• Title/Summary/Keyword: protease production

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Microbiological and Enzymological Studies on Takju Brewing (탁주(濁酒) 양조(釀造)에 관(關)한 미생물학적(微生物學的) 및 효소학적(酵素學的) 연구(硏究))

  • Kim, Chan-Jo
    • Applied Biological Chemistry
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    • v.10
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    • pp.69-100
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    • 1968
  • 1. In order to investigate on the microflora and enzyme activity of mold wheat 'Nuruk' , the major source of microorganisms for the brewing of Takju (a Korean Sake), two samples of Nuruk, one prepared at the College of Agriculture, Chung Nam University (S) and the other perchased at a market (T), were taken for the study. The molds, aerobic bacteria, lactic acid bacteria, and yeasts were examined and counted. The yeasts were classified by the treatment with TTC (2, 3, 5 triphenyltetrazolium chloride) agar that yields a varied shade of color. The amylase and protease activities of Nuruk were measured. The results were as the followings. a) In the Nuruk S found were: Aspergillus oryzae group, $204{\times}10^5$; Black Aspergilli, $163{\times}10^5$; Rhizogus, $20{\times}10^5$; Penicillia, $134{\times}10^5$; Areobic bacteria, $9{\times}10^6-2{\times}10^7$; Lactic acid bacteria, $3{\times}10^4$ In the Nuruk T found were: Aspergillus oryzae group, $836{\times}10^5$; Black Aspergilli, $286{\times}10^5$; Rhizopus, $623{\times}10^5$; Penicillia, $264{\times}10^5$; Aerobic bacteria, $5{\times}10^6-9{\times}10^6$; Lactic acid bacteria, $3{\times}10^4$ b) Eighty to ninety percent of the aerobic bacteria in Nuruk S appeared to belong to Bacillus subtilis while about 70% of those in Nuruk T seemed to be spherical bacteria. In both Nuruks about 80% of lactic acid bacteria were observed as spherical ones. c) The population of yeasts in 1g. of Nuruk S was about $6{\times}10^5$, 56.5% of which were TTC pink yeasts, 16% of which were TTC red pink yeasts, 8% of which were TTC red yeasts, 19.5% of which were TTC white yeasts. In Nuruk T(1g) the number of yeasts accounted for $14{\times}10^4$ and constituted of 42% TTC pink. 21% TTC red pink 28% TTC red and 9% TTC white. d) The enzyme activity of 1g Nuruk S was: Liquefying type Amylase, $D^{40}/_{30},=256$ W.V. Saccharifying type Amylase, 43.32 A.U. Acid protease, 181 C.F.U. Alkaline protease, 240C.F.U. The enzyme activity of 1g Nuruk T was: Liquefying type Amylase $D^{40}/_{30},=32$ W.V. Saccharifying type amylase $^{30}34.92$ A.U. Acid protease, 138 C.F.U. Alkaline protease 31 C.F.U. 2. During the fermentation of 'Takju' employing the Nuruks S and T the microflora and enzyme activity throughout the brewing were observed in 12 hour intervals. TTC pink and red yeasts considered to be the major yeasts were isolated and cultured. The strains ($1{\times}10^6/ml$) were added to the mashes S and T in which pH was adjusted to 4.2 and the change of microflora was examined during the fermentation. The results were: a) The molds disappeared from each sample plot since 2 to 3 days after mashing while the population of aerobic bacteria was found to be $10{\times}10^7-35{\times}10^7/ml$ inS plots and $8.2{\times}10^7-12{\times}10^7$ in plots. Among them the coccus propagated substantially until some 30 hours elasped in the S and T plots treated with lactic acid but decreased abruptly thereafter. In the plots of SP. SR. TP. and TR the coccus had not appeared from the beginning while the bacillus showed up and down changes in number and diminished by 1/5-1/10 the original at the end stage. b) The lactic acid bacteria observed in the S plot were about $7.4{\times}10^7$ in number per ml of the mash in 24 hours and increased up to around $2{\times}10^8$ until 3-4 days since. After this period the population decreased rapidly and reached about $4{\times}10^5$ at the end, In the plot T the lactic acid becteria found were about $3{\times}10^8$ at the period of 24 fours, about $3{\times}10$ in 3 days and about $2{\times}10^5$ at the end in number. In the plots SP. SR. TP, and TR the lactic acid bacteria observed were as less as $4{\times}10^5$ at the stage of 24 hours and after this period the organisms either remained unchanged in population or ceased to exist. c) The maiority of lactic acid bacteria found in each mash were spherical and the change in number displayed a tendency in accordance with the amount of lactic acid and alcohol produced in the mash. d) The yeasts had showed a marked propagation since the period of 24 hours when the number was about $2{\times}10^8$ ㎖ mash in the plot S. $4{\times}10^8$ in 48 hours and $5-7{\times}10^8$ in the end period were observed. In the plot T the number was $4{\times}10^8$ in 24 hours and thereafter changed up and