finally selected. Strains D2-2 and D12-5 produced poly-γ-glutamic acid and showed various enzyme activities, including α-glucosidase and ß-glucosidase. Growth properties of strains D2-2 and D12-5 included wide temperature and pH ranges, growth in up to 16% NaCl, and weak anaerobic growth, suggesting that they facilitate low-salt doenjang fermentation. Strains D2-2 and D12-5 were not hemolytic, carried no toxin genes, and did not produce biogenic amines. These results suggest that strains D2-2 and D12-5 can serve as appropriate starter cultures for fermenting low-salt doenjang with high quality and safety.

Isolation of the Inositol Phosphoceramide Synthase Gene (AUR1) from Stress-Tolerant Yeast Pichia kudriavzevii.

This study is the first report of the entire nucleotide sequence of an inositol phosphoceramide synthase gene from the stress-tolerant yeast Pichia kudriavzevii (PkAUR1). Sequence analysis revealed an open reading frame that spans 1,443 bp and encodes a 480-amino-acid-residue protein with the highest sequence similarity (41.7%) to Aur1 from Spathaspora passalidarum. A phenotypic assay with transformed S. cerevisiae and P. kudriavzevii indicated that two amino acid residues, Phe166 and Gly249, play crucial roles in the resistance to aureobasidin A, which is consistent with previous reports for other fungal Aur1s. The GenBank Accession No. for PkAUR1 is KP729614.

Optimization of Expression Conditions Enhances Production of Sepiapterin, a Precursor for Tetrahydrobiopterin Biosynthesis, in Recombinant Escherichia coli.

Sepiapterin is a precursor for the synthesis of tetrahydrobiopterin (BH4), which is a wellknown cofactor for aromatic amino acid hydroxylation and nitric oxide synthesis in higher mammals. In this study, a recombinant Escherichia coli BL21(DE3) strain harboring cyanobacterial guanosine 5'-triphosphate cyclohydrolase 1 (GCH1) and human 6- pyruvoyltetrahydropterin synthase (PTPS) genes was constructed to produce sepiapterin. The optimum conditions for T7 promoter-driven expression of GCH1 and PTPS were 30°C and 0.1 mM isopropyl-ß-D-thioglucopyranoside (IPTG). The maximum sepiapterin concentration of 88.1 ± 2.4 mg/l was obtained in a batch cultivation of the recombinant E. coli, corresponding to an 18-fold increase in sepiapterin production compared with the control condition (37°C and 1 mM IPTG).

Isolation of the Phosphoribosyl Anthranilate Isomerase Gene (TRP1) from Starch-Utilizing Yeast Saccharomycopsis fibuligera.

The nucleotide sequence of the TRP1 gene encoding phosphoribosyl anthranilate isomerase in yeast Saccharomycopsis fibuligera was determined by degenerate polymerase chain reaction and genome walking. Sequence analysis revealed the presence of an uninterrupted open-reading frame of 759 bp, including the stop codon, encoding a 252 amino acid residue. The deduced amino acid sequence of Trp1 in S. fibuligera was 43.5% homologous to that of Komagataella pastoris. The cloned TRP1 gene (SfTRP1) complemented the trp1 mutation in Saccharomyces cerevisiae, suggesting that it encodes a functional TRP1 in S. fibuligera. A new auxotrophic marker to engineer starch-degrading yeast S. fibuligera is now available. The GenBank Accession No. for SfTRP1 is KR078268.

Cysteine Protease Profiles of the Medicinal Plant Calotropis procera R. Br. revealed by de novo transcriptome analysis.

