Simplified enzymatic high-performance anion exchange chromatographic determination of total fructans in food and pet food-limitations and measurement uncertainty

A simplified method to determine total fructans in food and pet food has been developed and validated. It follows the principle of AOAC method 997.08, i.e., high-performance anion exchange chromatographic (HPAEC) determination of total fructose released from fructans (F(f)) and total glucose released from fructans (G(f)) after enzymatic fructan hydrolysis. Unlike AOAC method 997.08, calculation of total fructans is based on the determination of F(f) alone. This is motivated by the inherent difficulty to accurately determine low amounts of G(f) since many food and pet food products contain other sources of total glucose (e.g., starch and sucrose). In this case, a correction factor g can be used (1.05 by default) to take into account the theoretical contribution of G(f). At levels >5% of total fructans and in commercial fructan ingredients, both F(f) and G(f) can and should be accurately determined; hence, no correction factor g is required. The method is suitable to quantify total fructans in various food and pet food products at concentrations >or=0.2% providing that the product does not contain other significant sources of total fructose such as free fructose or sucrose. Recovery rates in commercial fructan ingredients and in selected food and pet food ranged from 97 to 102%. As part of a measurement uncertainty estimation study, individual contributions to the total uncertainty (u) of the total fructan content were identified and quantified by using the validation data available. As a result, a correlation between the sucrose content and the total uncertainty of the total fructan content was established allowing us to define a limit of quantitation as a function of the sucrose content. One can conclude that this method is limited to food products where the sucrose content does not exceed about three times the total fructan content. Despite this limitation, which is inherent to any total fructan method based on the same approach, this procedure represents an excellent compromise with regard to accuracy, applicability, and convenience.     

Fructan Content of Rye and Rye Products

The fructan content of Finnish rye grains (13 samples, seven cultivars, harvested in 1998‐2000) varied at 4.6–6.6 g/100 g (db). Commercial whole grain rye flour and rye flakes had fructan content of 4 g/100 g, light refined rye flour had fructan content of 3 g/100 g, and rye bran had fructan content of 7 g/100 g. Fructan content as high as 23 g/100 g was detected in the water‐extractable concentrate of rye bran. Finnish soft rye bread and rye crisp bread contained 2–3 g of fructan/100 g. According to the suggested new definition of dietary fiber, fructans are also classified as dietary fiber. This means that the dietary fiber content of some cereal foods such as rye products may be increased by as much as 20% due to the presence of fructans in the grain.     

Effect of storage on fructan and fructooligosaccharide of onion (Allium cepa L.)

The purpose of this study was a comparative examination of the fructan and fructooligosaccharide (FOS) content of different varieties of onions (Allium cepa L. cv. Sturon, Hysam, Durco, Grano de Oro, and Caribo) and the changes produced during their commercial storage. In fresh onions, the Grano de Oro variety presented a remarkably different behavior, showing low contents of total fructans and FOS and high levels of reducing sugars. In the other varieties, Sturon, Hysam, Durco, and Caribo, fructans were the main carbohydrates, the lowest polymerized FOS being the major oligomer. Storage period caused in these varieties important increased levels of free fructose attributed to fructan hydrolysis. Maleic hydrazide treatment had no significant effect in avoiding the hydrolysis of fructans during storage conditions for the Sturon variety. Varieties with >16% dry matter or 15% soluble solids contents could be stored for 6 months at 0 degrees C and 60-65% relative humidity.     

Molecular structures of fructans from Agave tequilana Weber var. azul

Agave plants utilize crassulacean acid metabolism (CAM) for CO(2) fixation. Fructans are the principal photosynthetic products generated by agave plants. These carbohydrates are fructose-bound polymers frequently with a single glucose moiety. Agave tequilana Weber var. azul is an economically important CAM species not only because it is the sole plant allowed for tequila production but because it is a potential source of prebiotics. Because of the large amounts of carbohydrates in A. tequilana, in this study the molecular structures of its fructans were determined by fructan derivatization for linkage analysis coupled with gas chromatography-mass spectrometry (GC-MS), nuclear magnetic resonance (NMR), and matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF-MS). Fructans were extracted from 8-year-old A. tequilana plants. The linkage types present in fructans from A. tequilana were determined by permethylation followed by reductive cleavage, acetylation, and finally GC-MS analysis. Analysis of the degree of polymerization (DP) estimated by (1)H NMR integration and (13)C NMR and confirmed by MALDI-TOF-MS showed a wide DP ranging from 3 to 29 units. All of the analyses performed demonstrated that fructans from A. tequilana consist of a complex mixture of fructooligosaccharides containing principally beta(2 --> 1) linkages, but also beta(2 --> 6) and branch moieties were observed. Finally, it can be stated that fructans from A. tequilana Weber var. azul are not an inulin type as previously thought.     

