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The synthesis of complex oligosaccharides is desired for their potential as prebiotics, and their role in the pharmaceutical and food industry. Levansucrase (LS, EC 2.4.1.10), a fructosyl-transferase, can catalyze the synthesis of these compounds. LS acquires a fructosyl residue from a donor molecule and performs a non-Lenoir transfer to an acceptor molecule, via β-(2→6)-glycosidic linkages. Genome mining was used to uncover new LS enzymes with increased transfructosylating activity and wider acceptor promiscuity, with an initial screening revealing five LS enzymes. The product profiles and activities of these enzymes were examined after their incubation with sucrose. Alternate acceptor molecules were also incubated with the enzymes to study their consumption. LSs from Gluconobacter oxydans and Novosphingobium aromaticivorans synthesized fructooligosaccharides (FOSs) with up to 13 units in length. Alignment of their amino acid sequences and substrate docking with homology models identified structural elements causing differences in their product spectra. Raffinose, over sucrose, was the preferred donor molecule for the LS from Vibrio natriegens, N. aromaticivorans , and Paraburkolderia graminis . The LSs examined were found to have wide acceptor promiscuity, utilizing monosaccharides, disaccharides, and two alcohols to a high degree.

Soybean oligosaccharides have been previously shown to be associated with the production of major odor-causing compounds in broilers, although little is known about the role of stachyose and raffinose, which are key components of soybean oligosaccharide, in broiler cecal microbiota and odor compound production. To this end, soybean oligosaccharide, stachyose, and raffinose were added to the birds' diets to investigate their effects on odor compound production and the microbial community characteristics of the cecum in broilers. A total of 300 one-day-old Arbor Acre broilers with similar initial live weight were randomly allocated into 5 dietary groups with 6 replicates of 10 birds. The diets included soybean meal (positive control), soybean meal-free (negative control), 0.6% soybean oligosaccharide, 0.6% stachyose, or 0.6% raffinose. After a 49-D feeding period, both ceca were aseptically removed postmortem, and the contents were collected and analyzed for skatole, indole, volatile fatty acids, and lactic acid by using high performance liquid chromatography. Bacterial communities were detected by using a high-throughput sequencing platform based on IlluminaMiSeq 2500. Levels of skatole and indole tended to be lower in the dietary supplementation of oligosaccharides. The lowest levels of skatole and indole were observed in the stachyose group (P < 0.05), while the highest levels were found in the negative control group (P < 0.05). Concentrations of acetic acid and propionic acid in the stachyose group were increased (P < 0.05) while those of butyric acid and lactic acid were decreased (P < 0.05) compared with the soybean oligosaccharide and raffinose groups. Firmicutes and Bacteroidetes were prevalent in all groups, the proportion of Bacteroidetes was slightly decreased in the stachyose group, and Verrucomicrobia was abundant in the raffinose group (P > 0.05). Bacterial genera Alistipes and Parabacteroides were comparably abundant in the stachyose group, while Bacteroides, Lactobacillus, and Akkermansia were more abundant in the negative control, stachyose, and raffinose groups, respectively. Collectively, these findings demonstrated that dietary oligosaccharide supplementation significantly reduced odor compound production by modulating the cecal microbial community. Compared with soybean oligosaccharide and raffinose, the addition of stachyose into diets may help improve gut fermentation and minimize odor compound generation in broilers., (Copyright © 2020. Published by Elsevier Inc.)

Metabolites, phytohormones, and genes involved in dehydration responses/tolerance have been predicted in several plants. However, metabolite/phytohormone-gene regulatory networks in soybean organs under dehydration conditions remain unclear. Here, we analyzed the organ specificity of metabolites, phytohormones, and gene transcripts and revealed the characteristics of their regulatory networks in dehydration-treated soybeans. Our metabolite/phytohormone analysis revealed the accumulation of raffinose, trehalose, and cis-zeatin (cZ) specifically in dehydration-treated roots. In dehydration-treated soybeans, raffinose, and trehalose might have additional roles not directly involved in protecting the photosynthetic apparatus; cZ might contribute to root elongation for water uptake from the moisture region in soil. Our integration analysis of metabolites-genes indicated that galactinol, raffinose, and trehalose levels were correlated with transcript levels for key enzymes (galactinol synthase, raffinose synthase, trehalose 6-phosphate synthase, trehalose 6-phosphate phosphatase) at the level of individual plants but not at the organ level under dehydration. Genes encoding these key enzymes were expressed in mainly the aerial parts of dehydration-treated soybeans. These results suggested that raffinose and trehalose are transported from aerial plant parts to the roots in dehydration-treated soybeans. Our integration analysis of phytohormones-genes indicated that cZ and abscisic acid (ABA) levels were correlated with transcript levels for key enzymes (cytokinin nucleoside 5'-monophosphate phosphoribohydrolase, cytokinin oxidases/dehydrogenases, 9-cis-epoxycarotenoid dioxygenase) at the level of individual plants but not at the organ level under dehydration conditions. Therefore, processes such as ABA and cZ transport, among others, are important for the organ specificity of ABA and cZ production under dehydration conditions., (© 2020 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

Bacteroides thetaiotaomicron (B. thetaiotaomicron), which resides in the human intestinal tract, has a number of carbohydrate enzymes, including glycoside hydrolase (GH) family 97. Only a few GH 97 enzymes have been characterized to date. In this study, a novel α-galactosidase (Bt&#95;3294) was cloned from B. thetaiotaomicron, expressed in Escherichia coli, and purified using affinity chromatography. This novel enzyme showed optimal activity at 60 °C and pH 7.0. Enzyme activity was reduced by 94.4% and 95.7% in the presence of 5 mM Ca 2+ and Fe 2+ , respectively. It is interesting that Bt&#95;3294 specifically hydrolyzed shorter α-galactosyl oligosaccharides, such as melibiose and raffinose. The D-values of Bt&#95;3294 at 40 °C and 50 °C were about 107 and 6 min, respectively. After immobilization of Bt&#95;3294, the D-values at 40 °C and 50 °C were about 37.6 and 29.7 times higher than those of the free enzyme, respectively. As a practical application, the immobilized Bt&#95;3294 was used to hydrolyze raffinose family oligosaccharides (RFOs) in soy milk, decreasing the RFOs by 98.9%., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Previous studies have demonstrated that the freezing tolerance (FT) of grapevine was enhanced by foliar application of exogenous abscisic acid (exo-ABA), a treatment which might be incorporated into cultural practices to mitigate cold damage in vineyards. To investigate the underlying mechanisms of this response, a two-year (2017 and 2018) study was conducted to characterize the effects of exo-ABA on greenhouse-grown 'Cabernet franc' grapevine. In control grapevines, both physiological (deeper dormancy) and biochemical (sugar accumulation in buds) changes occurred, indicating that grapevines initiated cold acclimation in the greenhouse. Compared to control, exo-ABA decreased stomatal conductance 2 h after application. Two weeks post application, exo-ABA treated grapevines showed accelerated transition of grapevine physiology during cold acclimation (increased depth of dormancy, decreased bud water content and enhanced bud FT), relative to control. Exo-ABA induced the accumulation of several sugars in buds including the raffinose family oligosaccharides (RFOs), and the RFO precursor, galactinol. The expression of raffinose and galactinol synthase genes was higher in exo-ABA treated grapevine buds, compared to control. The new findings from this study have advanced our understanding of the role of ABA in grapevine FT, which will be useful to develop future strategies to protect grapevines from cold damage., Competing Interests: Declaration of Competing Interest Authors have no competing interest to declare., (Copyright © 2020. Published by Elsevier B.V.)

