Fructan Assay Kit

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.

Procedures for the measurement of starch, starch damage (gelatinised starch), resistant starch and the amylose/amylopectin content of starch, β-glucan, fructan, glucomannan and galactosyl-sucrose oligosaccharides (raffinose, stachyose and verbascose) in plant material, animal feeds and foods are described. Most of these methods have been successfully subjected to interlaboratory evaluation. All methods are based on the use of enzymes either purified by conventional chromatography or produced using molecular biology techniques. Such methods allow specific, accurate and reliable quantification of a particular component. Problems in calculating the actual weight of galactosyl-sucrose oligosaccharides in test samples are discussed in detail.

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), α-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%.

An American Association of Cereal Chemists/AOAC collaborative study was conducted to evaluate the accuracy and reliability of an enzyme assay kit procedure for measurement of total starch in a range of cereal grains and products. The flour sample is incubated at 95 degrees C with thermostable alpha-amylase to catalyze the hydrolysis of starch to maltodextrins, the pH of the slurry is adjusted, and the slurry is treated with a highly purified amyloglucosidase to quantitatively hydrolyze the dextrins to glucose. Glucose is measured with glucose oxidase-peroxidase reagent. Thirty-two collaborators were sent 16 homogeneous test samples as 8 blind duplicates. These samples included chicken feed pellets, white bread, green peas, high-amylose maize starch, white wheat flour, wheat starch, oat bran, and spaghetti. All samples were analyzed by the standard procedure as detailed above; 4 samples (high-amylose maize starch and wheat starch) were also analyzed by a method that requires the samples to be cooked first in dimethyl sulfoxide (DMSO). Relative standard deviations for repeatability (RSD(r)) ranged from 2.1 to 3.9%, and relative standard deviations for reproducibility (RSD(R)) ranged from 2.9 to 5.7%. The RSD(R) value for high amylose maize starch analyzed by the standard (non-DMSO) procedure was 5.7%; the value was reduced to 2.9% when the DMSO procedure was used, and the determined starch values increased from 86.9 to 97.2%.

Microbial inulinases are an important class of industrial enzymes that have the ability to hydrolyze inulin into either fructose or fructooligosaccharides. Both of the products have commercial applications in many food and pharmaceutical industries. Inulinases have been reported from plants, animals, and microorganisms. They are present in much less quantity in plants and animals, which restricts their exploitation for industrial applications. Because of this limitation, microorganisms are considered as major sources of inulinases. A good number of microbial sources are reported for inulinases and a majority of them belong to fungi and yeasts. On the basis of the action pattern of inulinases on inulin, they are categorized into exoinulinases and endoinulinases. Exoinulinases hydrolyze the terminal linkages present in inulin and produce fructose, whereas endoinulinases act randomly on internal β-2,1 glycosidic linkages of inulin and produce fructooligosaccharides. Apart from the production of fructose and fructooligosaccharides, inulinases have also been used for the production of many other valuable products like bioethanol, single-cell oils, single-cell proteins, citric acid, etc. This chapter is a brief compilation of the production, purification, and applications of the inulinases.

Prebiotic carbohydrates are added as functional ingredients to a variety of processed foods. Data on the stability of prebiotics during food processing in complex matrices remain limited. The objective of this project was to determine the stability of fructooligosaccharides (FOS), inulin, galactooligosaccharides (GOS), and resistant starch (RS₂), when added as ingredients (1% w/w) to an extruded cereal and a low pH drink. The cereal was prepared using different screw speeds and barrel temperatures. GOS was not affected by any of the extrusion conditions, whereas inulin decreased significantly at 140 and 170°C. FOS levels decreased in all extrusion conditions, while resistant starch (RS) unexpectedly increased for each of the parameters. The low pH drink was prepared with different sucrose to corn syrup solids (S:CSS) ratios (1:2, 1:1, 2:1) at pH 3.0, 3.5, and 4.0. The 1:1 S:CSS drink at pH 3.0, negatively impacted FOS and inulin. Moreover, FOS levels decreased when exposed to 1:2 S:CSS (pH 3.5 and 4.0) and 1:1 S:CSS (pH 3.0). GOS and RS were unaffected by any drink formulations. As different conditions impact the stability of prebiotics differently, this study addresses the importance of developing product specific processes for each prebiotic when supplemented into a processed food.

