Analysis of carbohydrates by anion exchange chromatography and mass spectrometry

A versatile liquid chromatographic platform has been developed for analysing underivatized carbohydrates using high performance anion exchange chromatography (HPAEC) followed by an inert PEEK splitter that splits the effluent to the integrated pulsed amperometric detector (IPAD) and to an on-line single quadrupole mass spectrometer (MS). Common eluents for HPAEC such as sodium hydroxide and sodium acetate are beneficial for the amperometric detection but not compatible with electrospray ionisation (ESI). Therefore a membrane-desalting device was installed after the splitter and prior to the ESI interface converting sodium hydroxide into water and sodium acetate into acetic acid. To enhance the sensitivity for the MS detection, 0.5 mmol/l lithium chloride was added after the membrane desalter to form lithium adducts of the carbohydrates. To compare sensitivity of IPAD and MS detection glucose, fructose, and sucrose were used as analytes. A calibration with external standards from 2.5 to 1000 pmole was performed showing a linear range over three orders of magnitude. Minimum detection limits (MDL) with IPAD were determined at 5 pmole levels for glucose to be 0.12 pmole, fructose 0.22 pmole and sucrose 0.11 pmole. With MS detection in the selected ion mode (SIM) the lithium adducts of the carbohydrates were detected obtaining MDL's for glucose of 1.49 pmole, fructose 1.19 pmole, and sucrose 0.36 pmole showing that under these conditions IPAD is 3-10 times more sensitive for those carbohydrates. The applicability of the method was demonstrated analysing carbohydrates in real world samples such as chicory inulin where polyfructans up to a molecular mass of 7000 g/mol were detected as quadrupoly charged lithium adducts. Furthermore mono-, di-, tri-, and oligosaccharides were detected in chicory coffee, honey and beer samples.     

Hydrolytic Methods for the Quantification of Fructose Equivalents in Herbaceous Biomass

A low, but significant, fraction of the carbohydrate portion of herbaceous biomass may be composed of fructose/fructosyl-containing components (“fructose equivalents”); such carbohydrates include sucrose, fructooligosaccharides, and fructans. Standard methods used for the quantification of structural-carbohydrate-derived neutral monosaccharide equivalents in biomass are not particularly well suited for the quantification of fructose equivalents due to the inherent instability of fructose in conditions commonly used for hemicellulose/cellulose hydrolysis (>80% degradation of fructose standards treated at 4% sulfuric acid, 121°C, 1 h). Alternative time, temperature, and acid concentration combinations for fructan hydrolysis were considered using model fructans (inulin, β-2,1, and levan, β-2,6) and a grass seed straw (tall fescue, Festuca arundinacea) as representative feedstocks. The instability of fructose, relative to glucose and xylose, at higher acid/temperature combinations is demonstrated, all rates of fructose degradation being acid and temperature dependent. Fructans are shown to be completely hydrolyzed at acid concentrations well below that used for the structural carbohydrates, as low as 0.2%, at 121°C for 1 h. Lower temperatures are also shown to be effective, with corresponding adjustments in acid concentration and time. Thus, fructans can be effectively hydrolyzed under conditions where fructose degradation is maintained below 10%. Hydrolysis of the β-2,1 fructans at temperatures ≥50°C, at all conditions consistent with complete hydrolysis, appears to generate difructose dianhydrides. These same compounds were not detected upon hydrolysis of levan, sucrose, or straw components. It is suggested that fructan hydrolysis conditions be chosen such that hydrolysis goes to completion; fructose degradation is minimized, and difructose dianhydride production is accounted for.     

