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1.
The heat-induced aggregation of common buckwheat (Fagopyrum esculetum Moench) globulin (BWG) was studied using size-exclusion chromatography (SEC) combined with on-line multiangle laser light scattering (MALLS) and quasielastic light scattering (QELS). The unheated BWG was found to exist mainly as a hexamer, with an estimated weight-average molecular weight (M(w)) of 342 000, close to that deduced from the genomic cloned data of 13S buckwheat globulin. The QELS data predicted that the hexamer exists as two annular trimeric rings (diameter approximately 10.8 nm) placed on top of each other, forming an oblate cylinder (height approximately 9.1 nm). Upon heating, hexamers and trimers were dissociated and then associated to form extended small aggregates, finally forming compact, large macroaggregates. N-Ethylmaleimide would favor macroaggregate formation and increased the molar masses and hydrodynamic radii of the soluble aggregates, suggesting a different aggregation process in the presence of the sulfhydryl-blocking agent. A plot of log hydrodynamic radius versus log molar mass showed changes in the slope during heat treatment, suggesting conformational transformation in the heat-denatured and aggregated BWG molecules.  相似文献   

2.
Conjugation of the milk protein sodium caseinate and a protein-containing polysaccharide, gum arabic, was achieved through the use of the cross-linking enzyme transglutaminase. The extent of conjugation was monitored by size exclusion separation coupled with a multiangle laser light scattering detector. The elution times of gum arabic solutions incubated with transglutaminase were unchanged over time, whereas incubation of sodium caseinate with transglutaminase resulted in shorter elution times as reaction time increased, indicating the formation of cross-linked caseinate polymers. However, when mixtures of caseinate and gum arabic were incubated with transglutaminase, the elution times were decreased markedly, indicating conjugation between the protein and polysaccharide. The molecular masses of the conjugates increased from approximately 950 to 1600 kDa. This method of protein-polysaccharide conjugation offers noticeable advantages over previously used methods, and the conjugates produced may exhibit unique functional properties.  相似文献   

3.
Methylcellulose (MC) is ingested by humans in food and pharmaceutical formulations. The functional properties of MC like those of other linear polymers depend primarily on polymer length or molar mass for largely linear polymers. Although many studies in animals and humans have shown complete excretion of MC, in vitro human fecal fermentation studies indicate that MC can be degraded and presumably lose some of its functionality. In this study, MC polymer distribution in the feces from rats fed a diet containing 8% methylcellulose were compared to the fed MC. The water-soluble polymers in the feces were separated by a size exclusion chromatography (SEC) and the polymer distributions determined by multiple angle laser light scattering (MALLS). Detection of the fluorescent MC-calcofluor complex was used to confirm the identity of the eluting MC peak. All dietary MC was recovered in the feces. There is a small shift (P < 0.06) in the weight-averaged molecular weight of polymer distribution of MC extracted from the feces to 2.71 +/- 0.15 x 10(5) g/mol from 3.15 +/- 0.02 x 10(5) g/mol in the standard. There is also an increase in the polydispersity from 1.21 in the standard to 1.8 in the fecal extract. The distribution of the substituted methoxylated glucose monomers by gas chromatography also confirms the stability of MC fed to rats. The amount of actual hydrolysis is estimated to be about 0.1 glycosidic linkage/molecule. MC is not easily determined by standard dietary fiber methods, and SEC with MALLS and/or fluorescence may be a useful alternative.  相似文献   

4.
The role of disulfide bridges in the folding of Aspergillus niger phytase pH 2.5-optimum (PhyB) was investigated using dynamic light scattering (DLS). Guanidinium chloride (GuCl) at 1.0 M unfolded phytase; however, its removal by dialysis refolded the protein. The thiol reagent tris(2-carboxyethyl)phosphine (TCEP) reduces the refolding activity by 68%. The hydrodynamic radius (R(H)) of PhyB phytase decreased from 5.5 to 4.14 nm when the protein was subjected to 1.0 M GuCl concentration. The active homodimer, 183 kDa, was reduced to a 92 kDa monomer. The DLS data taken together with activity measurements could indicate whether refolding took place or not in PhyB phytase. The correlation between molecular mass and the state of unfolding and refolding is a very strong one in fungal phytase belonging to histidine acid phosphatase (HAP). Unlike PhyA phytase, for which sodium chloride treatment boosted the activity at 0.5 M salt concentration, PhyB phytase activity was severely inhibited under identical condition. Thus, PhyA and PhyB phytases are structurally very different, and their chemical environment in the active site and substrate-binding domain may be different to elicit such an opposite reaction to monovalent cations.  相似文献   

