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Volume 3 Special Issue 1 2009
Proceedings of the 6th International Fructan Symposium (IFS)
July 2008, Hokkaido, Japan
How to reference: Okada H, Kawazoe N, Yamamori A, Onodera S, Shiomi N (2009) Structural Analysis and Characteristics of Oligosaccharides Isolated from Fermented Beverage of Plant Extract. In: Benkeblia N, Shiomi N (Eds) Proceedings of the 6th International Fructan Symposium. Dynamic Biochemistry, Process Biotechnology and Molecular Biology 3 (Special Issue 1), 1-9
Guest Editors
Noureddine Benkeblia
University of the West Indies, Jamaica (www.mona.uwi.edu/lifesciences)
Norio Shiomi
Rakuno Gakuen University, Japan (www.rakuno.ac.jp/english/)
CONTENTS AND ABSTRACTS
Hideki Okada, Naoki Kawazoe, Akira Yamamori, Shuichi Onodera, Norio Shiomi (Japan) Structural Analysis and Characteristics of Oligosaccharides Isolated from Fermented Beverage of Plant Extract (pp 1-9)
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ABSTRACT
Invited Review: A fermented beverage of plant extract was prepared from about 50 kinds of fruits and vegetables. Natural fermentation was conducted by lactic acid bacteria (Leuconostoc spp.) and yeast (Zygosaccharomyces spp. and Pichia spp.). Eighteen kinds of oligosaccharides were isolated from this beverage, and their structures were confirmed by methylation analysis, MALDI-TOF-MS and NMR measurements. In these saccharides, eight novel oligosaccharides were found to be constructed by di- and trisaccharides with the fructosyl residue of pyranose form, and other trisaccharides with fructosyl residues of sucrose bond with the β-D-galactose and β-D-glucose. The characteristics of one of novel saccharide, O-β-D-fructopyranosyl-(2→6)-D-glucopyranose (Fp2-6G) were investigated, and it was shown to be non-cariogenicity and have low digestibility. Furthermore, the saccharide was selectively used by beneficial bacteria, Bifidobacterium adolescentis and B. longum, but was not used by unfavorable bacteria, Clostridium perfringens, Escherichia coli and Enterococcus faecalis that produce mutagenic substances.
Eri Fukushi, Hideki Okada, Akira Yamamori, Naoki Kawazoe, Shuichi Onodera, Jun Kawabata, Norio Shiomi (Japan) NMR Analysis of Oligosaccharides Containing Fructopyranoside (pp 10-15)
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ABSTRACT
Invited Mini-Review: This review focuses on the NMR methods for the oligosaccharides containing fructopyranoside that were previously isolated from the fermented beverage of an extract from 50 kinds of fruits and vegetables. The 1H and 13C-NMR signals of each saccharide were assigned using 2D-NMR including COSY, HSQC, HSQC-TOCSY, CH2-selected HSQC-TOCSY, and CT (constant time)-HMBC. The fructose in pyranosyl form showed different 13C chemical shifts from those of furanosyl form. Further confirmation of the pyranosyl form could be obtained from the HMBC correlation peak between C-2 and H-6 of fructose residue (Fru), whereas the C-2 of Fru in furanosyl form chould give the HMBC correlation peak between H-5 of Fru. Problems encountered were signal overlapping of protons and low peak separation. The key correlation peak between C-2 and H-6 of Fru was overlapped by the correlation peak indicating a glycosidic linkage between the C-2 of Fru and the H-6 of the glucose residue (Glc, or Glc’). These were solved using HSQC and CT-HMBC spectra rather than HMQC and conventional HMBC spectra, which have an inherent broad-line-shape in the carbon dimension.
