• Title/Summary/Keyword: amino acids

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Studies on Sclerotium rolfsii Sacc. isolated from Magnolia kobus DC. in Korea (목련(Magnolia kobus DC.)에서 분리한 흰비단병균(Sclerotium rolfsii Sacc.)에 관한 연구)

  • Kim Kichung
    • Korean journal of applied entomology
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    • v.13 no.3 s.20
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    • pp.105-133
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    • 1974
  • The present study is an attempt to solve the basic problems involved in the control of the Sclerotium disease. The biologic stranis of Sclerotium rolfsii Sacc., pathogen of Sclerotium disease of Magnolia kobus, were differentiated, and the effects of vitamins, various nitrogen and carbon sources on its mycelial growth and sclerotial production have been investigated. In addition the relationship between the cultural filtrate of Penicillium sp. and the growth of Sclerotium rolfsii, the tolerance of its mycelia or sclerotia to moist heat or drought and to Benlate (methyl-(butylcarbamoy 1)-2-benzimidazole carbamate), Tachigaren (3-hydroxy-5-methylisoxazole) and other chemicals were also clarified. The results are summarizee as follows: 1. There were two biologic strains, Type-l and Type-2 among isolates. They differed from each other in the mode of growth and colonial appearance on the media, aversion phenomenon and in their pathogenicity. These two types had similar pathogenicity to the Magnolia kobus and Robinia pseudoacasia, but behaved somewhat differently to the soybaen and cucumber, the Type-l being more virulent. 2. Except potassium nitrite, sodium nitrite and glycine, all of the 12 nitrogen sources tested were utilized for the mycelial growth and sclerotial production of this fungus when 10r/l of thiamine hydrochloride was added in the culture solution. Considering the forms of nitrogen, ammonium nitrogen was more available than nitrate nitrogen for the growth of mycelia, but nitrate nitrogen was better for sclerotia formation. Organic nitrogen showed different availabilities according to compounds used. While nitrite nitrogen was unavailable for both mycelial growth and sclerotial formation whether thiamine hydrochlioride was added or not. 3. Seven kinds of carbon sources examined were not effective in general, as long as thiamine hydrochloride was not added. When thiamine hydrochloride was added, glucose and saccharose exhibited mycelial growth, while rnaltose and soluble starch gave lesser, and xylose, lactose, and glycine showed no effect at all,. In the sclerotial production, all the tested carbon sources, except lactose, were effective, and glucose, maltose, saccharose, and soluble starch gave better results. 4. At the same level of nitrogen, the amount of mycelial growth increased as more carbon Sources were applied but decreased with the increase of nitrogen above 0.5g/1. The amount of sclerotial production decreased wi th the increase of carbon sources. 5. Sclerotium rolfsii was thiamine-defficient and required thiamine 20r/l for maximun growth of mycelia. At a higher concentration of more than 20r/l, however, mycelial growth decreased as the concentration increased, and was inhibited at l50r/l to such a degree of thiamine-free. 6. The effect of the nitrogen sources on the mycelial growth under the presence of thiamine were recognized in the decreasing order of $NH_4NO_3,\;(NH_4)_2SO_4,\;asparagine,\;KNO_3$, and their effects on the sclerotial production in the order of $KNO_3,\;NH_4NO_3,\;asparagine,\;(NH_4)_2SO_4$. The optimum concentration of thiamine was about 12r/l in $KNO_3$ and about 16r/l in asparagine for the growth of mycelia; about 8r/l in $KNO_3$ and $NH_4NO_3$, and 16r/l in asparagine for the production of sclerotia. 7. After the fungus started to grow, the pH value of cultural filtrate rapidly dropped to about 3.5. Hereafter, its rate slowed down as the growth amount increased and did not depreciated below pH2.2. 8. The role of thiamine in the growth of the organism was vital. If thiamine was not added, the combination of biotin, pyridoxine, and inositol did not show any effects on the growth of the organism at all. Equivalent or better mycelial growth was recognized in the combination of thiamine+pyridoxine, thiamine+inositol, thiamine+biotin+pyridoxine, and thiamine+biotin+pyridoxine+inositol, as compared with thiamine alone. In the combinations of thiamine+biotin and thiamine+biotin+inositol, mycelial growth was inhibited. Sclerotial production in dry weight increased more in these combinations than in the medium of thiamine alone. 9. The stimulating effects of the Penicillium cultural filtrate on the mycelial growth was noticed. It increased linearly with the increase of filtrate concentration up to 6-15 ml/50ml basal medium solution. 10. $NH_4NO_3$. as a nitrogen source for mycelial growth was more effective than asparasine regardless of the concentration of cultural filtrate. 