Purification and biochemical characteristics of pectinesterase from Malatya apricot (Prunus armeniaca L.)

Pectinesterase (PE) in Malatya apricot pulp (Prunus armeniaca L.) was extracted and purified through (NH(4))(2)SO(4) precipitation, dialysis, and DEAE-Sephadex gel filtration chromatography. The samples obtained from the dialysis procedure, named partially purified enzyme, were used for characterization of the apricot pectinesterase. The effect of various factors such as pH, temperature, heat, and storage stability on the partially purified apricot PE enzyme was investigated. Optimum pH value was 9.0 for PE with 1% pectin in 0.1 N NaCl (w/v). The optimum temperature for apricot PE was found to be 60 degrees C on standard analysis conditions. Heat inactivation studies showed a decrease in enzymatic activity at temperatures above 70 degrees C. Km and V(max) values were 0.77 mM and 1.75 micromol min(-1) mg(-1) for apricot PE. Five inhibitors were tested in the study; the most effective inhibitor was found to be sodium carbonate (100% inhibition). The order of inhibitory effectiveness was: Na(2)CO(3), iodine, lauril sulphate, AgNO(3), EDTA. Thermal inactivation data indicated that apparent activation energy with pectin substrate was 2.96 kcal mol(-1) for the enzyme. Ascorbic acid, CaCl(2), and KCl showed activatory effect on the apricot PE enzyme.     

Potential role of in vitro iron bioavailability studies in combatting iron deficiency: a study of the effects of phosvitin on iron mobilization from pinto beans

Iron deficiency and iron overload affect one billion people worldwide. Treatment of iron malnutrition can be enhanced by an understanding of iron bioavailability from the diet. We have focused on the development of in vitro methods for determining iron bioavailability in the hopes of providing both an understanding of the chemical basis leading to the inhibition or enhancement of iron absorption and the provision of methodologies which will allow nutritionists around the world to ascertain iron bioavailability of local foods and food combinations. The study reported here focuses on the effects of phosvitin, a suspected inhibitor of iron absorption found in egg yolks, on the chemistry of iron during the in vitro enzymatic digestion of pinto beans. Three basic types of information were obtained. First, the total soluble iron was determined during in vitro enzymatic digestion under simulated oral, gastric (pH 2) and duodenal (pH 6) conditions. Phosvitin was found to have a strong solubilizing effect at pH 6 and pH 2 when in the presence of ascorbate. Pyrophosphate also leads to high iron mobilization. A second approach is to determine the static Fe2+ and Fe3+ concentrations following in vitro enzymatic digestion of pinto beans at pH 2 and pH 6. Ascorbic acid enhanced the total soluble iron at both pH values, however, only at pH 2 was a large proportion of the iron found in the Fe2+ state and then only in the presence of phosvitin but not pyrophosphate. A third approach is to determine the amount of Fe2+ formed in the digestive supernatant during a 10-min incubation with ferrozine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fermentation of sunflower seed hull hydrolysate to ethanol by Pichia stipitis

Ethanol production from sunflower seed hull hydrolysate was evaluated using Pichia stipitis NRRL Y-7124. The hydrolysate prepared with 0.7 M H2SO4 at 90 degrees C was fermented as substrate in shaking bath experiments at 30 degrees C. In a group of experiments, the influence of various detoxification methods on the fermentability of hydrolysate was investigated at pH 6. Even though the ability of all employed pretreatments to enhance fermentation performance was close, the sequential application of overliming with sodium sulfite addition was the best detoxification method. Additional experiments were performed with detoxified hydrolysate to investigate the effect of shaking rate (70-130 rpm) and initial pH (5.5-7) on the fermentation. The highest ethanol level 11 gL(-1) was achieved at initial pH of 6 and 100 rpm shaking rate from a hydrolysate containing 48 gL(-1) total reducing sugar. The corresponding alcohol yield and volumetric productivity were 0.32 gg(-1) and 0.065 gL(-1)h(-1).     