down maintaining $2-5{\times}10^8$ in the range. e) Over 90% of the yeasts found in the mashes of S and T plots were TTC pink type while both TTC red pink and TTC red types held range of $2{\times}10-3{\times}10^7$ throughout the entire fermentation. f) The population of TTC pink yeasts in the plot SP was as $5{\times}10^8$ much as that is, twice of that of S plot at the period of 24 hours. The predominance in number continued until the middle and later stages but the order of number became about the same at the end. g) Total number of the yeasts observed in the plot SR showed little difference from that of the plot SP. The TTC red yeasts added appeared considerably in the early stage but days after the change in number was about the same as that of the plot S. In the plot TR the population of TTC red yeasts was predominant over the T plot in the early stage which there was no difference between two plots there after. For this reason even in the plot w hers TTC red yeasts were added TTC pink yeasts were predominant. TTC red yeasts observed in the present experiment showed continuing growth until the later stage but the rate was low. h) In the plot TP TTC pink yeasts were found to be about $5{\times}10^8$ in number at the period of 2 days and inclined to decrease thereafter. Compared with the plot T the number of TTC pink yeasts in the plot TP was predominant until the middle stage but became at the later stage. i) The productivity of alcohol in the mash was measured. The plot where TTC pink yeasts were added showed somewhat better yield in the earely stage but at and after the middle stage the difference between the yeast-added and the intact mashes was not recognizable. And the production of alcohol was not proportional to the total number of yeasts present. j) Activity of the liquefying amylase was the highest until 12 hours after mashing, somewhat lowered once after that, and again increased around 36-48 hours after mashing. Then the activity had decreased continuously. Activity of saccharifying amylase also decreased at the period of 24 hours and then increased until 48 hours when it reached the maximum. Since, the activity had gradually decreased until 72 hours and rapidly so did thereafter. k) Activity of alkaline protease during the fermentation of mash showed a tendency to decrease continusously although somewhat irregular. Activity of acid protease increased until hours at the maximum, then decreased rapidly, and again increased, the vigor of acid protease showed better shape than that of alkaline protease throughout. 3. TTC pink yeasts that were predominant in number, two strains of TTC red pink yeasts that appeared throughout the brewing, and TTC red yeasts were identified and the physiological characters examined. The results were as described below. a) TTC pinkyeasts (B-50P) and two strains of TTC red pink yeasts (B-54 RP & B-60 RP) w ere identified as the type of Saccharomyces cerevisiae and TTC pink red yeasts CB-53 R) were as the type of Hansenula subpelliculosa. b) The fermentability of four strains above mentioned were measured as follows. Two strains of TTC red pink yeasts were the highest, TTC pink yeasts were the lowest in the fermantability. The former three strains were active in the early stage of fermentation and found to be suitable for manufacturing 'Takju' TTC red yeasts were found to play an important role in Takju brewing due to its strong ability to produce esters although its fermentability was low. c) The tolerance against nitrous acid of strains of yeast was marked. That against lactic acid was only 3% in Koji extract, and TTC red yeasts showed somewhat stronger resistance. The tolerance against alcohol of TTC pink and red pink yeasts in the Hayduck solution was 7% while that in the malt extract was 13%. However, that of TTC red yeasts was much weaker than others. Liguefying activity of gelatin by those four strains of yeast was not recognized even in 40 days. 4. Fermentability during Takju brewing was shown in the first two days as much as 70-80% of total fermentation and around 90% of fermentation proceeded in 3-4 days. The main fermentation appeared to be completed during :his period. Productivity of alcohol during Takju brewing was found to be apporximately 65% of the total amount of starch put in mashing. 5. The reason that Saccharomyces coreanuss found be Saito in the mash of Takju was not detected in the present experiment is considered due to the facts that Aspergillus oryzae has been inoculated in the mold wheat (Nuruk) since around 1930 and also that Koji has been used in Takju brewing, consequently causing they complete change in microflora in the Takju brewing. This consideration will be supported by the fact that the original flavor and taste have now been remarkably changed.

