Measurement of Fructan and FOS

Transcription

1 Measurement of Fructan and FOS Updated Methodology to Include Levans as well as Inulin and Agave Fructan Barry McCleary and Lucie Charmier Megazyme

2 Fructan is defined as any compound where one or more fructosyl-fructose linkages constitute a majority of the linkages Fructans include: Inulin [containing exclusively the (2-1) fructosyl-fructose linkage] e.g. chicory inulin Branched fructan [containing both (2-1) and (2-6) fructosyl-fructose linkages in significant amounts] e.g. agave, wheat and onion fructans. Levan [containing mostly or exclusively the (2-6) fructosyl-fructose linkage] e.g. Timothy grass levan

3 Timothy Grass Levan Ion-Chromatography of Fructans Onion Fructan Chicory Inulin (Raftaline) Agave Fructan Hydrolysed Chicory Inulin (Raftilose P-95) Neosugars

4 FRUCTAN MEASUREMENT Megazyme K-FRUC (AOAC Method ) FRUCTAN + SUCROSE + STARCH Sucrase + ß-Amylase + Pullulanase + Maltase FRUCTAN + FRUCTOSE + GLUCOSE Sodium borohydride/acetic acid. FRUCTAN + Non-reducing, sugar alcohols exo-inulinase + endo-inulinase FRUCTOSE + GLUCOSE PAHBAH reducing sugar determination FRUCTAN CONTENT

5 Agave Fructan (Highly Branched) (Sample with 75% fructan content) Dionex Pattern; K-FRUC enzymes/0 min Dionex Pattern; K-FRUC enzymes/20 min HPLC (TSK); K-FRUC enzymes/0 min ~ 25% HPLC (TSK); K-FRUC enzymes/5 min HPLC (TSK); K-FRUC enzymes/20 min

6 Plant Levans also called Phleins

7 Measurement of Plant Levans using K-FRUC test kit The K-FRUC method was developed for the measurement of inulin-type and branched fructans. This method employs a mixture of exo-inulinase and endo-inulinase. exo-inulinase hydrolyses both (2-1) and (2-6) fructosyl-fructose linkages in lower DP oligosaccharides, but levan is not depolymerised by endo-inulinase. For quantitative measurement of levan-type polysaccharides [(2-6) fructosylfructose linkages] the enzyme mixture needs to include an endo-levanase. Such an enzyme has been produced by Megazyme and is now included in the K-FRUC fructanase enzyme mixture in the fructan assay kit. The enzyme mixture has been evaluated using a levan-type polysaccharide purified from Timothy grass.

8 Characterisation of Timothy grass Levan Size Exclusion Chromatography of Levan SEC profiles were prepared on a Superose 12 column with a linear detection range for carbohydrates between kda. Along with samples, several dextran standards were run to determine the MW of compounds present in the samples. Time RT Mp Mw Mn Đ SAMPLE Peak M range [Da] [Da] [Da] Levan Pk ~10,000 ~12,500 ~6, NMR-spectroscopy The sample (15 mg) was deuterium-exchanged by lyophilization in D 2 O. The dry sample was redissolved in 0.5 ml D 2 O (Cambridge Isotope Laboratories, 99.96% D). The solution was transferred into 5 mm OD NMR-tube. 1-D Proton spectra were acquired on a Varian Inova-600 MHz spectrometer at 25 C. Chemical shifts were measured relative to the residual HOD signal ( H =4.78 ppm). Comments: The protons detected in the CHO ring region of the 1D-NMR of Levan B were assigned based on the chemical shift, multiplicity and proton-proton coupling values. The individual proton peaks were deconvoluted in MNova and integrated. The integration of the peaks in the 1D-NMR data shows seven proton signals between ppm, which correspond to the protons on the ring of fructose. These results coincide with previously reported bacterial levans (Shih and Yu, J. Agri Food Chem, 2005; 53: ; Matsuzaki et al. Carbohydr. Res. 2017; 448:95-102), and indicates a polysaccharide of the levan type with a linkage of β (2 6) fructofuranoside. (Report from CCRC University of Georgia).

9 Hydrolysis of Inulin and Levan by endo-inulinase and endo-levanase HPLC on TSK columns Inulin no enzyme treatment Inulin + endo-inulinase for 10 min Inulin + endo-levanase for 60 min Levan no enzyme treatment Levan + endo-inulinase for 60 min Levan + endo-levanase for 10 min

10 FRUCTAN MEASUREMENT (AOAC Method ) FRUCTAN + SUCROSE + STARCH Sucrase + ß-Amylase + Pullulanase + Maltase FRUCTAN + FRUCTOSE + GLUCOSE Sodium borohydride/acetic acid. FRUCTAN + Non-reducing, sugar alcohols exo-inulinase + endo-inulinase + endo-levanase FRUCTOSE + GLUCOSE PAHBAH reducing sugar determination FRUCTAN CONTENT

11 Hydrolysis of Fructans by Fructanase Mixture endo-inulinase and endo-levanase and exo-inulinase Raftilose+ Enz/0 min Raftilose + Enz/20 min Neosugars + Enz/0 min Neosugars + Enz/20 min Agave fructan + Enz/0 min Agave fructan + Enz/20 min Timothy Levan + Enz/0 min Timothy Levan + Enz/20 min

12 Effect of the Incorporation of endo-levanase into the K-FRUC Fructan Assay Method on the Measurement of Levans Sample exo- and endo-inulinanases exo- and endo-inulinanases Plus endo-levanase Timothy grass (sample A) Timothy grass (sample B) Rye grass Oaten hay Barley MAX (grain) Pure levan from timothy grass Fructan content, % w/w (as is basis) Pure inulin from chicory

13 Repeatability of the Megazyme K-FRUC fructan assay procedure across a Range of Fructans (inulin, levan and highly branched) Sample Raftaline Inulin (native chicory fructan) Levan (from Timothy grass) P- LEV # Frutafit Agave fructan Timothy grass # Barley max Rye Grass Brett brother swine feed Barley Flour #60301a Purina GoCat dry cat food Top spec alfalfa Fructan, % (w/w), dwb mean ±2 SD, (CV, %) Day 1 Day 2 Day 3 Day 4 Interday mean, ±2 SD, (%RSDr, %) 99.8 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

14 Repeatability of the Megazyme K-FRUC fructan assay procedure across a Range of Fructans (inulin, levan and highly branched) AOAC SMPR Megazyme (K-FRUC) Operating range (% w/w) 0.2 to to 98.4 a Limit of quantitation (LOQ) (% w/w) b RSD r, % (0.2 to 1% w/w Fructan) RSD r, % (>1 to 10% w/w Fructan) RSD r, % (>10 to 100% w/w Fructan) AOAC SMPR - Standard Method Performance Requirements a Precise range dictated by fructan content in samples tested. b Based on replicate measurements for a sample with ~ 1% (w/w) fructan. Updated K-FRUC procedure: Conclusions Allows quantitative measurement of inulin-type fructans, highly branched fructans and levan-type fructans Employs ultra-pure, recombinant enzymes (exo-inulinase, endo-inulinase and endolevanase) and therefore is specific for fructan measurement The repeatability of this method (RSD r, % ) across various samples is excellent

15 Evaluation The Megazyme K-FRUC assay procedure for the measurement of all types of fructan in foods and feeds has been accepted as a Single Laboratory Validated (SLV) method by AOAC International (2018). The next step will be to coordinate a Multi-Laboratory Validation (MLV) later this year and into Would your laboratory like to be involved?