Characterization of Indigestible Carbohydrates in Various Fractions from Wheat Processing

Fractions rich in indigestible carbohydrates, such as fructan and arabinoxylan, are obtained as by‐products when ethanol, starch, and gluten are produced from wheat flour. Today, these fractions are used as animal feed. However, these components may have positive physiological effects in humans. In this study, the content of indigestible carbohydrates in distillers' grains and process streams from the wet fractionation of wheat flour was determined. The fractions were further characterized by ethanol extractability analysis, anion‐exchange chromatography, NMR, and size‐exclusion chromatography. One fraction from wet fractionation contained (g/100 g, db) 6.0 ± 1.0 fructan and 10.3 ± 1.1 dietary fiber (66 ± 4% arabinoxylan), while distillers' grains contained 20.7 g/100 g (db) dietary fiber (30% arabinoxylan). In addition to indigestible carbohydrates from wheat, distillers' grains contained β‐(1→3) and β‐(1→6) glucans and mannoproteins from the yeast and low molecular weight carbohydrates mainly composed of arabinose. The use of endoxylanase in wet fractionation decreased the molecular weight of the arabinoxylans and increased the arabinose to xylose ratio but had no effect on the fructans. In conclusion, waste streams from industrial wheat processing were enriched in fructan, arabinoxylan, and other indigestible carbohydrates. However, the physiological effects of these fractions require further investigation.     

钾对小麦茎和叶鞘碳水化合物含量及子粒淀粉积累的影响

田间条件下研究了钾素对冬小麦品种鲁麦22茎和叶鞘中碳水化合物含量变化以及灌浆过程中子粒淀粉积累的影响。结果表明,钾素有利于提高茎中果聚糖、蔗糖、果糖和葡萄糖在灌浆期间的积累,促进了灌浆后期果聚糖的降解及蔗糖、果糖和葡萄糖的输出。其中以K2处理的作用为最大;有利于提高灌浆过程中叶鞘果聚糖、果糖、蔗糖和葡萄糖的积累,但提高幅度小于茎中的提高幅度;同时钾素亦提高了灌浆期间子粒中可溶性总糖和蔗糖的供应,加速了淀粉积累速率,其中以K2处理为最高,最终获得了最高的产量(9411 15kg/hm2)和最大的钾素生产效率。     

Quantitation of N2-[1-(1-carboxy)ethyl]folic acid, a nonenzymatic glycation product of folic acid, in fortified foods and model cookies by a Stable isotope dilution assay

A stable isotope dilution assay (SIDA) for the quantitation of N(2)-[1-(carboxy)ethyl]folic acid (CEF) has been developed by using [(2)H(4)]CEF as the internal standard. After sample cleanup by anion exchange chromatography, the three-dimensional specifity of liquid chromatography-tandem mass spectrometry enabled unequivocal determination of the nonenzymatic glycation product of folic acid (FA). When CEF was added to cornstarch, the detection limit for CEF was found to be 0.4 microg/100 g, and a recovery of 98.5% was determined. In analyses of cookies, the intra-assay coefficient of variation was 8.0% (n = 5). Application of the SIDA to commercial cookies produced from wheat flour fortified with FA revealed CEF contents of up to 7.1 microg/100 g, which accounted for approximately 10-20% of the cookies' FA content. In baby foods, multivitamin juices, and multivitamin sweets, however, CEF was not detectable. Further studies on CEF formation during baking of cookies made from fortified flour and different carbohydrates revealed that fructose was most effective in generating CEF followed by glucose, lactose, and sucrose with 12.5, 3.9, 2.5, and 2.5 microg/100 g of dry mass, respectively. During baking, approximately 50% of FA was retained for both monosaccharides fructose and glucose, and 77% as well as 85% of its initial content was retained for the disaccharides lactose and sucrose, respectively. Of the degraded amount of FA, CEF comprised 28% for fructose as well as 18, 12, and 8% for sucrose, lactose, and glucose, respectively. Therefore, CEF can be considered an important degradation product of FA in baked foods made from fructose. To retain a maximum amount of FA, products should rather be baked with sucrose than with reducing carbohydrates.     

