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%.

Increasing the intake of dietary fiber from staple foods is a key strategy to improve the health of consumers. White bread is an attractive vehicle to deliver increased fiber as it is widely consumed and available to all socio-economic groups. However, fiber only accounts for about 4% of the dry weight of white flour and bread compared to 10-15% in whole grain bread and flour. We therefore discuss the challenges and barriers to developing and exploiting new types of wheat with high fiber content in white flour. These include defining and quantifying individual fiber components and understanding how they are affected by genetic and environmental factors. Rapid high throughput assays suitable for determining fiber content during plant breeding and in grain-utilizing industries are urgently required, while the impact of fiber amount and composition on flour processing quality needs to be understood. Overcoming these challenges should have significant effects on human health.

Roasted burdock root tea (BT), rich in inulin and chlorogenic acid, is currently being established as a tea beverage in Japan and a wider area. This study aimed to clarify the effects of BT on the gut environment and host health. BT was prepared using 5% (w/v) dried burdock root, and its effect on ammonia production was investigated using human faecal culture and Institute of Cancer Research mice fed with a high-sucrose and low-dietary fibre diet for 14 days. In human faecal cultures established using a medium prepared with BT, the pH significantly decreased from 6.5 to 4.6 ± 0.1. In vitro, BT significantly suppressed the production of ammonia and indole (p < 0.05). In vivo, BT increased the caecal acetate level from 34 ± 3 μmol/g to 49 ± 4 μmol/g and n-butyrate level from 5.1 ± 0.7 μmol/g to 14 ± 1 μmol/g and reduced the caecal pH from 7.0 ± 0.1 to 6.6 ± 0.1. Despite no significant changes in caecal ammonia levels, plasma ammonia levels in BT-treated mice decreased from 0.74 ± 0.07 to 0.50 ± 0.07 μmol/mL. Moreover, 16S rDNA (V4) amplicon sequencing of faeces revealed that BT increased the short-chain fatty acid-producing gut commensals Muribaculaceae and Clostridia UCG-014. These results suggest that BT has desirable functional properties correlated with short-chain fatty acid production and pH-lowering effects that inhibit ammonia production and absorption in the gut.

Background: Inulin and inulin-derived fructooligosaccharides (FOS) are well-known prebiotics for use in companion animals and livestock. The mechanisms by which FOS contribute to health has not been fully established. Further, the fine chemistry of fructan structures from diverse sources, such as graminan-type fructans found in cereal crops, has not been fully elucidated. New methods to study fructan structure and microbial responses to these complex carbohydrates will be key for evaluating the prebiotic potency of cereal fructans found in cattle feeds. As the rumen microbiome composition is closely associated with their metabolic traits, such as feed utilization and waste production, prebiotics and probiotics represent promising additives to shift the microbial community toward a more productive state. Results: Within this study, inulin, levan, and graminan-type fructans from winter wheat, spring wheat, and barley were used to assess the capacity of rumen-derived Bifidobacterium boum, Bifidobacterium merycicum, and Lactobacillus vitulinus to metabolize diverse fructans. Graminan-type fructans were purified and structurally characterized from the stems and kernels of each plant. All three bacterial species grew on FOS, inulin, and cereal crop fructans in pure cultures. L. vitulinus was the only species that could metabolize levan, albeit its growth was delayed. Fluorescently labelled polysaccharides (FLAPS) were used to demonstrate interactions with Gram-positive bacteria and confirm fructan metabolism at the single-cell level; these results were in agreement with the individual growth profiles of each species. The prebiotic potential of inulin was further investigated within naïve rumen microbial communities, where increased relative abundance of Bifidobacterium and Lactobacillus species occurred in a dose-dependent and temporal-related manner. This was supported by in situ analysis of rumen microbiota from cattle fed inulin. FLAPS probe derived from inulin and fluorescent in situ hybridization using taxon-specific probes confirmed that inulin interacts with Bifidobacteria and Lactobacilli at the single-cell level. Conclusion: This research revealed that rumen-derived Bifidobacteria and Lactobacilli vary in their metabolism of structurally diverse fructans, and that inulin has limited prebiotic potential in the rumen. This knowledge establishes new methods for evaluating the prebiotic potential of fructans from diverse plant sources as prebiotic candidates for use in ruminants and other animals.

