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%.

The complex of polysaccharides of the grain transforms during processing and modifies the physical and chemical characteristics of bread. The aim of the research was to characterize the changes of glucans, mannans and fructans in hull-less barley and wholegrain wheat breads fermented with spontaneous hull-less barley sourdough, germinated hull-less barley sourdough and yeast, as well as to analyze the impact of polysaccharides on the physical parameters of bread. By using the barley sourdoughs for wholegrain wheat bread dough fermentation, the specific volume and porosity was reduced; the hardness was not significantly increased, but the content of β-glucans was doubled. Principal component analysis indicates a higher content of β-glucans and a lower content of starch, total glucans, fructans and mannans for hull-less barley breads, but wholegrain wheat breads fermented with sourdoughs have a higher amount of starch, total glucans, fructans and mannans, and a lower content of β-glucans. The composition of polysaccharides was affected by the type of flour and fermentation method used.

Commercial fructans (inulin and oligofructose) are generally obtained from crops such as chicory, Jerusalem artichoke or agave. However, there are agricultural by-products, namely asparagus roots, which could be considered potential sources of fructans. In this work, the fructans extracted from asparagus roots and three commercial ones from chicory and agave were studied in order to compare their composition, physicochemical characteristics, and potential health effects. Asparagus fructans had similar chemical composition to the others, especially in moisture, simple sugars and total fructan contents. However, its contents of ash, protein and phenolic compounds were higher. FTIR analysis confirmed these differences in composition. Orafti^®GR showed the highest degree of polymerization (DP) of up to 40, with asparagus fructans (up to 25) falling between Orafti^®GR and the others (DP 10-11). Although asparagus fructan powder had a lower fructan content and lower DP than Orafti^®GR, its viscosity was higher, probably due to the presence of proteins. The existence of phenolic compounds lent antioxidant activity to asparagus fructans. The prebiotic activity in vitro of the four samples was similar and, in preliminary assays, asparagus fructan extract presented health effects related to infertility and diabetes diseases. All these characteristics confer a great potential for asparagus fructans to be included in the prebiotics market.

During microbial deterioration of sugarcane, a variety of extracellular polysaccharides (EPS, e.g., dextran, fructans) and other impurities (e.g., organic acids, sugar alcohols, alcohols) are produced. The microbial-derived EPS dextran (a polymer of glucose) has generally been considered the main problem in sugarcane processing, with its main production source being attributed to the bacterium Leuconostoc mesenteroides. Technical problems associated with the presence of dextran at the sugar factory can affect every step of sugar processing resulting in high viscosities, reduced efficiencies, elongation of sugar crystals, and the loss of sucrose to molasses. In recent years, fructans (a polymer of fructose) concentrations have been increasingly reported in both cane juice and molasses at Louisiana sugar factories. With the limited information that exists on the microbial origin of fructans at the sugar factories and its possible impact on Louisiana sugarcane processing, this research study aimed at identifying the microbial populations present in high numbers in the crusher juice or first extraction juice, and determining the effect of processing conditions on their ability to produce EPS. Our findings indicate that Lactobacillus, Lactococcus, Leuconostoc, Pantoea, Pseudomonas, and Saccharomyces were the microbial genera present in high cell numbers and that all isolates were capable of producing dextran and/or fructans. Leuconostoc had the most diverse number of species. A single isolate of L. suionicum A14 was identified and produced only fructans from sucrose. This is the first study to report on a fructan-only producing Leuconostoc isolate from sugarcane. EPS production was affected by temperature, sucrose concentrations, and medium pH.

Levan, a homopolysaccharide, has great potential as a functional biopolymer in foods, feeds, cosmetics, and the pharmaceutical and chemical industries. Two types of nanocarrier of levan obtained from Bacillus subtilis strain, as potential delivery system carriers, are studied in this work. The first type of polymer nanoparticles are created in water by a self-assembly process directly from fermentation, while the second type are created after precipitating a levan powder and dissolving it in water. Both types of the obtained nanoparticles were of a size in the range of 214.10 ± 0.70–238.27 ± 1.72 nm with low polydispersity. The nanoparticle systems were found to be visually and kinetically stable. There were differences in the surface morphology of the carriers: the first type had an irregular surface while the second type were smooth. Differences in their ability to scavenge free radicals were also demonstrated: nanoparticles from culture supernatant scoured 8.57% more radicals than nanoparticles from precipitated polymer. Both types are able to encapsulate hydrophobic compounds and penetrate the stratum corneum. Analysis using Raman spectroscopy showed that the nanoparticles increased hydration in the deeper layers of the skin. The diversity exhibited by the two types of levan nanoparticles would indicate a wide range of potential applications in the cosmetics industry.

