• Journal of Microbiology and Biotechnology
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• 제26권4호
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• pp.684-692
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• 2016

Acute respiratory virus infectious diseases are a growing health problem, particularly among children and the elderly. Much effort has been made to develop probiotics that prevent influenza virus infections by enhancing innate immunity in the respiratory tract until vaccines are available. Lactobacillus plantarum GB-LP2, isolated from a traditional Korean fermented vegetable, has exhibited preventive effects on influenza virus infection in mice. To identify the molecular basis of this strain, we conducted a whole-genome assembly study. The single circular DNA chromosome of 3,284,304 bp was completely assembled and 3,250 protein-encoding genes were predicted. Evolutionarily accelerated genes related to the phenotypic trait of anti-infective activities for influenza virus were identified. These genes encode three integral membrane proteins, a teichoic acid export ATP-binding protein and a glucosamine - fructose-6-phosphate aminotransferase involved in host innate immunity, the nonspecific DNA-binding protein Dps, which protects bacteria from oxidative damage, and the response regulator of the three-component quorum-sensing regulatory system, which is related to the capacity of adhesion to the surface of the respiratory tract and competition with pathogens. This is the first study to identify the genetic backgrounds of the antiviral activity in L. plantarum strains. These findings provide insight into the anti-infective activities of L. plantarum and the development of preventive probiotics.

• Journal of Microbiology and Biotechnology
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• 제19권7호
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• pp.727-733
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• 2009

Heat shock protein 70 kDa (hsp70), a molecular chaperone involved in folding of nascent proteins, has been studied for its ability to activate innate and specific immunity. High purity hsp70 preparation is generally required for immunization experiments, because endotoxins and other immunologically active contaminants may affect immune responses independently of hsp70. We have developed a novel modification of E. coli-expression medium that enabled a simple two-step production and purification method for endotoxin-free recombinant hsp70. During Ni-NTA-based affinity purification of hsp70, a contaminating protein from host E. coli cells, L-glutamine-n-fructose-6-phosphate aminotransferase (GFAT), was identified. By testing various compounds, supplementation of growth medium with a GFAT metabolite,N-acetylglucosamine, was found to reduce GFAT expression and increase the total hsp70 yield five times. The new protocol is based on column purification of His-tagged hsp70 protein produced by E. coli with the modified medium, followed by endotoxin removal by Triton X-114 extraction. This approach yielded hsp70 with high purity and minimal endotoxin contamination, making the final product acceptable for immunization experiments. In summary, a simple modification of growth medium allowed production of recombinant mouse hsp70 in high yield and purity, thus compatible with immunological studies. This protocol may be useful for production of other Histagged proteins expressed in E. coli.

• Journal of Animal Science and Technology
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• 제64권3호
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• pp.481-499
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• 2022

This study aims to determine the effects of D-methionine (D-Met) isomer and the methionine precursor 2-hydroxy-4-methylthiobutanoic acid i (HMBi) supplementation on milk protein synthesis on immortalized bovine mammary epithelial cell (MAC-T). MAC-T cells were seeded using 10-cm dishes and cultured in Dulbecco's modified Eagle's medium/F12 (DMEM/F12) basic medium. The basic medium of DMEM/F12 was replaced with the lactogenic DMEM/ F12 differentiation medium when 90% of MAC-T cells reached confluency. The best dosage at 0.6 mM of D-Met and HMBi and incubation time at 72 h were used uniformly for all treatments. Each treatment was replicated six times wherein treatments were randomly assigned in a 6-well plate. Cell, medium, and total protein were determined using a bicinchoninic acid protein assay kit. Genes, proteomics and metabolomics analyses were also done to determine the mechanism of the milk protein synthesis pathway. Data were analyzed by two-way analysis of variance (ANOVA) with supplement type and plate as fixed effects. The least significant difference test was used to evaluate the differences among treatments. The HMBi treatment group had the highest beta-casein and S6 kinase beta-1 (S6K1) mRNA gene expression levels. HMBi and D-Met treatments have higher gene expressions compared to the control group. In terms of medium protein content, HMBi had a higher medium protein quantity than the control although not significantly different from the D-Met group. HMBi supplementation stimulated the production of eukaryotic translation initiation factor 3 subunit protein essential for protein translation initiation resulting in higher medium protein synthesis in the HMBi group than in the control group. The protein pathway analysis results showed that the D-Met group stimulated fructose-galactose metabolism, glycolysis pathway, phosphoinositide 3 kinase, and pyruvate metabolism. The HMBi group stimulated the pentose phosphate and glycolysis pathways. Metabolite analysis revealed that the D-Met treatment group increased seven metabolites and decreased uridine monophosphate (UMP) production. HMBi supplementation increased the production of three metabolites and decreased UMP and N-acetyl-L-glutamate production. Taken together, D-Met and HMBi supplementation are effective in stimulating milk protein synthesis in MAC-T cells by genes, proteins, and metabolites stimulation linked to milk protein synthesis.

