• Title/Summary/Keyword: Rec assay

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Characterization of Lactobacilli with Tannase Activity Isolated from Kimchi

  • Kwon, Tae-Yeon;Shim, Sang-Min;Lee, Jong-Hoon
    • Food Science and Biotechnology
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    • v.17 no.6
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    • pp.1322-1326
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    • 2008
  • Tannase catalyzes the hydrolysis of gallic acid esters and hydrolysable tannins. Twenty-two Lactobacillus strains with tannase activity were isolated from 7 types of kimchi. A polymerase chain reaction-based assay targeting the recA gene assigned all isolates to either Lactobacillus plantarum or Lactobacillus pentosus. The tannase activities of isolates measured in whole cells and cell-free extracts varied even within each species. The activities of the isolates varied with the assay method, but both methods indicated that isolate LT7 (identified as L. pentosus) showed the highest activity. The results of thin layer chromatography and high performance liquid chromatography, respectively, showed that tannic acid and gallic acid degraded to pyrogallol in resting L. pentosus LT7 cells. Therefore, the putative biochemical pathway for the degradation of tannic acid by L. pentosus implies that tannic acid is hydrolyzed to gallic acid and glucose, with the formed gallic acid being decarboxylated to pyrogallol. This study revealed the possible production of pyrogallol from tannic acid by the resting cell reaction with L. pentosus LT7.

Protective Immunity of 56-kDa Type-Specific Antigen of Orientia tsutsugamushi Causing Scrub Typhus

  • Choi, Sangho;Jeong, Hang Jin;Ju, Young Ran;Gill, Byoungchul;Hwang, Kyu-Jam;Lee, Jeongmin
    • Journal of Microbiology and Biotechnology
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    • v.24 no.12
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    • pp.1728-1735
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    • 2014
  • Scrub typhus, caused by infection with Orientia tsutsugamushi, is a mite-borne zoonotic disease endemic to the Asian-Pacific region. In Korea, the incidence of this disease has increased with climate changes, and over 10,000 cases of infection were reported in 2013. Although this infection is treatable with antibiotics such as doxycycline and azithromycin, an effective prophylactic vaccine against O. tsutsugamushi would be more desirable for preventing scrub typhus in endemic areas. In this study, we investigated the 56-kDa type-specific antigen (TSA56), which is a major outer membrane protein of O. tsutsugamushi, as a vaccine candidate. Intranasal immunization of recombinant TSA56 (rec56) induced a higher level of TSA56-specific IgG than that induced by intramuscular immunization of tsa56-expressing DNA (p56). Both types of immunization induced a cell-mediated immune response to TSA56, as demonstrated by the splenic cell proliferation assay. Mice immunized with p56, followed by rec56 plus heat-labile enterotoxin B subunit from E. coli, had a stronger protection from a homologous challenge with the O. tsutsugamushi Boryong strain than with other combinations. Our preliminary results suggest that an effective human vaccine for scrub typhus can include either recombinant TSA56 protein or tsa56-expressing DNA, and provide the basis for further studies to optimize vaccine performance using additional antigens or different adjuvants.

Food Safety of Functional Neoglycoproteins Prepared by Covalent Attachment of Galactomannan to Food Proteins

  • Nakamura, Soichiro;Dokai, Kazumi;Matsuura, Megumi;Hata, Junya;Saeki, Hiroki
    • Preventive Nutrition and Food Science
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    • v.7 no.2
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    • pp.139-145
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    • 2002
  • Hen egg-white lysozyme, ovalbumin, egg-yolk phosvitin, acid-precipitated soy protein and $\alpha$$_{sl}$ milk casein were covalently linked with galactomannan through a controlled dry-heating at 6$0^{\circ}C$ under 79% relative humidity without any chemical reagent. Neoglycosylation by the covalent binding of polysaccharide chains brought a significant improvement into the surface functionalities of food proteins. Excellent emulsifying properties and foaming properties were observed in all protein-galactomannan conjugates. Bacterial mutagenesis tests and animal dose test were done to evaluate the food safety of the protein-galactomannan conjugates. The neo-glycoproteins were negative for Ames test using Salmonella typhimurium TA100 (hisG46) and TA98 (hisD3052) strains, and rec-assay using Bacillus subtilis Hl7 (rec) and M45 (re $c^{+}$) strains. All substances were also nontoxic for oral administration to rats. L $D_{50}$ 's of these substances were all more than 7.5 g/kg body-weight of rat. No effect was also observed in the weight increases and the concentrations of total cholesterol, triglyceride and phospholipids in blood serum of the administrated rats with 7.5 g/kg conjugates. Thus, Maillard-type protein-polysaccharide conjugates prepared by covalent attachment of galactomannan to food proteins were proposed to be useful as a safe functional biopolymer in this study.y.

