Optimization of the isoelectric precipitation method to obtain protein isolates from amaranth (Amaranthus cruentus) seeds

This research was conducted to evaluate the effect of extraction pH (7.8-9.2) and precipitation pH (4.3-5.7) on four selected quality attributes of protein isolates from amaranth seeds (Amaranthus cruentus) such as protein content (PC), whiteness index (WI), enthalpy of transition (EN), and denaturation temperature (DT). Ten different treatments involving extraction and precipitation pH combinations were analyzed by a central composite design; the experimental data were fitted by a second-order model using a least-squares method for each one of the four dependent variables. Response surface methodology was used for the optimization process; in addition, a common optimum value for the four dependent variables was obtained utilizing the desirability method. A confirmatory test showed that the generated regression equations could adequately predict performance of this isoelectric precipitation method. The results indicate that extraction pH and precipitation pH showed an important effect on PC, WI, and EN. However, the different combinations did not significantly affect the DT. Values of 9.2 and 8.0 for extraction pH and 5.7 for precipitation pH produced the best overall result for all responses. Finally, the results have shown that it is possible to obtain protein isolates from A. cruentus seeds at optimized values of extraction pH and precipitation pH, which presented a high protein content and good physicochemical properties.     

Lunasin and Bowman-Birk protease inhibitor concentrations of protein extracts from enzyme-assisted aqueous extraction of soybeans

Lunasin and Bowman-Birk protease inhibitor (BBI) are two soybean peptides to which health-promoting properties have been attributed. Concentrations of these peptides were determined in skim fractions produced by enzyme-assisted aqueous extraction processing (EAEP) of extruded full-fat soybean flakes (an alternative to extracting oil from soybeans with hexane) and compared with similar extracts from hexane-defatted soybean meal. Oil and protein were extracted by using countercurrent two-stage EAEP of soybeans at 1:6 solids-to-liquid ratio, 50 °C, pH 9.0, and 120 rpm for 1 h. Protein-rich skim fractions were produced from extruded full-fat soybean flakes using different enzyme strategies in EAEP: 0.5% protease (wt/g extruded flakes) used in both extraction stages; 0.5% protease used only in the second extraction stage; no enzyme used in either extraction stage. Countercurrent two-stage protein extraction of air-desolventized, hexane-defatted soybean flakes was used as a control. Protein extraction yields increased from 66% to 89-96% when using countercurrent two-stage EAEP with extruded full-fat flakes compared to 85% when using countercurrent two-stage protein extraction of air-desolventized, hexane-defatted soybean flakes. Extruding full-fat soybean flakes reduced BBI activity. Enzymatic hydrolysis reduced BBI contents of EAEP skims. Lunasin, however, was more resistant to both enzymatic hydrolysis and heat denaturation. Although using enzymes in both EAEP extraction stages yielded the highest protein and oil extractions, reducing enzyme use to only the second stage preserved much of the BBI and Lunasin.     

In vivo pharmacokinetics of hesperidin are affected by treatment with glucosidase-like BglA protein isolated from yeasts

Hesperidin is an abundant flavanone glycoside in citrus fruits and has been reported to possess a wide range of biological activities. However, hesperidin has poor bioavailability. Here, we tested the hypothesis that hesperetin found in chenpi will have a better bioavailability than hesperidin and that treatment of hesperidin with the glucosidase-like yeast Bg1A protein will increase its bioavailability. The results indicate that hesperidin in pure or extract form is hydrolyzed by BglA protein extracted from Sporobolomyces singularis or expressed in Escherichia coli BL21 (DE3). This biotransformation affected the plasma pharmacokinetics of total hesperetin in rats, in that the plasma T max was significantly shorter after administration of BglA protein-treated hesperidin than after administration of hesperidin extract. In addition, the area under the curve values for total hesperetin after administration of Bg1A-treated hesperidin were approximately 4-fold higher by oral administration and 3-fold higher by intravenous administration, respectively. In contrast, the plasma clearance value and volume of distribution after administration of Bg1A-treated hesperidin extract or pure hesperetin were significantly smaller than after administration of untreated hesperidin extract or pure hesperidin. This is the first study that systemically determines the absolute bioavailability of hesperidin and hesperetin simultaneously, shows clearly that hesperetin is more bioavailable than hesperidin regardless of the route of administration, and shows that prior transformation of hesperidin to hesperetin via fermentation should significantly increase its bioavailability because of the action of the yeast glycosidase-like protein BglA.     

