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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.

Until recently, only two AOAC Official MethodsSM have been available for the analysis of fructans: Method 997.08 and Method 999.03. Both are based on the analysis of the fructan component monosaccharides (glucose and fructose) after hydrolysis. The two methods have some limitations due to the strategies used for removing background interferences (such as from sucrose, α-glucooligosaccharides, and free sugars). The method described in this paper has been developed to overcome those limitations. The method is largely based on Method 999.03 and uses combined enzymatic and SPE steps to remove the interfering components without impacting the final analytical result. The method has been validated in two laboratories on infant formula and adult nutritionals. Recoveries were in the range of 86-119%, with most being in the range of 91-104%. RSDr values were in the range of 0.7-2.6%, with one exception when the fructan concentration was close to the LOQ, resulting in an RSDr of 8.9%. The performance is generally within the requirements outlined in the AOAC Standard Method Performance Requirements (SMPR® 2014.002), which specifies recoveries in the range of 90-110% and RSDr values below 6%.

A method for fructan analysis designed to comply with AOAC Standard Method Performance Requirements (SMPR®) 2014.002 is described. It is closely related to existing methods for fructan analysis, including AOAC 997.08 and 999.03, as well as a method previously published by Cuany et al. This new method achieves LOQ of 0.03% fructan on a ready-to-feed (RTF) basis with mean recoveries ranging from 93 to 108% in the presence of up to 9% sucrose (even at the 0.03% level of fructan). Repeatability ranged from 1.09 to 3.67%. Intermediate precision ranged from 2.46 to 6.79%. Sample preparation for quantitative analysis is simplified compared to some of the existing methodologies. The method incorporates a qualitative profile analysis to determine fructan size category. This allows assignment of appropriate correction factors without independent knowledge of fructan type.

A number of methods have previously been developed for the determination of fructans in food products, resulting in the frequently used AOAC Official Methods 997.08 and 999.03. Method 997.08 has a lower LOQ, but the accuracy may suffer if significant quantities of sucrose are present. Method 999.03 is less affected by sucrose, but the LOQ is not as low as that of the other method. In this work we have combined these two methods, using the sample preparation steps of AOAC Method 999.03 to remove sucrose and other free sugars, and the detection procedure of AOAC Method 997.08 to achieve a low LOQ. The resulting new method achieved an LOQ of 0.13 g/100 g with powdered products (using 1 g sample weight) and recoveries in the range of 83-103% in the presence of sucrose at 12 g/100 g. Expanded measurement uncertainties were calculated and ranged from +/-17.4% at oligofructose and fructooligosaccharide (FOS) concentrations of around 0.2 g/100 g to -/+21.0% at FOS concentrations of 0.6 g/100 g.

Fructans are water-soluble fructose oligomers and polymers that are based on sucrose, and have been implicated in protecting plants against water stress. Rice (Oryza sativa L.) is highly sensitive to chilling temperatures, and is not able to synthesize fructans. Two wheat fructan-synthesizing enzymes, sucrose:sucrose 1-fructosyltransferase, encoded by wft2, or sucrose:fructan 6-fructosyltransferase, encoded by wft1, were introduced into rice plants, and rice transformants that accumulate fructans were successfully obtained. The mature leaf blades of transgenic rice lines with wft2 or wft1 accumulated 16.2 mg g(-1) FW of oligo- and polysaccharides mainly composed of inulin oligomers of more than DP7, and 3.7 mg g(-1) FW of oligo- and polysaccharides, mainly composed of phlein oligomers of more than DP15, respectively. The transgenic rice seedlings with wft2 accumulated significantly higher concentrations of oligo- and polysaccharides than non-transgenic rice seedlings, and exhibited enhanced chilling tolerance. The oligo- and polysaccharide concentrations of seedlings expressing wft1 were obviously lower than those of lines expressing wft2, and no correlation between oligo- and polysaccharide concentrations and chilling tolerance was detected in wft1-expressing rice lines. The results suggest that transgenic rice lines expressing wheat-derived fructosyltransferase genes accumulated large amounts of fructans in mature leaf blades and exhibited enhanced chilling tolerance at the seedling stage. This is the first report owing that fructan accumulation enhanced tolerance to non-freezing low temperatures.

With the recognition that resistant starch (RS) and nondigestible oligosaccharides (NDO) act physiologically as dietary fiber (DF), a need has developed for specific and reliable assay procedures for these components. The ability of AOAC DF methods to accurately measure RS is dependent on the nature of the RS being analyzed. In general, NDO are not measured at all by AOAC DF Methods 985.29 or 991.43, the one exception being the high molecular weight fraction of fructo-oligosaccharides. Values obtained for RS, in general, are not in good agreement with values obtained by in vitro procedures that more closely imitate the in vivo situation in the human digestive tract. Consequently, specific methods for the accurate measurement of RS and NDO have been developed and validated through interlaboratory studies. In this paper, modifications to AOAC fructan Method 999.03 to allow accurate measurement of enzymically produced fructo-oligosaccharides are described. Suggested modifications to AOAC DF methods to ensure complete removal of fructan and RS, and to simplify pH adjustment before amyloglucosidase addition, are also described.

