Quantitative amino acid analysis of feedstuff hydrolysates by reverse phase liquid chromatography and conventional ion-exchange chromatography

Corn, soybean meal, and isolated soybean protein samples were acid-hydrolyzed and analyzed for amino acid content by reverse phase liquid chromatography (LC) and by conventional ion-exchange chromatography (IEC) using an amino acid analyzer. The former method employed pre-column derivatization with orthophthalaldehyde (OPTA)/ethanethiol and fluorescence detection. In the LC procedure, glycine and threonine were not resolved, and proline and cyst(e)ine were not detected. In general, amino acid values obtained by LC and IEC compared closely within and across feedstuffs, and both agreed well with published amino acid composition data. The notable exceptions were aspartic acid, glutamic acid, and alanine. Results of this study suggest that reverse phase LC with pre-column OPTA derivatization can be applied to accurately measure primary amino acids in individual feedstuffs.     

Hydrolysate preparation for analysis of amino acids in sorghum grains: effect of oxidative pretreatment

Sorghum samples were either untreated or oxidized with performic acid (PA) before hydrolysis, and their amino acid contents were determined by cation exchange chromatography using an amino acid analyzer. HCl was used to destroy excess PA. Oxidative pretreatment of the samples resulted in increased yields of Cys (as cysteic acid), Met (as Met dioxide), and His, destroyed Tyr and Phe, and resulted in the appearance of an extraneous peak which most likely consisted of halogenation by-products (HBP) of Tyr and Phe. The destruction of Tyr and Phe occurred despite the presence of phenol, a halogen scavenger, in both the PA and hydrolysis reagents. The higher His values observed in all oxidized samples most likely resulted from the co-elution of His with Tyr and Phe HBP. It was concluded that the complete (except Trp) amino acid content of a feedstuff cannot be accurately determined from only one oxidized hydrolysate preparation by using this particular procedure.     

Detection of Wheat Preharvest Sprouting Using a Pregelatinized Starch Substrate and Centrifugation

Wheat sprouting in the field before harvest is a serious negative quality attribute. Even low levels of preharvest sprouting affect the economic value of the grain. Unreleased test lines of wheat should be screened for resistance to preharvest sprouting. However, screening large numbers of test lines is relatively time‐consuming or expensive, depending on the existing method used. A new screening test for preharvest sprouting was developed and compared with the viscograph and α‐amylase activity (AAA) methods. The new method used the activity of sprout‐related elevation in α‐amylase to partially degrade added pregelatinized starch. The hydrolytic products were centrifuged and the weight of the centrifugate was expressed as a percentage of the original weight of the added pregelatinized starch plus the original meal or flour weight. The result reflected the AAA on pregelatinized starch (AAAPGS) as a measure of the degree of preharvest sprouting. The AAAPGS test had less standard error and was more sensitive at low levels of preharvest sprouting than the AAA method. Three grinders to produce wheat meal were compared for their effect on AAAPGS values. Flours produced slightly lower AAAPGS values than meals, but the coefficients of variation of each were comparable and both were less than that of the AAA method. The lowest levels of sensitivity to preharvest sprouting that could be detected by the AAA and AAAPGS methods were identified as areas of uncertainly, below which very low levels of preharvest sprouting could not be differentiated from sound, unsprouted background values. The new AAAPGS method was equally rapid and will be more economical than the AAA method or the viscograph when used for preharvest sprouting susceptibility of large numbers of samples.     