The Antimutagenic and Anticancer Effect of Taxus cuspidata Extracts (주목 추출물의 발암 억제효과 및 암세포에 미치는 영향)

  • 황병호;조국난;최근표;정성원;김은정;함승시
    • Journal of the Korean Society of Food Science and Nutrition
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    • v.25 no.6
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    • pp.1062-1068
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    • 1996
  • This study was Performed to determine the effects of antimutagenicity and anticancer of Taxus cuspidata produced in Korea. Extracts of Taxus cuspidata were obtained from leaves, barks and roots. All the samples tested had no effects on the mutagenicity by Ames test using Salmonella typhimurium TA98 and TA100 or rec-assay using Bacillus subtilis Hl7 and M45 strains. However the treatment of 50$\mu\textrm{g}$/plate of Taxus cuspidata extracts showed strong antimutagenicity with 98% inhibition against TA100 induced by MNNG and with 98% inhibition against TA98 and TA100 induced by 4NQO whereas 73~89% and 16~60% antimutagenic effect were shown against both strain induced by Trp-P-1 and $Benzo(\alpha)pyrene,$ respectively. The treatment of $0.5$\mu\textrm{g}$/{\mu}\ell$ root extracts had the highest cytotoxicity with 90% against liver cancer cell, Hep 3B, followed by bark extracts(87%) and leave extracts(72%), whereas $0.5$\mu\textrm{g}$/{\mu}\ell$ treatment of Taxus cuspidata extract had only 22~36% cytotoxicity on human normal liver cell WRL 68.

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Production and Characteristics of Fermented Soy Sauce from Mountain Herbs (산채류를 이용한 양조간장의 제조 및 특성)

  • Kang, Il-Jun;Ham, Seung-Shi;Chung, Cha-Kwon;Lee, Sang-Young;Oh, Deog-Hwan;Do, Jae-Joon
    • Korean Journal of Food Science and Technology
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    • v.31 no.5
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    • pp.1203-1210
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    • 1999
  • Soy sauce was fermented with the addition of mountain edible herbs, Ligularia fischeri, Codonopsis lanceolata and Symphytum officinale. In general, the total nitrogen content of soy sauce was increased with the increment of the amount of added mountain herbs. The mineral contents of calcium and potassium in the soy sauce after four months of aging at 20% substitution of Codonopsis lanceolata were increased by 1.3 and 1.5 times, respectively. With 10% substitution of mountain herb mixtures, the contents of tyrosine and arginine were increased by about 2 times as compared to the control. In the Rec assay system, antimutagenic effect of soy sauce with 10 and 20% substitution of Codonopsis lanceolata was higher than other samples. The results of sensory evaluation revealed that overall acceptability of soy sauce with 7% substitution of Codonopsis landeolata and 5% of mountain herb mixture exceeded other groups of samples.

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Application of an Interferometric Biosensor Chip to Biomonitoring an Endocrine Disruptor

  • Kim, Byung-Woo;Lim, Sung-Hyuk
    • Biotechnology and Bioprocess Engineering:BBE
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    • v.9 no.2
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    • pp.118-126
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    • 2004
  • Recombinant E.coli ACV 1003 (recA::lacZ) releasing ${\beta}$-galactosidase by a SOS regulon system, when exposed to DNA-damaging compounds, have been used to effectively monitor endocrine disruptors. Low enzyme activity of less than 10 units/mL, corresponding to a $\mu\textrm{g}$/L(ppb) range of an endocrine disruptor (tributyl tin, bisphenol A. etc.), can be rapidly determined, not by a conventional time-consuming and tedious enzyme assay, but by an alternative interferometric biosensor. Heavily boron-doped porous silicon for application as an interferometer, was fabricated by etching to form a Fabry-Perot fringe pattern, which caused a change in the refractive index of the medium including ${\beta}$-galactosidase. In order to enhance the immobilization of the porous silicon surface, a calyx crown derivative (ProLinker A) was applied, instead of a conventional biomolecular affinity method using biotin. This resulted in a denser linked formation. The change in the effective optical thickness versus ${\beta}$-galactosidase activity, showed a linear increase up to a concentration of 150 unit ${\beta}$-galactosidase/mL, unlike the sigmoidal increase pattern observed with the biotin.