Despite the recognition of Agave tequilana Weber var. Azul as raw material for producing tequila and obtaining prebiotics, there are other highly relevant Agave species in Mexico. Oaxaca contains a startlingly diverse range of Agave species; Agave angustifolia Haw. and Agave potatorum Zucc. are two classic specimens with great commercial potential. In this study, we examined the fructan fluctuation in these two species during their lifetime in the field (from 1 to 6 years old). First, we analyzed their morphological diversity based on vegetative characteristics. Subsequently, fructan extracts were analyzed by TLC, FT-IR, and HPAEC-PAD to identify carbohydrates. Multivariate analyses of the morphological parameters indicated a morphological divergence between the two species. Furthermore, we found that the concentration of simple carbohydrates and fructans, as well as the fructan DP, changed during plant development. Glucose, fructose, and fructooligosaccharides (FOS) were more abundant in A. potatorum, while A. angustifolia showed a greater amount of sucrose and fructans with a high DP. Fructan DP heatmaps were constructed using HPAEC-PAD profiles-the heatmaps were very helpful for establishing an easy correlation between age and the carbohydrate types present in the fructan extracts. This study is an important contribution to the agave fructan knowledge of the Mexican agave diversity.

Fructans are important biocompounds because of their health-promoting effects as dietary fiber and prebiotics and also because of their harmful effects as fermentable oligosaccharides, disaccharides, monosaccharides, and polyols (FODMAP) particularly in people suffering from irritable bowel syndrome (IBS) and inflammatory bowel disease (IBD), and recently as potential triggers of non-celiac wheat/gluten sensitivity. In this work, we have analyzed the fructan contents as well as its degree of polymerization (DP) in a genetically diverse set of wheat varieties, modern and landraces, from different commonly consumed species (N = 124). A significant variation in fructan contents within and between species was observed, with the following relationship: Triticum aestivum (Landraces) > Triticum aestivum (Modern) ≥ Triticum turgidum (Modern) = T. turgidum (Landraces) ≥ Triticum spelta. In addition, a substantial part of the fructans (>50%) showed a DP ≤ 6. Considering that wheat is a major source of fructans, our results can contribute to a better nutritional management of our diets and be a basis for targeted wheat breeding to alter fructan contents.

Young green barley (YGB) water extract has revealed a beneficial impact on natural killer (NK) cells’ ability to recognize and eliminate human colon cancer cells, without any side effects for normal colon epithelial cells. The direct anticancer effect of the tested compounds has been also shown. The mixture of oligosaccharides found in this extract was characterized by chemical analyses and via FT-IR spectroscopy and MALDI-TOF MS techniques. The YGB preparation contained 26.9% of proteins and 64.2% of sugars, mostly glucose (54.7%) and fructose (42.7%), with a small amount of mannose (2.6%) and galactose (less than 0.5%). Mass spectrometry analysis of YGB has shown that fructose oligomers contained from 3 to 19 sugar units. The number of fructans was estimated to be about 10.2% of the dry weight basis of YGB. The presented results suggest the beneficial effect of the consumption of preparations based on young barley on the human body, in the field of colon cancer prevention.

A diet low in fermentable oligosaccharides, disaccharides, monosaccharides, and polyols (FODMAPs) is a promising therapeutic approach to reduce gastrointestinal symptoms associated with irritable bowel syndrome (IBS). However, a shift toward a more sustainable, healthy diet with higher inclusion of whole-grain cereals (i.e., wheat, rye, barley) and pulses, naturally rich in FODMAPs, poses a severe challenge for susceptible individuals. Dietary restriction of fermentable carbohydrates (commonly called the "low FODMAP diet") has received significant consideration. Hence, the development of functional low FODMAP products is emerging in food science and the food industry. In this review, we evaluate the most promising yet neglected (bio)-technological strategies adopted for modulating the FODMAP contents in complex food systems and the extent of their uptake in the global food market. We extensively investigated the global low FODMAP market, contrasted with the status quo in food science and discussed the key principles and concomitant challenges of targeted FODMAP reduction strategies. Powerful tools are available which are based either on the use of ingredients where FODMAPs have been physically removed (e.g., by membrane filtration) or biotechnologically reduced during the food processing, mediated by added enzymes, microbial enzymes during a fermentation process, and seed endogenous enzymes. However, <10% of the small market of functional products with a low FODMAP claim (total ~800 products) used any of the targeted FODMAP reduction techniques. The global market is currently dominated by gluten-free products, which are naturally low in FODMAPs and characterized by inferior sensory attributes.