Amino acid analysis by capillary gas chromatography

Two developments have enabled major advancements in the use of capillary gas chromatography (GC), the result being its much more widespread use in investigations on a broad range of chemical and biological problems. The 2 technological developments were the introduction of fused silica capillary columns and the development of immobilized stationary phases for capillary GC columns. Because fused silica columns with immobilized stationary phases of varying polarities are offered by numerous vendors of chromatographic equipment, they have become widely used for many analytical tasks. We conducted a study to compare the effectiveness of commercially available fused silica capillary columns with the classical ion-exchange method in the separation and quantitation of amino acids. We selected the N-trifluoroacetyl (TFA) n-butyl and the N-heptafluorobutyryl (HFB) isobutyl ester derivatives for this study because of the extensive research and application of these derivatives during the past 20 years. The amino acid content of hydrolysates of 5 materials was measured: ribonuclease, beta-lactoglobulin, lysozyme, soybean meal, and a commercial poultry feed. Single 6N HCl hydrolysates of each material were prepared to minimize sample preparation differences, and 3 independent analyses of each hydrolysate were made by each of 3 techniques: the N-TFA n-butyl and N-HFB isobutyl ester methods using capillary gas chromatography and the ion-exchange chromatographic method using a Beckman 121 M amino acid analyzer. Our results clearly demonstrate that capillary GC analysis of amino acids using fused silica bonded-phase columns provides data with good precision and in general excellent agreement with ion-exchange analyses.     

Discrimination between arabica and robusta coffee species on the basis of their amino acid enantiomers

This work reports the results for the composition of robusta and arabica coffee species in terms of their amino acid enantiomers in the green and roasted states. The analyses were conducted for the free amino acids, as well as for the amino acids obtained after acid hydrolysis. The amino acids were extracted/hydrolyzed and isolated by SPE on strong cation exchange columns, derivatized to their N-ethoxycarbonylheptafluorobutyl esters, and analyzed by gas chromatography/FID on a Chirasil l-Val column. Multivariate analyses applied to the results showed that the free amino acids can be used as a tool for discrimination between coffee species, with a special reference to l-glutamic acid, l-tryptophan, and pipecolic acid. There is also some evidence that these compounds can be used for discrimination between green coffees subjected to different postharvest processes. It is also shown that the amino acid levels observed after acid hydrolysis can be used for the same purposes, although displaying less discriminatory power.     

鱼骨粉对土壤Cd污染钝化修复效应及其理化性质的影响

通过1年静态培养试验,研究了鱼骨粉对碱性和酸性Cd污染土壤钝化修复效应、基本理化性质和酶活性的影响。结果表明:随着鱼骨粉施加量的增加,酸性和碱性土壤中pH、有机质和阳离子交换量增加,而含水率则较对照降低了2.90%~6.19%和0.10%~1.81%。土壤中TCLP(toxicity characteristic leaching procedure)提取态Cd含量随鱼骨粉投加量增加而降低,较对照最大分别降低49.7%和17.0%。施加鱼骨粉后,碱性土壤中过氧化氢酶活性显著升高(p<0.05),过氧化物酶则受到抑制,脲酶活性仅在≤1%鱼骨粉处理时有所促进,而在酸性土壤中,过氧化氢酶、过氧化物酶和脲酶活性最大分别可增加90.5%,65.4%,75.8%。施用鱼骨粉改变了土壤有机碳官能团的数量,其中酚类化合物、脂肪C、芳香C官能团含量均有所增加。土壤中有效态Cd与土壤有机质含量、过氧化氢酶活性呈极显著负相关关系(p<0.01),与土壤pH、阳离子交换量呈显著负相关关系(p<0.05)。研究表明利用鱼骨粉钝化修复Cd污染土壤较为有效可行。     

Extraktion eines kupferbelasteten Bodens unter Verwendung eines aminosäurehaltigen Reststoffhydrolysats. 2. Elutionsdynamik und Bilanzierung der Aminosäuren nach kontinuierlichem Eintrag in Bodensäulen

Extraction of a copper contaminated soil material by the percolation of an amino acid containing residue hydrolysate. 2. Time course of amino acid elution and input/output balance of amino acids During 16 days an amino acid containing blood meal hydrolysate (amino acid concentration: 188 mMol·L^?1) was percolated through a column packed soil material (soil content per column: 4.1 kg dry weight, four parallels). The copper contaminated material (soil type: Typic Udifluvent, soil texture: sandy loam, loamy sand) was sampled from an area formerly used for cultivation of hop (Humulus lupus). Besides the investigation of the copper liberation the experiments aimed to determine the elution dynamic and input/output balance of amino acids (time span for amino acids balance 14 days). In total 11.7 L of hydrolysate, containing 2.2 Mol of amino acids, were introduced into each column. The mean amino acid output with the column effluent was 1.13 Mol. This corresponds to an elution degree of 51.2%, related to the sum of applicated amino acids, and to a mean substance specific elution degree of 48.4% reflecting the elution of 15 compounds. The substance specific elution ranged from 9.6% (serine) to 75.5% (valine). The highest concentrations of serine and threonine were determined in the effluents after two days, whereas the histidine concentration was highest at the last sampling. The differences in the percolation properties of the amino acids are discussed in terms of important retention and elimination processes (biodegradation, ad-/desorption, intercalation).     

