• Title/Summary/Keyword: protease treatment

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Production and characterization of lentivirus vector-based SARS-CoV-2 pseudoviruses with dual reporters: Evaluation of anti-SARS-CoV-2 viral effect of Korean Red Ginseng

  • Jeonghui Moon;Younghun Jung;Seokoh Moon;Jaehyeon Hwang;Soomin Kim;Mi Soo Kim;Jeong Hyeon Yoon;Kyeongwon Kim;Youngseo Park;Jae Youl Cho;Dae-Hyuk Kweon
    • 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.

Effect of partially replacing soybean meal with sunflower meal with supplementation of multienzymes on growth performance, carcass characteristics, meat quality, ileal digestibility, digestive enzyme activity and caecal microbiota in broilers

  • Yaqoob, Muhammad Umar;Yousaf, Muhammad;Imran, Safdar;Hassan, Safdar;Iqbal, Waqar;Zahid, Muhammad Umer;Ahmad, Naveed;Wang, Minqi
    • Animal Bioscience
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    • v.35 no.10
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    • pp.1575-1584
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    • 2022
  • Objective: An experiment was conducted to evaluate the effects of partially replacing soybean meal (SBM) with sunflower meal (SFM) with added exogenous multienzymes (MEs) on various biological parameters in broilers. Methods: One week-old, 400 broiler chicks were randomly divided into four treatments (control, 3SFM, 6SFM, and 9SFM) with 5 replicates/treatment (20 chicks/replicate). Control diet was without SFM and MEs, while diets of 3SFM, 6SFM, and 9SFM treatments were prepared by replacing SBM with SFM at levels of 3%, 6%, and 9%, respectively, and were supplemented with MEs (100 mg/kg). Feeding trial was divided into grower (8 to 21 day) and finisher phases (22 to 35 day). External marker method was used to measure the nutrient digestibility. At the end of trial, twenty birds (one birds per replicate) with similar body weight were slaughtered for samples collection. Results: No significant effect of dietary treatments was found on all parameters of growth performance and carcass characteristics, except relative weight of bursa. Weight (25.0 g) and length (15.80 cm) of duodenum were significantly (p<0.05) higher in 3SFM than control. Lowest (p<0.05) villus height/crypt depth ratio was found in 3SFM and 9SFM than control. Most of meat quality parameters remained unaffected, however, highest pH of breast meat (6.16) and thigh meat (6.44) were observed in 9SFM and 3SFM, respectively. Lowest (p<0.05) cook loss of thigh meat was found in 6SFM (31.76%). Ileal digestibility of crude protein was significantly (p<0.05) higher in 3SFM (72.35%) than control (69.46%). In addition, amylase (16.87 U/mg) and protease (85.18 U/mg) activities were significantly (p<0.05) higher in 3SFM than control. However, cecal microbial count remained unaffected. Conclusion: Partial replacement (up to 9%) of SBM with SFM, with added MEs can help to improve the nutrient digestibility, intestinal morphology, and digestive enzyme activities without affecting cecal microbial count and growth performance in broilers.

Effect of Natural Tenderizers or Phosphates on Quality Improvement of the Low-grade Seasoned Hanwoo Ribs (천연연화제 및 인산염의 첨가가 저급양념한우갈비의 품질개선에 미치는 효과)

  • Kim, K. J.;Min, J. S.;Lee, S. O.;Jang, A.;Jang, S. H.;Cheon, Y. H.;Lee, M.
    • Journal of Animal Science and Technology
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    • v.45 no.2
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    • pp.309-318
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    • 2003
  • In order to improve the quality of low-grade seasoned ribs, kiwi powder, pear powder and phosphates were studied. To seasoned ribs, 0.1%, 0.3% or 0.5% of kiwi powder was added and their MFI(Myofibrillar Fragmentation Index) values were 341.4, 368.3 and 405.1, respectively. As the amount of kiwi increased, MFI value increased(p<0.001). Also, when 0.5%, 1.0% or 3.0% of pear powder was added to seasoned ribs, their MFI values were increased as the addition levels of pear powder increased. As the amount of kiwi and pear powder were increased, WHC(Water Holding Capacity) decreased(p<0.001). On the other hand, as the amount of phosphates added increased, WHC increased. In drip loss, as the amount of kiwi and pear powder increased, it increased. However, drip loss was decreased as the amount of phosphates increased. For meat color, ‘L’ values of pear treatment groups were similar to those of control and ‘a’ values were higher in the treatment groups of 0.1${\sim}$0.3% kiwi powder, 0.5${\sim}$1.0% pear powder and 0.3${\sim}$0.5% phosphate than those of control. In sensory evaluation, treatment group of 0.3% kiwi powder and 0.5% phosphate showed the highest values in juiciness, tenderness and acceptability among the treatments(p<0.01).

