Two-Dimensional Nuclear Magnetic Resonance Studies of Starch and Starch Products

The use of two-dimensional Nuclear Magnetic Resonance (NMR) spectroscopy as applied to studies of starch, starch products and glycogen is reported. The use of both homonuclear correlation (COSY, relay-COSY and HOHAHA) and heteronuclear correlation (HMQC, HETCOR) experiments is discussed. This approach makes it possible to obtain complete assignments of the proton NMR spectra of these polysaccharides. This is not possible from 1-D spectra due to excessive overlap of the non-anomeric proton signals. The resulting assignments are useful in obtaining structural information regarding starch and related products. Moreover, the greater inherent resolution of the two-dimensional spectra can reveal the presence of low molecular weight carbohydrates (glucose, maltose etc.) in dextrin samples, thus providing information about carbohydrate composition.     

Carbon-13 and proton NMR studies of post-mortem metabolism in bovine muscles

Proton and carbon-13 NMR was used to investigate post-mortem metabolism in bovine muscles at 26°C during the first 10h after slaughter. WALTZ-16 decoupling was used to eliminate the proton couplings in the (13)C spectra and the 'jump and return' pulse sequence was used to suppress the water resonance in the (1)H-NMR experiments. With carbon-13 NMR the glycogen breakdown and the lactate development could be followed. This was compared with the lactate, creatine and phosphocreatine development as measured by proton NMR. The intracellular pH was estimated from the chemical shift of the abundant dipeptide, carnosine, as measured in the (1)H- and (13)C-NMR spectra. These were compared with similar measurements obtained earlier using phosphorus-31 NMR. The three independently determined pH profiles were in excellent agreement with one another, as well as with results obtained with the standard iodoacetate extraction method. In the course of these studies we observed that the post-mortem metabolism in cow and heifer was slow and that it took four more hours to complete compared to bull or young bull. After 10 h the pH was 5·9 in bull and 6·1 in cow. Phosphocreatine had completely disappeared after 3·5 h in bull samples while the lactate continued to increase even after 10h. The curves obtained by carbon-13 and proton NMR for the increase in lactate during the first 10 h post mortem were very similar. Moreover, plots for the increase in the lactate level versus the intracellular pH decrease showed a linear relationship, indicating that anaerobic glycolytic activity is the main determining cause for the intracellular pH decrease. Various other parameters, such as the ratio of unsatirated to saturated fatty acid side chains and the presence of amino acids and taurine, could be measured from the in vivo carbon-13 NMR spectra. However, no gross changes occured in any of these parameters during the first 10 h post mortem.     

Molecular Mobilities of Instant Starch Gels Determined by Oxygen-17 and Carbon-13 Nuclear Magnetic Resonance

The molecular mobility of both the water component and the starch component of three instant starches was determined by proton decoupled oxygen-17 (¹⁷O) and proton decoupled carbon-13 (¹³C) Nuclear Magnetic Resonance (NMR) spectroscopy. The effect of starch concentration and various storage conditions were studied. The molecular mobility of the water associated with each of the instant starches over all concentrations studied as measured by ¹⁷O NMR transverse relaxation rates was not much slower than that of free or bulk water. The effect of storage conditions on the molecular mobility and conformation of the starches, as monitored by proton decoupled ¹⁷O and ¹³C NMR, was in most cases small, despite the observed differences in retrogradation among the starches.     

Structural Studies of Pullulan by Nuclear Magnetic Resonance Spectroscopy

Nuclear Magnetic Resonance (NMR) spectroscopy was used to probe the primary structure of the glucan pullulan. Carbon-13, proton and phosphorus-31 NMR spectra in aqueous solution of the intact poly-saccharide obtained from three different sources indicate that significant structural differences can occur among these samples. In particular, two samples consist solely of α(1 → 6) linked maltotriose units, in accord with the generally accepted structure. However a third sample has fewer α(1 → 6) linkages and appears to consist of higher α(1 → 6) linked maltooligosaccharides in addition to maltotriose. Unlike the other samples, this sample was also phosphorylated. Enzymatic digestion of pullulan followed by NMR spectroscopy using pullulanase confirmed these results, as did HPLC analysis of the resulting digest, which showed the presence of maltooligosaccharides consisting of ten and more glucose units. However, HPLC analysis of acid hydrolysed pullulan indicated that no sugars other than glucose were present in any of the samples examined.     