Near-infrared reflectance spectroscopy enables the fast and accurate prediction of the essential amino acid contents in soy, rapeseed meal, sunflower meal, peas, fishmeal, meat meal products, and poultry meal

Near-infrared reflectance spectroscopy (NIRS) calibrations were developed to enable the accurate and fast prediction of the total contents of methionine, cystine, lysine, threonine, tryptophan, and other essential amino acids, protein, and moisture in the most important protein-rich feed ingredients. More than 1000 samples of global origin collected over four years were analyzed on amino acids following the official methods of the United States and the European Union. Detailed data and graphics are given to characterize the obtained calibration equations. NIRS was validated with independent samples for soy and meat meal products and compared to the amino acid predictions using linear crude protein regressions. With a few exceptions, validation showed that 85-98% of the amino acid variance in the samples could be explained using NIRS. NIRS predictions compared to reference results agree excellently, with relative mean deviations below 5%. Especially for meat and poultry meals, NIRS can predict amino acids much better than crude protein regressions. By enabling the amino acid analysis of many samples to be completed in a short time, NIRS can improve the accuracy of feed formulation and thus the quality and production costs of mixed feeds.     

Preventive effects of amino acids on the toxicity of copper to Daphnia magna

The protective role of 18 amino acids on the acute toxicity of Cu in a fresh water cladoceran, Daphnia magna is reported. One hundred percent mortality in 48 hr of exposure occured at 0.56 mg L^?1 of Cu or higher. A dose of each tested amino acid (10 mg L^?1 was found to significantly neutralize the toxic effect of Cu as evidenced by a decrease in mortality and a significant increase in the median survival time (LT50) value). The 48 hr LC50 value was 0.093 mg L^?1 for Cu alone, while the LC50s with Cu plus amino acids ranged from 0.438 to 2.516 mg L^?1 of Cu, suggesting a 4.7 to 27 fold decrease in acute toxicity of Cu. A significant difference between LC50 of Cu alone and Cu with amino acids was observed. The role of amino acids for protection against heavy metal pollution stress in aquatic animals is discussed.     

Reactions of allyl isothiocyanate with alanine, glycine, and several peptides in model systems

The nucleophilic addition reactions of allyl isothiocyanate (AITC) with alanine, glycine, and five alanine and/or glycine containing di- and tripeptides were investigated in model aqueous solutions of pH 6, 8, and 10 at 25 degrees C for 2-4 weeks. The formation of primary adducts, i.e., N-allylthiocarbamoyl amino acids (ATC-amino acids) or ATC-peptides, their transformation products, i.e., 3-allyl-2-thiohydantoins originating by cyclization of ATC-amino acids or by cleavage of ATC-peptides, and several other minor components were observed. The results revealed that both addition and cleavage rates rise proportionally to pH, whereas the formation of 2-thiohydantoins from ATC-amino acids is controlled by H(3)O(+) concentration. Depending on pH, differences in reaction rates of the additions are determined by either pK(a)(NH(2)) of amino compounds or electrical effects and steric hindrance of the molecules. The latter factors are crucial also for differences in cleavage rates of ATC-peptides. With regard to the pK(a) values and simultaneous AITC decomposition by aqueous nucleophiles, the reactions with amino acids and oligopeptides are predominant reaction pathways of AITC in solutions of pH 10 and 8, respectively. Reaction mechanism of the cleavage of 2-thiohydantoins from ATC-peptides in alkaline and mild acidic solutions is different from the conventional Edman scheme used for anhydrous acid medium.     

