Quantitative analysis of surface-located triacylglycerol in intact emulsion particles

The amount of triacylglycerol (TG) in the surface monolayer of intact phospholipid-stabilized emulsions was determined using (13)C nuclear magnetic resonance ((13)C NMR). (13)C NMR spectra of emulsions composed of bulk long-chain or medium-chain TG were prepared with [(13)C(3)]-carbonyl-enriched triolein, tripalmitin, or trioctanoin, and were analyzed and compared with NMR spectra of phosphatidylcholine vesicles with and without added TG. Identification of carbonyl peaks intermediate between those of phosphatidylcholine carbonyls and bulk TG confirmed the presence of surface TG in each emulsion. The surface of emulsions contained 2.2 mol % tripalmitin and 1.4 mol % triolein, but significantly more medium-chain TG, 9.1 mol % trioctanoin, as predicted by measurements of TG in phospholipid vesicles. Thus, medium-chain TGs are more accessible than long-chain TGs to enzymes or pro-oxidants in the continuous phase of phospholipid-stabilized emulsion systems. The quantitative determination of surface-located TG in intact particles will advance the understanding of emulsion colloidal properties, physicochemical stability, and metabolic behavior.     

(13)C NMR characterization of triacylglycerols of Moringa oleifera seed oil: an "oleic-vaccenic acid" oil

The composition of acyl chains and their positions in the triacylglycerols of the oil extracted from seeds of Moringa oleifera were studied by (13)C NMR spectroscopy. The unsaturated chains of M. oleifera seed oil were found to comprise only mono-unsaturated fatty acids and, in particular, two omega-9 mono-unsaturated acids, (cis-9-octadecenoic (oleic acid) and cis-11-eicosenoic acids) and one omega-7 mono-unsaturated acid (cis-11-octadecenoic acid (vaccenic acid)). The mono-unsaturated fatty acids were detected as separated resonances in the spectral regions where the carbonyl and olefinic carbons resonate according to the 1,3- and 2-positions on the glycerol backbone. The unambiguous detection of vaccenic acid was also achieved through the resonance of the omega-3 carbon. The (13)C NMR methodology enabled the simultaneous detection of oleate, vaccenate, and eicosenoate chains according to their positions on the glycerol backbone (1,3- and 2-positions) through the carboxyl, olefinic, and methylene envelope carbons of the triacylglycerol acyl chains.     

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.     

Fates of 13C from enriched glucose and glycine in an organic soil determined by solid-state NMR

The transformations of the indigenous ¹³C and the ¹³C from either uniformly enriched ¹³C-D-glucose or ¹³C-glycine added to an organic soil were followed during a 28-day incubation using cross polarization (CP) magic angle spinning (MAS) ¹³C nuclear magnetic resonance (NMR) spectroscopy and dipolar dephased (DDP) MAS ¹³C NMR. The C mineralization was determined from ¹³C remaining by mass spectrometry and from CO₂ evolution by gas chromatography. DDP MAS ¹³C NMR of the unamended soil indicated a transient increase in molecularly mobile ¹³C in the alkyl- and methyl-C over 5 days, which may be due to redistribution of ¹³C in the microbial biomass in response to perturbation. The added glucose-¹³C remaining declined to 43% after 7 days and 34% after 28 days. After 28 days the amount of added glucose-¹³C remaining was 6 times greater than the biomass C at the outset, while the microbial activity (CO₂ production) was 38% greater, indicating that a significant proportion of added glucose-¹³C was not in microorganisms. Added glycine-¹³C declined faster, such that 29% and 8% remained after 7 and 28 days, respectively. After 28 days’ incubation with ¹³C-glucose, the O-alkyl-C, the acetal- and ketal-C, and the methyl- and alkyl-C resonances in CP MAS ¹³C NMR spectra were all enhanced compared with the unamended soil. The calculated T₁ρH values of the O-alkyl-C and the acetal- and ketal-C resonances were less than those of crystalline glucose, indicating that there was no substantial reservoir of unreacted glucose. After 7 days’ incubation with ¹³C-glycine, none of the signals in the CP MAS ¹³C NMR spectra were enhanced when compared with the unamended soil, indicating that the added ¹³C remaining was distributed in undetectable quantities in a range of functionalities. The calculated T₁ρH values indicated that glycine ¹³C was in O-alkyl-C, acetal- and ketal-C and carbonyl-C. T₁ρH values may be more sensitive to changes in the distribution of ¹³C when ¹³C content is low. The DDP MAS ¹³C NMR spectra of both the ¹³C-glucose- and the ¹³C-glycine-amended soil showed that the molecularly mobile alkyl- and methyl-C increased compared with the unamended soil. Received: 27 February 1997     