Alternative polyadenylation (APA) is a pervasive mechanism for gene regulation in eukaryotes. Stachyose is the main assimilate translocated in the cucumber phloem. Stachyose synthase (CsSTS) catalyzes the last step of stachyose biosynthesis in cucumber leaves and plays a key role in the regulation of assimilate partitioning between source and sink. In this study, three CsSTS mRNAs with the same open reading frame and the 5`untranslated region (UTR), but differing in their 3`UTRs, named CsSTS1 (short), CsSTS2 (medium), and CsSTS3 (long), were identified. Southern blot and sequence analysis of the cucumber genome confirmed that these transcripts are regulated through APA from a single gene. No significant difference of in vitro translation efficiency was found among three mRNAs. However, the relative stabilities of three transcripts varied among different tissues and different leaf development stages of cucumber. CsSTS1 expression in cucumber calli was up-regulated by the raffinose (substrate of CsSTS) and down-regulated by stachyose (product of CsSTS), respectively. In cucumber plants, all three isoforms have considerable expression in non-fruit node leaves. However, in fruit-carrying node leaves, the expression of CsSTS2 and CsSTS3 was severely inhibited and only CsSTS1 was highly expressed, indicating fruit setting has a remarkable effect on the relative expression level of three transcripts. This "fruit setting" effect could be observed until at least 36 h after the fruit was removed from the node. Our results suggest that abundant expression of CsSTS1 is beneficial for stachyose loading in source leaves, and APA is a delicate mechanism for CsSTS to regulate cucumber source-sink balance., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interest., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

Soybean seeds produce valuable protein that is a major component of livestock feed. However, soybean seeds also contain the anti-nutritional raffinose family oligosaccharides (RFOs) raffinose and stachyose, which are not digestible by non-ruminant animals. This requires the proportion of soybean meal in the feed to be limited, or risk affecting animal growth rate or overall health. While reducing RFOs in soybean seed has been a goal of soybean breeding, efforts are constrained by low genetic variability for carbohydrate traits and the difficulty in identifying these within the soybean germplasm. We used reverse genetics Targeting Induced Local Lesions in Genomes (TILLING)-by-sequencing approach to identify a damaging polymorphism that results in a missense mutation in a conserved region of the RAFFINOSE SYNTHASE3 gene. We demonstrate that this mutation, when combined as a double mutant with a previously characterized mutation in the RAFFINOSE SYNTHASE2 gene, eliminates nearly 90% of the RFOs in soybean seed as a proportion of the total seeds carbohydrates, and results in increased levels of sucrose. This represents a proof of concept for TILLING by sequencing in soybean., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Cryopreservation is the universal technology used to enable long-term storage and continuous availability of cell stocks and tissues for regenerative medicine demands. The main components of standard freezing media are dimethyl sulfoxide (hereinafter Me 2 SO) and fetal bovine serum (FBS). However, for manufacturing of cells and tissue-engineered products in accordance with the principles of Good Manufacturing Practice (GMP), current considerations in regenerative medicine suggest development of Me 2 SO- and serum-free biopreservation strategies due to safety concerns over Me 2 SO-induced side effects and immunogenicity of animal serum. In this work, the effect of electroporation-assisted pre-freeze delivery of sucrose, trehalose and raffinose into human umbilical cord mesenchymal stem cells (hUCMSCs) on their post-thaw survival was investigated. The optimal strength of electric field at 8 pulses with 100 μs duration and 1 Hz pulse repetition frequency was determined to be 1.5 kV/cm from permeabilization (propidium iodide uptake) vs. cell recovery data (resazurin reduction assay). Using sugars as sole cryoprotectants with electroporation, concentration-dependent increase in cell survival was observed. Irrespective of sugar type, the highest cell survival (up to 80%) was achieved at 400 mM extracellular concentration and electroporation. Cell freezing without electroporation yielded significantly lower survival rates. In the optimal scenario, cells were able to attach 24 h after thawing demonstrating characteristic shape and sugar-loaded vacuoles. Application of 10% Me 2 SO/90% FBS as a positive control provided cell survival exceeding 90%. Next, high glass transition temperatures determined for optimal concentrations of sugars by differential scanning calorimetry (DSC) suggest the possibility to store samples at -80 °C. In summary, using electroporation to incorporate cryoprotective sugars into cells is an effective strategy towards Me 2 SO- and serum-free cryopreservation and may pave the way for further progress in establishing clinically safe biopreservation strategies for efficient long-term biobanking of cells., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The present work aims to dissect the underlying signaling pathways associated with soybean [ Glycine max (L.) Merrill] seed hormo-priming with ethephon (Eth). Our results demonstrated that soybean germination improved significantly upon Eth priming (Ethp). Phytohormone quantification shows relative enhanced endogenous gibberellin A 4 (GA 4 ) levels concomitant with impaired biogenesis and signaling of auxin, viz . , indole acetic acid (IAA) and abscisic acid (ABA). Phytochemical analysis revealed relative reduced levels of individual and total raffinose family oligosaccharide (RFO) components, starch, soluble sugars, and sucrose concomitant with enhanced levels of reducing sugars, glucose, cellular ATP, and acetyl-CoA pools. Secondary metabolite analysis revealed the activation of the mevalonate (MVA) pathway with a concomitant suppression of the plastidal 2-methyl-d-erythritol-4-phosphate/1-deoxy-d-xylulose-5-phosphate (MEP/DOX) and phenylpropanoid pathways, substantiated by relative reduced levels of total phenolics, tannins, and proanthocyanidin. Ethp also enhances the in vitro antioxidative activity (viz., 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging ability and ferric reducing antioxidant power (FRAP)) and endogenous antioxidants levels (viz., flavonoids, isoflavones, β - carotene, vitamin C, and vitamin E). Further quantitative real-time polymerase chain reaction (qRT-PCR) analysis showed transcriptional pattern of representative genes in agreement with these metabolic alterations.

Streptococcus pneumoniae is an opportunistic respiratory pathogen that can spread to other body sites, including the ears, brain, and blood. The ability of this bacterium to break down, import, and metabolize a wide range of glycans is key to its virulence. Intriguingly, S. pneumoniae can utilize several plant oligosaccharides for growth in vitro , including raffinose-family oligosaccharides (RFOs, which are α-(1→6)-galactosyl extensions of sucrose). An RFO utilization locus has been identified in the pneumococcal genome; however, none of the proteins encoded by this locus have been biochemically characterized. The enigmatic ability of S. pneumoniae to utilize RFOs has recently received attention because mutations in two of the RFO locus genes have been linked to the tissue tropism of clinical pneumococcal isolates. Here, we use functional studies combined with X-ray crystallography to show that although the pneumococcal RFO locus encodes for all the machinery required for uptake and degradation of RFOs, the individual pathway components are biochemically inefficient. We also demonstrate that the initiating enzyme in this pathway, the α-galactosidase Aga (a family 36 glycoside hydrolase), can cleave α-(1→3)-linked galactose units from a linear blood group antigen. We propose that the pneumococcal RFO pathway is an evolutionary relic that is not utilized in this streptococcal species and, as such, is under no selection pressure to maintain binding affinity and/or catalytic efficiency. We speculate that the apparent contribution of RFO utilization to pneumococcal tissue tropism may, in fact, be due to the essential role the ATPase RafK plays in the transport of other carbohydrates., (© 2019 Hobbs et al.)

This study compared two immobilization matrices like calcium-alginate and chitosan for immobilization of α-galactosidase and evaluated their potential for the removal of non-digestible raffinose family oligosaccharides from soy milk which cause abdominal discomfort. The pH optima of the free and immobilized enzymes were found to be similar at pH 4.0. The chitosan-immobilized α-galactosidase displayed higher optimal temperature (60°C) compared to alginate-immobilized enzyme (45°C) and free enzyme (50°C). The chitosan-immobilized and alginate-immobilized α-galactosidases displayed 93.7% and 97.6% hydrolysis of raffinose family oligosaccharides, respectively, while the free enzyme hydrolyzed only 30.3% oligosaccharides present in soy milk in 4 hr. Remarkably, both the immobilized enzymes showed complete removal of raffinose family oligosaccharides in 8 hr. Moreover, reusability studies indicate that even after five cycles of reuse, the chitosan and alginate-immobilized enzymes displayed 99% and 60% hydrolysis, respectively. PRACTICAL APPLICATIONS: In this study, we have used two inexpensive and non-toxic matrices for immobilizing α-galactosidase. We report that entrapment of α-galactosidase with chitosan significantly improved the optimal temperature of α-galactosidase, which is advantageous in food industry. The hydrolysis of raffinose family oligosaccharides in soy milk was also greatly enhanced after immobilization with chitosan and alginate. Thus, the results described in this study have relevance for development of safe, cost-effective and efficient method for removal of non-digestible soy oligosaccharides in food industry., (© 2019 Wiley Periodicals, Inc.)