Starch Functionalized by Chloroacetate Groups: Coupling of Bioactive Salicylic Acid

Starch partially functionalized with chloroacetate groups was obtained by reaction of starch with chloroacetyl chloride using the N,N‐dimethylacetamide/5%lithium chloride system as solvent and pyridine as catalyst. The coupling of salicylic acid to starch functionalized with chloroacetate groups was carried out by reaction with the sodium salt of salicylic acid. The structures of chloroacetylated starch and starch‐salicylic acid adducts were determined by means of FTIR, ¹H‐NMR and ¹³C‐NMR spectra. The degree of substitution per anhydroglucose unit (AGU) of chloroacetylated starch was calculated from the chloride content and ranged from 0.52 to 2.34, depending on the ratio of chloroacetyl chloride to starch. The hydrolysis of starch‐salicylic acid adducts in a heterogeneous phase showed that the release of sodium salicylate from tablets is dependent on the hydrophilic character of the adduct and on the pH value of the medium.     

Starch Phosphate Hydrogels. Part I: Synthesis by Mono‐phosphorylation and Cross‐linking of Starch

Starch‐based hydrogels were synthesized by cross‐linking monostarch monophosphates (MSMP) with different di‐ and tricarboxylic acids such as succinic, adipic or citric acid. Phosphorylation of starch was performed in a semi‐dry process using a mixture of primary and secondary sodium phosphates. The phosphorus content and degree of substitution of phosphate groups (DS_P) of the resulting MSMP were determined using a photometric method. For structural characterization of starch phosphates ³¹P NMR was used. Investigations showed that MSMP hydrogels were strong water absorbing polymer networks with a free swelling capacity (FSC) of up to 185 g water / g dry hydrogel.     

New Starch Phosphate Carbamides of High Swelling Ability: Synthesis and Characterization

The paper describes the synthesis of starch phosphate carbamides by reacting starch with phosphoric acid and urea. A solid state technique in vacuum at elevated temperatures is used. The degree of substitution of phosphate (DS_P) and carbamide groups (DS_C) can be adjusted by the molar ratio of starch: phosphoric acid: urea, the reaction temperature, and time. The starch derivatives prepared show remarkable swelling if the DS_P ranges between 0.2 and 0.3. With increasing content of urea in the reaction mixture the water‐holding ability is still significantly improved, which is explained by chemically and especially physically introduced urea into the starch polymer. Furthermore, the ratio of amylose: amylopectin of the starch samples influences both the DS values and the physicochemical properties, e.g., swelling power and multivalent metal ion adsorption of the products. The structure of the new polymers was determined by means of FTIR, ¹³C‐ and ³¹P NMR spectroscopy as well as by gel permeation chromatography (GPC).     

Physico‐chemical Characterization of Floridean Starch of Red Algae

The current work reports on isolation and physico‐chemical characterization of floridean starch from three species of agarophytic macro red algae. As determined by ¹H‐NMR spectroscopy, the average chain length and degree of branching frequency of this starch were 18 and 4.8, respectively. According to its amylopectin chain length distribution obtained by Dionex analysis, the crystalline polymorph of floridean starch from the red alga Gracilariopsis lemaneiformis was deduced to be C‐type and this was further supported from its X‐ray crystallographic pattern. Enzymatic analysis of its glucose 6‐phosphate content showed that floridean starch had a low level of covalently linked phosphate (1 nmol per milligram starch) and this was further confirmed by ³¹P‐NMR. The absorbance peak of floridean starch with iodine occurred at 527—530 nm and the blue value was low (0.1), indicating the absence of amylose, which was confirmed by differential scanning calorimetry (DSC). Floridean starch exhibited low gelatinization temperature, low viscosity, high clarity and little or no retrogradation upon repetitive freeze‐thaw cycles, as studied by DSC and rapid viscosity analysis (RVA). These results are discussed in light of the functional properties and the structure of floridean starch.     

Starch Derivatives of High Degree of Functionalization. 4. Homogeneous Tritylation of Starch and Subsequent Carboxymethylation

The synthesis of monomethoxytriphenylmethyl (MMtrityl) starch and the subsequent carboxymethylation of the 6‐O‐functionalized products were investigated. The trityIation both in N,N‐dimethylacetamide (DMA)/LiCl and in dimethyl sulfoxide (DMSO) occurred homogeneously. The highest degree of substitution of trityl groups (DS_TrityI) obtained after a single conversion step was 0.77 in both solvents. A complete functionalization of primary OH‐groups was achieved only with unsubstituted triphenylmethyl chloride in these reaction media. In case of monomethoxytriphenylmethyl chloride (MMTMC) as reagent an additional conversion step is necessary to synthesize products with a DS_TrityI = 1. The structure of the products was analyzed by FTIR‐ and ¹³C‐NMR spectroscopy. Subsequent carboxymethylation of the MMtrityl starch samples leads to products with a preferred functionalization of the unprotected secondary OH‐groups. After removal of the trityl moieties, the DS_CM and the distribution of carboxymethyl groups within the anhydroglucose unit was investigated by means of HPLC and ¹H‐NMR spectroscopy. The carboxymethylation was more effective at O‐2 than at O‐3. In case of ether products with DS_Trityl < 1 a partial substitution of the primary OH group took place as well.     

