Sphingolipid identification and skin barrier recovery capacity of a milk sphingolipid-enriched fraction (MSEF) from buttermilk powder.

OBJECTIVE: In this study, we isolated the milk sphingolipid-enriched fraction (MSEF) of sweet buttermilk powder and conducted a clinical trial for evaluating its efficacy in skin barrier recovery. METHODS: Milk sphingolipid-enriched fraction was isolated via solvent extraction of buttermilk powder, and further concentrated by removing the phospholipids and neutral lipids. A cream containing 1% MSEF was used during clinical trials to assess for water holding and skin barrier recovery capacities. RESULTS: The main components of the MSEF were sphingomyelin, glucosylceramide and lactosylceramide, confirmed by TLC, HPLC, MS and NMR. The MSEF cream-treated group had a significantly higher (P < 0.05) water holding capacity, compared with the base cream (vehicle) group. Compared with that in the base cream group, transepidermal water loss (TEWL) recovery increased in the presence of the sphingolipid-containing MSEF cream (MSEF group), with a significant difference (P < 0.05) recorded on day 14. CONCLUSION: The MSEF cream contributed to improving the water holding capacity and skin barrier recovery of damaged skin. Therefore, sphingolipid-containing MSEF can be useful for strengthening or repairing skin barrier function.

OBJECTIF: Dans la présente étude, nous avons isolé la fraction enrichie en sphingolipides du lait (milk sphingolipid-enriched fraction, MSEF) du lait de baratte doux en poudre et mené un essai clinique pour évaluer l'efficacité de la récupération de la barrière cutanée. MÉTHODES: La fraction enrichie en sphingolipides du lait est isolée par l'extraction de solvant du lait de baratte en poudre, et ensuite concentrée en enlevant les phospholipides et les lipides neutres. Une crème contenant un pourcent (1%) de la fraction enrichie en sphingolipides du lait (MSEF) est utilisée pendant des essais cliniques visant à évaluer les capacités de la rétention d'eau et de la récupération de la barrière cutanée. RÉSULTATS: Les principales composantes de la fraction enrichie en sphingolipides du lait (MSEF) étaient la sphingomyéline, la glucosylcéramide, et le lactosylcéramide, confirmées par la chromatographie sur couche mince (TLC), la chromatographie liquide à haute performance (CLHP), la spectrométrie de masse (MS) et la RMN(NMR). La groupe traitée par la crème enrichie en sphingolipides du lait (MSEF) avait une capacité de la rétention d'eau beaucoup plus élevée (P < 0.05), par rapport au groupe utilisant la crème de base (véhicule). Comparé au groupe utilisant la crème de base, la récupération de la perte d'eau transépidermique (TEWL) a augmenté en présence de la fraction enrichie en sphingolipides du lait (MSEF), avec une différence significative (P < 0.05) enregistrée au 14ème jour. CONCLUSION: La crème enrichie en sphingolipides du lait (MSEF) a contribué à améliorer les capacités de la rétention d'eau et de la récupération de la barrière cutanée de la peau endommagée. Par conséquent, la fraction enrichie en sphingolipides du lait (MSEF) peut être utile pour renforcer ou réparer la fonction de barrière cutanée.

Antimelanogenic effects of luteolin 7-sulfate isolated from Phyllospadix iwatensis Makino.