Strains of Lactobacillus reuteri are commonly used as probiotics due to their demonstrated therapeutic properties. Many strains of L. reuteri also utilize the prebiotic galactooligosaccharide (GOS), providing a basis for formulating synergistic synbiotics that could enhance growth or persistence of this organism in vivo In this study, in-frame deletion mutants were constructed to characterize the molecular basis of GOS utilization in L. reuteri ATCC PTA-6475. Results suggested that GOS transport relies on a permease encoded by lacS , while a second unidentified protein may function as a galactoside transporter. Two β-galactosidases, encoded by lacA and lacLM , sequentially degrade GOS oligosaccharides and GOS disaccharides, respectively. Inactivation of lacL and lacM resulted in impaired growth in the presence of GOS and lactose. In vitro competition experiments between the wild-type and ΔlacS ΔlacM strains revealed that the GOS-utilizing genes conferred a selective advantage in media with GOS but not glucose. GOS also provided an advantage to the wild-type strain in experiments in gnotobiotic mice but only on a purified, no sucrose diet. Differences in cell numbers between GOS-fed mice and mice that did not receive GOS were small, suggesting that carbohydrates other than GOS were sufficient to support growth. On a complex diet, the ΔlacS ΔlacM strain was outcompeted by the wild-type strain in gnotobiotic mice, suggesting that lacL and lacM are involved in the utilization of alternative dietary carbohydrates. Indeed, the growth of the mutants was impaired in raffinose and stachyose, which are common in plants, demonstrating that α-galactosides may constitute alternate substrates of the GOS pathway. IMPORTANCE This study shows that lac genes in Lactobacillus reuteri encode hydrolases and transporters that are necessary for the metabolism of GOS, as well as α-galactoside substrates. Coculture experiments with the wild-type strain and a gos mutant clearly demonstrated that GOS utilization confers a growth advantage in medium containing GOS as the sole carbohydrate source. However, the wild-type strain also outcompeted the mutant in germfree mice, suggesting that GOS genes in L. reuteri also provide a basis for utilization of other carbohydrates, including α-galactosides, ordinarily present in the diets of humans and other animals. Collectively, our work provides information on the metabolism of L. reuteri in its natural niche in the gut and may provide a basis for the development of synbiotic strategies., (Copyright © 2019 American Society for Microbiology.)

Background: α-Galactosidases are enzymes that act on galactosides present in many vegetables, mainly legumes and cereals, have growing importance with respect to our diet. For this reason, the use of their catalytic activity is of great interest in numerous biotechnological applications, especially those in the food industry directed to the degradation of oligosaccharides derived from raffinose. The aim of this work has been to optimize the recombinant production and further characterization of α-galactosidase of Saccharomyces cerevisiae., Results: The MEL1 gene coding for the α-galactosidase of S. cerevisiae (ScAGal) was cloned and expressed in the S. cerevisiae strain BJ3505. Different constructions were designed to obtain the degree of purification necessary for enzymatic characterization and to improve the productive process of the enzyme. ScAGal has greater specificity for the synthetic substrate p-nitrophenyl-α-D-galactopyranoside than for natural substrates, followed by the natural glycosides, melibiose, raffinose and stachyose; it only acts on locust bean gum after prior treatment with β-mannosidase. Furthermore, this enzyme strongly resists proteases, and shows remarkable activation in their presence. Hydrolysis of galactose bonds linked to terminal non-reducing mannose residues of synthetic galactomannan-oligosaccharides confirms that ScAGal belongs to the first group of α-galactosidases, according to substrate specificity. Optimization of culture conditions by the statistical model of Response Surface helped to improve the productivity by up to tenfold when the concentration of the carbon source and the aeration of the culture medium was increased, and up to 20 times to extend the cultivation time to 216 h., Conclusions: ScAGal characteristics and improvement in productivity that have been achieved contribute in making ScAGal a good candidate for application in the elimination of raffinose family oligosaccharides found in many products of the food industry.

Many genes of industrial relevance can be found in soil. In this study, metagenome sequencing of paddy soil was performed with 55.68 Gb sequences and 1,787,113 putative open reading frames (ORFs). The functional profiles and metabolic pathway of soil metagenomes were examined using Gene Ontology, Metagenomics RAST, and Kyoto Encyclopedia of Genes and Genomes. To verify the protein function and assembly of ORFs, a putative gene encoding α-galactosidase, namely GalR, which shares 65% identity with an unpublished glycoside hydrolase (GH) 27 family protein, was synthesized using its optimal codon for overexpression in Escherichia coli. GalR was successfully obtained and characterized. The optimal temperature and pH for GalR activity were 30°C and pH 9, respectively. Enzymatic activity indicated that GalR was alkaliphilic and different from acidophilic α-galactosidase in the GH 27 family. Furthermore, 50% of the relative activity of GalR can be attained for 1.7 and 0.7 h preincubation at 40°C and 50°C, respectively. Significant inhibition of GalR was observed in the presence of ethylenediaminetetraacetic acid (EDTA), MgCl 2 , sodium dodecyl sulfate (SDS), and H 2 O 2 ; however, it was resistant to 0.1% methanol and ethanol and was slightly activated with NaCl and KCl. The specific activity of GalR was achieved at 11.6 and 0.59 μmol/min/mg of protein using p-nitrophenyl-α-d-galactopyranoside and raffinose as substrates, respectively. Consequently, the metagenomic sequencing-based strategy can provide information for mining novel genes., (Copyright © 2019 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Galactose oxidase catalyzes a two-electron oxidation, mainly from the C6 hydroxyl group of D-galactose, with the concomitant reduction of water to hydrogen peroxide. This enzyme is secreted by Fusarium species and has several biotechnological applications. In this study, a screening of galactose oxidase production among species of the Fusarium fujikuroi species complex demonstrated Fusarium subglutinans to be the main producer. The truncated F. subglutinans gaoA gene coding for the mature galactose oxidase was expressed from the prokaryotic vector pTrcHis2B in the E. coli Rosetta™ (DE3) strain. The purified recombinant enzyme presented temperature and pH optima of 30 °C and 7.0, respectively, K M of 132.6 ± 18.18 mM, V max of 3.2 ± 0.18 µmol of H 2 O 2 /min, k cat of 12,243 s -1 , and a catalytic efficiency (k cat /K M ) of 9.2 × 10 4  M -1  s -1 . In the presence of 50% glycerol, the enzyme showed a T 50 of 59.77 °C and was stable for several hours at pH 8.0 and 4 °C. Besides D-(+)-galactose, the purified enzyme also acted against D-(+)-raffinose, α-D-(+)-melibiose, and methyl-α-D-galactopyranoside, and was strongly inhibited by SDS. Although the F. subglutinans gaoA gene was successfully expressed in E. coli, its endogenous transcription was not confirmed by RT-PCR.

Bacillus subtilis is a heterotrophic soil bacterium that hydrolyzes different polysaccharides mainly found in the decomposed plants. These carbohydrates are mainly cellulose, hemicellulose, and the raffinose family of oligosaccharides (RFOs). RFOs are soluble α-galactosides, such as raffinose, stachyose, and verbascose, that rank second only after sucrose in abundance. Genome sequencing and transcriptome analysis of B. subtilis indicated the presence of a putative α-galactosidase-encoding gene ( melA ) located in the msmRE-amyDC-melA operon. Characterization of the MelA protein showed that it is a strictly Mn 2+ - and NAD + -dependent α-galactosidase able to hydrolyze melibiose, raffinose, and stachyose. Transcription of the msmER-amyDC-melA operon is under control of a σ A -type promoter located upstream of msmR (P msmR ), which is negatively regulated by MsmR. The activity of P msmR was induced in the presence of melibiose and raffinose. MsmR is a transcriptional repressor that binds to two binding sites at P msmR located upstream of the -35 box and downstream of the transcriptional start site. MsmEX-AmyCD forms an ATP-binding cassette (ABC) transporter that probably transports melibiose into the cell. Since msmRE-amyDC-melA is a melibiose utilization system, we renamed the operon melREDCA IMPORTANCE Bacillus subtilis utilizes different polysaccharides produced by plants. These carbohydrates are primarily degraded by extracellular hydrolases, and the resulting oligo-, di-, and monosaccharides are transported into the cytosol via phosphoenolpyruvate-dependent phosphotransferase systems (PTS), major facilitator superfamily, and ATP-binding cassette (ABC) transporters. In this study, a new carbohydrate utilization system of B. subtilis responsible for the utilization of α-galactosides of the raffinose family of oligosaccharides (RFOs) was investigated. RFOs are synthesized from sucrose in plants and are mainly found in the storage organs of plant leaves. Our results revealed the modus operandi of a new carbohydrate utilization system in B. subtilis ., (Copyright © 2019 American Society for Microbiology.)

The low phytic acid ( lpa) soybean ( Glycine max L. Merr.) mutant Gm-lpa-TW-1-M, resulting from a 2 bp deletion in GmMIPS1, was crossed with a commercial cultivar. F 3 and F 5 progenies were subjected to nontargeted GC-based metabolite profiling, allowing analysis of a broad array of low molecular weight constituents. In the homozygous lpa mutant progenies the intended phytic acid reduction was accompanied by remarkable metabolic changes of nutritionally relevant constituents such as reduced contents of raffinose oligosaccharides and galactosyl cyclitols as well as increased concentrations in sucrose and various free amino acids. The mutation-induced metabolite signature was nearly unaffected by the cross-breeding and consistently expressed over generations and in different growing seasons. Therefore, not only the primary MIPS1 lpa mutant but also its progenies might be valuable genetic resources for commercial breeding programs to produce soybean seeds stably exhibiting improved phytate-related and nutritional properties.