11. In the series of fractionations of the cultural filtrate, mycelial growth occured in unvolatile, ether insoluble cation-adsorbed or anion-unadsorbed substance fractions among the fractions of volatile, unvolatile acids, ether soluble organic acids, ether insoluble, cation-adsorbed, cation-unadsorbed, anion-adsorbed and anion-unadsorbed. and anion-un-adsorbed substance tested. Sclerotia were produced only in cation-adsorbed fraction. 12. According to the above results, it was assumed that substances for the mycelial growth and sclerotial formation and inhibitor of sclerotial formation were include::! in cultural filtrate and they were quite different from each other. I was further assumed that the former two substances are un volatile, ether insotuble, and adsorbed to cation-exchange resin, but not adsorbed to anion, whereas the latter is unvolatile, ether insoluble, and not adsorbed to cation or anion-exchange resin. 13. Seven amino acids-aspartic acid, cystine, glysine, histidine, Iycine, tyrosine and dinitroaniline-were detected in the fractions adsorbed to cation-exchange resin by applying the paper chromatography improved with DNP-amino acids. 14. Mycelial growth or sclerotial production was not stimulated significantly by separate or combined application of glutamic acid, aspartic acid, cystine, histidine, and glysine. Tyrosine gave the stimulating effect when applied .alone and when combined with other amino acids in some cases. 15. The tolerance of sclerotia to moist heat varied according to their water content, that was, the dried sclerotia are more tolerant than wet ones. The sclerotia harvested directly from the media, both Type-1 and Type-2, lost viability within 5 minutes at $52^{\circ}C$. Sclerotia dried for 155 days at$26^{\circ}C$ had more tolerance: sclerotia of Type-l were killed in 15 mins. at $52^{\circ}C$ and in 5 mins. at $57^{\circ}C$, and sclerotia of Type-2 were killed in 10 mins. both at $52^{\circ}C$ or $57^{\circ}C$. 16. Cultural sclerotia of both strains maintained good germinability for 132 days at$26^{\circ}C$. Natural sclerotia of them stored for 283 days under air dry condition still had good germinability, even for 443 days: type-l and type-2 maintained $20\%$ and $26.9\%$ germinability, respectively. 17. The tolerance to low temperature increased in the order of mycelia, felts and sclerotia. Mycelia completely lost the ability to grow within 1 week at $7-8^{\circ}C$> below zero, while mycelial felts still maintained the viability after .3 weeks at $7-20^{\circ}C$ below zero, and sclerotia were even more tolerant. 18. Sclerotia of type-l and type-2 were killed when dipped into the $0.05\%$ solution of mercury chloride for 180 mins. and 240 mins. respectively: and in the $0.1\%$ solution, Type-l for 60 mins. and Type-2 for 30 mins. In the $0.125\%$ uspulun solution, Type-l sclerotia were killed in 180 mins., and those of Type-2 were killed for 90 mins. in the$0.125\%$solution. Dipping into the $5\%$ copper sulphate solution or $0.2\%$ solution of Ceresan lime or Mercron for 240 mins. failed to kill sclerotia of either Type-l or Type-2. 19. Inhibitory effect on mycelial growth of Benlate or Tachi-garen in the liquid culture increased as the concentration increased. 6 days after application, obvious inhibitory effects were found in all treatments except Benlate 0.5ppm; but after 12 days, distingushed diflerences were shown among the different concentrations. As compared with the control, mycelial growth was inhibited by $66\%$ at 0.5ppm and by $92\%$ at 2.0ppm of Benlate, and by$54\%$ at 1ppm and about $77\%$ at 1.5ppm or 2.0ppm of Tachigaren. The mycelial growth was inhibited completely at 500ppm of both fungicides, and the formation of sclerotia was checked at 1,000ppm of Benlate ant at 500ppm or 1,000ppm of Tachigaren. 20. Consumptions of glucose or ammonium nitrogen in the culture solution usually increased with the increment of mycelial growth, but when Benlate or Tachigaren were applied, consumptions of glucose or ammonium nitrogen were inhibited with the increment of concentration of the fungicides. At the low concentrations of Benlate (0.5ppm or 1ppm), however, ammonium nitrogen consumption was higher than that of the ontrol. 21. The amount of mycelia produced by consuming 1mg of glucose or ammonium nitrogen in the culture solution was lowered markedly by Benlate or Tachigaren. Such effects were the severest on the third day after their treatment in all concentrations, and then gradually recovered with the progress of time. 22. In the sand culture, mycelial growth was not inhibited. It was indirectly estimated by the amount of $CO_2$ evolved at any concentrations, except in the Tachigaren 100mg/g sand in which mycelial growth was inhibited significantly. Sclerotial production was completely depressed in the 10mg/g sand of Benlate or Tachigaren. 23. There was no visible inhibitory effect on the germination of sclerotia when the sclerotia were dipped in the solution 0.1, 1.0, 100, 1.000ppm of Benlate or Tachigaren for 10 minutes or even 20 minutes.