Purification and characterization of a novel polysaccharide involved in the pellicle produced by a thermotolerant Acetobacter strain

Acetobacter strains able to produce a thick pellicle at 37 degrees C were screened among many thermotolerant strains isolated from fruits in Thailand. As a result, Acetobacter sp. SKU 1100 was selected as the producer of a relatively thick pellicle even when cultured at higher temperatures such as 37 degrees C or 40 degrees C. This strain could produce a pellicle polysaccharide in a shaking submerged culture as well as under static culture conditions. The polysaccharide was found to be attached to the bacterial cells. Although the polysaccharide production was higher at 30 degrees C than at 37 degrees C in shaking submerged culture, the productivity in static culture was not decreased even at higher temperatures. The membrane-attached polysaccharide was purified from the SKU 1100 strain by cell disruptions using either ultrasonic treatment or lysozyme treatment, followed by ultracentrifugation, enzyme treatments, dialysis against SDS, DEAE-cellulose column chromatography, alcohol precipitation, and gel filtration chromatography. The polysaccharide purified by the sonic treatment and also by the mild conditions using lysozyme treatment had the same average molecular mass of 120 kDa. The purified polysaccharide was composed of three different monosaccharides; glucose, galactose, and rhamnose, in an approximately equimolar ratio of 1:1:1.     

Improvement of the enzymatic stability of a cytotoxic T-lymphocyte-epitope model peptide for its oral administration

Oral administration of peptide antigens, to provide proper mucosal and/or systemic immunity, is largely ineffective. This is mainly due to the very small quantity of antigen that survives degradation in the intestine and that crosses the intestinal absorption membrane. The present study focuses on the improvement of the enzymatic stability of a 13 amino acid long peptide containing a cytotoxic T-lymphocytes (CTL)-epitope. Within this study, it is shown, that simple chemical modification at the N- and C-terminus of the peptide can provide significant stability towards enzymatic attack by intestinal exopeptidases. Around 50% of the modified peptide resisted enzymatic attack on native porcine intestinal mucosa within 3h of incubation at pH 6.8 and 37 degrees C, whereas unmodified control peptide was almost completely degraded within the same time period. Additionally, a mucoadhesive drug carrier matrix with specific inhibitory properties towards luminally secreted endopeptidases has been generated. The incorporation of the simply modified peptide in this delivery system should enhance the amount of biologically active antigen being available at the mucosal site for further presentation to immunomodulating systems. This might open the door for a successful oral immunotherapy.     

Characterization of Caco-2 and HT29-MTX cocultures in an in vitro digestion/cell culture model used to predict iron bioavailability

Cocultures of two human cell lines, Caco-2 and HT29-MTX mucus-producing cells, have been incorporated into an in vitro digestion/cell culture model used to predict iron bioavailability. A range of different foods were subjected to in vitro digestion, and iron bioavailability from digests was assessed with Caco-2, Caco-2 overlaid with porcine mucin, HT29-MTX or cocultures of Caco-2 and HT29-MTX at varying ratios. It was found that increasing the ratio of HT29-MTX cells decreased the amount of ferritin formed and resulted in an overall decline in the ability of the model to detect differences in iron bioavailability. At the physiologically relevant ratios of 90% Caco-2/10% HT29-MTX and 75% Caco-2/25% HT29-MTX, however, a mucus layer completely covered the cell monolayer and the in vitro digestion model was nearly as responsive to changes in sample iron bioavailability as pure Caco-2 cultures. The in vitro digestion/Caco-2 cell culture model correlates well with human iron bioavailability studies, but, as mucus appears to play a role in iron absorption, the addition of a physiologically realistic mucus layer and goblet-type cells to this model may give more accurate iron bioavailability predictions.     

Analysis of the activation of pepsinogen in the presence of protein substrates and estimation of the intrinsic proteolytic activity of pepsinogen

Monkey pepsinogen A, monkey progastricsin, and porcine pepsinogen A were activated in the presence of two different protein substrates, namely, reduced and carboxymethylated lysozyme and hemoglobin. In each case, an extensive delay in activation was observed. The intermolecular activation reaction required for the generation of pepsin or gastricsin was strongly inhibited and this inhibition was essentially responsible for the delay. However, the intramolecular reaction required for the generation of the intermediate forms of the proenzymes was scarcely affected. The delay was longer at pH 3.0 than at pH 2.0. Irrespective of the delay in activation of pepsinogen, the digestion of substrates proceeded rapidly, evidence of the significant proteolytic activity of pepsinogen itself. Kinetic experiments demonstrated that pepsinogen changed from an enzymatically inactive species to an active species before the release of the activation segment. The proteolytic activity of the active pepsinogen was highest at pH 2.0, at 37 degrees C and the activity under these conditions was comparable to that of pepsin.     

