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%.

Irritable bowel syndrome (IBS) is a functional disorder of the gastrointestinal system. Adherence to a low-FODMAP (fermenting oligosaccharides, disaccharides, monosaccharides, and polyols) diet may be one of the solutions in this case. The major FODMAP carbohydrates found in wheat bread are fructans. The objective of this study was to produce wheat bread with a reduced fructans content. Breads were made from light and whole grain flour obtained from common wheat using two methods of dough development—I-stage method with the use of yeast, and II-stage method with the use of yeast and sourdough with a pure culture of Lactobacillus plantarum. Four different fermentation times were tested—60, 90, 120, and 150 min. Afterwards, quality attributes (loaf volume, crust and crumb color, and sensory properties) of the produced breads were evaluated, and the fructans content was determined. The results demonstrated that all the factors influenced the quality of wheat breads, as well as their fructans content. Breads made with the II-stage method and light flour had a lower content of fructans, which was decreased in breads along with extending the time of dough fermentation. The greatest impact on fructans content decrease in wheat bread was ascribed to the use of light flour, the II-stage method of dough development coupled with a dough fermentation time prolongation to 150 min.

Cereal-based products, such as bread, are staple foods in the western diet. Due to the nature of their basic ingredients and the diversity of recipes, the amount of fermentable short-chain carbohydrates (FODMAPs) in those products may be high. This study characterized the FODMAP-profiles of a broad range of cereal-product ingredients, serving as a basis for low FODMAP product development. Different cereals, pseudo-cereals, gluten-free flours, pulses, pulse protein ingredients, commercial sprouts, and other cereal-product ingredients were analyzed, using anion-exchange chromatography with electrochemical detection. Wheat and related cereals were high in fructans. Pulses, such as peas contained high galactooligosaccharides (GOS) amounts. Whereas GOS levels in pulse protein ingredients varied, depending on their production. Gluten-free flours, for instance, rice-flour, showed low FODMAP-profiles. Amongst those, buckwheat, which does not contain any of the FODMAPs investigated, contained high amounts of other soluble non-digestible carbohydrates, namely fagopyritols; these may have a similar effect on a sensitive gut as GOS. Finally, ingredients contained mainly high levels of fructans and GOS. Yet, the analysis of commonly consumed commercial cereal products, including bread, pasta, crackers and biscuits, highlighted the relevance of lactose, fructose in excess of glucose and polyols. These products serve as benchmarks for further product development.

Water-soluble dietary fiber such as inulin improves the beta diversity of the intestinal microbiota of mice fed with a high-fat diet (HFD). The circadian clock is the system that regulates the internal daily rhythm, and it affects the pattern of beta diversity in mouse intestinal microbiota. Burdock (Arctium lappa) root contains a high concentration of inulin/fructan (approximately 50%) and is a very popular vegetable in Japan. Arctium lappa also contains functional substances that may affect intestinal microbiota, such as polyphenols. We compared the effects of inulin and A. lappa powder on the diversity of the intestinal microbiota of HFD-fed mice. 16S rDNA from the intestinal microbiota obtained from feces was analyzed by 16S Metagenomic Sequencing Library Preparation. It was found to have a stronger effect on microbiota than inulin alone, suggesting that inulin has an additive and/or synergic action with other molecules in A. lappa root. We examined the effects of intake timing (breakfast or dinner) of A. lappa on intestinal microbiota. The intake of A. lappa root in the evening had a stronger effect on microbiota diversity in comparison to morning intake. Therefore, it is suggested that habitual consumption of A. lappa root in the evening may aid the maintenance of healthy intestinal microbiota.

Fructans are carbohydrates found in many plants, including wheat (Triticum aestivum L.), and they serve physiological roles in both plants and humans. Genomic selection (GS) could facilitate the rapid development of climate‐resilient, nutritionally improved wheat cultivars, such as high‐fructan cultivars, while decreasing resource‐intensive phenotyping requirements. However, few empirical studies have examined GS for nutritional quality breeding. Although GS can accelerate gain from selection, loss of genetic variation and inbreeding may limit the potential for long‐term gain. The objectives of this study were (a) to determine realized gain from GS for wheat grain fructan content with simple truncated selection (TS) and optimized contribution selection (OCS) methods, (b) to determine if gains agree with theoretical expectations, and (c) to compare impacts of selection on inbreeding, genetic variance, and indirect selection on agronomic characteristics. Over 2 yr, two cycles of GS were performed with equal contribution TS and inbreeding‐constrained OCS selection. Genomic selection with TS and OCS led to a 25 ± 12% and 34 ± 6.4% increase in wheat grain fructan content, respectively. Although positive gains from selection were observed for both populations, OCS populations exhibited these gains while simultaneously retaining greater genetic variance and lower inbreeding levels relative to TS populations. Selection for wheat grain fructan content did not change plant height but significantly decreased days to heading in OCS populations. In this study, GS effectively improved the nutritional quality of wheat, and OCS controlled the rate of inbreeding.