Characterization of a chitosanase isolated from a commercial ficin preparation

A chitosanolytic enzyme was purified from a commercial ficin preparation by affinity chromatographic removal of cysteine protease on pHMB-Sepharose 4B and cystatin-Sepharose 4B and gel filtration on Superdex 75 HR. The purified enzyme exhibited both chitinase and chitosanase activities, as determined by SDS-PAGE and gel activity staining. The optimal pH for chitosan hydrolysis was 4.5, whereas the optimal temperature was 65 degrees C. The enzyme was thermostable, as it retained almost all of its activity after incubation at 70 degrees C for 30 min. A protein oxidizing agent, N-bromosuccinimide (0.25 mM), significantly inhibited the enzyme's activity. The molecular mass of the enzyme was 16.6 kDa, as estimated by gel filtration. The enzyme showed activity toward chitosan polymers exhibiting various degrees of deacetylation (22-94%), most effectively hydrolyzing chitosan polymers that were 52-70% deacetylated. The end products of the hydrolysis catalyzed by this enzyme were low molecular weight chitosan polymers and oligomers (11.2-0.7 kDa).     

Use of three optical systems to obtain a microscopic profile of asbestos and other nonopaque particulate matter larger than 5 mum in parenteral drugs.

A comparative microscopic study of chrysotile fibers was conducted, using 3 sequential optical systems: plane polarized light (PL), phase contrast (PC), and Nomarski differential interference contrast (NDIC) for complementary image analyses. The introduction of NDIC for qualitative and quantitative analyses of particulate matter greater than 5 mum resolves the problem of diffraction halos encountered in PC microscopy. High optical contrast, the instrumental characteristic of NDIC, is particularly useful for the detection of chrysotile fibers at a magnification of 500 X.     

Plant and soil responses to a commercial blue-green algae inoculant

A commercial blue-green algae inoculant (Genesis II) for improving the N content of soils was applied to basalt and pumice soils in a greenhouse pot test with orchardgrass, pinegrass, Douglasfir, and ponderosa pine. Treatments were control (deionized water), live algae, killed algae, live algae plus P, K and S and P, K and S only. Plant biomass yields with live inoculant generally were significantly greater than with the control treatment but were the same as with killed inoculant. Live algae plus P, K and S further enhanced productivity. P, K and S alone produced no increase over the control treatment. Response to the inoculant compared to the control appears to be a result of addition of nutrients present in the inoculant stock solution. Further biomass increases with the P, K and S plus live algae treatment compared to live and dead algae treatments occurred because these nutrients became limiting as N was added. Total organic-N and total C in surface soils were similar in all treatments.When the stock solution was sampled via culture methods, the most prominent species found was Chlorella, a green alga. Anabaena, an N₂ fixing blue-green alga, produced less than one colony per plate on the average. Phormidium, a blue-green alga not found in the inoculant, was dominant on the soil surface at the end of the test suggesting a soil origin for this species. Results indicate that Genesis II probably has very limited potential to enhance blue-green algal populations and, consequently, N of the forest soils used in this test.     

Assessment of a laboratory method to obtain the equilibrium solution composition of forest soils

Equilibrium studies on soil require reliable estimates of ion concentrations in the soil solution under field conditions. We evaluated the previously described iterative method to approximate the equilibrium soil solution (ESS) with four acid forest soils. We examined for which ions the ESS is suitable, making use of the fact that concentrations in water extracts are functions of the soil: solution ratio. The electric conductivity, pH, and the concentrations of base cations, Mn²⁺, NO^?₃, SO₄²⁺, and dissolved organic carbon (DOC) were usually linear with the soil:solution ratio in water extracts, whereas no relation was observed for Al (with one exception) and Fe. Assuming that the ESS can be attributed the soil solution ratio of the field moist soil at the time of sampling, concentrations appeared as the continuation of the linear relation with the soil: solution ratio for base cations, pH, and the electric conductivity. This indicates that the ESS actually represents field conditions for these solution properties. For Al water extracts allowed no evaluation of the ESS result. The ESS underestimated SO₄^2? concentrations under field conditions, presumably because the lack of DOC in the solutions added distorts the balance amongst anions.     

