• 한국축산식품학회지
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• 제35권4호
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• pp.473-478
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• 2015

The aim of this study was to screen and In vitro characterize the properties of bacteriocin produced by lactic acid bacteria isolated from Vietnamese fermented pork (Nem chua). One hundred and fifty LAB were isolated from ten samples of Nem chua and screened for bacteriocin-producing lactic acid bacteria. Antimicrobial activity of bacteriocin was carried out by spot on lawn method against both gram positive and gram negative bacteria. One isolate, assigned as KL-1, produced bacteriocin and showed inhibitory activity against Lactobacillus sakei, Leuconostoc mesenteroides and Enterococcus faecalis. To characterize the bacteriocin-producing strain, optimum temperature, incubation period for maximum bacteriocin production and identification of bacteriocin-producing strain were determined. It was found that the optimum cultivation temperature of the strain to produce the maximum bacteriocin activity (12,800 AU/mL) was obtained at 30℃. Meanwhile, bacteriocin production at 6,400 AU/mL was found when culturing the strain at 37℃ and 42℃. The isolate KL-1 was identified as L. plantarum. Antimicrobial activity of cell-free supernatant was completely inhibited by proteolytic enzyme of trypsin, alpha-chymotrypsin and proteinase K. Bacteriocin activity was stable at high temperature up to 100℃ for 10 min and at 4℃ storage for 2 d. However, the longer heating at 100℃ and 4℃ storage, its activity was reduced.

• 한국축산식품학회지
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• 제38권5호
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• pp.1064-1079
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• 2018

In this study, the antimicrobial activity of a bacteriocin produced by Enterococcus faecalis KT11, isolated from traditional Kargı Tulum cheese, was determined, and bacteriocin KT11 was partially characterized. The results showed that bacteriocin KT11 was antagonistically effective against various Gram-positive and Gram-negative test bacteria, including vancomycin- and/or methicillin-resistant bacteria. The activity of bacteriocin KT11 was completely abolished after treatment with proteolytic enzymes (proteinase K, ${\alpha}$-chymotrypsin, protease and trypsin), which demonstrates the proteinaceous nature of this bacteriocin. Additionally, bacteriocin KT11 remained stable at pH values ranging from 2 to 11 and after autoclaving at $121^{\circ}C$ for 30 min. In addition, the activity of bacteriocin KT11 was stable after treatment with several surfactants (EDTA, SDS, Triton X-100, Tween 80 and urea) and organic solvents (chloroform, propanol, methanol, ethyl alcohol, acetone, hexane and ethyl ether). Cell-free supernatant of E. faecalis KT11 was subjected to ammonium sulfate precipitation and then desalted by using a 3.5-kDa cut-off dialysis membrane. The bacteriocin activity was determined to be 711 AU/mL in the dialysate. After tricine-SDS-PAGE analysis, one peptide band, which had a molecular weight of ~3.5 kDa, exhibited antimicrobial activity. Because the bacteriocin KT11, isolated from E. faecalis KT11, exhibits a broad antimicrobial spectrum, heat stability and stability over a wide pH range, this bacteriocin can be used as a potential bio-preservative in foods. Additionally, bacteriocin KT11 alone or in combination with conventional antibiotics may provide a therapeutic option for the treatment of multidrug-resistant clinical pathogens after further in vivo studies.

• 한국약용작물학회지
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• 제19권2호
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• pp.77-82
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• 2011

The antioxidative activity of various enzymatic extracts from Sarcodon aspratus (S. aspratus) was evaluated by measuring 1,1-diphenyl-2-picrylhydrazyl (DPPH), and alkyl radical scavenging activity using an electron spin resonance (ESR) spectrometer. For this study, the S. aspratus were enzymatically hydrolyzed by seven carbohydrases (Viscozyme, Celluclast, Dextrozyme, AMG, Promozyme, Maltogenase, and Termamyl) and eight proteases (${\alpha}$-chymotrypsin, Alcalase, Flavourzyme, Neutrase, papain, pepsin, Protamax, and trypsin). The DPPH radical scavenging activities of Viscozyme and pepsin extracts were the highest, and the half maximal inhibitory concentration ($IC_{50}$) values were 0.896 and 0.734mg/mL, respectively. The Celluclast and trypsin extracts showed the highest scavenging activities on alkyl radical, and their $IC_{50}$ values were 0.278 and 0.575mg/mL, respectively. The Celluclast extracts was decreased cell apoptosis in PC-12 cells against $H_2O_2$-induced oxidative damage. The findings of the present study suggest that enzymatic extracts of S. aspratus exhibit antioxidative activity against oxidative stress on PC-12 cells.

