• Title/Summary/Keyword: Protease enzyme assay

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Functional Cosmetic Effect of Porcine Placeta (Porcine Placenta의 기능성 화장품소재 특성)

  • Kim, Bo Young;Kim, Tagon;Kang, Whan Yul;Baek, Hyun;Cheon, Hae Young;Kim, Donguk
    • Korean Chemical Engineering Research
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    • v.48 no.3
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    • pp.327-331
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    • 2010
  • Porcine placenta was treated with alkali, acid and enzyme treatment to obtain extracts. Heavy metal contents such as Pb, As, and Hg were low enough to satisfy cosmetic agent standard. As a result of safety test(MTT assay), porcine placenta extracts showed over 80% of cell viability at $50{\mu}g/ml$, and cell toxicity was relatively lower. From antioxidation test using DPPH free radical scavenging assay, antioxidation effect was highest as 63% at $50{\mu}g/ml$ when porcine placenta was treated with alkali in pH 9. From whitening effect test using tyrosinase inhibition assay, tyrosinase inhibition effect was 30% at $50{\mu}g/ml$ concentration in alkali treated procine placenta, however, the efficiency was lower compared with arbutin or vitamin C. In anti-wrinkle effect test from elastase inhibition assay, elastase inhibition effects were 20~30% at $50{\mu}g/ml$ for 5 kinds of porcine placenta treatments, which was superior to standard, and especially, protease treated extracts showed best results. Skin formulation including 1% porcine placenta was made and the formulation was very stable for temperature and storage period. From this research, porcine placenta extract showed high potential for anti-wrinkle functional cosmetic agent.

Inhibitory Effect of Chitosan and Phosphate Cross-linked Chitosan against Cucumber Mosaic Virus and Pepper Mild Mottle Virus

  • Gangireddygari, Venkata Subba Reddy;Chung, Bong Nam;Cho, In-Sook;Yoon, Ju-Yeon
    • The Plant Pathology Journal
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    • v.37 no.6
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    • pp.632-640
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    • 2021
  • Cucumber mosaic virus (CMV) and Pepper mild mottle virus (PMMoV) causes severe economic loss in crop productivity of both agriculture and horticulture crops in Korea. The previous surveys showed that naturally available biopolymer material - chitosan (CS), which is from shrimp cells, reduced CMV accumulation on pepper. To improve the antiviral activity of CS, it was synthesized to form phosphate cross-linked chitosan (PCS) and compared with the original CS. Initially, the activity of CS and PCS (0.01%, 0.05%, and 0.1% concentration) compound against PMMoV infection and replication was tested using a half-leaf assay on Nicotiana glutinosa leaves. The total number of local lesions represented on a leaf of N. glutinosa were counted and analyzed with phosphate buffer treated leaves as a negative control. The leaves treated with a 0.1% concentration of CS or PCS compounds exhibited an inhibition effect by 40-75% compared with the control leaves. The same treatment significantly reduced about 40% CMV accumulation measured by double antibody sandwich enzyme-linked immunosorbent assay and increased the relative expression levels of the NPR1, PR-1, cysteine protease inhibitor gene, LOX, PAL, SRC2, CRF3 and ERF4 genes analyzed by quantitative reverse transcriptase-polymerase chain reaction, in chili pepper plants.

Catabolic Enzyme Activities and Physiological Functionalities of Lactic Acid Bacteria Isolated from Korean Traditional Meju (재래식 메주에서 분리한 유산균들의 각종 효소활성 및 기능성)

  • Jeong, Ji-Kang;Zheng, Yanfei;Choi, Hye-Sun;Han, Gwi-Jung;Park, Kun-Young
    • Journal of the Korean Society of Food Science and Nutrition
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    • v.39 no.12
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    • pp.1854-1859
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    • 2010
  • Three kinds of Korean traditional Meju were selected and the counts of microorganisms in these Meju were determined. The counts of total aerobic bacteria, lactic acid bacteria and yeasts and molds were 107~108, 106~108 and 107~108 cfu/g, respectively in three Meju and lactic acid bacteria were important microorganisms in the fermentation of Meju. Therefore, we isolated three kinds of dominant lactic acid bacteria from these Meju. They were identified as Leuconostoc mesenteroides (98%, Lm-SMm), Lactobacillus plantarum (99%, Lp-SMm) and Lactococcus lactis (98%, Ll-GAm). Then, enzyme activities and physiological functionalities of three lactic acid bacteria were investigated. Protease, lipase and $\alpha$-amylase activities were detected in three lactic acid bacteria, Ll-GAm showed relatively higher activities than other two lactic acid bacteria. Lm-SMm, Lp-SMm and Ll-GAm showed 45, 48 and 60% of antioxidative activity to 1,1-diphenyl-2-picryhydrazyl (DPPH), and exhibited 45, 67 and 70% of inhibitory effects in HT-29 human colon cancer cells, respectively. These results indicate that three lactic acid bacteria isolated from traditional Meju, especially Ll-GAm are applicable to Meju preparation for soybean paste industry.