The modifying effects of aqueous yacon extract (AYE) and Lactobacillus acidophilus CRL 1014 against colon carcinogenesis induced by 1,2-dimethylhydrazine (DMH) in male Wistar rats were investigated. Animals were allocated into five groups: G1: untreated group; G2: DMH-treated group; G3: DMH + L. acidophilus-treated group; G4: DMH + AYE-treated group; G5: DMH + L. acidophilus and AYE-treated group. A significant reduction (p < 0.05) in leukocyte DNA damage and in colonic cell proliferation was observed after the first DMH administration in G3 (probiotic), G4 (prebiotic) and G5 (synbiotic) groups. In this moment, a significant increase (p < 0.05) in colonic apoptosis was also observed in G3 (probiotic) and G5 (synbiotic) groups. In special, at five months after DMH administrations, a significant reduction (p < 0.05) in ACF development was observed in G3 (probiotic), G4 (prebiotic) and G5 (synbiotic) groups. Incidence of colon tumors was lower at five months in G4 (prebiotic) and G5 (synbiotic) groups but not in eight months after DMH administrations. In conclusion, the findings suggest that the oral treatments have potential effects as a chemopreventive agent against colon carcinogenesis on an early stage of tumor development.

The objective of this study was to determine the inulin-type fructan content in 20 selected food plants and other herbs commonly found in Sri Lanka. The inulin content of the selected plants were determined qualitatively and quantitatively using thin layer chromatography (TLC) and enzymatic spectrophotometric (ES) methods, respectively. The ES results showed that the inulin-type fructan contents based on fresh weight was highest in Allium sativum (18.62 % ± 1.55), followed by Asparagus falcatus (17.74 % ± 2.92), Asparagus racemosus (11.8 3% ± 0.87), Allium cepa (8.60 % ± 0.88), Allium ampeloprasum (6.20 % ± 0.23), Taraxacum javanicum (5.77 % ± 1.53) and Vernonia cinerea (4.55 % ± 0.93), respectively. Taraxacum javanicum and Vernonia cinerea plant extracts developed distinct blue black spots with the detection reagent on TLC plates similar to chicory inulin standard. However, Allium ampeloprasum, Allium cepa, Allium sativum, Asparagus falcatus and Asparagus racemosus developed thicker blue black streaks on TLC plates due to their higher inulin concentration, which confirmed the ES results. Aloe vera, Alpinia calcarata, Amophophallus campanulatus, Beta vulgaris, Canna indica, Diascorea alata and Sonchus oleraceus contained low levels (1 ≤ 0.5 g/100 g FW) of inulin while Caryota urens, Ipomoea batatas, Lasia spinosa and Maranta arundinacea contained very low levels or no (< 0.4 g/100 g FW) inulin.

Rice starch (RS) and fructo-oligosaccharides (FOS) were studied as substitutes for phosphates (STPP) and dextrose (Dex) in cooked hams using response surface methodology (RSM). RS, STPP, Dex and FOS were combined in 25 runs and applied to Biceps femoris (BF) and Semimembranosus (SM) muscles. Muscles were injected (120% of green weight), tumbled, netted, and steam cooked. Cook loss and yield were affected by STPP. Colour was predominantly affected by muscle type, but also by the ingredients studied; whereas texture was principally affected by STPP and RS. NMR and expressible moisture data showed higher retention of free water in samples containing RS. This was visualized by light microscopy as starch gel pockets. Despite some reductions in yield, it is feasible to substitute STPP with RS and obtain a satisfactory quality product. However, higher levels of added FOS would be required to warrant a health claim.

The ingestion of unavailable carbohydrates - functional ingredients - has presented an inverse relationship with the risk for chronic non-communicable diseases. The objective of this work was to evaluate the effects of addition of inulin to two ready-to-eat frozen meals on the release of gastrointestinal hormones and other parameters related to hunger and satiety. Prototypes of two different kinds of frozen meals were elaborated by the food industry: control meal (C1 and C2); and test meals, added inulin (T1 and T2). Three sequential clinical assays were performed with healthy volunteers: 1) evaluation of glycemic response (n = 16); 2) evaluation of gastrointestinal hormones related to satiety (n = 15); and 3) evaluation of satiety (by Visual Analogue Scale - VAS and energy intake) (n = 52). The meals showed low glycemic index and glycemic load, and T1 showed a decreased glycemic response peak compared to C1. The addition of inulin (~ 8 g) to the test meals (lunch) provided significant satiety, resulting in an decrease in energy intake of 419 (group 1) and 586 kJ (group 2) in the two subsequent meals (after 180 min and 360 min) and a decrease in hunger and increase in satiety at 120 and 180 min when comparing with control meals. A positive post-prandial variation was observed in the plasmatic levels of ghrelin and insulin in relation to the control meal (hormones related to hunger in high levels), after the intake of both two test meals. Inulin is an ingredient that presents several positive characteristics for the elaboration of products that stimulate healthy eating habits. These effects are currently being evaluated in medium-term trials.