David P. Livingston III, Tan Tuong, Shirley A. Owens (USA) Carbohydrate Changes in Winter Oat Crowns during Recovery from Freezing (pp 16-22)
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ABSTRACT
Original Research Paper: Fructan is an important cryoprotectant in plants but its exact mode of action is controversial. Much of the difficulty in identifying the mode of action is related to the lack of localization of fructan within tissues that are important for survival of the whole plant and a lack of studies on changes in fructan concentration after freezing, prior to the formation of new leaves. During recovery after freezing, fructan could ostensibly act as an energy source as well as a source for precursor molecules involved in cell wall synthesis/repair. We dissected 4 zones of the crown and quantified fructan and simple sugars. Winter oat plants which had been frozen and were in recovery contained about ½ the amount of total carbohydrate of those which had not been frozen, suggesting considerable utilization by the plant in the recovery process. The zone in which freezing had the biggest effect was the lowermost zone, above where the roots attach to the crown. The percentage of DP>5 fructan in this zone was significantly higher than unfrozen controls while the percentage of DP3 fructan and sucrose was significantly lower in plants recovering from freezing. Percentages of glucose and fructose were in many cases double what they were in unfrozen controls. These results suggest that carbohydrate re-allocation during recovery from freezing is an important part of overall winter hardiness. In addition, the biggest changes in CHO occurred in the first 3 days after freezing, suggesting that to better understand the metabolism of plants recovering from freezing, analysis should concentrate on the period just after freezing, prior to when new growth emerges.
Roberta Moretto, Carla Zuliani Sandrin, Nair Massumi Itaya, Marisa Domingos, Rita de Cássia Leone Figueiredo-Ribeiro (Brazil) Fructan Variation in Plants of Lolium multiflorum ssp. italicum ‘Lema’(Poaceae) Exposed to an Urban Environment Contaminated by High Ozone Concentrations (pp 23-28)
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ABSTRACT
Original Research Paper: Fructans are the main reserve carbohydrates in vegetative tissues of Poaceae from temperate regions. The synthesis and degradation of fructans vary according to physiological and ecological alterations, including high concentrations of air pollutants. Lolium multiflorum (Italian ryegrass) is a temperate grass species, cultivated as forage in the South of Brazil and has also been used for biomonitoring purposes since the plants accumulate heavy metals in the leaves, in addition to high concentrations of fructans. In this study, six week-old L. multiflorum plants were exposed during 28 consecutive days as well as for 24, 48 and 72 h in a polluted site affected by high levels of ozone (Ibirapuera Park) and in a glasshouse with filtered air (reference site) in the City of São Paulo (Brazil). After each exposure period, the content and composition of leaf carbohydrates were analysed. Plants from the polluted site contained higher concentrations of fructans, especially those with an intermediate degree of polymerization, when compared with plants maintained in the reference site. The pattern of fructan accumulation seemed to follow the diurnal pattern of ozone concentrations in the air, i.e., higher contents of fructose were found in the afternoon, when the levels of that pollutant were also high. The data obtained confirmed that fructan metabolism was affected by high concentrations of ozone, being a potential indicator of the stress imposed by tropical urban environments to plants of Italian ryegrass.
Renate Löppert, Anton Huber (Austria), Juan Villalvazo Naranjo (Mexico), Werner Praznik (Austria) Molecular and Physicochemical Characteristics of Fructan during Technological Processing of Agave tequilana Webervar. Azul (pp 29-33)
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ABSTRACT
Original Research Paper: Due to the high level of fructans, up to 72% of the dry matter, agave plants (Agavaceae) may be an excellent source to produce pure fructan powders as ingredients in functional food. Reliable analyses about content, quality profile and physicochemical properties are basic requirements for the development of appropriate sequences of processing steps. Samples of agave, harvested after 5-6 years (before flowering), from different plant sections (heart, basic and middle regions of leaves) and from consecutive steps of technological processing were investigated. Content of dry matter, protein and minerals were determined by means of AOAC methods. Content and composition of carbohydrates were analyzed using enzymatic and chromatographic methods. Solubility, viscosity behaviour and stability of solution were studied at different concentrations, pH and temperatures. The structure of fructan was investigated by methylation analysis. 28–32% DM was found in the heart of trunks and in the basic region of leaves. Total amount of carbohydrate was 65–83% of DM (up to 72% of fructan). Protein content was 4.3–5.0% and mineral content was 5.6–6.3% (high level of Ca2+ and Mg2+). Protein and mineral content was lost during processing. Solubility was approx. 80% with high storage stability. Structure analysis proved agave fructan to be a β-(2,1)-linked main chain with a branching degree of 0.22 (22 mol% of alditol derivatives), forming predominantly β-(2,6)-linkages in the side chains.
