Effects of mineral surface iron on the CPMAS 13C-NMR spectroscopic detection of organic matter from soil fractions in an agricultural topsoil with different amendments

The decrease of NMR visibility of the C signal in soil samples due to the association between organic carbon (OC) and the topsoil mineral surface was investigated. CPMAS ¹³C‐NMR spectra were obtained for soil particle‐size fractions (< 2 μm, 2–20 μm, > 20 μm) and bulk soils from an agricultural topsoil (Chernozem) that had received three different amendments (no fertilization, mineral fertilization (NPK), mineral (NPK) and organic (cattle manure) fertilizations) at Bad Lauchstädt, Germany. The soil organic carbon content of the three soils depended on the degree of soil fertilization. There was no constant relationship between the total NMR signal intensity and the total amount of organic carbon (TOC) for all size fractions. Indeed, a key role played in the C signal intensity by the paramagnetic ferric ion from the clay content in soil fractions and bulk soils was confirmed. Thus, we describe the variations of C signal intensity by taking into account the distribution of clay‐associated OC and non‐associated OC pools. Depending on the amendment, the C signal visibility was weakened by a factor of 2–4 for the clay‐associated OC. This estimation was rendered possible by combining mineral specific surface area (SSA) measurements with the N₂ gas adsorption method (BET method) and determination of TOC and iron concentrations. This approach contributes to the quantitative evaluation of the CPMAS ¹³C‐NMR detection.     

Combined high-field 13C CP MAS NMR and low-field NMR relaxation measurements on post mortem porcine muscles

Changes in postmortem muscle characteristics are investigated in muscles from eight pigs exposed to different combinations of preslaughter stress (exercise on treadmill) and stunning method (CO(2) vs electrical stunning). Solid-state (13)C cross-polarization (CP) magic-angle spinning (MAS) NMR experiments are carried out on a total of 16 rapidly frozen M. longissimus muscle biopsies taken in vivo the day before slaughter and at 45 min postmortem. Simultaneously, low-field NMR T(2) relaxation time measurements are carried out on samples from M. longissimus. Glycogen and lactate are estimated from the (13)C CP MAS spectra, and correlations of r = 0.89 and r = 0.70, respectively, to subsequent biochemical determinations using partial least squares regression (PLSR) are established. Moreover, PLSR reveals that, besides the 72 ppm signal (carbons in glycogen), a signal around 38 ppm, which increases concomitantly with lactate, is also significantly correlated to changes in glycogen/lactate. With the assumption that the 38 ppm signal reflects CH(2) in phosphocreatine/creatine, altered mobility of creatine as a result of dephosphorylation is indicated. Finally, PLSR on the 45 min (13)C CP MAS spectra also reveals correlation (r = 0.54) to the slowest relaxing T(2) population (50 min postmortem), known to reflect extra-myofibrillar water. Subsequently, evaluation of the loading plot in the PLSR analysis reveals that the correlation exclusively is associated to the 52 ppm resonance intensity. With the assumption that this resonance reflects methyl groups in choline/phosphatidyl choline, the intensity changes in the 52 ppm resonance imply alterations in membrane properties. Accordingly, the data indicate a relationship between membrane properties and the amount of water being expelled from muscle cells postmortem, which supports the hypothesis that disruption of membranes is implicated in the postmortem mobilization of muscle water.     

Differentiation of natural and synthetic phenylalanine and tyrosine through natural abundance 2H nuclear magnetic resonance

The natural abundance deuterium NMR characterization of samples of the amino acids tyrosine (1) and phenylalanine (2), examined as the acetylated methyl esters 4 and 6, has been performed with the aim to identify by these means the contribution in animals of the hydroxylation of the diet l-phenylalanine (2) to the formation of l-tyrosine (1), a feature previously revealed on the same samples through the determination of the phenolic delta(18)O values. The study, which includes also the NMR examination of benzoic acid (5) from 2 and of tyrosol (7) from 1, substantially fails in providing the required information because the mode of deuterium labeling of tyrosine samples of different origins is quite similar but indicates a dramatic difference in the deuterium labeling pattern of the two amino acids 1 and 2. The most relevant variation is with regard to the deuterium enrichments at the CH(2) and CH positions, which are inverted in the two amino acids of natural derivation. Moreover, whereas the diastereotopic benzylic hydrogen atoms of l-tyrosine (1) appear to be equally deuterium enriched, in l-phenylalanine (2) the (D/H)(3)(R)() > (D/H)(3)(S)(). Similarly, benzoic acid (5) shows separate signals for the aromatic deuterium nuclei, which are quite indicative of the natural or synthetic derivation. The mode of deuterium labeling of the side chain of 1 and 2 is tentatively correlated to the different origins of the two amino acids, natural from animal sources for l-tyrosine and biotechnological probably from genetically modified microorganisms for l-phenylalanine.     