草鱼肠道酸碱度的研究

胃肠道是一个复杂的消化系统, 每一部分都具有独特的生理特征。酸碱度(pH)是消化道重要的生理指标之一, 其对营养物质的消化、吸收和肠道微生物的生长等具有重要影响。为了研究草鱼在食物消化过程中, 肠道的酸碱度变化, 测定了草鱼肠道食物糜、肠液和黏膜的pH。结果显示, 随着食物的消化, 它们的pH都有下降的趋势。肠道食物糜pH在6.86±0.24到8.43±0.10之间, 肠液pH在7.14±0.22到8.63±0.02之间, 相同时间点相同肠段两者之间的pH差异很小, 并且在实验期间两者的pH变化趋势相同。黏膜pH在6.23±0.04到6.7±0.13之间, 为弱酸性。除了时间点12h外, 相同时间点和相同肠道部位黏膜的pH与食物糜、肠液的pH相比均有显著性差异(P<0.05)。分析发现草鱼摄食食物的pH与上述三相的pH之间均有显著性差异(P<0.05), 研究结果为草鱼消化生理及营养学研究提供了基础资料。     

A component of fluid absorption linked to passive ion flows in the superficial pars recta

We studied salt and water absorption in isolated rabbit superficial proximal straight tubules perfused and bathed with solutions providing oppositely directed transepithelial anion gradients similar to those which might obtain in vivo. The perfusing solution contained 138.6 mM Cl- 3.8 mM HCO-3 (pH 6.6) while the bathing solution contained 113.6 mM Cl- and 25 mM HCO-3 (pH 7.4); the system was bubbled with 95% O2-5% CO2. At 37 degrees C, net volume absorption (Jv nl min-1 mm-1) was 0.32 +/- 0.03 (SEM); Ve, the transepithelial voltage (millivolts; lumen to bath), was +3.1 +/- 0.2. At 21 degrees C, Ve rose to +3.7 +/- 0.1 and Jv fell to 0.13 +/- 0.01 (significantly different from zero at P less than 0.001); in the presence of 10(-4)M ouabain at 37 degrees C, Ve rose to +3.8 +/- 0.1 and Jv fell to 0.16 +/- 0.01 (P less than 0.001 with respect to zero). In paired experiments, the ouabain- and temperature-insensitive moieties of Jv and Ve became zero when transepithelial anion concentration gradients were abolished. Titrametric determinations net chloride flux at 21 degrees C or at 37 degrees C with 10(-4) M ouabain showed that chloride was the sole anion in an isotonic absorbate. And, combined electrical and tracer flux data indicated that the tubular epithelium was approximately 18 times more permeable to Cl- than to HCO-3. We interpret these results to indicate that, in these tubules, NaCl absorption depends in part on transepithelial anion concentration gradients similar to those generated in vivo and in vitro by active Na+ absorption associated with absorption to anions other than chloride. A quantitative analysis of passive solute and solvent flows in lateral intercellular spaces indicated that fluid absorption occurred across junctional complexes when the osmolality of the lateral intercellular spaces was equal to or slightly less than that of the perfusing and bathing solutions; the driving force for volume flow under these conditions depended on the fact that sigmaHCO3 exceeded sigmaCl.     

Physical factors affecting the production of organic solvent-tolerant protease by Pseudomonas aeruginosa strain K

The physical factors affecting the production of an organic solvent-tolerant protease from Pseudomonas aeruginosa strain K was investigated. Growth and protease production were detected from 37 to 45 degrees C with 37 degrees C being the optimum temperature for P. aeruginosa. Maximum enzyme activity was achieved at static conditions with 4.0% (v/v) inoculum. Shifting the culture from stationary to shaking condition decreased the protease production (6.0-10.0% v/v). Extracellular organic solvent-tolerant protease was detected over a broad pH range from 6.0 to 9.0. However, the highest yield of protease was observed at pH 7.0. Neutral media increased the protease production compared to acidic or alkaline media.     

