Impact of the Crystallinity on the Physical Properties of Inulin during Water Sorption

The impact of the crystallinity of spray-dried inulins on their stability and physical properties was investigated after a conditioning of 1 week at different relative humidity levels (0% to 94%) at 20 °C. An environmental scanning electron microscopy study showed that the amorphous powders hardened at a relative humidity storage between 59% and 75%; while their semi-crystalline counterparts were partially agglomerated but friable in the same conditions. Caking was observed when the glass transition temperature of the amorphous phase of the material dropped below the storage temperature of the powder. It resulted in a crystallization of the structural units of varying lengths composing inulin, but also an increase of the crystallinity of the semi-crystalline ones. This study showed the importance of the crystallinity of inulin on its stability and physical properties during storage which is of crucial importance for the shelf-life of food and pharmaceutical products in the dry state. Financial support was provided for this study by the Walloon Region of Belgium (DGTRE) and Cosucra Groupe Warcoing SA.     

Purification and characterization of a heat stable inulin fructotransferase (DFA I-producing) from Arthrobacter pascens a62-1

An inulin fructotransferase (DFA I-producing) [EC 2.4.1.200] from Arthrobacter pascens a62-1 was purified and the properties of the enzyme were investigated. The enzyme was purified from culture supernatant of the microorganism 58.5 fold with a yield of 8.32% using Super Q Toyopearl chromatography and butyl Toyopearl chromatography. It showed maximum activity at pH 5.5 and 45 °C and was stable up to 75 °C. This heat stability was highest in the inulin fructotransferases (DFA I-producing) reported until now. The molecular mass of the enzyme was estimated to be 37,000 by SDS-PAGE and 60,000 by gel filtration, and was considered to be a dimer. The N-terminal amino acid sequence (20 amino acid residues) was determined as Ala-Asn-Thr-Val-Tyr-Asp-Val-Thr-Thr-Trp-Ser-Gly-Ala-Thr-Ile-Ser-Pro-Tyr-Val-Asp.     

Trends in inulinase production – a review

This article highlights the research work carried out in the production of inulinases from various inulin substrates using strains of bacteria, yeast and fungi. Inulin is one of the numerous polysaccharides of plant origin that contains glucose or fructose. It is used as a substrate in industrial fermentation processes and in food industries due to its relatively cheap and abundant source for the microbiological production of high-fructose syrups, ethanol and acetone–butanol. The various oligosaccharides derived from inulin also find their application in the medical and dietary sector. The inulinase acts on the β-(2,1)-D-fructoside links in inulin releasing D-fructose. Hence, this article illustrates the capability of various microbes in hydrolyzing the carbon at its optimum nutrient concentration and operating condition towards inulinase production.     

Characterization of the Physical State of Spray-Dried Inulin

Modulated differential scanning calorimetry, wide angle x-ray scattering, and environmental scanning electron microscopy were used to investigate the physical and morphological properties of chicory root inulin spray dried under different conditions. When the feed temperature increased up to 80 °C, the average degree of polymerization of the solubilized fraction increased, leading to a higher glass transition temperature (Tg). Above 80 °C, the samples were completely amorphous, and the Tg did not change. The starting material was semicrystalline, and the melting region was composed of a dual endotherm; the first peak subsided as the feed temperature increased up to a temperature of 70 °C, whereas above 80 °C, no melting peak was observed as the samples were completely amorphous. To a lesser extent, the inlet air temperature of 230 °C allowed a higher amorphous content of the samples than at 120–170 °C but induced a blow-out of the particles.     

Influence of oligosaccharides on the viability and membrane properties of Lactobacillus reuteri TMW1.106 during freeze-drying