Study of the cultivar-composition relationship in Sicilian olive oils by GC,NMR, and statistical methods

The aim of this research is to find if there is direct evidence relating the fatty acid composition of olive oils to specific cultivars grown within a well-limited geographical region. To group olive oils according to their own cultivars,(13)C high-field nuclear magnetic resonance (NMR) and gas chromatography (GC) were used to analyze 60 extra virgin olive oils from the same Italian region (southwestern Sicily) obtained from four monovarietal cultivars. The (13)C NMR spectrum provides information about glycerol triesters of olive oils, i.e., about the acyl composition of major components and about the fatty acids' positional distribution on the glycerol moiety. GC gives the complete fatty acid profile of olive oil samples. Selection of NMR and GC peaks on the basis of their sensitivity to the different cultivars was performed by using multivariate analysis of variance (MANOVA). Principal component analysis, tree clustering analysis, multidimensional scaling (MDS), and linear discriminant analysis (LDA) were then performed on the MANOVA-selected peaks. Results obtained from (13)C NMR and GC techniques combined with the multivariate statistical procedure are in good agreement and prove the usefulness of fatty acids analysis to group the monovarietal olive oils belonging to the same cultivars. Grouping of olive oils according to their cultivars occurs for particular (13)C resonances all belonging to fatty chains in the sn 1,3 position of the glycerol moiety.     

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.     

Some characteristics of sugar ester nonionic microemulsions in view of possible food applications

This study explores some characteristics of microemulsions composed of sucrose monostearate (SMS), medium-chain triglycerides (MCT), or R-(+)-limonene, alcohols, and water. The systems are homogeneous, soft, and waxy solids at room temperature but liquefy and structure into homogeneous microemulsions when heated to >40 degrees C. The amount of solubilized water is enhanced as a function of the alcohol/oil ratio and is inversely proportional to the alcohol chain length. Over 60 wt % water can be solubilized in systems consisting of propanol/MCT/SMS at a weight ratio of 3:1:4 (initial weight ratio). These microemulsions are unique and differ from nonionic ethoxylated-based microemulsions in that their viscosity is very low and is reduced with increasing amounts of solubilized water. The electrical conductivity increases only slightly as a function of the water content and does not show typical bicontinuous or percolated behavior. The water in the core of the microemulsion strongly binds to the headgroups of the surfactant. Only at >15 wt % solubilization of water was free or bulk water detected in the core of the microemulsions. Such unique behavior of the core water might have a possible application in systems requiring monitoring of enzymatic (lipase) reactions carried out in the microemulsions as microreactors.     

Multivariate analysis of CPMAS 13C-NMR spectra of soils and humic matter as a tool to evaluate organic carbon quality in natural systems

A series of humic and fulvic acids isolated from different sources, size‐fractions separated from a humic acid, and three soils of different origin were subjected to CPMAS ¹³C‐NMR spectroscopy to obtain the distribution of their carbon contents. The relative areas of chemical shift regions in NMR spectra were used to apply a principal component analysis (PCA) to the three sets of samples. The multivariate analysis was successful in efficiently differentiating samples on the basis of the quality of their organic carbon content. The PC biplots based on two principal components distinguished objectively among samples as accurately as it was possible to do by subjective qualitative evaluation of the original spectra. In the case of the soils, a discriminant analysis (DA) was applied to build a classification model that allowed the validation of the three soils according to their origin. Percentage of validation in the classification model is expected to increase when a large number of NMR spectra are accumulated and/or the concentration of organic carbon in samples is enhanced. The multivariate analyses described are likely to become a useful tool to increase the importance of CPMAS ¹³C‐NMR spectra in the appraisal of natural organic matter variations in heterogeneous natural systems.     