In this study, twelve strains of acetic acid bacteria (AAB) belonging to five different genera were tested for their ability to produce levan, at 70 and 250 g/L of sucrose concentration, respectively. The fructan produced by the bacterial strains was characterized as levan by NMR spectroscopy. Most of the strains produced levan, highlighting intra- and inter-species variability. High yield was observed for Neoasaia chiangmaiensis NBRC 101099^T, Kozakia baliensis DSM 14400^T and Gluconobacter cerinus DSM 9533^T at 70 g/L of sucrose. A 12-fold increase was observed for N. chiangmaiensis NBRC 101099^T at 250 g/L of sucrose concentration. Levan production was found to be affected by glucose accumulation and pH reduction, especially in Ko. baliensis DSM 14400^T. All the Gluconobacter strains showed a negative correlation with the increase in sucrose concentration. Among strains of Komagataeibacter genus, no clear effect of sucrose on levan yield was found. Results obtained in this study highlighted the differences in levan yield among AAB strains and showed interdependence between culture conditions, carbon source utilization, and time of incubation. On the contrary, the levan yield was not always related to the sucrose concentration.

Cereal-based bioethanol stillage, the main by-product of industrial bioethanol production, is a potential substrate for fructans. However, the quantification of fructans in such complex sample matrices is still a challenge for the corresponding analytics to be overcome to allow for the identification and utilization of such unused fructan sources. Especially a possible utilization or rather the corresponding process development requires appropriate analytics first. Thus, this paper aims to develop and apply a method for fructan quantification in bioethanol stillage using common a high-performance liquid chromatography-refractive index detector.

Asparagus roots are by-products from asparagus cultivation and they could be considered one of the best sources of fructans. These polymers are interesting food ingredients for their prebiotic and immuno-stimulating characteristics. The aim of this work is to characterize the fructan profile from the roots of several asparagus varieties grown at different locations and pickled at three vegetative statuses in order to valorize these by-products as fructan source. Fructans were extracted with hot water and fractionated into three pools according to their molecular weight (MW). Their average MW was studied by HPSEC and their degree of polymerization by HPAEC. The fructan content was up to 12.5% on fresh weight basis, depending on variety and sampling date. The relative abundance of the three pools also depended on the picking moment as after the spear harvest period their total content and MW increased. The average MW of the three fractions was similar among varieties with 4.8, 8.4 and 9 sugar units, although fructans up to 30 units were identified by HPAEC. These characteristics make them similar to the commercialized Orafti^®-GR inulin, a common additive to food products. Therefore, the concept of asparagus roots as cultivation waste must be changed to a new feedstock for sustainable agriculture and industry.

Maitake (Grifola frondosa) is a medicinal mushroom known for its peculiar biological activities due to the presence of functional components, including dietary fibers and glucans, that can improve human health through the modulation of the gut microbiota. In this paper, a Maitake ethanol/water extract was prepared and characterized through enzymatic and chemical assays. The prebiotic potential of the extract was evaluated by the growth of some probiotic strains and of a selected probiotic consortium. The results revealed the prebiotic properties due to the stimulation of the growth of the probiotic strains, also in consortium, leading to the production of SCFAs, including lactic, succinic, and valeric acid analyzed via GC-MSD. Then, their beneficials effect were employed in evaluating the vitality of three different healthy and tumoral colorectal cell lines (CCD841, CACO-2, and HT-29) and the viability rescue after co-exposure to different stressor agents and the probiotic consortium secondary metabolites. These metabolites exerted positive effects on colorectal cell lines, in particular in protection from reactive oxygen species.

