Use of FT-IR spectroscopy as a tool for the analysis of polysaccharide food additives

This work purposes the characterisation of food additive polysaccharides such as starch, glucomannan and carrageenan by the use of the information of the principal components of the FT-IR spectra in the 1200–800 cm⁻¹ wavenumber region. The application of a PCA to this spectral region showed that several features could be obtained: (a) Separation between Glc and Gal, both monomeric and polymeric, and identification of their characteristic wavenumbers. (b) Identification of the specific absorbance wavenumbers for sucrose, Fru, Ara, and Man. (c) Distinction of pectic polysaccharides from the remaining carbohydrate samples. (d) Separation within κ-, ι-, and λ-carrageenans. These results show that the FT-IR spectroscopy in the 1200–800 cm⁻¹ wavenumber region can be a very reliable technique for food authentication of polysaccharide-based additives and be used for a quick screening of polysaccharides used as additives in foodstuffs.     

Rheological properties of barley β-glucan

Health benefits of cereal β-glucan are linked to its high viscosity. Although viscosity of β-glucan gum solutions has been reported previously, there are conflicting reports about its behavior at elevated temperatures. Therefore, the viscosity behavior of barley β-glucan gum obtained in a pilot plant (PP) or in a laboratory (LAB) was determined at different shear rates (1.29–129 s⁻¹) and temperatures (0.1–75 °C) in this study. Viscosity decrease with temperature was demonstrated for both gums and activation energy E_a was calculated from the Arrhenius equation. None of the fresh gum solutions exhibited thixotropic behavior at ≤1% (w/w) concentration, but the measurement demonstrated that increased shear rate is not applicable to polymer solutions of low viscosity. Information about rheological properties of β-glucan will lead to better understanding of its behavior under physiological and processing conditions.     

Relationships between conformation of β-lactoglobulin in solution and gel states as revealed by attenuated total reflection Fourier transform infrared spectroscopy

Attenuated total reflection Fourier transform infrared spectroscopy (ATR FT-IR) has been used to compare the structure of β-lactoglobulin, the major component of whey proteins, in solution and in its functional gel state. To induce variation in the conformation of β-lactoglobulin under a set of gelling conditions, the effect of heating temperature, pH, and high pressure homogenization on the conformation sensitive amide I band in the infrared spectra of both solutions and gels has been investigated. The results showed that gelification process has a pronounced effect upon β-lactoglobulin secondary structure, leading to the formation of intermolecular hydrogen-bonding β-sheet structure as evidenced by the appearance of a strong band at 1614 cm⁻¹ at the expense of other regular structures. These results confirm that this structure may be essential for the formation of a gel network as it was previously shown for other globular proteins. However, this study reveals, for the first time, that there is a close relationship between conformation of β-lactoglobulin in solution and its capacity to form a gel. Indeed, it is shown that conditions which promote predominance of intermolecular β-sheet in solution such as pH 4, prevent the formation of gel in conditions used by increasing thermal stability of β-lactoglobulin. On the basis of these findings, it is suggested that by controlling the extent of intermolecular β-structure of the protein in solution, it is possible to modify the ability of protein to form a gel and as a consequence to control the properties of gels.     

Effect of gamma-ray on activity and stability of alcohol-dehydrogenase from Saccharomyces cerevisiae

The effect of γ-ray irradiation on alcohol-dehydrogenase activity of yeast was investigated. The results suggested that low doses of γ-ray (10 and 20 Gy) significantly increased the enzyme activity. This work also describes the impact of irradiation on immobilization efficiency of biocatalyst entrapped on to alginate gel beads. When yeast irradiated to a dose of 20 Gy was immobilized, ADH stability was improved up to 1.4 times at 45 °C compared to the immobilized non-irradiated cells. Also, the irradiated biocatalyst, when immobilized, can be reused more than eight times in oxidation reaction of ethanol. This preparation also permitted to reach high yields of immobilization (79%) and activity (88%).     