Enzymatic degradation of oligosaccharides in pinto bean flour

The use of dry edible beans is limited due to the presence of flatulence factors, the raffinose oligosaccharides. Our objective was to investigate the process for the removal of oligosaccharides from pinto bean using enzymatic treatment and to compare it to removal by soaking and cooking methods. Crude enzyme preparation was produced by six fungal species on wheat bran- and okara-based substrates with soy tofu whey. The loss of raffinose oligosaccharides after soaking pinto beans for 16 h at the room temperature was 10%, after cooking for 90 min was 52%, and after autoclaving for 30 min was 58%. On the other hand, the treatment using crude alpha-galactosidase (60 U mL(-1)) produced by Aspergillus awamori NRRL 4869 from wheat bran-based substrate with soy tofu whey on pinto bean flour for 2 h completely hydrolyzed raffinose oligosaccharides. These results supported that the enzymatic treatment was the most effective among various processing methods tested for removing the raffinose oligosaccharides, and hence, crude alpha-galactosidases from fungi have potential use in the food industry.     

Sucrose metabolism and exopolysaccharide production in wheat and rye sourdoughs by Lactobacillus sanfranciscensis.

The exopolysaccharide (EPS) produced from sucrose by Lactobacillus sanfranciscensis LTH2590 is predominantly composed of fructose. EPS production during sourdough fermentation has the potential to affect rheological properties of the dough as well as the volume, texture, and keepability of bread. Its in situ production by L. sanfranciscensis LTH2590 was demonstrated during sourdough fermentation after the hydrolysis of water soluble polysaccharides. In wheat and rye doughs with sucrose addition the concentration of fructose in the hydrolysate of polysaccharides was significantly higher than that in the hydrolysate of control doughs or doughs without sucrose addition. EPS production by L. sanfranciscensis in wheat doughs was confirmed by the determination of delta (13)C values of water soluble polysaccharides after the addition of naturally labeled sucrose, originating from C(3)- and C(4)-plants. In rye doughs, evidence for EPS production with the isotope technique could be demonstrated only by the determination of delta (13)C values of fructose from water soluble polysaccharides. In addition to EPS formation from sucrose, sucrose hydrolysis by L. sanfranciscensis in wheat and rye sourdoughs resulted in an increase of mannitol and acetate concentrations and in accumulation of glucose. It was furthermore observed that flour arabinoxylans were solublized during the fermentation.     

Degree of polymerization of inulin-type fructans differentially affects number of lactic acid bacteria, intestinal immune functions, and immunoglobulin A secretion in the rat cecum

This study examined the role of degree of polymerization (DP) of inulin-fructans in modulating the interaction between lactic acid bacteria and IgA cecal secretion. Rats were fed a control diet or a diet containing one of the fructans with different DP. Consuming fructans increased the cecal IgA concentrations in the order DP4 > DP8 > DP16. Cecal lactobacilli counts were higher in DP4, DP8, and DP16, whereas bifidobacteria were higher in DP8, DP16, and DP23. Cecal IgA concentrations were correlated with cecal lactobacilli counts (P < 0.01). DP4, DP8, and DP16, but not DP23, significantly increased IgA-producing plasma cells in the cecal mucosa. IFN-γ and IL-10 production in the cecal CD4(+) T cells was enhanced solely in DP4. The results show that fructans with lower DP enhance cecal IgA secretion and increase the plasma cells and suggest that the increased lactobacilli may contribute to the stimulation of cecal IgA secretion.     