Freeze-drying is a process commonly used in starter culture preparation. To improve the survival rate of bacteria during the process, cryoprotectives are usually added before freezing. This study investigated the influence of the addition of sucrose, fructo-oligosaccharides (FOS), inulin and skim milk on the viability and membrane integrity of Lactobacillus reuteri TMW1.106 during freezing, freeze-drying and storage. The effect of drying adjuncts on survival was correlated to their interaction with bacterial membrane by determination of the parameters membrane fluidity and membrane lateral pressure. Sucrose, FOS and skim milk significantly enhanced survival of exponential-phase cells of L. reuteri during freeze-drying. Cellular viability during storage of exponential-phase cells remained highest for cells dried in the presence of skim milk and inulin. Membranes of these cells were completely permeabilized after freeze-drying. The application of FOS significantly improved survival of stationary phase cells of L. reuteri TMW1.106 after freeze-drying and storage. This increased viability of L. reuteri TMW1.106 in the presence of FOS correlated to improved membrane integrity. Fructo-oligosaccharides and fructans, but not gluco-oligosaccharides interacted with membrane vesicles prepared from L. reuteri TMW1.106 as indicated by increased membrane lateral pressure in the presence of FOS and fructans. Increased membrane integrity of stationary phase L. reuteri TMW1.106 was attributed to direct interactions between FOS and the membrane which leads to increased membrane fluidity and thus improved stability of the membrane during and rehydration.     

NMR structural study of fructans produced by Bacillus sp. 3B6, bacterium isolated in cloud water

Bacillus sp. 3B6, bacterium isolated from cloud water, was incubated on sucrose for exopolysaccharide production. Dialysis of the obtained mixture (MWCO 500) afforded dialyzate (DIM) and retentate (RIM). Both were separated by size exclusion chromatography. RIM afforded eight fractions: levan exopolysaccharide (EPS), fructooligosaccharides (FOSs) of levan and inulin types with different degrees of polymerization (dp 2–7) and monosaccharides fructose:glucose = 9:1. Levan was composed of two components with molecular mass ∼3500 and ∼100 kDa in the ratio 2.3:1. Disaccharide fraction contained difructose anhydride DFA IV. 1-Kestose, 6-kestose, and neokestose were identified as trisaccharides in the ratio 2:1:3. Fractions with dp 4–7 were mixtures of FOSs of levan (2,6-βFruf) and inulin (1,2-βFruf) type. DIM separation afforded two dominant fractions: monosaccharides with fructose: glucose ratio 1:3; disaccharide fraction contained sucrose only. DIM trisaccharide fraction contained 1-kestose, 6-kestose, and neokestose in the ratio1.5:1:2, penta and hexasaccharide fractions contained FOSs of levan type (2,6-βFruf) containing α-glucose. In the pentasaccharide fraction also the presence of a homopentasaccharide composed of 2,6-linked βFruf units only was identified. Nystose, inulin (1,2-βFruf) type, was identified as DIM tetrasaccharide. Identification of levan 2,6-βFruf and inulin 1,2-βFruf type oligosaccharides in the incubation medium suggests both levansucrase and inulosucrase enzymes activity in Bacillus sp. 3B6.     

Determination of the structure and degree of polymerisation of fructans from Echinacea purpurea roots

Highly water soluble fructans have been isolated from Echinacea purpurea (L.) Moench. roots by hot water extraction and precipitation at three different ethanol concentrations (80% v/v, 60% v/v and 40% v/v). The structure of the fructans has been characterised by three analytical methods: GC of silylated oxime derivatives and partially methylated alditol acetates, respectively, as well as 13C NMR analysis. The mean degree of polymerisation (mean DP) of each fructan has been determined by the glucose/fructose ratio. E. purpurea fructans represent linear inulin-type fructans with almost exclusively beta-(2-->1)-linked fructosyl units, terminal glucose and terminal fructose. Small proportions of beta-(2-->1,2-->6)-linked branch point residues were detected. The mean DP of the fructan fractions depends on the ethanol concentration used for precipitation: the lower the ethanol concentration the higher the mean DP. Corresponding results were found with all of the three analytical methods: 80% ethanol-insoluble fructan from E. purpurea shows an average mean DP of 35, 60% ethanol-insoluble fructan of 44 and 40% ethanol-insoluble fructan of 55. The applied methods provide sufficient sensitivity to determine not only the composition and structure but also the mean degree of polymerisation of fructans.     