Effect of γ‐irradiation on thermal and thermoluminescence properties of polystyrene/europium (III) oxide composite film

In the present study, polystyrene:europium (III) oxide polymer films at a ratio of 95:5 wt% were prepared using a solution casting technique. These polymeric films were irradiated with 5, 25 and 50 kGy γ‐radiation doses and their thermoluminescence (TL) and thermal properties were studied as a function of radiation dose. Analysis of Fourier transform infrared spectra revealed different modes of vibration and polymer–filler interaction. Reduction of vibrational modes with radiation dose was observed. The TL glow curve intensity was observed to increase with increasing radiation dose and to become broader in the 378 K and 444 K regions. Detrapping of electrons implied by the glow curve was caused by thermally induced macromolecular motion, concurrent with β‐relaxation in polystyrene. The TL glow curve parameters were computed using a glow curve deconvolution method. Differential scanning calorimetry analysis indicated that the glass transition temperature (T_g) increased with increase in dose, suggesting crosslinking of the polymer chain. Scanning electron microscopy analysis evidenced the change in surface morphology due to γ‐irradiation.     

Effects of γ‐Irradiation on the Molecular Structures and Functions of Human Serum Albumin

In this paper, we use spectroscopic methods (fluorescence spectroscopy, UV absorption spectroscopy, and circular dichroism (CD) spectroscopy) to elucidate the effects of reactive oxygen species generated by γ‐irradiation on the molecular properties of human serum albumin (HSA). The results of fluorescence spectroscopy indicated that oxidation by γ‐irradiation can lead to conformational changes of HSA. Data of CD spectra suggested that with the increase of radiation dose the percentage of α‐helix in HSA has decreased. The determination of protein hydrophobicity showed that the effective hydrophobicity of HSA decreased up to 62% compared to the native HSA solution due to the exposure to the γ‐irradiation. Furthermore, small changes in the esterase‐like activity of HSA were introduced because of oxidation. The content of bityrosine increased markedly, suggesting that the oxidized HSA was aggregated. Moreover, there was no obvious change in the molecular properties of HSA with low γ‐irradiation dose. Changes happened when the irradiation dose exceeded 200 Gy.     

Synthesis and characterization of cross-linked malonylchitosan microspheres for controlled release of acyclovir

The purpose of the present study was to obtain a polymeric system for delayed release of the drug acyclovir (ACV), which can be used for treatment of Herpes simplex and Varicella Zoster. The gelled chitosan (GCT) microspheres were obtained by coacervation-phase separation. They were treated with malonic acid to obtain malonylchitosan (MLCT) microspheres, which were characterized by, Fourier transform infrared spectroscopy (FT-IR), nuclear magnetic resonance spectroscopy (¹³C NMR), elemental analysis (CHN), thermogravimetric analysis (TG/DTG) and scanning electron microscopy (SEM). The drug was encapsulated in MLCT microspheres by a contact adsorption technique, and the final formulation (MLCT-ACV), was analyzed for loading efficiency, degree of swelling and in vitro release profiles. The results obtained support the N-substitution of malonyl groups in the MLCT microspheres. The loading efficiency increased with impregnation time and a major amount of drug was encapsulated after 24 h. The swelling rate was higher in acid pH. The median release time was 5.5 h in pH 1.2 and 6.8. The mechanism involved in release was non-Fickian (0.43 < n < 0.85, n = 0.8474) and Super Case II kinetics (n > 1, n = 1.0491) at pH 1.2 and 6.8, respectively.     

Fourier transform infrared spectroscopy used to evidence the prevention of beta-sheet formation of amyloid beta(1-40) peptide by a short amyloid fragment

Reflectance Fourier transform infrared (FT-IR) microspectroscopy was applied to study the prevention of β-sheet formation of amyloid β (Aβ)(1–40) peptide by co-incubation with a hexapeptide containing a KLVFF sequence (Aβ(15–20) fragment). Second-derivative spectral analysis was used to locate the position of the overlapping components of the amide I band of Aβ peptide and assigned them to different secondary components. The result indicates that each intact sample of Aβ(15–20) fragment or Aβ(1–40) peptide previously incubated in distilled water at 37 °C transformed their secondary structure from 1649 (1651) or 1653 cm⁻¹ to 1624 cm⁻¹, suggesting the transformation from -helix and/or random coil structures to β-sheet structure. By co-incubating both samples with different molar ratio in distilled water at 37 °C, the structural transformation was not found for Aβ(1–40) peptide after 24 h-incubation. But the β-sheet formation of Aβ(1–40) peptide after 48 h-incubation was evidenced from the appearance of the IR peak at 1626 cm⁻¹ by adding a little amount of Aβ(15–20) fragment. There was no β-sheet formation of Aβ(1–40) peptide after addition with much amount of Aβ(15–20) fragment, however, suggesting the higher amount of Aβ(15–20) fragment used might inhibit the β-sheet formation of Aβ(1–40) peptide. The more Aβ(15–20) fragment used made the more stable structure of Aβ(1–40) peptide and the less β-sheet formation of Aβ(1–40) peptide. The study indicates that the reflectance FT-IR microspectroscopy can easily evidence the prevention of β-sheet formation of Aβ(1–40) peptide by a short amyloid fragment.     