Raffinose, an oligosaccharide found in many seeds, plays an important role in seed vigor; however, the regulatory mechanism governing raffinose biosynthesis remains unclear. We report here that maize W22 wild type (WT) seeds, but not W22 viviparous1 ( zmvp1) mutant seeds, start accumulating galactinol and raffinose 28 days after pollination (DAP). Transcriptome analysis of the zmvp1 embryo showed that the expression of GALACTINOL SYNTHASE2 ( GOLS2) was down-regulated relative to WT. Further experiments showed that the expression of ZmGOLS2 was up-regulated by ZmABI5 but not by ZmVP1, and it was further increased by the coexpression of ZmABI5 and ZmVP1 in maize protoplasts. ZmABI5 interacted with ZmVP1, while ZmABI5, but not ZmVP1, directly binds to the ZmGOLS2 promoter. Together, all of the findings suggest that ZmVP1 interacts with ZmABI5 and regulates ZmGOLS2 expression and raffinose accumulation in maize seeds.

Lactic acid bacteria alone or with special adjunct probiotic strains are inevitable for the preparation of various specific functional foods. Moreover, because of their growth and metabolism, the final products are preserved for a certain time. Thus, growth dynamics of the lactic acid bacteria of the Fresco DVS 1010 culture ( Lactococcus lactis spp. lactis, Lactococcus lactis spp. cremoris, Streptococcus salivarius spp. thermophilus) during liquid-state fermentation of soya mashes and pH values within the process were analyzed in this study. Although milk is the most typical growth medium for the lactic acid bacteria, presumable viable counts of Fresco culture reached levels 10 9  CFU ml -1 after 8 h, representing 2-3 log increase in comparison to initial state (specific growth rates ranged from 1.06 to 1.64 h -1 ). After 21 days of storage period, the pH levels in the products were reduced to 4.50-4.70, representing a decrease of about 1.5-1.7 units. All prepared soybean products contained detectable amounts of raffinose-series oligosaccharides (0.25-0.68 g per 100 g) that were reduced in average by about 30.5% during period of 21 days.

Background: Soil salinity is one of the major serious factors that affect agricultural productivity of almost all crops worldwide, including the important oilseed crop sesame. In order to improve salinity resistance in sesame, it is crucial to understand the molecular mechanisms underlying the adaptive response to salinity stress., Results: In the present study, two contrasting sesame genotypes differing in salt tolerance were used to decipher the adaptive responses to salt stress based on morphological, transcriptome and metabolome characterizations. Morphological results indicated that under salt stress, the salt-tolerant (ST) genotype has enhanced capacity to withstand salinity stress, higher seed germination rate and plant survival rate, as well as better growth rate than the salt-sensitive genotype. Transcriptome analysis revealed strongly induced salt-responsive genes in sesame mainly related to amino acid metabolism, carbohydrate metabolism, biosynthesis of secondary metabolites, plant hormone signal transduction, and oxidation-reduction process. Especially, several pathways were preferably enriched with differentially expressed genes in ST genotype, including alanine, aspartate and glutamate metabolism, carotenoid biosynthesis, galactose metabolism, glycolysis/gluconeogenesis, glyoxylate and dicarboxylate metabolism, porphyrin and chlorophyll metabolism. Metabolome profiling under salt stress showed a higher accumulation degree of metabolites involved in stress tolerance in ST, and further highlighted that the amino acid metabolism, and sucrose and raffinose family oligosaccharides metabolism were enhanced in ST., Conclusions: These findings suggest that the candidate genes and metabolites involved in crucial biological pathways may regulate salt tolerance of sesame, and increase our understanding of the molecular mechanisms underlying the adaptation of sesame to salt stress.

Streptococcus pneumoniae is commonly carried asymptomatically in the human nasopharynx, but it also causes serious and invasive diseases such as pneumonia, bacteremia, and meningitis, as well as less serious but highly prevalent infections such as otitis media. We have previously shown that closely related pneumococci (of the same capsular serotype and multilocus sequence type [ST]) can display distinct pathogenic profiles in mice that correlate with clinical isolation site (e.g., blood versus ear), suggesting stable niche adaptation within a clonal lineage. This has provided an opportunity to identify determinants of disease tropism. Genomic analysis identified 17 and 27 single nucleotide polymorphisms (SNPs) or insertions/deletions in protein coding sequences between blood and ear isolates of serotype 14 ST15 and serotype 3 ST180, respectively. SNPs in raffinose uptake and utilization genes ( rafR or rafK ) were detected in both serotypes/lineages. Ear isolates were consistently defective in growth in media containing raffinose as the sole carbon source, as well as in expression of raffinose pathway genes aga , rafG , and rafK , relative to their serotype/ST-matched blood isolates. Similar differences were also seen between serotype 23F ST81 blood and ear isolates. Analysis of rafR allelic exchange mutants of the serotype 14 ST15 blood and ear isolates demonstrated that the SNP in rafR was entirely responsible for their distinct in vitro phenotypes and was also the determinant of differential tropism for the lungs versus ear and brain in a mouse intranasal challenge model. These data suggest that the ability of pneumococci to utilize raffinose determines the nature of disease. IMPORTANCE S. pneumoniae is a component of the commensal nasopharyngeal microflora of humans, but from this reservoir, it can progress to localized or invasive disease with a frequency that translates into massive global morbidity and mortality. However, the factors that govern the switch from commensal to pathogen, as well as those that determine disease tropism, are poorly understood. Here we show that capacity to utilize raffinose can determine the nature of the disease caused by a given pneumococcal strain. Moreover, our findings provide an interesting example of convergent evolution, whereby pneumococci belonging to two unrelated serotypes/lineages exhibit SNPs in separate genes affecting raffinose uptake and utilization that correlate with distinct pathogenic profiles in vivo This further underscores the critical role of differential carbohydrate metabolism in the pathogenesis of localized versus invasive pneumococcal disease., (Copyright © 2019 Minhas et al.)

The α-galactosidases, which can catalyze the removal of α-1,6-linked terminal galactose residues from galactooligosaccharide materials, have good potential for industrial applications. The high-level and efficient secretion of the α-galactosidases into the extracellular space has greatly simplified the downstream bioengineering process, facilitating their bioapplications. In this study, the effects of gene dosage and endoplasmic reticulum secretion-associated factors (ERSAs) on the secretory expression of an α-galactosidase gene derived from a Aspergillus oryzae strain were investigated by constructing multicopy expression cassettes and coexpressing the α-galactosidase gene with ERSAs. With the increase in the gene copy-number in the host genome, the expression of GalA was improved. However, the secretory expression level was not linearly related to the copy number. When the number was higher than four copies, the expression level of GalA gene declined. The ERSAs factors HAC1, PDI, and Ero1 improved the secretory expression of α-galactosidase, while Hsp40 inhibited its secretion. After methanol-induced expression in a bench-top bioreactor, Pichia recombinants carrying four copies of GalA genes reached 3520 U/mL in the supernatant of the culture. We further optimized the parameters for α-galactosidase to hydrolyze two types of galactooligosaccharides: raffinose and stachyose. This study has fulfilled the scale-up production of α-galactosidase, thus facilitating its industrial applications., (Copyright © 2018 Elsevier Inc. All rights reserved.)

A novel 57-kDa acidic α-galactosidase designated as HEG has been purified from the dry fruiting bodies of Hericium erinaceus. The isolation protocol involved ion-exchange chromatography and gel filtration on a Superdex75 column. The purification fold and specific activity were 1251 and 46 units/mg, respectively. A BLAST search of internal peptide sequences obtained by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis suggested that the enzyme belonged to the GH27 family. The activity of the enzyme reached its maximum at a pH of 6.0 or at 60 °C. The enzyme was stable within an acidic pH range of 2.2-7.0 and in a narrow temperature range. The enzyme was strongly inhibited by Zn 2+ , Fe 3+ , Ag + ions and SDS. The Lineweaver-Burk plot suggested that the mode of inhibition by galactose and melibiose were of a mixed type. N-bromosuccinimide drastically decreased the activity of the enzyme, whereas diethylpyrocarbonate and carbodiimide strengthened the activity slightly. Moreover, the isolated enzyme displayed remarkable resistance to acid proteases, neutral proteases and pepsin. The enzyme could also hydrolyse oligosaccharides and polysaccharides. In addition, acidic protease promoted the hydrolysis of RFOs by HEG. The K m values of the enzyme towards pNPGal, raffinose and stachyose were 0.36 mM, 40.07 mM and 54.71 mM, respectively. These favourable properties increase the potential of the enzyme in the food industry and animal feed applications., (Copyright © 2018. Published by Elsevier B.V.)

