Bioactivity of a pentapeptide isolated from corn gluten hydrolysate on Lolium perenne L.

Corn gluten hydrolysate (CGH) has been observed to inhibit root formation of germinating grass seeds and has the potential for use as a natural herbicide. Five dipeptides have been isolated from the aqueous solution of CGH and proved to have greater root-inhibiting activity than the crude extract of CGH. The objective of this study was to isolate and identify other biologically active compounds from CGH with herbicidal properties. A perennial ryegrass (Lolium perenne L.) Petri dish bioassay was used to test for the bioactivity. A pentapeptide, Leu-Ser-Pro-Ala-Gln, was isolated from CGH by using Sephadex G-15 gel filtration and two-step C18 reversed phase high performance liquid chromatography procedures. The compound suppressed growth of both the root and the shoot of germinating perennial ryegrass. It required 0.5 mg/mL of the pentapeptide to inhibit 50% of root length in the perennial ryegrass bioassay, and this compound is more active than any of the five dipeptides isolated previously from CGH.Abbreviations CGM corn gluten meal - CGH corn gluten hydrolysate - HPLC high performance liquid chromatography - RP reversed phase - MeOH methanol - AUFS absorbance units of full scale - Rt retention time - TFA trifluoroacetic acid - PTH phenylthiohydantoin - ANOVA analysis of variance     

Isolation and identification of root-inhibiting compounds from corn gluten hydrolysate

Interest has centered on the use of plantderived compounds as natural herbicides, and they are considered to represent an environmentally sound approach to weed control. Corn gluten hydrolysate, found to have a growth-regulating effect on the root system of germinating grass seeds, has been suggested as a natural herbicide. A protocol was developed to extract, isolate, and identify the root-inhibiting compounds from corn gluten hydrolysate aqueous solution and a perennial ryegrass (Lolium perenne L.). A Petri dish bioassay was used to test the root-inhibiting activity. Five bioactive dipeptides were isolated by using Sephadex G-15 gel filtration, solid-phase extraction, and C18 reversed-phase high-performance liquid chromatography procedures. The five dipeptides were glutaminyl-glutamine, alaninyl-asparagine, alaninyl-glutamine, glycinyl-alanine, and alaninyl-alanine. Their root-inhibiting activity on perennial ryegrass was demonstrated in Petri dish bioassays.Journal Paper No. J-15597 of Iowa Agriculture and Home Economics Experiment Station, Ames, IA Project No. 3149     

饲料原料中转基因成分的PCR检测

采用PCR检测方法从饲料的主要原料豆粕、玉米蛋白粉中成功地检出启动子35S (35S-promoter,originated from cauliflower mosaic virus)、终止子NOS (nopaline synthase-terminator,derived from Agrobacterium tumefaciens)、耐除草剂基因EPSPS(5-enolpyruvylshikimate-3-phosphate synthase)和抗虫基因CryIA（b）(delta-endotoxin,evolved from Bacillus thuringiensis subsp.kurstaki)等转基因成分,并通过扩增玉米自身蛋白基因Zein( a protein extracted from corn gluten)及大豆自身基因Lectin(chitin-binding protein)的引物和阴阳性对照、阴阳性质控,避免假阳性、假阴性结果。该方法已在口岸进口饲料原料转基因检测中得到初步应用。 Abstract:Based on the heterogenous genes usually used in transgenic crops,the PCR technique was performed with primers derived from CaMV 35S promoter(35S-promoter,originated from cauliflower mosaic virus),NOS terminator(nopaline synthase-terminator,derived from Agrobacterium tumefaciens),EPSPS(5-enolpyruvylshikimate-3-phosphate synthase) gene,and CryIA(b)(delta-endotoxin,evolved from Bacillus thuringiensis subsp.kurstaki)gene to detect transgenic agents from feed raw materials of soybean dregs and corn gluten meal,respectively.Endogenous corn Zein(a protein extracted from corn gluten) gene,soybean Lectin(chitin-binding protein) gene and negative,positive control were applied for avoiding false results.The method established here has been succeessfully applied in detecting transgenic elements in imported feed raw material.     