Fractional and structural characterization of lignins isolated by alkali and alkaline peroxide from barley straw.

A sequential treatment of dewaxed barley straw with sodium hydroxide, different concentrations of hydrogen peroxide, and potassium hydroxide/sodium borate degraded various proportions of the original lignin and solubilized different amounts of the original hemicelluloses. The isolated lignin fractions were subjected to comprehensive structural characterization by UV, FT-IR, and (13)C NMR spectroscopy, and their chemical compositions were analyzed by alkaline nitrobenzene oxidation. All of the lignin fractions were typical of grass lignins and had weight-average molecular weights between 1750 and 2190. It was found that the peroxide treatment at low concentrations (< or =2.0%) resulted in a slight increase in the amount of carboxyl groups, whereas the treatment at a relatively high concentration of alkaline peroxide, such as at 3.0% H(2)O(2), led to a noticeable oxidation of the lignins, as shown by an increase of carboxyl groups. Moreover, the results obtained indicated that the successive treatments with alkali and alkaline peroxide under the conditions used did not significantly affect the beta-O-4 structures of lignins. Substantial amounts of etherified ferulic acids were cleaved by the sequential treatments with alkaline peroxide, as shown in the (13)C NMR spectra. The results underscore the structural differences between alkali- and alkaline peroxide-soluble lignins from barley straw.     

Biflavonoids isolated from Selaginella lepidophylla inhibit photosynthesis in spinach chloroplasts

Through bioactivity-guided chemical analysis of Selaginella lepidophylla, biflavonoids robustaflavone (1), 2,3-dihydrorobustaflavone (2), and 2,3-dihydrorobustaflavone-5-methyl ether (3) were isolated and their structures confirmed by spectroscopic and spectrometric analyses. Their NMR resonances were unambiguously assigned from HMBC, NOESY, and NOESY-1D experiments, and absolute configurations of 2 and 3 were established. Compound 3 has not been reported, and although structure of 2 was described before, the (13)C NMR assignment does not correlate with the structure reported. Therefore, this is the first report of 2. All compounds inhibited ATP production. Compounds 1 and 2 behaved as Hill reaction inhibitors. 1 interacted with photosystem II, transforming the reaction centers to silent centers at 300 and 600 microM. The interaction and inhibition target of 2 was located on Cyt b6f to PC. The three compounds also behaved as energy transfer inhibitors, 3 being the most active.     

Production of saturated acyl L-ascorbate by immobilized lipase using a continuous stirred tank reactor

6-O-decanoyl, 6-O-dodecanoyl, or 6-O-tetradecanoyl L-ascorbate was continuously produced at 50 degrees C using a continuous stirred tank reactor (CSTR) with an immobilized lipase, Chirazyme L-2 C2, from Candida antarctica. Acetone was used as the reaction medium. For each saturated acyl L-ascorbate, the productivity of ca. 60 g/L reactor/day was achieved for at least 11 days. The solubility of the saturated acyl L-ascorbate in the soybean oil or water was measured at various temperatures. The solubilities in both the soybean oil and the water were higher for L-ascorbate with a shorter acyl chain. The acyl chain dependence of the solubility in water was stronger than that of the solubility in soybean oil. The temperature dependences of the solubility in both soybean oil and water could be expressed by the van't Hoff equation, and the dissolution enthalpy (DeltaH) values for the soybean oil and water were about 20 and 90 kJ/mol, respectively, irrespective of the acyl chain length. The radical scavenging activities of L-ascorbic acid and the saturated acyl L-ascorbates against 1,1-diphenyl-2-picrylhydrazyl free radical were ca. 95% for all of the compounds, and the introduction of a saturated acyl group to the L-ascorbic acid did not affect the activity.     