It has been proposed that delayed leaf senescence can extend grain filling duration and thus increase yields in cereal crops. We found that wheat (Triticum aestivum) NAM RNAi plants with delayed senescence carried out 40% more flag leaf photosynthesis after anthesis than control plants, but had the same rate and duration of starch accumulation during grain filling and the same final grain weight. The additional photosynthate available in NAM RNAi plants was in part stored as fructans in the stems, whereas stem fructans were remobilised during grain filling in control plants. In both genotypes, activity of starch synthase was limiting for starch synthesis in the later stages of grain filling. We suggest that in order to realise the potential yield gains offered by delayed leaf senescence, this trait should be combined with increased grain filling capacity.

Fructans are important in the survival of plants and also valuable for humans as potentially health promoting food ingredients. In this study fructan content and composition were determined in grains of 20 barley breeding lines and cultivars with a wide variation in chemical composition, morphology and country of origin, grown at one site in Chile. There was significant genotypic variation in grain fructan content ranging from 0.9% to 4.2% of grain dry weight. Fructan degree of polymerisation (DP) was analysed using high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Changes in the distribution of different chain lengths and the pattern of structures of fructan were detected with increasing amount of fructan in the different barleys. A positive correlation was found between fructan content and the relative amount of long chain fructan (DP > 9) (r = 0.54, p = 0.021). Our results provide a basis for selecting promising barley lines and cultivars for further research on fructan in barley breeding with the aim to produce healthy food products.

A large and diverse material collection of whole grain wheat samples (n = 129) was analysed for total dietary fibre (TDF) content and composition, including fructan (11.5–15.5%). Correlations between the dietary fibre components, associated bioactive components (e.g. tocols, sterols, phenolic acids and folates) and agronomic properties previously determined on the same samples were found with multivariate analysis (PCA). Samples from the same countries had similar characteristics. The first PC described variation in components concentrated in the starchy endosperm (e.g. starch, β-glucan and fructan) and the dietary fibre components concentrated in the bran (e.g. TDF, arabinoxylan and cellulose). The second PC described the variation in kernel weight and other bran components such as alkylresorcinols, tocols and sterols. Interestingly, there was no correlation among these different groups of bran components, which reflected their concentration in different bran tissues. The results are of importance for plant breeders who wish to develop varieties with health-promoting effects.

Asparagus racemosus Linn. (Fam. Liliaceae) is an ethno-pharmacologically acclaimed Ayurvedic medicinal plant. In the present study, aqueous extract of A. racemosus (ARC) was fractionated and screened for the polysaccharide fraction (ARP). The characterization was done by enzymatic, Size Exclusion, gas chromatography with flame ionization detector (GC–FID), high pressure anion exchange chromatography (HPAEC) and thin layer chromatographic analyses. Phyto-chemical evaluation confirmed the presence of 26.7% of 2 → 1 linked fructo-oligosaccharides (FOS). They have a degree of polymerization (DP) of nearly 7–8. Cytotoxicity evaluation on P388 cell lines was consistent with low cytotoxicity of the extracts. In vitro Natural Killer (NK) cell activity was evaluated using human peripheral blood mononuclear cells (PBMC) isolated from whole blood on a ficoll-hypaque density gradient. K562 a myeloid leukemia cell line, were used as target cells. ARC, tested over the range 0.2–50 μg/ml, showed a dose-related stimulation of NK cell activity with a peak increase of 16.9 ± 4.4% at 5.6 μg/ml. However, ARP demonstrated a higher stimulatory activity of 51.8 ± 1.2% at 25 μg/ml. The results indicate that the FOS from A. racemosus potentiates the NK cell activity and this could be an important mechanism underpinning the ‘Rasayana’ properties of this plant.