梨小食心虫的产卵选择性

为了准确掌握梨小食心虫的产卵特性,研究模拟室外条件下该虫在不同寄主果树的叶片、桃枝不同部位及不同品种桃果上的产卵偏好.结果表明: 梨小食心虫成虫对7种寄主果树叶片的产卵偏好由高至低依次为:桃＞樱桃＞苹果＞李＞梨＞海棠＞杏.在桃树叶片上的产卵量占总产卵量的33.5%,平均单叶卵量达8.3粒;虫卵在不同寄主叶片正、反两面的分布有所差异,苹果和海棠叶片正面卵量多于背面,桃、李、梨、杏叶背面卵量多于正面,桃叶背面卵量是正面的3.3倍,樱桃叶两面卵量差异不明显;该虫在桃枝上产卵部位选择顺序为:叶片＞托叶＞叶柄＞枝条,叶片是其主要产卵部位,占总产卵量的88.7%;梨小食心虫在桃枝上主要选择靠近顶端未展叶的前10片桃叶背面产卵,前10叶卵量占总产卵量的725%,其中第3片叶上产卵最多,占9.3%;第25叶以后仅占总产卵量的1.1%;在不同类型桃果上的产卵偏好次序为:油桃＞蟠桃＞毛桃;绒毛疏密及其特点是影响梨小食心虫在寄主果树叶片和果实上产卵选择的首要因素.     

梨小食心虫的产卵选择性

为了准确掌握梨小食心虫的产卵特性,研究模拟室外条件下该虫在不同寄主果树的叶片、桃枝不同部位及不同品种桃果上的产卵偏好.结果表明: 梨小食心虫成虫对7种寄主果树叶片的产卵偏好由高至低依次为:桃＞樱桃＞苹果＞李＞梨＞海棠＞杏.在桃树叶片上的产卵量占总产卵量的33.5%,平均单叶卵量达8.3粒;虫卵在不同寄主叶片正、反两面的分布有所差异,苹果和海棠叶片正面卵量多于背面,桃、李、梨、杏叶背面卵量多于正面,桃叶背面卵量是正面的3.3倍,樱桃叶两面卵量差异不明显;该虫在桃枝上产卵部位选择顺序为:叶片＞托叶＞叶柄＞枝条,叶片是其主要产卵部位,占总产卵量的88.7%;梨小食心虫在桃枝上主要选择靠近顶端未展叶的前10片桃叶背面产卵,前10叶卵量占总产卵量的725%,其中第3片叶上产卵最多,占9.3%;第25叶以后仅占总产卵量的1.1%;在不同类型桃果上的产卵偏好次序为:油桃＞蟠桃＞毛桃;绒毛疏密及其特点是影响梨小食心虫在寄主果树叶片和果实上产卵选择的首要因素.     

桃蛀果蛾寄主生物型分化的RAPD分析

以山茱萸蛀果蛾Carposina coreana Kim为外群,用44个随机引物对苹果、山楂、桃、杏、枣和酸枣等6种寄主植物上的桃蛀果蛾Carposina sasakii Matsumura进行了RAPD 分析。结果表明,不同寄主上的桃蛀果蛾之间遗传差异出现了较大的分化,桃蛀果蛾6个寄主生物型之间的平均遗传距离在0.040～0.637之间,尤其是杏生物型分化最大。经聚类分析,可将不同寄主上的桃蛀果蛾明显分成两个类群,杏生物型为一群,其余为一群。两个类群间遗传距离为0.465,亚群内部的遗传距离为0.040～0.369。推测杏树上的“桃蛀果蛾 ”已经分化成为不同的物种。     

Optimization of 7 alpha-hydroxysteroid dehydrogenase production by Escherichia coli 080

7 alpha-Hydroxysteroid dehydrogenase (EC 1.1.1.159) production by Escherichia coli strain 080 was highest when the organism was grown in brain heart infusion broth at pH 6.5 for 72-96 h with shaking at 37 degrees C. The oxygen consumption rate had a strong effect on the production of this constitutive enzyme. Glucose and lactose at 0.2-0.4%, detergents, and ethylenediaminetetra-acetic acid were found to increase the enzyme production.     