Erika Mellado-Mojica, Tania L. López-Medina, Mercedes G. López (Mexico) Developmental Variation in Agave tequilana Weber var. Azul Stem Carbohydrates (pp 34-39)
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ABSTRACT
Original Research Paper: Phenology studies the effect of seasonal and climatic variations on plant life cycles; and these variations are mainly due to temperature, light and precipitation changes among others. The content and type of carbohydrate are often used to establish seasonal, varietal, phenological and developmental stages of plants. The Agave genus is very appreciated due to its high adaptability under extreme conditions, such as water deficit and high and low temperatures, but nowadays it is more important due to its high fructan content. The purpose of this work was to establish the carbohydrate fluctuation in Agave tequilana Weber var. Azul of different ages or different stages of development. Fructans were extracted and separated into long- and short-DP fractions, and characterized by TLC and MALDI-TOF-MS. All Agaves presented glucose, fructose and sucrose like most plants, fructans and fructooligosaccharides (FOS) were always present. Qualitative and quantitative carbohydrate differences were observed at all ages. TLC results showed large differences between and within long- and short-DP fractions. The presence of neotype fructans was also observed by TLC in all samples, this FOS has always been found in all Agave plants. MALDI-TOF-MS analyses allowed the establishment that DPs range between 3 and 28, and the highest DP was found in the 8 years old plants. Fructan accumulation in A. tequilana presented a consecutive increment except for the 4 and 10 year-old. During these stages, A. tequilana Weber var. Azul reached two relevant physiological reproductive stages, shoots and inflorescence (seeds), for these reasons their overall carbohydrate content and structure might have changed. The main differences found for reducing carbohydrates and fructans are good indexes to evaluate the changes in the developmental stages of A. tequilana Weber var. Azul.
Iván Saldaña Oyarzábal (Mexico/UK), Tita Ritsema (Switzerland/The Netherlands), Stephen R. Pearce (UK) Analysis and Characterization of Fructan Oligosaccharides and Enzymatic Activities in the Leaves of Agave tequilana (Weber) var. ‘Azul’ (pp 40-51)
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ABSTRACT
Original Research Paper: The ability of Agave species to synthesize fructans has been poorly investigated in the past. Agave tequilana, a CAM plant of Mexican origin, accumulates fructans in their false stem or piña that are harvested and used as a source of sugars for the production of tequila. Synthesis of fructans occurs in the agave leaf and in the past it has been suggested that they are transported through the phloem to the storage organ. In this work the structural characteristics of oligosaccharides present in plant leaves are examined by different methods such as TLC, MALDI-TOF and HPAEC-PAD. Also, leaf protein extracts were tested for enzyme activities by incubation with different sugars to identify the machinery responsible for agave fructan structures. Results showed that sucrose represents the largest contributor (67%) followed by fructans (20%) up to DP 12 and monosaccharides (13%), respectively. Leaf oligofructan structures are comprised of a mixture of inulin, neoseries and branched fructans. All DP3 and DP4 fructans were linear molecules of the inulin and neoseries type, with the notable absence of 6-kestose or bifurcose, which is considered to be the primer oligosaccharide in grasses. Enzymatic assays confirmed the presence of the synthetic activities 1-SST, 1-FFT and 6G-FFT, but could not detect any 6-SFT activity, generally considered responsible of the branching fructans and synthesizer of 6-kestose or bifurcose that could not be found. From this study we conclude that the branching activity in Agave spp. is of different nature than 6-SFT already characterized in grasses. We proposed that an unknown 6-FFT activity may be responsible for the branched structure in Agave fructan.