Dietary inulin can affect the composition and metabolic activity of the gastrointestinal microbiota in piglets. To investigate whether the chain length of inulin may influence its stability in the gut and the bacterial community, 18 weaned piglets were housed 2 per cage, with 1 female and 1 castrated male animal each. The piglets received a control diet without or with 4% inulin, defined by an average degree of polymerisation (DP) of 31 (short-chain, I31) or 57 (long-chain, I57), with 6 piglets/diet. After a short feeding period of 6 d, fructan concentrations, selected bacterial groups, lactic acid, short-chain fatty acid concentrations, and the pH were determined in the digesta of different segments of the gastrointestinal tract. The results indicated that differences in the microbial degradation of inulin were depending on the DP. Compared to the short-chain inulin, the concentrations of the long-chain inulin were numerically greater in the small intestine and caecum, and greater in the digesta of the ascending colon. Differences were also observed in the bacterial composition of the digesta, showing greater cell numbers of enterococci (P=0.029), bifidobacteria (P=0.029), and Lactobacillus mucosae (P=0.028) in the ileum in group I57 compared to group I31. However, most bacteria tended to be numerically reduced in the ileum in group I31 compared to both control and I57 groups. Minor effects were observed in the ascending colon: L. reuteri and L. amylovorus were decreased in group I57 compared to the control group (P=0.031 and 0.034, respectively), and L. mucosae was decreased in group I31 compared to the control animals (P=0.029). The concentrations of bacterial metabolites were distinctively changed in the large intestine of the piglets fed inulin. The pH was lower in the rectum contents in group I57 compared to the control piglets (P=0.026), but lactic acid and total short-chain fatty acid concentrations were not affected. The molar ratios of propionic acid increased in the caecal contents (P=0.040) and in both, the ascending and descending colonic digesta (P=0.017 and 0.013, respectively) in group I57 compared to the control group, while acetic acid decreased (P<0.001) and n-valeric acid increased (PP=0.011, respectively) in the digesta of the ascending and descending colon in group I57. In conclusion, the microbial degradation of inulin was dependent on its chain length. Long-chain inulin affected the microbial fermentation more pronounced compared to short-chain inulin. The effects were already observed after 6 d, a relatively short application period, indicating that inulin may be used specifically during the sensitive post-weaning period for piglets.

BACKGROUND: Pasture (fresh or conserved as hay/haylage) forms the basis of most equid diets and contains varying amounts (0 to ≥ 200 g kg^-1 dry matter (DM) or more) of fructans. Over-consumption of fructan is associated with the onset of laminitis in equids, an agonizing condition that may necessitate euthanasia. To enable appropriate dietary management of animals susceptible to laminitis, it is essential that fructans can be properly quantified in fresh and conserved pasture. For research purposes, fructans are frequently quantified by high-performance liquid chromatography (HPLC), but these methods are costly for routine screening. However, an inexpensive colorimetric method for measuring fructans in human foods is commercially available. The aim here was to determine the suitability of the commercially available colorimetric method for determining the fructan content of pasture grasses for horses. RESULTS: Pasture grasses (Phleum pretense, Festuca rubra, Dactylis glomerata, Lolium perenne) managed for grazing (sampled from April to November) and a further set managed for conservation (sampled in July) were analysed for fructan content by HPLC and the colorimetric technique. HPLC values ranged from 83 to 299 g fructan kg^-1 DM (mean 154); corresponding colorimetric values were 5-238 g fructan kg^-1 DM (mean 82). Discrepancies in values between the two methods varied with time of sampling and plant species. Comparison of selected samples before and after incubation with the fructan hydrolases used in the colorimetric method revealed incomplete fructan hydrolysis from the pasture grasses, resulting in underestimates of their fructan content. CONCLUSION: The colorimetric technique was not a reliable substitute for HPLC to quantify the fructan content of pasture grasses.

The contents of fat, starch, pentosan, fructan, β-glucan and several mono- and oligosaccharides in grain were evaluated to find out the possible effects of the Wx-D1 gene of bread wheat using two sets of near-isogenic waxy and non-waxy lines and two low-amylose mutant lines with a common genetic background of Kanto 107. These materials have two non-functional Wx-A1b and Wx-B1b alleles in common. Waxy near-isogenic lines with a non-functional Wx-D1d allele showed consistently increased contents of fat, total fructan, β-glucan, glucose, fructose, sucrose, 1-kestose, 6-kestose, neokestose, nystose and bifurcose compared with non-waxy lines with a functional Wx-D1a allele throughout three growing/harvest seasons. Starch and total pentosan contents were inconsistently influenced by the allelic status of the Wx-D1 locus, while water-soluble pentosan and raffinose contents were not affected. The compositional changes of a low-amylose mutant line with an almost non-functional Wx-D1f allele were closely similar to those of waxy near-isogenic lines, while significantly different changes were barely observed in another low-amylose mutant line with a partly functional Wx-D1g allele in two seasons. These results showed that the Wx-D1 gene has pleiotropic effects on the fat and saccharide contents of bread wheat grain.