Effect of acetylation on the properties of corn starch

Acetylated corn starches with different degrees of substitution (DS 0.85, DS 1.78, DS 2.89) were synthesized by the reaction of corn starch with acetic anhydride in the presence of acetic acid under varying reaction temperatures. The product was characterized by FTIR spectroscopy, ¹H NMR, X-ray diffraction and contact angle measurement. Acid-base titration and ¹H NMR methods were employed to determine the degree of substitution of product. FTIR spectroscopic analysis showed that the characteristic absorption intensities of esterified starch increased with increase in the degree of substitution, and the characterized peak of hydroxyl group almost disappeared in the spectrum of DS 2.89 acetylated starch. The detailed chemical microstructure of native starch and acetylated starch was confirmed by ¹H NMR, ¹³C NMR and ¹³C–¹H COSY spectra. Analysis of ¹H NMR spectra of acetylated starches was assigned accurately. Strong peaks in X-ray diffraction of acetylated starch revealed that new crystalline regions were formed. Compared with native starch, the hydrophobic performance of acetylated starch esters was increased. The contact angle of acetylated starch with DS 2.89 was 68.2°.     

A Flow Injection System for the Determination of Starch in Starch from Different Origins with Immobilized α-Amylase and Amyloglucosidase Reactors

A heat stable α-amylase (Termamyl) was immobilized on controlled pore glass (CPG) with a pore size of 1 489Å and amyloglucosidase (AMG) was immobilized on a ceramic silica support (Micropil A) with a pore size of 300Å. These enzyme supports were packed into two separate immobilized enzyme reactors (IMERs) which together with a third reactor containing co-immobilized glucose dehydrogenase/mutarotase were incorporated into a flow injection (FI) system for the determination of the total glucose content of starch related poly- and oligosaccharides. Samples of maltose, a maltooligosaccharide mixture, three soluble starches, three amylopectins, two amyloses, glycogen, and native starches from different origins were injected. The degree of hydrolysis was determined by comparing the produced amount of glucose in the FI system with the calculated amount of glucose in the samples and with samples to which had been added and let to react for six hours soluble α-amylase and AMG before injected into the FI system also containing the IMERs. Virtually complete conversion to glucose was obtained for maltose, maltooligosaccharides, two soluble starches and native potato starch. Maximum enzymatic degradation by the starch hydrolyzing enzyme reactors was obtained in most instances except for glycogen (96%), native wheat (88%), rice (93%), and corn starch (83%).     

The structure of C-type Rhizoma Dioscorea starch granule revealed by acid hydrolysis method

Scanning electron microscope (SEM), X-ray powder diffraction (XRD) and cross polarisation/magic-angle spinning (CP/MAS) ¹³C nuclear magic resonance (NMR) have been used for the structural characterisation of C-type starch granule during acid hydrolysis. SEM shows that the amorphous areas mainly locate the core part of C-type starch granules, while the crystalline areas mainly exist in the peripheral region of starch granules. XRD analysis reveals that the B-type polymorph present in the C-type starch granule are preferentially degraded or degraded faster than the A-type polymorph. NMR spectra confirm that the amorphous regions in the starch granules are firstly hydrolysed and could be hydrolysed completely as long as the hydrolysis time is sufficient. After 40 days of hydrolysis, the acid-modified starch shows typical A-type characteristics upon analysis of the XRD pattern or the ¹³C CP/MAS NMR spectra.     

Synthesis of Higher Fatty Acid Starch Esters using Vinyl Laurate and Stearate as Reactants

This paper describes the synthesis of long‐chain fatty esters of corn starch (starch laurate and starch stearate) with a broad range in degree of substitution (DS = 0.24−2.96). The fatty esters were prepared by reacting the starch with vinyl laurate or vinyl stearate in the presence of basic catalysts (Na₂HPO₄, K₂CO₃, and Na acetate) in DMSO at 110°C. The yellowish products were characterized by ¹H‐, ¹³C‐NMR and FT‐IR. The DS of the products is a function of the carbon number of the fatty acid chain, vinyl ester to starch ratio and the type of catalyst. When performing the reactions using Na₂HPO₄ as the catalyst, the DS for the starch laurate compounds is higher than for the corresponding starch stearates. For low vinyl ester to starch ratios, an increase in the vinyl ester concentration leads to higher product DS values. At higher ratios, the DS decreases, presumably due to a reduction of the polarity of the reaction medium. K₂CO₃ and Na acetate are superior catalysts with respect to activity compared to Na₂HPO₄ and products with DS values close to 3 were obtained.     

