• Title/Summary/Keyword: In vitro screening

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Isolation of Probiotic Piliated Lactobacillus rhamnosus Strains from Human Fecal Microbiota Using SpaA Antiserum-Based Colony Immunoblotting

  • Yang, Zhen-quan;Xue, Yu;Rao, Sheng-qi;Zhang, Mi;Gao, Lu;Yin, Yong-qi;Chen, Da-wei;Zhou, Xiao-hui;Jiao, Xin-an
    • Journal of Microbiology and Biotechnology
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    • v.27 no.11
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    • pp.1971-1982
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    • 2017
  • Piliated Lactobacillus rhamnosus (pLR) strains possess higher adherent capacity than non-piliated strains. The objective of this study was to isolate and characterize probiotic pLR strains in human fecal samples. To this end, mouse polyclonal antiserum (anti-SpaA) against the recombinant pilus protein (SpaA) of L. rhamnosus strain GG (LGG) was prepared and tested for its reactivity and specificity. With the anti-SpaA, a method combining the de Man, Rogosa, and Sharpe (MRS) agar plating separation and colony immunoblotting (CIB) was developed to isolate pLR from 124 human fecal samples. The genetic and phenotypic characteristics of the resultant pLR isolates were compared by randomly amplified polymorphic DNA (RAPD) fingerprinting, and examination of adhesion to Caco-2 cells, hydrophobicity, autoaggregation, and in vitro gastrointestinal tolerance. Anti-SpaA specifically reacted with three pLR strains of 25 test strains, as assessed by western blotting, immunofluorescence flow cytometry, and immunoelectron microscopy (IEM) assays. The optimized MRS agar separation plus anti-SpaA-based CIB procedure could quantitatively detect $2.5{\times}10^3CFU/ml$ of pLR colonies spiked in $10^6CFU/ml$ of background bacteria. Eight pLR strains were identified in 124 human fecal samples, and were confirmed by 16S RNA gene sequencing and IEM identification. RAPD fingerprinting of the pLR strains revealed seven different patterns, of which only two isolates from infants showed the same RAPD profiles with LGG. Strain PLR06 was obtained with high adhesion and autoaggregation activities, hydrophobicity, and gastrointestinal tolerance. Anti-SpaA-based CIB is a rapid and inexpensive method for the preliminary screening of novel adherent L. rhamnosus strains for commercial purposes.

Probiotic Properties of Lactic Acid Bacteria Isolated Traditional Fermented Foods (전통발효식품 유래 유산균의 프로바이오틱스 특성 연구)

  • Kim, Eun-Ji;Jo, Seung-Wha;Kim, Jin-Kyeong;Jeong, Do-Youn
    • Journal of Life Science
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    • v.29 no.6
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    • pp.697-704
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    • 2019
  • This study performed to investigate the probiotic properties of lactic acid bacteria 200 strains isolated from traditional fermented foods. Based on being higher tolerance to bile salts and showing higher acid resistance, 4 LAB Strains were selected in the screening experiment; Lactobacillus plantarum SRCM 102224, Lb. plantarum SRCM102227, Lb. paracasei SRCM102329, Lb. paracasei SRCM102343. Antibacterial activity against various pathogens, acid and bile salt tolerance, hemolytic phenomenon, cell surface hydrophobicity, and antibiotic resistance were examined. Among the tested strains, SRCM 102343 (95.9%) was highly observed hydrophobicity compared to Lb. rhmanosus GG (13.4%) as control. In this study, the in vitro adhesion properties of 4 strains of LAB was investigated using human intestinal caco-2 cell cultures. SRCM102329 and SRCM102343showed higher adherence to caco-2 cells than Lb. rhamnosus GG. The antibacterial activities of 4 strains LAB were investigated. the 3 strains showing strongly antimicrobial activity against Escherichia coli ATCC10798, Staphylococcus aureus KCCM11593, Listeria invanovii KCTC3444, Bacillus cereus ATCC11778 and S. enterica serovar. Typhi KCTC1926. These results suggest that selected strains have good probiotic potential for application in functional foods.