Extractable dietary fiber (DF) plays an important role in nutrition. This study on porridge making with whole grain rye investigated the effect of rest time of flour slurries at room temperature before cooking and amount of flour and salt in the recipe on the content of DF components and molecular weight distribution of extractable fructan, mixed linkage (1→3)(1→4)-β-D-glucan (β-glucan) and arabinoxylan (AX) in the porridge. The content of total DF was increased (from about 20% to 23% of dry matter) during porridge making due to formation of insoluble resistant starch. A small but significant increase in the extractability of β-glucan (P = 0.016) and AX (P = 0.002) due to rest time was also noted. The molecular weight of extractable fructan and AX remained stable during porridge making. However, incubation of the rye flour slurries at increased temperature resulted in a significant decrease in extractable AX molecular weight. The molecular weight of extractable β-glucan decreased greatly during a rest time before cooking, most likely by the action of endogenous enzymes. The amount of salt and flour used in the recipe had small but significant effects on the molecular weight of β-glucan. These results show that whole grain rye porridge made without a rest time before cooking contains extractable DF components maintaining high molecular weights. High molecular weight is most likely of nutritional importance.

Background: Epidemiological studies show inverse relationship between intake of wholegrain cereals and several chronic diseases. Components and mechanisms behind possible protective effects of wholegrain cereals are poorly understood. Objective: To characterise commercial rye bran preparations, compared to wheat bran, regarding structure and content of nutrients as well as a number of presumably bioactive compounds. Design: Six different rye brans from Sweden, Denmark and Finland were analysed and compared with two wheat brans regarding colour, particle size distribution, microscopic structures and chemical composition including proximal components, vitamins, minerals and bioactive compounds. Results: Rye brans were generally greener in colour and smaller in particle size than wheat brans. The rye brans varied considerably in their starch content (13.2–28.3%), which reflected variable inclusion of the starchy endosperm. Although rye and wheat brans contained comparable levels of total dietary fibre, they differed in the relative proportions of fibre components (i.e. arabinoxylan, β-glucan, cellulose, fructan and Klason lignin). Generally, rye brans contained less cellulose and more β-glucan and fructan than wheat brans. Within small variations, the rye and wheat brans were comparable regarding the contents of tocopherols/tocotrienols, total folate, sterols/stanols, phenolic acids and lignans. Rye bran had less glycine betaine and more alkylresorcinols than wheat brans. Conclusions: The observed variation in the chemical composition of industrially produced rye brans calls for the need of standardisation of this commodity, especially when used as a functional ingredient in foods.

Structure and health effects of inulin-type fructans have been extensively studied, while less is known about the properties of the graminan-type fructans in wheat. Arabinoxylan (AX) is another important indigestible component in cereal grains, which may have beneficial health effects. In this study, the fructan content in milling fractions of two wheat cultivars was determined and related to ash, dietary fibre and AX contents. The molecular weight distribution of the fructans was analysed with HPAEC-PAD and MALDI-TOF MS using ¹H NMR and enzymatic hydrolysis for identification of fructans. The fructan content (g/100 g) ranged from 1.5 ± 0.2 in flour to 3.6 ± 0.5 in shorts and 3.7 ± 0.3 in bran. A correlation was found between fructan content and dietary fibre content (r = 0.93, P < 0.001), but with a smaller variation in fructan content between inner and outer parts of the grain. About 50% of the dietary fibre consisted of AX in all fractions. The fructans were found to have a DP of up to 19 with a similar molecular weight distribution in the different fractions.

• Leaf senescence can be described as the dismantling of cellular components during a specific time interval before cell death. This has the effect of remobilizing N in the form of amino acids that can be relocalized to developing seeds. High levels of carbohydrates have previously been shown to promote the onset of the senescence process. • Carbohydrate accumulation in barley (Hordeum vulgare) plants was induced experimentally by steam-girdling at the leaf base, occluding the phloem, and gene regulation under these conditions was investigated using the Affymetrix Barley GeneChip array and quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR). • Transcript levels of plastidial (aminopeptidases, cnd41) and vacuolar (thiol and serine) proteases clearly increase in girdled leaves. Of special interest are cnd41, a plastidial aspartyl peptidase that has been implicated in Rubisco degradation in tobacco; and cp-mIII, a highly upregulated carboxypeptidase. SAG12, hexokinases and other senescence-specific genes are also upregulated under these conditions. • Applying a genomic approach to the innovative experimental system described here significantly enhances our knowledge of leaf proteolysis and whole-plant N recycling.