Total Folate Contents of 15 Edible Plants Consumed in Korea Using Trienzyme Extraction Method (국내 소비되는 봄나물의 Trienzyme 추출법을 적용한 엽산 함량 분석)

  • Kim, Bo Min;Kim, So-Min;Oh, Ji Yeon;Cho, Young-Sook;Kim, Se-Na;Choi, Youngmin
    • Journal of the Korean Society of Food Science and Nutrition
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    • v.43 no.11
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    • pp.1796-1800
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    • 2014
  • Trienzyme digestion (AOAC Official Method 2004.05) procedure using protease, ${\alpha}$-amylase, and chicken pancreas conjugase was evaluated to determine its usefulness in the microbiological quantitation of total folate in foods. Folate values obtained by alkali hydrolysis (Korean Food Standards Codex) were compared to those obtained by the trienzyme method for four certified reference materials (CRM) representing diverse matrixes. Trienzyme treatment increased measurable folate from most CRM compared to levels found after alkali hydrolysis. The largest increases were observed with CRM 487 (pig liver, 5.8-fold) and CRM 121 (whole meal flour, 3.1-fold) after trienzyme digestion. Using trienzyme digestion method, total folate contents of raw and blanched edible plants were determined. Eleutherococcus senticosus ($146.9{\mu}g/100g$) showed the highest total folate content, followed by Aster glehni F. Schmidt ($142.8{\mu}g/100g$) and Ledebouriella seseloides H. Wolff ($140.4{\mu}g/100g$) on a wet weight basis. Blanching of samples resulted in an insignificant decrease in folate content for five samples and 11~63% reduction for nine samples. Our finding suggests that trienzyme digestion method is accurate for the determination of food folate in leafy vegetables.

Screening of 94 Plant Species Showing ACE Inhibitory Activity (식물자원으로부터 Angiotensin Converting Enzyme 저해활성 탐색)

  • Yun, Jeong-Sik;Chung, Byung-Hee;Kim, Na-Young;Seong, Nak-Sul;Lee, Hyeon-Yong;Lee, Jin-Ha;Kim, Jong-Dai
    • Korean Journal of Medicinal Crop Science
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    • v.11 no.3
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    • pp.246-251
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    • 2003
  • Angiotensin converting enzyme(ACE) belongs to the class of zinc protease and plays an important role in the regulation of blood pressure. In this experiment, we investigated the inhibitory activities of ninety four plant extracts on ACE. The extracts were prepared by water and refluxing with 70% and 100% methanol. Among the extracts, two plant extracts such as Cassia tora, Persicaria cochinchinensis Kitagawa showed more than 60% inhibitory activities, and Foeniculum vulgare Gaertner, Scutellaria baicalensis Georgl, Caragana sinica (Buchoz) Rehder, Inula britannica var. chinensis showed $45.2{\sim}49.7%$ inhibitory activities. Twenty eight plant extracts such as Hemerocallis fulva L, Camptotheca acuminata Decne, Inula britannica var. chinensis, Xanthium strumarium, Polygonatum odoratum, Phellodendron amurense Rupr, Coix lachryma-jobi var. mayuen, Prunus ansu, Hibiscus mutabilis L, Thchosanthes kirilowii, Helianthus annuus, Juglans sinensis showed $30.3{\sim}39.7%$ Inhibitory activities. These results suggest that plant extracts which contain high ACE inhibitory activities may be useful as anti-hypertension agents and to the treatment of hypertension.

Biochemical Characterizations of Phenylalanine Ammonia-Lyase and its Mutants to Develop an Enzymatic Therapy for Phenylketonuria (페닐케톤뇨증의 효소치료 개발을 위한 phenylalanine ammonia-lyase 및 유전자 변이형의 생화학적 특성)