Fructan and free fructose content of common Australian vegetables and fruit

Fructans are not digested in the small intestines of humans. While many health benefits have been attributed to these carbohydrates, they can cause gastrointestinal symptoms in some individuals. We measured the total fructans in 60 vegetables and 43 fruits using the Megazyme fructan assay. Vegetables with the highest quantity of fructans included garlic, artichoke, shallots, leek bulb, and onions (range, 1.2-17.4 g/100 g fw). Fruits with low, but detectable, fructans included longon, white peach, persimmon, and melon (range, 0.21-0.46 g/100 g fw). The fructan assay was modified to provide an estimate of the average chain length (degree of polymerization) for high fructan vegetables. d-Fructose can also be malabsorbed in the small intestine of humans, so the d-fructose content in some foods was measured to supplement the current food tables. Research in this area will be facilitated through the availability of more comprehensive food composition data.     

Effect of extraction procedure on measured sugar concentrations in onion (Allium cepa L.) bulbs

Bulb samples from a range of onion cultivars grown over three consecutive years were freeze-dried and the resulting powders extracted using three previously reported methods. The extracts were analyzed for fructose, glucose, and sucrose content using HPLC coupled with ELSD, and for fructans using MALDI-MS. The three methods gave differing results, indicating that the extraction procedure is crucial in the determination of the concentration and ratios of nonstructural carbohydrates in onion bulbs. O'Donoghue et al.'s method (O'Donoghue, E. M.; Somerfield, S. D.; Shaw, M.; Bendall, M.; Hedderly, D.; Eason, J.; Sims, I. J. Agric. Food Chem. 2004, 52, 5383-5390), which utilized a more polar solvent (62.5% (v/v) aqueous methanol) and also had the benefit of shorter extraction times and lower temperatures, was far superior to 80% (v/v) ethanol-based methods in extracting significantly greater amounts of fructose, glucose, and sucrose from all onion bulbs tested. Discrepancies between and within cultivars tested also demonstrated that the ratio of monosaccharides to sucrose was affected by extraction method, such that some caution should be given to interpreting some previous work on elucidating the nonstructural carbohydrate composition in onion.     

Water-soluble carbohydrates and fructan structure patterns from Agave and Dasylirion species

Fructans, storage carbohydrates with beta-fructofuranosyl linkages, are found in approximately 15% of higher plants. The metabolic flexibility of those molecules allows them easily to polymerize and depolymerize to soluble carbohydrates according to plant development stage and environmental conditions. In this work, water-soluble carbohydrates, including fructan structure patterns, were compared among Agave and Dasylirion species grown in different environmental regions in Mexico. Fructans were the main storage carbohydrate present in Agave stems, in addition to other carbohydrates related to its metabolism, whereas Dasylirion spp. presented a different carbohydrate distribution. A good correlation of water-soluble carbohydrate content with climatic conditions was observed. Fructans in Agave and Dasylirion genera were found in the form of polydisperse molecules, where structural heterogeneity in the same plant was evidenced by methylation linkage analysis and chromatographic methods. Fructans from the studied species were classified into three groups depending on DP and linkage-type abundance. These storage carbohydrates share structural characteristics with fructans in plants that belong to the Asparagales members. Agave and Dasylirion fructans can be categorized as graminans and branched neo-fructans, which we have termed agavins.     

Effect of Tempering Conditions on Milling Performance and Flour Functionality

Tempering conditions of wheat grain change the quality of the flour, yet most experimental milling systems use a standard tempering without optimization. The effect of tempering condition on milling performance and flour functionality for soft red winter (SRW) wheat grain was tested by measuring flour yield, ash, polyphenol oxidase (PPO), and solvent retention capacity (SRC) in grain samples from three SRW cultivars (Roane, Cyrus, and Severn). Tempering was conducted with a full factorial design of initial wheat moisture, tempered wheat moisture, tempering temperature, and tempering time at two levels. Tempered wheat moisture had the largest effect on milling performance and flour functionality. Flour yield was more reduced for all samples tempered at 15% moisture than for samples tempered to 12% moisture. Flour quality of the 15% tempered sample was better than the 12% tempered samples due to less bran contamination as measured by flour ash and PPO. Increasing the tempering moisture increased flour sucrose SRC and lactic acid SRC but reduced sodium carbonate SRC for samples. Changing tempered wheat moisture changed flour yield and quality much more than did changing the length of time for tempering, the temperature at wheat is tempered, or differences in the initial moisture of the wheat before tempering. The last three effects could be used to improve flour yield in both the 12 and 15% tempered wheat treatment but the detrimental effects of these treatments on flour quality were minimal when combined with the 15% tempered wheat moisture treatment.     