BACKGROUND: Abnormal deposition of melanin may cause an aesthetic skin problem; therefore, the control of unwanted excessive melanin synthesis is the major goal of cosmetic research. OBJECTIVES: To identify novel tyrosinase (TYR) inhibitors from marine plants and examine their cellular antimelanogenic effects. METHODS: The extracts of 50 marine plants endemic to Korea were screened against human TYR. Active constituents were then isolated from the selected plant extracts that showed potential and their chemical structures elucidated. Furthermore, their antimelanogenic effects were examined using murine melanoma B16/F10 cells and human epidermal melanocytes (HEM). RESULTS: Among the tested extracts, that of Phyllospadix iwatensis Makino exhibited the strongest human TYR inhibitory activity. The active constituents were purified from the butanol fraction of the P. iwatensis extract and identified as hispidulin 7-sulfate and luteolin 7-sulfate. Luteolin 7-sulfate inhibited human TYR more strongly than hispidulin 7-sulfate, luteolin, hispidulin and arbutin. Furthermore, luteolin 7-sulfate showed lower cytotoxicity than luteolin in both B16/F10 cells and HEM. Luteolin 7-sulfate attenuated cellular melanin synthesis more effectively in B16/F10 cells and HEM stimulated by α-melanocyte-stimulating hormone and l-tyrosine than arbutin. CONCLUSIONS: This study demonstrates that luteolin 7-sulfate isolated from P. iwatensis is a human TYR inhibitor with advantageous antimelanogenic properties, and would be useful for development as a therapeutic agent for the control of unwanted skin pigmentation.

The effect of mirodenafil on the penile erection and corpus cavernosum in the rat model of cavernosal nerve injury.

Impotence is one of the common complications after the radical prostatectomy. One of the main reasons of this complication is due to the dysfunction of the veins in corpus cavernosum. Recent studies have shown that the erectile function is improved after the long-term therapy of phosphodiesterase type 5 inhibitor among patients with post-prostatectomy erectile dysfunction. In this study, we evaluated the effects of mirodenafil on the penile erection and corpus cavernosum tissues in the rat model of cavernosal nerve injury. Rats were divided into four groups: (1) control group, (2) bilateral cavernosal nerve injury group, (3) mirodenafil 10 mg therapy group after the nerve injury and (4) mirodenafil 20 mg therapy group after the nerve injury. After we identified the nerve from the pelvic nerve complex on the lateral side of the prostate, the rats in the control group were sutured without causing any nerve injury and in other groups we damaged the nerve by compressing it with a vessel clamp. Then, 10 and 20 mg kg(-1) of mirodenafil were orally administered to two experimental groups. After 8 weeks, the intracavernosal pressure (ICP) was recorded. The immunohistochemical staining and western blot were performed, and the effect of mirodenafil on the expression of cyclic guanosine monophosphate (cGMP) was evaluated through enzyme-linked immunosorbent assay. The ICP of nerve-injured group was decreased compared with the control group; however, the ICP of the mirodenafil-administered groups was improved compared with the nerve-injured group. The Masson's trichrome staining confirmed that the smooth muscle (SM) component was increased in the mirodenafil-administered groups. The nitric oxide synthase expression and cGMP of mirodenafil-administered groups was increased compared with the nerve-injured group. Long-term therapy of mirodenafil may improve the erectile function after the radical prostatectomy by preserving the SM content and inhibiting the fibrosis of the corpus cavernosum.

Vitamin C increases the fecal fat excretion by chitosan in guinea-pigs, thereby reducing body weight gain.

The aims of this study were to investigate the antiobesity properties of chitosan on its own, as well as in the presence of vitamin C, in vivo. Hartley guinea-pigs were divided into Control (normal diet), F-control (high fat diet), Chitosan (high fat diet with 5.0% chitosan) and Chito-vit C (high fat diet with 5.0% chitosan containing 0.5% vitamin C) groups, respectively. The effects of chitosan, both alone and in the presence of vitamin C, on body weight, total fecal weight, fecal composition and plasma lipid level were studied for 5 weeks. The results of this study indicated that the fat-binding and water-holding capacity of chitosan might decrease body weight by reducing the absorption of cholesterol and fat, subsequently increasing total fecal weight, fecal fat excretion and fecal water excretion. Vitamin C increased the fecal fat excretion by chitosan in guinea-pigs, thereby reducing body weight gain.

Biological activities of thermo-tolerant microbes from fermented rice bran as an alternative microbial feed additive.