• Journal of Microbiology
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• 제38권3호
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• pp.160-168
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• 2000

A-factor I a microbial hormone that can positively control cell differentiation leading to spore formation and secondary metabolite formation in Streptomyces griseus. to identify a protease that is deeply involved in the morphological and physiological differentiation of Streptomyces, the proteases produced by Streptomyces griseus IFO 13350 and its A-factor deficient mutant strain, Streptomyces griseus HH1, as well as Streptomyces griseus HH1 transformed with the afsA gene were sturdied. In general Streptomyces griseus showed a higher degree of cell growth and protease activity in proportion to its ability to produce a higher amount of A-factor. In particular, the specific activity of the trypsin of Streptomyces griseus IFO 13350 was greatly enhanced more than twice compared with that of Streptomyces griseus HH1 in the later stage of growth. The specific activity of the metalloprotease of Streptomyces griseus HH1 was greatly enhanced more than twice compared with that of Streptomyces griseus IFO 13350, and this observation was reversed in the presence of thiostreptione, However, Streptomyces griseus HH1 transformed with the afsA gene showed a significantly decreased level of trypsin and metalloprotease activity compared with that of the HH1 strain. There was no significant difference between Streptomyces griseus IFO 13350 and HH1 strain in their chymotrypsin and thiol protease activity, yet the level of leu-amionpeptidase activity was 2 times higher in Streptomyces griseus HH1 than in strain IFO 13350 . Streptomyces griseus HH1 harboring afsA showed a similar level of enzyme activity , however, all the three protease activities sharply increased and the thiol protease activity was critically increased at the end of the fermentation. When a serine protease inhibitor, pefabloc SC, and metalloprotease inhibitor, EDTA, were applied to strain IFO 13350 to examine the in vivo effects of the protease inhibitors on the morpholofical differentiation, the formation of aerial meycelium and spores was delayed by two or three days.

• BMB Reports
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• 제33권2호
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• pp.112-119
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• 2000

Three kinds of serine protease inhibitors, members of the Bowman-Birk trypsin inhibitor, were purified from Dolichos lablab seeds and named Dolichos protease inhibitor 1, 2 and 3 (DI-1, DI-2 and DI-3), respectively. Each inhibitor showed a single band with gel mobility at around 15.9, 12.1 and 14.6 kDa on 20% SDS-PAGE under reducing conditions. To characterize inhibitory specificity, the inhibition constant (Ki) for these inhibitors was measured against several known serine proteases. All three Dolichos protease inhibitors (DI-1, DI-2 and DI-3) inhibited the activity of trypsin and plasmin, but had no effect on thrombin and kallikrein (either for human plasma kallikrein or for porcine pancreas kallikrein). DI-1 inhibited chymotrypsin most effectively (Ki = $3.6{\times}10^{-9}\;M$), while DI-2 displayed inhibitory activity for porcine pancreatic elastase (Ki = $6.2{\times}10^{-8}\;M$). Pre-treatment of the 33 mg/kg of DI-mixture (active fractions from $C_{18}$ open column chromatography that included DI-1, DI-2 and DI-3) inhibited the induction of pseudomonal elastase-induced septic hypotension and prevented an increase in bradykinin generation in pseudomonal elastase-treated guinea pig plasma. Also, the increase of kallikrein activity, by injection of pseudomonal elastase, was inhibited by the pretreatment of the DI-mixture in a guinea pig. Since the DI-mixture had no inhibitory effect on kallikrein activity when Z-Phe-Arg-MCA was used as a substrate in vitro, its inhibitory activity in the pseudomonal elastase-induced septic hypotension model might not be due to a direct inhibition of plasma kallikrein in the activation cascade of the Hageman factor and prekallikrein system. These results suggest that the Dolichos DI-mixture might be used as an inhibitor in pathogenic bacterial protease-induced septic shock.