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.     

Dissolved organic matter sorption on sub soils and minerals studied by 13C-NMR and DRIFT spectroscopy

Sorption on the mineral matrix is an important process restricting the movement of dissolved organic matter (DOM) in soils. In this study, we aimed to identify the chemical structures responsible for the retention of DOM by sorption experiments with total DOM and acidic humic substances (AHS), containing humic and fulvic acids, on soil samples and minerals (goethite, ferrihydrite, and amorphous Al(OH)₃). The AHS remaining in solution after sorption were studied by ¹³C nuclear magnetic resonance (NMR) analysis, and total DOM and AHS for bed on the surfaces of minerals by diffuse reflectance Fourier-transform infrared (DRIFT) spectroscopy. The soil samples were taken from strongly sorbing Bw horizons of two Inceptisols rich in pedogenetic Fe (29 and 35 g kg ^?1) and containing little C (7 and 22 g kg^?1). The ¹³C-NMR spectra showed that sorption causes a preferential removal of aromatic and carboxyl C from the solution, whereas alkyl-C accumulates in the solution. No change was observed for O-alkyl C. The DRIFT spectra of sorbed total DOM and AHS showed a relative increase of the band intensity of carboxyl groups compared to DOM in the initial solution, confirming the importance of those groups for the sorption to mineral surfaces. The spectra also indicated reactions of carboxyl groups with metals at the mineral surfaces. The extent to which the carboxyl groups are bound depended on the surface coverage with DOM and the type of mineral.     

The [13C]nuclear magnetic resonance spectrum of a methylated humic acid

A humic acid has been investigated by [¹³C]NMR spectroscopy using the methylated humic acid dissolved in chloroform which gave a relatively simple spectrum.Estimates of different types of C show that 21% of the C is aromatic, 35% consists of methylene chains and methyl groups, 3%, as the carbonyl C of methyl esters, and 13% as the methoxyl C of methylated phenols and alcohols. The rest, 28%, is not accounted for and may be mostly C bound to O as in polysaccharides or in peptides. No ketonic or quinonic groups were detected.The [¹³C]NMR spectrum of the methylated humic acid is entirely different from those of lignin and its derivatives thus suggesting that lignin residues play no significant role in the structure of the humic acid.     

Tryptophan-N-glucoside in fruits and fruit juices

In extracts prepared from various fruits as well as in fruit juices a single tryptophan glycoconjugate was detected by HPLC-MS analysis. Product ion spectra demonstrated the N-glycosidic linkage of a hexose moiety to the indole nitrogen. For structure elucidation, the novel tryptophan glycoside was isolated from pear juice and identified as N(1)-(beta-D-glucopyranosyl-(4)C(1))-L-tryptophan by (1)H, HH-COSY and (13)C NMR spectroscopy. Finally, we disclosed the biosynthetic origin of the novel tryptophan metabolite by demonstrating the enzymatic glycosylation of deuterium-labeled tryptophan, which was applied to pear fruit.     

Nondestructive observation of bovine milk by NMR spectroscopy: analysis of existing States of compounds and detection of new compounds

In this study were successfully observed the one- (1H, 13C) and two-dimensional (1H-13C, 1H-15N, 1H-31P) NMR spectra of milk directly without any pretreatment. The signals of each NMR spectrum were assigned, and their existing states were also analyzed. Lactose existed in a free state in milk. The signals due to the butyric acid chain can be assigned among the other fatty acid chains. Monounsaturated fatty acid (oleic acid chains) and polyunsaturated fatty acid chains (linoleic and linolenic acid) were assigned by their characteristic signals. The signals from citrate, N-acetylcarbohydrates, and lecithin could be observed directly in the 1H-13C HSQC NMR spectra; the assignment of their signals was made through the 1H-13C, 1H-15N, and 1H-31P HMBC spectra of extracted milk. Signals from creatine and N-acetylcarbohydrates were detected for the first time.     