Preparation of corn gluten hydrolysate with angiotensin I converting enzyme inhibitory activity and its solubility and moisture sorption

This study attempted to prepare a hydrolysate of corn gluten containing powerful ACE inhibitory activity and higher solubility, which could be used as a physiologically functional food material. The digestion of starch at 80 °C resulted in more removal of the starch from the corn gluten than that at 60 °C. More than 95% reducing sugars as enzymic digests of starch was removed by washing three-times. As for the thermal treatment effect on the increase of degree of hydrolysis (DH), degree of increase of protein content (slope) was 6.30 mg/ml min at 100 °C, 4.40 mg/ml min at 80 °C and 2.29 mg/ml min without heat treatment. After 45 min of heat treatment the increased ratio of protein content was greater than those by other heat treatments. After the pretreatment of corn gluten, hydrolysis with Flavourzyme, among six commercial proteases, resulted in the production of the hydrolysate with the highest ACE inhibitory activity, and with Protamax, the second highest ACE inhibitory activity was obtained. Flavourzyme hydrolysate of corn gluten at the 4% level was easily and completely soluble between pH 2 and 9. Water sorption of the hydrolysate slowly increased up to 0.8 of water activity and greatly increased at higher than 0.8 of water activity.     

Incidence of aflatoxins and aflatoxin producing fungi in animal feedstuffs

Fifty four samples including 5 of broken rice, 8 of corn grains, 8 of corn gluten feed, 13 of cottonseed cake and 4 each of rice polish, corn gluten, sesame oil cake, guar meal and wheat bran were screened for the presence of aflatoxins. Among all the samples, 14 were damaged and 40 apparently undamaged. The incidende of aflatoxins was found to be 60, 25, 25, and 23 per cent in broken rice, corn grains, corn gluten feed and cottonseed cake. Aflatoxins were not detected from rice polish, corn gluten, sesame oil cake, guar meal and wheat bran. Damaged sample revealed a much higher incidence i.e. 50 per cent as compared to undamaged ones i.e. 7.5 per cent. Mean concentration of aflatoxin B and G was found to be 15.5 and 12.2 ppb respectively.Cultural examination of aflatoxin positive feedstuffs yielded 39 isolates of different fungi including 21 of Aspergillus, 7 of Mucor, 6 of Rhizopus, 4 of Fusarium and one of Penicillium. These strains when tested for aflatoxin producing ability, revealed this property in only one isolate, identified as Aspergillus parasiticus.     

Comparison of [14C]oryzalin Uptake in Root Segments of a Sensitive and a Resistant Species

Uptake of the dinitroaniline herbicide oryzalin (3,5-dinitro-N⁴,N⁴-dipropylsulfamlamide) and its effect on root growth werestudied using 5 mm corn (Zea mays L.) and pea (Pisum sativum)root apices. Pea root growth was much less susceptible to oryzalinthan corn root growth. Uptake studies showed that pea root apicesalso accumulated much less [¹⁴C]oryzalin and had a lower bindingaffinity for this herbicide. [¹⁴C]oryzalin was not metabolizedin root apices from either species. Thus, the differential susceptibilityto oryzalin in the case of corn versus pea can be explained,at least in part, by differences in oryzalin uptake and accumulationby roots. Oryzalin, dinitroaniline herbicides, Zea mays, Pisum sativum     

Production of hydrolysate with antioxidative activity by enzymatic hydrolysis of extruded corn gluten

Hydrolysate of extruded corn gluten with higher solubility and antioxidative property was prepared. Extrusion and starch removal of corn gluten were applied as pretreatment before enzymatic hydrolysis by Alcalase. The amylase hydrolysis of starch at 70°C for 3 h resulted in the removal of the starch from the extruded corn gluten. The best hydrolysis results can be obtained by conducting the hydrolysis at 60°C with water addition 20 g/g protein, enzyme addition 0.048 Ansen units/g protein, pH 8.5, and 120 min. Degree of hydrolysis of extruded and nonextruded corn gluten reached 39.54 and 31.16%, respectively, under the optimal condition. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of the optimal hydrolysate revealed that proteolysis of extruded corn gluten was more extensive than proteolysis of its counterpart which was not subjected to extrusion. The molecular weight of the peptides in the optimal hydrolysate was mainly over 3,710–660 Da as determined by gel filtration chromatography. The hydrolysates displayed good solubility and antioxidative activity. The separation profile of the hydrolysate on an ion exchange chromatography of Q-Sepharose Fast Flow showed that many kinds of peptides had antioxidative effect. A new peptide with antioxidative activity was purified, and its amino acid sequence was Phe-Pro-Leu-Glu-Met-Met-Pro-Phe, which was identified by Q-TOF2 mass spectrometry.     