• The Korean Journal of Physiology and Pharmacology
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• 제3권4호
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• pp.447-454
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• 1999

The production of nitric oxide (NO) and the expression of inducible nitric oxide synthase (iNOS) are known to be modulated by a variety of factors. Recent study showed that endotoxin-induced NO synthesis and iNOS expression were greatly enhanced by elevation of extracellular glucose concentration in murine macrophages. Although this was suggested to be due to the activation of protein kinase C (PKC) via sorbitol pathway, there was lack of evidence for this speculation. This study was performed to delineate the underlying intracellular mechanisms of glucose-enhancing effect on endotoxin-induced NO production in Raw264.7 macrophages. The levels of NO release induced by lipopolysaccharide (LPS) significantly increased by the treatment of glucose in a concentration dependent manner and also, this effect was observed in LPS-preprimed cells. Concurrent incubation of cells with PKC inhibitors, H-7 or chelerythrine, and LPS resulted in the diminution of NO production regardless of glucose concentration but this was not in the case of LPS-prepriming, that is, chelerythrine showed a minimal effect on the glucose- enhancing effect. PMA, a PKC activator, did not show any significant effect on glucose-associated NO production. Modulation of sorbitol pathway with zopolrestat, an aldose reductase inhibitor, did not affect LPS-induced NO production and iNOS expression under high glucose condition. And also, sodium pyruvate, which is expected to normalize cytosolic $NADH/NAD^+$ ratio, did not show any significant effect at concentrations of up to 10 mM. Glucosamine marginally increased the endotoxin-induced nitrite release in both control and high glucose treated group. 6-diazo-5-oxonorleucine (L-DON) and azaserine, glutamine: fructose- 6-phosphate amidotransferase (GFAT) inhibitors, significantly diminished the augmentation effect of high glucose on endotoxin-induced NO production. On the other hand, negative modulation of GFAT inhibitors was not reversed by the treatment of glucosamine, suggesting the minimal involvement, if any, of glucosamine pathway in glucose-enhancing effect. In summary, these results strongly suggest that the hexosamine biosynthesis pathway and the activation of PKC via sorbitol pathway do not contribute to the augmenting effect of high glucose on endotoxin induced NO production in macrophage-like Raw264.7 cells.

• 미생물학회지
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• 제30권5호
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• pp.391-396
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• 1992

광자가영양이며 질소고정는을 갖는 사상체의 남조 Anabaena variabilis ATCC 29413 Allen & Arnon (1/8) 의 무질소 최소배지에서 배양하여, 이형세포를 유도하고 생장과 질소고정활성의 변화와 각종 조절요인의 영향을 분석하였다. 질소결핍의 배지에서 생장과 질소고정활성의 증가율은 비례하였고, 생장에 따한 배지내 질산 ($NO^{3}$-N) 의 축적을 보였으며, 배양 6일 후에도 생장은 지속되었으나, 질소고정은 최대활성을 보인후 급격한 감소를 보여 질산의 저해농도와 일치하였다. 중요 환경요인의 영향은 혐기적 조건, 10,000 lux pH 8 및 $30^{\circ}C$ 에서 최적의 생장과 활성을 보였고, 질소화합물은 0.1 mM 이상의 저농도에서 현저히 활성을 저해하였으며, 탄산은 5 mM 에서 활성을 촉진하였으나 보다 높은 농도에서 저해를 보였다. 내염성을 NaCl 20mM 이하이었고 중금속이온은 $Hg^{2+}$>$Cd^{2+}$> $Co^{2+}$>$Zn^{2+}$>$Pb^{2+}$의 순으로 0.3~10 ppm 의 범위에서 활성을 저해하였다. 탄수화물은 0.5-1.0% 에서 생장과 활성을 촉진하였고, 설탕>과당>포도당의 순이었다.