Raffinose has become a major focus of research interest and recent studies have shown that besides beneficial bifidobacteria and lactobacilli, Escherichia coli, Enterococcus faecium and Streptococcus pneumoniae can also utilize raffinose and raffinose might lead to flatulence in some hosts. Therefore, it is required to find out the raffinose-metabolizing bacteria in the gut and the bacteria responsible for the flatulence. The BLASTP search results showed that the homologous proteins of glycosidases related to raffinose utilization are widely distributed in 196 of the 528 gut bacterial strains. Fifty-nine bacterial strains belonging to nine species of five genera were isolated from human feces and were found to be capable of utilizing raffinose; of these species, Enterococcus avium and Streptococcus salivarius were reported for the first time. High-performance liquid chromatography (HPLC) analysis of the supernatants of the nine species revealed that the bacteria could utilize raffinose in different manners. Glucose and melibiose were detected in the supernatants of Enterococcus avium E5 and Streptococcus salivarius B5, respectively. However, no resulting saccharides of raffinose degradation were detected in the supernatants of other seven strains, indicating that they had different raffinose utilization types from Enterococcus avium E5 and Streptococcus salivarius B5. Gas was produced with raffinose utilization by Escherichia coli, Enterococcus faecium, Streptococcus macedonicus, Streptococcus pasteurianus and Enterococcus avium. Thus, more attention should be paid to the raffinose-utilizing bacteria besides bifidobacteria and further studies are required to reveal the mechanisms of raffinose utilization to clarify the relationship between raffinose and gut bacteria.

Raffinose family oligosaccharides (RFOs) accumulate during seed development, and have been thought to be associated with the acquisition of desiccation tolerance (DT) by seeds. Here, comprehensive approaches were adopted to evaluate the changes of DT in developing Arabidopsis seeds of wild type, overexpression (OX-AtGS1/GS2/RS5), and mutant lines by manipulating the expression levels of the GALACTINOL SYNTHASE (GS) and RAFFINOSE SYNTHASE (RS) genes. Our results indicate that seeds of the double mutant (gs1, gs2) and rs5 delayed the timing of DT acquisition as compared to wild type. Subsequent detection confirmed that seeds from OX-AtGS1/GS2 plants with high levels of galactinol, raffinose, and stachyose, and OX-AtRS5 plants possess more raffinose and stachyose but less galactinol compared to wild type. These lines all showed greater germination percentage and shorter time to 50% germination after desiccation treatment at 11 and 15 days after flower (DAF). Further analysis revealed that the role of RFOs is time limited and mainly affects the middle stage (9-16 DAF) of seed development by enhancing seed viability and the ratio of GSH to GSSH in cells, but there is no significant difference in DT of mature seeds. In addition, RFOs could reduce damage to seeds caused by oxidative stress. We conclude that GALACTINOL SYNTHASE and RAFFINOSE SYNTHASE play important roles in DT acquisition during Arabidopsis seed development, and that galactinol and RFOs are crucial protective compounds in the response of seeds to desiccation stress., (Copyright © 2018 Elsevier GmbH. All rights reserved.)

Fructans, homopolymers of fructose produced by fructosyltransferases (FTs), are emerging as intriguing components in halophiles since they are thought to be associated with osmotic stress tolerance and overall fitness of microorganisms and plants under high-salinity conditions. Here, we report on the full characterization of the first halophilic FT, a levansucrase from Halomonas smyrnensis AAD6 T (HsLsc; EC 2.4.1.10). The encoding gene (lsc) was cloned into a vector with a 6xHis Tag at its C-terminus, then expressed in Escherichia coli. The purified recombinant enzyme (47.3 kDa) produces levan and a wide variety of fructooligosaccharides from sucrose, but only in the presence of high salt concentrations (> 1.5 M NaCl). HsLsc showed Hill kinetics and pH and temperature optima of 5.9 and 37 °C, respectively. Interestingly, HsLsc was still very active at salt concentrations close to saturation (4.5 M NaCl) and was selectively inhibited by divalent cations. The enzyme showed high potential in producing novel saccharides derived from raffinose as both fructosyl donor and acceptor and cellobiose, lactose, galactose, and ʟ-arabinose as fructosyl acceptors. With its unique biochemical characteristics, HsLsc is an important enzyme for future research and potential industrial applications in a world faced with drought and diminishing freshwater supplies.

The aim of the study was to investigate the metabolism of non-digestible oligo- and polysaccharides by fecal microbiota, using isothermal microcalorimetry. The five tested substrates were raffinose, melibiose, a mixture of oligo- and polysaccharides produced from raffinose by levansucrase, levan synthesized from raffinose, and levan from timothy grass. Two inocula were comprised of pooled fecal samples from overweight or normal-weight children, from healthy adult volunteers and a pure culture of Bacteroides thetaiotaomicron as a reference bacterium for colon microbiota. The growth was analyzed based on the heat evolution curves, and the production of organic acids and gases. Taxonomic profiles of the microbiota were assessed by 16S rDNA sequencing. Raffinose and melibiose promoted the growth of bifidobacteria in all fecal pools. Several pool-specific substrate-related responses to raffinose and melibiose were revealed. Lactate-producing bacteria (Streptococcus and Enterococcus) became enriched in the pool of overweight children resulting in lactic acid as the major fermentation product on short saccharides. Acetic and butyric acids were prevalent at fermentation in the normal-weight pool coinciding with the enrichment of Catenibacterium. In the adult pool, the specific promotion of Bacteroides and Lachnospiraceae by levans was disclosed. In the fecal pool of normal-weight children, levans stimulated the growth of Senegalimassilia and Lachnoclostridium and this particular pool also showed the highest maximum heat production rate at levan fermentation. Levans and raffinose-derived oligosaccharides, but not raffinose and melibiose were completely fermented by a pure culture of Bacteroides thetaiotaomicron. The main conclusion from the study is that fecal microbiota of normal and overweight children have different compositions and they respond in specific manners to non-digestible oligo- and polysaccharides: raffinose, melibiose, raffinose-derived oligosaccharides and levans. The potential of the tested saccharides to support a healthy balance of colon microbiota requires further studies., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Soy, meat (mixture of pork and beef), and fish proteins were fed to rats with and without prebiotic raffinose (RAF), and the composition and fermentation of gut microbiota were examined. Bifidobacterium spp. populations were higher, and propionic acid concentration was lower in soy protein-fed than meat protein-fed rats. Likewise, Enterobacteriaceae populations were higher in fish protein-fed rats than other rats. RAF feeding increased Bifidobacterium spp. and decreased Faecalibacterium prausnitzii populations regardless of the dietary protein source. Interactions between dietary proteins and RAF were shown for Lactobacillus spp. and Clostridium perfringens group; the increase of Lactobacillus spp. populations by RAF was seen only for soy protein-fed rats, whereas the reduction of C. perfringens group by RAF was evident in fish and meat protein-fed rats. It is concluded that dietary proteins may differentially modulate the effects of prebiotic oligosaccharides on gut fermentation and microbiota, with differences observed between plant and animal proteins.

Lactobacilli derive metabolic energy mainly from carbohydrate fermentation. Homofermentative and heterofermentative lactobacilli exhibit characteristic differences in carbohydrate transport and regulation of metabolism, however, enzymes for carbohydrate transport in heterofermentative lactobacilli are poorly characterized. This study aimed to identify carbohydrate active enzymes in the L. reuteri strains LTH2584, LTH5448, TMW1.656, TMW1.112, 100-23, mlc3, and lpuph by phenotypic analysis and comparative genomics. Sourdough and intestinal isolates of L. reuteri displayed no difference in the number and type of carbohydrate-active enzymes encoded in the genome. Predicted sugar transporters encoded by genomes of L. reuteri strains were secondary carriers and most belong to the major facilitator superfamily. The quantification of gene expression during growth in sourdough and in chemically defined media corresponded to the predicted function of the transporters MalT, ScrT and LacS as carriers for maltose, sucrose, and lactose or raffinose, respectively. The genotype for sugar utilization matched the fermentation profile of 39 sugars for L. reuteri strains, and indicated preference for maltose, sucrose, raffinose and (iso)-malto-oligosaccharides, which are available in sourdough and in the upper intestine of rodents. Pentose utilization in L. reuteri species was strain-specific but independent of the origin or phylogenetic position of isolates. Two glycosyl hydrolases, licheninase (EC 3.2.1.73) and endo-1, 4-β-galactosidase (EC 3.2.1.89) were identified based on conserved domains. In conclusion, the study identified the lack of PTS systems, preference for secondary carriers for carbohydrate transport, and absence of carbon catabolite repression as characteristic features of the carbohydrate metabolism in the heterofermentative L. reuteri., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Raffinose family oligosaccharides (RFOs) accumulate under stress conditions in many plants and have been suggested to act as stress protectants. To elucidate the metabolic process of RFOs under cold stress, levels of RFOs, and related carbohydrates, the expression and activities of main metabolic enzymes and their subcellular compartments were investigated during low-temperature treatment and during the recovery period in cucumber leaves. Cold stress induced the accumulation of stachyose in vacuoles, galactinol in vacuoles and cytosol, and sucrose and raffinose in vacuoles, cytosol, and chloroplasts. After cold stress removal, levels of these sugars decreased gradually in the respective compartments. Among four galactinol synthase genes (CsGS), CsGS1 was not affected by cold stress, while the other three CsGSs were up-regulated by low temperature. RNA levels of acid-α-galactosidase (GAL) 3 and alkaline-α-galactosidase (AGA) 2 and 3, and the activities of GAL and AGA, were up-regulated after cold stress removal. GAL3 protein and GAL activity were exclusively located in vacuoles, whereas AGA2 and AGA 3 proteins were found in cytosol and chloroplasts, respectively. The results indicate that RFOs, which accumulated during cold stress in different subcellular compartments in cucumber leaves, could be catabolized in situ by different galactosidases after stress removal., (Copyright © 2018 Elsevier GmbH. All rights reserved.)