Calotropis procera R. Br., a traditional medicinal plant in India, is a promising source of commercial proteases, because the cysteine proteases from the plant exhibit high thermo-stability, broad pH optima, and plasma-clotting activity. Though several proteases such as Procerain, Procerain B, CpCp-1, CpCp-2, and CpCp-3 have been isolated and characterized, the information of their transcripts is limited to cDNAs encoding their mature peptides. Due to this limitation, in this study, to determine the cDNA sequences encoding full open reading frame of these cysteine proteases, transcripts were sequenced with an Illumina Hiseq2000 sequencer. A total of 171,253,393 clean reads were assembled into 106,093 contigs with an average length of 1,614 bp and an N50 of 2,703 bp, and 70,797 contigs with an average length of 1,565 bp and N50 of 2,082 bp using Trinity and Velvet-Oases software, respectively. Among these contigs, we found 20 unigenes related to papain-like cysteine proteases by BLASTX analysis against a non-redundant NCBI protein database. Our expression analysis revealed that the cysteine protease contains an N-terminal pro-peptide domain (inhibitor region), which is necessary for correct folding and proteolytic activity. It was evident that expression yields using an inducible T7 expression system in Escherichia coli were considerably higher with the pro-peptide domain than without the domain, which could contribute to molecular cloning of the Calotropis procera protease as an active form with correct folding.

Genome-wide screening of Saccharomyces cerevisiae genes regulated by vanillin.

During pretreatment of lignocellulosic biomass, a variety of fermentation inhibitors, including acetic acid and vanillin, are released. Using DNA microarray analysis, this study explored genes of the budding yeast Saccharomyces cerevisiae that respond to vanillin-induced stress. The expression of 273 genes was upregulated and that of 205 genes was downregulated under vanillin stress. Significantly induced genes included MCH2, SNG1, GPH1, and TMA10, whereas NOP2, UTP18, FUR1, and SPR1 were down regulated. Sequence analysis of the 5'-flanking region of upregulated genes suggested that vanillin might regulate gene expression in a stress response element (STRE)-dependent manner, in addition to a pathway that involved the transcription factor Yap1p. Retardation in the cell growth of mutant strains indicated that MCH2, SNG1, and GPH1 are intimately involved in vanillin stress response. Deletion of the genes whose expression levels were decreased under vanillin stress did not result in a notable change in S. cerevisiae growth under vanillin stress. This study will provide the basis for a better understanding of the stress response of the yeast S. cerevisiae to fermentation inhibitors.

Cloning of the transketolase gene from erythritol-producing yeast Candida magnoliae.

The entire nucleotide sequence of the TKL1 gene encoding transketolase (TKL) in an erythritolproducing yeast of Candida magnoliae was determined by degenerate polymerase chain reaction and genome walking. Sequence analysis revealed an open reading frame of C. magnoliae TKL1 (CmTKL1) that spans 2,088 bp and encodes 696 amino acids, sharing 61.7% amino acid identity to Kluyveromyces lactis TKL. Functional analysis showed that CmTKL1 complemented a Saccharomyces cerevisiae tkl1 tkl2 double mutant for growth in the absence of aromatic amino acids and restored transketolase activity in this mutant. An enzyme activity assay and RT-PCR revealed that the expression of CmTKL1 is induced by fructose, H2O2, and KCl. The GenBank accession number for C. magnoliae TKL1 is KF751756.

Enhanced production of ε-caprolactone by coexpression of bacterial hemoglobin gene in recombinant Escherichia coli expressing cyclohexanone monooxygenase gene.

Baeyer-Villiger (BV) oxidation of cyclohexanone to epsilon-caprolactone in a microbial system expressing cyclohexanone monooxygenase (CHMO) can be influenced by not only the efficient regeneration of NADPH but also a sufficient supply of oxygen. In this study, the bacterial hemoglobin gene from Vitreoscilla stercoraria (vhb) was introduced into the recombinant Escherichia coli expressing CHMO to investigate the effects of an oxygen-carrying protein on microbial BV oxidation of cyclohexanone. Coexpression of Vhb allowed the recombinant E. coli strain to produce a maximum epsilon-caprolactone concentration of 15.7 g/l in a fed-batch BV oxidation of cyclohexanone, which corresponded to a 43% improvement compared with the control strain expressing CHMO only under the same conditions.

Characterization of Acetobacter pomorum KJY8 isolated from Korean traditional vinegar.

