The expression of the Paenibacillus sp. A11 cyclodextrinase (CDase) gene using the pUC 18 vector in Escherichia coli JM 109 resulted in the formation of an insoluble CDase protein in the cell debris in addition to a soluble CDase protein in the cytoplasm. Unlike the expression in Paenibacillus sp. A11, CDase was primarily observed in cytoplasm. However, by adding 0.5 M sorbitol as an osmolyte, the formation of insoluble CDase was prevented while a three-fold increase in cytoplasmic CDase activity was achieved after a 24 h-induction. The recombinant CDase protein was purified to approximately 14-fold with a 31% recovery to a specific activity of 141 units/mg protein by 40-60% ammonium sulfate precipitation, DEAE-Toyopearl 650 M, and Phenyl Sepharose CL-4B chromatography. It was homogeneous by non-denaturing and SDS-PAGE. The enzyme was a single polypeptide with a molecular weight of 80 kDa, as determined by gel filtration and SDS-PAGE. It showed the highest activity at pH 7.0 and $40^{\circ}C$. The catalytic efficiency ($k_{cat}/K_m$) values for $\alpha$-, $\beta$-, and $\gamma$-CD were $3.0{\times}10^5$, $8.8{\times}10^5$, and $5.5{\times}10^5\;M^{-1}\;min^{-1}$, respectively. The enzyme hydrolyzed CDs and linear maltooligosaccharides to yield maltose and glucose with less amounts of maltotriose and maltotetraose. The rates of hydrolysis for polysaccharides, soluble starch, and pullulan were very low. The cloned CDase was strongly inactivated by N-bromosuccinimide and diethylpyrocarbonate, but activated by dithiothreitol. A comparison of the biochemical properties of the CDases from Paenibacillus sp. A11 and E. coli transformant (pJK 555) indicates that they were almost identical.
Meat provides high quality proteins, lipids, minerals and vitamins. The meat protein is especially high in essential amino acids that are crucial for human health, growth & development and for the formation of enzymes, hormones and antibodies. Relatively cheap and nutritionally sound vegetable proteins that are similar to animal proteins are being developed to replace the animal proteins in texture, nutrition and food characteristics. In this study a nutritionally sound meat lipid replacing food Oatrim that has been produced by converting oat starch into maltodextrin by ${\alpha}$-amylase, have been partially substituted for beef and general component analysis, texture measurement and sensory tests have been conducted. The results are 1. Water content of the non-treated (0% treated) was 67.1% and the treated (10% treated) was 77%. The treated showed better water holding capacity. 2. Protein content of the non-treated was 21.2 g/100 g; the 4% treated, 18.4 g/100 g; the 6% treated, 18.2 g/100 g; the 8% treated, 17.2 g/100g; and the 10% treated, 16.0 g/100 g. The protein content tended significant. 3. Amino acid analysis results showed that glutamic acid content was the highest in Oatrim and as its amino acid make up is exellent, it is valuable as a fine low fat protein food. 4. Sensory tests show that the increased Oatrim content increased the appearance quality but food characteristics were high only in the 4% and 6% treated groups, indicating that the replacement ratio should not exceed 10%. 5. Texture measurement analysis results show that the higher the replacement content, lower the springness, cohesiveness, hardness, chewiness and gumminess, resulting in relatively soft overall texture. However, in order to better the food characteristics, more studies must be continuously done, and so by being able to increase vegetable substitution over meat, it may be able to contribute to the prevention of adult disease.