Investigation of stability,consistency, and oil oxidation of emulsion filled gel prepared by inulin and rice bran oil using ultrasonic radiation

Inulin, rice bran oil and rosemary essential oil were used to produce high quality emulsion filled gel (EFG) using ultrasonic radiation. Response surface methodology was used to investigate the effects of oil content, inulin content and power of ultrasound on the stability and consistency of prepared EFG. The process conditions were optimized by conducting experiments at five different levels. Second order polynomial response surface equations were developed indicating the effect of variables on EFG stability and consistency. The oil content of 18%; inulin content of 44.6%; and power of ultrasound of 256 W were found to be the optimum conditions to achieve the best EFG stability and consistency. Microstructure and rheological properties of prepared EFG were investigated. Oil oxidation as a result of using ultrasonic radiation was also investigated. The increase of oxidation products and the decrease of total phenolic compounds as well as radical scavenging activity of antioxidant compounds showed the damaging effect of ultrasound on the oil quality of EFG.     

Complexation of maltodextrin-based inulin and green tea polyphenols via different ultrasonic pretreatment

Ultrasound has been applied in food processing for various purpose, showing potential to advance the physical and chemical modification of natural compounds. In order to explore the effect of ultrasonic pretreatment on the complexation of inulin and tea polyphenols (TPP), different frequencies (25, 40, 80 kHz) and output power (40, 80, 120 W) were carried out. According to the comparison in particle size distribution and phenolic content of different inulin-TPP complexes, it was indicated that high-intensity ultrasonic (HIU) treatment (25 kHz, 40 W, 10 min) could accelerate the interaction of polysaccharides and polyphenols. Moreover, a series of spectral analysis including UV–Vis, FT-IR and NMR jointly evidenced the formation of hydrogen bond between saccharides and phenols. However, the primary structure of inulin and the polysaccharide skeleton were not altered by the combination. Referring to field emission scanning electron microscopy (FESEM), the morphology of ultrasound treated-complex presented a slight agglomeration in the form of bent sheets, compared to non-treated sample. The inulin-TPP complex also revealed better stability based on thermogravimetric analysis (TGA). Thus, it can be speculated from the identifications that proper ultrasonic treatment is promising to promote the complexation of some food components during processing.     

Inulin Hydrolysis by Immobilized Inulinase on Functionalized Magnetic Nanoparticles Using Soy Protein Isolate and Bovine Serum Albumin

Inulin hydrolysis was performed by inulinase from Aspergillus niger covalently immobilized on magnetite nanoparticles (Fe₃O₄) covered with soy protein isolate (Fe₃O₄/SPI) functionalized by bovine serum albumin (Fe₃O₄/SPI/BSA) nanoparticles as a new bio‐functional carrier. The specific activity and protein content of the immobilized enzyme were 25.99 U/mg and 3.52 mg/mL, respectively, with 80% enzyme loading. The immobilized inulinase showed maximum activity at 45 °C, which is 5 °C higher than the optimum temperature of the free enzyme. Also, the optimum pH of the immobilized enzyme shifted from 6 to 5.5, which is more acidic compared to that of the free enzyme. The K_m value of immobilized inulinase decreased to 2.03 mg/mL. Thermal stability increased considerably at 65 and 75 °C, and a 5.13‐fold rise was detected in the enzyme half‐life at 75 °C after immobilization. Moreover, 80% of initial activity of immobilized inulinase remained after 10 cycles of hydrolysis.     

Isolation and identification of inulooligosaccharides resulting from inulin hydrolysis

In this study, inulooligosaccharides (F_n-type inulin) resulting from the endo-inulinase hydrolysis of globe artichoke inulin were purified and characterized. The aim was to produce F_n oligomer standards with the intention of identifying them in the complex inulin chromatogram. Inulin was extracted from globe artichoke and presented a high average degree of polymerization (DP) of about 80 as determined by high-performance anion exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD). This inulin was hydrolyzed by a commercial endo-inulinase yielding a product with a very high F_n/GF_n molecule ratio, thus limiting the interference of GF_n during the purification process. High-performance size exclusion chromatography was used to individually isolate and collect each retention peak corresponding to a specific oligomer. The purity of these fractions was checked by HPAEC-PAD and showed that relatively pure molecules were produced. Electrospray ionization mass spectrometry allowed the molecular weight determination of these purified oligomers and ascertained their DP as F₂, F₃ and F₄. These F_2-4 standards were used with glucose, fructose, sucrose and GF_2-4 (commercially available) to spike commercial oligofructose products in order to determine the elution profile in the HPAEC-PAD chromatogram.     