Yacon is a little-known, non-starchy Andean root crop recognized as a rich source of β-(2-1) fructooligosaccharides. The aim of this research is to study different drying conditions to obtain yacon flour with high color and bioactive conservation to be used as a functional ingredient. Three different types of pre-drying processing were tested: pulped, cut as cubes and sliced with addition of bisulfite, and drying for 16 h at 65°C. Dried sliced-yacon remained light-yellow with the lowest anti-browning index. The flour obtained from this sample also showed high reducing sugar content, and good fructans conservation (around 60% compared to freeze-dried) and the highest total polyphenol content (≈450 mg GAE/100 g) and antioxidant activity. Starch-based gluten-free muffins were made with 5% and 10% of substitution of corn starch by flour obtained from dried sliced-yacon with minimal effect on crust and crumb color. Addition of yacon flour made muffins less firm and more cohesive with a decrease in chewiness as compared to control. Its addition also generated a larger number of smaller pores in the muffin's microstructure. The results highlight that yacon flour may be produced by different methods and this powder represents an ingredient with considerable potential for food enrichment.

BARLEYmax^® is a unique barley rich in dietary fiber and resistant starch, which are considered to decrease the risk of developing type II diabetes mellitus. In this study, we evaluated the postprandial glycemic response for boiled BARLEYmax and determined its glycemic index in a Japanese population. Eleven healthy subjects were administered a 50 g/150 mL glucose drink twice and boiled BARLEYmax containing 50 g available carbohydrate after a wash-out period. Postprandial blood glucose, its change from baseline over 90 min, and the incremental area under the curve for BARLEYmax were statistically lower than those for the glucose drink. The glycemic index of the BARLEYmax was 24.3 (standard error of the mean, 3.5). These results suggest that in Japanese individuals, boiled BARLEYmax contributes to improving the postprandial glycemic response, which is one of the possible risk factors for the development of type II diabetes mellitus.

A rising global population necessitates continued genetic improvement of wheat (Triticum spp.), but not without monitoring of unintended consequences to processors and consumers. Our objectives were to re-establish trends of genetic progress in agronomic and milling traits using a generational meter stick as the timeline rather than cultivar release date, and to measure correlated responses in flour quality and human wheat-sensitivity indicators. Grain yield and kernel size showed stepwise increases over cycles, whereas wheat protein content decreased by 1.1 g/100 g. Reduced protein content, however, did not result in lower dough strength pertinent to bread baking. A novel method of directly testing gluten elasticity via the compression-recovery test indicated a general increase in gluten strength, whereas the ratio of total polymeric to total monomeric proteins remained stable. Also showing no change with genetic progress in yield were flour levels of gluten epitopes within the key immunotoxic 33-mer peptide. The oligosaccharide fructan, present in milled and wholemeal flours, increased with increasing grain yield potential. While yield improvement in U.S. bread wheat was not accompanied by a decline in gluten strength or systematic shift in a key wheat sensitivity parameter, the unanticipated rise in total fructans does implicate potentially new dietary concerns.

Browning is the major problem associated with yacon [Smallanthus sonchifolius (Poepp.) H. Rob.] processing as it takes place rapidly and intensely after tissue damage. This study investigated the effects of different blanching conditions on the browning parameters and some chemical components of yacon. Whole yacon tubers were blanched in hot water under different temperature and time conditions and immediately cooled in ice water. Increasing blanching temperature and time significantly reduced the polyphenol oxidase (PPO) activity but resulted in an undesirable change in firmness; lightness (L^*) decreased approaching to a black color flesh of yacon, and the a^* and b^* values decreased representing a tendency to a green and blue colors, respectively. Blanching the whole yacon at 60⁰C for 20 mins was found to be the most effective and can be used as preprocessing treatment to reduce browning of the product. At this condition, PPO activity was reduced to 70%, firmness was retained, and further browning was decelerated when the cut surface of yacon was allowed to stand at room temperature. Moreover, the total phenolic content (TPC), antioxidant activity, glucose, sucrose and fructose were not significantly different from unblanched yacon but fructooligosaccharide (FOS) content was reduced from 51.36 to 36.29% on dry weight basis.