Antioxidant, Antibacterial and Anti-inflammatory Effects of Stachys sieboldii Extract (초석잠 추출물의 항산화, 항균 및 항염 활성)

  • Lee, Jeong Ho
    • Korean Journal of Plant Resources
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    • v.34 no.5
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    • pp.420-432
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    • 2021
  • This study was conducted to evaluate antioxidant, antibacterial, anti-inflammatory, and digestive enzyme activity in water extract (SAW) and 60% ethanol extract (SAE) from Stachys sieboldii. As the treatment concentration of each extract S. sieboldii extract increased, antibacterial and antioxidant activity increased. The total polyphenol and total flavonoid contents of SAW were 106.25 ± 0.94 mgGAE/g, 24.4 ± 0.24 mgQE/g and SAE were 124.61 ± 1.11 mgGAE/g, 45.2 ± 3.52 mgQE/g, respectively. The 400 ㎍/mL of SAW and SAE performed more than 53% protective effects against oxidative stress in HepG2 cell lines. All extracts were not showed cytotoxicity to RAW 264.7 cell line at 100 ㎍/mL. NO production was reduced to 44.3 ± 1.4% for SAW and 45.1 ± 1.0% for SAE at a concentration of 100 ㎍/mL. The production of inflammatory cytokines each TNF-α, IL-1β, and IL-6 was inhibited in a concentration-dependent. S. sieboldii extract did not showed Caco-2 cells cytotoxicity and inhibited NO production in concentration-dependent. As the concentration of the S. sieboldii extract increased of α-amylase and protease enzymes activity, which are digestive enzyme. As a result of the experiment, it is judged that it can be used as basic data for the development of health food using S. sieboldii.

Identification of Microorganisms, Cladosporium sp. and Sterigmatomyces sp., Proliferated on the Surface of Traditional Soy Sauce, and the Effect of NaCl Concentration on Their Enzymatic Activity (전통간장으로부터 분리한 Cladosporium sp.과 Sterigmatomyces sp. 미생물의 동정 및 NaCl 농도에 따른 미생물 효소활성 분석)

  • Lee, Nam-Keun;Ryu, Young-Jun;Yeo, In-Cheol;Park, Sung-Joon;Kwon, Ki-Ok;Cha, Chang-Jun;Hahm, Young-Tae
    • Korean Journal of Food Science and Technology
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    • v.44 no.4
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    • pp.488-492
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    • 2012
  • Two strains, traditionally referred to as rock flower (Bawhi-kot) and buckwheat flower (Memil-kot or Chile-Kot), were isolated from stored traditional soy sauce and were identified by using the 18S ITS1/4 region sequences. The rock flower strain showed 99% of similarity with Cladosporium sp. and buckwheat flower strain was 99% identical with yeast Sterigmatomyces halophilus. Both strains were tentatively named Cladosporium sp. NK1 and Sterigmatomyces halophilus NK2, respectively. The optimal growth pHs and temperatures of both strains in a YPD broth medium were in the range of pH 5.0 to 7.0 and 22 to $27^{\circ}C$. Both strains were able to grow in more than 20% of NaCl. In the enzyme activity assay, high protease activity of Cladosporium sp. NK1 and S. halophilus NK2 were obtained in YPD containing 10% of NaCl. High amylase activities of both stains were in 15% and 5% of NaCl, respectively. Lipase activity was, however, not detected in both strains.

