• Applied Biological Chemistry
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• v.10
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• pp.69-100
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• 1968

1. In order to investigate on the microflora and enzyme activity of mold wheat 'Nuruk' , the major source of microorganisms for the brewing of Takju (a Korean Sake), two samples of Nuruk, one prepared at the College of Agriculture, Chung Nam University (S) and the other perchased at a market (T), were taken for the study. The molds, aerobic bacteria, lactic acid bacteria, and yeasts were examined and counted. The yeasts were classified by the treatment with TTC (2, 3, 5 triphenyltetrazolium chloride) agar that yields a varied shade of color. The amylase and protease activities of Nuruk were measured. The results were as the followings. a) In the Nuruk S found were: Aspergillus oryzae group, $204{\times}10^5$; Black Aspergilli, $163{\times}10^5$; Rhizogus, $20{\times}10^5$; Penicillia, $134{\times}10^5$; Areobic bacteria, $9{\times}10^6-2{\times}10^7$; Lactic acid bacteria, $3{\times}10^4$ In the Nuruk T found were: Aspergillus oryzae group, $836{\times}10^5$; Black Aspergilli, $286{\times}10^5$; Rhizopus, $623{\times}10^5$; Penicillia, $264{\times}10^5$; Aerobic bacteria, $5{\times}10^6-9{\times}10^6$; Lactic acid bacteria, $3{\times}10^4$ b) Eighty to ninety percent of the aerobic bacteria in Nuruk S appeared to belong to Bacillus subtilis while about 70% of those in Nuruk T seemed to be spherical bacteria. In both Nuruks about 80% of lactic acid bacteria were observed as spherical ones. c) The population of yeasts in 1g. of Nuruk S was about $6{\times}10^5$, 56.5% of which were TTC pink yeasts, 16% of which were TTC red pink yeasts, 8% of which were TTC red yeasts, 19.5% of which were TTC white yeasts. In Nuruk T(1g) the number of yeasts accounted for $14{\times}10^4$ and constituted of 42% TTC pink. 21% TTC red pink 28% TTC red and 9% TTC white. d) The enzyme activity of 1g Nuruk S was: Liquefying type Amylase, $D^{40}/_{30},=256$ W.V. Saccharifying type Amylase, 43.32 A.U. Acid protease, 181 C.F.U. Alkaline protease, 240C.F.U. The enzyme activity of 1g Nuruk T was: Liquefying type Amylase $D^{40}/_{30},=32$ W.V. Saccharifying type amylase $^{30}34.92$ A.U. Acid protease, 138 C.F.U. Alkaline protease 31 C.F.U. 2. During the fermentation of 'Takju' employing the Nuruks S and T the microflora and enzyme activity throughout the brewing were observed in 12 hour intervals. TTC pink and red yeasts considered to be the major yeasts were isolated and cultured. The strains ($1{\times}10^6/ml$) were added to the mashes S and T in which pH was adjusted to 4.2 and the change of microflora was examined during the fermentation. The results were: a) The molds disappeared from each sample plot since 2 to 3 days after mashing while the population of aerobic bacteria was found to be $10{\times}10^7-35{\times}10^7/ml$ inS plots and $8.2{\times}10^7-12{\times}10^7$ in plots. Among them the coccus propagated substantially until some 30 hours elasped in the S and T plots treated with lactic acid but decreased abruptly thereafter. In the plots of SP. SR. TP. and TR the coccus had not appeared from the beginning while the bacillus showed up and down changes in number and diminished by 1/5-1/10 the original at the end stage. b) The lactic acid bacteria observed in the S plot were about $7.4{\times}10^7$ in number per ml of the mash in 24 hours and increased up to around $2{\times}10^8$ until 3-4 days since. After this period the population decreased rapidly and reached about $4{\times}10^5$ at the end, In the plot T the lactic acid becteria found were about $3{\times}10^8$ at the period of 24 fours, about $3{\times}10$ in 3 days and about $2{\times}10^5$ at the end in number. In the plots SP. SR. TP, and TR the lactic acid bacteria observed were as less as $4{\times}10^5$ at the stage of 24 hours and after this period the organisms either remained unchanged in population or ceased to exist. c) The maiority of lactic acid bacteria found in each mash were spherical and the change in number displayed a tendency in accordance with the amount of lactic acid and alcohol produced in the mash. d) The yeasts had showed a marked propagation since the period of 24 hours when the number was about $2{\times}10^8$ ㎖ mash in the plot S. $4{\times}10^8$ in 48 hours and $5-7{\times}10^8$ in the end period were observed. In the plot T the number was $4{\times}10^8$ in 24 hours and thereafter changed up and down maintaining $2-5{\times}10^8$ in the range. e) Over 90% of the yeasts found in the mashes of S and T plots were TTC pink