Physicochemical properties and in vitro digestibility of flour and starch from pea (Pisum sativum L.) cultivars

Flours and isolated starches from three different cultivars (1544-8, 1658-11 and 1760-8) of pea grown under identical environmental conditions were evaluated for their physicochemical properties and in vitro digestibility. The protein content, total starch content and apparent amylose content of pea flour ranged from 24.4 to 26.3%, 48.8 to 50.2%, and 13.9 to 16.7%, respectively. In pea starches, the 1760-8 showed higher apparent amylose content and total starch content than the other cultivars. Pea starch granules were irregularly shaped, ranging from oval to round with a smooth surface. All pea starches showed C-type X-ray diffraction pattern with relative crystallinity ranging between 23.7 and 24.7%. Pea starch had only a single endothermic transition (12.1-14.2 J/g) in the DSC thermogram, whereas pea flour showed two separate endothermic transitions corresponding to starch gelatinization (4.54-4.71 J/g) and disruption of the amylose-lipid complex (0.36-0.78 J/g). In pea cultivars, the 1760-8 had significantly higher setback and final viscosity than the other cultivars in both pea flour (672 and 1170 cP, respectively) and isolated starch (2901 and 4811 cP). The average branch chain length of pea starches ranged from 20.1 to 20.3. The 1760-8 displayed a larger proportion of short branch chains, DP (degree of polymerization) 6-12 (21.1%), and a smaller proportion of long branch chains, DP ≥ 37 (8.4%). The RDS, SDS and RS contents of pea flour ranged from 23.7 to 24.1%, 11.3 to 12.8%, and 13.2 to 14.8%, respectively. In pea starches, the 1760-8 showed a lower RDS content but higher SDS and RS contents. The expected glycemic index (eGI), based on the hydrolysis index, ranged from 36.9 to 37.7 and 69.8 to 70.7 for pea flour and isolated pea starch, respectively.     

Amylose content and chemical modification effects on thermoplastic starch from maize – Processing and characterisation using conventional polymer equipment

A design of experiments was performed on extruded starch based materials studied in a recently published article [Chaudhary, A. L., Miler, M., Torley, P. J., Sopade, P. A., & Halley, P. J. (2008). Amylose content and chemical modification effects on the extrusion of thermoplastic starch from maize. Carbohydrate Polymers, 74(4), 907–913] highlighting the effects of amylose content, chemical modification and extrusion on a range of maize starches. An investigation into the effects of starch type (unmodified 0–80% amylose starch; hydroxypropylated 80% amylose starch), screw speed and ageing after moulding on final product properties such as mechanical properties (Young’s modulus, maximum stress and strain at break), moisture absorption, morphology and retrogradation are included. A full factorial design was used to study these starch type, processing and final product property relationships. Microscopy was used to observe any morphological difference between the various starch types in thermoplastic starch (TPS) blends and X-ray diffraction (XRD) was used to observe changes in crystallinity over time (retrogradation). The results show that 0% amylose (waxy maize) and hydroxypropylated 80% amylose thermoplastic starches have mechanical properties comparable to that of low density polyethylene (LDPE) and high density polyethylene (HDPE), therefore these materials have the potential to be an environmentally friendly alternative to current polymer resins.     

Debranched cassava starch crystallinity determination by Raman spectroscopy: Correlation of features in Raman spectra with X-ray diffraction and C CP/MAS NMR spectroscopy

Because starch crystallinity influences the physical, mechanical, and technological aspects of numerous starch-based products during production and storage, rapid techniques for its assessment are vital. Samples of different levels of crystallinity were obtained by debranching gelatinized cassava starch, followed by subjection to various hydrothermal treatments. The recrystallized products were further subjected to partial hydrolysis with a mixture of α-amylase and glucoamylase prior to freeze-drying. Crystallinities were determined using X-ray diffraction (XRD) and ¹³C CP/MAS NMR spectroscopy, and correlated with FT-Raman spectra features. XRD crystallinities ranged between 0 and 58%, and agreed with crystalline-phase fractions (R² = 0.99) derived from the respective ¹³C CP/MAS NMR spectra. A strong linear correlation was found between crystallinities and integrated areas of the skeletal mode Raman band at 480 cm⁻¹ (R² = 0.99). With appropriate calibration, FT-Raman spectroscopy is a promising tool for rapid determination of starch crystallinity.     

