• Applied Biological Chemistry
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• v.39 no.4
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• pp.255-259
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• 1996

In order to improve the quality of commercially manufactured Doenjang, yeast florae, gas and alcohol formation during fermentation of Doenjang were periodically examined. Candida rugosa, Candida zeylanoides, Pichia farinosa Saccharomyces cerevisiae and Zygosaccharomyces rouxii were isolated and identified from Doenjang at various fermentation stage. S. cerevisiae and Z. rouxii showed distintive gas and alcohol formation activities and the distribution ratio of Z. rouxii was 26% at 14 days and 76% as prevailed yeast strain after 30 days fermentation, respectively. Ethanol content of Doenjang was gradually increased into 2.19% at final stage of fermentation. The amount of gas generated during fermentation was 9.75 ml/g after 14 days, 4.5 ml/g after 30 days and decreased into negligible amount after 45 days fermentation. These inhibitory effects on gas generation by fermentation period would be ascribed to the ethanol Produced for fermentation. This results suggest that gas generation in commercially manufactured Doenjang could be eliminated through the effective control of fermentation by yeast without application of any preservatives.

• Preventive Nutrition and Food Science
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• v.21 no.3
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• pp.221-226
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• 2016

A new type of doenjang was manufactured by mixing soaked soybean, koji (Rhizopus oryzae), cheonggukjang (Bacillus amyloliquefaciens MJ1-4 and B. amyloliquefaciens EMD17), and Pichia farinosa SY80 as a yeast, salt, and water, followed by fermentation with koji that was made by fermenting whole wheat with R. oryzae. The mixed culture doenjang was designed to have a more palatable flavor and stronger biological activities than the conventional product. The extract of mixed culture doenjang showed higher antioxidant activity than the commercial doenjang as evaluated by the ferric reducing antioxidant power assay although it was not significantly different from the commercial product in 2,2-diphenyl-1-picrylhydrazyl and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) radical scavenging activities. Further, the mixed culture doenjang reduced intracellular reactive oxygen species levels and protected cells from glutamate-induced cytotoxicity more efficiently in human hippocampal HT22 neuroblastoma cells than the commercial doenjang. In conclusion, a newly-developed mixed culture doenjang had a strong antioxidant activity in vitro and cultured cell model systems, exhibited a potential to prevent oxidative stress-associated disorders although animal and clinical studies are needed to confirm its in vivo efficacy.

• Journal of Microbiology and Biotechnology
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• v.22 no.3
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• pp.331-338
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• 2012

A novel gene coding for an endo-${\beta}$-1,4-mannanase (manA) from Bacillus subtilis strain G1 was cloned and overexpressed in P. pastoris GS115, and the enzyme was purified and characterized. The manA gene consisted of an open reading frame of 1,092 nucleotides, encoding a 364-aa protein, with a predicted molecular mass of 41 kDa. The ${\beta}$-mannanase showed an identity of 90.2-92.9% ${\leq}95%$) with the corresponding amino acid sequences from B. subtilis strains deposited in GenBank. The purified ${\beta}$-mannanase was a monomeric protein on SDS-PAGE with a specific activity of 2,718 U/mg and identified by MALDI-TOF mass spectrometry. The recombinant ${\beta}$-mannanase had an optimum temperature of $45^{\circ}C$ and optimum pH of 6.5. The enzyme was stable at temperatures up to $50^{\circ}C$ (for 8 h) and in the pH range of 5-9. EDTA and most tested metal ions showed a slightly to an obviously inhibitory effect on enzyme activity, whereas metal ions ($Hg^{2+}$, $Pb^{2+}$, and $Co^{2+}$) substantially inhibited the recombinant ${\beta}$-mannanase. The chemical additives including detergents (Triton X-100, Tween 20, and SDS) and organic solvents (methanol, ethanol, n-butanol, and acetone) decreased the enzyme activity, and especially no enzyme activity was observed by addition of SDS at the concentrations of 0.25-1.0% (w/v) or n-butanol at the concentrations of 20-30% (v/v). These results suggested that the ${\beta}$-mannanase expressed in P. pastoris could potentially be used as an additive in the feed for monogastric animals.

