Levan

Traditional enzyme-based methods for measurement of fructan were designed to measure just inulin and branched-type (agave) fructans. The enzymes employed, namely exo-inulinase and endo-inulinase, give incompletely hydrolysis of levan. Levan hydrolysis requires a third enzyme, endo-levanase. This paper describes a method and commercial test kit (Megazyme Fructan Assay Kit) for the determination of all types of fructan (inulin, levan, and branched) in a variety of animal feeds and pet foods. The method has been validated in a single laboratory for analysis of pure inulin, agave fructan, levan, and a range of fructan containing samples. Quantification is based on complete hydrolysis of fructan to fructose and glucose by a mixture of exo-inulinase, endo-inulinase, and endo-levanase, followed by measurement of these sugars using the PAHBAH reducing sugar method which gives the same color response with fructose and glucose. Before hydrolysis of fructan, interfering sucrose and starch in the sample are specifically hydrolyzed and removed by borohydride reduction. The single-laboratory validation (SLV) outlined in this document was performed on commercially available inulin (Raftiline) and agave fructan (Frutafit^©), levan purified from Timothy grass, two grass samples, a sample of legume hay, two animal feeds and two barley flours, one of which (Barley MAX^©) was genetically enriched in fructan through plant breeding. Parameters examined during the validation included working range, target selectivity, recovery, LOD, LOQ, trueness (bias), precision (repeatability and intermediate precision), robustness, and stability. The method is robust, quick, and simple.

Scope: Fructans are a group of dietary fibers which are known to have many beneficial effects including immune-modulating effects. A family of fructans are β-(2,6)-linked levan-type fructans that are known to serve as exopolysaccharides in the cell wall of many species of bacteria including commensal bacteria and probiotics. It is still largely unknown whether and how they can serve as immunomodulating molecules. Results: Microbial β-(2,6)-fructans were found to induce TLR-dependent activation of THP-1 cells, in a dose-dependent fashion. Low molecular weight (M_w), medium M_w and high M_w β-(2,6)-fructans activated both TLR2 and 4 in a dose- and molecular weight-dependent fashion. In addition, it was found that β-(2,6)-fructans were able to inhibit signalling of various TLRs with the strongest effect on TLR5 and 8, which were inhibited by all the β-(2,6)-fructans in a dose- and molecular weight-dependent fashion. The final effect of this activation and inhibition of TLRs on cytokine responses in human dendritic cells (DCs) was minor which may be explained by the counter-activating effects of the different β-(2,6)-linked levan-type fructans on inhibition of TLR signalling in the DCs. Conclusion: A mechanism by which exopolysaccharide levan β-(2,6)-fructans can be immune-modulating is by impacting TLR signalling. This knowledge could lead to food in which exopolysaccharide levan β-(2,6)-fructans are added for preventing disorders where TLR-signalling is modulated.

Fructans are water-soluble polymers of fructose in which fructose units are linked by β-(2 → 1) and/or β-(2 → 6) linkages. In plants, they are synthesized in the vacuole but have also been reported in the apoplastic sap under abiotic stress suggesting that they are involved in plasmalemma protection and in plant-microbial interactions. However, the lack of fructan-specific antibodies currently prevents further study of their role and the associated mechanisms of action, which could be elucidated thanks to their immunolocalization. We report the production of two monoclonal antibodies (named BTM9H2 and BTM15A6) using mice immunization with antigenic compounds prepared from a mixture of plant inulins and levans conjugated to serum albumin. Their specificity towards fructans with β-(2 → 1) and/or β-(2 → 6) linkage has been demonstrated by immuno-dot blot tests on a wide range of carbohydrates. The two mAbs were used for immunocytolocalization of fructans by epifluorescence microscopy in various plant species. Fructan epitopes were specifically detected in fructan-accumulating plants, inside cells as well as on the surface of root tips, confirming both extracellular and intracellular localizations. The two mAbs provide new tools to identify the mechanism of extracellular fructan secretion and explore the roles of fructans in stress resistance and plant-microorganism interactions.

The Hessian fly is an obligate parasite of wheat causing significant economic damage, and triggers either a resistant or susceptible reaction. However, the molecular mechanisms of susceptibility leading to the establishment of the larvae are unknown. Larval survival on the plant requires the establishment of a steady source of readily available nutrition. Unlike other insect pests, the Hessian fly larvae have minute mandibles and cannot derive their nutrition by chewing tissue or sucking phloem sap. Here, we show that the virulent larvae produce the glycoside hydrolase MdesGH32 extra-orally, that localizes within the leaf tissue being fed upon. MdesGH32 has strong inulinase and invertase activity aiding in the breakdown of the plant cell wall inulin polymer into monomers and converting sucrose, the primary transport sugar in plants, to glucose and fructose, resulting in the formation of a nutrient-rich tissue. Our finding elucidates the molecular mechanism of nutrient sink formation and establishment of susceptibility.

Fermentable oligosaccharides, disaccharides, monosaccharides, and polyols (FODMAPs) trigger symptoms of irritable bowel syndrome (IBS). Fructan degradation during bread making reduces FODMAPs in bread while maintaining the content of dietary fiber. This study explored the presence of the fructanases FruA in lactobacilli and characterized its use in bread making. FruA was exclusively present in vertebrate-adapted lactobacilli. In Lactobacillus crispatus DSM29598, FruA was located in cell wall fractions and includes a SLAP domain. FruA hydrolyzed levan or inulin; expression of fruA was not subject to catabolite repression. Fructans in bread were reduced by less than 50% in a straight dough process; conventional sourdough fermentation reduced fructans in bread by 65-70%. Sourdough fermentation with L. crispatus reduced fructans in bread by more than 90%. In conclusion, reduction of FODMAP by sourdough fermentation may improve tolerance in many IBS patients. Fermentation with FruA-expressing L. crispatus DSM29598 produces a low FODMAP bread.