• 한국임상수의학회지
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• 제19권1호
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• pp.80-85
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• 2002

This study was performed to investigate the freezomg condition especially focused on extender composition to achieve good post-thaw viability and motility of canine sperm. Semen were collected from 6 male dogs which had been proved to be fertile in the past and were treated for freezing. Equex-STM paste was contained in both the 1st(3%) and the 2nd(7%) diluent and the 2nd diluent was added to the 1st diluent following glycerol equilibration for an hour and a half. To investigate the effect of Equex-STM paste in the extender on post-thaw canine sperm characteristics, the post-thaw viability, motility, and HOS(Hypoosmotic swelling) values were evaluated according to the different composition of extender with or without Equex-STM paste, thawing conditions, and different thawing media added to thawed semen. 1. Canine sperm removed from seminal plasma and frozen )n Sweden extender containing Equex showed higher post-thaw viability, motility, and HOS values than those frozen in the extender containing Equex-STM paste with seminal plasma and those frozen in the extender without Equex and seminal plasma. 2. Canine sperm frozen in Sweden extender containing Equex-STM paste with 5% glycerol showed higher post-thaw viability, motility, and HOS values than those frozen with 3%, 8% glycerol or 5% DMSO. 3. The canine semen frozen in Sweden extender with 5% glycerol and Equex-STM paste showed higher viability, motility, and HOS values when thawed at $70^{\circ}C$ for 8 seconds than when thawed at $37.5^{\circ}C$ for 1 min and at $18-20^{\circ}C$ for 5 min. 4. TFC (tris -fructose-citrate) and PB S (phosphate buffered saline) medium added immediately to thawed canine semen brought better viability, motility, and HOS values for the sperm than those semen added with TGC(tris-glucose-citrate) and no medium. These results indicated that Equex-STM paste in Sweden extender for freezing the canine sperm which were removed from seminal plasma brought good post-thaw viability and motility of canine sperm. Also of the freezing conditions of canine sperm with the same extender containing Equex, the concentration of 5% glycerol, the thawing condition at $70^{\circ}C$ for 8 sec, and TFC and PBS medium added to the thawed semen brought better post-thaw viability and motility of canine sperm than the other conditions used in this study.

• Journal of Microbiology and Biotechnology
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• 제32권5호
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• pp.630-637
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• 2022

The objective of this study was to optimize industrial-grade media for improving the biomass production of Weissella cibaria JW15 (JW15) using a statistical approach. Eleven variables comprising three carbon sources (glucose, fructose, and sucrose), three nitrogen sources (protease peptone, yeast extract, and soy peptone), and five mineral sources (K₂HPO₄, potassium citrate, ⳑ-cysteine phosphate, MgSO₄, and MnSO₄) were screened by using the Plackett-Burman design. Consequently, glucose, sucrose, and soy peptone were used as significant variables in response surface methodology (RSM). The composition of the optimal medium (OM) was 22.35 g/l glucose, 15.57 g/l sucrose, and 10.05 g/l soy peptone, 2.0 g/l K₂HPO₄, 5.0 g/l sodium acetate, 0.1 g/l MgSO₄·7H₂O, 0.05 g/l MnSO₄·H₂O, and 1.0 g/l Tween 80. The OM significantly improved the biomass production of JW15 over an established commercial medium (MRS). After fermenting OM, the dry cell weight of JW15 was 4.89 g/l, which was comparable to the predicted value (4.77 g/l), and 1.67 times higher than that of the MRS medium (3.02 g/l). Correspondingly, JW15 showed a rapid and increased production of lactic and acetic acid in the OM. To perform a scale-up validation, batch fermentation was executed in a 5-l bioreactor at 37℃ with or without a pH control at 6.0 ± 0.1. The biomass production of JW15 significantly improved (1.98 times higher) under the pH control, and the cost of OM was reduced by two-thirds compared to that in the MRS medium. In conclusion, OM may be utilized for mass producing JW15 for industrial use.