5.
"Mapuey" tubers in Venezuela are staple food for indigenous peoples from the Caribbean coast and Amazon regions. Noticeable differences between genotypes of yam starches were observed. Granules were large, triangular, or shell-shaped with monomodal particle size distribution between 24.5 and 35.5 μm. Differential scanning calorimetry (DSC) analyses revealed onset gelatinization temperatures from 69.1 to 73.4 °C with high gelatinization enthalpy changes from 22.4 to 25.3 J g(-1). All X-ray diffractograms of starches exhibit B-type crystallinity. Crystallinity degrees varied from 24% to 40%. The highest crystallinity was found for the genotype having the highest amylose content. Iodo-colorimetric, amperometric, and DSC amylose determinations varied from 1.4 to 8.7%, 2.2 to 5.9%, and 1.4 to 3.5% for Amazonian genotypes, in comparison with commercial Mapuey starches: 12.0, 9.5, and 8.7%, respectively. Solubility and swelling power at 90 °C varied from 2.1 to 4.4% and 20.5 to 37.0%, respectively. Gel clarity fluctuated from 22.4 to 79.2%, and high rapid visco analyzer (RVA) viscosity was developed at 5% starch suspension (between 1430 and 2250 cP). Amylopectin weight average molar mass M(w), radius of gyration R(G), hydrodynamic coefficient ν(G), and apparent molecular density d(Gapp) were determined using high-performance size exclusion chromatography (HPSEC) and asymmetrical flow field flow fractionation (A4F) techniques coupled with multiangle laser light scattering (MALLS) on the Dioscorea trifida genotypes exhibiting the lowest and highest amylose contents. Amylopectins showed very similar molecular conformations. M(w) values were 1.15 × 10(8) and 9.06 × 10(7) g mol(-1) using HPSEC and A4F, respectively, thus, 3-5 times lower than those reported with the same techniques for other yam species, and very close to those of potato and cassava amylopectins. This discovery of a new natural amylose-free starch in the neglected yam "Mapuey" could present some potential for the food industry.  相似文献   

6.
Proteolytic digestion of dried bonito muscle with thermolysin produces a hydrolysate with strong angiotensin-converting enzyme (ACE) inhibitory activity and is the basis of a dietary supplement with antihypertensive activity. A major portion of the ACE activity was shown previously to arise from the peptide Leu-Lys-Pro-Asn-Met (LKPNM). A straightforward method to quantify this peptide was developed using one-step C18 solid-phase extraction (SPE) followed by LC-MS/MS quantification. The SPE step resulted in a hydrolysate that was still crude, as illustrated by combined size-exclusion chromatography/multi-angle laser light scattering detection that showed that a major fraction of oligopeptides were in the 2-20 kDa range. This fraction has a weight-average molecular weight (M(w)) of approximately 5.0 kDa. Method validation for specificity, linearity, accuracy, precision, and reproducibility showed that standard additions of synthetic LKPNM to bonito extract with SPE enrichment followed by LC-MS/MS is a suitably robust procedure for the determination of LKPNM content. The method was also successful for encapsulated powders in which the excipients used are insoluble in water and could be removed by centrifugation.  相似文献   

7.
Dimethyl sulfoxide (DMSO), with either 50 mM LiBr, 10% water, or both, was used as solvent for multi-angle laser-light scattering (MALLS) batch mode analysis of rice starch, and amylopectin and amylose weight-average molecular weight (Mw). DMSO/50 mM LiBr was a better solvent for these measurements than was DMSO/10% water, based on this solvent's ability to dissolve starch and to reduce the size of starch aggregates. Starch concentration decreased and amylose:amylopectin ratio increased when starch suspended in DMSO was centrifuged or filtered prior to size-exclusion chromatography (SEC)-MALLS analysis. A higher amylose:amylopectin ratio made starch more soluble, and the higher this ratio, the lower the Mw of eluted amylopectin. For SEC analysis of Mw, fractions of starch amylopectin and amylose dispersed in DMSO-based solvents yielded better results than starch dispersed directly into the solvents, because dispersion of these fractions decreased starch aggregation. When these two starch components were fractionated and then dissolved separately in DMSO/50 mM LiBr, the Mw of dispersed amylopectin ranged from 40 to 50 million, and that of amylose was ca. 3 million, whereas starch from three rice varieties of varying amylose content ranged from 60 to 130 million. We recommend that SEC evaluation of amylopectin and amylose be accomplished with fractionated samples as in this study; such evaluations were superior to evaluations of natural mixtures of amylopectin and amylose.  相似文献   