Mechanism of reduction of exchangeable aluminum by gypsum application in acid andosols

Mechanism of reduction of exchangeable aluminum in acid Andosols treated with gypsum was studied by using cation exchange resin methods to determine the amount of polymerized aluminum. Two types of acid Andosols were used as test soils: Kitakami light colored Andosol (fine, mixed, mesic, Andic Dystrochrept) and Kawatabi thick high humic Andosol (medial, mesic mixed Alic Pachic Melanudand). Polymerization of aluminum in the soil solution of both Kitakami and Kawatabi Andosols treated with gypsum was suggested based on an analysis using cation exchange resin methods, whereas that in monomer aluminum solution was not detected. Accumulation of polymerized aluminum in both Kitakami and Kawatabi Andosols was determined by using cation exchange resin, and the amounts of polymer aluminum trapped by the resin and the ratio of polymer aluminum to monomer aluminum were increased with the incubation time. The values of CEC which decreased in the Kitakami Andosol after gypsum treatment were almost equivalent to the amounts of cation exchange sites occupied by polymer aluminum ions which were calculated based on the decrease of the values of Y _l. We conclude that the mechanism of reduction of exchangeable aluminum in strongly acid Andosols treated with gypsum is as follows: firstly, exchangeable aluminum adsorbed on the cation exchange sites of soils may be released into the soil solution due to the increase in the ion strength caused by gypsum application, and then monomer aluminum in soil solution may be polymerized in the presence of soil colloidal materials. Consequently, the polymer aluminum formed in the soil solution may be selectively and irreversibly fixed on the cation exchange sites of 2 : 1 clay minerals.     

Analysis of products of animal origin in feeds by determination of carnosine and related dipeptides by high-performance liquid chromatography

Products of animal origin such as meat meal were commonly used as sources of protein and amino acids for the production of compound feeds. Because the feeding of such products is prohibited in Germany, the official feedstuff control of the government must evaluate feeds for the forbidden use of products of animal origin. Microscope examination is the official method to prove animal-originated adulterations of feeds. This paper proposes a high-performance liquid chromatography method for the determination of the dipeptide carnosine and related dipeptides (anserine and balenine) and shows the dependence of the contents of anserine, balenine, and carnosine in compound feeds on the content of meat meal in feeds. The presented method can complete and confirm the result of the microscopic method for evidence of components of animal origin in feeds.     

Metabolic characteristics of microbial communities of Aeolian sandy soils induced by saline water drip irrigation in shelter forests

The microbial metabolic activities of soils sampled at eight depths (0–5, 5–15, 15–30, 30–50, 50–70, 70–100, 100–150 and 150–200 cm) in shelter forests (comprising the tree species, Haloxylon sp.) along the Tarim Desert Highway under drip irrigation with different saline waters were investigated by using Biolog technology. Variance analysis and principal component analysis were conducted. The average well colour development (AWCD) values for single carbon source use by microorganisms increased with the incubation time. The microorganisms reached a lag phase within 24 hours of incubation and entered exponential and stationary phases after 48 and 216 hours of incubation, respectively; however, the death phase was not obvious. As the salinity of drip irrigation water increased, the AWCD values significantly decreased; the AWCD value in the land drip‐irrigated with lowest saline water (S8) was roughly 20% larger than that of the land drip‐irrigated with highest saline water (S1), which was mainly caused by the differences in the use of carboxylic acids, amino acids and phenolic compounds by soil microorganisms. The vertical differences in carbon source utilization by soil microbial communities were obvious: these may be caused by the vertical differences in soil organism abundance associated with distribution of roots in the shelter forest. The largest and smallest AWCD values were noted in the 70–100‐cm soil layer and 0–5‐cm topsoil layer, respectively. The correlation coefficients between the AWCD values and soil organic matter, total nitrogen, total phosphorus, total potassium, available potassium, available phosphorus, pH, cation exchange capacity and bulk density were > 0.85. Thus, the differences in carbon source metabolism activities of soil microorganisms were caused mainly by the soil chemical properties, with total nutrients being the main driving factor. Furthermore, the amounts of carboxylic acids, amino acids and polymers provided sensitive markers for distinguishing the ability of soil microorganisms to use carbon sources under drip irrigation with different saline waters. Saline water irrigation affected the soil microbial community in shelter forest and produced obvious differences among the shelter forests irrigated with different saline waters.     