Study on the Whitening Efficacy and Skin Barrier by Lysosome-related Organelle Extract (LOE) from Egg White (난백(Egg White)에서 추출한 리소좀 추출물(LOE)의 미백 효능 및 피부장벽에 관한 연구)

  • Choi, Da Hee;Jeon, Gyeongchan;Yoon, Jihee;Min, Jiho;Park, Si Jun;Kim, Jung Su;Hwang, Ee Taek;Hwang, Hyung Seo
    • Journal of the Society of Cosmetic Scientists of Korea
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    • v.45 no.4
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    • pp.389-397
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    • 2019
  • Lysosomes are cellular organelles involved in energy metabolism and intracellular digestion in eukaryotic cells, including protease, nuclease, glycosidase, lipase, and phosphatase. Our previous studies have confirmed that egg white lysosomes had melanin decolorization and reduction activity. However, there have been few studies on skin barrier and skin regeneration as well as inhibition of melanin production by egg white lysosomes on B16F10 melanocyte cell line. In this study, we attempted to identify the effect of lysosome-related organelle extract (LOE) extracted from egg white on the melanin content change and skin barrier enhancement in cells. First, cytotoxicity evaluation was performed on B16F10 melanocyte cell line to confirm the whitening efficacy of LOE. Cytotoxicity by LOE was not observed at 20 mg/mL concentration, but cytotoxicity was observed at 40 mg/mL, and the maximum concentration value was set to 20 mg/mL in all subsequent experiments. LOE samples of 5, 10, 20 mg/mL inhibited melanin production by 61.5 ± 4.0%, 61.4 ± 7.3%, 58.3 ± 8.3%, respectivly, compared to α-MSH, a negative control in melanin contents assay. MITF mRNA expression was reduced by about 39.7 ± 3.2% compared to the α-MSH treatment group. TEER assay using HaCaT showed that LOE increased TEER resistance in a dose-dependent manner, indicating that LOE is involved in strengthening the skin barrier. LOE also increased the TEER resistance under TNF-α treatment. Skin barrier was normally restored by LOE even under the condition of inflammation. LOE had a positive effect on cell division and cell migration promotion, confirmed by the observing the effect of promoting cell migration by LOE through cell migration assay. Taken together, we expect that LOE can be developed as a cosmetic material to enhance has effects on skin regeneration and skin barrier strengthening as well as whitening function if enzyme stabilization and formulation technology are combined.

Correlation of expression and activity of matrix metalloproteinase-9 and -2 in human gingival cells of periodontitis patients

  • Kim, Kyung-A;Chung, Soo-Bong;Hawng, Eun-Young;Noh, Seung-Hyun;Song, Kwon-Ho;Kim, Hanna-Hyun;Kim, Cheorl-Ho;Park, Young-Guk
    • Journal of Periodontal and Implant Science
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    • v.43 no.1
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    • pp.24-29
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    • 2013
  • Purpose: Matrix metalloproteinases (MMPs) are capable of degrading extracellular matrix, and they are inducible enzymes depending on an inflammatory environment such as periodontitis and bacterial infection in periodontal tissue. Gingival inflammation has been postulated to be correlated with the production of MMP-2 and MMP-9. The objective of this study was to quantify the expression and activity of MMP-9 and -2, and to determine the correlation between activity and expression of these MMPs in human gingival tissues with periodontitis. Methods: The gingival tissues of 13 patients were homogenized in $500{\mu}L$ of phosphate buffered saline with a protease inhibitor cocktail. The expression and activity of MMP-2 and -9 were measured by enzyme-linked immunosorbent assay and Western blot analysis, and quantified by a densitometer. For the correlation line, statistical analysis was performed using the Systat software package. Results: MMP-9 was highly expressed in all gingival tissue samples, whereas MMP-2 was underexpressed compared with MMP-9. MMP-9 activity increased together with the MMP-9 expression level, with a positive correlation (r=0.793, P=0.01). The correlation was not observed in MMP-2. Conclusions: The expression of MMP-2 and -9 might contribute to periodontal physiological and pathological processes, and the degree of MMP-9 expression and activity are predictive indicators relevant to the progression of periodontitis.