Continuous thermal processing (CTP) is a common method for sterilizing food. However, it can result in an uneven temperature distribution, which can lead to a varying degree of processing intensity. Ohmic heating (OH) can be advantageous in this regard, as it enables volumetric heating for more homogenous treatments. However, evaluating the processing intensity distribution inside the equipment for OH is challenging due to the complex interaction between electrical, mechanical and thermal phenomena. Furthermore, the comparison of OH and conventional heating treatments often lack a profound basis of comparable treatment intensity considerations. To gain a deeper mechanistic understanding of the technology, a numerical computational fluid dynamics model for the OH sterilization of a clear carrot juice from the heating region to the cooling process was developed. The model was validated with thermal and electrical measurements and showed an error rate below 2.5% in its prediction capacities. Moreover, the model was implanted for the validation of the products sterilization and compared to a conventional validation approach, reviling a 33.3% underestimation of the thermal load by conventional manners, which can lead to faulty sterilization of the food product. Additionally, the model was expanded to also be able to predict the microbial inactivation ratio of the system with an average error of 1.10±0.74%. In addition, results indicate that the numerical calculation of the F₀ values and their validation with the microbial inactivation ratio have a notable potential for localization and evaluation of hotspots in OH simulations. Therefore, it can be seen as a promising step for establishing a foundation for computer-assisted optimization of CTP and targeted processing.

Inulin, a prebiotic utilized in the food and pharmaceutical industries, promotes the growth of beneficial bacteria in the colon, thereby enhancing human health. Although inulin is commercially produced from chicory and artichoke, Inula helenium roots offer a high potential for inulin production. The aim of this study is to investigate the prebiotic activity of inulin (inulin-P) from I. helenium roots on Lactobacillus rhamnosus, as well as its ability to produce synbiotic microcapsules and the effects on probiotic viability during freeze-drying, in vitro gastrointestinal (GI) digestion, and storage. First, the effect of inulin-P on L. rhamnosus viability and short-chain fatty acid (SCFA) production was compared to other commonly utilized prebiotics. The findings revealed that inulin-P remarkably promoted the growth and SCFA yield of L. rhamnosus for 48 h of fermentation and 28 days of storage. Then, L. rhamnosus was encapsulated with inulin-P and commercial inulin to compare its survival throughout storage and the GI tract. Inulin-P microcapsules outperformed in terms of viability during storage (7.98 log CFU/g after 30 days at 4°C). Furthermore, inulin-P microcapsules were heat-resistant and protected L. rhamnosus from GI conditions, resulting in a high survival rate (89.52%) following large intestine simulation, which is ideal for increasing customer benefits. Additionally, inulin-P microcapsules exhibited similar physical characteristics to commercial inulin. Consequently, this study revealed that inulin-P, which is easy to produce, low-cost, and has industrial application potential, could be used as a good carrier for the synbiotic encapsulation of L. rhamnosus.

Prebiotics are defined as non-digestible dietary components that promote the growth of beneficial gut microorganisms. In many cases, however, this capability is not systematically evaluated. Here, we develop a methodology for determining prebiotic-responsive bacteria using the popular dietary supplement inulin. We first identify microbes with a capacity to bind inulin using mesoporous silica nanoparticles functionalized with inulin. 16S rRNA gene amplicon sequencing of sorted cells revealed that the ability to bind inulin was widespread in the microbiota. We further evaluate which taxa are metabolically stimulated by inulin and find that diverse taxa from the phyla Firmicutes and Actinobacteria respond to inulin, and several isolates of these taxa can degrade inulin. Incubation with another prebiotic, xylooligosaccharides (XOS), in contrast, shows a more robust bifidogenic effect. Interestingly, the Coriobacteriia Eggerthella lenta and Gordonibacter urolithinfaciens are indirectly stimulated by the inulin degradation process, expanding our knowledge of inulin-responsive bacteria.

Fructans found in agave are called agavins, highly branched neo-fructans. They are essential on the yield and quality of Tequila production. The need for agave specimens with higher accumulation of agavins became essential before the growing demand of such products. To get such specimens, understanding agavins metabolism is a quintessential requirement. For this, a more efficient biological model is required. The recently reclassified Agave amica possesses the potential to gather the requirements for becoming such a model. Therefore, this study dealt with the characterization of carbohydrates in the bulbs of A. amica focusing on fructans. Moreover, it tested and described its feasibility as model for the accelerated study of agavins. Infrared analysis unveiled potential content of fructans in the bulbs of A. amica. Furthermore, high performance thin layer chromatography detected fructooligosaccharides. High performance anion exchange chromatography confirmed a polydisperse mixture of branched fructans. Gas chromatography–mass spectrometry analysis demonstrated agavins like structures in the bulbs of A. amica. Moreover, total fructan content and multivariate data analysis through bulb’s age demonstrated their correlation. Thus, the presence of agavins, their correlation with phenology, and their technical advantages highlighted the feasibility of this species as a potential new biological model for the study of agavins’ metabolism.