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A Novel Human BTB-kelch Protein KLHL31, Strongly Expressed in Muscle and Heart, Inhibits Transcriptional Activities of TRE and SRE

  • Yu, Weishi;Li, Yongqing;Zhou, Xijin;Deng, Yun;Wang, Zequn;Yuan, Wuzhou;Li, Dali;Zhu, Chuanbing;Zhao, Xueying;Mo, Xiaoyang;Huang, Wen;Luo, Na;Yan, Yan;Ocorr, Karen;Bodmer, Rolf;Wang, Yuequn;Wu, Xiushan
    • Molecules and Cells
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    • v.26 no.5
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    • pp.443-453
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    • 2008
  • The Bric-a-brac, Tramtrack, Broad-complex (BTB) domain is a protein-protein interaction domain that is found in many zinc finger transcription factors. BTB containing proteins play important roles in a variety of cellular functions including regulation of transcription, regulation of the cytoskeleton, protein ubiquitination, angiogenesis, and apoptosis. Here, we report the cloning and characterization of a novel human gene, KLHL31, from a human embryonic heart cDNA library. The cDNA of KLHL31 is 5743 bp long, encoding a protein product of 634 amino acids containing a BTB domain. The protein is highly conserved across different species. Western blot analysis indicates that the KLHL31 protein is abundantly expressed in both embryonic skeletal and heart tissue. In COS-7 cells, KLHL31 proteins are localized to both the nucleus and the cytoplasm. In primary cultures of nascent mouse cardiomyocytes, the majority of endogenous KLHL31 proteins are localized to the cytoplasm. KLHL31 acts as a transcription repressor when fused to GAL4 DNA-binding domain and deletion analysis indicates that the BTB domain is the main region responsible for this repression. Overexpression of KLHL31 in COS-7 cells inhibits the transcriptional activities of both the TPA-response element (TRE) and serum response element (SRE). KLHL31 also significantly reduces JNK activation leading to decreased phosphorylation and protein levels of the JNK target c-Jun in both COS-7 and Hela cells. These results suggest that KLHL31 protein may act as a new transcriptional repressor in MAPK/JNK signaling pathway to regulate cellular functions.