常压下酸催化菊糖水解反应动力学研究

研究了菊芋中菊糖的酸催化水解。通过改变常压下菊糖酸催化水解的反应条件研究了反应动力学 ,得出该反应符合一级串连反应规律的结论     

Purification and Characterization of Extracellular Inulinase from a Marine Yeast <Emphasis Type="Italic">Cryptococcus aureus</Emphasis> G7a and Inulin Hydrolysis by the Purified Inulinase

The extracellular inulinase in the supernatant of the cell culture of the marine yeast Cryptococcus aureus G7a was purified to homogeneity with a 7.2-fold increase in specific inulinase activity compared to that in the supernatant by ultrafiltration, concentration, gel filtration chromatography (Sephadex™ G-75), and anion exchange chromatography (DEAE sepharose fast flow anion exchange). The molecular mass of the purified enzyme was estimated to be 60.0 kDa. The optimal pH and temperature of the purified enzyme were 5.0 and 50 °C, respectively. The enzyme was activated by Ca²⁺, K⁺, Na⁺, Fe²⁺, and Zn²⁺. However, Mg²⁺, Hg²⁺, and Ag⁺ acted as inhibitors in decreasing the activity of the purified inulinase. The enzyme was strongly inhibited by phenylmethanesulphonyl fluoride (PMSF), iodoacetic acid, EDTA, and 1,10-phenanthroline. The K _m and V _max values of the purified enzyme for inulin were 20.06 mg/ml and 0.0085 mg/min, respectively. A large amount of monosaccharides were detected after the hydrolysis of inulin with the purified inulinase, indicating the purified inulinase had a high exoinulinase activity.     

Effect of diffusion in solid acid catalyzed inulin hydrolysis

Hydrolysis of inulin was investigated employing various solid acids as hydrolytic catalysts. The catalytic performances of several ion exchange resins were compared. The effects of the particle size and porosity on the hydrolysis reaction were found to be significant, indicating that the intraparticle diffusion of reactants controls the reaction rate. Theoretical models were developed, therefore, to analyze the effects of intraparticle diffusion occurring in a sequential reaction. The results were found to be consistent with experimental observations regarding the adverse effects of pore diffusion and the reaction pattern.     

Purification and characterization of an exoinulinase from Aspergillus fumigatus

An extracellular exoinulinase was purified from the crude extract of Aspergillus fumigatus by ammonium sulfate precipitation, followed by successive chromatographies on DEAE-Sephacel, Sephacryl S-200, concanavalin A-linked amino-activated silica, and Sepharose 6B columns. The enzyme was purified 25-fold, and the specific activity of the purified enzyme was 171 IU/mg of protein. Gel filtration chromatography revealed a molecular weight of about 200 kDa, and native polyacrylamide gel electrophoresis (PAGE) showed an electrophoretic mobility corresponding to a molecular weight of about 176.5 kDa. Sodium dodecyl sulfate-PAGE analysis revealed three closely moving bands of about 66, 62.7, and 59.4 kDa, thus indicating the heterotrimeric nature of this enzyme. The purified enzyme appeared as a single band on isoelectric focusing, with a pI of about 8.8. The enzyme activity was maximum at pH 5.5 and was stable over a pH range of 4.0–9.5, and the optimum temperature for enzyme activity was 60°C. The purified enzyme retained 35.9 and 25.8% activities after 4 h at 50 and 55°C, respectively. The inulin hydrolysis activity was completely abolished with 1 mM Hg⁺⁺, whereas EDTA inhibited about 63% activity. As compared to sucrose, stachyose, and raffinose, the purified enzyme had lower K _m (0.25 mM) and higher V _max (333.3 IU/mg) values for inulin.