Quantave Measurement of Fructan (Inulin, Levan and Branched) in Animal Feeds Barry V. McCleary and Lucie M. J. Charmier Megazyme, Bray Business Park, Bray, Co. Wicklow, Ireland

Fructans are recognised health-promong carbohydrates, composed of fructosyl Assay improvement and performance units with a terminal D-glucosyl residue. These carbohydrates may be linear or endo-Levanase was added to the exisng fructanase mixture containing exo- branched with varying degrees of polymerisaon (DP) and complexity. They are and endo-inulinases in the standard K-FRUC procedure. The modified classified based on the glycosidic linkages present, with three major types procedure was validated on a range of pure fructans from various sources to 1 idenfied. demonstrate that quantave hydrolysis was obtained (Figure 4). A) B) Inulin [containing Branched fructan exclusively (2-1) [containing both (2-1) and fructosyl-fructose (2-6) fructosyl-fructose linkages] e.g. linkages in significant chicory inulin amounts] e.g. agave, wheat and onion fructans.

Levan [containing mostly or exclusively (2-6) fructosyl-fructose linkages] e.g. Timothy grass levan. C) D)

Two methods have tradionally been used to measure fructan; AOAC 999.03, an enzymac/spectrophotometric procedure, and AOAC 997.08, an enzymac/ HPAEC-PAD based alternave. The Fructan Assay Kit as developed by Megazyme (K-FRUC ; AOAC Method 999.03) 2,3 was designed to measure inulin-type fructans. The method accurately measures inulin and branched fructans, but significantly underesmates the levan-type fructans as present in pasture grasses4. To extend the applicaon of this method, Megazyme has produced an endo-levanase and added this to the exo-/endo-inulinase mixture that is currently used in the assay. Figure 4: HPLC analysis of hydrolysis of different types of fructans A) Railose (β-2,1 linked fructooligosaccharides with DP 2-8), B) Agave (β-2,1 linked fructan containing extensive β-2,6 branch points with ~DP ≤30), C) Timothy levan (DP β-2,6 linked fructan with DP~70) and D) Neosugars (β-2,1 linked fructooligosaccharides with DP 2-5). Fructan content was determined in various grass samples with exo- and endo- inulinases, both with and without added endo-levanase.

Fructan content, % w/w (as is basis) Sample exo- and endo-inulinases exo- and endo-inulinases plus endo-levanase Timothy grass (sample A) 4.9 13.8 Timothy grass (sample B) 3.2 6.2 Rye grass 8.9 9.9 Oaten hay 10.7 10.9 Barley MAX (grain) 12.8 12.8 Pure levan from mothy grass 59.2 91.2 Pure inulin from chicory 95.0 92.3 + endo-Levanase

Figure 5: Fructan content of various samples determined using exo- and endo-inulinases with and without endo-levanase added.

Assay reproducibility/applicability/robustness

Figure 1: Schemac representaon of Megazyme K-FRUC method (AOAC 999.03 method); showing the point of inclusion of endo-levanase Fructan content was determined in various samples matrices such as pure linear and The current fructan method (K-FRUC, Figure 1) quanvely measures inulin and branched fructans; forages and grasses and animal feed and ingredients to assess highly branched fructans. For commercial Agave fructan (which contains branched reproducibility, applicability and robustness of this method fructan), a fructan content of just 75% is obtained. This is not due to incomplete hydrolysis of the fructan, but rather to the high levels of fructose, glucose and sucrose in this material (Figure 2) (which are not fructan, so are not measured).

Figure 2: HPLC analysis showing 0, 5 and 20 minute incubaons of commercial Agave fructan with the endo-/exo-inulinase mixture. The highlighted region between 33-38 minutes corresponds to mono- and disaccharides. The K-FRUC assay procedure (AOAC Method 999.03) underesmates levan. To address this limitaon and allow quantave measurement of this fructan, a recombinant endo-levanase was produced and added into the fructanase mixture in the K-FRUC assay kit. Levan polysaccharide was purified from Timothy grass (Phleum pretense), and incubaons have been performed to determine the extent of hydrolysis of Timothy levan (Figure 3). Timothy levan is completely hydrolysed to mono- and di-saccharides Figure 6: Repeatability studies across a variable range of matrices. by endo-levanase, but is not hydrolysed by endo-inulinase.

Updated K-FRUC procedure is a validated, robust and reliable method that: • Allows quantave measurement of inulin-type fructans, highly branched fructans and levan-type fructans – i.e. all types of fructan • Employs ultra-pure, recombinant enzymes (exo-inulinase, endo-inulinase and endo- levanase) and therefore is specific for fructan measurement

Figure 3: HPLC analysis of incubaons of levan polysaccharide with A) no enzyme treatment, B) endo-levanase aer 10 min and C) endo-inulinase 1. Lewis, D. H. (1993). In: New Phytologist 124, 583-595.. 3. McCleary, B. V. et al (2000). In: J. AOAC Internaonal, 83, 356-364. aer 60 minutes. 2. McCleary, B. V et al (1999). In: Cereal Foods World, 44,. 398-406 4. Longland, A. C. et al (2011): In: J. Sci. Food Agric, 92, 1878-188