Oropetium thomaeum is a desiccation tolerant grass and acquisition of desiccation tolerance is correlated with changes in carbohydrate metabolism. Here we address the question whether the changes in carbohydrate metabolism are specific to the dehydration process or whether other environmental factors such as high temperature, low temperature, hypoxia, salinity or exogenous ABA application trigger the same or different changes in the sugar metabolism. Fifteen different sugar metabolites were identified by GC/MS, including erythritol, arabinose, fructose, galactose, glucose, myo-inositol, sedoheptulose, sucrose, trehalose, galactinol, maltose, raffinose, manninotriose and stachyose. Together with starch, these sugars were placed into the pathways of sucrose metabolism and raffinose family oligosaccharides (RFOs) metabolism, as well as into the group of rare sugars. By comparing the changes of sugars under various stresses, we concluded that the changes in the sugar metabolism are both convergent and divergent in response to different stresses. Except for the general response to stress, such as starch degradation, the changes of specific sugar metabolites reflect a stress-specific response of O. thomaeum. Erythritol seems to be specific for dehydration, myo-inositol for salt stress and trehalose for hypoxia stress. Similar as dehydration, low temperature, salt stress and ABA application resulted in the accumulation of sucrose and RFOs in O. thomaeum, which indicates that these stresses share high similarity with dehydration. Thus it is proposed that sucrose and RFOs have a general protective role under these stresses. In contrast sucrose and RFOs did not accumulate in response to high temperature or hypoxia whose effects tend to be consumptive and destructive. The accumulation of galactose, melibiose and manninotriose demonstrate that RFOs are degraded under stress. The accumulation of these sugar metabolites might result from the reaction of RFOs and stress-produced hydroxyl radicals, which supports a possible role of RFOs in stress defense. In addition, ABA application led to substantial synthesis of stachyose which occurs only in response to dehydration, indicating that stachyose synthesis is possibly closely related to ABA in O. thomaeum., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Grapevine (Vitis vinifera L.) is a widely cultivated fruit crop whose growth and productivity are greatly affected by low temperatures. On the other hand, wild Vitis species represent valuable genetic resources of natural stress tolerance. We have isolated and characterized a MYB-like gene encoding a putative GARP-type transcription factor from Amur grape (V. amurensis) designated as VaAQUILO. AQUILO (AQ) is induced by cold in both V. amurensis and V. vinifera, and its overexpression results in significantly improved tolerance to cold both in transgenic Arabidopsis and in Amur grape calli. In Arabidopsis, the ectopic expression of VaAQ increased antioxidant enzyme activities and up-regulated reactive oxygen species- (ROS) scavenging-related genes. Comparative mRNA sequencing profiling of 35S:VaAQ Arabidopsis plants suggests that this transcription factor is related to phosphate homeostasis like their Arabidopsis closest homologues: AtHRS1 and AtHHO2. However, when a cold stress is imposed, AQ is tightly associated with the cold-responsive pathway and with the raffinose family oligosaccharides (RFOs), as observed by the up-regulation of galactinol synthase (GoLS) and raffinose synthase genes. Gene co-expression network (GCN) and cis-regulatory element (CRE) analyses in grapevine indicated AQ as potentially regulating VvGoLS genes. Increased RFO content was confirmed in both transgenic Arabidopsis and Amur grape calli overexpressing VaAQ. Taken together, our results imply that AQ improves cold tolerance through promoting the accumulation of osmoprotectants.

Sesame (Sesamum indicum L.) is an important oilseed crop. However, multiple abiotic stresses severely affect sesame growth and production. Raffinose family oligosaccharides (RFOs), such as raffinose and stachyose, play an important role in desiccation tolerance of plants and developing seeds. In the present study, three types of key enzymes, galactinol synthase (GolS), raffinose synthase (RafS) and stachyose synthase (StaS), responsible for the biosynthesis of RFOs were identified at the genome-wide scale in sesame. A total of 7 SiGolS and 15 SiRS genes were identified in the sesame genome. Transcriptome analyses showed that SiGolS and SiRS genes exhibited distinct expression profiles in different tissues and seed developmental stages. Comparative expression analyses under various abiotic stresses indicated that most of SiGolS and SiRS genes were significantly regulated by drought, osmotic, salt, and waterlogging stresses, but slightly affected by cold stress. The up-regulation of several SiGolS and SiRS genes by multiple abiotic stresses suggested their active implication in sesame abiotic stress responses. Taken together, these results shed light on the RFOs-mediated abiotic stress resistance in sesame and provide a useful framework for improving abiotic stress resistance of sesame through genetic engineering.

Due to their prebiotic potential indigestible oligosaccharides became a major focus of research interest. In this study the growth of selected probiotic strains including lactobacilli, bifidobacteria, Lactococcus lactis, Streptococcus salivarius ssp. thermophilus, Pediococcus ssp. and Enterococcus faecium with the, raffinose family oligosaccharides (RFOs) raffinose, stachyose and verbascose and galactomannan from guar bean Cyamopsis tetragonoloba (total guar carbohydrates, oligosaccharides (dp 2-4) and polysaccharides (dp > 5), obtained by size exclusion chromatography) were tested by means of turbidity measurements. RFOs were used by 75% of all strains, with some delay for the trisaccharide raffinose and the tetrasaccharide stachyose and a limited fermentation of the pentasaccharide verbascose. L. reuteri, P. pentosaceus and B. lactis HNO19™ were able to ferment not only raffinose and stachyose but also verbascose. Guar oligosaccharides were fermented by 15 out of 20 strains; P. acidilactici, L. acidophilus, L. rhamnosus GG and B. animalis ssp. lactis BB12 metabolized them comparably well as glucose or galactose. Isolated guar polysaccharides were not fermented whereas total guar carbohydrates were fermented by 7 strains, apparently caused by the oligosaccharide content. The findings of this study may be important for functional food products especially for indigestible oligosaccharides which may cause adverse effects in the gut when not cleaved.

A novel α-galactosidase gene (agaB) from Bacillus megaterium 3-7 was cloned and expressed in Escherichia coli. The gene coded for a protein with 741 amino acids and a calculated molecular mass of 85.4kDa. The native structure of the recombined AgaB was determined to be a homotrimer. AgaB showed the highest identity of 57% with the characterized glycosyl hydrolase family 36 α-galactosidase from Clostridium stercorarium F-9. The enzyme exhibited a specific activity of 362.6U/mg at 37°C and pH 6.8. The enzyme showed strong resistance to proteases and great tolerance to galactose (K i =12.5mM). AgaB displayed wide substrate specificity toward pNPGal, melibiose, raffinose and stachyose, with a K m of 0.42, 12.1, 17.0 and 25.4mM, respectively. Furthermore, AgaB completely hydrolyzed raffinose and stachyose present in soybean milk at 37°C within 4h when combined with trypsin. These favorable properties make AgaB a potential candidate for applications in the food and feed industries., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The protective role of hypoxia-inducible factor-1 (HIF-1) against liver ischemia-reperfusion injury has been well proved. However its role in liver donation and preservation from donation after cardiac death (DCD) is still unknown. The objective of this study was to test the hypothesis that pharmaceutical stabilization of HIF-1 in DCD donors would result in a better graft liver condition. Male SD rats (6 animals per group) were randomly given the synthetic prolyl hydroxylase domain inhibitor FG-4592 (Selleck, 6 mg/kg of body weight) or its vehicle (dimethylsulfoxide). Six hours later, cardiac arrest was induced by bilateral pneumothorax. Rat livers were retrieved 30 min after cardiac arrest, and subsequently cold stored in University of Wisconsin solution for 24 h. They were reperfused for 60 min with Krebs-Henseleit bicarbonate buffer in an isolated perfused liver model, after which the perfusate and liver tissues were investigated. Pretreatment with FG-4592 in DCD donors significantly improved graft function with increased bile production and synthesis of adenosine triphosphate, decreased perfusate liver enzyme release, histology injury scores and oxidative stress-induced cell injury and apoptosis after reperfusion with the isolated perfused liver model. The beneficial effects of FG-4592 is attributed in part to the accumulation of HIF-1 and ultimately increased PDK1 production. Pretreatment with FG-4592 in DCD donors resulted in activation of the HIF-1 pathway and subsequently protected liver grafts from warm ischemia and cold-stored injury. These data suggest that the pharmacological HIF-1 induction may provide a clinically applicable therapeutic intervention to prevent injury to DCD allografts., (© 2017 International Center for Artificial Organs and Transplantation and Wiley Periodicals, Inc.)