Enantioselective Degradation of (2RS,3RS)‐Paclobutrazol in Peach and Mandarin Under Field Conditions

In this study we investigated the enantioselective degradation of (2RS,3RS)‐paclobutrazol in peach and mandarin fruits under field conditions after foliar treatment at 500 mg active ingredient/L using a Lux Cellulose‐1 chiral column on a reverse‐phase liquid chromatography–tandem mass spectrometry system. Degradations of paclobutrazol in both fruits followed first‐order kinetics, with half‐lives of about 9 days. Initial deposits were 1.63 mg/kg on peach and 1.99 mg/kg on mandarin; terminal concentrations were lower than 0.05 mg/kg, which was acceptable in most cases. As anticipated, paclobutrazol levels in peels of mature mandarin were about 6.3 times higher than in pulp, indicating the potential risk of peel consumption. We also observed that paclobutrazol degradation in mature mandarin was relatively slow, indicating it might not be efficient enough to hold mandarin fruits on trees for lowering paclobutrazol concentrations. Significant enantioselectivity was observed: the (2R,3R)‐enantiomer was preferentially degraded in mandarin (whole fruit, peels, and pulp) but enriched in peach. Because of its more rapid degradation in mandarin and the lower levels observed in pulp compared with peels, potential endocrine‐related side effects due to the (2R,3R)‐enantiomer pose less of a risk in mandarin than in peach. Chirality 26:400–404, 2014. © 2014 Wiley Periodicals, Inc.     

Pyridoxal phosphate protects against an irreversible temperature-dependent inactivation of hepatic delta-aminolevulinic acid synthase

The stability of hepatic delta-aminolevulinic acid synthase (ALAS), the first and rate-limiting enzyme of the heme biosynthetic pathway, was investigated. Incubation of the mitochondrial matrix fraction obtained from either control or allylisopropylacetamide-induced rats at 37 degrees C in Tris-Cl, pH 7.4, EDTA, and dithiothreitol resulted in a rapid decrease in ALAS activity such that 50-70% of the activity was lost after 30 min. Similar decreases in ALAS activity were observed when a cytosolic fraction from the induced animals was incubated at 37 degrees C. Addition of 0.1 mM pyridoxal-P, the cofactor of ALAS, to the preincubation medium completely prevented the observed loss of activity; however, dialysis of the inactive matrix fraction against several changes of buffer containing pyridoxal-P did not restore activity, suggesting that the inactivation was irreversible. These decreases in ALAS activity in the absence of pyridoxal-P were temperature dependent, as a 55% loss of ALAS activity was observed after a 60-min incubation at 30 degrees C, while the enzyme was completely stable when preincubated at 22 degrees C for 60 min. This inactivation of ALAS does not appear to involve proteolytic digestion, as addition of a wide spectrum of protease inhibitors to the preincubation medium in the absence of pyridoxal-P did not protect against the inactivation. The suggestion is made that the cofactor, pyridoxal-P, may dissociate from the enzyme during the preincubation and, consequently, the apoenzyme may be irreversibly inactivated at temperatures above 22 degrees C.     

Factors affecting the removal of low-molecular-weight fractions (LMWF) from egg yolk and seminal plasma in extended semen by dialysis: effect on post-thaw sperm survival

Three factors affecting dialysis of bovine semen were studied. These factors were (1) dialysis rates of egg yolk, seminal plasma, and glycerol, (2) temperature (37 degrees C, 5 degrees C, and while cooling from 37 to 5 degrees C), and (3) dialysis ratios between retentate and dialysate (1:1, 1:10, 1:20, 1:50, and 1:100). Ninety percent of the low-molecular-weight fraction (LMWF) from seminal plasma, egg yolk, and glycerol was removed from the retentate in a 2-hr period at 5 degrees C, and only slight changes were detected after the third hour of dialysis. Temperature affected dialysis and was faster at 37 degrees C. It was also found that a 1:20 dialysis ratio was sufficient to obtain 90% clearance of the LMWF. The effect of sperm dilution ratio, dialysis ratio, and exchange of the LMWF from egg yolk and/or seminal plasma for buffer systems was also studied. An improvement in post-thaw motility of spermatozoa (P less than 0.05) was obtained when the LMWF from both seminal plasma and egg yolk were replaced. A third experiment was conducted to study the effect of different combinations between the buffer systems, TEST and Na citrate, in the dialysate. The results indicated that a 1:1 combination of iso-osmotic solutions (320-325 mOsm/Kg, pH 7.0) between these two buffers, with 5% glycerol (v/v), yielded significant (P less than 0.05) sperm post-thaw motility as compared with the individual use of TEST-glycerol or Na citrate-glycerol. Dialyzed samples also yielded sperm post-thaw motility higher than that of the nondialyzed samples. Colloidal materials in the dialysate did not affect survival of spermatozoa.     