• BMB Reports
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• v.40 no.1
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• pp.22-28
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• 2007

MyoD, expressed in skeletal muscle lineages of vertebrate embryo, is one of muscle-specific basic helix-loop-helix (bHLH) transcription factors, which plays a key role in the determination and differentiation of all skeletal muscle lineages. In this study, a cDNA of grass carp MyoD was cloned and characterized from total RNA of grass carp embryos by RT-PCR. The full-length cDNA of grass carp MyoD is 1597 bp. The cDNA sequence analysis reveals an open reading frame of 825 bp coding for a protein of 275 amino acids, which includes a bHLH domain composed of basic domain (1-84th amino acids) and HLH domain (98-142th amino acids), without signal peptide. Then the MyoD cDNA of grass carp was cloned to yeast expression vector pPICZ$\alpha$A and transformed into P. pastoris GS115 strain, the recombinant MyoD protein with a molecular weight of about 31KD was obtained after inducing for 2d with 0.5% methanol in pH 8.0 BMGY medium, and the maximum yield was about 250 mg/L in shaking-flask fermentation. The results were expected to benefit for further studies on the crystal structure and physiological function of fish MyoD.

• Mycobiology
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• v.43 no.3
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• pp.280-287
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• 2015

In this study, transcriptome analysis of twelve laccase genes in Pleurotus ostreatus revealed that their expression was differentially regulated at different developmental stages. Lacc5 and Lacc12 were specifically expressed in fruiting bodies and primordia, respectively, whereas Lacc6 was expressed at all developmental stages. Lacc1 and Lacc3 were specific to the mycelial stage in solid medium. In order to investigate their biochemical characteristics, these laccases were heterologously expressed in Pichia pastoris using the pPICHOLI-2 expression vector. Expression of the laccases was facilitated by intermittent addition of methanol as an inducer and sole carbon source, in order to reduce the toxic effects associated with high methanol concentration. The highest expression was observed when the recombinant yeast cells were grown for 5 days at $15^{\circ}C$ with intermittent addition of 1% methanol at a 12-hr interval. Investigation of enzyme kinetics using 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) as a substrate revealed that the primordium-specific laccase Lacc12 was 5.4-fold less active than Lacc6 at low substrate concentration with respect to ABTS oxidation activity. The optimal pH and temperature of Lacc12 were 0.5 pH units and $5^{\circ}C$higher than those of Lacc6. Lacc12 showed maximal activity at pH 3.5 and $50^{\circ}C$, which may reflect the physiological conditions at the primordiation stage.

• Food Science of Animal Resources
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• v.27 no.4
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• pp.538-542
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• 2007

This study was carried out to investigate the toxicity of exopolysaccharides (EPS) from kefir toward MA104 cells and evaluate the inhibitory effects of kefir EPS on rotavirus infection. The results obtained are summarized as follows: Lactic acid bacteria (Lactobacillus fermentum, L. acidophilus, L. brevis) and yeasts (Candida kefyr, Cryptococcus albidus, Pichia ohmeri) were isolated and identified from kefir grain and culture. At 1% EPS, the inhibitory effects of EPS on the infection of MA-104 cells using the MTT assay were $72.52{\pm}6.48%$ for human rotavirus (KU), $36.06{\pm}7.63%$ for bovine rotavirus (NCDV), and $81.66{\pm}1.11%$ for porcine rotavirus (OSU). At 1/128% EPS, the effects were $24.98{\pm}4.58%$ for human rotavirus (KU), $4.71{\pm}6.16%$ for bovine rotavirus (NCDV), and $4.05{\pm}14.90%$ forporcine rotavirus (OSU). EPS isolated from kefir have inhibitory effects on rotaviruses of various serotypes and rotaviruses from different animals.