Apparent digestion coefficients for dry matter, protein and essential amino acids in terrestrial ingredients for Pacific shrimp Litopenaeus vannamei (Decapoda: Penaeidae)

Protein quality mainly depends on the essential amino acid (EAA) profile, but also on its bioavailability, because EAA digestibility is generally lower than the analyzed amounts. This information is needed in the aquaculture industry for aquafeed formulation. For this purpose, the apparent digestibility coefficients of dry matter, protein, and essential amino acids of eight feedstuffs of terrestrial origin were determined for the juvenile whiteleg shrimp Litopenaeus vannamei (15-19 g), using 1% chromic oxide as an inert marker. A reference diet was formulated and produced in the laboratory. Eight experimental diets were prepared each with 30% of one of the experimental ingredients added to the reference diet: casein, porcine byproduct meal poultry byproduct meal, corn meal, wheat gluten meal, soybean paste, sorghum meal, and wheat meal. The experiment consisted of a single-factor, completely randomized design with three replicates per treatment. Samples of ingredients, diets and feces were analyzed for nitrogen and amino acids. For amino acid assay, we used reverse-phase high performance liquid chromatography. To avoid partial loss of methionine and cystine, samples of ingredients, diets, and feces were oxidized with performic acid to methionine sulfone and cysteic acid prior to acid hydrolysis. The apparent dry matter and protein digestive utilization coefficients varied from 68% to 109% and from 70% to 103%, respectively. Apparent digestibility of protein for casein, soy paste, wheat meal and wheat gluten were very high (over 90%), corn gluten and poultry byproducts meal showed high protein digestibility (over 80%), but porcine byproducts meal and sorghum meal had low digestibility (76% and 70%, respectively). There was a reasonable, but not total, correspondence between apparent protein digestibility and average essential amino acid digestibility coefficients, except for arginine in corn gluten, phenylalanine and leucine in sorghum meal, phenylalanine in soy paste and lysine in wheat meal and poultry by-product meal. The most digestible feed ingredients for whiteleg shrimp were: wheat gluten, wheat meal and soy paste; poultry byproduct meal and corn gluten were less digestible and the lowest digestibility occurred in porcine byproduct meal and sorghum meal. Feedstuffs exhibited great variability in dry matter, protein and amino acid digestive utilization coefficients, which should be considered when formulating shrimp feeds.     

Microscopic analysis of corn fiber using starch- and cellulose-specific molecular probes

Ethanol is the primary liquid transportation fuel produced from renewable feedstocks in the United States today. The majority of corn grain, the primary feedstock for ethanol production, has been historically processed in wet mills yielding products such as gluten feed, gluten meal, starch, and germ. Starch extracted from the grain is used to produce ethanol in saccharification and fermentation steps; however the extraction of starch is not 100% efficient. To better understand starch extraction during the wet milling process, we have developed fluorescent probes that can be used to visually localize starch and cellulose in samples using confocal microscopy. These probes are based on the binding specificities of two types of carbohydrate binding modules (CBMs), which are small substrate-specific protein domains derived from carbohydrate degrading enzymes. CBMs were fused, using molecular cloning techniques, to a green fluorescent protein (GFP) or to the red fluorescent protein DsRed (RFP). Using these engineered probes, we found that the binding of the starch-specific probe correlates with starch content in corn fiber samples. We also demonstrate that there is starch internally localized in the endosperm that may contribute to the high starch content in corn fiber. We also surprisingly found that the cellulose-specific probe did not bind to most corn fiber samples, but only to corn fiber that had been hydrolyzed using a thermochemical process that removes the residual starch and much of the hemicellulose. Our findings should be of interest to those working to increase the efficiency of the corn grain to ethanol process.     