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Microbiological and Enzymological Studies on the Flavor Components of Sea Food Pickles (젓갈등속(等屬)의 정미성분(呈味成分)에 관(關)한 미생물학적(微生物學的) 및 효소학적(酵素學的) 연구(硏究))

  • Lee, Ke-Ho
    • Applied Biological Chemistry
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    • v.11
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    • pp.1-27
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    • 1969
  • More than thirty kinds of sea food pickles have been eaten in Korea. Out of these salted yellow tail pickle, salted clam pickle, salted oyster pickle, and salted cuttlefish pickle were employed for the analysis of their components, identification of main fermenting microbes, and determination of enzyme characteristics concerned. Also studied was the effect of enzymic action of microbes, which are concerned with the fermenting of pickles, on the production of flavorous 5'-mononucleotides and amino acids. The results are summarized as follows: 1. Microflora observed in the pickles are: (a) Total count of viable cells after 1-2 months of pickling was found to be $10^7$ and that after 6 months decreased to $10^4$. (b) Microbial occurence in the early stage of pickling was observed to be 10-20% Micrococcus spp., 10-20% Brevibacterium spp., 0-30% Sarcina spp., 20-30% Leuconostoc spp., ca 30% Bacillus spp., 0-10% Pseudomonas spp., 0-10% Flavobacterium spp., and 0-20% yeast. (c) Following the early stage of pickling, mainly halophilic bacteria such as Bacillus subtilis, Leuconostoc mesenteroides, Pediococcus halophilus and Sarcina litoralis, were found to exhibit an effect on the fermentation of pickle and their enzyme activities were in direct concern in fermentation of pickles. (d) Among the bacteria participating in the fermentation, Sarcina litoralis 8-14 and 8-16 strains were in need of high nutritional requirement and the former was grown only in the presence of purine, pyrimidine and cystine and the latter purine, pyrimidine and glutamic acid. 2. Enzyme characteristics studied in relation to the raw materials and the concerned microbes isolated are as follows: (a) A small amount of protease was found in the raw materials and 30-60% decrease in protease activity was demonstrated at 7% salt concentration. (b) Protease activity of halophilic bacteria, Bacillus subtilis 7-6, 11-1, 3-6 and 9-4 strains, in the complete media decreased by 10-30% at the 7% salt concentration and that of Sarcina litoralis 8-14 and 8-16 strains decreased by 10-20%. (c) Proteins in the raw materials were found to be hydrolyzed to yield free amino acids by protease in the fermenting microbes. (d) No accumulation of flavorous 5'-mononucleotides was demonstrated because RNA-depolymerase in the raw materials and the pickles tended to decompose RNA into nucleoside and phosphoric acid. (e) The enzyme produced in Bacillus subtilis 3-6 strain isolated from the salted clam pickles, was ascertained to be 5'-phosphodiesterase because of its ability to decompose RNA and thus accumulating 5'-mononucleotide. (f) It was demonstrated that the activity of phosphodiesterase in Bacillus subtilis 3-6 strain was enhanced by some components in the corn steep liquor and salted clam pickle. The enzyme activity was found to decrease by 10-30% and 40-60% at the salt concentration of 10% and 20%, respectively. 