Dynamic Biochemistry, Process Biotechnology and Molecular Biology

Dynamic Biochemistry, Process Biotechnology and Molecular Biology
Abbreviation: Dyn. Biochem. Process Biotech. Mol. Biol.
Print: ISSN 1749-0626
Frequency and Peer status: Biannual, Peer reviewed
Scope and target readership: Dynamic Biochemistry, Process Biotechnology and Molecular Biology receives papers in which
biochemical, molecular biology, biophysical, bioinformatic, genomic and proteomic approaches (preferably multidisciplinary) to study
any aspect of biotechnology:
1) Biochemical and bioprocess engineering; Industrial processes/new products; Modelling and scale-up of laboratory processes;
2) Biominerals (metal ions, metal chelates, siderophores, metal-containing proteins) in biology, biochemistry and medicine;
3) Biotechniques and medical biotechnology (new techniques for cell culture or in vitro systems, environmental control, flow cytometry/
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4) Biotherapy and bioengineering (production of enzymes, vitamins, and other biologically active substances; studies on the processing of raw
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and artificial implementation of biological information processing and self-organizing principles);
6) Cytotechnology (a) derivation, genetic modification, characterization of cell lines, genetic and phenotypic regulation, control of cellular
metabolism, cell physiology and biochemistry related to cell function, performance and expression of cell products; (b) Cell culture
techniques, substrates, environmental requirements and optimization, cloning, hybridization and molecular biology, including genomic and
proteomic tools; (c) Cell culture systems, processes, reactors, bio-reactors, scale-up, and industrial production (up- and down-stream).
Descriptions of the design or construction of equipment, media or quality control procedures, that are ancillary to cellular research. (d) The
application of cells in differentiation, cancer research, immunology, genetics, senescence, inflammatory and viral disease and other medical
and veterinary investigations, including application in gene therapy and tissue engineering. (e) The use of cell cultures as a substrate for
bioassay, cytotoxicity and pharmacology measurement, biomedical applications and in particular as a replacement for animal models;
7) Metabolomics and molecular biology (metabolite target analysis, metabolic profiling and metabolic fingerprinting; improvements in data
preparation, storage, curation and analyses; comparative integrated studies with transcriptomics and proteomics including within a systems
biology context; and the application of metabolomics as it relates to man, animals and plants;
8) Nanoscience;
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micromachines, micro-robots, neural networks, neurocomputers, neurocomputing technologies and applications, virtual engineering, and
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10) Space research;
11) Sustainable (bio)production systems;
12) Systems biology;
13) Tissue banking (quality assurance and control of banked cells/tissues, effects of preservation and sterilisation methods on cells/tissues,
biotechnology, clinical applications; standards of practice in procurement, processing, storage and distribution of cells/tissues; ethical issues;
medico-legal issues);
14) Xenotransplantation (organ and tissue transplantation across species barriers): controversial theological, ethical, legal and psychological
implications.
Editor-in-Chief
Jaime A. Teixeira da Silva, Kagawa University, Japan
Technical Editor
Kasumi Shima, Japan
Statistics Advisor
Marcin Kozak, Warsaw University of Life Sciences, Poland
Editorial Board and Advisory Panels (Listed alphabetically)
Emil Alexov, Clemson University, USA
Michèle Amouyal, France
Abdolkarim Chehregani, Bu Ali Sina University, Iran
Anjali Dash, Banaras Hindu University, India
Riad El-Mohamedy, National Research Center, Egypt
Vicenza Faraco, University of Naples Federico II, Italy
Domingo J. Iglesias, Instituto Valenciano de Investigaciones Agrarias,
Spain
Mohsen Jahanshahi, Babol University of Technology, Iran
Babu Joseph, Allahabad Agricultural Institute, India
Parigi Ramesh Kumar, Central Food Technological Research Institute,
India
Nikos E. Labrou, Agricultural University of Athens, Greece
Andreas Liese, Technical University of Hamburg-Harburg, Germany
Ramamurthy Mahalingam, Oklahoma State University, USA
Dragomira Majhen, Ruđer Bošković Institute, Croatia
Reda Moghaieb, Cairo University, Egypt
Gopi K. Podila, The University of Alabama in Huntsville, USA
Pratap C. Pullammanappallil, University of Florida, USA
David J. Timson, The Queen's University of Belfast, UK
Valentina Tosato, International Centre for Genetic Engineering and
Biotechnology, Italy