type while both TTC red pink and TTC red types held range of $2{\times}10-3{\times}10^7$ throughout the entire fermentation. f) The population of TTC pink yeasts in the plot SP was as $5{\times}10^8$ much as that is, twice of that of S plot at the period of 24 hours. The predominance in number continued until the middle and later stages but the order of number became about the same at the end. g) Total number of the yeasts observed in the plot SR showed little difference from that of the plot SP. The TTC red yeasts added appeared considerably in the early stage but days after the change in number was about the same as that of the plot S. In the plot TR the population of TTC red yeasts was predominant over the T plot in the early stage which there was no difference between two plots there after. For this reason even in the plot w hers TTC red yeasts were added TTC pink yeasts were predominant. TTC red yeasts observed in the present experiment showed continuing growth until the later stage but the rate was low. h) In the plot TP TTC pink yeasts were found to be about $5{\times}10^8$ in number at the period of 2 days and inclined to decrease thereafter. Compared with the plot T the number of TTC pink yeasts in the plot TP was predominant until the middle stage but became at the later stage. i) The productivity of alcohol in the mash was measured. The plot where TTC pink yeasts were added showed somewhat better yield in the earely stage but at and after the middle stage the difference between the yeast-added and the intact mashes was not recognizable. And the production of alcohol was not proportional to the total number of yeasts present. j) Activity of the liquefying amylase was the highest until 12 hours after mashing, somewhat lowered once after that, and again increased around 36-48 hours after mashing. Then the activity had decreased continuously. Activity of saccharifying amylase also decreased at the period of 24 hours and then increased until 48 hours when it reached the maximum. Since, the activity had gradually decreased until 72 hours and rapidly so did thereafter. k) Activity of alkaline protease during the fermentation of mash showed a tendency to decrease continusously although somewhat irregular. Activity of acid protease increased until hours at the maximum, then decreased rapidly, and again increased, the vigor of acid protease showed better shape than that of alkaline protease throughout. 3. TTC pink yeasts that were predominant in number, two strains of TTC red pink yeasts that appeared throughout the brewing, and TTC red yeasts were identified and the physiological characters examined. The results were as described below. a) TTC pinkyeasts (B-50P) and two strains of TTC red pink yeasts (B-54 RP & B-60 RP) w ere identified as the type of Saccharomyces cerevisiae and TTC pink red yeasts CB-53 R) were as the type of Hansenula subpelliculosa. b) The fermentability of four strains above mentioned were measured as follows. Two strains of TTC red pink yeasts were the highest, TTC pink yeasts were the lowest in the fermantability. The former three strains were active in the early stage of fermentation and found to be suitable for manufacturing 'Takju' TTC red yeasts were found to play an important role in Takju brewing due to its strong ability to produce esters although its fermentability was low. c) The tolerance against nitrous acid of strains of yeast was marked. That against lactic acid was only 3% in Koji extract, and TTC red yeasts showed somewhat stronger resistance. The tolerance against alcohol of TTC pink and red pink yeasts in the Hayduck solution was 7% while that in the malt extract was 13%. However, that of TTC red yeasts was much weaker than others. Liguefying activity of gelatin by those four strains of yeast was not recognized even in 40 days. 4. Fermentability during Takju brewing was shown in the first two days as much as 70-80% of total fermentation and around 90% of fermentation proceeded in 3-4 days. The main fermentation appeared to be completed during :his period. Productivity of alcohol during Takju brewing was found to be apporximately 65% of the total amount of starch put in mashing. 5. The reason that Saccharomyces coreanuss found be Saito in the mash of Takju was not detected in the present experiment is considered due to the facts that Aspergillus oryzae has been inoculated in the mold wheat (Nuruk) since around 1930 and also that Koji has been used in Takju brewing, consequently causing they complete change in microflora in the Takju brewing. This consideration will be supported by the fact that the original flavor and taste have now been remarkably changed.