Effect of high external pressures on the vibrational spectra of crystalline dichloro(1,5-cycloctadiene)platinum(II), Pt(η-C8H12)Cl2

The effects of high external pressures on the principal IR and Raman bands of crystalline dichloro(1,5-cycloctadiene)platinum(II), Pt(COD)Cl₂ (COD = η⁴-C₈H₁₂), have been investigated for pressures up to ∼30 kbar by diamond-anvil cell microspectroscopy. This square-planar Pt(II) complex does not undergo any pressure-induced structural change throughout the pressure range investigated and the pressure dependences (dν/dP) for the vibrational modes range from −0.18 to 0.79 cm⁻¹ kbar⁻¹. The negative dν/dP value observed for the IR band at 1426 cm⁻¹ (−0.18 cm⁻¹ kbar⁻¹) suggests that this band is chiefly associated with a CC stretching mode of the Pt-COD group. This observation provides yet another indication that high-pressure vibrational spectroscopy may indeed be a general method for establishing the presence of π-backbonding in organometallic complexes, such as metal carbonyls and alkenes.     

A possible structure of retrograded maize starch speculated by UV and IR spectra of it and its components

“Retrogradation” has been used to describe the changes that occur in starch after gelatinization, from an initially amorphous state to a more ordered or crystalline state, which has a significant impact on starch application in food, textiles and materials fields. But mechanism of starch retrogradation is still unclear until now and there is no breakthrough in this area. Here we are speculating a possible structure of retrograded maize starch by UV (binding with iodine) and IR spectra of it and its compositions. We speculate that nucleation of retrograded starch origins from combination of reducing end of amylopectin and non-reducing end of amylose, and retrogradation terminates at combining of non-reducing end of amylopectin and reducing end of amylose. The chain length of resistant digestion retrograded starch should be nearly same. The hydroxyl associated with sixth carbon atoms of glucan must form hydrogen bond with other hydroxyl of starch.     

Studies of the retrogradation process for various starch gels using Raman spectroscopy

The retrogradation of untreated wild-type starches (potato, maize, and wheat), waxy maize starches, and one pregelatinized, modified amylose-rich starch was investigated continuously using Raman spectroscopy. The method detects conformational changes due to the multi-stage retrogradation, the rate of which differs between the starches. The pregelatinized, modified amylose-rich starch shows all stages of retrogradation in the course of its Raman spectra. In comparison to amylose, the retrogradation of amylopectin is faster at the beginning of the measurements and slower in the later stages. The untreated starches can be ranked in the order of their rate of retrogradation as follows: potato>maize>wheat.     

Structural and physicochemical properties of starch gels prepared from partially modified starches using Thermus aquaticus 4-α-glucanotransferase

The modified starch gels prepared from partial enzyme treatments (1, 3, and 6 U/g starch; 2-h incubation) of the corn and rice starch pastes using Thermus aquaticus 4-α-glucanotransferase (TAαGT) were investigated for their molecular characteristics, microstructures, and physicochemical properties. Unlike the native and partially modified normal starches, the native and partially modified waxy starches could not form gels strong enough for textural analysis after 24 h for gel setting. Features of the partially modified normal starches were the specific apparent amylose contents and maximum iodine absorption wavelength (λ_max, ∼567 nm), as well as the tri-modal molecular weight profiles and flatter side-chain distributions. Also, the partially modified normal starch gels possessed fractured surfaces with discontinuous crystalline fibrous assembly that differed from the native starch gels’ porous continuous network, which resulted in more brittle, rigid, and resilient gels compared with the native gels.     

Determination of the degree of N-acetylation (DA) of chitin and chitosan in the presence of water by first derivative ATR FTIR spectroscopy

A new method for the determination of the degree of N-acetylation (DA) of chitin and chitosan is described using first derivative diamond ATR FTIR spectroscopy. Applying the derivative values of the amide III band at 1327 cm⁻¹ and the CH deformation band of the N-acetyl group at 1383 cm⁻¹ as measure of the N-acetyl content of the sample in relation to the derivative value of the bridge oxygen vibration at 1163 cm⁻¹ as internal standard, a linear correlation to the results of first derivative UV spectroscopy was obtained and confirmed by elemental analysis and Raman spectroscopy. The described method allows the determination of the degree of N-acetylation of chitosan and chitin in the presence of water thus making drying procedures unnecessary.     