Screening and prevention of the mutagenicity for fishes accordind to cookery and storage (어류의 가열조리 및 보존에 의해 생성되는 변이원성 물질의 정량적 해석과 제어법)

  • 홍이진;이준경;구성자
    • Korean journal of food and cookery science
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    • v.16 no.6
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    • pp.652-662
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    • 2000
  • This study was performed to measure the mutagenicity of fish by cooking and storage. Mutagenicity of the fish extract was measured by Ames test(Salmonella typhimurium reversion assay with TA 100) in vitro and by micro-nucleus test in vivo. The fish samples screened in this study were white fish(Trichiurus, Croaker, Salted Croaker) and red fish(Saury pike, Mackerel, Yellowtail, Salmon). The number of revertants of red fish were significantly higher than that of white fish. And the mutagenicity of mackerel was higher than other red fish, so followed experiment was made by using the extract of mackerel. Mutagenicity of the samples cooked on microwave oven was the lowest, whereas there was no significant difference between the samples cooked on gas grill and the ones on electric grill. In the presence of S9 mixture, the methanol extract of mackerel showed 2∼4 times high values of mutagenicity in comparison with the extract without S9. The extract of mackerel cooked with various vegetable juices showed inhibitory effects on the mutagenicity in the order of green tea, ginger, and radish. Also, the number of revertants was increased in the stored samples. Mutagenicity of the samples stored in the refrigerator was higher than that of the freezer. In micronucleus test, the methanol extract treated with vegetable juice inhibited micro-nucleus formation in bone marrow by cyclophosphamide in the order of ginger, green tea, and radish. In TBA test, there was a tendency that TBA values were increased as the storage time increased. Also, the rancidity of sample were stored in the refrigerator was higher value than sample stored in the freezer. Samples cooked on microwave oven showed the highest value in rancidity. When the antioxidant effect of vegetable juice was measured by electron donating ability(EDA) of mackerel cooked with vegetable juice to DPPH, the samples treated with onion showed the highest value of EDA(%), and the samples treated with green tea, ginger and cabbage also showed the antioxidant effect.

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Preliminary Screening of Some Jeju Island Native Plants for Whitening and Antioxidant Activity (제주도 자생식물들에 대한 미백 및 항산화 효능 탐색)

  • Yoo, Byoung-Sam;Moon, Ji-Young;Kim, Ju-Ho;Hyun, Jin-Won;Kang, Kyoung-Ah;Koh, Jea-Sook;Seo, Young-Kyoung;Baek, Ji-Hwoon;Park, Deok-Hoon;Lee, Jong-Sung;Jung, Eun-Sun
    • Journal of the Society of Cosmetic Scientists of Korea
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    • v.32 no.3 s.58
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    • pp.161-171
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    • 2006
  • In this study, we investigated the whitening and anti-oxidant activity of 37 Jeju island native plants. The active ingredients of the plants were prepared by methanol extraction. Whitening activity of plant extracts was examined from the inhibitory effect of tyrosinase and the inhibition of melanin synthesis of the B16-F1 cell line. Their anti-oxidant activity was measured by electron donating ability of DPPH (1,1-diphenyl-2-picrylhydrazyl) and ROS (reactive oxygen species) scavenging activity in V79-4 lung fibroblast cells using DCF-DA (dichlorofluorescin diacetate). Cytotoxicity of the extracts on cell s based experiments was investigated by MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) assay. Also, the toxicity test using a rabbit and the human skin patch test were carried out for examining the safety of the extracts which showed the high whitening activity. It is interesting that the extracts of Lespedeza cuneata, Ligustrum lucidum (stem), Morus bombycis (stem) and Prunella vulgaris var. lilacina showed both potent whitening and anti-oxidant activities.

Screening for inhibitory effect on nine CYP isoforms by 20 herbal medications (고속 스크리닝 기법을 이용한 한약제제의 cytochrome P45O 저해능 탐색)

  • Kim, Hyun-Mi;Liu, Kwang-Hyeon
    • Journal of Life Science
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    • v.17 no.3 s.83
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    • pp.334-339
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    • 2007
  • We evaluated the potential of 20 herbal medications (HMs), commonly used in Korea, to inhibit the catalytic activities of several cytochrome P450 (CYP) isoforms. The abilities of 500 ${\mu}g/ml$ of aqueous extracts of 20 HMs to inhibit phenacetin O-deethylation (CYP1A2), coumarin 6-hydroxylation (CYP2A6), bupropion hydroxylation (CYP2B6), rosiglitazone hydroxylation (CYP2C8), tolbutamide 4-methylhydroxylation (CYP2C9), S-mephenytoin 4'-hydroxylation (CYP2C19), dextromethorphan O-demethylation (CYP2D6), chlorzoxazone 6-hydroxylation (CYP2E1), and midazolam 1'-hydroxylation (CYP3A) were tested using human liver microsomes. The HMs Woohwangcheongsimwon suspension and Hwanglyeonhaedok-Tang strongly inhibited CYP2B6 and CYP2D6 isoform activity, respectively. These results suggest that some of the HMs used in Korea have potential to inhibit CYP isoforms in vitro. Although the plasma concentrations of the active constituents of the HMs were not determined, some herbs could cause clinically significant interactions because the usual doses of those individual herbs are several grams of freeze-dried extracts.