Characteristics of Angiotensin-I Converting Enzyme Inhibitors Derived from Fermented Fish Product -2. Characteristics of Angiotensin-I Converting Enzyme Inhibitors of Fish Sauce Prepared from Sardine, Sardinops melanosticta- (수산발효식품 중의 Angiotensin-I 전환효소 저해제의 특성 -2. 정어리 어간장 중의 Angiotensin-I 전환효소 저해제의 특성-)

  • YEUM Dong-Min;LEE Tae-Gee;DO Jeong-Ryong;KIM Oi-Kyung;PARK Young-Beom;KIM Seon-Bong;PARK Young-Ho
    • Korean Journal of Fisheries and Aquatic Sciences
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    • v.26 no.5
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    • pp.416-423
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    • 1993
  • Fish sauces prepared from sardine, Sardinops melanosticta were tested for inhibitory activity against angiotensin-I converting enzyme(ACE). Three kinds of fish sauces were prepared from scrap(S), meat(M) and round(R) of sardine, respectively. ACE inhibitory activity of sardine sauce S and R decreased with the elapse of fermentation period, whereas that of sardine sauce M increased to 30 days and thereafter decreased. ACE inhibitory activity of sardine sauce M fermented with koji was higher than that without koji. And occurrence of $5\%$ TCA soluble peptide-nitrogen was similar to tendancy of the ACE inhibitory activity. The ACE inhibitory activity increased with an increment of amounts added and was stable at heat treatment in boiling water bath for 5hrs. $IC_{50}\%$ (Amounts of inhibitors need for $50\%$ inhibition) of the sardine sauce S, M and R fermented with(without) koji during 90 days was $125{\mu}g(140{\mu}g),\;200{\mu}g(100{\mu}g)$ and $125{\mu}g(135{\mu}g)$, respectively. From the profiles of fractionation of the sardine sauce R fermented without koji for 90 days, the molecular weight of most active fraction was about 1,400 and the amino acids of Glu, Ala, Leu and Lys were found in abundance.

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Effects of Extruded Corn in Nursery and Finishing Pigs (자돈 및 비육돈에 있어 옥수수의 가공 효과)

  • Han, Y.K.;Kim, I.H.;Hong, J.W.;Kwon, O.S.;Min, B.J.;Lee, W.B.;Shon, K.S.;Lee, J.H.
    • Journal of Animal Science and Technology
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    • v.45 no.6
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    • pp.949-956
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    • 2003
  • For the Exp. 1, a total of sixty pigs(15.95${\pm}$0.09kg average initial body weight) were used in a 28-d growth assay to determine the effects of extruded chinese corn on growth performance and nutrient digestibility in nursery pigs. Dietary treatments included 1) UCORN(U.S. corn-SBM based diet), 2) CCORN(Chinese corn-SBM based diet) and 3) ECCORN(Extruded Chinese corn-SBM based diet). Overall period, average daily gain of pigs fed ECCORN diet was higher than that of pigs fed CCORN diet(547 vs 522 g/d), however, there was not significant difference. On day 10 of the experiment, pigs fed UCORN and ECCORN diet had significantly increased in DM and DE digestibilities compared to pigs fed CCORN diet(P〈0.05). Also, on day 24 of the experiment, pigs fed UCORN and ECCORN diet had a significant increase in DM digestibility compared to pigs fed CCORN diet(P〈0.05). Pigs fed ECCORN diet had significantly increased DE digestibility compared to pigs fed CCORN diet(P〈0.05). For the Exp. 2, three cannulated barrows(54.09kg average initial body weight) were used to determine the apparent ileal digestibilities of amino acids and nutrient digestibility of extruded corn in finishing pigs. Dietary treatments were the same as in Exp. 1. Apparent ileal digestibility of cystine was greater for UCORN and ECCORN than for CCORN(P〈0.05). Apparent digestibility of DM at the total tract was greater for UCORN and ECCORN than for CCORN(P〈0.05). Pigs fed UCORN and ECCORN diet had a significant increase in apparent total tract digestibility of N compared to pigs fed CCORN diet(P〈0.05). In conclusion, the results obtained from these feeding trials suggest that the extruded corn for nursery pigs had affected growth performance and DM and DE digestibilities. In finishing pigs, extruded corn was an effective means to improve apparent total tract digestibilities of DM and N.