FODMAPs are fermentable oligo-, di, and monosaccharides and polyols, which can have adverse effects on human health. Especially in individuals with gastrointestinal disorders such as irritable bowel syndrome, ingestion of FODMAPs may trigger or aggravate symptoms like abdominal pain, bloating, or diarrhea. Our objectives were to investigate the FODMAP content in the flour of 21 wheat varieties and in their 42 yeast-leavened breads prepared with a short (110 min) or a long dough fermentation (24 h), the latter according to a typical baker's recipe. Fructan, glucose, fructose and excess fructose were measured using enzymatic kits. On average, the breads of both dough fermentations had a FODMAP content reduced by > 65% compared to that of the whole grain wheat flour. Average FODMAP content of both dough fermentations was 0.22 g per 100 g fresh bread. FODMAP content tended to be lower in the long compared to the short dough fermentation, however, the difference was not statistically significant. We also found that the 21 wheat varieties differed up to five times in their potential to form FODMAPs in bread. In conclusion, the choice of a low fructan wheat variety coupled with long dough fermentation with sourdough and yeast is best to minimize FODMAPs in breads.

Jerusalem artichokes contain high amounts of inulin, which is a prebiotic that supports digestive health, as well as a variety of insoluble fibers and caffeoylquinic acid. The individual impact of these components on gut microbiota is well known; however, the combinatorial effects are less clear. In this investigation, we fractionated Jerusalem artichokes into three parts (water-soluble extract, insoluble extract, and organic extract) and powdered them. Mice were fed a high-fat diet that included one or more of these extracts for 10 days, and then their cecal pH, cecal short-chain fatty acids (SCFAs), and fecal microbiota were evaluated. The combination of the water-soluble and organic extract decreased cecal pH and increased the concentration of SCFAs and led to dynamic changes in the composition of the gut microbiota. These results demonstrate that both the water-soluble and organic extracts in Jerusalem artichokes are bioactive substances that are capable of changing SCFA production and the composition of gut microbiota. Powdered Jerusalem artichokes, rather than inulin supplements, may be superior for promoting a healthy gut.

Date fruits (Phoenix dactylifera) of ten varieties, collected in the United Arab Emirates, were studied to determine their dietary fiber content and composition. Fourier transform infrared (FTIR) spectroscopy indicated that the dietary fiber components in all the date fruit varieties was similar. The major dietary fiber components, including cellulose, hemicellulosic components, lignin, and pectin, were analyzed by the Uppsala method. The total dietary fiber content in the date fruits analyzed (5.2%-8.3%) is comparable to commonly consumed dried fruits and is correlated with the content of lignin. The lignin was the main determinant of the total dietary fiber content in dates and its content was higher in semi-hard and hard fruit varieties.

Yacon syrup is a rich source of fructooligosaccharides (FOS); however, its diet supplementation effect on subjective sensation and appetite biomarkers in human is still unknown. This study aimed to evaluate the acute postprandial effect of yacon syrup ingestion on appetite. The double-blind crossover clinical trial was carried out with 40 adult women: 20 eutrophic and 20 obese grade I. On each day, the first blood collection was performed after a 12-h fast. Then, the volunteers ingested either intervention A (breakfast + 40 g of placebo) or intervention B (breakfast + 40 g of yacon syrup, containing 14 g of FOS). New aliquots of blood were collected at 45, 60, 90, 120, and 180 min. Appetite was assessed by estimating ghrelin and glucagon-like peptide-1 (GLP-1) levels and by assessing subjective appetite sensation. Analysis was performed using two-way ANOVA, followed by Bonferroni's multiple comparison test. No effect of yacon syrup was observed on postprandial ghrelin and GLP-1 levels at all times evaluated. Similar observations were made after stratifying the analysis by BMI (body mass index) (eutrophic and obese). The effect of yacon syrup on postprandial subjective sensations of hunger, satiety, fullness, and desire to eat was not evident in the total group of women evaluated and even after BMI stratification. We concluded that yacon syrup had no effect on postprandial ghrelin and GLP-1 levels and on the subjective appetite sensation in young adult women.