Characterization of rhizosphere products especially 2-ketogluconic acid

Examination of rhizosphere products of wheat seedlings using a variety of Chromatographie techniques established that 2-ketogluconic acid was the only acid present in significant amounts. Elemental analysis of crystallized potassium 2-ketogluconate isolated from the rhizosphere products and data obtained by X-ray diffraction, i.r. spectrometry and specific rotation determinations confirmed the identification of the 2-ketogluconic acid. Spectrophotometric and specific rotation measurements showed that 2-ketogluconic acid represented about 20% of the rhizosphere products. Furthermore, the 2-ketogluconic acid was present in fractions of rhizosphere products shown to release phosphate from calcium phosphates. Pure 2-ketogluconic acid released phosphate from apatite rock, di- and tri-calcium phosphates. In each case there was an associated decrease in pH of the suspensions compared with controls.About 38% of the rhizosphere products was carbohydrate, and glucose and fructose were the dominant free sugars present. Since 2-ketogluconic acid is known to be a product of microbial action on glucose and the organisms involved occur in the rhizosphere it is likely that the 2-ketogluconic acid was produced from glucose in the rhizosphere.     

不同制粉方式对运黑161全麦粉加工特性的影响

为探讨不同制粉方式及不同粒度对黑小麦流变学特性及加工品质的影响,将运黑161黑小麦经直接粉碎法和回添法制得的全麦粉分别过80、100、120目筛,以运黑161黑小麦面粉为对照,分析全麦粉淀粉含量、水合特性、发酵特性以及流变学特性。结果表明,直接粉碎且过80目筛的全麦粉支链淀粉含量最高,为80.18%,破损淀粉含量最低,为9.93%,溶解度、膨润力最小,分别为2.5%和3%。回添法制备的全麦粉面团发酵高度、持气率小于直接粉碎法。直接粉碎且过80目筛全麦粉的粉质质量指数最高,为59.5,形成时间和稳定时间分别为3.45和3.2 min,较接近于黑小麦面粉,且该全麦粉的阻力值、最大阻力值在醒发时间135 min时最大,用该全麦粉制作的面包感官得分最高。综上,以直接粉碎且过80目筛制备的全麦粉为原料制作的全麦面包品质较好。本研究结果为黑小麦全麦面包研发提供了理论基础。     

Starch Properties and Stability of Club and Soft White Winter Wheats from the Pacific Northwest of the United States

Based on examination of 192 club and soft white winter (SWW) wheat samples, club and SWW wheat flours showed comparable levels of starch damage and flour peak viscosity, while differing significantly in starch content. Varietal differences and growing conditions had strong influence on the characteristics of both classes of wheat flour. Club wheat flour exhibited better stability in starch content and starch damage than did SWW wheat flour. A significant correlation between starch damage and cookie diameter in both club and SWW wheat was observed (r = -0.480, P < 0.0001 for club wheat and r = -0.430, P < 0.0001 for SWW wheat). Sponge cake volume was positively correlated with starch content in both classes of wheat (r = 0.362, P < 0.01 for club wheat and r = 0.181, P < 0.05 for SWW wheat). When wheat samples were grown in one location over three years, club and SWW wheat flours had comparable starch content. However, flour and prime starch peak viscosities were significantly different in club than in SWW wheat. Club wheat flour had lower starch damage and amylose content, as measured by high-performance size-exclusion chromatography (HPSEC), than did SWW wheat flour. Crop year and varietal differences had significant effect on amylose content, starch damage, and flour and starch peak viscosities, but not on starch content, in both classes of wheat flour. When wheat samples were grown in one year over seven locations, club wheat flour was higher in starch content, lower in starch damage, and comparable in amylose to SWW wheat flour. Both flour and prime starch viscosities were significantly higher in club wheat than in SWW wheat. Varietal differences and growing location had strong influence on starch properties in both classes of wheat. Peak viscosity of the isolated starch did not correlate well with the corresponding flour, indicating that flour pasting property does not reflect the pasting property of starch. The fine structure of isoamylase-debranched amylopectins from club and SWW wheats had a similar tri-modal pattern, with maximum at ≈DP 15 and two valleys at ≈DP 20 and 45, respectively. Although wheat flour samples differed widely in their prime starch peak viscosity, no significant difference between debranching patterns was obtained. These results indicate that the fine structure of amylopectin might not be responsible for the large differences in prime starch pasting property.     