The Intra-cellular Localization of the Herbicide Diphenamid in Corn Root .Tip

The intra-cellalar localization of the herbicide N,N-dimethyl–2,2-diphenylacetamide (diphenamid) was examined in 10 mm root tips of corn seedlings (Zea mays L. cv. Shawnee). Experiments are described which culminate in the observation that a significant, measurable effect on root growth occurs within 12 hours after treatment with diphenamid. Corn seedlings were then treated with 14-C-diphenamid for 12 hours and the 10 mm root tips excised and homogenized. By use of differential centrifugation and gel filtration, the root homogenate was separated into fractions; as follows; cell wall, mitochondria, microsomal, protein and eluate. The highest concentration of the herbicide was found in the root tips. Furthermore, most of the radioactivity was found in the eluate. Results are discussed as to localization of diphenamid in the microsomal and protein fraction and possible mode of action.     

Phytase production byAspergillus ficuum on semisolid substrate

Summary Phytase production byAspergillus ficuum was studied using solid state cultivation on several cereal grains and legume seeds. The microbial phytase was used to hydrolyze the phytate in soybean meal and cotton seed meal. Wheat bran, soybean meal, cottonseed meal and corn meal supported good fungal growth and yielded a high level of phytase when an adequate amount of moisture was present. The level of phytase production on solid substrate was higher than that obtained by submerged liquid fermentation. Higher levels of phosphorus (more than 10 mg Pi/100 g substrate) in the growth medium (static culture) inhibited phytase synthesis, and the degree of phosphorus inhibition was less apparent in semisolid medium than in liquid medium. A static cultivation on semisolid substrate produced a higher level of phytase (2-20-fold) than that obtained by agitated cultivation. The minimal amount of water required for growth and enzyme production on those substrates was about 15%, while the optimum level for phytase production was between 25 and 35% and that for cell growth was above 50%. Optimum pH for phytase production was between 4 and 6.A ficuum grew well on raw (unheated) substrate containing a minimal amount of water and produced as much phytase as on heated substrate. About half of the phytic acid in soybean meal and cottonseed meal was hydrolyzed by treatment withA. ficuum phytase.     

Outdoor evaluation of herbicide resistant strains of Anabaena variabilis as biofertilizer for rice plants

Application of diazotrophic cyanobacteria, Anabaena variabilis, as biofertilizer for rice cultivation has a beneficial effect on crop productivity and maintenance of soil fertility. However, periodic applications of herbicides used to obtain high crop productivity are not only detrimental to weeds but to biofertilizer strains of cyanobacteria also. Therefore, research was undertaken to isolate four herbicide resistant strains (Arozin-R, Alachlor-R, Butachlor-R and 2,4-D-R) and a multiple herbicide resistant strain (MHR) of natural isolates of A. variabilis exhibiting resistance against these common rice field herbicides. The outdoor survivability of mutant strains and the productivity of rice crop (IR-36) were evaluated by inoculating the wild type and herbicide resistant mutant strains of A. variabilis in the presence and absence of recommended field dosages of test herbicides. No difference in survival and biofertilizer potentials of the herbicide resistant strains was observed in herbicide treated or in untreated conditions. Highest survivability (87%) was exhibited by MHR relative to other mutants. Highest growth and grain yield (76%) were recorded in plants treated with MHR as compared to uninoculated control rice plants. In conclusion, the mutant strains of A. variabilis had stable resistance to herbicides under outdoor conditions in flooded soils. Not only did the herbicide resistance strains increase growth of rice relative to the uninoculated pots, they were more beneficial for rice growth than the wild type strain. Responsible Editor: Richard W. Bell.     

Opimization of Conditions for Production of Uridine by a Mutant of Bacillus subtilis

This work was designed to examine culture variables influencing the production of uridine by the Bacillus subtilis mutant No. 556. A combination of corn steep liquor (4%) + corn gluten meal (0.7%) + urea (2%) was the most suitable nitrogen source for the production of uridine; a high level of ammonium ions in the medium brought about abundant cell growth in the early stage of incubation and was detrimental to the fermentation. The addition of calcium salts, such as CaCO₃ and CaCl₂, was also essential for obtaining high yields of uridine. Among the carbon sources tested, glucose gave the best result. The optimum temperature and oxygen transfer rate for producing uridine were around 38°C and 20 × 10^?7 mol O₂/min/ml, respectively. Under optimum conditions in a 6000-liter fermentor, strain No. 556 produced 65mg/ml of uridine from 18% glucose.     