• Journal of Microbiology and Biotechnology
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• v.31 no.1
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• pp.163-170
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• 2021

Enzyme replacement therapy for lysosomal storage diseases usually requires recombinant enzymes containing mannose-6-phosphate (M6P) glycans for cellular uptake and lysosomal targeting. For the first time, a strategy is established here for the in vitro mannosyl-phosphorylation of high-mannose type N-glycans that utilizes a recombinant Mnn14 protein derived from Saccharomyces cerevisiae. Among a series of N-terminal- or C-terminal-deleted recombinant Mnn14 proteins expressed in Pichia pastoris, rMnn1477-935 with deletion of N-terminal 76 amino acids spanning the transmembrane domain (46 amino acids) and part of the stem region (30 amino acids), showed the highest level of mannosyl-phosphorylation activity. The optimum reaction conditions for rMnn1477-935 were determined through enzyme assays with a high-mannose type N-glycan (Man8GlcNAc2) as a substrate. In addition, rMnn1477-935 was shown to mannosyl-phosphorylate high-mannose type N-glycans (Man7-9GlcNAc2) on recombinant human lysosomal alpha-glucosidase (rhGAA) with remarkably high efficiency. Moreover, the majority of the resulting mannosyl-phosphorylated glycans were bis-form which can be converted to bis-phosphorylated M6P glycans having a superior lysosomal targeting capability. An in vitro N-glycan mannosyl-phosphorylation reaction using rMnn1477-935 will provide a flexible and straightforward method to increase the M6P glycan content for the generation of "Biobetter" therapeutic enzymes.

• Journal of the Korean Society of Food Culture
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• v.25 no.1
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• pp.61-69
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• 2010

This study was carried out to investigate the effects of various kinds of commercial salts, including sun-dried (Korea), purified, and traditional salts on the chemical and sensory properties and growth of microorganisms involved in kimchi fermentation. Kimchi was prepared by salting in 10% NaCl solution for 2 hours followed by addition of other spices and fermentation at $20^{\circ}C$. The decreases in pH suggested that kimchi fermentation can be classified into 3 steps: initial, intermediate, and final stages. In texture analysis, the hardness and fracturability of traditional salt kimchi were higher than those of regular kimchi. From the sensory evaluation test for kimchi, sensory scores were high for traditional salt addition, especially taste, overall preference and texture. Among various microorganisms related to kimchi fermentation, the growth of Leuconostoc mesenteroides, Lactobacillus plantarum, Pichia membranaefaciens and Escherichia coli were examined. Based on the conditions of kimchi fermentation, a 2% and 5% concentration of each salt were studied. Also, the conditions of the cultures at $37^{\circ}C$ were examined. There was no considerable difference in the growth of Leuconostoc mesenteroides, Lactobacillus plantarum, Escherichia coli in the different kinds of salts. However, the growth of Pichia membranaefaciens was strongly inhibited by a 5% concentration of traditional salt during incubation at $37^{\circ}C$.

• Applied Biological Chemistry
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• v.13 no.2
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• pp.171-180
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• 1970