Purification and identification of a protease inhibitor from glassfish (Liparis tanakai) eggs

Two protease inhibitors of 67 and 18 kDa, respectively, were purified from glassfish, Liparis tanakai, eggs by affinity chromatography. The smaller protein was purified with a yield and purity of 0.25% and 49.69-fold, respectively, and was characterized for further study. The glassfish egg protease inhibitor exhibited stability between 50 and 65 degrees C in an alkaline environment (pH 8). It was shown to be a noncompetitive inhibitor against papain, with an inhibitor constant (Ki) of 4.44 nM. Potent glassfish protease inhibitor with N-Val-Gly Ser-Met-Thr-Gly-Gly-Phe-Thr-Asp-C amino acid residues was synthesized and its inhibitory activity was compared. Moreover, the 18-kDa protein inhibited cathepsin, a cysteine protease, more effectively than did egg white protease inhibitor, whereas the reverse was true for papain. Glassfish egg protease inhibitor is classified as a member of the family I cystatins.     

Chlorpyrifos decline curves and residue levels from different commercial formulations applied to oranges

Residue levels and degradation rates of chlorpyrifos in orange fruits, orange leaves, and soil were investigated by using three different formulation types, that is, emulsifiable concentrate (EC), wettable granules (WG), and microencapsulates (ME). The pesticide degradation was studied for a period 131 days in orange fruits and for 161 days in orange leaves and soil. The experimental data were used to establish a mathematical model for the decline curves of chlorpyrifos residues as a function of time and to determine the relevant parameters describing such a process. Field trials showed a different degradation rate for EC and WG formulations as compared to ME formulation. For the first two formulations, the dissipation of chlorpyrifos in orange fruits was fast during the first phase and became much slower during the later period. Residue levels of chlorpyrifos from ME remained almost constant for approximately 65 days and then began to decrease. A similar behavior was observed for the three chlorpyrifos formulations on orange leaves and soil. Although microencapsulation of pesticides leads to improved handling safety, additional risks for the consumers, the agriculture workers, and the environment should be taken into account due to prolonged persistence of high residue levels in fruits as well as in leaves and soil.     

Use of Power Plant Ash to Remove and Solidify Heavy Metals from a Metal-finishing Wastewater

This laboratory-scale study investigated initially the potential of heavy metal removal from a metal-finishing wastewater using fly and bottom ash from a power plant as coagulants. It was found that the maximum heavy metal content in the ash–sludge mix was obtained at a fly ash-to-bottom ash ratio of 1.5:1 and a stirring time of 3 h, which resulted in heavy metal removal (i.e., Cr, Ni, Cu, Zn, Cd, and Pb) in excess of 99%, with effluent concentrations below the corresponding regulatory standards of Thailand. Furthermore, the feasibility of using fly ash as an admixture to stabilize and solidify the ash–sludge mix generated previously was explored. Results indicated that the stabilization/solidification process can achieve a high level of heavy metal removal efficiency from the ash–sludge mix. The optimum ratio regarding chromium leaching was found to be 1:0.75:0.75 (cement:fly ash:ash–sludge). In addition, the compressive strength and the chromium leaching concentration of the solidified sludge were within acceptable levels for secure landfill disposal and/or use as a construction material.     

Interactions among protein molecules in freeze-gel of soymilk and protein structures in heated soymilk during cooling

To estimate the interactions forming in soymilk freeze-gel, lyophilized gel was extracted successively with various solvents. A mixture of urea, sodium dodecyl sulfate (SDS), and 2-mercaptoethanol (2-ME) dissolved the proteins effectively. The thiol-disulfide exchange reactions and hydrophobic interactions were shown to have a complex relation with a three-dimensional network. The addition of SDS or 2-ME resulted in an incomplete gel or no precipitation of soymilk. In our previous paper (Shimoyamada et al. Food Sci. Technol. Res. 1999, 5, 284-288), the significance of precooling to form small, homogeneously distributed ice crystals in soymilk was reported. In this study, precooling was shown to maintain the partially denatured structures of soybean proteins in soymilk that had unfolded due to heat treatment. These phenomena were considered to be other important functions of precooling in freeze-gelation.     