Reciprocal Antagonism between the Herbicides, Diclofop-Methyl and 2,4-D, in Corn and Soybean Tissue Culture

The antagonistic interaction between the grass herbicide, diclofopmethyl (methyl 2-[4(2′,4′-dichlorophenoxy)phenoxy]propanoate) (DM), and 2,4-dichlorophenoxyacetic acid (2,4-D), was demonstrated in DM-resistant soybean (Glycine max [L.] Merr.) and DM-susceptible corn (Zea mays L.). 2,4-D caused root shortening and thickening, and induced callus growth in soybean and corn root tissue cultures at 1 and 10 micromolar. Normal soybean root growth was unaffected by 10 micromolar DM whereas corn root growth was inhibited completely by 1 to 10 micromolar DM. DM at 10 micromolar reversed completely the induction of callus growth by 1 micromolar 2,4-D in soybean roots. In corn, 10 micromolar 2,4-D reversed the growth inhibiting activity of 1 micromolar DM and induced callus growth. The antagonistic interaction between DM and 2,4-D was reciprocal and the activity of either compound depended upon the relative concentration of the other. 2,4-D did not antagonize or decrease the activity of DM by decreasing its uptake by root tissues or increasing the rate of its detoxication. The antagonistic interaction between DM and 2,4-D probably involves involves cellular activity associated with actively growing and proliferating cells and requires the presence of both compounds at the sensitive site.     

A Biosurfactant/Polystyrene Polymer Partition System for Remediating Coal Tar-Contaminated Sediment

A sustainable, green chemistry process is proposed for the cleanup of coal tar impacted sediment in under 2 hr. A mixture of proteins and polypeptides, extracted from corn gluten meal and hemp, when mixed with sediment and polystyrene foam pellets (PFPs), serves to mobilize tar, which sorbs onto PFP. Since the sorbent floats, coal tar is easily extracted from the agitation vessel. An empirically derived 4-dimensional surface response model accurately predicts removal rates of the tar and operational costs of the system under various experimental conditions. At optimum relative to cost, 81% of the two to six ring polycyclic aromatic hydrocarbons (PAHs) and 73% of the total tar mass are removed despite high sediment organic carbon content (16.4%) and silty fines (～85%). Multiple PFP extractions (n = 2) of the same sediment/biosurfactant mixture yielded 94% extraction of PAH. Scanning electron microscope images illustrate free-phase tar (globule) sorption onto the foam. A field pilot was conducted in which 25 kg of sediment was processed. Results were in excellent agreement with both lab (10 g) experiments and model predictions. The process is considered sustainable and green because the active ingredients are derived from renewable crop materials, recycled polystyrene is used, and the biosurfactant is recyclable which reduces water demand and treatment costs, with the recovered tar used as fuel and sediment as beneficial reuse material.     

Digestibility of phosphorous in cereals and co-products for animal feed

Feed ingredients used in swine diets contain various levels and availabilities of nutrients. Nutritional precision evaluation of each ingredient is necessary for formulating diets of pigs. Especially, phosphorous (P) is one of important nutrients for metabolism. However, current data of P digestibility were most apparent digestibility. Therefore, this study was aimed to estimate the coefficient of total tract standardized digestibility (CTTSD) of P in cereals and various co-products used in pig diet. Twelve barrows (initial BW?±?SD, 46.70?±?3.21?kg) were used in this experiment. The experimental design was a 12?×?8 incomplete Latin square with 12 diets and 8 periods. Experimental diets were consisted of barley, wheat, lupine kernel (LK), soybean meal (SBM), almond meal (AM), corn gluten meal (CGM), corn gluten feed from China (CGF-C), corn gluten feed from Korea (CGF-C), wheat bran (WB), rice bran (RB), lupine hull (LH) and P-free diet. The CTTAD of Ca was higher in AM than RB and CGF-K. The LK and CGM showed greater CTTSD of P than RB and LH. In conclusion, our results indicated that the cereals and co-products as P sources were the ideally used as an ingredient in mixed diets of the growing-finishing pigs.     