Galactinol synthase (GolS) is a key enzyme in the biosynthetic pathway of raffinose family oligosaccharides (RFOs), which play roles in carbon storage, signal transduction, and osmoprotection. The present work assessed the evolutionary history of GolS genes across the Rosaceae using several bioinformatic tools. Apple (Malus × domestica) GolS genes were transcriptionally characterized during bud dormancy, in parallel with galactinol and raffinose measurements. Additionally, MdGolS2, a candidate to regulate seasonal galactinol and RFO content during apple bud dormancy, was functionally characterized in Arabidopsis. Evolutionary analyses revealed that whole genome duplications have driven GolS gene evolution and diversification in Rosaceae speciation. The strong purifying selection identified in duplicated GolS genes suggests that differential gene expression might define gene function better than protein structure. Interestingly, MdGolS2 was differentially expressed during bud dormancy, concomitantly with the highest galactinol and raffinose levels. One of the intrinsic adaptive features of bud dormancy is limited availability of free water; therefore, we generated transgenic Arabidopsis plants expressing MdGolS2. They showed higher galactinol and raffinose contents and increased tolerance to water deficit. Our results suggest that MdGolS2 is the major GolS responsible for RFO accumulation during apple dormancy, and these carbohydrates help to protect dormant buds against limited water supply.

The prebiotic effects of GOS (galactooligosaccharides) are known to depend on the glycosidic linkages, degree of polymerization (DP), and the monosaccharide composition. In this study, a novel form of α-GOS with a potentially improved prebiotic effect was synthesized using bifidobacterial α-galactosidase (α-Gal) purified from recombinant Escherichia coli. The carbohydrate produced was identified as α-d-galactopyranosyl-(1→6)-O-α-d-glucopyranosyl-(1→2)-[α-d-galactopyranosyl-(1→6)-O-β-d-fructofuranoside] and was termed stachyobifiose. Among 17 nonprobiotics, 16 nonprobiotics showed lower growth on stachyobifiose than β-GOS. In contrast, among the 16 probiotics, 6 probiotics showed higher growth on stachyobifiose than β-GOS. When compared with raffinose, stachyobifiose was used less by nonprobiotics than raffinose. Moreover, compared with stachyose, stachyobifiose was used less by Escherichia coli, Enterobacter cloacae, and Clostridium butyricum. The average amounts of total short-chain fatty acids (SCFA) produced were in the order of stachyobifiose > stachyose > raffinose > β-GOS. Taken together, stachyobifiose is expected to contribute to beneficial changes of gut microbiota.

Mitochondria are dynamic organelles that fuse and divide. These changes alter the number and distribution of mitochondrial structures throughout the cell in response to developmental and metabolic cues. We have demonstrated that mitochondrial fission is essential to the maintenance of mitochondrial DNA (mtDNA) under changing metabolic conditions in wild-type Saccharomyces cerevisiae. While increased loss of mtDNA integrity has been demonstrated for dnm1-∆ fission mutants after growth in a non-fermentable carbon source, we demonstrate that growth of yeast in different carbon sources affects the frequency of mtDNA loss, even when the carbon sources are fermentable. In addition, we demonstrate that the impact of fission on mtDNA maintenance during growth in different carbon sources is neither mediated by retrograde signaling nor mitophagy. Instead, we demonstrate that mitochondrial distribution and mtDNA maintenance phenotypes conferred by loss of Dnm1p are suppressed by the loss of Sod2p, the mitochondrial matrix superoxide dismutase.

This study was performed to determine the efficacy of raffinose on the growth, non-specific immunity, intestinal morphology and microbiota of juvenile hybrid sturgeon, (Acipenser baeri Brandt ♀ × A. schrenckii Brandt ♂). Hybrid sturgeons were divided into 2 groups and each group was fed with diets supplemented with or without raffinose for 56 days. Hybrid sturgeon fed diet supplemented with raffinose had significantly higher final body weight (FBW), specific growth rate (SGR), and weight gain ratio (WGR) than fish fed the control diet (P < 0.05). Raffinose in diet had no negative effect on feed intake (FI) and feed conversion ratio (FCR) (P > 0.05). Compared with the control diet, the myeloperoxidase (MPO) and respiratory burst (NBT) activitives were significantly higher in sturgeon fed the raffinose supplemented diet (P < 0.05). The increasing of intestinal villi area and mucosal folds were observed in intestinal tract of sturgeon when they fed the raffinose supplemented diet. Meanwhile, the residual bait of intestinal tract was relatively lower in sturgeon with raffinose treatment. High-throughput sequencing revealed that majority of reads derived from the sturgeon digesta were constituted by members of Proteobacteria, Firmicutes, Fusobacteria and Actinobacteria. Shannon's diversity index existed significant difference among dietary treatments indicating that the overall microbial community was modified to a large extent by dietary raffinose. In conclusion, supplementation of the diet with raffinose is capable of improving hybrid sturgeon growth performances and intestinal morphology, modifying the intestinal microbial composition., (Copyright © 2017. Published by Elsevier Ltd.)

Raffinose family oligosaccharides (RFOs) participate in various aspects of plant physiology, and galactinol synthase (GolS; EC 2.4.1.123) catalyzes the key step of RFO biosynthesis. Stress-induced accumulation of RFOs, in particular galactinol and raffinose, has been reported in a few plants; however, their precise role and mechanistic insight in stress adaptation remain elusive. In the present study, we have shown that the GolS activity as well as galactinol and raffinose content are significantly increased in response to various abiotic stresses in chickpea. Transcriptional analysis indicated that the CaGolS1 and CaGolS2 genes are induced in response to different abiotic stresses. Interestingly, heat and oxidative stress preferentially induce CaGolS1 over CaGolS2. In silco analysis revealed several common yet distinct cis-acting regulatory elements in their 5'-upstream regulatory sequences. Further, in vitro biochemical analysis revealed that the CaGolS1 enzyme functions better in stressful conditions than the CaGolS2 enzyme. Finally, Arabidopsis transgenic plants constitutively overexpressing CaGolS1 or CaGolS2 exhibit not only significantly increased galactinol but also raffinose content, and display better growth responses than wild-type or vector control plants when exposed to heat and oxidative stress. Further, improved tolerance of transgenic lines is associated with reduced accumulation of reactive oxygen species (ROS) and consequent lipid peroxidation as compared with control plants. Collectively, our data imply that GolS enzyme activity and consequent galactinol and raffinose content are significantly increased in response to stresses to mitigate stress-induced growth inhibition by restricting excessive ROS accumulation and consequent lipid peroxidation in plants., (© The Author 2017. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Raffinose family oligosaccharides (RFOs) accumulate in seeds during maturation desiccation in many plant species. However, it remains unclear whether RFOs have a role in establishing seed vigor. GALACTINOL SYNTHASE (GOLS), RAFFINOSE SYNTHASE (RS), and STACHYOSE SYNTHASE (STS) are the enzymes responsible for RFO biosynthesis in plants. Interestingly, only raffinose is detected in maize seeds, and a unique maize RS gene (ZmRS) was identified. In this study, we found that two independent mutator (Mu)-interrupted zmrs lines, containing no raffinose but hyperaccumulating galactinol, have significantly reduced seed vigor, compared with null segregant controls. Unlike maize, Arabidopsis thaliana seeds contain several RFOs (raffinose, stachyose, and verbascose). Manipulation of A. thaliana RFO content by overexpressing ZmGOLS2, ZmRS, or AtSTS demonstrated that co-overexpression of ZmGOLS2 and ZmRS, or overexpression of ZmGOLS2 alone, significantly increased the total content of RFOs and enhanced Arabidopsis seed vigor. Surprisingly, while overexpression of ZmRS increased seed raffinose content, its overexpression dramatically decreased seed vigor and reduced the seed amounts of galactinol, stachyose, and verbascose. In contrast, the atrs5 mutant seeds are similar to those of the wild type with regard to seed vigor and RFO content, except for stachyose, which accumulated in atrs5 seeds. Total RFOs, RFO/sucrose ratio, but not absolute individual RFO amounts, positively correlated with A. thaliana seed vigor, to which stachyose and verbascose contribute more than raffinose. Taken together, these results provide new insights into regulatory mechanisms of seed vigor and reveal distinct requirement for RFOs in modulating seed vigor in a monocot and a dicot., (Copyright © 2017 The Author. Published by Elsevier Inc. All rights reserved.)

