Verbascose

Microarrays are powerful tools for high throughput analysis, and hundreds or thousands of molecular interactions can be assessed simultaneously using very small amounts of analytes. Nucleotide microarrays are well established in plant research, but carbohydrate microarrays are much less established, and one reason for this is a lack of suitable glycans with which to populate arrays. Polysaccharide microarrays are relatively easy to produce because of the ease of immobilizing large polymers noncovalently onto a variety of microarray surfaces, but they lack analytical resolution because polysaccharides often contain multiple distinct carbohydrate substructures. Microarrays of defined oligosaccharides potentially overcome this problem but are harder to produce because oligosaccharides usually require coupling prior to immobilization. We have assembled a library of well characterized plant oligosaccharides produced either by partial hydrolysis from polysaccharides or by de novo chemical synthesis. Once coupled to protein, these neoglycoconjugates are versatile reagents that can be printed as microarrays onto a variety of slide types and membranes. We show that these microarrays are suitable for the high throughput characterization of the recognition capabilities of monoclonal antibodies, carbohydrate-binding modules, and other oligosaccharide-binding proteins of biological significance and also that they have potential for the characterization of carbohydrate-active enzymes.

Germinating seeds of lucerne, guar, carob and soybean initially depleted raffinose series oligosaccharides and then galactomannan. This depletion was accompanied by a rapid increase and then a decrease in α-galactosidase levels. Lucerne and guar contained two α-galactosidase activities, carob three and soybean four. One of these in each plant, from its location in the endosperm, time of appearance and kinetic behaviour, appeared to be primarily involved in galactomannan hydrolysis. This enzyme in lucerne had MW of 23 000 and could not be separated from β-mannanase by (NH₄)₂SO₄ fractionation, DEAE, CM or SE-cellulose chromatography or gel filtration, but only by polyacrylamide gel electrophoresis. In guar, carob and soybean, it could be separated by ion-exchange chromatography and gel filtration. In lucerne, carob and guar most of the total increase in activity was due to this enzyme. The other α-galactosidases had MWs of about 35 000 and could be separated from β-mannanase by dissection, ion exchange cellulose chromatography and gel filtration. They were located in the cotyledon-embryo and appeared to be primarily involved in galactosylsucrose oligosaccharide hydrolysis.

Faba bean (Vicia faba L.) is a potential, sustainable protein alternative. Germination behavior of Vicia faba L. var. minor and Vicia faba L. var. major needs further studies in order to enable larger scale bioprocessing. In this study, early-stage water uptake of two distinct faba bean varieties was assessed by hyperspectral imaging. Nutritional and technological functionality of germinated faba bean ingredients as such and in combination with fermentation were evaluated. Hyperspectral imaging revealed that early-stage water uptake in faba beans occurred evenly from the different sides of the beans. Germination on petri dishes and in pilot-scale showed that smaller faba beans moistened and germinated significantly (p < 0.05) faster and retained water better through germination than larger faba beans. Germinated faba flour of minor-type variety resulted in 72% higher dextran production in Weissella confusa VTT E-143403 fermentation than respective native faba flour. With both types of faba bean varieties, germination as such, and with minor-type variety, germination as combined with fermentation decreased notably the content of raffinose family oligosaccharides. These bioprocessing methods also improved functionality of faba bean ingredients by increasing protein separation efficiency in air classification, protein solubility, foaming capacity, and foam stability. Based on this study, minor- and major-type or small- and large-seeded faba bean varieties set different requirements to the germination process. In addition, germination alone or as combined with fermentation was proved to improve the nutritional and technological quality of faba bean material promoting its use in several food applications including also gluten-free products.