3. Quantitative data for free amino acids in the pickles are as follows: (a) Amounts of acidic amino acids such as glutamic and aspartic acids in salted clam pickle, were observed to be 2-10 times other pickles and it is considered that the abundance in these amino acids may contribute significantly to the specific flavor of this food. (b) Large amounts of basic amino acids such as arginine and histidine were found to occur in salted yellow tail pickle. (c) It is much interesting that in the salted cuttlefish pickle the contents of sulfur-containing amino acids were exceedingly high compared with those of others: cystine was found to be 17-130 times and methionine, 7-19 times. (d) In the salted oyster pickle a high content of some essential amino acids such as lysine, threonine, isoleucine and leucine, was demonstrated and a specific flavor of the pickle was ascribed to the sweet amino acids. Contents of alanine and glycine in the salted oyster pickle were 4 and 3-14 times as much as those of the others respectively. 4. Analytical data for 5'-mononucleotides in the pickles are as follows: (a) 5'-Adenylic acid and 3'-adenylic acid were found in large amounts in the salted yellow tail pickle and 5'-inosinic acid in lesser amount. (b) 5'-Adenylic acid, especially 3'-adenylic acid predominated in amount in the salted oyster pickle over that in the other pickles. (c) The salted cuttlefish pickle was found to contain only 5'-adenylic acid and 3'-adenylic acid. It has become evident from the above fact that clam and the invertebrate lack of adenylic deaminase and contain high content of adenylic acid. Thus, they were demonstrated to be the AMP-type. (d) 5'-Inosinic acid was contained in the salted yellow tail pickle in a significant concentration, and it might be considered to be IMP-type. 5. Comparative data for flavor with regard to the flavorous amino acids and the contents of 5'-mononucleotides are: (a) A specific flavor of salted yellow tail pickle was ascribed to the abundance in glutamic acid and aspartic acid, and to the existence of a small amount of flavorous 5'-inosinic acid. The combined effect of these components was belived to exhibit a synergistic action in producing a specific fiavor to the pickle. (b) A specific flavor of salted clam pickle has been demonstrated to be attributable to the richness in glutamic acid and aspartic acid rather than to that of 5'-mononucleotides.

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Activities of the Hydrolytic Enzymes Produced by Plant Pathogenic Fungi, Sclerotium rolfsii, Sclerotinia Sclerotinia and Sclerotiorum, and Helminthosporium sigmoideum var. irregulare (수종의 식물병원균(흰비단병균$\cdot$균핵병균 및 좀검은 균핵병균)이 생산하는 가수분해효소의 활성)