Nonenzymatic browning reaction of essential amino acids: effect of pH on caramelization and Maillard reaction kinetics.

The interaction between glucose and essential amino acids at 100 degrees C at pH values ranging from 4.0 to 12.0 was investigated by monitoring the disappearance of glucose and amino acids as well as the appearance of brown color. Lysine was the most strongly destroyed amino acid, followed by threonine which induced very little additional browning as compared with that undergone by glucose. Around neutrality, the nonenzymatic browning followed pseudo-zero-order kinetics after a lag time, while the glucose and amino acid losses did not follow first-order kinetics at any of the pH values tested. Glucose was more strongly destroyed than all of the essential amino acids, the losses of which are really small at pH values lower than 9.0. However, glucose was less susceptible to thermal degradation in the presence of amino acids, especially at pH 8.0 with threonine and at pH 10.0 with lysine. The contribution of the caramelization reaction to the overall nonenzymatic browning above neutrality should lead to an overestimation of the Maillard reaction in foods.     

无机酸处理对褐煤腐殖酸含量及特性的影响

研究了不同浓度的各种无机酸对褐煤中腐殖酸含量、CEC及其光学性质变化规律的影响。试验结果表明:H_3PO_4和HCl处理能在一定程度上提高阳离子交换能力,但对腐殖酸含量及光学性质基本无影响。一定强度的HNO_3能使腐殖酸含量增加25％以上,阳离子交换能力有了很大程度提高,且随硝酸强度的变化过程要明显滞后于腐殖酸含量的变化过程,光学性质发生了很大变化。不同褐煤达到最佳处理效果的条件也不尽相同。     

Physical properties of homoionic montmorillonite and kaolinite complexed with amino acids and peptides

X-ray diffractometry was used to investigate whether various amino acids and peptides expanded montmorillonite (M) homoionic to different cations, and the electrophoretic mobility (EPM) was measured to determine whether the binding of these amino acids and peptides influenced the net surface charge of M and kaolinite (K). Neither aspartic acid, adsorbed at equilibrium at concentrations of 20–250 nM mg^?1 M homoionic to Ca or Zn, nor cysteine, at 50–830 nM mg^?1 M homoionic to Zn or Al, caused expansion, whereas proline, at 90–870 nM mg^?1 clay, caused expansion of M homoionic to Ca or Zn, and both proline and arginine, at 60–1150 nM mg^?1 clay, caused expansion of M homoionic to H or Al. The extent of expansion caused by proline and arginine was related to the concentration of the amino acids and the exchangeable cation on the homoionic M. Only M-A1 complexed with arginine remained expanded after extensive washing of the clay-amino acid complexes. The expansion of M was apparently dependent on both the type of exchangeable cation on the clay and the characteristics of the amino acid (i.e. the functional groups, dielectric constant increment, dipole moment and concentration). M homoionic to H, Na, Zn, Al or La was not expanded by a series of di- to tetrapeptides bound on these clays and none of the homoionic K-amino acid or peptide complexes was expanded. The EPM of the homoionic M and K and of the corresponding clay-amino acid or peptide complexes was identical, indicating that the concentration of the amino acids or peptides bound was not sufficient to cover enough of the surface of the clays to alter significantly their net charge. The lack of differences in the bulk pH of the clays after binding with amino acids or peptides (with the exception of complexes with arginine) also indicated limited coverage of the clays by these organics.     

Color characteristics of monascus pigments derived by fermentation with various amino acids

Various pigment colors were produced by Monascus fermentations with separate addition of 20 amino acids. The color characteristics and structures of the pigment derivatives were investigated. When each amino acid was added to the fermentation broth as a precursor, pigment extracts with different hue and chroma values were obtained depending on the content ratios of yellow, orange, and red colors in the fermentation broth. The yellow and orange pigments were identical regardless of amino acid addition. The red compounds varied on the basis of the type of amino acid added. LC-MS and (1)H and (13)C NMR structural analyses confirmed that the derivative pigments contained the moieties of the added amino acids. L, a, and b values of the CIELAB color system for the derivative pigments were measured. Values of hue and chroma were then calculated. The colors of the derivative pigments were in the range of orangish red to violet red. The hydrophilicities/hydrophobicities of the derivative pigments could be predicted from their log P values, which were estimated using computer programs.     