一株产高温蛋白酶耐热菌BY25的产酶条件与酶学性质研究

对菌株BY25的生长条件、产酶条件及其产生的蛋白酶的酶学性质进行了研究。结果发现,BY25的最高生长温度为55℃,最适生长温度为50℃,最佳产酶温度为30℃,最佳培养起始pH为8.0,最佳C源为葡萄糖,高通气量明显提高菌株产酶能力。在以上条件下培养52 h,上清液蛋白酶活力达4 170 U/ml。酶学性质研究表明：该蛋白酶为高温中性金属蛋白酶,最适反应pH为7.0,最适反应温度为55℃,具有良好的pH耐受性和较好的热稳定性;EDTA能强烈抑制酶活力,而Fe2+、Cu2+对酶活力也具有一定抑制作用。     

Plight of the bumble bee: Pathogen spillover from commercial to wild populations

Pathogen spread or ‘spillover’ can occur when heavily infected, domestic hosts interact with closely-related wildlife populations. Commercially-produced bumble bees used in greenhouse pollination often have higher levels of various pathogens than wild bumble bees. These pathogens may spread to wild bees when commercial bees escape from greenhouses and interact with their wild counterparts at nearby flowers. We examined the prevalence of four pathogens in wild bumble bee populations at locations near and distant to commercial greenhouses in southern Ontario, Canada. Bumble bees collected near commercial greenhouses were more frequently infected by those pathogens capable of being transmitted at flowers (Crithidia bombi and Nosema bombi) than bees collected at sites away from greenhouses. We argue that the spillover of pathogens from commercial to wild bees is the most likely cause of this pattern and we discuss the implications of such spillover for bumble bee conservation.     

Characterization of three chitosanase isozymes isolated from a commercial crude porcine pepsin preparation

Three chitosanases designated PSC-I, PSC-II, and PSC-III were purified from commercial pepsin preparation by sequentially applying pepstatin A-agarose affinity chromotography, DEAE-Sephacel ion-exchange chromatography, Mono Q column chromatography, and Mono P chromatofocusing. With respect to chitosan hydrolysis, the optimal pHs were 5.0, 5.0, and 4.0 for PSC-I, PSC-II, and PSC-III, respectively; optimal temperatures were 40, 40, and 30 degrees C; and the Km's were 5.2, 4.0, and 5.6 mg/mL. The molecular masses of the three isozymes were approximately 40 kDa, as estimated by both gel filtration and SDS-PAGE, and the isoelectric points were 4.9, 4.6, and 4.4, respectively, as estimated by isoelectrofocusing electrophoresis. All three chitosanase isozymes showed activity toward chitosan polymer and N,N",N' "-triacetylchitotriose oligomer. Most effectively hydrolyzed were chitosan polymers that were 68-88% deacetylated.     

Purification and characterization of a cysteine protease inhibitor from chum salmon (Oncorhynchus keta) plasma

A cysteine protease inhibitor (CPI) in chum salmon ( Oncorhynchus keta) plasma (CSP) was detected after performing inhibitory activity staining against papain under nonreducing condition. The CPI was purified from CSP by affinity chromatography with a yield and purification ratio of 0.94% and 30.36-fold, respectively. CSP CPI had a molecular mass of 70 kDa based on the results of SDS-PAGE and Sephacryl S-100 gel filtration. CSP CPI was a glycoprotein based on the periodic acid-Schiff (PAS) staining of the SDS-PAGE gel and classified as a kininogen. CSP CPI was stable in the pH range of 6.0-9.0 with maximal stability at pH 7.0. CSP CPI presented thermal stability at temperatures below 50 degrees C and exhibited maximal activity at temperatures of 20-40 degrees C. CSP CPI was determined to be a noncompetitive inhibitor against papain, with an inhibitor constant (Ki) of 105 nM.     

小流域尺度土壤养分的空间分布特征及其与土地利用的关系

以四川省资阳市松涛镇的响水村小流域为研究背景,以地理信息系统为技术手段,研究了小流域尺度土壤养分的空间分布特征,并结合数理统计的分析方法,进一步探讨了土地利用对土壤养分的影响。结果表明:研究区域土壤表层养分含量均呈现较明显的空间分布格局,总趋势大都具有条带状和斑块状的特点,旱地、果园、裸地的土壤全氮、有机质普遍缺乏,特别是处于坡顶的裸地,应成为今后的控制重点,此外,旱地、果园的水溶态养分高值分布区也应给予高度重视。方差分析与均值比较表明:土地利用对全氮、有机质、全磷、水溶性磷含量都有显著影响(P<0.01),其影响程度为有机质>全氮>全磷>水溶性磷,而全钾、硝态氮则不受土地利用的显著影响,究其原因可能是紫色土钾素含量更倾向于土壤母质中所含矿物的控制,而土壤硝态氮的情况比较复杂,可能有更为重要的因素(如施肥、降雨、径流)制约着硝态氮的含量。     