  • Cho B. H.;Kim K.
    • Korean journal of applied entomology
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    • v.16 no.4 s.33
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    • pp.199-208
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    • 1977
  • Activities of various hydrolytic enzymes produced by three plant pathogenic fungi, Sclerotium rolfsii Sacc., Sclerotinia sclerotiorum (Lieb.) deBary and Helminthosporium sigmoideum var. irregulare Crallery et Tullius, were measured. Activties and amounts of the enzymes in mycelia, cultural filtrates, and sclerotia(except of sclerotia of H. sigmoideum var. irregulare) were estimated at various pH levels in order to find out optimal pH for their enzymatic activities. Enzymes such as cellulase (ex), invertase, xylanase, $\beta-amylase$, polymethylgalacturonase, polygalacturonase, phosphatase and protease were estimated. Culture solution for production of enzymes was prepared by adding of 10g, D-glucose, 1.3g $NH_4NO_3,\; 0.5g\; MgSO_4,\;7H_2O,\; and\; 1.0g\; KH_2PO_4$ into 1 liter of potato decoction plus 2ml of micro element solution consisting of 0.2mg. Fe, 0.2mg Zn, and 0.1mg Mn as the sulphates into 1 liter of distilled water. All tested mycelia and cultural filtrates were obtained from the cultures incubarted in previous solution for ten days at $25^{\circ}C$, and sclerotia were harvested from PDA plates of 3. days old, The crude enzyme solutions were prepared according to the method of Miyazaki etal. Ten days after incubation, activities of Cx produced by Scl. sclerotiorum were higher than those of the other fung and each of Cx from three fungi showed different pH optima, such as S. rolfsii and Scl. schlerotiorum in acid side (around pH 3.0), H. sigmoideum var. irregulare in neutral side (around pH 6.3). Invertase activities of S. rolfsii were 20 times higher than those of the other fungi in all samples. All tested fungi, however, showed no significant difference between the enzymatic activities of their cultural filtrate and mycelia and the activities in sclerotia of S. rolfsii and Scl. sclerotiorum were hardly recognized. There were multiple peaks on the xylanase activity curves of three fungi in terms of pH values. High activities of the xylanase were revealed in sclerotia of S. rolfsii and Scl. sclerotiorum, and in mycelia of H. sigmoideum var. irregulare. The highest activities of $\beta-amylase$ were shown both in mycelia and cultural filtrate of H. sigmoideum var. irregulae among the tested fungi, and their optimal pH was 6.2 in both mycelia and cultural filtrate. In the S. rofsii and Sel. sclerotiorum, however, the activities of cultural filtrates were higher than those of the other fungi, and optimal pH was 3.0 and 6.2 for cultural filtrate and both mycelia and sclerotia, respectively. Activities of PMG were high in cultural filtrates of all tested fungi, especially in Scl. sclerotiorum and H. sigmoideum var. irregulare. Mycelia of themalso showed the considerable activities. Optimal pH for enzymatic activities were variable with thekind of fungi or with the samples measured. The highest activities of PG were presented by mycelia of S. rolfsii and Scl. sclerotiorum. $9.l\mu /min.\; and\; 9.5\mu g/min.$, respectively. Optimal pH for activity of PG in mycelia was around 4.5 in S. rolfsii and around 3.0 in Scl. sclerotiorum. Phosphatase of S. rolfsii and Scl. sclerotiorum was more active in acid side (optimal PH3. 5) and that of H. sigmoideum var. irregulare showed one peak each in acid, neutral and alkaline side. But the highest peak was at pH 9.5. Protease of all tested fungi was more active at pH 10.0, especially that of the cultural filtrate of H. sigmoideum var. irregualre.

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A Novel Ubiqutin C-terminal Hydrolase (UCH-9) from Chick Skeletal Muscle: Its Purification and Charaterization

  • U, Seong-Gyun;Baek, Seong-Hui;Sin, Dong-Hun;Kim, Hye-Seon;Yu, Yeong-Jun;Jo, Jung-Myeong;Gang, Man-Sik;Jeong, Jin-Ha
    • Animal cells and systems
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    • v.1 no.2
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    • pp.323-328
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    • 1997
  • We have previously shown that chick muscle extracts contained at least 10 different ubiquitin C-terminal hydrolases (UCHs). In the present studies, one of the enzymes, called UCH-9, was purified by conventional chromatographic procedures using $^{125}l$-labeled ubiquitin-${\alpha}$NH-MHISPPEPESEEEEE HYC (Ub-PESTc) as a substrate. The purified enzyme behaved as a 27-kDa protein under both denaturing and nondenaturing conditions, suggesting that it consists of a single polypeptide chain. It was maximally active at pHs between 7 and 8.5, but showed little or no activity at pH below 6 and above 10. Lice other UCHs, its activity was strongly inhibited by sulfhydryl blocking reagents, such as iodoacetamide, and by Ub-aldehyde. In addition to Ub-PESTc, UCH-9 hydrolyzed Ub-aNH-protein extensions, including Ub-${\alpha}NH$-carboxyl extension protein of 80 amino acids and Ubo-${\alpha}NH$-dihydrofolate reductase. However, this enzyme was not capable of generating free Ub from mono-Ub-${\varepsilon}NH$-protein conjugates and from branched poly-Ub chains that are ligated to proteins through ${\varepsilon}NH$-isopeptide bonds. This enzyme neither could hydrolyze poly-His-tagged di-Ub. These results suggest that UCH-9 may play an important role in production of free Ub and ribosomal proteins from their conjugates.