FT-Raman spectroscopic simultaneous determination of fructose and glucose in honey

A new method for mass percentage determination of fructose and glucose based on FT-Raman spectroscopy is evaluated with a standard HPLC-based method. FT-Raman spectra manipulation is done via the spectrometer software, and a PLS (partial least squares) method is developed with the TQ Analyst software (Ver 1. 1a). The simultaneous quantitative determination uses an input range from 1700 to 700 cm(-1) without correction or baseline factors. The standards used in the PLS method are honey samples previously analyzed by HPLC to obtain their mass percentage concentrations in fructose and glucose. The returned results are statistically tested with those of the HPLC method. Both methods appear to score equally in terms of reproducibility. The honey content of the two sugars in total was found up to 40-74%. The honey samples content in fructose and glucose was determined by HPLC (24.1-42.9% and 16.2-33.1%, respectively) and FT-Raman (24.0-40.8% and 21.1-32.2%, respectively).     

Quantitative characterization of nonstructural carbohydrates of mezcal Agave (Agave salmiana Otto ex Salm-Dick)

Fructans are the reserve carbohydrates in Agave spp. plants. In mezcal factories, fructans undergoes thermal hydrolysis to release fructose and glucose, which are the basis to produce this spirit. Carbohydrate content determines the yield of the final product, which depends on plant organ, ripeness stage, and thermal hydrolysis. Thus, a qualitative and quantitative characterization of nonstructural carbohydrates was conducted in raw and hydrolyzed juices extracted from Agave salmiana stems and leaves under three ripeness stages. By high-performance liquid chromatography (HPLC), fructose, glucose, sucrose, xylose, and maltose were identified in agave juice. Only the plant fraction with hydrolysis interaction was found to be significant in the glucose concentration plant. Interactions of the fraction with hydrolysis and ripeness with hydrolysis were statistically significant in fructose concentration. Fructose concentration rose considerably with hydrolysis, but only in juice extracted from ripe agave stems (early mature and castrated). This increase was statistically significant only with acid hydrolysis.     

大蒜果聚糖的水解方法及其定量分析

为了建立大蒜果聚糖含量的快速高效检测方法,本试验以乐都紫皮大蒜为试材,首先通过热水、超声、超声-热水法提取大蒜总糖,分别利用不同的水解试剂类型、pH值、浓度、水解时间将其中的果聚糖彻底水解,通过响应面分析筛选了果聚糖降解的最优条件;随后利用分光光度计和高效液相法测定水解前后果聚糖降解产物(蔗糖、葡萄糖、果糖)的含量并计算差值,得到大蒜组织中所有果聚糖的总含量;最后利用方法学验证对确立的方法进行了优化和评价。结果表明,热水法更有利于提取大蒜可溶性总糖;大蒜果聚糖最佳水解工艺条件为水解温度90℃、HCl浓度3 mol·L^-1、水解时间1 h,经此条件水解后大蒜果聚糖含量预测值为627.903 mg·g^-1 FW,与实际测定值(620.470 mg·g^-1 FW)差异不显著,所得回归模型拟合良好。方法学验证结果显示,该果聚糖定量方法精密度、重复性及稳定性良好,三者的相对标准偏差(RSD)均小于1.5%;平均回收率为98.45%~101.04%,其RSD为0.59%~1.75%。利用此方法鉴定大蒜资源果聚糖性质发现,不同品种大蒜鳞茎中果聚糖含量差异显著。本研究筛选确立的检测方法准确性较高,结果可靠,适用于大蒜果聚糖含量的测定分析。本研究结果为大蒜资源果聚糖性状的深入评价和开发利用提供了理论依据。