Effects of cellulose, carboxymethylcellulose and inulin fed to rats as single supplements or in combinations on their caecal parameters

We compared the effect of diets containing different nondigestible carbohydrates: cellulose (C), inulin (IN) and carboxymethylcellulose (CMC) as single supplements or in dietary combination on caecal physiology of rats. Sixty male Wistar rats (Rattus norvegicus) were divided into five groups and for 4 weeks were fed a casein diet with the compared carbohydrates (4% of diet) or a combination of IN+C or IN+CMC (both 4+4%). Diet intake and FCR index remained unaffected by the treatments, whereas IN improved the body weight gain of rats compared to CMC. Compared to C group, all diets containing IN and CMC decreased the caecal pH as well as enlarged the caecum, thus increasing the weights of contents and tissue, especially upon CMC treatment. Rats given carboxymethylcellulose (CMC and IN+CMC groups) had watery caecal digesta, and some of them suffered from diarrhoea. In the case of CMC, the caecal enlargement was due to tissue hypertrophy and digesta accumulation mostly in response to an increased bulk of contents. Unlike C+IN, the dietary combination of CMC- and inulin-enhanced fermentation in the caecum of rats, however the proportion of acetate, propionate and butyrate was less beneficial. Compared to CMC, inulin gave a higher concentration of SCFA, especially of butyrate and propionate. The action of inulin in the caecum of rats could be pronounced by dietary treatment combined with CMC.     

Purification and characterization of fructan: fructan fructosyl transferase from chicory (Cichorium intybus L.) roots

Fructan: fructan fructosyl transferase (FFT, EC 2.4.1.100) was purified from chicory (Cichorium intybus L. var. foliosum cv. Flash) roots by a combination of ammonium sulfate precipitation, concanavalin A affinity chromatography, and anion- and cation-exchange chromatography. This protocol produced a 60-fold purification and a specific activity of 14.5 mol·(mg protein) ^–1·min^–1. The mass of the enzyme was 69 kDa as estimated by gel filtration. On sodium dodecyl sulfatepolyacrylamide gel electrophoresis and mass spectrometry, 52-kDa and 17-kDa fragments were found, suggesting that the enzyme was a heterodimer. Optimal activity was found between pH 5.5 and 6.5. The enzyme used 1-kestose, 1,1-nystose, oligofructan and commercial chicory root inulin (degree of polymerization 10) as donors and acceptors. Sucrose was the best acceptor but could not be used as a donor. However, at higher concentrations sucrose acted as a competitive inhibitor for donors of FFT. 1-Kestose was the most efficient and 1,1-nystose the least efficient donor. The purified enzyme exhibited -fructosidase activity, specially at higher temperatures and lower substrate concentrations. The synthesis of fructans from 1-kestose decreased at higher temperatures (5–50°C). Therefore enzyme assays were performed at 0°C. The same fructan oligosaccharides, with a distribution similar to that observed in vivo, were obtained upon incubation of the enzyme with sucrose and commercial chicory root inulin.Abbreviations Con A concanavalin A - DP degree of polymerization - FFT fructan: fructan fructosyl transferase - Fru fructose - Glc glucose - Kes 1-kestose - MALDI-TOF MS matrix-assisted laser desorption ionisation time of flight mass spectrometry - Nys 1,1-nystose - pI isoelectric point - SST sucrose: sucrose fructosyl transferase - Suc sucrose The authors would like to thank E. Nackaerts for valuable assistance. W. Van den Ende is also grateful to the National Fund for Scientific Research (NFSR Belgium) for giving a grant for research assistants. P. Verhaert is a research associate of the NFSR. This work was also supported by grant OT/91/18 from the Research Fund K.U. Leuven.     

Quantification of the permeability of the blood-CSF barrier to alpha-MSH in the rat

The ability of alpha-MSH to cross the blood-CSF barrier of the rat was assessed by measurement of the rate of appearance of immunoreactive alpha-MSH in a cerebrospinal fluid (CSF) perfusate following intravenous injection of peptide. Comparisons were made with the rate of appearance of a simultaneously administered dose of 14C-inulin which is poorly permeable at the blood-CSF barrier. Concentrations of drugs measured in plasma were fitted to two-compartment pharmacokinetic models, and those measured in the CSF perfusate to one-compartment open systems receiving an input from the plasma compartment. The rate constant for entry of alpha-MSH into CSF was 0.00087 min-1, which was not significantly different from that for inulin of 0.00055 min-1. As alpha-MSH penetrated into CSF at a rate comparable to inulin, it was concluded that the limited entry of peptide was by aqueous diffusion along with other water-soluble macromolecules.