Measurement of the potentially available charge and the dissociation behaviour of humic acid from cobaltihexammine adsorption

The use of a new method for characterizing humic acids based on the measurement of the cobaltihexammine cation exchange capacity (CEC) vs. pH is examined. The method was first verified on a macroporous weak-acid cation exchange resin: charge vs. pH curves and pK values from cobaltihexammine adsorption identify with results from discontinuous acid-base titrations at high ionic strength (5 M). Similar agreement was obtained for humic acids from a Podzol soil: the cobaltihexammine-CEC identifies with the macromolecular charge (from continuous acid-base titrations) at ionic strength of 3 and 5 M, but slightly underestimates (5%) the humic acid charge at 1 M ionic strength. The cobaltihexammine method is suitable for determining the potentially available charge in the pH range 2 to 12 and the intrinsic pK values of humic acids.     

Effects of amino-acid chemistry and soil properties on the behavior of free amino acids in acidic forest soils

Free amino acids (FAAs) in soil solution are increasingly recognized as a potentially important source of nitrogen (N) for plants, yet we are just beginning to understand the behavior of FAAs in soil. I investigated the effects of amino-acid chemistry and soil properties on mineralization, microbial assimilation and sorption of amino-acid N in soils from three ecosystems representing the two endpoints and mid point of a temperate forest fertility gradient ranging from low mineral N availability/high FAA oak forests to high mineral N availability/low FAA maple-basswood forests. Soils were amended with six ¹⁵N-labeled amino-acid substrates that ranged widely in chemical properties, including molecular weight, C:N ratio, average net charge, hydrophobicity, and polarity: Arginine (Arg), Glutamine (Gln), Glutamate (Glu), Serine (Ser), Glycine (Gly) and Leucine (Leu). Mineralization of amino-acid N accounted for 7-45% (18% avg.) of the added label and was most strongly affected by soil characteristics, with mineralization increasing with increasing soil fertility. Mineralization of amino-acid N was unrelated to amino-acid C:N ratio, rather, I observed greater N mineralization from polar FAAs compared to non-polar ones. Assimilation of amino-acid N into microbial biomass accounted for 6-48% (29% avg.) of the added label, and was poorly predicted by either intrinsic amino-acid properties or soil properties, but instead appeared to be explicable in terms of compound-specific demand by soil micoorganisms. Sorption of amino-acid N to soil solids accounted for 4-15% (7% avg.) of the added label and was largely controlled by charge characteristics of individual amino acids. The fact that both positively- and negatively-charged amino acids were more strongly sorbed than neutral ones suggests that cation and anion exchange sites are an important factor controlling sorption of FAAs in these acid forest soils. Together, the findings from this study suggest that there may be important differences in the behavior of free amino acids in sandy, acidic forest soils compared to generalizations drawn from finer-textured grassland soils, which, in turn, might affect the availability of some FAAs in soil solution.     

Complete amino acid analysis in hydrolysates of foods and feces by liquid chromatography of precolumn phenylisothiocyanate derivatives

The amino acid analysis method using precolumn phenylisothiocyanate (PITC) derivatization and liquid chromatography was modified for accurate determination of methionine (as methionine sulfone), cysteine/cystine (as cysteic acid), and all other amino acids, except tryptophan, in hydrolyzed samples of foods and feces. A simple liquid chromatographic method (requiring no derivatization) for the determination of tryptophan in alkaline hydrolysates of foods and feces was also developed. Separation of all amino acids by liquid chromatography was completed in 12 min compared with 60-90 min by ion-exchange chromatography. Variation expressed as coefficients of variation (CV) for the determination of most amino acids in the food and feces samples was not more than 4%, which compared favorably with the reproducibility of ion-exchange methods. Data for amino acids and recoveries of amino acid nitrogen obtained by liquid chromatographic methods were also similar to those obtained by conventional ion-exchange procedures.