  • Kim, Woo-Mi
    • Journal of Life Science
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    • v.19 no.9
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    • pp.1226-1231
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    • 2009
  • Enzyme substitution with recombinant phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) is currently being explored for treatment of phenylketonuria (PKU), an autosomal recessive genetic disorder with mutations of the gene encoding phenylalanine-4-hydroxylase (EC 1.14.16.1). However, oral administration of PAL is limited because of proteolytic digestion in the gastrointestinal tract. The aim of this study was to determine the biochemical properties of PAL and delinate the susceptibility of wild-type PAL to pancreatic proteolysis by exploring several mutants, and to develop therapeutic drugs with PAL for PKU. The specific activity of PAL was assayed and its optimal pH, temperature stability, and intestinal protease susceptibility were investigated. Its $V_{max}$ values for phenylalanine and tyrosine were 1.77 and $0.47{\mu}mol$/ min/mg protein, respectively, and its $K_m$ values were $4.77{\times}10^{-4}$ and $4.37{\times}10^{-4}\;M$, respectively. PAL showed an optimal pH at 8.5, corresponding to the average pH range of the small intestine. It showed no loss of activity at $-80^{\circ}C$ for 5 months and possessed 93.4% of its activity under $4^{\circ}C$ for 4 wks. PAL was susceptible to chymotrypsin digestion and, to a lesser extent, to trypsin, elastase, carboxypeptidase A, and B. The trypsin and chymotrypsin cleaving sites were mutated to investigate protection from pancreatic digestion and the specific activities of these mutants were evaluated. The six mutants displayed low specific activities compared to the wild-type, suggesting that the primary trypsin and chymotrypsin cleaving sites may be essential for catalytic reaction. The PAL mutants could therefore be applied as a pretreatment modality without susceptibility to proteolytic attack, however, additional modification for enhancing enzymatic activity is needed to reduce the Phe levels effectively.

Studies on Rheological Characterization of Barley ${\beta}-Glucan$ [mixed-linked $(1-3),(1-4)-{\beta}-D-Glucan$] (보리 ${\beta}-Glucan$ [mixed-linked $(1-3),(1-4)-{\beta}-D-Glucan$의 리올로지 특성)

  • Kim, Mi-Ok;Cha, Hee-Sook;Koo, Sung-Ja
    • Korean Journal of Food Science and Technology
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    • v.25 no.1
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    • pp.15-21
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    • 1993
  • Crude ${\beta}-glucan$ extracted from Barley was purified by stepwise enzyme treatment (Thermostable ${\alpha}-amylase$, amyloglucosidase, protease). The Intrinsic Viscosity $[{\eta}]$ of the purified ${\beta}-glucan$ was determined by Cannon Fenske Capillary Viscometer (size 50, Cannon Instruments, State, College pa.) at different pH (2, 4, 7, 9, 11) and various salt concentration (0.01 M, 0.03 M, 0.05 M, 0.07 M, 0.1 M and 0.2 M). The $[{\eta}]$ of purified ${\beta}-glucan$ was ranged from $0.997{\sim}2.290\;dl/g$. The $[{\eta}]$ of purified ${\beta}-glucan$ at both alkali, acid condition were lower than those at pH 7. However, the alkali condition of puified ${\beta}-glucan$ solution showed less $[{\eta}]$ than the acid condition of this solution. From 0 M to 0.2 M salt concentration, the $[{\eta}]$ of purified ${\beta}-glucan$ solution was decreased to 0.03 M then increased to 0.05 M NaCl and remained constant to 0.2 M NaCl. The chain stiffness parameter of purified ${\beta}-glucan$ was not affected by temperature from $15^{\circ}C$ to $65^{\circ}C$. The shear rates of various ${\beta}-glucan$ conditions were determined by Bohlin Rheometer (Lund, Sweden). The ${\beta}-glucan$ concentration of 1.0 g/dl and 2.0 g/dl behaved as Newtonian fluid. However, above the concentration of 3.0 g/dl ${\beta}-glucan$ solution, it showed thixotropic and psedoplastic characteristics. Barley ${\beta}-glucan$ appears a damping at 0.5 frequency for the 4.0 g/dl solution. Below 0.5 frequency, it appears a viscous behavior property and above 0.5 frequency, it appears a elastic behavior property.

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Studies on the Mutation of Aspergillus niger (흑국균(黑麴菌)의 인공변이(人工變異)에 관(關)한 연구(硏究))

  • Park, Yoon-Joong;Sohn, Cheon-Bae
    • Korean Journal of Food Science and Technology
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    • v.14 no.1
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    • pp.72-79
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    • 1982
  • Several mutants were isolated from the parent strain of Aspergillus niger CF: the first mutant strain CF-11 was obtained by UV irradiation, and the second mutant strain CF-21 and CF-22 were from NTG (N-methyl-N'-nitroso-N-nitroso-guanidine) treatment on the CF-11. These mutants were characterized, and their enzyme and acid production on wheat bran Koji and wheat flour Koji were studied. Asp. niger CF-22 mutant appeared to be tan type which conidial heads were discolored. It's glucoamylase activity was inreased approximately two times and its ${\alpha}-amylase$ about 50 percent as compared with that of the parent strain of Asp. niger CF, when grown on wheat bran Koji under the optimal conditions. Asp. niger CF-21 mutant showed slower growth rate and poor sporulation than the wild type, although its conidial heads were not discolored. Approximately 4-fold increment in its acid production was observed as compared with the weld type. The activities of glucoamylase and ${\alpha}-amylase$ of the Asp. niger CF-22 and CF-21 mutants were higher than those of the wild type, but their protease activity was rather lower. The maximum production of glucoamylase by the Asp. niger CF-22 mutant was obtained after 2 to 3 days incubation on wheat bran at 30 to $35^{\circ}C;$ ${\alpha}-amylase$2 days incubation at 30 to $35^{\circ}C$. The maximal levels of acid production by the mutant CF-21 was appeared after 2 days incubation on wheat bran Koji, and after 3 days on wheat flour Koji at $30^{\circ}C$. Little differences in the levels of acid production were observed between on wheat bran and flour Koji.