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Effects of Supplementary Threonine, Canola Oil or Enzyme on Nutrient Digestibility, Performance and Carcass Traits of Growing-finishing Pigs Fed Diets Containing Wheat Distillers Grains with Solubles

  • Thacker, P.A.
    • Asian-Australasian Journal of Animal Sciences
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    • v.22 no.12
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    • pp.1676-1685
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    • 2009
  • This trial was conducted to determine the effects of various feed additives on nutrient digestibility, performance and carcass traits of growing-finishing pigs fed diets containing wheat distiller' grains with solubles (WDGS). Seventy-two, individually fed pigs (19.7${\pm}$2.6 kg), were assigned to one of six dietary treatments in a 6${\times}$2 (treatment${\times}$sex) factorial design (N = 12). The control diet was based on wheat and soybean meal while the five experimental diets contained 20% WDGS during the growing period and 12% WDGS during the finishing period. One 20% WDGS diet was unsupplemented while the remaining diets were supplemented with either 0.1% threonine, 5% canola oil, 0.2% enzyme (0.1% Endofeed W containing 1,250 units/g of xylanase and 385 units/g of $\beta$-glucanase and 0.1% Vegpro containing 7,700 HUT/g protease and 75 CMC/g cellulase), or a combination of the three additives at the same levels as those fed separately. The digestibility of dry matter, crude protein and energy were all significantly higher in the control diet than the unsupplemented diet containing 20% WDGS. None of the feed additives improved nutrient digestibility. In addition, none of the additives had any significant effect on gain or feed intake during the growing (19.7 to 43.6) or finishing (43.6 to 114.3 kg) periods or overall (19.7 to 114.3 kg). During the growing period, feed conversion was significantly improved for pigs fed the combination of additives compared with the unsupplemented WDGS diet. During the finishing period and overall, feed conversion was significantly improved for pigs fed 5% canola oil alone or in combination with the other additives. None of the supplements had any effect on carcass traits. These results indicate that WDGS can be successfully used as a partial replacement for soybean meal in diets fed to growingfinishing pigs. However, due to its low energy content, there may be some merit in including high energy ingredients such as canola oil when diets containing WDGS are fed.

Utilization of Fruit Processing Wastes in the Diet of Labeo rohita Fingerling

  • Deka, Abani;Sahu, N.P.;Jain, K.K.
    • Asian-Australasian Journal of Animal Sciences
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    • v.16 no.11
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    • pp.1661-1665
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    • 2003
  • A feeding trial was conducted for 60 days to study the utilization of fruits processing wastes as feed ingredient in the diet of Labeo rohita fingerlings. One hundred and sixty fingerlings (av. body weight, $1.65g{\pm}0.03$) were equally distributed in four experimental groups having 4 replicates each. Four different experimental diets were prepared by replacing wheat flour and rice bran with either orange (T2) (Cirtus qurantium), pineapple (T3) (Ananas spp. and Pseudananas spp.) or sweet lime (T4) (Citrus sinensis) wastes to the basal diet along with the control (T1, without any fruit wastes) keeping the CP level at around 40%. The water quality parameters like DO, $CO_2$, pH, total alkalinity, total hardness, ammonia and water temperature were recorded within the optimum range. The diet containing 25% pineapple wastes (T3) showed significantly higher growth in terms of SGR (1.50), FCR (2.09) and PER (1.19) than the other groups. However, growth of T4 and T2 groups were not significantly different than the control group (T1). Protease activity (17.17 unit/mg protein), protein digestibility (91.57%) and carbohydrate digestibility (41.62%) were not significantly different among the different groups. Survival of the fingerlings were not significantly different among the experimental groups. It concludes that waste of orange, pineapple and sweet lime can be used at 25% level as a substitute of wheat flour and rice bran in the diet of Labeo rohita.