Nutritional Properties of ChOl-PyOn Preparation by Adding Mugwort and Pine leaves (쑥과 솔잎을 첨가한 절편의 영양학적 특성)

  • 김종군
    • Korean journal of food and cookery science
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    • v.11 no.5
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    • pp.446-455
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    • 1995
  • The nutritional properties of the Chol-Pyon were investigated with changing the materials (mugwort and pine leaves). In proximate composition, rice powder added mugwort and pine leaves showed the lligher con-tents of crude protein, crude lipid and crude ash than in rice powder. Ihe pH of rice powder, mugwort and pine leaves was 6.4, 6.8 and 3.5, respectively. The rice powder added pine leaves showed the lowest pH value. The content of the free sugar in raw materials for ChOl-PyOn preparation was 0.9% in rice powder, 0.3% in mugwort and 2.7% in pine leaves. Eighteen kinds of amino acids were determined in raw materials for ChOl-fyOn preparation and their contents were 4.8% in mugwort, 4.2% in rice powder and 2.8% in pine leaves. The major minerals of raw materials for ChOl-PyOn preparation was 0.9% increased in the order of K> Na > Mg > Ca in rice powder, Mg > K > Ca > Na in mugwort, and K > Ca > Mg > Na in pine leaves. Both of mugwort and pine leaves additives showed the higher contents of 8 kinds of minerals (Ca, Mg, K, Na, Mn, Fe, Cu, Zn) than in rice powder. In relation to changes in the texture of ChOl-PyOn, hardness, fracturability and adhesiveness at 25${\pm}$1$^{\circ}C$ were measured to be highest in white ChOl-PyOn. Cohesiveness was shown to be highest at 15% in case of mugwort and 2.5% in case of pine leaves. Elasticity was measured to be highest at 0.99 in case that 7.5% mugwort was added to raw materials for ChOl-PyOn. As a result of estimating the sensory qualities of the ChOl-PyOn prepared to which the additives were added in differing amounts, immediately after its preparation the mugwort additive of 7.5% showed the superior sensory qualities Chol-PyOn (p < 0,01).

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Discrimination of African Yams Containing High Functional Compounds Using FT-IR Fingerprinting Combined by Multivariate Analysis and Quantitative Prediction of Functional Compounds by PLS Regression Modeling (FT-IR 스펙트럼 데이터의 다변량 통계분석을 이용한 고기능성 아프리칸 얌 식별 및 기능성 성분 함량 예측 모델링)

  • Song, Seung Yeob;Jie, Eun Yee;Ahn, Myung Suk;Kim, Dong Jin;Kim, In Jung;Kim, Suk Weon
    • Horticultural Science & Technology
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    • v.32 no.1
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    • pp.105-114
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    • 2014
  • We established a high throughput screening system of African yam tuber lines which contain high contents of total carotenoids, flavonoids, and phenolic compounds using ultraviolet-visible (UV-VIS) spectroscopy and Fourier transform infrared (FT-IR) spectroscopy in combination with multivariate analysis. The total carotenoids contents from 62 African yam tubers varied from 0.01 to $0.91{\mu}g{\cdot}g^{-1}$ dry weight (wt). The total flavonoids and phenolic compounds also varied from 12.9 to $229{\mu}g{\cdot}g^{-1}$ and from 0.29 to $5.2mg{\cdot}g^{-1}$dry wt. FT-IR spectra confirmed typical spectral differences between the frequency regions of 1,700-1,500, 1,500-1,300 and $1,100-950cm^{-1}$, respectively. These spectral regions were reflecting the quantitative and qualitative variations of amide I, II from amino acids and proteins ($1,700-1,500cm^{-1}$), phosphodiester groups from nucleic acid and phospholipid ($1,500-1,300cm^{-1}$) and carbohydrate compounds ($1,100-950cm^{-1}$). Principal component analysis (PCA) and subsequent partial least square-discriminant analysis (PLS-DA) were able to discriminate the 62 African yam tuber lines into three separate clusters corresponding to their taxonomic relationship. The quantitative prediction modeling of total carotenoids, flavonoids, and phenolic compounds from African yam tuber lines were established using partial least square regression algorithm from FT-IR spectra. The regression coefficients ($R^2$) between predicted values and estimated values of total carotenoids, flavonoids and phenolic compounds were 0.83, 0.86, and 0.72, respectively. These results showed that quantitative predictions of total carotenoids, flavonoids, and phenolic compounds were possible from FT-IR spectra of African yam tuber lines with higher accuracy. Therefore we suggested that quantitative prediction system established in this study could be applied as a rapid selection tool for high yielding African yam lines.