8.
The structural characteristics of the gum exudate of Acacia senegal (gum arabic) have been investigated by monitoring the composition and physicochemical properties before and after treatment with proteolytic enzyme and various alkaline systems. Molecular mass ( M w) and radius of gyration ( R g) measurements were performed using gel permeation chromatography (GPC) coupled to refractive index, UV absorbance, and multiangle light scattering detectors and indicated that the macromolecules present have a compact structure. It was found that treatment with proteolytic enzyme caused the arabinogalactan-protein component (AGP) with average molecular mass approximately 2 x 10 (6) Da to degrade, yielding material of molecular mass approximately 4 x 10 (5) Da, whereas the bulk of the material corresponding to the protein-deficient arabinogalactan component (AG) with molecular mass 4 x 10 (5) remained unaffected. Barium hydroxide was found to hydrolyze the polysaccharide component (AG) itself in addition to the proteinaceous component as demonstrated in control experiments using dextran. However, sodium borohydride/sodium hydroxide treatments were unable to hydrolyze dextran and were assumed to hydrolyze only the proteinaceous component of gum arabic. The AGP component was completely degraded, yielding material of molecular mass approximately 4.5 x 10 (4) Da. It has been concluded, therefore, that the enzyme did not fully hydrolyze all of the protein present and that the AGP component of gum arabic consists of carbohydrate blocks of approximately 4.5 x 10 (4) Da linked to a polypeptide chain consistent with the wattle blossom structure. Because the AGP was degraded to differing extents using a mild and more severe sodium borohydride/sodium hydroxide treatment, it was concluded that the polysaccharide moieties were linked through both O-serine and O-hydroxyproline residues. The gum arabic sample was deglycosylated by treatment with anhydrous hydrogen fluoride and revealed the presence of two putative core proteins of approximately 3 x 10 (4) and approximately 5 x 10 (3) Da, respectively, which correspond to proteins of approximately 250 and 45 amino acids in length. A new model for the structure of the AGP component has been proposed.  相似文献   

9.
Our research on several proteins indicates that accurate molecular weights cannot be determined by Superose 12 column chromatography. In support of this statement, we present data on molecular weights of purified red kidney bean alpha-amylase inhibitor (RKB alphaAI) and white kidney bean alpha-amylase inhibitor (WKB alphaAI) to document this problem. The molecular weight of purified RKB alphaAI determined by Sephadex G-100 gel filtration, polyacrylamide gel electrophoresis, Superose 12 gel filtration and cDNA were 49.0, 51.0, 22.9, and 49.805 kDa (not glycosylated), respectively. The molecular weights of WKB alphaAI by several methods were as follows: Sephadex G-100 gel filtration, 51.0 kDa; Superose 12 gel filtration in 0.2 M NaCl buffer, 23.1 kDa; polyacrylamide gel electrophoresis (PAGE), 51.0 kDa; sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), 45.0 kDa; multiangle laser light scattering (MALLS), 49.940 kDa; laser-assisted time-of-flight mass spectrometry (LATOFMS), 56.714 kDa; and cDNA sequence (with 12.2% carbohydrate), 55.9 kDa. The data indicate there is ionic interaction between proteins and the matrix of Superose 12 in low ionic strength buffers and hydrophobic interaction at higher ionic strength buffers. Researchers should be cautious when using Superose 12 columns for molecular weight determinations.  相似文献   