• Microbiology and Biotechnology Letters
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• v.41 no.1
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• pp.17-25
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• 2013

The thermotolerant methylotrophic yeast Hansenula polymorpha is an attractive model organism for various fundamental studies, such as the genetic control of enzymes involved in methanol metabolism, peroxisome biogenesis, nitrate assimilation, and resistance to heavy metals and oxidative stresses. In addition, H. polymorpha has been highlighted as a promising recombinant protein expression host, especially due to the availability of strong and tightly regulatable promoters. In this study, we investigated the possibility of employing human serum albumin (HSA) as the fusion tag for the secretory expression of heterologous proteins in H. polymorpha. A set of four expression cassettes, which contained the methanol oxidase (MOX) promoter, translational HSA fusion tag, and the terminator of MOX, were constructed. The expression cassettes were also designed to contain sequences for accessory elements including His8-tag, $2{\times}(Gly_4Ser_1)$ linkers, tobacco etch virus protease recognition sites (Tev), multi-cloning sites, and strep-tags. To determine the effects of the size of the HSA fusion tag on the secretory expression of the target protein, each cassette contained the HSA gene fragment truncated at a specific position based on its domain structure. By using the Green fluorescence protein gene as the reporter, the properties of each expression cassette were compared in various conditions. Our results suggest that the translational HSA fusion tag is an efficient tool for the secretory expression of recombinant proteins in H. polymorpha.

• Journal of Microbiology and Biotechnology
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• v.10 no.3
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• pp.349-354
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• 2000

Abstract A facultative alkalophilic gram-negative Vibrio metschnikovii strain RH530, isolated from the wastewater, produced several alkaline proteases (VAP) including six alkaline serine proteases and a metalloprotease. From this strain, high yielding YAP mutants were isolated by NTG treatment. The isolated mutant KS1 showed nine times more activity than the wild-type after optimization of the culture media. The production was regulated by catabolite repression when glucose was added to the medium. The effects of several organic nitrogen sources on the production of the YAP were investigated to avoid catabolite repression. The combination of 4% wheat gluten meal (WGM), 1.5% cotton seed flour (eSF), and 5% soybean meal (SBM) resulted in the best production when supplemented with 1% NaCl. The YAP showed a resistance to surfactants such as $sodium-{\alpha}-olefin$ sulfonate (AOS), polyoxy ethylene oxide (POE), and sodium dodecyl sulfate (SDS), yet not to linear alkylbenzene sulfonate (LAS). However, the activity of the YAP was restored completely when incubated with LAS in the presence of POE or $Na_2SO_4$. The YAP was stable in a liquid laundry detergent containing 6.6% SLES (sodium lauryl ether sulfate), 6.6% LAS, 19.8% POE, and stabilizing agents for more than two weeks at $40^{\circ}C$, but the stability was sharply decreased even after 1 day when incubated at $60^{\circ}C$. A washing performance test with the YAP exhibited it to be a good washing power by showing 51 % and 60% activity at $25^{\circ}C{\;}and{\;}40^{\circ}C$, respectively, thereby indicating that the YAP also has a good detergency at a low temperature. All the results suggest that the YAP produced from the mutant strain KSI has suitable properties for use in laundry detergents.rgents.