To evaluate the commercial potential of new microbial feed additive, Issatchenkia orientalis Y266 and Bacillus subtilis B266 from commercial fermented rice bran were tested for their tolerance or resistance to pH, bile, oxgall, and temperature. It was found that the strains grew very well up to pH 3.0 and resistant to relatively high concentrations of bile salt and oxgall. I. orientalis and B. subtilis are extremely tolerant in range of 70-90 degrees C in solid medium. B. subtilis B266 also has excellent tolerant property up to 90 degrees C in liquid medium. The health indexes (the microflora in the small intestines and the antibody titer to Newcastle disease virus) of chicks were significantly improved in the fermented rice bran with these strains (0.25% addition to diet) in comparison with the Avilamycin (20 mg/kg diet)-fed group (p < 0.05). The fermented rice bran-fed group showed a better microbial flora in the small intestines. Accordingly, it would appear that the fermented rice bran with these strains may be a potential candidate for an alternative microbial feed additive.

Effects of a 2-, 3- and 4-electrode stimulator design on current dispersion on the surface and into the limb during electrical stimulation in controls and patients with wounds.

Electrical stimulation is a widely used modality in the field of physical therapy and exercise physiology. The most common method for the application of electrical stimulation is a two-electrode system where one electrode is the source and the other is a reference. However, recent studies report that a more effective delivery system can be achieved if more than two electrodes are used. In the present investigation, the circuitry to deliver electrical stimulation through a 2-, 3- or 4-electrode delivery system was designed. The system was evaluated by its ability to deliver current on the surface of the skin as well as deep into the quadriceps muscle in six control subjects and in and around wounds in six other subjects. The results of the experiments showed that much better depth of penetration was achieved in a 4-electrode system (one electrode was on the opposite side of the limb and three electrodes were on top of the limb) than in either a 2- or a 3-electrode delivery system. In non-wounded skin, given the same current from the stimulator, the current in the quadriceps muscle was found to be double with a 4-electrode versus a 2-electrode system. In wounds, this same finding was seen. Here, blood flow, an indicator of the effectiveness of electrical stimulation in wounds, was three times higher if a multi-channel stimulator was used versus a 2-channel stimulator. Thus a multi-channel electrical stimulation system is more effective than a 2-electrode system.

Appetite suppressive effects of yeast hydrolysate on nitric oxide synthase (NOS) expression and vasoactive intestinal peptide (VIP) immunoreactivity in hypothalamus.

To investigate the effects of yeast hydrolysate on appetite regulation mechanisms in the central nervous system, nitric oxide synthase (NOS) expression and vasoactive intestinal peptide (VIP) immunoreactivity in the paraventricular nucleus (PVN) and ventromedial hypothalamic nucleus (VMH) of the hypothalamus were examined. Male Sprague-Dawley (SD) rats were assigned to five groups: control (normal diet), BY-1 and BY-2 (normal diet with oral administration of 0.1 g and 1.0 g of yeast hydrolysate <10 kDa/kg body weight, respectively), AY-1 and AY-2 (normal diet with oral administration of 0.1 g and 1.0 g of yeast hydrolysate 10-30 kDa/kg body weight, respectively). The body weight gain in the BY groups was less than that in the control. In particular, the weight gain of the BY-2 group (133.0 +/- 5.1 g) was significantly lower (p < 0.05) than that of the control group (150.1 +/- 3.7 g). Among the test groups, the BY-2 group was shown to have significantly lower triacylglycerol (TG) levels (p < 0.05) than the other groups. The staining intensities and optical densities of NOS neurons in the PVN of the AY group were significantly higher (p < 0.05) than in the control and BY groups. The staining intensities and optical densities of VIP immunoreactivity in the PVN and VMH of the BY groups were higher than those of the AY groups and the control. In conclusion, these results indicated that yeast hydrolysate of <10 kDa reduced the body weight gain and body fat in normal diet-fed rats and increased the lipid energy metabolism by altering the expression of NOS and VIP in neurons.

A multi-channel stimulator and electrode array providing a rotating current whirlpool for electrical stimulation of wounds.