To study effects of methanol extract of prosomillet on lopid metabolism , five groups of male Sprang-Dawley rats weighing 116$\pm$9 g were fed test diets for four weeks. The five diets consisted of one low fat(5% w/w) diet containing starch as carbohydrate source(normal) and four high fat diets(15% w/w) containing 40.5%(w/w)sucrose(control) and additional 80% nethanol extractof prosomillet at the levels of 0.3% and 1%(w/w) or prosomillet powder at the level of 20%(w/w). Serum level of total cholesterol was a little higher but that of triglyceride was 41% lower in 20% (w/w) prosomillet powder group than in the control group. The cholesterol levels of two Liver cholesterol levels were lower and phospolipid levels higher in all three prosomillet powder group . Fecal excretionof bile acid was most increased in the prosomillet powder group among all five test groups. Acitivity of liver microsomal 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase was significantly lower in 0.3% methanol extract fed group than the control and also appeared to be reduced in 1% extract fed one, wherease those of 20 cholesterol 7$\alpha$-hydroxylase were not different among the five groups. Activities of liver cytosilic glucose-6-phosphate dehydrogenase(G6PDH) and malic enzyme were decreased in 0.3% prosomillet methanol extract and 20% powder groups. The results indicate that in addition to fiber, certain active components in prosomillet have potential to exert hypolipidemic effects via regulating hepatic cholesterogenesis and lipogenesis.
This study was designed to investigate whether Loranthus parasiticus extract (LPE) could inhibit the activities of carbohydrate digestive enzymes and alleviate postprandial hyperglycemia in diabetic mice. Lyophilized L. parasiticus was extracted with 80% ethanol and concentrated. The inhibitory effects of LPE on carbohydrate digestive enzymes were evaluated by examining α-glucosidase and αamylase, and it was seen to inhibit the activities of both enzymes in a dose-dependent manner. More specifically, the IC50 values of LPE against α-glucosidase and α-amylase were 0.121±0.007 and 0.157±0.004 mg/ml, respectively, significantly lower than those of acarbose, showing that LPE has stronger inhibitory effects than the positive control. These results suggest that LPE strongly inhibits the activities of these digestive enzymes. Blood glucose levels in the control group of diabetic mice increased to 490.00±28.52 mg/dl and 474.60±25.30 mg/dl at 60 and 120 min after a meal, respectively. However, when LPE was added to starch, postprandial blood glucose levels were significantly reduced (463.0±23.73 and 418.5±24.50 mg/dl at 60 and 120 min, respectively; p<0.05). The area under the curve also significantly decreased following administration of LPE, with no cytotoxicity. These results therefore indicate that LPE could be used as an α-glucosidase and α-amylase inhibitor and delay carbohydrate digestion and, thus, glucose absorption after a meal.
Two experiments with growing pigs were conducted to investigate the effects of two distinct multienzyme preparations on nutrient digestibility, growth performance and blood profiles. In Exp. 1, a total of 96 pigs ($29.7{\pm}0.69\;kg$) were utilized in a 42-day performance and digestibility trial using four dietary treatments: CON (control diet), ENDO (control+0.10% Endopower), NSPase1 (control+0.10% NSPase) and NSPase2 (control+0.20% NSPase). Endopower was a commercial multienzyme preparation which contained ${\alpha}$-galactosidase, galactomannase, xylanase and ${\beta}$-glucanase. NSPase mainly contained ${\alpha}$-1,6-${\beta}$-galactosidase, ${\beta}$-1,4-mannanase and ${\beta}$-1,4-mannosidase. There were six replication pens per treatment with four pigs per pen. Pigs fed NSPase1 diet had a higher ADG (p<0.05) and G:F (p<0.05) than those fed the control diet. There were no significant differences in growth performance among the multienzyme treatments (p>0.05). Compared with CON, apparent digestibility of DM was increased (p<0.05) by ENDO treatment. N digestibility was improved (p<0.05) in response to multienzyme treatments during the experimental period. Blood urea nitrogen (BUN) was higher (p<0.05) in ENDO treatment than in CON and NSPase1 treatments at the end of the experiment, while the glucose level improved (p<0.05) due to ENDO and NSPase2 treatments. In Exp. 2, four ileal-cannulated, growing barrows ($20.17{\pm}1.31\;kg$) were housed in individual metabolism crates and randomly assigned to 1of 4 treatments (same as Exp. 1) within a $4{\times}4$ Latin square design. Enzyme supplementations improved the majority of apparent ileal amino acid digestibilities (p<0.05). It is concluded that the supplementation of NSPase1 improved growth performance as well as N digestibility and partially improved apparent ileal amino acid digestibility in growing pigs fed a diet based on corn and soybean meal.