Impact of annealing and heat-moisture treatment on rapidly digestible,slowly digestible and resistant starch levels in native and gelatinized corn,pea and lentil starches

Impact of annealing (ANN) and heat-moisture treatment (HMT) on rapidly digestible starch (RDS), slowly digestible starch (SDS), resistant starch (RS), and expected glycemic index (eGI) of corn, pea, and lentil starches in their native and gelatinized states were determined. ANN was done for 24 h at 70% moisture at temperatures 10 and 15 °C below the onset (T_o) temperature of gelatinization, while HMT was done at 30% moisture at 100 and 120 °C for 2 h. The swelling factor (SF), amylose leaching (AML) and gelatinization parameters of the above starches before and after ANN and HMT were determined. SF and AML decreased on ANN and HMT (HMT > ANN). The gelatinization temperatures increased on ANN and HMT (HMT > ANN). However, the gelatinization temperature range decreased on ANN but increased on HMT. Birefringence remained unchanged on ANN but decreased on HMT. The Fourier transform infrared (FT-IR) absorbance ratio of 1047 cm^?1/1022 cm^?1 increased on ANN but decreased on HMT. ANN and HMT increased RDS, RS and eGI levels and decreased SDS levels in granular starches. HMT had a greater impact than ANN on RDS, RS, and SDS levels. In gelatinized starches, ANN and HMT decreased RDS and eGI, but increased SDS and RS levels. These changes were more pronounced on HMT. This study showed that amylopectin structure and interactions formed during ANN and HMT had a significant impact on RDS, SDS, RS and eGI levels of starches.     

Heat–moisture treatment under mildly acidic conditions alters potato starch physicochemical properties and digestibility

Potato starch was subjected to heat–moisture treatment (HMT; 120 °C, 3 h) under mildly acidic conditions (pH 5, 6, or 6.5 [control]) at moisture levels of 15, 20 or 25%. HMT starches exhibited significantly delayed pasting times and reduced overall paste viscosities, amylose leaching, and granular swelling characteristics relative to native starch, as well as enhanced levels of thermo-stable resistant starch (≈24%). HMT appeared to alter/enhance short-range chain associations (FT-IR) within amorphous and/or crystalline regions of starch granules. However, the extent of physicochemical change and RS enhancement during HMT was most facilitated by a mildly acidic condition (pH 6) at higher treatment moisture levels (20 or 25%). These conditions promoted limited hydrolysis of amylopectin molecules, primarily at α-(1 → 6) branch points, likely enhancing mobility and interaction of starch chains during HMT. Thus, a slightly acidic pH might reduce conditions and/or timeframe needed to impart physicochemical changes and reduced digestibility to potato starch.     

Pasting viscosity and in vitro digestibility of retrograded waxy and normal corn starch powders

Pasting viscosity and in vitro digestibility of oven-dried powders of waxy and normal corn starch gels (40% solids) retrograded under an isothermal (4 °C) or temperature cycled (4/30 °C) storage were investigated. Temperature cycling induced higher onset temperature for melting of amylopectin crystals than isothermal storage under a differential scanning calorimeter whereas little difference in crystalline type was observed under X-ray diffraction analysis. Temperature cycling caused higher pasting temperature and viscosity for the retrograded starches than isothermal storage. The retrograded waxy corn starch powders exhibited pasting behaviors similar to that of native waxy corn starch. However, the retrograded normal corn starch powders showed very much different pasting patterns with lower pasting viscosity but higher pasting temperature than native starch counterpart. The retrogradation increased slowly digestible starch content without changing resistant starch content, more effectively by the temperature cycling than the isothermal storage.     

Quantification of blockiness in pectins—A comparative study using vibrational spectroscopy and chemometrics

The gelling properties of pectins are related not only to the degree of esterification (DE), but also to the distribution of the ester groups. In this study, we have examined an experimentally designed series of 31 pectins originating from the same mother pectin and de-esterified using combinations of two different enzymatic mechanisms. The potential of using infrared (IR), Raman, and near infrared (NIR) spectroscopies combined with chemometrics for reliable and rapid determination of the DE and distribution patterns of methyl ester groups in a designed set of pectin powders was investigated. Quantitative calibration models using partial least squares (PLS) regression were developed and compared. The calibration models for prediction of DE obtained on extended inverse signal correction (EISC)-treated spectra of all three spectroscopic methods yielded models with cross-validated prediction errors (RMSECV) between 1.1%p and 1.6%p DE and correlation coefficients of 0.99. A calibration model predicting degree of random de-esterification (R) and block de-esterification (B) was developed for each spectroscopic method, yielding RMSECV values between 4.4 and 6.7 and correlation coefficients (r) between 0.79 and 0.92. Variable selection using interval PLS (iPLS) significantly improved the prediction of R for IR spectroscopy, yielding RMSECV of 3.5 and correlation coefficients of 0.95. All three spectroscopic methods were able to distinguish the spectral patterns of pectins with different enzyme treatments in simple classification models by principal component analysis (PCA). Extended canonical variate analysis revealed one specific signal in the Raman (1045 cm⁻¹) spectrum and one significant area (1250-1400 cm⁻¹) in the IR spectrum which are able to classify the pectin samples according to the four different enzyme treatments. In both Raman and IR spectra, the signal intensity decreased in the sequence R-B > B > B-R > R > re-methylated pectin.     