When electrical stimulation is used on wounds, the electrical current has difficulty penetrating areas where there is necrotic tissue. Further, for an irregularly shaped wound, current distribution is poor in some areas of the wound since conventional two-electrode delivery systems provide the greatest current in a line directly between the electrodes. A new stimulator and electrode system is described which uses three electrodes spaced around a wound to disperse current more evenly. The stimulator senses tissue impedance and then redirects current by altering its Thevenin's output impedance for each electrode; each of the three electrodes becomes the active one in sequence while the remaining are the sink electrodes. Eight subjects were examined to test the stimulator. Electrical stimulation was applied to the skin above the quadriceps muscle at currents of 15 mA in six subjects without wounds and in two subjects with wounds. The relationship between electrode position and current dispersion on the skin was examined with a two-electrode vs. a three-electrode system to set stimulation parameters for the computer. The results showed that the three-electrode system could (1) detect areas of the skin with high impedance; (2) compensate by altering the Thevenin's output impedance at each of the three electrodes to shift current to high impedance areas; (3) provide uniform current across the skin as assessed by skin current and blood flow measurements with a laser Doppler flow imager.

Hypolipidemic effect of lactobacillus ferment as a functional food supplement.

The Lactobacillus ferment used in this study was composed of Lactobacillus fermented wheat, barley and kefir grains. Fermentation increased the CFU of lactic acid bacteria with a reduction in pH value and in the contents of dietary fi ber and glucan. Male SD rats were fed a high fat diet with or without 10% Lactobacillus ferment for 4 weeks. In the Lactic-F group (group fed high-fat diet with Lactobacillus ferment) there was a significantly reduced increase of body weight compared with the HF-control (group fed high-fat diet without Lactobacillus ferment). The food efficiency ratio (FER) tended to be decreased in the Lactic-F group, but there was no significant difference between the Lactic-F and HF-control groups. The perirenal and the epididymal fat weights in the normal dietary group (control) and in the Lactic-F group were significantly lower than those of the HF-control. The serum HDL-cholesterol and the total cholesterol in the Lactic-F group were similar to the control group, and were significantly different from those of the HF-control. These results indicated that the Lactobacillus ferment is a functional material having antiobesity effects, with use as a supplement in functional, health-favoring food.

Estimated daily intakes of butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) and tert-butyl hydroquinone (TBHQ) antioxidants in Korea.

The study was conducted to establish the estimated daily intake (EDI) of antioxidants such as butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) and tert-butyl hydroquinone (TBHQ) in Korea. The EDIs were obtained from two sources. One of the estimations was based on the analytical determination of BHA, BHT and TBHQ in 12 food categories (ten food categories for TBHQ) and on individual dietary intake data obtained from the National Health and Nutrition Survey in 1998 (n=11 525, age > 1 year). The other EDIs of BHA, BHT and TBHQ were based on the maximum permitted levels specified in national food standards in Korea and on individual dietary intake data obtained from the National Health and Nutrition Survey in 1998 (n=11 525, age > 1 year). To establish the EDIs based on the analytical determination and on individual dietary intake data, 133 food samples in 12 food categories were selected from the foods considered to be representative sources of BHA, BHT and TBHQ in the Korean diet. Selected samples were analysed by GC with FID. BHA was not detected in any of the samples analysed. BHT and TBHQ were detected in the samples, but the levels were significantly lower than their maximum limits. The EDIs1 of BHT, and TBHQ for average consumers were 0.0156(-3), and 0.0012(-3) mg kg(-1) body weight bw day(-1) and as a proportion of the ADI were 0.0052 and 0.0002%, respectively. For 95th percentile consumers, the EDIs of BHT and TBHQ were 0.0080 and 0.0006 mg kg(-1) bw day(-1), and as a proportion of the ADI were 2.67 and 0.09%, respectively. EDIs for BHA, BHT and TBHQ based on the maximum permitted levels and on individual dietary intake data were 0.04, 0.04 and 0.04 mg kg(-1) bw day(-1), respectively. The EDIs of BHA, BHT and TBHQ for average consumers ranged from 6.00 to 14.42% of the ADI of each antioxidant. According to these results, the EDIs of BHA, BHT and TBHQ in Korea were significantly lower than ADI of these antioxidants established by the JECFA.