Patricia Araceli Santiago-García, Mercedes G. López (Mexico) Prebiotic Effect of Agave Fructans and Mixtures of Different Degrees of Polymerization from Agave angustifolia Haw (pp 52-58)
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ABSTRACT
Original Research Paper: Agave fructans are complex and highly branched molecules, which cannot be digested in the upper gastrointestinal tract. As a result, when they reach the large intestine, they serve as fermentative substrates for bacterial growth. Previous reports have shown that fructans of the inulin-type, through both in vitro and in vivo assessments, are effective prebiotics, increasing the content of bifidobacteria and lactobacilli in the gut and, consequently, inhibiting the growth of pathogenic bacteria. Oaxaca has the largest diversity of Agave species in Mexico. Agave angustifolia contains high amount of fructans with potential health benefit for humans. The aim of this work was to investigate the growth rate of six bifidobacteria and four lactobacilli strains when fructans and mixtures with different degrees of polymerization (DP) from A. angustifolia were used as an energy source. We observed that agave fructans stimulated the growth of bifidobacteria and lactobacilli more efficiently (2-fold) that commercial inulins. Bacterial growth, pH drop and SCFA’s production, mainly acetate, were different among strains; while in vitro fermentation revealed that mixtures of different degrees of polymerization and short-DP (< 10) fructans were highly fermented. Biomass and pH drop were larger when the substrate contained mostly short-DP fructans. In conclusion, the presence in the mixtures of short-DP fructans, influenced significantly the rate of fermentation by the probiotic bacteria.
Judith E. Urías-Silvas, Mercedes G. López (Mexico) Agave spp. and Dasylirion sp. Fructans as a Potential Novel Source of Prebiotics (pp 59-64)
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ABSTRACT
Original Research Paper: Prebiotics of the inulin-type fructans have been studied for many years under a wide range of conditions, including concentration, degree of polymerization and variety of probiotics. This work is the first that addresses the potential of Agave spp. and Dasylirion sp. fructans as prebiotics. Fructans from five different Agave species and from Dasylirion sp. grown in six different geographic areas were tested with six different bifidobacteria and four lactobacilli strains, with commercial inulin-type fructans used as positive controls. Results indicate that bifidobacteria and lactobacilli grew using species of Agave and Dasylirion fructans as a carbon source. Most fructans stimulated the growth of both genera more efficiently than commercial inulin, as indicated by the absorbance and pH values. Fructans of Dasylirion sp. from Chihuahua and Agave tequilana from Guanajuato were the most effective, followed by Raftilose®Synergy1, a commercial inulin. This study supports previous reports that acetic, formic, and lactic acids were the main detected acids in all cases. This work further proves the potential of Agave and Dasylirion fructans as prebiotics.
Ken-ichi Tamura, Akira Kawakami, Yasuharu Sanada, Kazuhiro Tase, Toshinori Komatsu, Midori Yoshida (Japan) Enzymatic and Expression Analysis of Timothy PpFT1 Encoding a Fructosyltransferase for Synthesis of Highly Polymerized Levans (pp 65-69)
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ABSTRACT
Original Research Paper: Although fructosyltransferase (FT) genes have been isolated in a range of plant species, sucrose:fructan 6-fructosyltransferase (6-SFT) cDNAs have been functionally characterized in only few species, such as barley and wheat. In this study we characterized the cDNA of an FT homolog, PpFT1, from timothy (Phleum pratense L.) which accumulates high-DP fructans. A recombinant PpFT1 protein expressed in Pichia pastoris showed 6-SFT and sucrose:sucrose 1-fructosyltransferase (1-SST) activities with optimal pH of 5.2. The recombinant enzyme produced linear β(2,6)-linked levans from sucrose with higher DPs (>50) than those of fructans produced by wheat recombinant 6-SFT at low temperature (7°C). We also confirmed that excised timothy leaves had elevated levels of PpFT1 transcripts during the accumulation of fructans under an illuminated condition. Our results suggest that timothy PpFT1 is involved in the synthesis of highly polymerized levans with unique enzymatic properties different from those of previously cloned plant 6-SFTs.