A 45-kDa monomeric acidic α-galactosidase with a specific activity of 193.12 units/mg was isolated from the fruiting bodies of Agaricus bisporus. Blast search of internal peptide sequences suggested that it is a member of GH family 27 and it is most similar to hypothetical protein AGABI2DRAFT&#95;70106. The enzyme displayed maximal activity at pH 4.0 and 60°C, respectively. The enzyme remained stable within the pH range 2.0-9.0 but its activity was markedly suppressed in the presence of Cu 2+ , Hg 2+ , Fe 3+ and Ag + ions. It displayed resistance to α-chymotrypsin and neutral protease. Moreover, it manifested degradative activity toward both oligosaccharides and polysaccharides. The enzyme manifested Km values of 0.30mM, 10.65mM and 19.21mM, toward pNPGal, stachyose and raffinose respectively. These results suggest that Agaricus bisporus α-galactosidase is a promising candidate for elimination of raffinose oligosaccharides (RFOs) in biotechnological applications., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Cold acclimation proceeds sequentially in response to decreases in photoperiod and temperature. This study aimed at assessing the impact of photoperiod prior to cold acclimation on freezing tolerance and related biochemical and molecular responses in two alfalfa cultivars. The fall dormant cultivar Evolution and semi-dormant cultivar 6010 were grown in growth chambers under different photoperiods (8, 10, 12, 14 or 16h) prior to cold acclimation. Freezing tolerance was evaluated as well as carbohydrate concentrations, levels of transcripts encoding enzymes of carbohydrate metabolism as well as a K-3dehydrin, before and after cold acclimation. The fall dormant cultivar Evolution had a better freezing tolerance than the semi-dormant cultivar 6010. The effect of photoperiod prior to cold acclimation on the level of freezing tolerance differed between the two cultivars: an 8h-photoperiod induced the highest level of freezing tolerance in Evolution and the lowest in 6010. In Evolution, the 8h-induced superior freezing tolerance was associated with higher concentration of raffinose-family oligosaccharides (RFO). The transcript levels of sucrose synthase (SuSy) decreased whereas those of sucrose phosphatase synthase (SPS) and galactinol synthase (GaS) increased in response to cold acclimation in both cultivars. Our results indicate that RFO metabolism could be involved in short photoperiod-induced freezing tolerance in dormant alfalfa cultivars., (Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.)

In many species, Suc en route out of the leaf migrates from photosynthetically active mesophyll cells into the phloem down its concentration gradient via plasmodesmata, i.e. symplastically. In some of these plants, the process is entirely passive, but in others phloem Suc is actively converted into larger sugars, raffinose and stachyose, and segregated (trapped), thus raising total phloem sugar concentration to a level higher than in the mesophyll. Questions remain regarding the mechanisms and selective advantages conferred by both of these symplastic-loading processes. Here, we present an integrated model-including local and global transport and kinetics of polymerization-for passive and active symplastic loading. We also propose a physical model of transport through the plasmodesmata. With these models, we predict that (1) relative to passive loading, polymerization of Suc in the phloem, even in the absence of segregation, lowers the sugar content in the leaf required to achieve a given export rate and accelerates export for a given concentration of Suc in the mesophyll and (2) segregation of oligomers and the inverted gradient of total sugar content can be achieved for physiologically reasonable parameter values, but even higher export rates can be accessed in scenarios in which polymers are allowed to diffuse back into the mesophyll. We discuss these predictions in relation to further studies aimed at the clarification of loading mechanisms, fitness of active and passive symplastic loading, and potential targets for engineering improved rates of export., (© 2017 American Society of Plant Biologists. All Rights Reserved.)

We describe here the identification and characterization of a copper radical oxidase from auxiliary activities family 5 (AA5&#95;2) that was distinguished by showing preferential activity toward raffinose. Despite the biotechnological potential of carbohydrate oxidases from family AA5, very few members have been characterized. The gene encoding raffinose oxidase from Colletotrichum graminicola ( Cg RaOx; EC 1.1.3.-) was identified utilizing a bioinformatics approach based on the known modular structure of a characterized AA5&#95;2 galactose oxidase. Cg RaOx was expressed in Pichia pastoris , and the purified enzyme displayed the highest activity on the trisaccharide raffinose, whereas the activity on the disaccharide melibiose was three times lower and more than ten times lower activity was detected on d-galactose at a 300 mM substrate concentration. Thus, the substrate preference of Cg RaOx was distinguished clearly from the substrate preferences of the known galactose oxidases. The site of oxidation for raffinose was studied by 1 H nuclear magnetic resonance and mass spectrometry, and we confirmed that the hydroxyl group at the C-6 position was oxidized to an aldehyde and that in addition uronic acid was produced as a side product. A new electrospray ionization mass spectrometry method for the identification of C-6 oxidized products was developed, and the formation mechanism of the uronic acid was studied. Cg RaOx presented a novel activity pattern in the AA5 family. IMPORTANCE Currently, there are only a few characterized members of the CAZy AA5 protein family. These enzymes are interesting from an application point of view because of their ability to utilize the cheap and abundant oxidant O 2 without the requirement of complex cofactors such as FAD or NAD(P). Here, we present the identification and characterization of a novel AA5 member from Colletotrichum graminicola As discussed in the present study, the bioinformatics approach using the modular structure of galactose oxidase was successful in finding a C-6 hydroxyl carbohydrate oxidase having substrate preference for the trisaccharide raffinose. By the discovery of this activity, the diversity of the CAZy AA5 family is increasing., (Copyright © 2017 American Society for Microbiology.)

The putative gene cluster involved in the degradation of the raffinose family oligosaccharides (RFO) was identified in Caldicellulosiruptor bescii. Within the cluster, the gene encoding a putative α-galactosidase (CbAga36) was cloned and expressed in Escherichia coli. Size exclusion chromatography of the purified rCbAga36 indicated that the native form was a tetramer. Its primary sequence was similar to the family of glycoside hydrolase 36. The purified recombinant CbAga36 (rCbAga36) was optimally active at pH 5.0 and 70°C and had a half-life of 15 h and 10 h at 70°C and 80°C, respectively. rCbAga36 showed high activity with the artificial substrate (p-nitrophenyl α-d-galactopyranoside, pNPαGal) exhibiting lower K m and higher k cat than natural substrates such as melibiose and raffinose. Although rCbAga36 demonstrated preferential activity toward the hydrolysis of RFO such as raffinose and stachyose, it did not degrade the polymeric galactomannans. Our results imply that CbAga36 may play a role in the degradation of RFO, transported into the cytoplasm via a transporter into galactose, which is further utilized as an energy source in C. bescii. Furthermore, its ability to synthesize novel oligosaccharides by transglycosylation renders this enzyme potentially useful for the production of dietary oligosaccharides with novel function., (Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

The aim of the present study was to investigate how the enrichment of chitosan films with anti-fibronectin aptamers could enhance scaffold colonization by osteoblasts, by improving their adhesion and accelerating their proliferation. Chitosan discs were enriched with excess of anti-fibronectin aptamer. Aptamer adsorption on chitosan was monitored by measuring aptamer concentration in the supernatant by spectrophotometry, as well as its release, while functionalization was confirmed by labelling aptamers with a DNA intercalating dye. Chitosan samples were then characterized morphologically with atomic force microscopy and physically with contact angle measurement. Chitosan enrichment with fibronectin was then investigated by immunofluorescence and Bradford assay. 2% chitosan discs were then enriched with increasing doses of aptamers and used as culture substrates for MC3T3-E1 cells. Cell growth was monitored by optical microscopy, while cell viability and metabolic activity were assessed by chemiluminescence and by Resazurin Sodium Salt assay. Cell morphology was investigated by cytofluorescence and by scanning electron microscopy. Chitosan films efficiently bound and retained aptamers. Aptamers did not affect the amount of adsorbed fibronectin, but affected osteoblasts behavior. Cell growth was proportional to the amount of aptamer used for the functionalization, as well as aptamers influenced cell morphology and their adhesion to the substrate. Our results demonstrate that the enrichment of chitosan films with aptamers could selectively improve osteoblasts behavior. Furthermore, our results support further investigation of this type of functionalization as a suitable modification to ameliorate the biocompatibility of biomaterial for hard tissue engineering applications.