• Korean Journal of Microbiology
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• v.49 no.3
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• pp.275-281
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• 2013

Probiotics have been reported to benefit human health by modulating immunity, lowering cholesterol, improving lactose tolerance, and preventing some cancer. Once ingested, probiotic microorganisms have to survive harsh conditions such as low pH, protease-rich condition, and bile salts during their passage through the gastro-intestinal (GI) tract colonize and proliferate to exert their probiotic effects. The dual coating technology, by which the bacteria are doubly coated with peptides and polysaccharides in consecutive order, was developed to protect the ingested bacteria from the harsh conditions. The aim of the study was to evaluate the viable stability of a doubly coated blend of four species of Bifidobacterium by comparing its bile/acid resistance and heat viability in vitro with that of the non-coated blend. After challenges with acid, bile salts, heat, and viable cell counts (VVCs) of the dual coated and non-coated blend were determined by cultivation on agar plates or flow cytometric measurement after being stain with the BacLigtht kit$^{TM}$. The results showed that the dual coated blend was much higher resistant to the acidic or bile salt condition than the non-coated blend and heat viability was also higher, indicating that the dual coating can improve the survival of probiotic bacteria during their transit through the GI tract after consumption.

• The Korean Journal of Pesticide Science
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• v.14 no.4
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• pp.421-426
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• 2010

Many bacterial strains inhabit strong saline condition, such as tidal mudflat and salted seafoods, were identified and reported for the proposed protease activities and salt resistance; however antifungal activities against plant fungal pathogen have not well been studied until now. In this study, primary screening was performed for the isolation of promising strains against major plant pathogens like Sclerotinia sclerotiorum, Fusarium oxysporum, Phytophthora capsici, Botrytis cineria, Collectotrichum acutatum and Pythium ultimum. Totally 423 bacterial strain were isolated from laboratory media which was based on different morphological characteristics and all the strains were dual cultured against major fungal pathogens on PDA, finally 40 strains were selected as antifungal bacterial strain and identified by fatty acid phylogenic difference analysis from MIDI shorlock gas chromatography system. As a result, antifungal strains from tidal mudflat were 10 species of 6 genus. Paenibacillus macerans was dominant species; 5 strains among the 17 isolates from tidal mudflat. Antifungal strains from salted seafoods were 7 species of 3 genus and Bacillus atrophaeus was dominant species; 12 strains among the 23 isolates from salted fishes.

• Journal of Microbiology and Biotechnology
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• v.21 no.8
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• pp.861-868
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• 2011

A xylanase gene, xyn7c, was cloned from Paenibacillus sp. 12-11, an alkalophilic strain isolated from the alkaline wastewater sludge of a paper mill, and expressed in Escherichia coli. The full-length gene consists of 1,296 bp and encodes a mature protein of 400 residues (excluding the putative signal peptide) that belongs to the glycoside hydrolase family 10. The optimal pH of the purified recombinant XYN7C was found to be 8.0, and the enzyme had good pH adaptability at 6.5-8.5 and stability over a broad pH range of 5.0-11.0. XYN7C exhibited maximum activity at $55^{\circ}C$ and was thermostable at $50^{\circ}C$ and below. Using wheat arabinoxylan as the substrate, XYN7C had a high specific activity of 1,886 U/mg, and the apparent $K_m$ and $V_{max}$ values were 1.18 mg/ml and 1,961 ${\mu}mol$/mg/min, respectively. XYN7C also had substrate specificity towards various xylans, and was highly resistant to neutral proteases. The main hydrolysis products of xylans were xylose and xylobiose. These properties make XYN7C a promising candidate to be used in biobleaching, baking, and cotton scouring processes.