• Journal of Nutrition and Health
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• 제26권3호
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• pp.268-276
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• 1993

Coffee is known to increase pancreatic secretion of digestive enzymes. The mutagen, aflatoxin B1(AFB1) is contained in fermented foods and known to increase the specific activities of pancreatic chymotrypsin, trypsi, amylase, and lipase. Nowadays, coffee intake is increased among Koreans who have consumed relatively high amount of traditional fermented foods. Therefore, this study was performed to examine the effect of coffee and AFB1 on pancreatic exocrine function and structure. Rats were divided into 10 experimental groups. The first five groups were W(control group), LD(0.2g decaffeinated coffee/Kg B.W), HD(3g decaffeinated coffee/Kg B.W), LC(0.2g coffee/Kg B.W), and HC(3g coffee/Kg B.W). The second five groups were WA, LDA, HDA, LCA, HCA, same as first five groups in caffieine level but treated with AFB1. The result of this experiment showed that the caffeine intake did not influence significantly on the growth and feed efficiency. But water intake was increased by caffeine intake and AFB1 treatment. The weights of pancreas and liver were increased as the caffeine intake was increased. Trypsin activities were tend to increase in concentrated coffee groups(HD, HC). AFB1 treated groups showed the higher trypsin level than the AFB1 untreated groups. Amylase activities were tend to increase in concentrated coffee groups(HD, HC) of AFB1 untreated animals. AFB1 treated did not show the additional effect on the stimulated amylase secretion by coffee. Lipase activities were tend to decrease in concentrated coffee groups(HD, HC) of AFB1 untreated animals. Lipase activities were increased in the order named WA group, coffee groups, decaffeinated coffee groups in AFB1 treated animals. AFB1 treated groups showed the higher lipase level than AFB1 untreated groups. In the histologic observation of pancreas HCA group showed more dense compound tubuloalveolar glands and proliferation of nuclei than normal. The result suggested a development of a atypia which is ongoing phase to a cancer.

• 한국응용약물학회:학술대회논문집
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• 한국응용약물학회 1996년도 춘계학술대회
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• pp.217-217
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• 1996

The putative role of vasoactive intestinal polypeptide (VIP) as non-adrenergic non-cholinergic (NANC) neurotransmitter has been studied in rabbit corpus cavernosum. In the presence of atropine and guanethidine the short and prolonged electrical field stimulation (EFS, 2～16 ㎐) induced a frequency-dependent relaxation which was abolished by tetrodotoxin (0.3 ${\mu}$M), a nerve conductance blocker. The neurogenic relaxant reponses were not affected in the presence of VIP-inactivating peptidase, ${\alpha}$-chymotrypsin (2 units/$m\ell$), whereas VIP-induced relaxation were completely abolished. Inhibition of nitric oxide synthase by N$\^$G/-nitro-L-arginine (10～100 ${\mu}$M) caused concentration-dependent inhibition to the neurogenic relaxant responses and at 100 ${\mu}$M the relaxations were virtually abolished. In contrast NO (3～30 ${\mu}$M) and VIP (0.001～l ${\mu}$M)-induced relaxation were unaffected. The inhibitory effect of L-NNA was reversed in the presence of L-arginine (5 mM), the precursor of the NO biosynthesis. Hemog1obin (20～60 ${\mu}$M), sequestering NO in the extracellular space, abolished the NO-evoked relaxation and also caused a concentration-dependent inhibition to the neurogenic relaxation. These observation indicate that NANC relaxation induced by prolonged EFS of rabbit corpus cavernosum is also mediated mainly by nitric oxide as same as that of short EFS, and suggest that VIP is not involved in NANC relaxation of rabbit corpus cavernosum and NO would not be produced by VIP in this tissue.

• 한국가축번식학회지
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• 제17권2호
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• pp.93-101
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• 1993

