• KSBB Journal
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• v.32 no.1
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• pp.71-82
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• 2017

To verify anti-diabetic effect of oligopeptide with His-Pro repeats (mHP peptide), the oligopeptide was first secreted and optimized using the secretion vector, pRBAS with alkaline protease gene promoter and the signal sequence in Bacillus subtilis and directly the anti-diabetic effect of the mHP peptide was investigated in insulinoma cell, RINm5F cell line. The oligopeptide gene was obtained by annealing oligonucleotides with repeated His-Pro sequence and finally was constructed as 18 dipeptides (108 bp and 4.0 kDa) coding gene, named oligopeptide with His-Pro repeats (mHP peptide) to make cyclo(His-Pro) known to be anti-diabetic effects. The region encoding the oligopeptide gene was subcloned into the pRBAS secretion vector (E.coli-Bacillus shuttle vector) after PCR amplification using the designed primers including initiation and termination codons and His tag, named pRBAS-mHP (6.56 kb). To optimize secretion of the oligopeptide, various culture conditions were investigated in Bacillus subtilis LKS. As a result, the secreted oligopeptide was maximally measured (approximately $59.6{\mu}g/mL$) in 3 L batch culture and the highest secretion was achieved at $30^{\circ}C$, PY medium, and carbon sources (particularly barley and glycerol). In the RINm5F cells treated with 2 mM STZ, the oligopeptide treatment (0.1 mg/mL) restored the cell viability (10%) and reduced the nitric oxide (NO) generation (35%) and DNA fragmentation (90%). And also, insulin secretion level was increased to 17% higher than in STZ-treated RINm5F cells. These results suggest that the oligopeptide with His-Pro repeats could be a candidate material for anti-diabetic agent against STZ-induced diabetes.

• Korean Journal of Food Science and Technology
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• v.41 no.4
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• pp.423-427
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• 2009

This study investigated antithrombotic and hypocholesterolemic activities of defatted soybean grits (DSG) and fermented DSG (FD). The FD was prepared by the solid state fermentation using Bacillus subtilis NUC1 at $40^{\circ}C$ for 24h. The water extracts of fermented DSG (FDW) exhibited higher fibrinolytic activity and inhibition of platelet aggregation induced by ADP than water extracts of DSG (DW). However, the DW and FDW inhibited HMG-CoA reductase activity and significantly decreased the intracellular cholesterol contents in HepG2 cells. In addition, DW treatment did not show any cholesterol adsorption capacity, while FDW demonstrated the highest cholesterol adsorption by 90%. The results suggest that fermented DSG have significant antithrombotic and hypocholesterolemic effects in vitro and these activities were improved during fermentation by B. subtilis NUC1.

• Journal of agriculture & life science
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• v.50 no.4
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• pp.147-155
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• 2016

This study presented a measure for turning by-products, released from land farming sites, into resources. The measure involved adding food by-products such as rice bran and nonfat soybean to the sludge, released from the eel farming sites, inoculating the lactic acid bacteria, Aspergillus oryzae, and Bacillus subtilis by step, fermenting them, and measuring the changed ingredients of the fermented fodder. The water content of the fermented fodder by the step of preparation was the first-step fermented product (14.6%) using the lactic acid bacteria, and the second and third-stage fermented product (33.0% and 34.0% respectively) using Aspergillus oryzae and Bacillus subtilis. The pH level was found to be 5.38 in the first-step fermented product due to the secretion of lactic acid caused by the lactic acid bacteria, and the pH level of the second and third-stage fermented products was 5.66 and 7.26, respectively, showing that the pH level increased. The phytic acid content was 0.126g/100g in the first-step fermented product, 0.004g/100g in the second-stage fermented product, and 0.093g/100g in the third-stage fermented product. The measurement of nitrogen content revealed that the amino nitrogen content was high with 1226.37mg% in the second-stage fermented product, and a little lower with 710.18mg% in the third-stage fermented product. The ammonium nitrogen content increased from 0.988mg/kg in the first-stage fermented product to 1.502mg/kg in the third-stage fermented product. Total nitrogen content increased to 2.78% in the first-stage fermented product, 4.08% in the second-stage fermented product, and 4.85% in the third-stage fermented product. As fermentation continued with the three microbes, the phytic acid decreased, and the protein decomposition rate increased. Also, due to the 3 step fermentation, the low-molecule nitrogen ingredient content increased, suggesting that the fodder was developed to offer high digestion and absorption.