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Biological Control of Root-knot Nematode by Streptomyces sampsonii KK1024 (Streptomyces sampsonii KK1024를 이용한 뿌리혹선충 (Root-knot nematode)의 생물학적 방제)

  • Kim, Sang-Su;Kang, Seon-I;Kim, Jin-Si;Lee, Yong-Sung;Hong, Sung-Hyun;Naing, Kyaw Wai;Kim, Kil-Yong
    • Korean Journal of Soil Science and Fertilizer
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    • v.44 no.6
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    • pp.1150-1157
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    • 2011
  • Streptomyces sampsonii KK1024 having strong chitinolytic activity was isolated from crab-shell rich soil at Muan, Jeolanamdo. The KK1024 produced chitinase, protease, gelatinase and lipase. When 50% of KK1024 culture broth was treated to juveniles and eggs of root-knot nematode, juvenile mortality at 3 days was 81.67% and egg hatch rate at 5 days was 2.00%. When $183.7{\mu}g\;mL^{-1}$ of crude enzyme produced by KK1024 was treated, juvenile mortality at 3 days was 96.00% and egg hatch rate at 5 days was 5.33%. At 1% of butanol extract from KK1024, juvenile mortality was highest with 90.00% and egg hatch rate was lowest with 0%. The comparison of the effect of KK1024 culture broth with only medium, synthetic fertilizer, and commercial nematicide on tomato growth and nematode infection was examined in pot trials. KK1024 culture broth showed lower number of egg mass and gall in plant, and population of juveniles in soil compared with only medium and synthetic fertilizer treatment, but not in commercial nematicide. However, the highest shoot weight and length was discovered in KK1024 culture broth. These results suggest that Streptomyces sampsonii KK1024 producing lytic enzymes and nematicidal compounds can be one of candidates for biocontrol agents against root-knot nematodes.

Inhibitory Effects of Rosa rugosa Crude Extract and Solvent Fractions on Adipogenic Differentiation of 3T3-L1 Preadipocytes (해당화 추출물이 3T3-L1 지방세포 분화에 미치는 영향)

  • Lee, Hyunjung;Yang, Jiho;Choi, Mi-na;Jeon, Seongeun;Zhou, Xianrong;Oh, Jung Hwan;Karadeniz, Fatih;Seo, Youngwan;Kong, Chang-Suk
    • Journal of Life Science
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    • v.32 no.12
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    • pp.979-988
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    • 2022
  • Halophytes have been reported to possess a variety of physiological activities, such as anti-cancer, anti-oxidant, anti-diabetes, anti-inflammatory, and anti-obesity. Studies on the roots of the halophyte Rosa rugosa, in particular, have shown a variety of physiological activities and are known to be effective for nursing diabetic complications in traditional Korean medicine. In this study, the effect of R. rugosa on adipogenesis was investigated in 3T3-L1 pre-adipocytes treated with crude extract and solvent fractions (H2O, n-BuOH, 85% aq. MeOH, and n-Hex) obtained from R. rugosa roots. Treatment with extract and the solvent fractions inhibited the formation of intracellular lipid droplets in differentiated 3T3-L1 adipocytes compared to the untreated group. In particular, n-BuOH and 85% aq. MeOH fractions effectively decreased the expression of adipogenic transcription factors: peroxisome proliferator activated receptor-γ (PPARγ), CCAAT/enhancer-binding protein α (C/EBPα), and sterol regulatory element-binding protein 1c (SREBP1c) in both mRNA and protein levels. In conclusion, these results suggest that R. rugosa contains anti-adipogenic molecules that can be utilized as a nutraceutical against obesity. Further refining of n-BuOH and 85% aq. MeOH fractions and analysis of their action mechanisms could yield potential therapeutic agents with anti-adipogenic effects.