Decoloration of chitosan by UV irradiation

Decoloration of chitosan by UV irradiation, which was used to replace a bleaching step during chitosan preparation, was evaluated under four separate treatments (effect of irradiation time, chitosan/water ratio, stirring speed, and UV light source). The optimal decoloration condition was defined as that producing white chitosan with higher viscosity. Decoloration of chitosan could be achieved effectively using a UV-C light by stirring unbleached chitosan in water (1:8, w/v) for 5 min at 120 rpm. UV irradiation applied under the optimal conditions could be used to produce chitosan with desirable white color (L^* = 76.95, a^* = −0.37, and b^* = 14.04) and high viscosity (1301.7 mPa s at 0.5% w/v in 1.0% v/v acetic acid).     

Study of the interaction of artemisinin with bovine serum albumin

The study on the interaction of artemisinin with bovine serum albumin (BSA) has been undertaken at three temperatures, 289, 296 and 303 K and investigated the effect of common ions and UV C (253.7 nm) irradiation on the binding of artemisinin with BSA. The binding mode, the binding constant and the protein structure changes in the presence of artemisinin in aqueous solution at pH 7.40 have been evaluated using fluorescence, UV–vis and Fourier transform infrared (FT-IR) spectroscopy. The quenching constant K_q, K_sv and the association constant K were calculated according to Stern–Volmer equation based on the quenching of the fluorescence of BSA. The thermodynamic parameters, the enthalpy (ΔH) and the entropy change (ΔS) were estimated to be −3.625 kJ mol⁻¹ and 107.419 J mol⁻¹ K⁻¹ using the van’t Hoff equation. The displacement experiment shows that artemisinin can bind to the subdomain IIA. The distance between the tryptophan residues in BSA and artemisinin bound to site I was estimated to be 2.22 nm using Föster's equation on the basis of fluorescence energy transfer. The decreased binding constant in the presence of enough common ions and UV C exposure, indicates that common ions and UV C irradiation have effect on artemisinin binding to BSA.     

X-ray inactivation, Weigle reactivation, and Weigle mutagenesis of the lysogenic Vibrio kappa phage

Vibrio cholerae lysogenic kappa phage was inactivated by X-ray (60 kV) in a dose-dependent manner, the inactivation dose leading to 37% survival (D37) in phosphate-buffered saline (PBS), pH 7.4, being 0.36 kGy. The phages were significantly protected against X-ray irradiation when histidine or cysteine or both were present in PBS or when phages were irradiated in nutrient broth. Maximum protection was offered when both histidine (10.0 mM) and cysteine (10.0 mM) were present in PBS (dose enhancement factor being 4.17). The X-irradiated kappa phages also underwent a small but significant Weigle reactivation and also Weigle mutagenesis in the UV-irradiated V. cholerae host H218Smr. The Weigle factor or the frequency of clear-plaque mutants increased with increasing UV dose, attained a maximum at a UV dose of 2.4 J m-2, and thereafter decreased gradually with a further increase of the UV dose. The X-ray dose (D)--survival (S) curves could be empirically described by the equation S = exp[-(aD + bD2)], where a and b are constants depending on the irradiation conditions, and a good agreement between the theoretical curves and experimental data was obtained.     

Incorporation of the antibacterial agent, miconazole nitrate into a cellulosic fabric grafted with β-cyclodextrin

The aim of the study was to investigate the incorporation of the antibacterial agent, miconazole nitrate into cyclodextrin cavities covalently bonded onto cloth fibers. The cellulosic fabric was grafted with β-cyclodextrin molecules through reaction with monochlorotriaziny β-cyclodextrin (MCT-β-CD). The suitable bonded reaction conditions were found to be MCT-β-CD 60–100 g/L, catalyst Na₂CO₃ 50–60 g/L, the reaction temperature 150–160 °C and the reaction time 5–8 min.

The modified and unmodified fabrics were characterized by UV spectrophotometry. The level of miconazole nitrate entrapped in the fabrics were determined by HPLC and was founded to be much higher (0.458% w/w) for the textile functionalized with MCT-β-CD compared to the unmodified fabric (0.056% w/w). The antibacterial abilities measured by shaker flask method showed that the antibacterial property was markedly enhanced by impregnation with miconazole nitrate of the MCT-β-CD grafted textile. The finished fabric kept the antibacterial abilities more than 70% even after washing 10 times, while the antibacterial activity of the unmodified textile was almost lost.     