Daily intake assessment of saccharin, stevioside, D-sorbitol and aspartame from various processed foods in Korea.

This study was carried out to estimate the daily intakes (EDIs) of artificial sweeteners such as saccharin, stevioside, D-sorbitol and aspartame in order to evaluate the safety of the artificial sweeteners in Korea. A total of 274 food samples were selected from the foods considered to be representative sources of artificial sweeteners in the Korean diet and analysed by using HPLC with evaporative light scattering and ultraviolet detectors. In case of aspartame, the reference values were used without instrumental analysis. The EDIs of saccharin, stevioside, D-sorbitol and aspartame for average consumers were 0.028, 0.008, 4.9 and 0.14 mg kg-1 body weight day-1, respectively, and as a proportion of the acceptable daily intake (ADI) were not higher than 1% of ADI of the Joint FAO/WHO Expert Committee on Food Additives (JECFA). For 90th percentile consumers, the EDIs of saccharin, stevioside, D-sorbitol and aspartame were 2.0, 0.20, 141 and 4.6 mg kg-1 body weight day-1, respectively, and as a proportion of the ADI, the EDIs of saccharin and aspartame were 40.7% and 11.4% of the ADI set by the JECFA, respectively. Because JECFA did not assign ADIs for stevioside and D-sorbitol, the values for these sweeteners were not compared. According to these results, the EDIs of artificial sweeteners such as saccharin and aspartame in Korea are significantly lower than ADI set by the JECFA.

Macrophage-stimulating activity of exo-biopolymer from cultured rice bran with Monascus pilosus.

To find a new use of rice bran, five fungi were examined for the production of exo-biopolymer with macrophage-stimulating activity from rice bran. Among the exo-biopolymers produced from the cultures, Monascus pilosus had the most potent macrophage stimulating activity in a liquid culture rather than in a solid culture. In order to improve the yield of exo-biopolymer with macrophage-stimulating activity, a suitable medium for exo-biopolymer was tested in submerged culture of M. pilosus. The highest amount of exo-biopolymer (13.9 mg/mL) was obtained in a medium containing rice bran as an only carbon source followed by media with additional maltose and sucrose (13.8 and 13.7 mg/mL, respectively). The addition of peptone resulted in the production of high amount of exo-biopolymer (15.1 mg/mL), meanwhile the addition of ammonium chloride resulted in 264.0 microg/mL of glucosamine content. Among eight different kinds of inorganic salts tested, potassium phosphate (0.1%) was the most effective inorganic salt for the mycelial growth and exo-biopolymer production. Therefore the optimal medium composition was as follows (g/L): 20 g of rice bran, 5 g of peptone, and 1 g of KH2PO4. The optimal culture pH and time for mycelial growth and exo-biopolymer production was pH 5.0 and 25 degrees C, respectively. The maximum exo-biopolymer (20.1 mg/mL) was observed at the fourth day of cultivation. Exo-biopolymer, a crude polysaccharide fraction, mainly contained neutral sugar (81.8%) with considerable amounts of uronic acid (18.2%). Component sugar analysis showed that the active fraction consisted mainly of arabinose, galactose, glucose, which was digested from starch of rice bran during cultivation, and uronic acid (molar ratio; 0.8:1.0:0.7:0.8).

Yeast hydrolysate reduces body fat of dietary obese rats.