The role of storage carbohydrates in plant carbon economy is currently disputed as possibly passive accumulation when other resources are limiting growth, or part of a conservative growth strategy as insurance for regrowth and stress response. One indication may be the fate of carbohydrates in senescing rhizomes, as either translocated to be retained in the live and growing end of the rhizome or kept within the senescing rhizome end and lost into the soil for it to decompose. To examine carbohydrate storage in senescing rhizomes, eight rhizomatous species were grown in a split-pot design with one compartment containing the forward-growing and younger end of the rhizome and another containing the older end. Both compartments were either watered (control) or the older one was left un-watered (drought treatment) to trigger rhizome senescence and potential carbohydrate translocation. Plant growth, root traits, and non-structural carbohydrate types and concentrations were assessed in four sequential harvests. Drought treatment plants had higher rhizome dry matter content. Younger rhizome parts produced higher new rhizome and above-ground biomass than older rhizome parts. Carbohydrate concentrations in rhizomes remained consistent for both treatments, younger and older rhizome parts, and all harvests, probably because of the translocation of water from the watered to the dry compartment to prevent senescence and rhizome loss. Contrary to expectations, the experimental treatment did not trigger rhizome senescence: plants responded by conserving the rhizome and resources within, rather than by losing their older parts. The invariant composition and concentration of carbohydrates within the rhizome suggest that rhizomes are essential plant organs and the storage carbohydrates they contain are necessary for regrowth after stress.

Six cross-bred barley lines developed by a breeding strategy with the target to enhance the fructan synthesis activity and reduce the fructan hydrolysis activity were analyzed together with their parental lines, and a reference line (Gustav) to determine whether the breeding strategy also affected the content and molecular structure of amylopectin and β-glucan. The highest fructan and β-glucan content achieved in the novel barley lines was 8.6 % and 12 %, respectively (12.3-fold and 3.2-fold higher than in Gustav). The lines with low fructan synthesis activity had higher starch content, smaller building blocks in amylopectin, and smaller structural units of β-glucans than the lines with high-fructan synthesis activity. Correlation analysis confirmed that low starch content was associated with high amylose, fructan, and β-glucan content, and larger building blocks in amylopectin.

Recent studies have highlighted the prebiotic effect of inulin through the selective promotion of colon-residing bacteria, modulation of the composition of the gut microbiome, and consequent generation of beneficial effects on gastrointestinal inflammation, diabetes, and cancer. However, as a water-soluble polysaccharide, the prebiotic effect of inulin is limited by low delivery efficiency and short retention time within the colon. In this study, inulin microparticles (MPs) were produced by the electrospray method, and their material properties and bioavailability were evaluated. Inulin was electrosprayed with poly(vinyl) alcohol (PVA) (MW = 89,000-98,000 g/mol) to improve its processability and mucoadhesive properties. MPs produced at PVA:Inulin mass ratio 1:3 were of diameter 0.42 ± 0.46 μm. FTIR and confocal laser scanning microscopy confirmed the presence and colocalization of the PVA and inulin in the particles. MP suspensions exhibited a time dependent viscoelastic rheological response that trended with time toward the response of the inulin suspension. Additionally, MP suspensions exhibited greater viscosity and shear thinning behavior than their individual components and two-component mixtures. The gut retention of inulin in mice was prolonged when delivered in these MP suspensions relative to inulin suspensions and PVA-inulin two-component mixtures. The increased retention is hypothesized to be a result of the effect of PVA on rheological and mucoadhesive properties. The increased retention of inulin leads to improved availability of inulin for gut microbiota which can support applications in drug delivery and foods.

Consumption of fermentable oligo-, di-, and monosaccharides and polyols (FODMAPs) can promote gut health in individuals with a healthy gastrointestinal tract. However, FODMAPs, as well as amylase-trypsin inhibitors (ATIs), have been identified as potential triggers of intestinal symptoms in irritable bowel syndrome (IBS) and non-celiac wheat sensitivity (NCWS) patients. Wheat is a major staple worldwide, and hence, accounts for a large proportion of the intake of FODMAPs and ATIs. Thus, this paper aims to provide an overview of the strategies utilized in reducing the levels of FODMAPs and ATIs in wheat.