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.     

Front-face fluorescence spectroscopy study of globular proteins in emulsions: influence of droplet flocculation

Measurement of the intensity (I(MAX)) and/or wavelength (lambda(MAX)) of the maximum in the tryptophan (TRP) emission spectrum using front-face fluorescence spectroscopy (FFFS) can be used to provide information about the molecular environment of proteins in nondiluted emulsions. Many protein-stabilized emulsions in the food industry are flocculated, and therefore, we examined the influence of droplet flocculation on FFFS. Stock oil-in-water emulsions stabilized by bovine serum albumin were prepared by high-pressure valve homogenization (30 wt % n-hexadecane, 0.35 wt % BSA, pH 7). These emulsions were used to create model systems with different degrees of droplet flocculation, either by changing the pH, adding surfactant, or adding xanthan. Emulsions (21 wt % n-hexadecane, 0.22 wt % BSA) with different pH (5 and 7) and molar ratios of Tween 20 to BSA (R = 0-131) were prepared by dilution of the stock emulsion. As the surfactant concentration was increased, the protein was displaced from the droplet surfaces, which caused an increase in both I(MAX) and lambda(MAX), because of the change in TRP environment. The dependence of I(MAX) and lambda(MAX) on surfactant concentration followed a similar pattern in emulsions that were initially flocculated (pH 5) and nonflocculated (pH 7). Relatively small changes in FFFS emission spectra were observed in emulsions (21 wt % n-hexadecane, 0.22 wt % BSA, pH 7) with different levels of depletion flocculation induced by adding xanthan. These results suggested that droplet flocculation did not have a major impact on FFFS. This study shows that FFFS is a powerful technique for nondestructively providing information about the molecular environment of proteins in concentrated and flocculated protein-stabilized emulsions. Nevertheless, in general the suitability of the technique may also depend on protein type and the nature of the physicochemical matrix surrounding the proteins.     

Molecular characterization around a glassy transition of starch using (1)H cross-relaxation nuclear magnetic resonance

The aim of this work was to characterize the glassy-rubbery transition in starch gels using molecular (NMR) techniques. Proton cross-relaxation ((1)H CR) NMR spectra of gelatinized starch ( approximately 50% mc) were obtained by cooling stepwise from 20 to -30 degrees C. A significant line broadening was observed in the CR spectra between 0 and -10 degrees C. Deconvolution of the spectra into its component curves (broad and narrow) yielded a peak amplitude, width at half-height, and peak area for each curve. Between 0 and -10 degrees C (temperatures around T(g)), a significant line width change in the broad component (rigid solid) was apparent. These observed qualitative changes may be evidence of a glassy-rubbery transition at a molecular (short-range) level which are strengthened by a similar transition temperature range found previously with (13)C CP-MAS and DMA tan delta(T) measurements. However, the increase in the relative quantity of rigid protons observed by (1)H CR NMR spectra could also be attributed to ice. The (1)H CR NMR method showed its potential application for probing solid components in gels using a simple and economical NMR spectrometer, without the need for a solid-state instrument.     

Characterization and esterification of hemicelluloses from rye straw

Hemicelluloses were extracted with 10% KOH/0.5% Na(2)B(4)O(7). 10H(2)O from delignified rye straw. Esterification of the hemicelluloses with various acyl chlorides was performed in a homogeneous N,N-dimethylformamide and lithium chloride system using 4-(dimethylamino)pyridine catalyst and triethylamine as a neutralizer. The degree of substitution was controlled between 0.37 and 1.65. Under an optimum condition (sample 14, molar ratio 3:1), >90% of the free hydroxyl groups in native hemicelluloses were stearoylated at 75 degrees C for 40 min. Meanwhile, the products were characterized by FT-IR and GPC techniques as well as their solubilities. The molecular mass measurements (31400-123300 g mol(-)(1)) showed only a minimal degradation of the macromolecular hemicelluloses during rapid reactions at 48-75 degrees C for 20-40 min.