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Guest Editors
Prof. Noureddine Benkeblia
Department of Life Sciences, University of West Indies, Jamaica
Prof. Norio Shiomi
Department of Food and Nutrition Sciences, Graduate School, Rakuno Gakuen University
Official Proceedings of the 6th International Fructan Symposium (IFS)
Sapporo, Hokkaido, Japan, 27-31 July 2008
Cover photos/figures: Top left: Two dimensional (2D) HSQC spectrum of saccharide 1 (Fukushi et al., pp 10-15). Top
right: Assignment of a sucrose phosphate synthase (SPS4) marker to A. cepa chromosomes using A. fistulosum - shallot
AALs (1A-8A); Bottom right: Genetic mapping of the SPS locus to chromosome 8 in the ‘A. cepa x A. roylei’ population
and alignment with the onion linkage map (Yaguchi et al., pp 70-77). Bottom left: Schematic proposed model of fructan
syntetic pathway for Agave species (Saldaña Oyarzábal et al., pp 40-51).
Disclaimers: All comments, conclusions, opinions, and recommendations are those of the author(s), and do not necessarily
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manuscript, and accepts product descriptions and details to be an integral part of the scientific content.
Printed in Japan on acid-free paper.
Published: December, 2009.

The Guest Editors
Dr. Noureddine Benkeblia
Noureddine Benkeblia is a Professor involved in postharvest plant biochemistry and
physiology including preservation technologies for horticultural crops. His main work is
devoted to the metabolism of the carbohydrate, fructooligosaccharides (FOS), during plant
development and storage periods. A few years ago, he introduced the new concept of system
biology –Metabolomics – to investigate the mechanisms of biosynthesis and accumulation
of FOS in Liliaceous plants. Prof. Benkeblia, first received his BSc, MSc and Doctor in
Agricultural Sciences (PhD) from the Institut National Agronomique, Algiers, and also Doctor in Agriculture (PhD) from
Kagoshima University. After few years teaching in Algeria, He joined INRA, Avignon (France) as Postdoctoral Scientist
from 2001. From 2002 to 2007, he worked as a Visiting Professor at the University of Rakuno Gakuen, Ebetsu (Japan) and
also as a Research Associate at Hokkaido University from 2005 to 2007. Prof. Benkeblia joined the Department of Life
Sciences, University of West Indies (Jamaica) in 2008, continuing his work on the physiology, the biochemistry and
metabolomics of fructan-containing plants in Jamaica. He also works on the postharvest physiology and biochemistry of
local fruits. Prof. N. Benkeblia is teaching plant physiology, horticulture and postharvest management of crops.
Dr. Norio Shiomi
Shiomi Norio received his MSc of Agriculture in 1967 and his PhD in Agriculture in 1980 from
Hokkaido University where he was working on the enzymatic synthesis of fructooligosaccharides
in asparagus and onion plants as a graduate student and an assistant professor.
In 1985, Dr Shiomi moved to Rakuno Gakuen University where he was appointed as Associate
Professor and was promoted to the rank of Professor in 1989. He visited Department of
Nutrition Science, University of California at Berkeley during 1990-1991. Dr. Shiomi acted as
the chair of the Graduate School, Rakuno Gakuen University from 1999 to 2007. In 2007, he
received The Award of Merit for Study of Food Biochemistry on Fructans and Related Synthesis Enzymes from The
Japanese Society of Applied Glycoscience. From 2008, Dr. Norio Shiomi was appointed Professor Emeritus in the
Department of Food and Nutrition Sciences, Graduate School, Rakuno Gakuen University.