Journal of the Korean Society of Food Science and Nutrition
/
v.19
no.6
/
pp.625-635
/
1990
The purpose of this study was focused on investigation of biochemical properties of amylases in germinating corn(Zea mays L.) the amylase(I), (II) and (III) from germinating corn seeds were partially purified by ammonium sulfate precipitation, DEAE-Sephadex A-50 ion exchange column chromatography and Sephadex G-100 gel filtration. The last step was effective for separation of the corn amylases to a homogeneous slate. the purified amylase(I) was identified as a kind of $\alpha$-amylase from the fact that 5% starch solution was hydrolysed into mainly maltose and maltotetrose by it, and amylase(II) and amylase(III) were enzymes producing maltotetrose as main product. The molecular weight and specific activity of the amylase(I), (II) and (III) were determined to be 54,000 and 70.47 unit/mg, 39,000 and 62.98 unit/mg, and 51,000 and 80.39 unit/mg, respectively. It showed a tendency to increase the amylases activities in presence of Ba, Ca, Co and Fe groups, but inhibits in that of Ag, Sn, Hg and Zn groups, and amylase(I), (II) and (III) remained stable at pH 5-6 and 2$0^{\circ}C$ for 40 days in containing of 1 mM CaCl$_2$. The optimum pH and optimum temperatures were pH 6, pH 5 and pH 6 and 35$^{\circ}C$, 55$^{\circ}C$ and 55$^{\circ}C$, respectively. These results suggest that the amylase(I), (II) and (III) were different amylases.
The purpose of this study was to investigate the hypoglycemic effect of Saururus chinensis Baill in vitro and in vivo. Methanol extract of S. chinensis Baill inhibited yeast ${\alpha}$-glucosidase activity by 49.8%, which was twice as strong as that of acarbose at a concentration of 0.5 mg/mL in vitro. The effect of S. chinensis Baill methanol extract on the postprandial increase in blood glucose levels was studied in streptozotocin-induced diabetic rats using a carbohydrate load test. Oral administration of S. chinensis Baill extract (500 mg/kg) significantly decreased incremental blood glucose levels at 60 and 90 min (p<0.05) after oral ingestion of starch (1 g/kg). The area under the glucose response curve of the S. chinensis Baill group was significantly decreased compared to that of the control group (p<0.05). The effect of prolonged feeding of S. chinensis Baill was studied in an animal model of type 2 diabetes. Three-week-old db/db mice were fed an AIN-93G diet or a diet containing 0.5% S. chinensis Baill extract for 7 weeks after 1 week of adaptation. Plasma glucose, insulin, and blood glycated hemoglobin levels of the mice fed S. chinensis Baill extract were significantly lower than those of the control group (p<0.05). Therefore, we conclude that S. chinensis Baill is effective in controlling hyperglycemia in animal models of diabetes mellitus.
This study was designed to investigate whether extracts from Oxya chinensis sinuosa Mistshenk (an edible insect considered a grasshopper) could inhibit the activity of carbohydrate digestive enzymes and alleviate postprandial hyperglycemia in diabetic mice. Oxya chinensis sinuosa Mistshenk was extracted with 80% ethanol (OEE) or water (OWE) and then concentrated. The carbohydrate digestive enzyme-inhibiting activity of the resulting extracts was evaluated by examining α-glucosidase and α-amylase. The IC50 values of OEE against α-glucosidase and α-amylase were 0.229 mg/ml and 0.106 mg/ml, respectively. This result indicated that OEE has stronger inhibitory effects than OWE and positive control. The blood glucose levels of the diabetic control mice increased after one meal. However, when OEE (300 mg/kg) was added to starch, this increase in postprandial blood glucose levels was significantly suppressed. The area under the curve also significantly decreased following the administration of OEE, which exhibited no cytotoxicity. These results indicate that OEE is more efficacious than OWE and may be used as a carbohydrate digestive enzyme inhibitor, delay carbohydrate digestion and glucose absorption, and thus alleviate postprandial hyperglycemia caused by dietary carbohydrates.