10.
A water-soluble neutral polysaccharide (AF1) was extracted from Auricularia (A.) auricula-judae with 0.15 M aqueous NaCl at 80-100 °C. Its chemical components and structure were analyzed by GC, GC-MS, and NMR. AF1 was identified as a β-(1→3)-D-glucan with two β-(1→6)-D-glucosyl residues for every three main chain glucose residues, showing a comb-branched structure. The M(w) values of AF1 in both aqueous solution and DMSO determined by LLS and SEC-LLS were in the narrow range of 2.07-2.15 × 10(6), indicating AF1 existed as single chains in the two solvents. The high intrinsic viscosity [η] of 1753 mL/g and the structure-sensitive parameter ρ (≡R(g)/R(h)) value of 2.3 in water revealed that AF1 existed as stiff chain conformation. Moreover, we directly observed the extended stiff chain conformation by AFM. The branching structure led to the water solubility of AF1, and the intramolecular hydrogen bonds sustained the stiff chain conformation. The rheological results showed that this polysaccharide aqueous solution had higher viscosity than even xanthan, a pronounced thickening agent. This work provided important information for developing new thickeners in food fields, and how neutral polysaccharides can be used as good candidates.  相似文献   

11.
The lignin component found in both water insoluble (WI) and water and alkali insoluble (WIA) fractions derived from SO(2)-impregnated steam-exploded eucalyptus chips (SEE) was isolated and characterized. Dioxane lignins with a sugar content lower than 2% (w/w) were obtained after each material was treated with commercial cellulases. The C9 formulas of both SEE-WI and SEE-WIA dioxane lignins were C(9)H(6.83)N(0.04)O(2.24)(OCH(3))(1.21)(OH(aro))(0.56)(OH(ali))(0. 77) and C(9)H(8.65)N(0.29)O(1.97)(OCH(3))(0.90)(OH(aro))(0. 46)(OH(ali))(1.02), respectively. The weight-average molecular weight (M(w)) of the SEE-WI lignin corresponded to 3.85 kDa, whereas the SEE-WIA lignin had an M(w) of 3.66 kDa for the same polydispersity of 2.4. The SEE-WIA lignin was shown to be more thermally stable than the SEE-WI lignin, requiring temperatures in the range of 520 degrees C for complete degradation. FTIR and (1)H NMR analyses of both untreated and peracetylated lignin fractions showed that (a) the alkali insoluble lignin contained a relatively higher degree of substitution in aromatic rings per C9 unit and that (b) alkaline extraction removed lignin fragments containing appreciable amounts of phenolic hydroxyl groups.  相似文献   

12.
Study of thermal aggregation of oat globulin by laser light scattering   总被引：1，自引：0，他引：1  
The heat-induced aggregation of oat globulin was studied using size-exclusion chromatography (SEC) combined with on-line multiangle laser light scattering (MALLS) and quasi-elastic light scattering (QELS). The unheated oat globulin exists as a mixture of hexamer (>95%), trimer, and dimer forms of hexamer. The molecular weight of the hexamer was estimated by MALLS to be 330 000, close to that deduced from the genomic cloned data of the acidic and basic polypeptides of oat globulin. From QELS measurements, it can be predicted that the hexamer exists as two annular trimeric rings, with a diameter of 11.8 nm, placed on top of each other, forming an oblate cylinder with a height of about 8.5 nm. Upon heating at 100 degrees C, the oat globulin hexamers and trimers were dissociated into monomers. The heat-denatured monomers, probably assuming an extended structure, were associated to form small aggregates, which were further aggregated to high molecular weight complexes. Upon further heating (60 min), the soluble aggregates were associated to form insoluble aggregates. Aggregation of oat globulin occurred at a much faster rate at 110 degrees C. The results indicate that the SEC-MALLS-QELS system is suitable for studying thermal aggregation of food proteins.  相似文献   

13.
Meat and bone meal (MBM) is a high protein agricultural commodity that currently has few applications other than as an animal feed. Unmodified MBM has poor functional properties, due to its low solubility. Our results from pilot plant trials demonstrate that MBM can be extrusion-processed along with sodium caseinate to produce a useful plastic material. We developed this material for use as a dog chew toy. For this application, elastic modulus (stiffness) is a key characteristic. Our results detail the relationship between ambient relative humidity and equilibrium moisture content (MC) in the material. The influence of MC on the glass transition temperature and elastic modulus reflects the plasticization of this material by water. On the basis of a comparison to a commercially available dog chew, the range of stiffness achievable with our material, 0.25-2.50 GPa, encompasses the values appropriate for a dog chew. Our results show that a particular desired stiffness can be maintained by applying an edible moisture barrier to the surface of the material.  相似文献   