UV resonance Raman studies on the activation mechanism of human hematopoietic prostaglandin D2 synthase by a divalent cation, Mg

The Mg²⁺ ion-assisted activation mechanism of the active site Tyr8 of a human hematopoietic prostaglandin D₂ synthase (H-PGDS) was studied by ultraviolet resonance Raman (UVRR) spectroscopy. Addition of Mg²⁺ to the native H-PGDS at pH 8.0 resulted in the Y8a Raman band of Tyr8 shifting from 1615 cm⁻¹ to 1600 cm⁻¹. This large shift to lower energy of the tyrosine Y8a vibrational mode is caused by the deprotonation of the tyrosine phenol group promoted by binding of Mg²⁺. Upon subsequent addition of glutathione (GSH), the Mg²⁺/H-PGDS solution showed the Tyr8 Raman band shifted to 1611 cm⁻¹, which is 11 cm⁻¹ higher than the frequency of the Mg²⁺ complex of H-PGDS, but 4 cm⁻¹ lower than the Mg²⁺ free enzyme. These UVRR observations suggest that the deprotonated Tyr8 in the presence of Mg²⁺ is re-protonated by the abstraction of H⁺ from the thiol group of GSH, and that the re-protonated Tyr8 species forms a hydrogen bond with the thiolate anion of GSH. Density functional theory calculations on several model complexes of p-cresol were also performed, which suggested that the pK_a and vibrational frequencies of the Tyr8 phenol group are affected by the degree and structure of hydration of the Tyr8 residue.     

Photochemical and photophysical properties of ruthenium(II) bis-bipyridine bis-nitrile complexes: Photolability

The electrochemical and photophysical properties of two bis-nitrilo ruthenium(II) complexes formulated as [Ru(bpy)₂(L)₂](PF₆)₂, where bpy is 2,2′-bipyridine and L is AN = CH₃CN and sn = NC-CH₂CH₂-CN, have been investigated. Electrochemical data are typical of Ru-bpy complexes with two reversible reduction peaks located near −1.3 and −1.6 V assigned to each bipyridine ligand and one Ru^II/Ru^III oxidation wave centered at approximately +1.5 V. The sn derivative is both IR and Raman active with its coordinated CN stretch appearing at 2277 cm⁻¹ and 2273 cm⁻¹, respectively. The UV/Vis absorption spectrum of the sn derivative is dominated by an intense (ε_max ∼ 58700 M⁻¹ cm⁻¹) absorption band at 287 nm assigned as a LC (π → π∗) transition. The peak observed at 418 nm (ε ∼ 10 400 M⁻¹ cm⁻¹) is an MLCT band while the one at 244 nm (ε ∼ 23 600 M⁻¹ cm⁻¹) is of LMLCT character. The AN derivative behaves similarly. Both complexes show low-temperature emission at around 537 nm with a lifetime near 10.0 μs. ¹H and ¹³C assignments are consistent with the formulation of the complexes. The complexes undergo photosubstitution of solvent with quantum efficiencies near one. Calculated and experimental results support replacement of the nitrile ligands by solvent. Based on DFT calculations, the electron density of the HOMO lies on the metal center, the bipyridine ligands and the nitrile ligands and electron density of the LUMO resides primarily on the bipyridine ligands. The electronic spectra obtained from TDDFT calculations closely match the experimental ones.     

Resistant glutarate starch from adlay: Preparation and properties

Reaction conditions were optimized to increase the content of resistant starch in adlay starch using esterification with glutaric acid, and the physicochemical properties of the prepared glutarate starches were investigated. Different amounts of glutaric acid (0.1–0.5 g/g starch, dry weight basis) were reacted with adlay starch at various temperatures (70–130 °C) and reaction times (3–9 h). The resistant starch levels increased with increased glutaric acid content, reaction temperature, and reaction time. The color difference was mainly affected by reaction time. The highest resistant starch content (RS 66%) was obtained using conditions of 0.4 g glutaric acid/g starch, 115 °C, and 7.5 h, with a color difference of 10.24. After digestion with α-amylase and amyloglucosidase, the water-soluble fraction of glutarate starch had more oligosaccharides than high-amylose maize starch (RS 43%). FT-IR and solid-state NMR detected carbonyl groups in the glutarate starch, indicating the formation of cross-linkages through esterification. The granular structure of the glutarate starches was not destroyed and they retained birefringence. After heating with an excess of water, the granules kept their shape but lost their birefringence. The glutarate starches had low solubility in both cold and hot water, and the resistant starch contents were unchanged after heating due to the restriction of swelling by cross-linking. The glutarate starches had a similar chain-length distribution to raw starch, indicating that acid hydrolysis took place at branching points in the amorphous region. Furthermore, the glutarate starches possessed a weaker crystalline region, more diverse double helical chains, and lower enthalpy than raw starch.     