Acetobacter sp. strains were isolated from traditional vinegar collected in Daegu city and Gyeongbuk province. The strain KJY8 showing a high acetic acid productivity was isolated and characterized by phenotypic, chemotaxonomic, and phylogenetic inference based on 16S rRNA sequence analysis. The chemotaxonomic and phylogenetic analyses revealed the isolate to be a strain of Acetobacter pomorum. The isolate showed a G+C content of 60.8 mol%. It contained LL-diaminopimelic acid (LL-A2pm) as the cell wall amino acid and ubiquinone Q9 (H6) as the major quinone. The predominant cellular fatty acids were C18:1w9c, w12t, and w7c. Strain KJY8 grew rapidly on glucose-yeast extract (GYC) agar and formed pale white colonies with smooth to rough surfaces. The optimum cultivation conditions for acetic acid production by the KJY8 strain were 20°C and pH 3.0, with an initial ethanol concentration of 9% (w/v) to produce an acetic acid concentration of 8% (w/v).

Overexpression, crystallization and preliminary X-ray crystallographic analysis of a putative xylose isomerase from Bacteroides thetaiotaomicron.

Bacteroides thetaiotaomicron BT0793, a putative xylose isomerase, was overexpressed in Escherichia coli, purified and crystallized using polyethylene glycol monomethyl ether 550 as the precipitant. X-ray diffraction data were collected to 2.10 Šresolution at 100 K using synchrotron X-rays. The crystal was found to belong to space group P1, with unit-cell parameters a=96.3, b=101.7, c=108.3 Å, α=82.8, ß=68.2, γ=83.0°. The asymmetric unit contained eight subunits of xylose isomerase with a crystal volume per protein weight (VM) of 2.38 Å3 Da(-1) and a solvent content of 48.3%.

Addition of ethanol to supercritical carbon dioxide enhances the inactivation of bacterial spores in the biofilm of Bacillus cereus.

Supercritical carbon dioxide (SC-CO2) was used to inactivate Bacillus cereus spores inside biofilms, which were grown on stainless steel. SC-CO2 treatment was tested using various conditions, such as pressure treatment (10-30 MPa), temperature (35-60°C), and time (10-120 min). B. cereus vegetative cells in the biofilm were completely inactivated by treatment with SC-CO2 at 10 MPa and at 35°C for 5 min. However, SC-CO2 alone did not inactivate spores in biofilm even after the treatment time was extended to 120 min. When ethanol was used as a cosolvent with SC-CO2 in the SC-CO2 treatment using only 2-10 ml of ethanol in 100ml of SC-CO2 vessel for 60-90 min of treatment time at 10 MPa and 60°C, B. cereus spores in the biofilm were found to be completely inactivated in the colony-forming test. We also assessed the viability of SC-CO2-treated bacterial spores and vegetative cells in the biofilm by staining with SYTO 9 and propidium iodide. The membrane integrity of the vegetative cells was completely lost, while the integrity of the membrane was still maintained in most spores. However, when SC-CO2 along with ethanol was used, both vegetative cells and spores lost their membrane integrity, indicating that the use of ethanol as a cosolvent with SC-CO2 is efficient in inactivating the bacterial spores in the biofilm.

Growth and fermentation characteristics of Saccharomyces cerevisiae NK28 isolated from kiwi fruit.

The influences of glucose concentration, initial medium acidity (pH), and temperature on the growth and ethanol production of Saccharomyces cerevisiae NK28, which was isolated from kiwi fruit, were examined in shake flask cultures. The optimal glucose concentration, initial medium pH, and temperature for ethanol production were 200 g/l, pH 6.0, and 35oC, respectively. Under this growth condition, S. cerevisiae NK28 produced 98.9 ± 5.67 g/l ethanol in 24 h with a volumetric ethanol production rate of 4.12 ± 0.24 g/l·h. S. cerevisiae NK28 was more tolerant to heat and ethanol than laboratory strain S. cerevisiae BY4742, and its tolerance to ethanol and fermentation inhibitors was comparable to that of an ethanologen, S. cerevisiae D5A.