The purpose of this study was to assess the antiobesity effect of the yeast hydrolysate (DNF) on the body weight, body fat and plasma lipids levels of high-fat fed rats. The weight gain of the HF (high fat diet) (162.58 +/- 6.68 g) was significantly (p < 0.05) greater than that of DNF-1, DNF-2, (high fat diet with DNF of 0.5 and 1.0 g/kg body weight, respectively) and control groups (143.19 +/- 7.33 g, 139.20 +/- 8.36 g, 130.23 +/- 8.02 g, respectively). The wet weight of the epididymal fat and the perirenal fat pads of the DNF-1, DNF-2 and control groups were reduced significantly (p < 0.05). A significant (p < 0.05) increase of HDL-cholesterol level of the DNF-2 and control groups was observed. However, there was no significant difference between DNF-1 and DNF-2. It was also found that the triacylglycerol (TG) levels decreased significantly (p < 0.05) in the DNF-2 group from that of the HF, but there was no significant (p < 0.05) difference between DNF-1 and DNF-2.

Pharmacological effects of fermented red pepper.

The pharmacological effects were investigated of fermented red pepper (HF-S), which consisted of 14.7% carbohydrate, 1.5% lipid, 4.9% protein, 0.3% ash, 78.2% moisture content, with 0.15% capsaicin and 0.06% dihydrocapsaicin. Oral administration of 0.25 mL HF-S for 3 weeks produced significant changes of the perirenal fat pad weight compared with the HF-control group, suggesting a suppressive effect on lipid accumulation and a significant decrease in the risk of arteriosclerosis. The HF-S (0.25 mL) group also showed a lower plasma TG, TC level and atherogenic index than that of the HF-control. In addition, the HF-S (0.25 mL) group showed a marked increase in the production of glutathione, which is the major endogenous antioxidant, and a decrease in the production of lipid peroxide as the product of chemical damage by oxygen free radicals. It is assumed that the effect of HF-S might relate to high glutathione production on the suppression of lipid peroxidation. HF-S stimulated not only the proliferation of macrophages (as high as the positive control, LPS at 1000 microg/mL) but also mitogenic activity (1.2-fold of LPS at 100 microg/mL).

Pharmacological activities of stromata of Cordyceps scarabaecola.

The chemical components of freeze-dried stromata from Cordyceps scarabaecola were examined. The stromata consisted of crude carbohydrates (55.1%) and crude proteins (14.2%). The stromata were also composed of a low content of crude ash (6.6%) and fat (1.5%). The composition of the carbohydrate in the stromata included a large quantity of glucose (46.6%), mannose (35.4%) and galactose (18.0%). The acidic amino acids such as glutamic acid (32.1 mg/g) and aspartic acid (24.7 mg/g) were present in a large quantity. The extracts of stromata did not reveal any inhibitory activity for AChE in vitro. It was observed that a hot-water extract (HW) of the stromata contributed significantly to the anticoagulant activity (60 s coagulating time) and anticomplementary activity (62% of ITCH50 value). The MeOH-soluble fraction (M) from the freeze-dried stromata inhibited TPA-induced O2- generation as effectively as the positive control, genistine 27%. The hot-water extract (HW) showed the most potent intestinal immune system modulation activity and the MeOH-soluble fraction (M) had intermediate activity.

Anti-stress and anti-fatigue effect of fermented rice bran.

The anti-stress and anti-fatigue effects of a hot water extract of fermented rice bran (FRB) were investigated with Saccharomyces cerevisae IFO 2346 on rats or mice. Oral administration (1 g/kg/day) of a hot water extract of FRB inhibited major changes in weight of the adrenal, thymus, spleen and thyroid, showing the anti-stress effect. A hot water extract of FRB also inhibited the increase of GPT and LDH activity, cholesterol and serum glucose levels. Administration (1 g/kg/day) for 2 weeks significantly prolonged the swimming time, resulting in an increase of the anti-fatigue effect. From these results, it can be considered that FRB has an anti-stress and anti-fatigue effect.

Optimization of submerged culture conditions for the production of angiotensin converting enzyme inhibitor from Flammulina velutipes.