Starch biosynthesis: experiments on how starch granules grow in vivo

Four varieties of starch granules from potato, wheat, maize, and rice were fractionated into homogeneous 10-μm-sized ranges. The size with the largest amount of granules was reacted with ADP-[¹⁴C]Glc, washed, and peeled into 7−9 layers, using a controlled peeling process, involving 90:10 volume proportions of Me₂SO-H₂O at 10 °C. All of the starches showed biosynthesis of starch throughout the granules. Starch synthase activities were determined for each of the layers. Three of the starches had a relatively large amount of synthase activity in the second layer, with only a small amount in the first layer. Potato starch had the largest amount of activity in the first layer. Starch synthase activity was found to alternate between higher and lower activities throughout all of the varieties of granules, showing that the synthesis was not uniform and also was not exclusively occurring at the surface of the starch granules, which had previously been hypothesized. From these results and our previous studies on the mechanism of starch chain elongation by the addition of d-glucose to the reducing end of a growing chain that is covalently attached to the active site of starch synthase, a hypothesis is proposed for how starch granules grow in vivo.     

Non-invasive label-free monitoring the cardiac differentiation of human embryonic stem cells in-vitro by Raman spectroscopy

Background

Online label-free monitoring of in-vitro differentiation of stem cells remains a major challenge in stem cell research. In this paper we report the use of Raman micro-spectroscopy (RMS) to measure time- and spatially-resolved molecular changes in intact embryoid bodies (EBs) during in-vitro cardiogenic differentiation.

Methods

EBs formed by aggregation of human embryonic stem cells (hESCs) were cultured in defined medium to induce differentiation towards cardiac phenotype and maintained in purpose-built micro-bioreactors on the Raman microscope for 5 days (between days 5 and 9 of differentiation) and spatially-resolved spectra were recorded at 24 h intervals.

Results

The Raman spectra showed that the onset of spontaneous beating of EBs at day 7 coincided with an increase in the intensity of the Raman bands at 1340 cm^− 1, 1083 cm^− 1, 937 cm^− 1, 858 cm^− 1, 577 cm^− 1 and 482 cm^− 1. The spectral maps corresponding to these bands had a high positive correlation with the expression of the cardiac-specific α-actinin obtained by immuno-fluorescence imaging of the same EBs. The spectral markers obtained here are also in agreement with previous studies performed on individual live hESC-derived CMs.

Conclusions

The intensity profile of these Raman bands can be used for label-free in-situ monitoring of EBs to estimate the efficacy of cardiogenic differentiation.

General significance

As the acquisition of the time-course Raman spectra did not affect the viability or the differentiation potential of the hESCs, this study demonstrates the feasibility of using RMS for on-line non-invasive continuous monitoring of such processes inside bioreactor culture systems.     

Improved cold starch hydrolysis with urea addition and heat treatment at subgelatinization temperature

We explore how the presence of urea can influence the kinetics of amylolysis, with a long-term objective of developing practical and energy efficient bioconversion protocols. In this study, triticale and corn starches were hydrolyzed by a granular starch hydrolyzing enzyme with or without addition of urea and a pre-heating treatment at subgelatinization temperature. Differential scanning calorimetry showed that the gelatinization parameters of triticale and corn starches were negatively correlated with the urea concentration in the starch suspension. Addition of urea did not significantly affect starch amylolysis by the granular starch hydrolyzing enzyme at 30 °C. However when pre-heating at a higher yet sub-gelatinization temperature (50 °C for triticale and 61 °C for corn, 5 °C below the onset of starch gelatinization) for 30 min, the presence of urea greatly facilitated the amylolysis of both tricticale and corn starches. Scanning electron microcopy revealed starch granule mophological changes to a porous structure in residual starch granules/fragments rich in resistant starch. This means that the amylolysis pattern in the presence of urea was fundamentally changed, and urea disrupts starch hydrogen bonds effectively with heating treatment at a sub-gelatinization temperature. This treatment combination increased both starch hydrolysis rate and extent. Since extra energy was not necessary to gelatinize starch, this method may benefit starch and bio-enthanol industries to reduce the costs of starch hydrolysis.