Raffinose family oligosaccharides are non-digestible compounds considered as dietary prebiotics with health-related properties. Hence, it is important to develop highly specific methods for their determination. An analytical method is developed in this study for oligosaccharide identification and quantification using liquid chromatography-tandem mass spectrometry equipped with a triple quadrupole analyser operating in Multiple Reaction Monitoring mode. Raffinose, stachyose and verbascose are separated in a 10-minute run and the method is validated over a broad concentration range, showing good linearity, accuracy, precision and high sensitivity. A low-cost, short eco-friendly procedure for oligosaccharide extraction from legumes, with a high recovery rate extraction, good repeatability and reproducibility is also proposed. No plant-matrix effects were demonstrated. The method applied to the screening of 28 different legumes revealed species-related traits for oligosaccharide distribution, highlighting Pisum sativum (9.22 g/100 g) as the richest source of these prebiotics and its suitability as a functional food ingredient.

A sustainable dry processing method to obtain nutritional and functional chickpea products was developed, yielding protein concentrates suited to prepare products without additives. Chickpeas were milled and air-classified into protein and starch-enriched concentrates. Subsequently, spontaneous solid state fermentation (SSF) with daily back-slopping was performed at 37°C. The dominant autochthonous lactic acid bacteria (LAB) strains in chickpea flour and enriched fractions were Pediococcus pentosaceus and Pediococcus acidilactici. Strains were selected on their ability to metabolise flatulence-causing α-galactosides. SSF reduced the pH of the doughs in 24h from 6.6 to 4.2. After 72 h, concentrations of raffinose and stachyose were reduced by 88.3-99.1%, while verbascose became undetectable. Moreover, phytic acid reduced with 17% while total phenolic contents increased with 119%. Besides the observed differences in smell, texture and colour, the sourdoughs showed 67% higher water-holding capacity. This natural route to produce chickpea concentrates thus increases both the nutritive and technical functionality.

Fava bean flour is regarded as a potential plant-based protein source, but the addition of it at high concentration is restricted by its poor texture-improving ability and by anti-nutritional factors (ANF). Exopolysaccharides (EPS) produced by lactic acid bacteria (LAB) are regarded as good texture modifiers. In this study, fava bean flour was fermented with Leuconostoc spp. and Weissella spp. with or without sucrose addition, in order to evaluate their potential in EPS production. The contents of free sugars, organic acids, mannitol and EPS in all fermented fava bean doughs were measured. Rheological properties of sucrose-enriched doughs, including viscosity flow curves, hysteresis loop and dynamic oscillatory sweep curves, were measured after fermentation. As one of the ANF, the degradation of raffinose family oligosaccharides (RFO) was also studied by analyzing RFO profiles of different doughs. Quantification of EPS revealed the potential of Leuconostoc pseudomesenteroides DSM 20193 in EPS production, and the rheological analysis showed that the polymers produced by this strain has the highest thickening and gelling capability. Furthermore, the viscous fava bean doughs containing plant proteins and synthesized in situ EPS may have a potential application in the food industry and fulfill consumers' increasing demands for “clean labels” and plant-originated food materials.

The content of raffinose family oligosaccharides (RFOs) in pea seeds constrains their usage in feeding humans and animals. In our research, the content of soluble carbohydrates—particularly α-D-galactosides of sucrose (RFOs)—was analyzed. The materials were as follows: 248 accessions from the Polish Pisum Genebank including representatives of taxa (from species to convarietas), type lines for genes controlling seed characters, and breeding materials and cultivars. Accessions were divided into homogeneous groups considering content of total soluble carbohydrates, total RFOs and individual RFOs: raffinose, stachyose and verbascose. The highest content of total soluble carbohydrates and total RFOs were stated for accessions with wrinkled seeds (r and rb genes) and the lowest content for seeds of the wild species P. fulvum Sibth. et Sm. Accessions valuable for breeding (for further decreasing of anti-nutritional compounds) were found among domesticated taxa (P. sativum subsp. sativum convar. vulgare Alef. And speciosum (Dierb.) Alef.), breeding lines, and some wild taxa. Accessions with decreased content of a total RFOs and verbascose are particularly valuable. It was found that the content of total RFOs was the most highly, frequently, and positively correlated with a stachyose and verbascose content. However, in P. fulvum seeds with the lowest content of RFOs and verbascose, a high correlation between the content of total RFOs and stachyose was revealed. Contents of all oligosaccharides were substantially lower in lines with dominant alleles of main pea seed genes (R, A, and I). It can be assumed that wild, primitive peas were characterized by not-all-to-high (rather not high) content of oligosaccharides; then recessive mutations in mentioned genes resulted in an increased content of RFOs. It seems to be an interesting observation from an evolutionary point of view.