Expression of Epidermal Growth Factor Receptor in the Inflamed Gingival Epithelium and the Dental Follicle (염증성 치은 상피와 치낭의 표피성장인자 수용체의 발현 및 실험적 치아이동에 미치는 영향에 관한 연구)

  • Kim, Young Ho;Bae, Chang
    • The korean journal of orthodontics
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    • v.27 no.2
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    • pp.349-357
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    • 1997
  • Epidermal growth factor(EGF), a single chain polypeptide of 53 amino acids with a molecular weight of 6,045 Da, was first isolated from the male mouse submandibular glands. EGF stimulates cellular proliferation and differentiation in several tissues and accelerates the rate of wound healing. EGF is bound to the specific receptor(EGFR) on the cell membrane of its target cell. EGFR is a transmembrane glycoprotein with a molecular weight of 170,000 Da and is detectable on a large variety of cell types and tissues. The authors investigated the expression of EGFR in the normal and inflamed human gingival epithelium to study the role of EGFR in the inflammation of the gingival epithelium, and the expression of EGFR in the dental follicle by using in situ mRNA hybridization and immunohistochenistry. The results weree as follows : 1. The expression of EGFR mRNA in the normal gingival epithelium on in situ mRNA hybridization was mainly localized on the basal cell layer, and the spinous layer was weakly positive The granular and cornified layers were negative 2. The expression of EGFR protein in the normal gingival epithelium on inmunohistochemistry was localized on the cornified and granular layers, and the spinous layer was weakly positive. The basal cell layer was completely negative 3. The expression of EGFR mRNA in the inflamed gingival epithelium on in situ mRNA hybridization was evenly and homogeneously distributed in the whole layers of the gingival epithelium except the cornified layer. The staining intensity appeared to increase progressively from the basal cell layer to the cornified layer. 4. The expression of EGFR protein in the inflamed gingival epithelium on immunohistochemistry was evenly and homogeneously distributed in the whole layers of the gingival epithelium. The staining intensity appeared to increase progressively from the cornified layer to the basal cell layer. 5. Strong positive reaction was seen in the epithelial cell rests of Malassez, whereas only background staining was seen in other cells of the dental follicle. In conclusion, the up-regulation of EGFR in the inflamed gingival epithelium and the high amounts of EGFR in the epthelial cell rests of Malassez in the dental follicle can be regarded as responses to the possible damages to the oral environment to maintain the homeostatic conditions.

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Variations of Properties and Microbial Community during Fermentation of Makgeollies by Isolated Yeasts from Traditional Makgeollies (전통막걸리에서 분리한 효모균주를 이용한 막걸리 발효과정 중의 물성 및 미생물 군집의 변화)

