1,1,1-Kestopentaose

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.

Levansucrases are key enzymes responsible for the synthesis of β-2,6-linked fructans, found in plants and microbes, especially in bacteria. Levansucrases have been applied in the production of levan biopolymer and fructooligosaccharides (FOSs) using sucrose as a substrate as well as in reducing sugar levels in fruit juice. As a result, levansucrases that are active at low temperatures are required for industrial applications to maintain product stability. Therefore, this work firstly reports the novel cold-active levansucrase (SacBPk) isolated from a sucrolytic bacterial strain, P. koreensis HL12. The SacBPk was classified into glycoside hydrolase family 68 subfamily 1 (GH68_1) and comprised a single catalytic domain with the Asp104/Asp267/Glu362 catalytic triad. Interestingly, the recombinant SacBPk demonstrated cold-active levansucrase activity at low temperatures (on ice and 4–40 °C) with the highest specific activity (167.46 U/mg protein) observed at 35 and 40°C in 50 mM sodium phosphate buffer pH 6.0. SacBPk mainly synthesized levan polymer as the major product (129 g/L, corresponding to 25.8% of total sugar) with a low number of short-chain FOSs (GF2-4) (12.8 g/L, equivalent to 2.5% of total sugar) from 500 g/L sucrose after incubating at 35 °C for 48 h. These results demonstrate the industrial application potential of SacBPk levansucrase for levan and FOSs production.

Fermentable oligosaccharides, di- and monosaccharides, and polyols defined as FODMAPs readily trigger the symptoms of irritable bowel syndrome (IBS), which affects up to 23% of the population, through several mechanisms. A low-FODMAP diet is a short-term solution due to significant nutrient deficiencies, especially in dietary fibre (DF). IBS patients must avoid cereals, especially wholegrain cereals such as wheat and rye, which are an important natural source of DF and therefore FODMAPs (part of soluble DF). This study is the first of its kind to employ biotechnological tools for the creation of wholegrain low-FODMAP cookies and crackers based on wholegrain wheat and rye flours with high FODMAP contents. Endogenous enzymes activated via prolonged dough resting and exogenously activated enzymes originating from chicory extract, wheat malt, and baker’s yeast were employed. The prolonged dough resting time and the addition of wheat malt reduced the FODMAP content in the wholegrain wheat and rye cookies by 46% and 99.5%, respectively. The best result was achieved in the wholegrain wheat crackers, with a FODMAP content reduction of 59.3% based on the combination of a prolonged dough resting time and the addition of wheat malt and baker’s yeast. In the wholegrain rye crackers, a prolonged resting time alone was sufficient to achieve an 83.6% reduction in the total oligosaccharide content.

Early sprouting is a main cause of onion spoilage during storage. However, limited knowledge is available on which factors trigger sprouting. Here, this was studied in the Hyfive and Exhibition cultivars, which largely differ in sprouting time. Sprouting progress was compared to the fructan and abscisic acid (ABA) profiles in the bulb scales and basal plates. Fructan concentrations decreased in the scales from harvest time onwards in the late-sprouting cultivar Hyfive, while remaining constant in the cultivar Exhibition until internal sprouting. In the basal plates, fructan concentrations increased in both cultivars from approximately one month after harvest, but reached maximum concentrations at moments that could not be related to the difference in internal sprouting. ABA levels generally decreased in the scales of both cultivars, while increasing in their basal plates. Nevertheless, for fructans, the measured variation in ABA concentrations was not consistently associated with differences in internal sprouting. A subsequent perturbation of internal sprouting by Maleic Hydrazide treatment in the cultivar Hyfive confirmed a lack of correlation. Altogether, this indicates that fructan and ABA levels in the scales and basal plate tissue change independent of internal sprouting and cannot be regarded as predictive markers for sprouting and storability.

Lactic acid bacteria (LAB) are being used for probiotic and starter cultures to prevent global damage to microbial cells. To retain the benefits of LAB in the commercially used powdered form, highly efficient cryoprotective agents are required during the manufacturing process. This study suggests a novel cryoprotective agent derived from Jerusalem artichoke (JA; Helianthus tuberous L.) and describes the mechanism of cryoprotective effect improvement by sonication treatment. The cryoprotective effect of JA extract was verified by examining the viability of Leuconostoc mesenteroides WiKim33 after freeze-drying (FD). Sonication of JA extract improved the cryoprotective effect. Sonication reduced fructose and glucose contents, which increased the induction of critical damage during FD by 15.84% and 46.81%, respectively. The cryoprotective effects of JA and sonication-treated JA extracts were determined using the viable cell count of Leu. mesenteroides WiKim33. Immediately after FD and storage for 24 weeks, the viability of Leu. mesenteroides WiKim33 with JA extract was 82.8% and 76.3%, respectively, while that of the sonication-treated JA extract was 95.2% and 88.8%, respectively. Our results show that reduction in specific monosaccharides was correlated with improved cryoprotective effect. This study adopted sonication as a novel treatment for improving the cryoprotective effect and verified its efficiency.