Isolation and characterization of anti-Salmonella lactic acid bacteria from the porcine gastrointestinal tract

AIMS: To identify lactic acid bacteria (LAB) of porcine intestinal origin with anti-Salmonella activity. METHODS AND RESULTS: Samples were obtained from pig faeces and caeca and screened for the presence of anti-Salmonella LAB. The 11 most promising isolates were identified as belonging to the genera Lactobacillus and Pediococcus. The LAB exhibited large variation in their ability to survive in simulated gastric juice at pH 1.85. While Lactobacillus johnsonii species survived at levels of 80% for up to 30 min, Lactobacillus pentosus species declined to <0.001% in that time. All isolates tolerated porcine bile at a concentration of 0.3% (w/v), with some isolates capable of growth in the presence of up to 5% (w/v) bile. The ability of the LAB isolates to prevent Salmonella invasion of intestinal epithelial HT-29 cells varied, with reductions of between 30% (Lact. pentosus) and 80% (Lactobacillus murinus spp.) observed. CONCLUSIONS: LAB of porcine origin were observed to survive simulated passage through the GIT and inhibit growth of Salmonella and its invasion of the intestinal epithelium. SIGNIFICANCE AND IMPACT OF THE STUDY: The data demonstrate that some porcine intestinal LAB isolates may offer potential as probiotics for the reduction of Salmonella carriage in pigs.     

Purification and characterization of carboxymethyl cellulase from Bacillus sp. isolated from a paddy field

A microorganism hydrolyzing carboxymethyl cellulose was isolated from a paddy field and identified as Bacillus sp. Production of cellulase by this bacterium was found to be optimal at pH 6.5, 37 degrees C and 150 rpm of shaking. This cellulase was purified to homogeneity by the combination of ammonium sulphate precipitation, DEAE cellulose, and sephadex G-75 gel filtration chromatography. The cellulase was purified up to 14.5 fold and had a specific activity of 246 U/mg protein. The enzyme was a monomeric cellulase with a relative molecular mass of 58 kDa, as determined by SDS-PAGE. The enzyme exhibited its optimal activity at 50 degrees C and pH 6.0. The enzyme was stable in the pH range of 5.0 to 7.0 and its stability was maintained for 30 min at 50 degrees C and its activity got inhibited by Hg2+, Cu2+, Zn2+, Mg2+, Na2+, and Ca2+.     

模拟胃肠环境对3株光合细菌存活率影响的实验研究

目的模拟胃肠环境对3株光合细菌(Photosynthetic bacteria,PSB)存活率影响进行体外实验研究。方法测定3株PSB分别在pH2.0、3.0和4.0的模拟胃液及含有或不含有0.45%牛胆盐(pH8.0)的模拟小肠液中不同时间的存活率。结果pH2.0时模拟胃液处理不同时间对3株PSB存活率有显著影响(P0.01);pH3.0时,模拟胃液处理60、90和180 min对菌株rub存活率有显著影响(P0.01),处理90、180 min对菌株Ga存活率有显著影响(P0.05),处理180 min对菌株2C存活率无显著影响(P0.05);pH4.0时模拟胃液处理180 min对3株PSB存活率无显著影响(P0.05)。模拟小肠液处理240 min对3株PSB菌株活菌数无显著影响(P0.05)。结论模拟胃液对3株PSB存活率影响具有菌株特异性和pH依赖性,而模拟小肠液对3株PSB存活率无显著影响。