Structural characterization of cold extracted fraction of soluble sulfated polysaccharide from red seaweed Gracilaria birdiae

Water soluble polysaccharide from Gracilaria birdiae cultivated along the northeast coast of Brazil was characterized by infrared (FT-IR) and nuclear magnetic resonance (NMR) spectroscopy. The composition of the polysaccharide in wt% was determined as: β-d-galp (50.3%), 3,6-anhydro--l-galp (40.5%) and --l-galp-6 sulfate (9.2%). The ratio of l/d units (β-d-galp units and 3,6-anhydro--l-galp + -l-galp-6 sulfate) is that of an ideal agarose. The sulfate content calculated by S% accounts for 6.4%. 1D and 2D NMR techniques were employed in order to assign the spin system of polysaccharide without partial degradation. The structure is composed of → 4-3,6-anhydro--l-galp (1 → 3)β-d-galp 1 → segments, with the possibility of a -l-galp unit substituted at the 6-position by sulfate ester.     

Reactivity of Cartilage and Selected Carbohydrates with Hydroxyl Radicals: An NMR Study to Detect Degradation Products

It was investigated to what extent isolated, monomeric and polymeric carbohydrates as well as cartilage specimens are affected by hydroxyl radicals generated by γ-irradiation or Fenton reaction and what products can be detected by means of NMR spectroscopy. Resonances of all protons in glucose and other monosac-charides as well as carbon resonances in ¹³C-enriched glucose were continuously diminished upon γ-irradiation. Formate and malondialdehyde were found as NMR detectable products in irradiated glucose solutions under physiologically relevant (aerated) conditions. In polysaccharide solutions (e.g. hyaluronic acid) γ-irradiation and also treatment with the Fenton reagent caused first an enhancement of resonances according to mobile N-acetyl groups at 2.02 ppm. This indicates a breakdown of glycosidic bonds in polysac-charides. Using higher radiation doses or higher concentrations of the Fenton reagent formate was also detected. The same sequence of events was observed upon treatment of bovine nasal cartilage with the Fenton reagent. First, glycosidic linkages in cartilage polysaccharides were cleaved and subsequently formate was formed. In contrast, collagen of cartilage was affected only to a very low extent. Thus, HO-radicals caused the same action on cartilage as on isolated polymer solutions, inducing a fragmentation of polysaccharides and the formation of formate.     

Esr and Spin-Trapping Study of Free Radicals in γ-Irradiated Solid Lysozyme

Free radicals produced by γ-irradiation of solid lysozyme were investigated by a technique combining ESR, spin-trapping and enzymatic digestion. MNP and DMPO were used as spin-trapping reagents. The solid lysozyme was first γ-irradiated and then dissolved in an aqueous solution containing the spin-trapping reagent to stabilize free radicals. The spin adducts of lysozyme were digested to oligopeptides to get ESR spectra having a well-resolved hyperfine structure. The ESR spectra obtained showed that carbon-centered radicals, CH-, at the side chains of amino acids, and thiyl radicals, -CH₂-_-S^-, at disulfide bridges were produced in γ-irradiated solid lysozyme.     

Effect of gamma irradiation on rheological properties of polysaccharides exuded by A. fluccosus and A. gossypinus

In this study, Iranian gum tragacanth (GT) exudates from Astragalus fluccosus (AFG) and Astragalus gossypinus (AGG) were irradiated at 3, 7, 10 and 15 kGy. Fourier transform infrared spectroscopy (FTIR) data showed that irradiation did not induce changes in the chemical structure of either type of gum. Although particle size distribution and both steady shear and dynamic rheological properties were considerably affected by the irradiation process, the magnitude of the effect of irradiation on each of the rheological and size variables was different for the hydrocolloids. For instance, for AGG, increasing the irradiation dose from 3 to 10 kGy, the d(0.5) and D[3,2] values were reduced by one-sixth to one-eighth fold. Colour measurement revealed that the radiation process led to an increase in the yellow index and b* values for both types of GT in powder form, but it was more pronounced for AGG samples. Irradiation led to an approximate 13-fold increase in redness in AFG. Surface and shape changes of the gum crystals were studied by scanning electron microscope (SEM) and a smoother surface for irradiated samples was detected. The notable changes in functional properties of each variety of irradiated gum should be taken into consideration before using the radiation technology as a commercial tool for sterilisation.     