• Journal of Life Science
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• v.20 no.4
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• pp.589-596
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• 2010

We investigated antimicrobial activities of various pine (Pinus densiflora) needle extracts against antibiotic resistant strains of Staphylococcus aureus. Hot water extract showed the highest antimicrobial activity against normal and methicillin resistant Staphylococcus aureus (MRSA), however, it exhibited no antimicrobial activity against penicillin resistant S. aureus (PRSA). Hot water-hexane (HWH), hot water-ethanol (HWE), hexane, and ethanol extracts showed antimicrobial activity against S. aureus, PRSA and MRSA. Minimum inhibitory concentrations (MIC) of HWH, HWE, hexane, and ethanol extracts were 0.05, 0.05, 0.5 and 5 mg/ml, respectively, and HWH and HWE extracts showed the strongest antimicrobial activity among these extracts. Antimicrobial activities of pine needle extracts were stable after heating at $121^{\circ}C$ for 20 min. These results suggested that pine needle extracts can be used as an effective natural antimicrobial agent for food and medical industries.

• Journal of Life Science
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• v.18 no.2
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• pp.287-290
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• 2008

In order to obtain yeast cells producing a bacteriocin, Subpeptin JM4-A or Subpeptin JM4-B, the 48 bp oligonucleotides corresponding to Subpeptin JM4-A and Subpeptin JM4-B genes including codon for start and stop were chemically synthesized and cloned into pAUR123, an yeast expression vector. Transformed yeast cells exhibited growth inhibition of Bacillus subtilis, Escherichia coli and Pseudonomas aeruginosa. This result indicates that yeast cells producing Subpeptin JM4-A or Subpeptin JM4-B possess bacteriocidal properties against both Gram positive B. subtilis and Gram negative E. coli and P. aeruginosa cells. The recombinant yeast strains constructed in this study can be applied in the food preservative or. animal foodfeed.

• Journal of the Korean Society of Food Science and Nutrition
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• v.39 no.6
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• pp.872-879
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• 2010

Textured vegetable protein (TVP) was fermented by the solid-state fermentation using Bacillus subtilis HA and biologically active compounds were produced by fermentation for 7 days. The longer fermentation time resulted in the color change of fermented TVP with strong dark red and yellow color. Melanoidin production rapidly increased until fermentation for 48 hr, but did change afterwards. The 70% ethanol extract of TVP fermented for 24 hr showed higher DPPH radical scavenging effect with $IC_{50}$ of 0.99 mg/mL but longer fermentation did not increase its activity. Also, 70% ethanol extract of TVP fermented for 72 hr indicated higher ABTS radical scavenging effect with $IC_{50}$ of 1.68 mg/mL. Consistency index in TVP fermented for 48 hr was the highest values with 7.89 $Pa{\cdot}s^n$. Viscoelastic properties of TVP fermented for 48 hr were maximally enhanced, and viscous value (G") is higher than the elastic value (G'). The $\gamma$-polyglutamic acid (PGA) content was increased by increasing fermentation time with 37.72% of $\gamma$-PGA at 168 hr. However, levan content and molecular weight of PGA were decreased with increasing fermentation time from 7.83% to 3.91% and 1649.3 kDa to 1286.8 kDa, respectively.

• Korean Journal of Food Science and Technology
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• v.19 no.3
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• pp.212-219
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• 1987