Shigenori Yaguchi (Japan), John McCallum, Martin Shaw, Meeghan Pither-Joyce (New Zealand), Tran Thi Minh Hang (Vietnam), Hikaru Tsukazaki, Vu Quynh Hoa, Shin-ichi Masuzaki, Tadayuki Wako, Shuichi Onodera, Norio Shiomi, Naoki Yamauchi, Masayoshi Shigyo (Japan) Chromosome Engineering Techniques Modify Contents and Constituents of Fructans in Cultivated Allium Species (pp 70-77)
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ABSTRACT
Original Research Paper: Onion (Allium cepa L.) and shallot (A. cepa Aggregatum group) exhibit wide variation in bulb fructan content and the Frc locus on chromosome 8 conditions much of this variation. To understand the biochemical basis of Frc we conducted biochemical and genetic analyses of Allium fistulosum (FF) - shallot alien monosomic addition lines (AALs; FF+1A-FF+8A), onion mapping populations and shallot - A. fistulosum addition lines. Sucrose and fructan levels in leaves of FF+2A were significantly lower than FF throughout the year. FF+8A showed significantly higher winter sucrose accumulation and sucrose phosphate synthase (SPS) activity. Markers for additional candidate genes for sucrose metabolism were obtained by cloning a major SPS expressed in onion leaf and exhaustively mining onion EST resources. SPS and sucrose synthase (SuSy) loci were assigned to chromosome 8 and 6 respectively using AALs and linkage mapping. Further loci were assigned, using AALs, to chromosomes 1 (sucrose phosphate phosphatase), 2 (SuSy and 3 invertases) and 8 (neutral invertase). The shallot - A. fistulosum AAL (AA+8F) also showed the high fructan accumulation. The concordance between chromosome 8 localization of SPS and elevated leaf sucrose levels conditioned by high fructan alleles at the Frc locus in bulb onion or alien monosomic additions of chromosome 8 in A. fistulosum and in A. cepa suggest that the Frc locus may condition variation in SPS activity.
Midori Yoshida, Akira Kawakami, Yutaka Sato (Japan) Growth and Characteristics of Fructan-Accumulating Transgenic Rice: Potential for Utilization in Forage (pp 78-84)
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ABSTRACT
Original Research Paper: The feasibility of using rice as whole crop silage and biomass material has recently been investigated in Japan. Some plants in Graminae such as wheat and temperate grasses accumulate fructan, whereas rice is not able to synthesize fructan. In order to increase the quality of rice for forage and biomass material, we introduced wheat fructosyltransferase genes into rice and succeeded in obtaining transgenic rice that accumulate fructan in tissues such as leaves, sheaths and seeds. A rice transformant with the 1-SST (sucrose:sucrose 1-fructosyltransferase) gene accumulates β(2,1) linkage oligomer (DP3-5), and a rice transformant with the 6-SFT (sucrose:fructan 6-fructosyltransferase) gene accumulates mainly β(2,6) linkage fructan. A rice transformant carrying both 1-SST and 6-SFT genes showed additional accumulation of fructan with β(2,6)-linked fructosyl units attached to bifurcose. When the plants were grown in a greenhouse under summer light conditions, lines expressing 1-SST and both genes accumulated a considerable amount of fructan in tissues, and the content of total soluble sugar also increased in these lines. This fructan-accumulating rice has the potential for utilization for forage in northern regions.