The purpose of this study is to prepare extruded rice flours suitable for baking rice cookies. The extruded rice flours were prepared at 100 and 130$^{\circ}C$ temperature and 25 and 27% moisture content in a co-rotating twin screw extruder. The rice extrudates were dried at 100$^{\circ}C$ for 18 hr and subsequently ground into the fine flour. Characteristics of the extruded rice flours were examined by rapid visco analysis, hydration property analysis, differential scanning calorimetry (DSC), and in vitro digestion test. Water absorption, solubility, and swelling power of all extruded rice flours were higher than those of native rice flour. DSC analysis showed that native rice flour had a peak at about 65$^{\circ}C$ while all extruded rice flours did not show any peaks since they were already gelatinized during the extrusion proess. Viscosity of the extruded rice flours decreased with increasing temperature and lowering moisture content in the extrusion proess. The extruded rice flours prepared at 130$^{\circ}C$ exhibited lower viscosity than those prepared at 100$^{\circ}C$. The operating temperature of the extrusion proess was critical for the starch digestion in vitro. The extruded rice flours prepared at 130$^{\circ}C$ showed a rapid decrease in digestible starch content while an increased level of slowly digestible starch content was observed compared to those treated at 100$^{\circ}C$ in the extruder. Cookies were prepared with a mixture of wheat flour and extruded rice flours at the ratio of 7 to 3. The cookies made with the extruded rice flours had lower spread factor and darker yellow color than those prepared with wheat flour only. Hardness of the extruded rice flour-added cookies was similar to that of the wheat flour cookie whereas their overall acceptance was better. Therefore the rice cookies partially supplemented with extruded rice flours may have a potential as early childhood foods which require soft texture and allergy reduction.
Strain A-3, an amylase-producing bacteria, was isolated from coastal seawater near Daecheon in the Republic of Korea. It was seen to possess a single polar flagella and grow well, on ASW-YP agar plates, at temperatures of between $20-37^{\circ}C$. However, it grew more slowly at the temperatures of $15^{\circ}C$ and $40^{\circ}C$. Similarly, it was observed to grow abundantly, in an Artificial Sea Water-Yeast extract-Peptone (ASW-YP) liquid medium, in a pH range of 6-9, but not grow at pHs of 4-5 and a pH of 10. Strain A-3 was noted as being close to Pseudoalteromonas phenolica O-$BC30^T$, Pseudoalteromonas luteoviolacea $NCIMB1893^T$, Pseudoalteromonas rubra $ATCC29570^T$, and Pseudoalteromonas byunsanensis $FR1199^T$, with 98.30%, 97.86%, 97.78%, and 97.25% similarities respectively, in its 16S rRNA sequence. A phylogenetic tree revealed that strain A-3 and P. phenolica O-$BC30^T$ belong to a clade. However, strain A-3 differed from P. phenolica O-$BC30^T$ in relation to a number of physiological characteristics. Strain A-3 exhibited no growth above 5% NaCl concentrations, no utilization of D-glucose, D-mannose, D-maltose, or D-melibose, and no lipase (C-14) activity. All of these properties strongly indicate that strain A-3 is distant from P. phenolica O-$BC30^T$ and thus led us to name it Pseudoalteromonas sp. A-3. Pseudoalteromonas sp. A-3 produces ${\alpha}$-amylase throughout growth. Maximal amylase activities of 144.48 U/mL and 149.20 U/mL were seen at pH 7.0 and $37^{\circ}C$, respectively. Pseudoalteromonas sp. A-3's high, stable production of ${\alpha}$-amylase in addition to its biochemical features, such as alkalitolerance, suggest that it is a good candidate for industrial applications.
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