Effect of paramagnetic cations on solid state 13C nuclear magnetic resonance spectra of natural organic materials

Abstract

The effect of cation (Zn²⁺, Cu²⁺, Pr³⁺) amendment on the solid state ¹³C nuclear magnetic resonance (NMR) spectral properties of organic materials was investigated. The organic materials were chosen to represent structures found in natural organic matter (NOM) from soils, waters, sediments, sewage sludges and plant residues, and included cellulose, pectin, chitin, collagen, a commercial humic acid, and charcoal. Cation amendment was shown to have little effect on the observability of ¹³C NMR signal, except for the paramagnetic amended pectin samples, for which observability was decreased from near 100% in the unamended sample to 19% for the Cu²⁺ amended sample and 71% for the Pr³⁺ amended sample. NMR relaxation parameters (T_1p H, T₁H) were more sensitive to cation amendment. For a number of the samples, a decrease in relaxation rate (increase in T_lpH and T₁H) was observed on amendment with Zn²⁺. This was ascribed to a decrease in molecular motion due to the chelating effects of Zn²⁺. An increase in relaxation rate (decrease in T_1pH and T₁H) was generally observed on amendment with Cu²⁺. The effects of amendment with Pr³⁺ varied. T₁H was more sensitive to the presence of paramagnetic species than was T_1pH. These results suggest that bound paramagnetic cations will only decrease the observability of ¹³C NMR signal in NOM samples (or domains within NOM samples) at high paramagnetic cation concentrations (>3%). There is great potential for the use of paramagnetic cation amendment to differentiate relaxation rates of domains within NOM samples, subspectra for which can then be generated using the proton spin relaxation editing (PSRE) technique.     

长期施有机肥和化肥对潮土胡敏酸结构特征的影响

以河南封丘长期肥料试验为平台,选择有机肥(OM)、化肥NPK(NPK)和不施肥(CK)3种处理,研究了不同施肥措施对潮土胡敏酸(HA)结构特征的影响。HA样品经提取纯化后,采用一系列固态~(13)C核磁共振(NMR)技术并结合元素分析和稳定碳同位素(δ~(13)C)技术对其结构进行定量表征。其中NMR技术包括高速多重倾斜幅度交叉极化/魔角自旋(multiCP/MAS)、偶极相移、化学位移各向异性过滤和质子碳编辑技术。结果表明:长期施有机肥和NPK肥增加了HA的饱和程度、氧化程度和极性,但缩合程度降低;HA的δ~(13)C值降低,表明有新的有机碳进入HA中。潮土HA以脂肪族化合物为主,烷基比例最高,占全碳的24.1%~26.3%;δ64~44处同时存在含氮烷基和甲氧基,且含氮烷基比例更高;另外异头碳和烷氧基中的非质子碳比例均很低。HA的芳香族成分中,δ142~113芳香碳以质子碳为主,且有少量质子芳香碳存在于δ113~93。结果还表明施肥均降低了HA的羧基和δ142~113芳香碳比例,增加了烷氧基和甲氧基比例;施有机肥还增加了酚碳和含氮烷基比例,降低了烷基比例。本研究表明长期施用有机肥和NPK肥使潮土HA结构向着疏水性程度和分解程度降低的方向发展;OM和NPK处理均使糖类物质增加,OM处理还使木质素和多肽增加,脂类物质降低。不同施肥模式下HA的形成机制不同。     

Lignin changes after steam explosion and laccase-mediator treatment of eucalyptus wood chips

Eucalyptus globulus chips were steam exploded followed by treatment with a laccase-mediator system (LMS) under different experimental conditions. Removal of hemicelluloses and, to a lesser extent, lignin was observed. Thermogravimetic analyses of whole meal obtained from chips before and after steam explosion indicated an increase in lignin degradation temperature due to lignin condensation. In contrast, application of LMS treatment caused a reduction in lignin and polysaccharide degradation temperatures. Lignins were isolated from wood samples before and after each treatment and analyzed by 2D NMR and (13)C NMR. An increase in carboxyl and phenolic hydroxyl groups and a significant decrease in β-O-4 structures were found in steam-exploded samples. The most relevant changes observed after laccase treatment were increased secondary OH and degree of condensation.     