Selection of Salt-Tolerant Silage Rice Through in vitro Screening and Saltol QTL Analysis (기내 선발과 Saltol QTL 분석을 통한 내염성 증진 사료용 벼 선발)

  • Cho, Chuloh;Kim, Kyung Hwa;Ahn, Eok-Keun;Park, Hyangmi;Choi, Man-Soo;Chun, Jaebuhm;Seo, Mi-Suk;Jin, Mina;Kim, Dool-Yi
    • KOREAN JOURNAL OF CROP SCIENCE
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    • v.65 no.3
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    • pp.214-221
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    • 2020
  • Salinity is one of the major abiotic stressors that inhibits the growth, yield, and productivity of crop plants. Therefore, it is necessary to develop crops with increased salt tolerance for cultivation in saline soils such as is found in reclaimed land. The objective of this study was to develop a salt-tolerant silage rice line that grows on reclaimed land. In order to develop this salt-tolerant silage rice, we transferred Saltol, a major QTL associated with salt tolerance, from IR64-Saltol, a salt-tolerant indica variety, into Mogyang, a susceptible elite japonica variety. To determine the effect of salt stress, Mogyang and IR64-Saltol cultivars were grown on a medium containing various concentrations of NaCl in in vitro conditions. Shoot length was found to decrease with increasing salt concentrations, and root growth was almost arrested at NaCl concentrations over 50 mM in the Mogyang cultivar. Based on these preliminary results, we screened five salt-tolerant lines showing superior growth under salt stress conditions. Polymerase chain reaction and sequencing results showed that the introgression types of Saltol QTL were derived from the IR64-Saltol cultivar in almost all selected lines. Based on the observed growth and physiological characteristics, the new Saltol introgression lines showed higher salt tolerance compared to the Mogyang parental cultivar. The salt-tolerant lines identified in this study could be used as a genetic resource to improve rice salt tolerance.

Development of an in vitro culture method for harvesting the free-living infective larvae of Strongyloides venezuelensis (베네수엘라분선충 (Strongyloides venezuelensis Brumpt, 1934) 자유생활형 유충의 시험관 내 배양 기술 개발)

  • ;M.
    • Parasites, Hosts and Diseases
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    • v.36 no.1
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    • pp.15-22
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    • 1998
  • An in uitro culture technique was established for harvesting Strongwloides venezuelensis free-living infective larvae using a nutrient broth medium as a substitute for rat-feces in polyvinyl culture bags ($10{\;}{\times}{\;}12{\;}cm$). The egg hatch rate V) in sterile saline at different incubation temperatures (X) was expressed as the quadratic function, Y = $-0.192X^2$ + 8.673x - 19.550 (r = 0.901). The highest (100%) egghatch rate was observed at $25^{\circ}C$. A significant difference (p<0.05) in development rate W) of free-living infective larvae was observed between different concentrations of nutrient broth (X) which was highest (20.6%1 in 0.12% nutrient broth concentrations, incubated at $20^{\circ}C$ for 5 days [Y = $-864.032X^2$ + 245.995X- 0.560 (r = 0.875)]. Yields (Y) of infective larvae were observed relatively high when the culture medium was incubated at higher temperatures (X) which peaked at $25^{\circ}C$ (20.0%) than at lower temperatures. $15^{\circ}C$M (10.9%) and $20^{\circ}C$ (18.1%) [Y = $-0.189X^2$ + 8.387x- 72.795 (r = 0.981)]. The period W) required for the development of infective larvae decreased with higher incubation temperatures (X) [Y = $0.035X^2$ - 2.025X + 32.375 (r = 0.995)] The highest yield (19.2%) of infective larvae was obtained from culture bag inoculated with 15.000 eggs than with below and over 15,000 eggs in 0.12% nutrient broth and incubated at $25^{\circ}C$ for 4 days. The newly adapted culture method (from egg to third-stage larva) may be useful as a bio-bar/bioassay system for screening new chemical products, anthelmintics and pesticides, as well as for parasito immunological studies with Strongwloides species.