We isolated two cDNAs of winter wheat (Triticum aestivum L.), designated wft1 and wft2, which encoded sucrose:fructan 6-fructosyltransferase (6-SFT) and sucrose:sucrose 1-fructosyltransferase (1-SST; EC 2.4.1.99), respectively, which are involved in the synthesis of fructan in wheat. wft1 and wft2 were cloned by screening of a cDNA library with probed-cDNA fragments corresponding to plant fructosyltransferase and invertase. The identity of the clones was verified by functional characterization of recombinant proteins expressed in methylotrophic yeast, Pichiapastoris. Northern blotting showed that the level of wft2 transcripts increased from autumn to early winter in the crown tissues of all field-grown wheat cultivars examined. Higher levels of wft1 and wft2 transcripts were found in leaf tissues of snow mold-resistant cultivars, which accumulated more fructan than other cultivars. Our results showed that Wft1 and Wft2 were important in fructan accumulation during cold hardening of winter wheat.

A study was made of the effect of the activity and purity of enzymes in the assay of total dietary fiber (AOAC Method 985.29) and specific dietary fiber components: resistant starch, fructan, and beta-glucan. In the measurement of total dietary fiber content of resistant starch samples, the concentration of alpha-amylase is critical; however, variations in the level of amyloglucosidase have little effect. Contamination of amyloglucosidase preparations with cellulase can result in significant underestimation of dietary fiber values for samples containing beta-glucan. Pure beta-glucan and cellulase purified from Aspergillus niger amyloglucosidase preparations were used to determine acceptable critical levels of contamination. Sucrose, which interferes with the measurement of inulin and fructooligosaccharides in plant materials and food products, must be removed by hydrolysis of the sucrose to glucose and fructose with a specific enzyme (sucrase) followed by borohydride reduction of the free sugars. Unlike invertase, sucrase has no action on low degree of polymerization (DP) fructooligosaccharides, such as kestose or kestotetraose. Fructan is hydrolyzed to fructose and glucose by the combined action of highly purified exo- and endo-inulinases, and these sugars are measured by the p-hydroxybenzoic acid hydrazide reducing sugar method. Specific measurement of beta-glucan in cereal flour and food extracts requires the use of highly purified endo-1,3:1,4 beta-glucanase and A. niger beta-glucosidase. Beta-glucosidase from almonds does not completely hydrolyze mixed linkage beta-glucooligosaccharides from barley or oat beta-glucan. Contamination of these enzymes with starch, maltosaccharide, or sucrose-hydrolyzing enzymes results in production of free glucose from a source other than beta-glucan, and thus an overestimation of beta-glucan content. The glucose oxidase and peroxidase used in the glucose determination reagent must be essentially devoid of catalase and alpha- and beta-glucosidase.

An AOAC collaborative study was conducted to evaluate the accuracy and reliability of an enzyme assay kit procedure for measuring oligofructans and fructan polysaccharide (inulins) in mixed materials and food products. The sample is extracted with hot water, and an aliquot is treated with a mixture of sucrase (a specific sucrose-degrading enzyme), alpha-amylase, pullulanase, and maltase to hydrolyze sucrose to glucose and fructose, and starch to glucose. These reducing sugars are then reduced to sugar alcohols by treatment with alkaline borohydride solution. The solution is neutralized, and excess borohydride is removed with dilute acetic acid. The fructan is hydrolyzed to fructose and glucose using a mixture of purified exo- and endo-inulinanases (fructanase mixture). The reducing sugars produced (fructose and glucose) are measured with a spectrophotometer after reaction with para-hydroxybenzoic acid hydrazide. The samples analyzed included pure fructan, chocolate, low-fat spread, milk powder, vitamin tablets, onion powder, Jerusalem artichoke flour, wheat stalks, and a sucrose/cellulose control flour. Repeatability relative standard deviations ranged from 2.3 to 7.3%; reproducibility relative standard deviations ranged from 5.0 to 10.8%.

D-Fructans synthesized from sucrose by cell-free systems of strains of Streptococcus mutans and Strep. salivarius have been shown by methylation and enzymatic studies to have different glycosidic linkages. The cold water-insoluble D-fructans from Strep. mutans strain BHT and JC-1 have inulin-type structures consisting beta-(2 leads to 1)-D-fructofuranosidic linkages, with average repeating units of 8 and 27 sugar residues, respectively, whereas the water-soluble fructan from Strep. salivarius strain HHT has a levan-type branched structure consisting beta-(2 leads to 6)-D-fructofuranosidic linkages with an average repeating unit of 9 sugar residues. The solubility properties of these fructants are discussed on the basis of the structural differences.