  • Jeon, Myong Je;Jang, Min Kyung;Lee, Sol Jee;Park, Sung Hwan;Kim, Mihyang;Sohn, Jae Hak;Lee, Han-Seung;Lee, Dong-Geun;Lee, Sang-Hyeon
    • Journal of Life Science
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    • v.23 no.6
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    • pp.796-803
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    • 2013
  • Property changes and bacterial characterizations by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) were investigated during the fermentation of Makgeollies by 5 isolated yeast strains. Changes of pH were large between day 0 (pH 6) and day 2 (pH 3) and showed less variation after then. ANOVA analyses revealed that pHs were statistically different with fermentation times (p<0.001), while strains (p=0.60) did not. Acidities were changed from 0.19 to 1.04% and showed rather high increase from day 2, and fermentation times (p<0.001) and strains (p=0.006) represented statistical differences. All strains showed less than 0.150% at amino-type nitrogen contents except S strain showed 0.442% at day 8, and there were no statistical differences with fermentation times (p=0.4558) and strains (p=0.3513). Saccharinities of C strain were higher from day 4, and fermentation times (p<0.0001) and strains (p=0.007) showed statistical differences. Large variation of alcohol concentrations (%) were observed between day 0 (0%) and day 2 (10%) and showed less variation after day 2, and there was no statistical difference with strains. Dominant prokaryotes were Lactobacillus fermentum and Pediococcus pentosaceus, which producing acids and functional materials. Dominant eukaryote was Saccharomyces cerevisiae, which might be resulted from addition of yeasts.

Evolutionary Explanation for Beauveria bassiana Being a Potent Biological Control Agent Against Agricultural Pests

  • Han, Jae-Gu
    • 한국균학회소식:학술대회논문집
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    • 2014.05a
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    • pp.27-28
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    • 2014
  • Beauveria bassiana (Cordycipitaceae, Hypocreales, Ascomycota) is an anamorphic fungus having a potential to be used as a biological control agent because it parasitizes a wide range of arthropod hosts including termites, aphids, beetles and many other insects. A number of bioactive secondary metabolites (SMs) have been isolated from B. bassiana and functionally verified. Among them, beauvericin and bassianolide are cyclic depsipeptides with antibiotic and insecticidal effects belonging to the enniatin family. Non-ribosomal peptide synthetases (NRPSs) play a crucial role in the synthesis of these secondary metabolites. NRPSs are modularly organized multienzyme complexes in which each module is responsible for the elongation of proteinogenic and non-protein amino acids, as well as carboxyl and hydroxyacids. A minimum of three domains are necessary for one NRPS elongation module: an adenylation (A) domain for substrate recognition and activation; a tholation (T) domain that tethers the growing peptide chain and the incoming aminoacyl unit; and a condensation (C) domain to catalyze peptide bond formation. Some of the optional domains include epimerization (E), heterocyclization (Cy) and oxidation (Ox) domains, which may modify the enzyme-bound precursors or intermediates. In the present study, we analyzed genomes of B. bassiana and its allied species in Hypocreales to verify the distribution of NRPS-encoding genes involving biosynthesis of beauvericin and bassianolide, and to unveil the evolutionary processes of the gene clusters. Initially, we retrieved completely or partially assembled genomic sequences of fungal species belonging to Hypocreales from public databases. SM biosynthesizing genes were predicted from the selected genomes using antiSMASH program. Adenylation (A) domains were extracted from the predicted NRPS, NRPS-like and NRPS-PKS hybrid genes, and used them to construct a phylogenetic tree. Based on the preliminary results of SM biosynthetic gene prediction in B. bassiana, we analyzed the conserved gene orders of beauvericin and bassianolide biosynthetic gene clusters among the hypocrealean fungi. Reciprocal best blast hit (RBH) approach was performed to identify the regions orthologous to the biosynthetic gene cluster in the selected fungal genomes. A clear recombination pattern was recognized in the inferred A-domain tree in which A-domains in the 1st and 2nd modules of beauvericin and bassianolide synthetases were grouped in CYCLO and EAS clades, respectively, suggesting that two modules of each synthetase have evolved independently. In addition, inferred topologies were congruent with the species phylogeny of Cordycipitaceae, indicating that the gene fusion event have occurred before the species divergence. Beauvericin and bassianolide synthetases turned out to possess identical domain organization as C-A-T-C-A-NM-T-T-C. We also predicted precursors of beauvericin and bassianolide synthetases based on the extracted signature residues in A-domain core motifs. The result showed that the A-domains in the 1st module of both synthetases select D-2-hydroxyisovalerate (D-Hiv), while A-domains in the 2nd modules specifically activate L-phenylalanine (Phe) in beauvericin synthetase and leucine (Leu) in bassianolide synthetase. antiSMASH ver. 2.0 predicted 15 genes in the beauvericin biosynthetic gene cluster of the B. bassiana genome dispersed across a total length of approximately 50kb. The beauvericin biosynthetic gene cluster contains beauvericin synthetase as well as kivr gene encoding NADPH-dependent ketoisovalerate reductase which is necessary to convert 2-ketoisovalarate to D-Hiv and a gene encoding a putative Gal4-like transcriptional regulator. Our syntenic comparison showed that species in Cordycipitaceae have almost conserved beauvericin biosynthetic gene cluster although the gene order and direction were sometimes variable. It is intriguing that there is no region orthologous to beauvericin synthetase gene in Cordyceps militaris genome. It is likely that beauvericin synthetase was present in common ancestor of Cordycipitaceae but selective gene loss has occurred in several species including C. militaris. Putative bassianolide biosynthetic gene cluster consisted of 16 genes including bassianolide synthetase, cytochrome P450 monooxygenase, and putative Gal4-like transcriptional regulator genes. Our synteny analysis found that only B. bassiana possessed a bassianolide synthetase gene among the studied fungi. This result is consistent with the groupings in A-domain tree in which bassianolide synthetase gene found in B. bassiana was not grouped with NRPS genes predicted in other species. We hypothesized that bassianolide biosynthesizing cluster genes in B. bassiana are possibly acquired by horizontal gene transfer (HGT) from distantly related fungi. The present study showed that B. bassiana is the only species capable of producing both beauvericin and bassianolide. This property led to B. bassiana infect multiple hosts and to be a potential biological control agent against agricultural pests.