Solid-State 13C CP/MAS NMR studies on aging of starch in white bread

The effects of storage methods and glycerol on the aging of breadcrumbs were studied using solid-state (13)C CP/MAS NMR. After baking, a shift in C(1) peaks from triplet (A-type) to singlet (V-type) was observed. Addition of glycerol reduced the carbon peak intensities of fresh and aged breads, which correlated well with the DSC amylopectin "melting" enthalpy (r(2) = 0.91). Upon storage of bread with crust in hermetically sealed containers (when moisture migrated from the crumbs to the crust), the (13)C CP/MAS NMR peak intensity increased more rapidly during aging than when the bread was stored without crust. Although addition of glycerol retarded the starch retrogradation, as observed by (13)C CP/MAS NMR and DSC, it accelerated the firming rate. Therefore, bread firming in this case was controlled not only by starch retrogradation but also by other events (such as local dehydration of the matrix or gluten network stiffening).     

Quantitative solid-state 13C NMR spectroscopy of organic matter fractions in lowland rice soils

Spin counting on solid‐state ¹³C cross‐polarization (CP) nuclear magnetic resonance (NMR) spectra of two humic fractions isolated from tropical lowland soils showed that only 32–81% of potential ¹³C NMR signal was detected. The observability of ¹³C NMR signal (C_obs) was higher in the mobile humic acid (MHA) than in the calcium humate (CaHA) fraction, and increased with increasing intensity of irrigated rice cropping. NMR observability appeared to be related to the nature of the organic carbon, with phenol‐ and methoxyl‐rich samples having the higher values of C_obs. The Bloch decay (BD) technique provided more quantitatively reliable ¹³C NMR spectra, as evidenced by values of C_obs in the range 91–100% for seven of the eight humic fractions studied. The BD spectra contained considerably more aryl and carbonyl signal, and less O–alkyl and alkyl signal, with the greatest differences between CP and BD spectra observed for the samples with low C_obs(CP). The causes of low CP observability were investigated using the spectral editing technique RESTORE ( RE storation of S pectra via T _CH and T O ne R ho (T_1ρH) E diting). Rapid T_1ρH relaxation was found to be primarily responsible for the under‐representation of carbonyl carbon, whereas inefficient cross‐polarization was primarily responsible for the under‐representation of aryl carbon in CP spectra. Proton NMR relaxation rates T_1H and T_1ρH were found to correlate with other NMR properties and also with cropping management. Non‐uniform rates of T_1H relaxation in two of the CaHA fractions enabled the generation of proton spin relaxation editing subspectra.     

Characterization of milled wood lignin (MWL) in Loblolly pine stem wood, residue, and bark

Milled wood lignin samples from Loblolly pine stem wood, forest residue, and bark were isolated and characterized by quantitative (13)C and (31)P nuclear magnetic resonance (NMR), Fourier transform infrared spectroscopy (FT-IR), and gel permeation chromatography (GPC) for molecular weight determination. Results from (13)C NMR show the stem wood and forest residue samples have similar functional group contents. However, the bark has fewer methoxyl groups, β-O-4 structures, dibenzodioxocin, and side chains than the other two lignins. The bark lignin has the highest amounts of p-hydroxyphenyl (h) and C-5 condensed lignin, stem wood has the lowest, and the residue lies between. (31)P NMR analysis indicates that bark lignin contains more C-5 substituted phenolics and fewer aliphatic hydroxyl groups than the lignin isolated from stem wood or residue. The molecular weight distribution analysis indicates the bark lignin has higher weight-average molecular weight (M(w)) and polydispersity index than the lignin recovered from stem wood or residue.     