The authors used mesoporous silica microspheres as a support for the immobilization of inulinase from Aspergillus brasiliensis MTCC 1344 by the process of cross-linking. Under optimized operating conditions of pH 6.0, particle/enzyme ratio of 2.0:1.0 and glutaraldehyde concentration of 7 mM, a maximum immobilization yield of 90.7% was obtained after a cross-linking time of 12.25 h. Subsequently, the cross-linked inulinase was utilized for the hydrolysis of 5% inulin, and a maximum fructose concentration of 31.7 g/L was achieved under the optimum conditions of pH 6.0 and temperature 60°C in 3 h. Furthermore, on performing reusability studies during inulin hydrolysis, it was observed that the immobilized inulinase could be reused up to 10 subsequent cycles of hydrolysis, thus providing a facile and commercially attractive process of high-fructose syrup production.

Background and Aims: Although the plant economic spectrum seeks to explain resource allocation strategies, carbohydrate storage is often omitted. Belowground storage organs are the centre of herb perennation, yet little is known about the role of their turnover, anatomy, and carbohydrate storage in relation to the aboveground economic spectrum. Methods: We collected aboveground traits associated with the economic spectrum, storage organ turnover traits, storage organ inner structure traits, and storage carbohydrate concentrations for approximately eighty temperate meadow species. Key Results: The suites of belowground traits were largely independent from one another, but there was significant correlation between the aboveground traits with both inner structure and storage carbohydrates. Anatomical traits diverged according to leaf nitrogen concentration on one side and vessel area and dry matter content on the other; carbohydrates separated along leaf nitrogen and plant height. Conclusions: Contrary to our expectations, aboveground traits and not storage organ turnover were correlated with anatomy and storage carbohydrates. Belowground traits associated with the aboveground economic spectrum also did not fall clearly within the fast-slow economic continuum, thus indicating the presence of a more complicated economic space. Our study implies that the generally over-looked role of storage within the plant economic spectrum represents an important dimension of plant strategy.

We profiled the grain oligosaccharide content of 154 two-row spring barley genotypes and quantified 27 compounds, mainly inulin- and neoseries-type fructans, showing differential abundance. Clustering revealed two profile groups where the ‘high’ set contained greater amounts of sugar monomers, sucrose, and overall fructans, but lower fructosylraffinose. A genome-wide association study (GWAS) identified a significant association for the variability of two fructan types: neoseries-DP7 and inulin-DP9, which showed increased strength when applying a novel compound ratio-GWAS approach. Gene models within this region included three known fructan biosynthesis genes (fructan:fructan 1-fructosyltransferase, sucrose:sucrose 1-fructosyltransferase, and sucrose:fructan 6-fructosyltransferase). Two other genes in this region, 6(G)-fructosyltransferase and vacuolar invertase1, have not previously been linked to fructan biosynthesis and showed expression patterns distinct from those of the other three genes, including exclusive expression of 6(G)-fructosyltransferase in outer grain tissues at the storage phase. From exome capture data, several single nucleotide polymorphisms related to inulin- and neoseries-type fructan variability were identified in fructan:fructan 1-fructosyltransferase and 6(G)-fructosyltransferase genes. Co-expression analyses uncovered potential regulators of fructan biosynthesis including transcription factors. Our results provide the first scientific evidence for the distinct biosynthesis of neoseries-type fructans during barley grain maturation and reveal novel gene candidates likely to be involved in the differential biosynthesis of various types of fructan in barley.

Inulin-type fructans have been used as an ingredient to partially replace starchy flours in foods such as pasta, confectionery, breads, sauces and desserts, as they have the potential to improve technological properties, nutritional quality of the products and reduce glycemic response. Therefore, this study aimed evaluating the impact of inulin on the physical properties, proximate composition, glycemic response and sensory acceptance of corn breakfast cereals. A mixture of corn grits and inulin was extruded in a single screw extruder, at a feed rate of 247 g/min, temperatures in the shearing zones at 140°C and screw speed of 192 rpm. The addition of inulin did not impair the physical properties of breakfast cereals, such as expansion ratio, density, instrumental force and color. Moreover, inulin did not affect the acceptance of products, as well as having a positive impact on them. Extrudates with inulin presented a high fiber level and moderate glycemic load, in contrast to the control extrudate that had low fiber level and high glycemic load. Therefore, the addition of inulin in breakfast cereals is promising and may contribute to increasing the consumption of products with aggregated nutritive value.