Nuclear Magnetic Resonance Studies of Homopolysaccharides Related to Starch

One- and two-dimensional ¹H and ¹³C NMR spectra were obtained for a variety of homopolysaccharides, including the glucans dextran and pullulan, and the fructans levan and inulin. The NMR results provided information about the primary structure of these polysaccharides. Our results are in agreement with those determined previously by methylation analysis and other classical methods. The number of α(1 → 44) and α(1 → 6) linkages could be quantitated for pullulan. The presence of a single nonreducing glucose residue in the polyfructose inulin could also be demonstrated. In the case of the branched polysaccharides dextran and levan, it was possible to determine the degree of branching. In addition for lower molecular weight polysaccharides such as inulin and partially hydrolyzed dextran, it was possible to detect signals arising from reducing and non-reducing end groups. Their approximate molecular weight could be determined by quantitation of these signals.     

Studies on Chemical Modifications in Heat-processed Starch and Wheat Flour

1,6-Anhydro-β-D -glucopyranose (levoglucosan) and oligosaccharides with these end units were present in 80% aqueous ethanol extracts from heat-processed starch and wheat flour. Evidence was adduced that the 1,6-anhydro-D -glucose units react further with starch and starch fragmentation products by external trans-glycosidation reactions to give branched structures, resistant to amylolytic enzymes. High performance liquid chromatography (HPLC), 13-carbon nuclear magnetic resonance (¹³C-NMR) and gas-liquid chromatography (GLC) were invaluable tools for fractionation, identification and quantification of 1,6-anhydrosaccharides, respectively. The analytical dietary fibre value comprising non-starch polysaccharides and Klason lignin, increased with the extent of oven-heating and extrusion cooking of wheat flour. This increase seemed to originate from mainly three types of reactions, namely: (1) chemical modification of starch to enzyme-resistant glucan structures by external transglycosidation, (2) polymers formed by the Maillard reaction and analysed as Klason lignin, and (3) strong retrogradation of part of the starch (“resistant starch”). Another effect was that the water soluble part of the arabinoxylan in wheat flour increased.     

Evaluation of the Degree of Starch Gelatinization by a New Enzymatic Method

In this work, a new enzymatic method is proposed to evaluate the degree of starch gelatinization in starchy food and feed. The procedure developed is based on the fact that the gelatinization process enhances the chemical reactivity of inert starch granules towards amylolytic enzymes. Aqueous suspensions of maize starch were treated at different temperatures to obtain different degrees of gelatinization, from 25 °C (control without gelatinization) until 95 °C. Heated samples were then incubated with a glucoamylase. The enzymatic activity was measured by the glucose released during the digestion time by using a standard glucose oxidase method. The initial velocity value of the enzymatic reaction (V_i) was selected as the parameter to quantify the degree of starch gelatinization (DG). Changes in granule morphology and the starch available for hydrolysis were evaluated by photomicrographs. The new method was standardized and compared with DSC and viscosity measurements in order to check its efficiency, considering the DG observed by photomicrographs. A good agreement was observed between the DG calculated by V_i and by DSC (correlation coefficient r = 0, 97), thus V_i reflect the degree of starch gelatinization as well as DSC. These results show that the developed enzymatic procedure is an effective method to evaluate the DG in starchy foods and feeds.     

Characterization of Hydroxyethyl Starch by Polymer Analysis for Use as a Plasma Volume Expander

Hydroxyethyl starch is currently finding increasing use as a basis material for plasma volume expanders. In clinical applications it is desirable to have a precise knowledge of the steric and chemical structure, as these affect the pharmacokinetics and pharmacology. Characterization involved the determination of the mean molar masses and distribution functions of various hydroxyethyl starches, with molar masses ranging from 40,000 g/mol to 200,000 g/mol and degrees of substitution from 0.38 to 0.64, by means of size exclusion chromatography followed by double detection (MALLS/RI). Hydrodynamic data (Staudinger indices, Huggins constants and equivalent diameters) were determined by viscometric means. The chemical structure of the hydroxyethyl starches were clarified by {¹H}-¹³C NMR spectroscopy. Signal assignment for the {¹H}-¹³C NMR spectra made it possible to carry out an absolute determination of the molar, mean and partial degrees of substitution and the degree of branching. The partial degrees of substitution of the carbon atom C-2 was found to constitute between 60 and 80% of the total degree of substitution. This value is significantly larger than the partial degrees of substitution at the atoms C-3 and C-6, which were found to contribute up to approximately 10% and 20% respectively of the total degree of substitution. Degrees of branching ranging from 3.1% to 5.5% were detected.     