Nondestructive quantification of organic compounds in whole milk without pretreatment by two-dimensional NMR spectroscopy

In this study, various organic compounds in commercial whole milk were quantified simultaneously by 1H 1D and 1H - 13C HSQC 2D NMR spectra without any pretreatment. 2D NMR spectroscopy was applied to quantification of milk compounds for the first time. Milk fat content was easily determined to be 3.6 +/- 0.1%, and the lactose content was 47.8 +/- 1.0 mg/mL by 1H NMR spectra. From 1H-13C HSQC spectra, the concentrations of citrate, N-acetylcarbohydrates, and trimethylamine were determined to be 3.2 +/- 0.2, 2.9 +/- 0.1, and 4.0 +/- 0.6 mM, respectively. The latter two compounds were quantified in milk for the first time. Butyric acid, total monounsaturated fatty acids, and total polyunsaturated fatty acids of triacylglycerols were 6.2 +/- 0.5, 9.1 +/- 0.9, and 2.9 +/- 0.3 mM, respectively. The fatty acid compositions (mol %) of triacylglycerols were then calculated and were observed to be in good agreement with reference values. The results indicated that 1H 1D and 1H-13C HSQC 2D NMR spectroscopy is useful for the rapid and nondestructive determination of various compounds in milk.     

Chemical structures of corn stover and its residue after dilute acid prehydrolysis and enzymatic hydrolysis: insight into factors limiting enzymatic hydrolysis

Advanced solid-state NMR techniques and wet chemical analyses were applied to investigate untreated corn stover (UCS) and its residues after dilute acid prehydrolysis (DAP) and enzymatic hydrolysis (RES) to provide evidence for the limitations to the effectiveness of enzyme hydrolysis. Advanced solid-state NMR spectral-editing techniques as well as 1H-13C two-dimensional heteronuclear correlation NMR (2D HETCOR) were employed. Our results indicated that dilute acid prehydrolysis selectively removed amorphous carbohydrates, increased aromatic CH/other protonated -C═C- and enriched alkyl CH and CH2 components. Cinnamic acids were increased, and proteinaceous materials and N-containing degradation or condensation compounds were absorbed or coprecipitated in RES. 2D HETCOR experiments indicated a close association between lignin and the residual carbohydrates. Ketones/aldehydes were not detected in the DAP, in contrast to a report in which an appreciable amount of ketones/aldehydes was generated from the acid pretreatment of a purified cellulose in the literature. This suggested that acid pretreatment may modify the structure of purified cellulose more than biomass and that biomass may be a better substrate than model biopolymers and compounds for assessing structural changes that occur with industrial processing. On the basis of NMR and wet chemical analyses, we found the following factors could cause the limitations to the effectiveness of enzymatic hydrolysis: (1) chemical modification of carbohydrates limited the biologically degradable carbohydrates available; (2) cinnamic acids in the residue accumulated; (3) accessibility was potentially limited due to the close association of carbohydrates with lignin; and (4) proteinaceous materials and N-containing degradation or condensation compounds were absorbed or coprecipitated.     

Post-mortem changes in porcine M. longissimus studied by solid-state 13C cross-polarization magic-angle spinning nuclear magnetic resonance spectroscopy

Solid-state (13)C cross-polarization (CP) magic-angle spinning (MAS) nuclear magnetic resonance (NMR) experiments are carried out for the first time on rapidly frozen muscle biopsies taken in M. longissimus in vivo and at 1 min, 45 min, and 24 h post-mortem from three pigs. Two of the pigs were CO(2)-stunned (control animals), and one was pre-slaughter-stressed (treadmill exercise) followed by electrical stunning to induce difference in metabolism post-mortem. (13)C resonance signals from saturated and unsaturated carbons in fatty acids, carboxylic carbons, and carbons in lactate and glycogen are identified in the solid-state NMR spectra. The (13)C CP MAS spectra obtained for post-mortem samples of the stressed, electrically stunned pig differ significantly from the post-mortem control samples, as the intensity of a resonance line appearing at 30 ppm, assigned to carbons of the methylene chains, is reduced for the stressed pig. This spectral difference is probably due to changes in lipid mobility and indicates altered membrane properties in the muscle of the stressed/electrically stunned animal when compared with the control animals already 1 min post-mortem. In addition, the post-mortem period changes in glycogen carbons can be estimated from the (13)C CP MAS spectra, yielding a correlation of r = 0.74 to subsequent biochemical determination of the glycogen content.     

