• KSBB Journal
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• v.9 no.4
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• pp.385-394
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• 1994

Large scale purification to get antifungal antibiotic KRF-001 of 90% purity, was investigated using preparative HPLC. Crude KRF-001 was purified by XAD-7 adsorption chromatography, acid precipitation and microfiltration. Microfiltration was the most effective isolation method of crude KRF-001. The purification methods using C18 chromatography was convenient compared with the conventional methods. Delta PAK C18 column and Bonda PAK C18 column were adapted large scale purification of KRF-001. Gradient system of prep HPLC using Delta PAK C18 column was more effective. With these conditions, final recovery of KRF-001 yielded 77%.

• KSBB Journal
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• v.9 no.4
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• pp.378-384
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• 1994

An improved method for the selective isolation of high-yielding mutant strains for the production of antifungal antibiotic KRF-001 was investigated. The mutant strain U. V 4, which produces high titer of KRF-001, was selected on the high potency agar plate after ultraviolet light irradiation. The U. V 4 strain produced 2-fold more KRF-001 than the mother strain in production media. Large scale fermentation was performed using the U. V 4 strain in 100$\ell$ fermenter. The antifungal antibiotic KRF-001 secreted into culture broth was detected by HPLC in 24hrs of fermentation.

• Microbiology and Biotechnology Letters
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• v.19 no.6
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• pp.614-618
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• 1991

Antifungal compound, KRF-001, was produced by Bacillus subtilis subsp. krictiensis isolated from soil. Physico-chemical factors affecting cell growth and bioactivity were examined to improve the production yield. Nutrient composition, temperature, pH and phosphate ion concentration were proved to be important factors for the production of KRF-001. Mutation was performed to select high yielding strains. First, mutation was performed with ultra-violet light, and the second mutation process was conducted by MNNG (N-Methyl-N'-nitro-N-nitrosoguanidine) resulting in three high yielding strains.

• Microbiology and Biotechnology Letters
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• v.19 no.4
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• pp.337-342
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• 1991

A bacterium producing the antifungal compound KRF-001 isolated from soil was selected and identified as Bacillus subtilis. The antibiotic KRF-001 was active against various fungi. Effecacy of KRF-001 at various concentration for controlling leaf blast of rice in the paddy field was evaluated and compared with recommended rates of kasugamycin, blasticidin- s and tricyclazole. KRF-001 caused no irritation on the skin of rabbits and LD50 for mice was deduced more than 5000 mg/kg which indicates the possibility of low toxicity or no toxicity.

• Microbiology and Biotechnology Letters
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• v.19 no.6
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• pp.598-603
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• 1991

An antifungal mixture of six members (component A to F), KRF-001 produced by Bacillzts subtilis subsp. krictiensis was isolated from the fermentation broth. Molecular weight of component A to F was determined by FAB-MS to be 1042, 1056, 1056, 1070, 1070 and 1084 respectively. Various instrumental analyses (amino acid analysis, GC-MS, $^1H-NMR, ^1HH$ COSY NMR) revealed that the mixture was a homologous cyclic peptide composed of each one mole of glutamine, proline, tyrosine, serine, unusual $\beta$-amino acid and three moles of asparagine. The structural differences of component A to F were found in carbon number and terminal structure of the unusual $\beta$-amino acid. After determination of the sequence and stereochemistry of those amino acids, the tentative structure of KRF-001 was determined.

• Korean Journal of Veterinary Research
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• v.48 no.1
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• pp.53-60
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• 2008

We have investigated efficiency of a recombinant subunit Pasteurella multocida toxin (PMT) that was mixed with a vaccine consisted of inactivated whole cells of Bordetella bronchiseptica, P. multocida (types A and D). For verification of the efficacy of the vaccine, all experimental pigs (suckling piglets, sow and gilts) in the three farms were vaccinated. Antibody titers against B. bronchiseptica and P. multocida type A of the vaccinated pigs by microplate agglutination were significantly higher than those of the control pigs (p < 0.05). Similar patterns were observed in the analysis of anti- PMT neutralizing antibody by serum neutralizing method using Vero cell (p < 0.05). Anti- P. multocida type D antibody titer of the vaccinated sows and gilts by ELISA showed significant differences with those of the non-vaccinated pigs (p < 0.05). Although antibody titers increased, it was unable to find out the difference in the clinical signs between the vaccinated and non-vaccinated pigs. However, the increase in body weight of the vaccinated piglets was observed in comparison with the non-vaccinated piglets on a farm. At slaughtering of the pigs, pathological lesions in the turbinate bones of the vaccinated pigs were significantly lower than those of the non-vaccinated pigs (p < 0.001). These results suggested that efficacy of the vaccine in pigs demonstrated to protect against atrophic rhinitis in Korea.

• Journal of Microbiology and Biotechnology
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• v.18 no.6
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• pp.1179-1185
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• 2008

The immune-stimulating activities of Bordetella bronchiseptica antigens containing dermonecrotoxin (BBD) loaded in chitosan microspheres (CMs) have already been reported in vitro and in vivo with a mouse alveolar macrophage cell line (RAW264.7) and mice. Therefore, this study attempted to demonstrate the successful induction of mucosal immune responses after the intranasal administration of BBD loaded in CMs (BBD-CMs) in colostrum-deprived pigs. The BBD was introduced to the CMs using an ionic gelation process involving tripolyphosphate (TPP). Colostrum-deprived pigs were then directly immunized through intranasal administration of the BBD-CMs. A challenge with a field isolate of B. bronchiseptica was performed ten days following the final immunization. The BBD-specific IgG and IgA titers, evident in the nasal wash and serum from the vaccinated pigs, increased with time (p<0.05). Following the challenge, the clinical signs of infection were about 6-fold lower in the vaccinated pigs compared with the nonvaccinated pigs. The grades for gross morphological changes in the turbinate bones from the vaccinated pigs were also significantly lower than the grades recorded for the nonvaccinated pigs (p<0.001). Therefore, the mucosal and systemic immune responses induced in the current study would seem to indicate that the intranasal administration of BBD-CMs may be an effective vaccine against atrophic rhinitis in pigs.