The yeasts in the soy sauce mash were isolated and identified, and they were classified by coloring with the treatment of TTC(2, 3, 5, triphenyltetrazolium chloride) agar and counted in process of time. The results obtained were as follows: a) The number of ordinary and osmophilic yeasts in 1 ml. of the soy sauce mash showed a tendency to be increased from the mashing to the mature stages and to decrease in the aging stages: $127{\times}10^3$ immediately after mashing, $83{\times}10^3$ 1 month after, $356{\times}10^3$ 3 months after, $1250{\times}10^3$ 6 months after and $65{\times}10^3$ 2 years after mashing in the case of ordinary yeasts, and 0 after mashing, $40{\times}10^3$ 1 month after, $81{\times}10^3$ 3 months after, $358{\times}10^3$ 6 month after and $23{\times}10^3$ 2 years after mashing in the case of osmophilic yeasts. b) 50 strains of yeasts were isolated from the soy sauce mash optionally in process of fermentation period, and they were identified as 7 genera and 18 species: 10 strains of Saccharomyces rouxii, 1 strain of Saccharomyces marxianus, 3 strains of Saccharomyces rosei, 1 strain of Saccharomyces fermentati, 6 strains of Saccharomyces mellis, 1 strain of Saccharomyces acidifaciens, 1 strain of Saccharomyces pastori, 3 strains of Pichia polymorpha, 2 strains of Hansenula anomala, 1 strain of Hansenula saturnus, 2 strains of Hansenula suaveolens, 5 strains of Nadsonia fulvescens, 8 strains of Debaryomyces hasenii, 1 strain of Debaryomyces nicotianae, 1 strain of Debaryomyces kloeckeri, 2 strains of Torulopsis sake, 1 strain of Torulopsis holmii and 1 strain of Candida pelliculasa. c) Distribution of yeasts according to the fermentation period was as follows: i) Saccharomyces rouxii, Saccharomyces marxianus, Saccharoymces rosei, Pichia polymorpha, Debaryomyces hansenii, Torulopsis sake, Candida pelliculosa, Debaryomyces nicotianae, Nadsonia fulvescens, Hansenula suaveolens and Hansenula saturnus were found in the early stages of fermentation. ii) Saccharomyces rouxii, Saccharomyces rosei, Saccharomyces fermentati, Saccharomyces mellis, Saccharomyces pastori, Hansenula anomala, Saccharomyces acidifaciens and Debaryomyces hansenii appeared in the mature stages. iii) Saccharomyces rouxii, Saccharomyces mellis, Nadsonia fulvescenes, Dedaryomyces hansenii, Debaryomyces kloeckeri, Torulopsis sake and Torulopsis holmii were distributed in the aging stages. d) TTC white yeasts were found in abundance in the early stages of fermentation and TTC red yeasts appeared more than 50 per cent in the mature and aging stages. e) The yeasts belonging to Saccharomyces mellis and Saccharomyces pastori were classified as TTC red yeasts, Saccharomyces acidifaciens were reel pink, Hansenula saturnus Debaryomyces kloeckeri, and Torulopsis holmii were pink, Saccharomyces marxianus and Nadsonia fulvescens were white and the others were the same as the description in the previous report. Saccharomyces rouxii ware classified for the most part as TTC red yeasts, and while some of them were red pink. f) Species of yeasts in the soy sauce mash were similar to those in the soy sauce koji, but the latter were not osmophilic and in the former case, the osmophilic yeasts were increased in process of fermentation period.

• Food Science and Preservation
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• v.24 no.3
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• pp.472-482
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• 2017

To improve functionality and palatability of Korean Campbell Early wine. Campbell Early and aronia were fermented by either individually or at 5:5 (v/v) mixed culture of Saccharomyces cerevisiae Fermivin and Pichia anomala JK04. Blending was carried out using those two wines with different mixing ratio. Antioxidant activity analysis and sensory evaluation of blending wines were conducted. The Campbell Early wine and aronia wine blended with 9:1 (v/v) ratio showed excellent antioxidant activity and sensory scores. Total anthocyanin compound, DPPH radical scavenging activity and total phenolic compound of blending wines were higher than those of Campbell Early wine (control). Hue and intensity values increased in the order of A, B, C and D, E, F depending on P. anomala JK04 use. Anonia wine contributed the increase in a and b values of blending wine. Although blending wines fermented by P. anomala JK04 increased small amounts of aldehyde and acid compound, ester compound, the most important factor for wine aroma was also increased sharply. Adding aronia wine fermented by single culture of P. anomala JK04 (A, D) got higher color, taste, sourness and overall preference scores than other wines in the sensory evaluation. All of blending wines showed higher flavor scores than control did. This research shows a possibility of blending and utilizing non-Saccharomyces yeast for Korean wine industry.