Chemical properties of humic matter as related to induction of plant lateral roots

A series of humic matter samples isolated from a soil sequence, different oxisols, size‐fractionated from a vermicompost humic acid and subjected to chemical modifications, were characterized by CPMAS ¹³C‐NMR spectroscopy. The relative signal areas in chemical shift regions of NMR spectra of the four sets of samples were analysed by principal component analysis (PCA). Hierarchical cluster analysis (HCA) was applied to build a classification model, which allowed the recognition of humic matter according to its origin. The relationship between carbon species and biological activity of humic acids, as promoters of lateral root emergence, was obtained by applying PLS multivariate analysis. This showed that lateral root emergence was mostly related to NMR parameters such as the hydrophobicity index (HB/HI) and the 40–110 and 160–200 ppm chemical shift regions (hydrophilic carbon HI), while the content of hydrophobic (HB) carbon in humic samples was negatively correlated with induction of lateral root hair. Our results represent a step further in the structure‐bioactivity relationship of natural humic substances and confirm their role as plant root growth promoters.     

Elucidation of protein-lipid interactions in a lysozyme-corn oil system by Fourier transform Raman spectroscopy

Lysozyme (25% in D2O, corn oil, and their emulsions (10% w/w oil/D2O solution) were examined by Fourier transform Raman spectroscopy. Emulsions showed three layers, namely, top oil, middle cream, and bottom aqueous layers. Raman spectral analysis revealed hydrophobic interactions involving both protein and lipid components. Compared to lysozyme in D2O, the difference spectrum obtained after subtraction of oil from the cream layer spectrum showed reduced intensity of tryptophan bands at 760, 1013, 1340, and 1360 cm(-)(1), reduced intensity ratio of the tyrosine doublet at 850 and 830 cm(-)(1), and increased intensity of the C-H bending band at 1455 cm(-)(1). Compared to corn oil, the difference spectrum after subtraction of lysozyme from the cream layer spectrum indicated decreased intensity at 2855 cm(-)(1) (lipid CH(2) symmetric stretch) and 3011 cm(-)(1) (unsaturated fatty acid hydrocarbon chain =C-H stretch) and a higher intensity ratio of the C-H stretching band at 2900 cm(-)(1) to bands at 2885 and 2933 cm(-)(1). Spectra of the top and bottom layers resembled corn oil and lysozyme, respectively, except for changes in the D2O band. Raman spectroscopy can be used to detect structural changes in proteins, lipids, and D2O due to protein-lipid interactions.     

Differentiation of extractive and synthetic salicin. The 2H aromatic pattern of natural 2-hydroxybenzyl alcohol

The natural abundance deuterium NMR characterization of four commercially available samples (Kahlbaum, Aldrich, Fluka, and Extrasynthese) of salicin 1 in comparison with two extractive samples from Salix humboldiana and Salix purpurea L. and with a synthetic material, performed on the pentaacetate derivative 2 and on diacetyl salicyl alcohol 4, is reported. Product 2 from S. humboldiana and the sample from salicin Kahlbaum show mean (D/H)aromatic values of 117 and 121 ppm, whereas, for the remaining, values of 146, 154, 153, and 150 ppm are observed, thus suggesting that salicin Kahlbaum is from extractive origin. The (D/H) values at positions 5' and 6' of the sugar moiety suggest a hypothesis on the origin of the glucose residue discriminating between those deriving from C3 or C4 plants. The analysis of 4, obtained from 3, formed in the beta-glucosidase hydrolysis of salicin 1, reveals in the natural samples from S. purpurea and from Kahlbaum the trend (D/H)4(para) > (DH)3(meta) approximately (D/H)5(meta) > (D/H)6(ortho), the first example of deuterium pattern of an ortho-oxygen-substituted phenylpropanoid. The three samples derived from commercial 1 (Aldrich, Fluka, and Extrasynthese) and the synthetic sample show almost identical deuterium content at positions 4 and 6 (around 153 ppm), whereas for the two meta positions (D/H)3 > (D/H)5 (ca. 162 and 140 ppm, respectively). Product 4, obtained from 3 submitted to acid-catalyzed deuteration, shows different deuterium incorporations in the two meta positions (which are ortho/para to the activating phenolic hydroxyl group), suggesting that possibly the deuterium abundance at the two meta positions may be affected by exchange phenomena with the medium.     