14.
The weight average molar mass (Mw) and root mean square radii of starches from waxy maize (Amioca), waxy rice flour, cassava, Hylon V, Hylon VII, and potato amylose were determined by size-exclusion chromatography (SEC) and multiple-angle laser light scattering (MALLS). Dimethylsulfoxide (DMSO) containing 50 mM LiBr was used to dissolve the starches and also served as the mobile phase. SEC with large particle size polystyrene divinylbenzene packing materials and MALLS instrumentation were evaluated for the ability to separate and determine molar mass (MM) of starch polymers, respectively. The determination of Mw by MALLS is necessary because the Mw of many cereal starches exceeds the available molecular standards by one or two orders of magnitude. The Mw depends on the method of calculation. The Mw (Berry method) of starch from waxy corn was 2.27 × 108 Da, waxy rice 8.9 × 107 Da, cassava 5.7 × 107 Da, Hylon V 2.7 × 107 Da, Hylon VII 4.8 × 106 Da, and potato amylose 1.9 × 105 Da. Recovery dropped dramatically for molecules with root mean square radii >200 nm.  相似文献   

15.
The suitability of a one-step derivatization procedure using N-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide for the simultaneous assay of 22 free amino acids and its application for their analysis in six animal source foods (pork, dry cured ham, chicken stock, fresh cheese, ripened cheese, and dry salted sardine) by GC-MS were studied. All 22 free amino acid derivatives were correctly detected and resolved. Reproducibility (%RSD) of the method was in the range of 1.9-12.2%. Detection and quantitation limits of the analytical procedure ranged from 0.01 to 0.46 mg/100 g dry weight and from 0.02 to 1.55 mg/100 g dry weight, respectively. The calibration curves were linear within the range 0.1-15.0 mg/100 g with correlation coefficient values (R(2)) from 0.9891 to 0.9983. All analyzed food products showed free amino acid contents similar to those found in the scientific literature. The proposed GC-MS method for the determination of free amino acids in animal source food can be used in routine for both analytical and research purposes.  相似文献   

16.
The effects of heat at temperatures in the range of 80-90 degrees C on mixtures of reconstituted skim milk powder (RSMP) and sodium caseinate have been determined. In the absence of caseinate, the action of heat on RSMP produces soluble complexes of whey proteins and kappa-casein, as well as complexes of whey protein with the casein micelles. When sodium caseinate was added to RSMP at levels of 0.5 and 1.0%, the denaturation of the whey protein and the production of the soluble complexes in the serum were hardly affected, either in rate or in amount. However, during the heating, the caseinate disappeared from the serum. Further studies on model mixtures of the different components showed that it was probable that the bulk of the caseinate associated with the casein micelles during heating, probably by binding inside the surface layer of kappa-casein, because no increase in the diameters of the casein micelles could be observed.  相似文献   

17.
Cyclopia genistoides, normally used for the preparation of an herbal tea, honeybush, is a good source of the bio-active compounds mangiferin and hesperidin and is in demand for the preparation of xanthone-enriched extracts. Near-infrared spectroscopy (NIRS) was used to develop calibration models to predict the mangiferin and hesperidin contents of the dried green plant material. NIRS measurements of plant material and pure compounds were performed in diffuse reflectance mode. The calibration sets for mangiferin and hesperidin contents ranged from 0.7 to 7.21 and 0.64-4.80 g/100 g, respectively. Using independent validation, it was shown that the NIRS calibration models for the prediction of mangiferin (SEP=0.46 g/100 g; R2=0.74; and RPD=1.96) and hesperidin (SEP=0.38 g/100 g; R2=0.72; and RDP=1.90) contents of the dried plant material are adequate for screening purposes, based on RPD values.  相似文献   