Expression of Various Pattern Recognition Receptors in Gingival Epithelial Cells

  • Shin, Ji-Eun;Ji, Suk;Choi, Young-Nim
    • International Journal of Oral Biology
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    • v.33 no.3
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    • pp.77-82
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    • 2008
  • Innate immune response is initiated by the recognition of unique microbial molecular patterns through pattern recognition receptors (PRRs). The purpose of this study is to dissect the expression of various PRRs in gingival epithelial cells of differentiated versus undifferentiated states. Differentiation of immortalized human gingival epithelial HOK-16B cells was induced by culture in the presence of high $Ca^{2+}$ at increased cell density. The expression levels of various PRRs in HOK-16B cells were examined by realtime reverse transcription polymerase chain reaction (RTPCR) and flow cytometry. In addition, the expression of human beta defensins (HBDs) was examined by real time RT-PCR and the amounts of secreted cytokines were measured by enzyme linked immunosorbent assay. In undifferentiated HOK-16B cells, NACHT-LRR-PYDcontaining protein (NALP) 2 was expressed most abundantly, and toll like receptor (TLR) 2, TLR4, nucleotide-binding oligomerization domain (NOD) 1, and NOD2 were expressed in substantial levels. However, TLR3, TLR7, TLR8, TLR9, ICE protease-activating factor (IPAF), and NALP6 were hardly expressed. In differentiated cells, the levels of NOD2, NALP2, and TLR4 were different from those in undifferentiated cells at RNA but not at protein levels. Interestingly, differentiated cells expressed the increased levels of HBD-1 and -3 but secreted reduced amount of IL-8. In conclusion, the repertoire of PRRs expressed by gingival epithelial cells is limited, and undifferentiated and differentiated cells express similar levels of PRRs.

Selection of Biocontrol Agent of Tomato Gray Mold Disease from Flower and Pollinator Hive (토마토 꽃과 수정용 벌집으로부터 잿빛곰팡이병 방제 길항균주 선발)

  • Kim, Da-Ran;Lee, Jun-Taek;Kim, Hye sun;Jeon, Chang Wook;Kwak, Youn-Sig
    • The Korean Journal of Pesticide Science
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    • v.21 no.1
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    • pp.90-96
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    • 2017
  • Gray mold disease, cause by Botrytis cinerea, occurs severe damage on varieties of fruit and vegetable production, and have no a critical control method. In case of chemicals control, it is a trigger emergence of drug resistance strains due to using them continuously. In addition, the pathogen is difficult to control naturally because it is possible to survive regardless of host status. In this study, microorganisms were isolated from tomato flower and hive samples and in order to select suitable microbial control agents for tomato gray mold disease. During six-months study, we isolated 1,004 isolates from flower and 925 isolates from pollinator hive samples. Among them, 6 strains were selected based on result of antifungal activity test. The selected strains showed not only strong antifungal activity against gray mold pathogen, but also cellulase and protease enzyme activities. The selected strains were identified as Paenibacillus polymyxa. In plant assay, P. polymyxa prevented the gray mold disease occurrence near 75%.

The Identification and Physiological Properties of Lactobacillus plantarum JK-01 Isolated from Kimchi (김치로부터 분리한 Lactobacillus plantarum JK-01의 동정 및 생리적 특성)

  • Cho, Jin-Koo;Li, Guan-Hao;Cho, Sung-Jin;Yoon, Yoh-Chang;Hwang, Seong-Gu;Heo, Kang-Chil;Choe, Il-Shin
    • Food Science of Animal Resources
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    • v.27 no.3
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    • pp.363-370
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    • 2007
  • In order to identify probiotic microorganisms, 25 isolates of Lactobacillus sp. were selected from kimchi based on their growth rates, lactic acid production and salt tolerance. The isolate JK-01 was identified as Lactobacillus plantarum by the API kit and 16S rDNA analysis (99.9% of homology), and named as L. plantarum JK-01. The maximum number of L. plantarum JK-01 was reached at 18 hr fermentation in MRS broth and the pH gradually decreased to 4.5. L. plantarum JK-01 showed high enzyme activities for xylanase, amylase, protease, and phytase on MRS agar plates containing each substrate. L. plantarum JK-01 showed high resistance to acidic pH and bile salts, and grew well even at pH 2.0 and 1.0% bile salt. In particular, L. plantarum JK-01 showed high heat stability as shown by $3.3{\times}10^3$ CFU/mL at $60^{\circ}C$. The isolate showed remarkable antimicrobial activity against E. coli in MRS broth based on its disappearance after 18 hr and clear zone formation using a paper disk assay. These results suggest that L. plantarum JK-01 may be probiotic in nature.