Relationship between the glass transition of soy protein and molecular structure

The change in molecular structure of the soy protein samples as a result of the microbial transglutaminase treatment was studied using solid-state (13)C NMR spectroscopy and circular dichroism (CD), and the relation to the glass transition temperature (T(g)) was examined. From NMR measurements, the structure of the local region of the C(alpha) methine was observed to change, and the region had relatively high mobility. From CD measurements, the structural change seemed to be caused by the change in the secondary structure (disintegration of the beta-structure). By comparison with the T(g) of another protein, the state of the secondary structure of a protein was suggested to be a key in determining its T(g).     

Improvement of 13C and 15N CPMAS NMR spectra of bulk soils, particle size fractions and organic material by treatment with 10% hydrofluoric acid

The small organic matter content of mineral soils makes it difficult to obtain ¹³C and ¹⁵N nuclear magnetic resonance (NMR) spectra with acceptable signal-to-noise ratios. Subjecting such samples to hydrofluoric acid removes mineral matter and leads to a relative increase in organic material. The effect of treatment with 10% hydrofluoric acid on bulk chemical composition and resolution of solid-state ¹³C NMR spectra was investigated with six soils, some associated particle size fractions, plant litter and compost. The treatment enhanced the signal-to-noise ratio of the solid-state ¹³C NMR spectra. The improvement in spectrum quality was greatest in the clay fraction of soil contaminated with coal ash. The removal of paramagnetic compounds associated with the ash may be the main reason for the improvement. Based on total C, total N, C/N ratio and intensity distribution of the solid-state ¹³C NMR spectra, no changes in organic matter composition could be detected, except for a possible loss of carbohydrates. After treatment with HF, solid-state ¹⁵N NMR spectra of particle size fractions were obtained and indicated that the observable nitrogen is present mostly as peptides and free amino groups. Extraction with hydrofluoric acid is recommended as a routine treatment prior to solid-state ¹³C and ¹⁵N NMR on soil containing little C or N and soil samples containing paramagnetic compounds from natural or anthropogenic sources.     

Isolation and identification of molecular species of phosphatidylcholine and lysophosphatidylcholine from jojoba seed meal (Simmondsia chinensis)

A mixture of lysophosphatidylcholine (LPC) and phosphatidylcholine (PC) has been isolated by column chromatography from a jojoba meal (Simmondsia chinensis) extract. The molecular species of both classes could be separated and isolated by C18 reversed phase HPLC. The two major compounds were identified by 1D and 2D (1)H and (13)C NMR, by MS, and by GC-MS as 1-oleoyl-3-lysophosphatidylcholine and 1,2-dioleoyl-3-phosphatidylcholine. Eight other molecular species of LPC and four other molecular species of PC could be assigned by comparison of the mass spectra of the isolated compounds with the spectra of the two major compounds. Complete characterization of the individual molecular species was achieved by GC and GC-MS analysis of the fatty acyl composition from the isolated compounds. The PC/LPC proportion in the phospholipid mixture from three different samples is 1.6 +/- 0.1. LPC is considered to be an important bioactive compound; the results of this study suggest further research for the evaluation of potential health benefits of jojoba meal phospholipids.     

NMR and molecular mechanics study of pyrethrins I and II.

Bioassay-directed fractionation of the organic extract of the Kenyan pyrethrum flowers (Chrysanthemum cinerariaefolium Vissiani) resulted in the isolation of two natural pyrethrin esters, pyrethrin I (PI) and pyrethrin II (PII) as the major constituents. These esters elicited inhibition of the multiple drug resistant (MDR) Mycobacterium tuberculosis. The high-field (1)H and (13)C nuclear magnetic resonance (NMR) chemical shifts of PI and PII were unequivocally assigned using modern two-dimensional (2D) proton-detected heteronuclear multiple-quantum coherence (HMQC) and heteronuclear multiple-bond correlation (HMBC) experiments. The conformations of both esters were deduced from (1)H-(1)H vicinal coupling constants and confirmed by 2D nuclear Overhauser effect spectroscopy (NOESY). Computer molecular modeling (MM) studies revealed that PI and PII molecules adopt a "love-seat" conformation in chloroform (CDCl(3)) solution.