The rec-assay on Bacillus subtilis strains H17$({Rec}^+)$ and M45$({Rec}^-)$, the Ames test with modification of preincubation on Salmonella typhimurium TA98 and TA100 and DNA-breaking test on double strand calfthymus DNA were carried out using enzymatically browned substances obtained from the reaction of Prunus salicina (Red) enzyme and polyphenols. The spore rec-assay of enzymatic browning reaction products of pyrogallol, hydroxyhydroquinone. 3,4-dihydrohyoluene and chlorogenic acid showed non-mutagenic activity The spore rec-assay showed a little influence of ${Zn}^{2+}$ and ${Ni}^{2+}$ on the action of four kinds of enzymatic browning reaction products. The enzymatic browning reaction products of polyphenols did not show DNAbreaking activity. ${Cu}^{2+}$ of various metal ions influenced on DNA-breaking of enzymatic browning reaction products of pyrogallol. However, enzymatic browning reaction products of chlorogenic acid inhibited on DNA-breaking activity. Four kinds of enzymatic browning reaction products showed non-mutagenic activity on Salmonella typhimurium TA98 and TA100 with S-9 mix. In the mutagenicity on Salmonella typhimurium TA98 and TA100 with S-9 mix in the presence of benzo$({\alpha})$pyrene which is the carcinogenic substances, four kinds of enzymatic browning reaction products showed desmutagenic activity.

• Journal of Mushroom
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• v.15 no.4
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• pp.237-243
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• 2017

In order to develop fermented silkworm "Dongchunghacho" (Paecilomyces tenuipes) with improved absorption and increased effectiveness, we fermented Dongchunghacho using four kinds of microorganisms, viz., lactic acid bacteria, Bacillus subtilis, Natto bacillus, and yeast. A total of 15 samples were fermented using a combination of microbial inoculation culture and conditions to produce fermentation products. The contents of basic components such as sugar, reducing sugar, protein, total polyphenol, and total flavonoid were examined as well as the antioxidant, tyrosinase inhibitory, and thrombolytic activities of the fermented products were analyzed. We observed that reducing sugar and protein contents decreased in most of the fermented products, but the products fermented using yeast exhibited higher sugar content and, thus, higher sweetness. Total polyphenol content and antioxidant activity did not increase in fermented products compared to non-fermented Dongchunghachos, but total flavonoid content and tyrosinase inhibitory and thrombolytic activities increased by fermentation. In particular, total flavonoid content and tyrosinase inhibitory and thrombolytic activities primarily increased in the products fermented using yeast and lactic acid bacteria. However, it was not possible to confirm the increase in these activities in samples fermented by single fermentation using only yeast. Therefore, we propose that it will be possible to develop fermented food from silkworm Dongchunghacho (P. tenuipes) with excellent health benefits through additional study of multiple fermentation conditions using lactic acid bacteria and yeast.

• Microbiology and Biotechnology Letters
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• v.36 no.2
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• pp.101-105
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• 2008

In order to obtain yeast cells producing a bacteriocin OR-7, the 180 bp polynucleotide corresponding to the OR-7 gene including codons for start and stop was chemically synthesized and cloned into pAUR123, an yeast expression vector. Transformed yeast cells exhibited growth inhibition of Bacillus subtilis, Campylobacter jeuni, Escherichia coli and Pseudomonas aeruginosa. This result indicates that yeast cells producing OR-7 possess bacteriocidal properties against both Gram positive B. subtilis and Gram negative C. jejuni, E. coli and P. aeruginosa cells. The recombinant yeast strain constructed in this study can be applied in the food preservative or animal feed.

• Microbiology and Biotechnology Letters
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• v.16 no.6
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• pp.468-475
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• 1988

The cloned B. subtilis cdd gene encoding cytidine/2'deoxycytidine deaminase (EC 3.5.4.5) was expressed in the cdd deficient B. subtilis mutant ED40. The gene was isolted from the cdd complementing plasmid pSO21, and inserted into the EcoR1/Pvu1 sites of pGB215-110 ΔB, which is a temperature sensitivie E. coli-B. subtilis shuttle vector. In the transformed B. subtilis ED4O harboring the resulting plasmid pSO100, cdd was expressed at several hundred fold elevated levels, and the cytidine deaminase activity in E. coli containing pSO100 was twice the level in B. subtilis/pSO0100. The Km value for cytidine of the partially purified enzyme is 1.88$\times$10$^{-4}$M at pH 7.0 and the V$_{max}$ = 11.1 $\mu$mol/min/mg of protein. The enzyme was completely inhibited by 0.1M mercaptoethanol and HgCl$_2$. The inhibition by p-chrolomercurybenzoic acid showed a Ki = 5 uM. These results suggest that sulfhydryl reagents block an active site thiol group, and/or disturb the formation of the tetrameic holoenzyme.