Atsuko Miki, Ryusuke Sugita, Jun Watanabe, Hiroyuki Ito, Tatsuya Morita, Kei Sonoyama (Japan) Elimination Mechanism of Candida albicans in the Colon of BALB/c Mice by Dietary Fructo-oligosaccharide (pp 85-89)
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ABSTRACT
Original Research Paper: To test whether dietary fructo-oligosaccharides (FOS) eliminate Candida albicans from the gastrointestinal tract, BALB/c mice were inoculated intragastrically with C. albicans (1 × 107 cells/mouse) and then fed either a control diet or diet supplemented with a mixture of 1-kestose, nystose, and fructosylnystose, which is referred to as Meioligo-P, for four weeks. Recovery of organisms in the colon was significantly lower in mice fed Meioligo-P than in mice fed the control diet. This difference was abolished by ampicillin administration (1 mg/ml in drinking water). Meioligo-P increased total anaerobic and bifidobacteria attached to colon tissue and concentrations of short-chain fatty acids (SCFAs) in cecal contents. Although SCFAs suppressed hyphal formation of C. albicans in vitro, levels of hyphal formation of organismscultured in the cecal contents of mice fed Meioligo-P did not differ from those of mice fed the control diet. These data suggest that FOS are prebiotics that reduce C. albicans in the colon. This action may be not attributed to SCFAs.
Tsuneyuki Oku, Mariko Nakamura, Michiru Hashiguchi-Ishiguro, Kenichi Tanabe, Sadako Nakamura (Japan) Bioavailability and Laxative Threshold of 1-kestose in Human Adults (pp 90-95)
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ABSTRACT
Original Research Paper: 1-kestose is a trisaccharide and is one of the components of fructooligosaccharide (FOS). Although FOS is a typical non-digestible oligosaccharide with several beneficial health effects that have been clarified, the detailed properties of 1-kestose itself remain unknown. We first determined the digestibility of 1-kestose using rat and human small intestinal homogenates and the inhibition of 1-kestose to intestinal disaccharidases activity using rat small intestinal brush border membrane vesicles (BBMV). Thereafter, we estimated the bioavailability of 1-kestose based on the results from incremental blood glucose and insulin levels and breath hydrogen excretion after the oral ingestion of 5 and 30 g of 1-kestose in healthy human subjects. 1-kestose was hardly hydrolyzed by the rat and human intestinal disaccharidases, and competitively inhibited trehalase. When human subjects ingested 1-kestose, the blood glucose and insulin did not respond, and the excretion of breath hydrogen in a dose-dependent manner was markedly observed. The permissive dose for transitory diarrhea was estimated to be 0.24 g/kg of body weight in male subjects and 0.34 g/kg of body weight in female subjects. These results demonstrate that 1-kestose is a candidate to be the prebiotic agent in addition to FOS, and that the available energy of 1-kestose was estimated at 2 kcal/g based on the calculation method of the Health Promotion Act in Japan.
Rosemeire A. B. Pessoni, Kelly Simões, Marcia R. Braga, Rita de Cássia L. Figueiredo-Ribeiro (Brazil) Effects of Substrate Composition on Growth and Fructo-Oligosaccharide Production by Gliocladium virens (pp 96-101)
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ABSTRACT
Original Research Paper: Several species of filamentous fungi isolated from the rhizosphere of Asteraceae from the Brazilian cerrado have been shown to produce and metabolise fructose-containing sugars. Among them is Gliocladium virens, isolated from the rhizosphere of Vernonia herbacea, an inulin-accumulating species. In the present work, we investigated the ability of G. virens to produce fructo-oligosaccharides (FOS) when sucrose was used as the carbon source. We also studied the growth and FOS production in cultures of this fungus fed with different nitrogen sources. Significant increases in mycelium dry matter and production of FOS were observed when the sucrose concentration in the culture medium was increased to 3%. 1-Kestose, nystose, and 1-F-fructofuranosylnystose were the main FOS detected in fluids of G. virens cultured up to 18 d on 3% sucrose-containing media. The addition of complex sources of nitrogen, such as corn and yeast extracts, increased biomass production and reduced the content of extracellular proteins when G. virens was cultured in a sucrose-containing medium. Production of FOS was detected during the fungal growth cycle and was not affected by the nitrogen source. Although the production of oligo-fructans has gained tremendous commercial importance, only few microorganisms have the potential for industrial application. In this context, it is worth to find microbes from unexplored environments with the ability to synthesise these products. G. virens isolated from the rhizosphere of tropical plants has shown the ability to produce FOS, indicating that the Brazilian cerrado represents a profitable environment to search these microbes.
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