Gamma-ray crosslinking of poly(3-hydroxyoctanoate-co-undecenoate)

The gamma-ray crosslinking of films made of poly(3-hydroxyoctanoate) containing undecenoate moieties (up to 33 mole%) were studied. X-ray diffraction, thermal analysis, dynamic mechanical analysis (DMA), solid state nuclear magnetic resonance (NMR) spectroscopy and degree of crosslinking (swelling analysis) as a function of irradiation dose were evaluated for treatments in air or in N(2) atmosphere. After uncrosslinked material was isolated by CHCl(3) extraction, solid state NMR data suggested that only a small percentage of the double bonds took part in the formation of irradiation crosslinks. Crosslinking in N(2) was more efficient than in air and a 20 kGy dose was sufficient for optimal crosslinking. The X-ray diffraction patterns of the polymer films were unaffected by moderate irradiation. The use of sodium hypochlorite to isolate poly(3-hydroxyoctanoate-co-undecenoate) samples resulted in partial chlorination of the double bonds and considerable depolymerization.     

Effect of γ-irradiation on pasting and emulsification properties of octenyl succinylated rice starches

Octenylsuccinylated (OS) starches from waxy rice or high-amylose rice (28.1% amylose) (DS 0.023 and 0.025, respectively) were gamma-irradiated at 10, 30, or 50kGy and their pasting and thermal properties, crystallinity, and emulsification property were examined. When the OS starches were irradiated, the degrees of substitution gradually decreased as irradiation dose increased. A significant decrease in pasting viscosity was observed with an increase in irradiation dose, indicating the presence of chain degradation induced by the radiation. The melting temperature and enthalpy determined by differential scanning calorimetry increased slightly by irradiating at 10 or 30kGy. Little change in crystallinity was observed in the X-ray diffraction analysis for the OS high-amylose rice starch regardless of irradiation doses, whereas a decrease in crystallinity was observed with the OS waxy starch irradiated at 50kGy. Chain degradation induced by irradiation occurred mainly in the amorphous regions, but some loss of crystallinity occurred when the irradiation was excessive. The OS starches showed greater emulsion capacity and stability than the native counterparts due to their amphipathic nature. The irradiation further improved the emulsification properties of OS starches. The irradiation at 10kGy was optimal, and treating at higher doses decreased the emulsion capacity and stability of the OS starches.     

Molecular changes in UV-induced and γ-ray-induced mutations in human lymphoblastoid cells

We have characterized the structural changes in the hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene of 14 UV-induced, 15 γ-ray-induced and 17 spontaneous mutants of human lymphoblastoid cells selected for 6-thioguanine (6TG) resistance. Southern blot analysis using the full-length HPRT cDNA as a probe revealed that 29% (5/17) of the spontaneous mutants contained detectable alterations in their restriction fragment patterns. Among the 15 mutants induced by γ rays, 7 (47%) had such alterations indicative of large deletions in the HPRT gene. In contrast, all 14 UV-induced mutants exhibited hybridization patterns indistinguishable from those of the wild-type cells. These results suggest that UV is likely to induce point mutations at the HPRT locus on the human chromosome and that the molecular mechanism of UV-induced mutation is quite different from that of ionizing radiation-induced mutation or spontaneous mutation in human cells.     

Production, purification and properties of γ-glutamyltranspeptidase from a newly isolated Bacillus subtilis NX-2

Production, purification and properties of γ-glutamyltranspeptidase from a newly isolated Bacillus subtilis NX-2 was investigated. At the optimum conditions for enzyme formation, a high level, 3.2 U/ml of γ-GTP was obtained. The extracellular γ-GTP from this strain was purified 111.15-fold to homogeneity from the culture supernatant by acetone precipitation, hydrophobic interaction chromatography and ion exchange chromatography. The purified enzyme was a heterodimer consisting of one large subunit (43 kDa) and one small subunit (32 kDa), and exhibited high activity at 40–60 °C, pH 8.0. It preferred basic amino acids as γ-glutamyl acceptor in transpeptidation, and the stereochemistry of the γ-glutamyl acceptor had no influence on the enzyme activity, which was different from other γ-GTPs reported. Furthermore, it was proved that γ-GTP of this strain could catalyze the transfer of l-glutamine to glycylglycine to synthesize Gln–Gly–Gly, which was promising for the synthesis of valuable γ-glutamyl peptides.