18.
Aminocyclopyrachlor sorption/desorption was investigated in 14 soils from Brazil, representing a range of pH, and organic carbon (OC) and clay contents. The Freundlich equation adequately described behavior of aminocyclopyrachlor in soil. Freundlich sorption coefficient (K(f)) values ranged from 0.06 to 1.64 and 1/n values for ranged from 0.9 to 1.0. Sorption was correlated to OC (K(f,oc) ranged from 11 to 64) and clay contents. The lowest sorption was found for soils with very low OC contents (0.50-0.65%) and loamy-sand to sand textures. The 1/n values for desorption were lower than those observed for sorption, suggesting that aminocyclopyrachlor sorption by soil was not reversible; hysteresis coefficients ranged from 0.13 to 0.74. The results suggest that although aminocyclopyrachlor would be very mobile based on its sorption coefficients, its potential depth of leaching may be overestimated due to the hysteretic desorption.  相似文献   

19.
Hydrogen cyanide (HCN) is a toxic chemical that can potentially cause mild to severe reactions in animals when grazing forage sorghum. Developing technologies to monitor the level of HCN in the growing crop would benefit graziers, so that they can move cattle into paddocks with acceptable levels of HCN. In this study, we developed near-infrared spectroscopy (NIRS) calibrations to estimate HCN in forage sorghum and hay. The full spectral NIRS range (400-2498 nm) was used as well as specific spectral ranges within the full spectral range, i.e., visible (400-750 nm), shortwave (800-1100 nm) and near-infrared (NIR) (1100-2498 nm). Using the full spectrum approach and partial least-squares (PLS), the calibration produced a coefficient of determination (R(2)) = 0.838 and standard error of cross-validation (SECV) = 0.040%, while the validation set had a R(2) = 0.824 with a low standard error of prediction (SEP = 0.047%). When using a multiple linear regression (MLR) approach, the best model (NIR spectra) produced a R(2) = 0.847 and standard error of calibration (SEC) = 0.050% and a R(2) = 0.829 and SEP = 0.057% for the validation set. The MLR models built from these spectral regions all used nine wavelengths. Two specific wavelengths 2034 and 2458 nm were of interest, with the former associated with C═O carbonyl stretch and the latter associated with C-N-C stretching. The most accurate PLS and MLR models produced a ratio of standard error of prediction to standard deviation of 3.4 and 3.0, respectively, suggesting that the calibrations could be used for screening breeding material. The results indicated that it should be feasible to develop calibrations using PLS or MLR models for a number of users, including breeding programs to screen for genotypes with low HCN, as well as graziers to monitor crop status to help with grazing efficiency.  相似文献   

20.
A protocol has been developed to fractionate sugar beet pectin using hydrophobic affinity chromatography. Three samples eluted from the column using 4 M NaCl as solvent (fractions 1A, 1B, and 1C), two fractions eluted using 2 M NaCl (fractions 2A and 2B), and one fraction eluted using water (fraction 3). The fractions were shown to be very polydisperse, and differences between the GPC refractive index and UV absorbance (214 nm) elution profiles demonstrated chemical heterogeneity. They were found to contain significantly different proportions of protein (1A, 2.79%; 1B, 0.97%; 1C, 0.77%; 2A, 1.41%; 2B, 5.09%; and 3, 5.89%) and ferulic acid (approximately 1A, 0.5%; 1B, 0.5%; 1C, 0.9%; 2B, 1.5%; and 3, 2%). The weight-average molecular mass, M(w), of the fractions also varied (1A, 153 kDa; 1B, 155 kDa; 1C, 306 kDa; 2A, 562 kDa; 2B, 470 kDa; 3, 282 kDa). Three fractions, that is, 1A, 1B, and 3, produced orange oil emulsions with a relatively small droplet size that were stable over a period of weeks. The other three fractions (1C, 2A, and 2B with higher M(w) values) produced emulsions with an initially larger droplet size, and the droplet size increased considerably over time. The increased droplet size may be influenced by the viscosity of the aqueous continuous phase. There was no simple relationship between protein or ferulic acid content and emulsification ability. For example, fraction 1B, which contained the lowest proportion of both protein and ferulic acid, produced stable emulsions of similar droplet size to fraction 3, which contained the largest proportion of protein and ferulic acid. The role of protein in the emulsification process was investigated by measuring the amount of protein in the aqueous phase before and after emulsification. It was clearly demonstrated that proteinaceous material adsorbed at the oil-water interface. It is evident that the emulsification properties of sugar beet pectin are influenced by the accessibility of the protein and ferulic acid groups to the surface of the oil droplets, the proportion of ester groups, and the molecular mass distribution of the fractions.  相似文献   

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