Effect of Feeding Ficus infectoria Leaves on Rumen Microbial Profile and Nutrient Utilization in Goats

  • Singh, B.;Chaudhary, L.C.;Agarwal, N.;Kamra, D.N.
    • Asian-Australasian Journal of Animal Sciences
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    • v.24 no.6
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    • pp.810-817
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    • 2011
  • A feeding trial was conducted to study the effect of tannin rich Pakar (Ficus infectoria) leaves on microbial profile, rumen fermentation and nutrient utilization in goats. Eight goats divided in two groups were fed pakar leaves (experimental group) and green oats (control group) as sole roughage source along with a fixed quantity of concentrate mixture for a period of 3 months. Two metabolic trials of six days duration were conducted after 30 and 90 days of experimental feeding. The dry matter intake was significantly higher (p<0.05) and digestibility's of DM, OM, CP, EE, NDF and ADF were reduced in experimental as compared with the control group. The TDN intake was similar (236.52 vs. 240.39 g/d) in both the groups. All the animals were in positive nitrogen balance. The concentration of ammonia nitrogen, TVFA, lactic acid and activities of xylanase and protease were reduced in pakar leaves fed goats. The rumen microbial profile as obtained by MPN technique showed no change in total bacterial population but total fungi and cellulolytic bacteria were reduced (p<0.05), whereas, tannin degrading/tolerant bacteria increased with the feeding of pakar leaves. Real time PCR data revealed a decrease in Ruminococcus flavefaciens, an increase in methanogens and no change in the Fibrobacter succinogenes population by feeding of pakar leaves.

Monoclonal Antibody-Based Indirect-ELISA for Early Detection, Diagnosis and Monitoring of Epiphytic Didymella bryoniae in Cucurbits.

  • Lee, Seon-Chul;Shim, Chang-Ki;Kim, Dong-Kil;Bae, Dong-Won;Kyo, Seo-Il;Kim, Hee-Kyu
    • Proceedings of the Korean Society of Plant Pathology Conference
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    • 2003.10a
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    • pp.133.1-133
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    • 2003
  • Gummy stem blight, caused by Didymella bryoniae occurs exclusively on cucurbits. This fungus has been known not to produce its pycnidium in vitro unless irradiated. Through this study, we optimized cultural conditions for mass-production of pycnidiospore by Metal Halide Lamp irradiation. In brief, the mycelial was cultured at $26^{\circ}C$ on PDA, for 2 days under the darkness, and then the plate was illuminated with MH lamp continuously for 3-4 days at $26^{\circ}C$, a great number of pycnidia was simultaneously formed. Thus produced pycnidiospores were used as immunogen. From fusions of myeloma cell (v-653) with splenocytes from immunifed mice were car ried out. And, two hybridoma cell lines that recognized the immunogen Didymella bryoniae were obtained. One Monoclonal Antibody, Db1, recognized the supernatant and the other monoclonal antibody, Db15, recognized the spore. Two clones were selected which were used to produce ascite fluid two MAb Db1 and Db15, were immunotyped and identified as IgG1 and IgG2b, respectively. Titer of MAb Db1 and MAb Db15 was measured absorbance exceeded 0.5 even at a $10^{-5}$ dilution. The MAbs reacted positively with Didymella bryoniae but none reacted with other of fungi and CMV, CGMMV Sensitivity of MAb was precise enough to detect spore concentration as low as $10^{3}$ well by indirect ELISA characterization of the MAb Db1, Db15 antigen by heat and protease treatments show that the epitope recognized by the MAb Bb1, Db15 were a glycoprotein.