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Physicochemical Quality Characteristics of Traditional Kamju Using Extruded Rice Collet Powder (압출 쌀 콜렛 분말을 사용한 전통 감주의 제조 및 이화학적 품질 특성)

  • Je, Hae-Soo;Kang, Kyung-Hun;Park, Si-Young;Choi, Byeong-Dae;Kang, Young-Mi;Kim, Jeong-Gyun
    • Korean Journal of Food Science and Technology
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    • v.48 no.2
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    • pp.147-152
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    • 2016
  • This study was conducted to investigate the physicochemical quality properties and provide basic data for the activation of traditional Kamju of juice type product prepared by mixing malt and extruded rice collet powder. Malt extracts were prepared by extracting the mixture of malt and water at a weight ratio of 25:75 after soaking for 2 h at $45^{\circ}C$. Rice collet powder was prepared by adjusting the barrel temperature to $95^{\circ}C$, screw speed to $3.07{\times}g$, discharge port diameter to 7 mm and a raw material input to 50 kg/h, the powder was then ground to a particle size of 80 mesh. The physicochemical characteristics (pH, color, viscosity, reducing sugars, number of viable cells, free amino acids) and sensory evaluations were conducted at various time points during the saccharification and at different mixing ratios of the extruded rice collet powder to malt extract (5:95, 15:85, 25:75, 35:65, each at $55^{\circ}C$ for 9 h). As a result, with an increase in the proportion of the extruded rice collet powder and saccharification time, the physicochemical properties of traditional Kamju significantly improved (p<0.05). A mixing ratio of 35:65 rice collet powder to malt extract and a saccharification time of 9 h were found to be the most desirable conditions. However, based on the sensory evaluation, a mixing ratio of rice collet powder and malt extract of 25:75 and a saccharification time of 5 h resulted in the most preferable palatability of traditional Kamju (p<0.05). Therefore, the mixing ratio and saccharification time should be determined to provide a better choice with respect to the taste and economic aspects of traditional Kamju.