Background: FODMAPs (Fermentable oligosaccharides, disaccharides, monosaccharides, and polyols) intake is associated with the onset of irritable bowel syndrome symptoms. FODMAPs in wheat-derived baked goods may be reduced via bioprocessing by endogenous enzymes and/or microbial fermentation. Because of the inherent enzyme activities, bread made by baker’s yeast and sourdough may result in decreased levels of FODMAPs, whose values are, however, not enough low for people sensitive to FODMAPs. Results: Our study investigated the complementary capability of targeted commercial enzymes and metabolically strictly fructophilic lactic acid bacteria (FLAB) to hydrolyze fructans and deplete fructose during wheat dough fermentation. FLAB strains displayed higher fructose consumption rate compared to conventional sourdough lactic acid bacteria. Fructose metabolism by FLAB was faster than glucose. The catabolism of mannitol with the goal of its reuse by FLAB was also investigated. Under sourdough conditions, higher fructans breakdown occurred in FLAB inoculated doughs compared to conventional sourdough bacteria. Preliminary trials allowed selecting Apilactobacillus kunkeei B23I and Fructobacillus fructosus MBIII5 as starter candidates, which were successfully applied in synergy with commercial invertase for low FODMAPs baking. Conclusions: Results of this study clearly demonstrated the potential of selected strictly FLAB to strongly reduce FODMAPs in wheat dough, especially under liquid-dough and high oxygenation conditions.

The benefits of a diet, low in fermentable oligo-, di- and monosaccharides and polyols (FODMAP) for patients suffering from irritable bowel syndrome (IBS) has been well established. Thus, the exact knowledge of the amount and composition of FODMAPs in foods is of vital importance for these patients. This study investigated the possibilities of FODMAP reduction by adjusting the processing parameters, which are feasible in practise, while still producing marketable breads. Therefore, the impact of prolonged proofing and the addition of sourdough on the FODMAPs in the final products was evaluated. High performance anion exchange chromatography was used for qualitative and quantitative analysis. A prolonged proofing time resulted in reduction of the fructan content and in consequence of the total FODMAPs. In contrast, the addition of sourdough only altered the FODMAP composition, by reducing the fructan content but increasing the mannitol content. While all the breads produced from refined wheat flour meet the low-FODMAP criteria, the breads from rye and whole meal wheat flour have to be considered as high-FODMAP, regardless of the processing conditions investigated. Breads produced from rye flour exceeded the limits for fructans and mannitol, while whole meal wheat breads were found to exceed the threshold for excess fructose. Overall, the production of low-FODMAP rye breads was identified as the biggest challenge for future research, since it could not be achieved using conventional processing parameter.

Whole grains and pulses are rich in nutrients but often avoided by individuals with gastrointestinal disorders, due to high levels of fermentable oligo-, di-, monosaccharides and polyols (FODMAPs). This study investigated the impact of malting as delivery-system for endogenous enzymes. Malts from barley and wheat (naturally high in fructans), lentils and chickpeas (high in galactooligosaccharides), oat and buckwheat (low in FODMAPs) were produced. While barley and wheat malts had slightly elevated fructan-levels, in oat malt 0.8 g/100 g DM fructans were de novo synthesized. In lentils and chickpeas galactooligosaccharides diminished by 80–90 %. Buckwheat did not contain any FODMAPs commonly investigated, but fagopyritols which may have a similar physiological effect. Also fagopyritols were degraded. While malted pulses and buckwheat are directly suitable for low FODMAP applications, using the combined approach of malting and fermentation, malted cereals could contribute to high nutritional values of such products.

To further our understanding of how sustained changes in temperature affect the carbon economy of rice (Oryza sativa), hydroponically grown plants of the IR64 cultivar were developed at 30°C/25°C (day/night) before being shifted to 25/20°C or 40/35°C. Leaf messenger RNA and protein abundance, sugar and starch concentrations, and gas‐exchange and elongation rates were measured on preexisting leaves (PE) already developed at 30/25°C or leaves newly developed (ND) subsequent to temperature transfer. Following a shift in growth temperature, there was a transient adjustment in metabolic gene transcript abundance of PE leaves before homoeostasis was reached within 24 hr, aligning with R_dark (leaf dark respiratory CO₂ release) and A_n (net CO₂ assimilation) changes. With longer exposure, the central respiratory protein cytochrome c oxidase (COX) declined in abundance at 40/35°C. In contrast to R_dark, A_n was maintained across the three growth temperatures in ND leaves. Soluble sugars did not differ significantly with growth temperature, and growth was fastest with extended exposure at 40/35°C. The results highlight that acclimation of photosynthesis and respiration is asynchronous in rice, with heat‐acclimated plants exhibiting a striking ability to maintain net carbon gain and growth when exposed to heat‐wave temperatures, even while reducing investment in energy‐conserving respiratory pathways.