The angiotensin-converting enzyme (ACE) inhibitory effect was tested in the culture broth from submerged mycelial cultures of 20 basidiomycetes. The ACE inhibitory effect of culture broth from Flammulina velutipes strain 414 was the highest (52.8%), followed by Lentinus edodes strains 2 (44.4%) and 16 (41.3%). Nutritional requirements for the production of ACE inhibitory substance from F. velutipes were studied. Sucrose, ammonium acetate, and glutamic acid were chosen for the maximum production of ACE inhibitory substance. The optimal medium composition was (g/l): sucrose 20, ammonium acetate 5, glutamic acid 2, KH(2)PO(4) 3, MgSO(4).7H(2)O 0.8, and yeast extract 0.5. Under optimal culture conditions, the ACE inhibitory effect was more than 80%.

Biological activities of Saccharomyces cerevisiae and fermented rice bran as feed additives.

AIMS: The objective of the present study was to examine for the various biological activities for feed additives of Saccharomyces cerevisiae and fermented rice bran. METHODS: The feed additives (100 g of each: YE, brewery yeast; PM, mixture of YE and SF; SF, fermented rice bran) were decocted with 2 l water at 100 degrees C to a half volume. RESULTS: The hot-water extract of PM was found to increase the stimulation of the macrophage activation (2.0-fold) and the intestinal immune modulating activity (1.8-fold), compared with those of control. When the hot-water extracts of PM, SF and YE were fed to the rats for 8 d (1 g kg-1 d-1), PM and SF showed the more effective anti-stress effect on spleen, thyroid and thymus gland. SIGNIFICANCE AND IMPACT OF THE STUDY: The hot-water extract of PM can have a significant impact on strengthening the living body's immune system and proliferating anti-stress activities with relation to enhance immunity.

Characterization of a keratinolytic metalloprotease from Bacillus sp. SCB-3.

A keratinolytic protease-producing microorganism was isolated from soybean paste waste and was identified as a strain of Bacillus sp. The keratinase was purified by polyethylene glycol precipitation and two successive column chromatographies with DEAE-Toyopearl 650C and Sephacryl S-200 HR. The purified enzyme had overall 11 purification folds with an 18% yield. The results of sodium dodecyl sulfate polyacrylamide gel electrophoresis and gel filtration on Sephacryl G-200 indicated that the purified enzyme was monomeric and had a molecular weight of 134 kDa. The optimum temperature and pH were 40 degrees C and 7.0, respectively. This enzyme was completely inhibited by EDTA and EGTA, and it was restored by the addition of Ca2+ and Mg2+. These results suggested that it is a metalloprotease. The stimulated enzyme activity by reducing agents indicated that the reducing condition was important in the expression of the activity.

Purification and properties of extracellular phytase from Bacillus sp. KHU-10.

Bacillus species producing a thermostable phytase was isolated from soil, boiled rice, and mezu (Korean traditinal koji). The activity of phytase increased markedly at the late stationary phase. An extracellular phytase from Bacillus sp. KHU-10 was purified to homogeneity by acetone precipitation and DEAE-Sepharose and phenyl-Sepharose column chromatographies. Its molecular weight was estimated to be 46 kDa on gel filtration and 44 kDa on SDS-polyacrylamide gel elctrophoresis. Its optimum pH and temperature for phytase activity were pH 6.5-8.5 and 40 degrees C without 10 mM CaCl2 and pH 6.0-9.5 and 60 degrees C with 10 mM CaCl2. About 50% of its original activity remained after incubation at 80 degrees C or 10 min in the presence of 10 mM CaCl2. The enzyme activity was fairly stable from pH 6.5 to 10.0. The enzyme had an isoelectric point of 6.8. As for substrate specificity, it was very specific for sodium phytate and showed no activity on other phosphate esters. The Km value for sodium phytate was 50 microM. Its activity was inhibited by EDTA and metal ions such as Ba2+, Cd2+, Co2+, Cr3+, Cu2+, Hg2+, and Mn2+ ions.