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Screening the Antioxidant Components and Antioxidant Activitiy of Extracts Derived from Five Varieties of Edible Spring Flowers (식용 봄꽃(개나리꽃, 진달래꽃, 목련꽃, 벚꽃) 추출물의 항산화성분과 항산화활성 검색)

  • Kim, Su-Min;Kim, Do-Yeon;Park, Hye-Ri;Seo, Ji-Hye;Yeom, Bo-Min;Jin, Yoo-Jung;Pyo, Young-Hee
    • Korean Journal of Food Science and Technology
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    • v.46 no.1
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    • pp.13-18
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    • 2014
  • This study was designed to investigate the in vitro antioxidant potentials and the antioxidant components of 80% ethanolic extracts prepared from five edible spring flowers. The contents of total phenols (14.1-18.9 mg gallic acid equivalents/g of dry weight; DW), flavonoids (0.3-4.9 mg catechin equivalents), total carotenoids ($1.9-578.1{\mu}g$ ${\beta}$-carotene equivalents), and proanthocyanidins (2.8-23.5 mg catechin equivalents), were determined by spectrophotometry. In addition, the content of ascorbic acid (0.71-1.31 mg) was determined using HPLC with UV detection. All varieties of the flower examined showed antioxidant potential (1.2-46.1 mM Trolox equivalents/g DW), which was measured by the radical scavenging activity and the reducing power. Of the five flowers analyzed, Prunus avium L. showed the highest antioxidant activity. A strong correlation was found between the ascorbic acid ($r^2=0.75$), flavonoids ($r^2=0.71$), and total phenolic content ($r^2=0.64$) and the antioxidant parameters, suggesting that these components are likely significant contributors to the antioxidant capacity of the five spring flowers.

Screening of Traditional Herbal Medicines to Develop New Materials for Anti-aging and Anti-wrinkle in the Skin (전통 생약재에서의 항노화와 주름 개선 활성 신소재 탐색)

  • Lee Sang Eun;Son Dong Wook;Yoon Year Pill;Lim Tae Young;Cho Ja Wun;Kim Haen Su
    • Journal of the Society of Cosmetic Scientists of Korea
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    • v.31 no.2 s.51
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    • pp.147-152
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    • 2005
  • As a part of searching tot the natural components which inhibit the skin aging and wrinkle formation, the $80\%$ methanolie extracts of 121 species of traditional herbal medicines used to treat lung and skin disease were investigated for their in vitro anti-oxidative activity against 1,1-diphenyl-2-picrylhydrazyl (DPPH) and superoxide radicals, and inhibitory activity against elastase. We selected 9 kinds of the traditional herbal medicines showing inhibitory activities of winkle formation. The effective concentrations of 9 candidates for anti-wrinke/skin firming activity was less than 0.1 mg/mL, and there is no toxicity to cell viability at these concentrations. Through analysis of human skin primary patch test data, the traditional herbal medicines represented non-irritant materials. We suggest that these 9 candidates with ability to help anti-wrinkle/skin firming may be useful for functional cosmetic materials.

Screening of Antifungal Activities of Plant Extracts against Phytopathogenic Fungi (식물추출물의 식물병원성 곰팡이 포자에 대한 발아억제 활성)

  • Park, Sang-jo;Rhu, Young Hyun;Bae, Soo Gon;Seo, Dong Hwan
    • Korean Journal of Plant Resources
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    • v.30 no.4
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    • pp.343-351
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    • 2017
  • Plant extracts were screened for antifungal activity against major plant pathogens, Botrytis sp., Collectotrichum sp., Alternaria sp. and Cylindrocarpon sp. using 96-well microdilution method. Among the 662 methanol extracts from 401 plant species, 36 extracts showed complete inhibition of spore germination against at least one of four pathogenic fungi. Extracts of Morus alba twig and Sophora flavescens root showed minimum inhibition concentration (MIC) at $1,250{\mu}g/ml$ against Botrytis sp.. Extracts of Chloranthus japonicus root showed MIC at $1,250{\mu}g/ml$ against Collectotrichum sp.. Extracts of Glycyrrhiza uralensis aerial part, Inula helenium root and Menispermum dauricum root showed MIC between 625 and $1,250{\mu}g/ml$ against Alternaria sp.. G. uralensis aerial part and I. helenium root showed MIC at $1,250{\mu}g/ml$ against Cylindrocarpon sp.. Specifically, the extracts of Agrimonia pilosa root, Angelica tenuissima root, Asarum sieboldii root, Campsis grandifolia leaf and twig, Cnidium officinale root, Dictamnus dasycarpus root, G. uralensis aerial part, I. helenium root and M. alba twig completely inhibited spore germination at lower than $5,000{\mu}g/ml$ against all of four pathogenic fungi. Two methanol extracts from G. uralensis aerial part and M. alba twig may used as a candidate to develop into effective disease management materials in plant cultivation.