These experiments were carried out to investigate whether zona hardening affect the efficiency of in vitro fertilization in mouse oocytes. The soluble properties for zona pellucida of oocytes matured in vivo, aged oocytes, and ovarian oocytes matured in vitro have been analyzed with proteolytic enzyme, 3mg/ml of $\alpha$-chymotrypsin. The mean solubility(t50) for the zona of unfertilized oocytes, oocytes not fertilized at the first inseminati and in vitro produced zygotes were 10.1, 20.3 and 32.3min., respectively. The t50 for zona lysis of fertilized oocytes was significantly difference than those observed for unfertilized oocytes and oocytes not fertilized at the first insemination(P<0.01). In addition, the t50 of zona in ovulated oocytes with and without cumulus cells incubated for 0, 3, 6, 9 and 12 hr in vitro, t50 were 13.9, 11.1, 20.7 and 28.0min., and 22.3, 21.0, 30.0 and 33.5min., respectively. In these experiments, the zona pellucida showed a gradual increase in resistance to dissolution by $\alpha$-chyjotrypsin with in vitro aging for more than 6 hrs. This effect was greater in cumulus-free as compared to cumulus-intact oocytes. Finally, in cumulus-intact and cumulus-free ovarian oocytes matured for 0, 5, 10 and 15 hr in vitro the t50 of zona pellucida were 3.0, 10.6, 18.4 and 24.5 min., and 3.0, 14.0, 26.2 and 32.0 min., respectively. Clear differences in solubility between the zona pellucida of oocytes matured in vivo and in vitro. This data were found suggest that under in vitro conditions there is a gradual change in the soluble properties of the zona pellucida, particularly in the absence of the cumulus cells.

• Fisheries and Aquatic Sciences
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• 제15권2호
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• pp.137-143
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• 2012

The abalone Haliotis discus hannai, is one of the economically important species in the fisheries industry. Abalone intestines are one of the by-products of its processing. To investigate its bioactive potential, abalone intestine was digested using an in vitro gastrointestinal (GI) digestion system containing pepsin, trypsin, and ${\alpha}$-chymotrypsin. The abalone intestine G1 digests (AIGIDs) produced by the GI digestion system were fractionated into AIGID I (> 100 kDa), AIGID II (10-100 kDa), and AIGID III (1-10 kDa) using an ultrafiltration membrane system. Of the three digests, AIGID II and AIGID III exhibited inhibitory effects against matrix metalloproteinase-2 and -9 (MMP-2, MMP-9) in HT1080 human fibrosarcoma cells. Both fractions potently inhibited gelatine digestion by MMP-2 and MMP-9 treated with phorbol 12-myristate 13-acetate (PMA) and migration of HT1080 cells in dose dependently. Furthermore, AIGID II and III attenuated expression of p65, a component of nuclear transcription factor kappa B. These results indicate that of the abalone intestine digests inhibit MMP-2 and MMP-9. Thus, the AIGIDs or their active components may have preventive and therapeutic potential for diseases associated with MMP-2 and MMP-9 activation in fibrosarcoma cells.

• Clinical and Experimental Reproductive Medicine
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• 제21권1호
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• pp.99-110
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• 1994

One consequence of fertilization in mammals is an increased resistance of the zona pellucida (ZP) to proteases and various chemical reagents. This phenomenon has been called 'zona pellucida hardening' (ZPH), and it is generally accepted that it is caused by the secretory products of cortical granules released by the egg at fertilization. ZP of mouse oocytes maturing in vitro in a chemically defined medium becomes progressively more resistant to solubilization by chymotrypsin ("Spontaneous" ZP hardening). In the present study, it was aimed to find the specificity of spontaneous ZPH in relation to its possible relevance to the cortical reaction and the physiological block to polyspermy. When a maturation inhibitors, cAMP analog(dbcAMP) and phosphodiesterase inhibitor (IBMX) was added to culture medium, it prevent spontaneous ZPH of mouse oocyte during in vitro culture. Thus spontaneous ZPH requires GVBD, since it is prevented by those agents, which inhibit GVBD in vitro. However, culture for 3 hours in the presence of PMA(lOng/ml), a protein kinase C activator, resulted in ZPH without GVBD, thus suggesting that ZPH may be regulated independently apart from the event of GVBD. Pretreatment of mouse oocyte with FBS result in partially inhibitory effect on subsequent spontaneous ZPH. Induction of GVBD in vivo had a inhibitory effect on the spontaneous ZPH, but subsequent spontaneous ZPH. Induction of GVBD in vivo had a inhinbitory effect on the spontaneous ZPII, but had no inhibitory effect on PMA-induced ZPH. Treatment with a microfilament formation blocker(cytochalasin-B) at 1${\mu}g$/ml concentration, resulted in the excellent inhibitory effect on spontaneous ZPH. However cytochalasin-B did not inhibit PMA-induced ZPH. Thus this suggesting that spontaneuse ZPH had a different mechanism from PMA-induced ZPH.