Optimization and validation of evaporative light scattering detector (ELSD), aided by response surface methodology (RSM), has been developed for the liquid chromatography analysis of a wide molecular weight (MW) range of carbohydrates, including polysaccharides and oligosaccharides. Optimal experimental parameters for the ELSD detection were: 88.8°C evaporator temperature, 77.9°C nebulizer temperature and 1.1 standard litres per minute nitrogen flow rate. Optimal ELSD detection, used together with high performance size exclusion chromatography (HPSEC) of carbohydrates, gave a linear range from 250 to 1000 mg L^-1 (R² > 0.998), with limits of detection and quantitation of 4.83–11.67 and 16.11–38.91 mg L^-1, respectively. Relative standard deviation was lower than 1.8% for intra-day and inter-day repeatability for apple pectin, inulin, verbascose, stachyose and raffinose. Recovery ranged from 103.7% to 118.3% for fructo-oligosaccharides, α-galacto-oligosaccharides and disaccharides. Optimized and validated ELSD detection is proposed for the analysis of high- to low-MW carbohydrates with high sensitivity, precision and accuracy.

The accumulation of Late Embryogenesis Abundant (LEA) proteins in plants is associated with tolerance against stresses such as freezing and desiccation. Two main functions have been attributed to LEA proteins: membrane stabilization and enzyme protection. We have hypothesized previously that LEA7 from Arabidopsis thaliana may stabilize membranes because it interacts with liposomes in the dry state. Here we show that LEA7, contrary to this expectation, did not stabilize liposomes during drying and rehydration. Instead, it partially preserved the activity of the enzyme lactate dehydrogenase (LDH) during drying and freezing. Fourier-transform infrared (FTIR) spectroscopy showed no evidence of aggregation of LDH in the dry or rehydrated state under conditions that lead to complete loss of activity. To approximate the complex influence of intracellular conditions on the protective effects of a LEA protein in a convenient in-vitro assay, we measured the activity of two Arabidopsis enzymes (glucose-6-P dehydrogenase and ADP-glucose pyrophosphorylase) in total soluble leaf protein extract (Arabidopsis soluble proteome, ASP) after drying and rehydration or freezing and thawing. LEA7 partially preserved the activity of both enzymes under these conditions, suggesting its role as an enzyme protectant in vivo. Further FTIR analyses indicated the partial reversibility of protein aggregation in the dry ASP during rehydration. Similarly, aggregation in the dry ASP was strongly reduced by LEA7. In addition, mixtures of LEA7 with sucrose or verbascose reduced aggregation more than the single additives, presumably through the effects of the protein on the H-bonding network of the sugar glasses.

Immature seeds of five bean cultivars (flageolet-type and those intended for dry-seed production) were assessed for changes in water-soluble carbohydrates including raffinose family oligosaccharides (RFOs) due to boiling, sterilization, and storage of the sterilized product. About 100 g fresh weight of edible portion of fresh bean seeds contained 2449.3–3182.6 mg total soluble sugars, of which RFOs comprised 44–49%. The highest amounts of these compounds were found in the seeds of the cultivars Laponia and Mona. The dominant oligosaccharide was stachyose. Boiling fresh seeds to consumption consistency reduced total soluble sugars and RFOs: average values were 57% and 55%, respectively. Sterilization in cans resulted in 65% reductions of both total soluble sugars and RFOs. In general, there were no changes in the content of soluble sugars in canned and sterilized products stored for 12 months.