Use of Rice Husk Ash as an Admixture to Remove Chromium from a Tannery Waste

This laboratory-scale study explored the use of rice husk ash (RHA) as an admixture to stabilize and solidify the waste sludge from a chrome-tanning process. Various ratios of RHA and tannery sludge were used, ranging from 0.25 to 1.0 by weight of the cement content, resulting in an experimental matrix of 16 runs. It was observed that although the compressive strength of solidified sludge improved at longer curing times, the increase was moderate after 7 days of curing. In addition, higher RHA and/or sludge ratios progressively reduced the strength of the samples. On the other hand, curing time had a positive effect on chromium leachability since the leaching concentration decreased considerably with an increase in curing time, up to 28 days. Furthermore, both the RHA and/or sludge content affected leachability, producing acceptable results, from a Thai regulatory perspective, at ratios of 0.50 or lower. Finally, the solidified sludge can be disposed of in a secure landfill and/or used as a construction material in a number of applications.     

Use of the test-mineral technique to distinguish simple acidolysis from acido-complexolysis in a Podzol profile

A test vermiculite was inserted in different horizons of a Podzol profile to study its behavior in contact with the organic solutes in the soil solution. After incubation in the soil for three years, the ECEC showed a clear negative correlation with the contents of Tamura extracted Al. XRD analyses confirmed the presence of Al-polymers blocking the minerals interlayer. The formation of these stable Al-polymers is in contradiction to the theory that acido-complexolysis and not simple acidolysis is one of the major soil forming processes in Podzols, preventing Al-interlayering in open 2:1 phyllosilicates by the strong complexing power of the organic acids present. The hypothesis that podzolization is no longer active in the studied profile so that the complexing acids, typical of the acido-complexolysis are no longer present in large enough quantities to prevent “aluminization” of the vermiculites had to be rejected after studying the chemical composition of the soil solutions. Significant amounts (∼ 75%) of dissolved aluminium proved to be strongly bound to organic acids, indicating an active podzolization process. The behaviour of test-minerals as a criterion to distinguish acidolysis from acido-complexolysis must thus be used with great care and should never be interpreted without detailed analysis of the soil solution. Where aluminium is present in the soil solution, the use of test-minerals is likely to fail to distinguish simple acidolysis from acido-complexolysis, as the conditions in the field are then too different from those during the laboratory experiments from which the theory on simple acidolysis/acido-complexolysis originally was derived and where the sole source of Al was the mineral itself.     

Apparent Use Efficiency of Nitrogen and Phosphorus from Litter Applied to Bermudagrass

More than 80% of broiler (chicken, Gallus gallus domesticus) litter produced annually is applied as a plant nutrient source, particularly for nitrogen (N) and phosphorus (P), to pastures. However, N losses during the process of litter N mineralization limit availability of N to crops. This study determined broiler litter N and P availability and apparent use efficiency (ANUE, APUE) to bermudagrass [Cynodon dactylon] during the first year after litter application. Treatments consisted of three litter rates (3.3, 6.6, and 13.2 Mg ha^?1), a commercial N fertilizer rate that provided 358 kg N ha^?1 as ammonium nitrate (NH₄NO₃), and an untreated control. Results showed bermudagrass dry-matter (DM) yield increased significantly with increase in litter rate. Commercial N fertilizer produced significantly greater DM yield than 3.3 and 6.6 Mg ha^?1 of litter but produced less DM yield than 13.2 Mg ha^?1 of litter. The overall average of ANUE from litter was 39% compared to the 59% from fertilizer. The mean litter N availabilities to bermudagrass during the first year after litter application were 48.5, 112.5, and 222 kg ha^?1, corresponding to the 3.3, 6.6, and 13.2 Mg ha^?1 litter rates, respectively. The overall mean of litter N mineralization, which was surface broadcast to bermudagrass plots during the first year, was 59.5% of the total litter N applied. The APUE, averaged across the rate and locations, was 13.6%, which was quite smaller than the ANUE of 39%. This finding of small APUE also validates the potential for P accumulation in soil after long-term animal manure application.