Foreword
Fructans have a history of more than 150 years, and many has been reported on their general history research. Ancient
peoples used fructans containing plants as food, feed or medicine, and onion is one of the most and old vegetable used by
Pharaoh civilization in their rituals. The modern history of fructans began with their discovery by Rose (1804) and known at
the turn of the past century considerable development with Edelman’s proposal concerning their metabolism in higher plant.
During the last decades of the 20 th century, fructans research has known a considerable progress, especially with the
molecular biology tools, and the scope of fructans research expended considerably. Presently, fructans are considered food
rather than food ingredients, and are found in more than 500 food products resulting in significant daily human intake. On
the other hand, the science of nutrition also changed, and fructans are now considered under the new concept “functional
foods”, and are nowadays used as food and feed additives. This passionate history of fructans concerning their safety and
health benefits continues to arise interest of scientists who discover every day their potentials as food and ingredient.
The presence of fructan metabolism in higher plants is extensively studied and during the last few decades a
considerable amount of information has been gathered in a number of reviews focusing on the distribution, structure,
physiology, metabolism and, more recently, on the molecular biology of fructans. The interest in studying fructan-containing
species is greatly due to their agricultural use and to their application in food and pharmaceutical industry. The general
scientific interest in fructan metabolism arises partly from (i) their distribution in flora, and (ii) from their physiological
roles in the adaptation of plants to environmental stresses. The investigation has focused on many species, mainly of
economic interests, while a lot remains to discover on other species.
From this point of view, fructans in plants occur in many prominent orders like Asterales, Liliales and Poales,
representing the most economic plants such as wheat, barley and onion. Chemically, fructans are classified according to
their differences in glycosidic linkage type, and account linear fructans (inulins) typically found in Asterales (e. g. chicory),
and branched and/or mixed fructans types occurring in onion, garlic, asparagus, wheat and barley, although these branched
fructans are of more complex structures. In plants, fructans mainly serves not only as a reserve carbohydrate, but have also
physiological roles such osmoregulation and frost resistance. Beside the huge literature reported on the biochemistry,
physiology, enzymology, and molecular biology of fructans, many remains not understood and is still unclear such their
accumulation and translocation, the physiological factors triggering and regulating their degradation, as well as their
chemistry in the different cell compartments.
This symposium aimed to answer at least few of the numerous questions researchers have on fructans and where the
science led. The results reported by these papers are of great pertinence, but we are quite sure that we will rise many other
questions again. However, this exciting work would be an additional booster to go “inside” the fructans world and make
possible to answer these and additional questions.
Professor Noureddine BENKEBLIA
Professor Norio SHIOMI
December, 2009

SPECIAL ISSUE: CONTENTS
Hideki Okada, Naoki Kawazoe, Akira Yamamori, Shuichi Onodera, Norio Shiomi (Japan) Structural Analysis and
Characteristics of Oligosaccharides Isolated from Fermented Beverage of Plant Extract
Eri Fukushi, Hideki Okada, Akira Yamamori, Naoki Kawazoe, Shuichi Onodera, Jun Kawabata, Norio Shiomi (Japan)
NMR Analysis of Oligosaccharides Containing Fructopyranoside
David P. Livingston III, Tan Tuong, Shirley A. Owens (USA) Carbohydrate Changes in Winter Oat Crowns during Recovery
from Freezing
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
Renate Löppert, Anton Huber (Austria), Juan Villalvazo Naranjo (Mexico), Werner Praznik (Austria) Molecular and
Physicochemical Characteristics of Fructan during Technological Processing of Agave tequilana Weber var. Azul
Erika Mellado-Mojica, Tania L. López-Medina, Mercedes G. López (Mexico) Developmental Variation in Agave tequilana
Weber var. Azul Stem Carbohydrates
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’
Patricia Araceli Santiago-García, Mercedes G. López (Mexico) Prebiotic Effect of Agave Fructans and Mixtures of Different
Degrees of Polymerization from Agave angustifolia Haw
Judith E. Urías-Silvas, Mercedes G. López (Mexico) Agave spp. and Dasylirion sp. Fructans as a Potential Novel Source of
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
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
Midori Yoshida, Akira Kawakami, Yutaka Sato (Japan) Growth and Characteristics of Fructan-Accumulating Transgenic
Rice: Potential for Utilization in Forage
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
Tsuneyuki Oku, Mariko Nakamura, Michiru Hashiguchi-Ishiguro, Kenichi Tanabe, Sadako Nakamura (Japan)
Bioavailability and Laxative Threshold of 1-kestose in Human Adults
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
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Dynamic Biochemistry, Process Biotechnology and Molecular Biology
SPECIAL ISSUE: Proceedings of the 6 th International Fructan Symposium (IFS), July 2008, Hokkaido Japan
(http://fructan2008.agr.hokudai.ac.jp/index.html). Guest Editors: Dr. Noureddine Benkeblia (University of the West
Indies, Jamaica) and Prof. Norio Shiomi (Department of Food and Nutrition Science, Graduate School of Dairy Science
Research, Rakuno Gakuen University, Japan) ~ December, 2009
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)
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)
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 1 H and 13 C-NMR
signals of each saccharide were assigned using 2D-NMR including COSY, HSQC, HSQC-TOCSY, CH 2-selected HSQC-TOCSY,
and CT (constant time)-HMBC. The fructose in pyranosyl form showed different 13 C 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)
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

etter 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)
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 Weber var. Azul (pp 29-33)
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 Ca 2+ and Mg 2+ ). 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)
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)
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)
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)
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)
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)
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)
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)
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 × 10 7 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 organisms cultured 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)
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)
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.