Interactions between secreted GRA proteins and host cell proteins across the parasitophorous vacuolar membrane in the parasitism of Toxoplasma gondii

  • Ahn, Hye-Jin;Kim, Sehra;Kim, Hee-Eun;Nam, Ho-Woo
    • Parasites, Hosts and Diseases
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    • v.44 no.4 s.140
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    • pp.303-312
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    • 2006
  • Interactions between GRA proteins of dense granules in Toxoplasma gondii and host cell proteins were analyzed by yeast two-hybrid technique. The cMyc-GRA fusion proteins expressed from pGBKT7 plasmid in Y187 yeast were bound to host cell proteins from pGADT7-Rec-HeLa cDNA library transformed to AH109 yeast by mating method. By the selection procedures, a total of 939 colonies of the SD/-AHLT culture, 348 colonies of the $X-\alpha-gal$ positive and PCR, 157 colonies of the $X-\beta-gal$ assay were chosen for sequencing the cDNA and finally 90 colonies containing ORF were selected to analyze the interactions. GRA proteins interacted with a variety of host cell proteins such as enzymes, structural and functional proteins of organellar proteins of broad spectrum. Several specific bindings of each GRA protein to host proteins were discussed presumptively the role of GRA proteins after secreting into the parasitophorous vacuoles (PV) and the PV membrane in the parasitism of this parasite.

Enhancement of Sensitivity in Interferometric Biosensing by Using a New Biolinker and Prebinding Antibody

  • Park, Jae-Sook;Lim, Sung-Hyun;Sim, Sang-Jun;Chae, Hee-Yeop;Yoon, Hyun-C.;Yang, Sang-Sik;Kim, Byung-Woo
    • Journal of Microbiology and Biotechnology
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    • v.16 no.12
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    • pp.1968-1976
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    • 2006
  • Recombinant E. coli ACV 1003 (recA:: lacZ) was used to measure low concentrations of DNA-damaging chemicals, which produce $\beta$-galactosidase via an SOS regulon system. Very low $\beta$-galactosidase activities of less than 0.01 unit/ml, $\beta$-galactosidase produced through an SOS response corresponding to the 10 ng/ml (ppb) of DNA damaging chemicals in the environment, can be rapidly determined by using an alternative interferometric biosensor with optically flat thin films of porous silicon rather than by the conventional time-consuming Miller's enzyme assay as well as the ELISA method. fu order to enhance the sensitivity in the interferometry, it needs to obtain more uniform distribution and higher biolinking efficiency, whereas interferometric sensing is rapid, cheap, and advantageous in high throughput by using a multiple-well-type chip. In this study, pore size adjusted to 60 nm for the target enzyme $\beta$-galactosidase to be bound on both walls of a Si pore and a calyx crown derivative was apllied as a more efficient biolinker. Furthermore, anti-$\beta$-galactosidase was previously functionalized with the biolinker for the target $\beta$-galactosidase to be specifically bound. When anti-$\beta$-galactosidase was bound to the calyx-crown derivative-linked surface, the effective optical thickness was found to be three times as high as that obtained without using anti-$\beta$-galactosidase. The resolution obtained was very similar to that afforded by the time-consuming ELISA method; however, the reproducibility was still unsatisfactory, below 1 unit $\beta$-galactosidase/ml, owing to the microscopic non-uniform distribution of the pores in the etched silicon surface.

Identification and Antimicrobial Activity Detection of Lactic Acid Bacteria Isolated from Corn Stover Silage