• Journal of Life Science
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• v.32 no.9
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• pp.734-741
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• 2022

Currently, around 40 million people worldwide are living with human immunodeficiency virus (HIV) infection making HIV a critical global health risk. Present therapies for HIV infection consist of drug cocktails that target different steps of the HIV life cycle to prevent infection, replication, and release of the virus. Due to its mutating nature, drug resistance coupled with side-effects of long-term drug use, novel strategies, and pharmaceuticals to treat and manage HIV infection are constant needs and continuously being studied. Plants allocate a major repertoire of chemical diversity and are therefore regarded as an important source of new bioactive agents that can be utilized against HIV. Since the early 1990s, upon recommendations of the World Health Organization, numerous studies reported phytochemicals from different structural classes such as flavonoids, coumarins, tannins and terpenes with strong inhibitory effects against HIV infection. The present review gathered and presented recent research (2021-present) on plant extracts and phytochemicals that exhibit anti-HIV properties with the aim of providing insights into future studies where ethnomedical and underutilized plant sources may yield important natural products against HIV. Considering the relation and importance of HIV treatment with current viral infection risks such as SARS-CoV-2, screening plants for anti-HIV agents is an important step towards the discovery of novel antivirals.

• Journal of Ginseng Research
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• v.47 no.1
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• pp.123-132
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• 2023

Background: Pseudotyped virus systems that incorporate viral proteins have been widely employed for the rapid determination of the effectiveness and neutralizing activity of drug and vaccine candidates in biosafety level 2 facilities. We report an efficient method for producing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pseudovirus with dual luciferase and fluorescent protein reporters. Moreover, using the established method, we also aimed to investigate whether Korean Red Ginseng (KRG), a valuable Korean herbal medicine, can attenuate infectivity of the pseudotyped virus. Methods: A pseudovirus of SARS-CoV-2 (SARS-2pv) was constructed and efficiently produced using lentivirus vector systems available in the public domain by the introduction of critical mutations in the cytoplasmic tail of the spike protein. KRG extract was dose-dependently treated to Calu-3 cells during SARS2-pv treatment to evaluate the protective activity against SARS-CoV-2. Results: The use of Calu-3 cells or the expression of angiotensin-converting enzyme 2 (ACE2) in HEK293T cells enabled SARS-2pv infection of host cells. Coexpression of transmembrane protease serine subtype 2 (TMPRSS2), which is the activator of spike protein, with ACE2 dramatically elevated luciferase activity, confirming the importance of the TMPRSS2-mediated pathway during SARS-CoV-2 entry. Our pseudovirus assay also revealed that KRG elicited resistance to SARS-CoV-2 infection in lung cells, suggesting its beneficial health effect. Conclusion: The method demonstrated the production of SARS-2pv for the analysis of vaccine or drug candidates. When KRG was assessed by the method, it protected host cells from coronavirus infection. Further studies will be followed for demonstrating this potential benefit.

• Korean Journal of Microbiology
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• v.49 no.1
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• pp.64-70
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• 2013

The greenhouse whitefly, Trialeurodes vaporariorum, is an economically important pest for greenhouse crops because they cause direct damage by feeding on plant nutrients and indirect damage as transmits many virus vectors. It has recently become a serious problem because of the continuous use of insecticide resulting in resistance among greenhouse whitefly population. To overcome these problems, in this study, the biological characteristics and virulence of an entomopathogenic fungus isolated from the cadaver of nymph greenhouse whitefly were investigated. Isolated fungus was identified as Isaria fumosorosea by morphological examinations and genetic identification using sequences of the ITS, ${\beta}$-tubulin, and EF1-${\alpha}$ regions. This fungus was named as I. fumosorosea SDTv and tested for the virulence against nymphs T. vaporariorum and the cold activity, the thermotolerance and the stability of UV-B irradiation on conidia. Mortality rate of greenhouse whitefly showed from 84 to 100% and the virulence increased with increasing conidial concentrations, $1{\times}10^5$ to $10^8$ conidia/ml. Conidia were stable at $35^{\circ}C$, 0.1 $J/cm^2$ of UV irradiation and germinated after 8 days at $4^{\circ}C$. Additionally, the activities of chitinases and proteases produced by I. fumosorosea SDTv were varied according to the medium. In conclusion, I. fumosorosea SDTv which showed high mortality rate against greenhouse whitefly will be used effectively in the integrated pest management programs against the greenhouse whitefly.