Purification and evaluation of a novel antioxidant peptide from corn protein hydrolysate

In the present study, corn protein hydrolysate (CPH) with antioxidant activity was obtained by enzymatic hydrolysis. Corn gluten meal (CGM) was hydrolyzed using two proteases (Alcalase and Protamex) to produce the antioxidant peptide. Extrusion and starch removal of corn protein were used as pretreatment procedures before proteolysis. Hydrolysis by Alcalase has more remarkable digesting efficiency on corn protein than that by Protamex. Therefore, the hydrolysate catalyzed by Alcalase was fractionated by ultrafiltration, and peptide with the highest antioxidant activity was purified from <6 kDa molecular weight fraction. The amino acid sequence of the novel peptide was Gln-Gln-Pro-Gln-Pro-Trp as identified by a quadrupole time-of-flight mass spectrometer (Q-TOF2), with molecular weight of 782.34 Da, which was matched to γ-zein f (50–55). The new peptide was further synthesized by Fmoc solid-phase method. It showed scavenging activity against DPPH, ABTS, and hydroxyl free radicals in dose dependent manner with EC₅₀ values of 0.95, 0.0112 and 4.43 mg/mL, respectively. It also exhibited notable reducing power of 0.54 at 2.0 mg/mL, but showed weaker Fe²⁺-chelating capacity with EC₅₀ value of 6.27 mg/mL. These results suggest that the hexapeptide is a potential natural antioxidant that can be used as drug or functional food ingredient.     

The use of canola meal as a substrate for xylanase production by Trichoderma reesei

Summary Tests made utilizing canola meal as a substrate for the production of xylanase indicate that Trichoderma reesei produced this enzyme in similar or better yields from canola meal than from Solka-floc, xylan or glucose. The maximum xylanase activity obtained from canola meal was 210 IU/ml in 9–12 days. The enzyme system produced using canola meal also contained a higher proportion of acetyl-xylan esterase, cellulase, and xylosidase activities. This system was more than or equally efficient as that produced using Solka-floc in hydrolysing canola meal, corn cobs, corn and wheat brans, straw, and larchwood xylan to fermentable sugars. Offprint requests to: Z. Duvnjak     

Influence of metham sodium on suppression of collar rot disease of peanut, in vitro antibiosis, siderophore production and root colonization by a fluorescent pseudomonad strain FPO4.

A hydroxamate type siderophore producing fluorescent Pseudomonas strain, isolated from the rhizoplane of paddy root showing plant growth promoting activity, exhibited a decreased in vitro antibiosis, production of siderophore and suppression of collar rot in presence of metham sodium. Use of herbicide had a detrimental effect on the plant growth promoting activity of this organism. The multiple drug resistant mutant strain derived from this rhizobacteria colonized the roots, but the herbicide application had a negative effect on their population. HPLC analysis of the siderophore showed five peaks of which the peak number three confirmed the antifungal activity.     

Allelopathic activity of different plant parts of <Emphasis Type="Italic">Peganum harmala</Emphasis> L. and identification of their growth inhibitors substances

This study was conducted to evaluate the inhibitory potential of P. harmala leaf, stem and root extract on germination and growth of Avena fatua L. and Convolvulus arvensis L., as well as identification of the phytotoxic substances responsible for this activity. According to our results, the degree of toxicity of different P. harmala plant parts can be arranged in the following order: leaves > stems > roots. The two test species differed in their sensitivity to P. harmala extracts. Inhibitory effect on shoot length and seedling dry weight was more pronounced in C. arvensis, whereas higher reduction in germination, root length and total chlorophyll content occurred in A. fatua. A significant amount of water-soluble phenolic acids were found in P. harmala plant extracts. Total phenolic acids content was higher in leaf extracts when compared to that of stem or root extracts. Seven phenolic acids including gallic acid, vanillic acid, 4-hydroxybenzoic acid, 3,4-dihydroxybenzoic acid, caffeic acid, syringic acid and ferulic acid were found in P. harmala leaf extracts. On the other hand, we identified four phenolic acids from stem (galllic acid, vanillic acid, 3,4-dihydroxybenzoic acid and caffeic acid) and root (galllic acid, 4-hydroxybenzoic acid, syringic acid and cinnamic acid) extracts. The greater number of growth inhibitors detected in the leaves might explain the stronger inhibitory activity. Overall, our results suggest that P. harmala might be used as a natural herbicide for weed control and consequently reduce dependence on synthetic herbicides.