Hydrothermal Changes of Starch Monitored by Combined NMR and DSC Methods

The thermal, dynamic, and structural properties of wheat starch–water systems with different levels of water content (11, 35, 40, 42, 45, and 50%, wet basis) were investigated. ¹H time domain nuclear magnetic resonance (TD-NMR) spectroscopy was used to interpret and quantify the water transfer and starch transformations in terms of water uptake, granule swelling, amylose leaching, and melting of starch polymers in relation to the different levels of water content. Complementary differential scanning calorimetry (DSC) experiments were performed to study the effects of water content on the degree of starch gelatinization. In particular, this twofold approach was applied to the first endotherm to study the mechanisms of gelatinization with a common heating range both in NMR and DSC. It was shown that the trend of the enthalpy changes in the first phase transition in starch–water (SW) mixtures was strongly correlated with the loss of solid content measured by NMR in the corresponding temperature range (55–70 °C). Based on the evolution of the relative amplitudes of T ₂, structural transformations of starch were shown to occur in both crystalline and amorphous regions within SW samples, supporting the fact that the amorphous phase of starch also plays a significant role in the phase transition of granules during gelatinization. This dynamic and hydrothermal approach provided the first NMR-based interpretation of the first endotherm measured by DSC.     

Effective Approaches for Estimating the Functionalization Pattern of Carboxymethyl Starch of Different Origin

Three types of differently prepared carboxymethyl starches were analyzed by HPLC and ¹H-NMR spectroscopy after chain degradation. In derivatives obtained in the conventional manner with values of the degree of substitution (DS) up to 0.85; glucose, mono- and di-O-carboxymethyl glucose were detected as building units. Comparison with statistic calculations revealed an even distribution of functional groups along the chain. ¹H-NMR studies confirmed a preferred substitution at the 2 position of the repeating unit. Comparable results were obtained for the carboxymethyl ether of amylose, amylopectin and β-cyclodextrin. The analysis of carboxymethylated starch samples prepared using a new synthesis concept via a reactive microstructure revealed a high DS achieved in a one-step synthesis as well as a non-statistic distribution of carboxymethyl groups along the chain. A significant amount of 2,3,4,6-tetra-O-functionalization, caused by the branched structure of starch, was found. Moreover, carboxymethylation of 6-O-triphenylmethyl starch and subsequent detritylation yields a regioselectively functionalized polymer consisting not only of the expected mono- and di-O-carboxymethylated repeating units but also containing a significant amount of 2,3,4-tri-O-functionalized anhydro-glucose units.     

Long- and short-range structural changes of recrystallised cassava starch subjected to in vitro digestion

The conformational and structural order of resistant starch type III made from cassava starch was studied. The gelatinized starch was debranched using pullulanase and then recrystallised by annealing, temperature-cycling or heat-moisture treatment. Subsequently, the recrystallised products were subjected to in vitro digestion using porcine pancreatic α-amylase and amyloglucosidase. The undigested and digested products were analyzed for polymer chain distribution, crystallinity, molecular order, structural conformations and thermal stability using high performance anion exchange chromatography, wide angle X-ray diffraction, Fourier transform infrared spectroscopy, ¹³C CP/MAS nuclear magnetic resonance and differential scanning calorimetry, respectively. Average degree of polymerisation increased from 20 to 22 glucose units upon digestion. Both the undigested and digested starches comprised mixtures of A, B and V crystalline types. Percentage of crystallinities by X-ray diffraction were 40.9%, 50.7% and 56.2% in annealed, temperature-cycled and heat-moisture treated starches, respectively. These values increased to 47.9%, 54.4% and 58.2%, respectively, in the digested products. The ordered fractions in the undigested annealed, temperature-cycled and heat-moisture treated starches were 69.3%, 71.4% and 79.2%, respectively, as determined by ¹³C CP/MAS nuclear magnetic resonance. However, the disordered phase was indistinct in the digested products although the contents of non-crystalline conformations were significantly (p < 0.01) higher. The melting enthalpies of the digested residues increased by factors of 2.50 in annealed, 2.53 in temperature-cycled and 2.06 in heat-moisture treated starches, suggesting molecular rearrangement in a manner related to the enzyme susceptibility of the initial materials.