  • Li, Dongxia;Ni, Kuikui;Pang, Huili;Wang, Yanping;Cai, Yimin;Jin, Qingsheng
    • Asian-Australasian Journal of Animal Sciences
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    • v.28 no.5
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    • pp.620-631
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    • 2015
  • A total of 59 lactic acid bacteria (LAB) strains were isolated from corn stover silage. According to phenotypic and chemotaxonomic characteristics, 16S ribosomal DNA (rDNA) sequences and recA gene polymerase chain reaction amplification, these LAB isolates were identified as five species: Lactobacillus (L.) plantarum subsp. plantarum, Pediococcus pentosaceus, Enterococcus mundtii, Weissella cibaria and Leuconostoc pseudomesenteroides, respectively. Those strains were also screened for antimicrobial activity using a dual-culture agar plate assay. Based on excluding the effects of organic acids and hydrogen peroxide, two L. plantarum subsp. plantarum strains ZZU 203 and 204, which strongly inhibited Salmonella enterica ATCC $43971^T$, Micrococcus luteus ATCC $4698^T$ and Escherichia coli ATCC $11775^T$ were selected for further research on sensitivity of the antimicrobial substance to heat, pH and protease. Cell-free culture supernatants of the two strains exhibited strong heat stability (60 min at $100^{\circ}C$), but the antimicrobial activity was eliminated after treatment at $121^{\circ}C$ for 15 min. The antimicrobial substance remained active under acidic condition (pH 2.0 to 6.0), but became inactive under neutral and alkaline condition (pH 7.0 to 9.0). In addition, the antimicrobial activities of these two strains decreased remarkably after digestion by protease K. These results preliminarily suggest that the desirable antimicrobial activity of strains ZZU 203 and 204 is the result of the production of a bacteriocin-like substance, and these two strains with antimicrobial activity could be used as silage additives to inhibit proliferation of unwanted microorganism during ensiling and preserve nutrients of silage. The nature of the antimicrobial substances is being investigated in our laboratory.

강원도 고랭지 산채자원의 이용기술 개발

  • 오덕환
    • Proceedings of the Korean Society of Postharvest Science and Technology of Agricultural Products Conference
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    • 1999.04a
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    • pp.29-52
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    • 1999
  • 본 연구는 강원도 고랭지에서 자생되는 산채류를 이용하여 총질소함량을 기준으로 소맥대신 이들 산채류를 일정비율로 대체하여 더덕 및 산채혼합 간장을 제조하여 그 특성을 조사하였으며 또한, 감마선조사 또는 블랜칭처리가 참나물 즙액의 선도유지연장에 미치는 영향을 조사하였다. 제조된 간장의 총 질소함량은 일반적으로 산채 대체량의 증가에 따라 증가하였다. 숙성 4개월 후의 무기질 함량에 있어 더덕 20% 대체간장은 Ca, K 함량이 각각 1.3배, 1.5배의 증가율을 나타내었으며, 산채 혼합 10% 대체간장도 Ca 함량이 1.5배 증가하였다. 대체량 별로 아미노산 함량 변화를 살펴보면 더덕간장 10% 대체군의 경우 threonine과 aspartic acid가, 산채 혼합 10% 대체군은 tyrosine과 arginine이 높은 증가율을 나타내었다. Rec assay system을 이용한 항돌연변이 시험에서, 더덕간장은 10%와 20% 대체군이, 산채혼합 간장은 7%와 10% 대체군이 다른 시료에 비해 높은 항돌연변이성을 나타내었다. 관능검사 결과, 전체적 기호도에 있어 더덕간장은 7% 대체군, 산채혼합 간장은 5% 대체군이 가장 우수하였다. 미생물의 생육변화는 감마선 조사를 하였을 때 두처리구 모두 0.5 kGy 조사에서도 무처리구에 비하여 현저한 생육감소를 나타내었으며 감마선 처리된 녹즙을 저온에서 저장하였을 때는 미생물의 생육억제효과가 높았으나 상온에서는 감마선 효과가 매우 감소함을 나타내었다. 색도변화는 블랜칭한 녹즙이 블랜칭하지 않은 녹즙보다 색깔이 훨씬 선명하고 맑은 녹색을 띄었으며 시간이 경과함에 따라 감마선 조사구가 시간이 경과함에 따라 무처리구에 비하여 L값, a값 및 b값이 훨씬 적게 변화함을 나타내었다. 비타민 C의 변화는 블랜칭 처리한 녹즙의 비타민 C의 함량변화는 블랜칭 처리를 하지않은 녹즙보다 적었으며 감마선 처리구와 거의 차이가 없었으며 온도가 낮을수록 두 처리구 모두 비타민 C의 손실율이 적게 나타났다. 감마선을 조사하였을 때 블랜칭하지않은 녹즙에서는 무처리구와 비교하여 볼 때 밝기, 풀냄새, 쓴맛, 신선감 및 수용성에서 차이가 없었으며 블랜칭한 녹즙에서는 무처리구가 감마선처리구에 비하여 풀냄새와 쓴맛이 증가하였고 신선감과 수용성이 감소되었다.

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