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Correlation of expression and activity of matrix metalloproteinase-9 and -2 in human gingival cells of periodontitis patients

  • Kim, Kyung-A;Chung, Soo-Bong;Hawng, Eun-Young;Noh, Seung-Hyun;Song, Kwon-Ho;Kim, Hanna-Hyun;Kim, Cheorl-Ho;Park, Young-Guk
    • Journal of Periodontal and Implant Science
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    • v.43 no.1
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    • pp.24-29
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    • 2013
  • Purpose: Matrix metalloproteinases (MMPs) are capable of degrading extracellular matrix, and they are inducible enzymes depending on an inflammatory environment such as periodontitis and bacterial infection in periodontal tissue. Gingival inflammation has been postulated to be correlated with the production of MMP-2 and MMP-9. The objective of this study was to quantify the expression and activity of MMP-9 and -2, and to determine the correlation between activity and expression of these MMPs in human gingival tissues with periodontitis. Methods: The gingival tissues of 13 patients were homogenized in $500{\mu}L$ of phosphate buffered saline with a protease inhibitor cocktail. The expression and activity of MMP-2 and -9 were measured by enzyme-linked immunosorbent assay and Western blot analysis, and quantified by a densitometer. For the correlation line, statistical analysis was performed using the Systat software package. Results: MMP-9 was highly expressed in all gingival tissue samples, whereas MMP-2 was underexpressed compared with MMP-9. MMP-9 activity increased together with the MMP-9 expression level, with a positive correlation (r=0.793, P=0.01). The correlation was not observed in MMP-2. Conclusions: The expression of MMP-2 and -9 might contribute to periodontal physiological and pathological processes, and the degree of MMP-9 expression and activity are predictive indicators relevant to the progression of periodontitis.

Intestinal Development and Function of Broiler Chickens on Diets Supplemented with Clinoptilolite

  • Wu, Q.J.;Zhou, Y.M.;Wu, Y.N.;Wang, T.
    • Asian-Australasian Journal of Animal Sciences
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    • v.26 no.7
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    • pp.987-994
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    • 2013
  • The purpose of this study was to evaluate the effect of natural clinoptilolite (NCLI) and modified clinoptilolite (MCLI) on broiler performance, gut morphology, intestinal length and weight, and gut digestive enzyme activity. A total of 240 d-old male chicks were randomly assigned to 3 treatments, each of which comprised 8 pens of 10 chicks per pen. Birds in the control group were fed the basal diet, while those in the experimental groups were fed diets supplemented with NCLI at 2% (NCLI group), or MCLI at 2% (MCLI group), respectively, for 42 d. Compared with the control, supplementation with NCLI or MCLI had no significant (p>0.05) effects on productive parameters from d 1 to 42. Supplementation with NCLI or MCLI had no influence on the relative length and weight of small intestine at d 1 to 21. But supplementation with NCLI or MCLI significantly reduced the relative weight of duodenum. Supplementation with MCLI and NCLI was associated with greater (p<0.05) villus height in the jejunal and ileal mucosa compared with those areas in the controls from d 1 to 42. However, supplementation with NCLI and MCLI had no significant (p>0.05) influence on the crypt depth in the jejunal and ileal mucosa compared with those in the controls. The addition of either NCLI or MCLI to the diet improved the activities of total protease, and amylase in the small intestinal contents. In conclusion, supplementation with NCLI or MCLI in diets improved intestinal morphology, increased the intestinal length and weigh and gut digestive enzyme activity.