• 한국미생물·생명공학회지
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• 제41권2호
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• pp.190-197
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• 2013

전통 장류에서 점성물질을 분비하는 유산균을 분리하고 높은 활성을 보이는 N45-10, K6-7 및 N58-5의 3균주를 선발하였다. 선발한 3균주의 16S rDNA의 염기서열을 분석한 결과 N45-10은 Leuc. citreum, K6-7, N58-5는 Leuc. mesenteroides로 동정되었다. 산 저항성과 인공위액 저항성은 3균주 중에서 Leuc. citreum N45-10이 높은 생균수($10^5-10^6$ CFU/ml)를 나타내어 산 저항성과 인공위액 저항성이 가장 우수하게 나타났으며, 인공 담즙 저항성은 Leuc. citreum N45-10은 높은 생균수($10^3-10^4$ CFU/ml)를 유지하여 담즙액 저항성이 우수하게 나타났다. 3균주들 중에서 Leuc. citreum N45-10은 EPS 생성량이 가장 많았고, MRS 배지에서 배양하여 EPS가 생성되지 않았을 때보다 슈크로오스 배지에서 배양하여 EPS를 생성하였을 때 생균수가 더 높게 나타났다. 이것은 EPS 생성이 유산균의 세포벽 주위에 보호막으로 작용한 결과로 생각된다. 선발 유산균 3주의 EPS 생산량은 슈크로오스 액체배지에서 각각 16.173, 8.167, 3.652 g/L였으며, Leuc. citreum N45-10은 글루코오스로만 이루어진 homo-polysaccharide, Leuc. mesenteroides K6-7과 N58-5는 프락토오스, 글루코오스로 구성된 hetero-polysaccharides로 동정되었다. 선발 유산균 3주 모두는 용혈 음성반응을 나타내고, 젤라틴 액화능을 나타내지 않아 안전성이 확인되었다.

• 한국식품영양과학회지
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• 제30권6호
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• pp.1060-1067
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• 2001

깍두기에 여러 가지 점증제를 첨가하여 2$0^{\circ}C$에서 숙성시키면서 이화학적, 관능적 특성을 측정한 결과는 다음과 같다. 산도는 점증제 첨가군이 대조군보다 높았으나 숙성 말기 (숙성 7일)에 xanthan gum 첨가군과, 산처리 전분은 대조군보다 낮았다. 유리당 함량은 점증제 첨가군이 담금 직후에는 대조군보다 높았으나 숙성됨에 따라 크게 감소하여 숙성 7일에는 xanthan gum을 제외한 모든 점증제가 대조군보다 낮았다. 주요 유리당으로는 대조군과 점증제 첨가군 모두 포도당과 과당이었으며, 이들 당의 함량은 숙성 기간이 경과됨에 따라 감소하였으나, 만니톨은 증가하였다. 점도는 담금직 후에는 점증제 첨가군이 대조군에 비해 높았으나 숙성초기에 급격히 감소하여 대조군과 유사하였으나 xanthan gum 첨가군의 점도는 대조군보다 매우 높았고 숙성이 진행되어도 점도의 감소정도가 적어 초기 점도를 유지하였다. 경도는 숙성이 진행되면서 감소하였으나 찹쌀, 인산가교전분, xanthan gum 첨가군은 숙성 7일에 대조군에 비해 유의적으로 높았다(p<0.05). 관능적 특성으로 신냄새, 신맛, 감칠맛은 모든 처리군에서 유의적인 차이는 없었다. 군냄새는 숙성 5일에 찹쌀, 밀가루, 산처리 전분 첨가군은 대조군에 비해 유의적으로 높았으며, xanthan gum, 인산가교 전분, 초산아디피산 전분은 유의적인 차이가 없었다. 전분맛은 xanthan gum 첨가군이 숙성 3일 이후에 대조군에 비해 유의적으로 높았다 (p<0.05). 점도는 숙성이 경과됨에 따라 유의적으로 낮아졌 으나 인산가교 전분과 xanthan gum 첨가군은 유의적으로 높았다. 경도는 찹쌀, 초산아디피산 전분, 인산가교 전분 첨가군이 유의적으로 낮았고, xanthan gum 첨가군은 숙성기간이 경과되어도 경도가 높게 유지되었다(p<0.05). 전반적인 기호도는 숙성 7일에 찹쌀, 밀가루, 옥수수 전분 첨가군이 대조군에 비해 유의적으로 낮은 점수를 나타내었고, 산처리 전분, 인산가교 전분, xanthan gum 첨가군은 대조군과 차이가 없었다.