Due to the health-promoting effects and functional properties of inulin-type fructooligosaccharides (I-FOS), the global market for I-FOS is constantly growing. Hence, there is a continuing demand for new, efficient biotechnological approaches for I-FOS production. In this work, crude inulosucrase InuGB-V3 from Lactobacillus gasseri DSM 20604 was used to synthesize I-FOS from sucrose. Supplementation with 1 mM CaCl₂, a pH of 3.5-5.5, and an incubation temperature of 40°C were found to be optimal production parameters at which crude inulosucrase showed high conversion rates, low sucrose hydrolysis, and excellent stability over 4 days. The optimal process conditions were employed in cell-free bioconversion reactions. By elevating the substrate concentration from 570 to 800 g L⁻¹, the I-FOS concentration and the synthesis of products with a low degree of polymerization (DP) could be increased, while sucrose hydrolysis was decreased. Bioconversion of 800 g L⁻¹ sucrose for 20 h resulted in an I-FOS-rich syrup with an I-FOS concentration of 401 ± 7 g L⁻¹ and an I-FOS purity of 53 ± 1% [w/w]. I-FOS with a DP of 3-11 were synthesized, with 1,1-kestotetraose (DP4) being the predominant transfructosylation product. The high-calorie sugars glucose, sucrose, and fructose were removed from the generated I-FOS-rich syrup using activated charcoal. Thus, 81 ± 5% of the initially applied I-FOS were recovered with a purity of 89 ± 1%.

Fructooligosaccharides (FOS) can be used as feed prebiotics, but are limited by high production costs. In this study, low-cost sugarcane molasses was used to produce whole-cell biocatalysts containing transfructosylating enzymes by Aureobasidium pullulans FRR 5284, followed by FOS production from molasses using the whole-cells of A. pullulans. A. pullulans in molasses-based medium produced cells and broth with a total transfructosylating activity of 123.6 U/mL compared to 61.0 and 85.8 U/mL in synthetic molasses-based and sucrose-based media, respectively. It was found that inclusion of glucose in sucrose medium reduced both transfructosylating and hydrolytic activities of the produced cells and broth. With the use of pure glucose medium, cells and broth had very low levels of transfructosylating activities and hydrolytic activities were not detected. These results indicated that A. pullulans FRR 5284 produced both constitutive and inducible enzymes in sucrose-rich media, such as molasses while it only produced constitutive enzymes in the glucose media. Furthermore, treatment of FOS solutions generated from sucrose-rich solutions using an invertase-deficient Saccharomyces yeast converted glucose to ethanol and acetic acid and improved FOS content in total sugars by 20–30%. Treated FOS derived from molasses improved the in vitro growth of nine probiotic strains by 9-63% compared to a commercial FOS in 12 h incubation. This study demonstrated the potential of using molasses to produce FOS for feed application.

The use of whole-cell biocatalyst for production of fructo-oligosaccharide (FOS) eliminates the need for costly enzyme recovery and purification. In this study, a novel Aureobasidium pullulans strain (FRR 5284) was identified from seven A. pulllulans strains as an efficient whole-cell biocatalyst for FOS production. The strain had a specific intracellular transfructosylating activity of 4.44 ± 0.18 U/mg dry cells, one of the highest transfructosylating activities thus far reported for whole-cell biocatalyst. Under optimal conditions (pH 5.5 and 55°C), only 5 g/L of dry cells and 3 h reaction time were required to achieve a FOS yield of 61 % from 50 % (w/v) sucrose. Incubation of sugarcane molasses with an invertase-free Saccharomyces cerevisiae prior to addition of A. pullulans whole-cell biocatalyst eliminated glucose inhibition and increased FOS yield from 44 % to 56 % in 1 h. This study has demonstrated that the novel A. pullulans FRR 5284 was an efficient source of whole-cell biocatalyst for FOS production and a promising strategy for transformation of sucrose and sugarcane molasses into a higher-value prebiotic.

Cereals and pulses often contribute to the intake of Fermentable Oligo-, Di-, Monosaccharides, and Polyols (FODMAPs) due to high amounts of fructans or galactooligosaccharides (GOS). FODMAPs can trigger symptoms of Irritable Bowel Syndrome (IBS) and therefore, the development of foods and beverages with a lower FODMAP-content are favourable for IBS patients. Enzyme technology is a promising tool to reduce the FODMAP-content in foods and to maintain product quality. This fundamental study investigates the efficiency of invertase, inulinase, and α-galactosidase as potential food additives to reduce the total FODMAP content of food ingredients. Extracts of high FODMAP ingredients, such as wheat and lentil, and standard solutions of various fructans and GOS were incubated with invertase, inulinase and α-galactosidase for 1 h and 2 h. Contents of oligosaccharides before and after treatment and related IBS-triggering reaction products were quantified using ion chromatography. Inulinase showed a high degradation yield (over 90% of degradation) for both GOS and fructans. For invertase only low degradation yields were measured. α-Galactosidase showed the highest efficiency in decomposing GOS (100% of degradation) and led to non-IBS triggering degradation products. This indicates a high potential for a combined inulinase/α-galactosidase treatment for products containing both fructans and GOS.

The thermostable β-fructosidase (BfrA) from the bacterium Thermotoga maritima converts sucrose into glucose, fructose, and low levels of short–chain fructooligosaccharides (FOS) at high substrate concentration (1.75 M) and elevated temperatures (60-70°C). In this research, FOS produced by BfrA were characterized by HPAE-PAD analysis as a mixture of 1–kestotriose, 6^G-kestotriose (neokestose), and to a major extent 6-kestotriose. In order to increase the FOS yield, three BfrA mutants (W14Y, W14Y-N16S and W14Y-W256Y), designed from sequence divergence between hydrolases and transferases, were constructed and constitutively expressed in the non-saccharolytic yeast Pichia pastoris. The secreted recombinant glycoproteins were purified and characterized. The three mutants synthesized -kestotriose as the major component of a FOS mixture that includes minor amounts of tetra- and pentasaccharides. In all cases, sucrose hydrolysis was the predominant reaction. All mutants reached a similar overall FOS yield, with the average value 37.6% (w/w) being 3–fold higher than that of the wild–type enzyme (12.6%, w/w). None of the mutations altered the enzyme thermophilicity and thermostability. The single mutant W14Y, with specific activity of 841 U mg⁻¹, represents an attractive candidate for the continuous production of FOS–containing invert syrup at pasteurization temperatures.