Fermentative synthesis and hydrolysis of fructans

The review of the experimental papers dealing with some aspects of fermentative biosynthesis of fructans with different degrees of polymerization from microorganisms and plants is presented. The role of sucrose 1F-fructosyltransferase and 1,2-beta-fructan 1F-fructosyltransferase in the biosynthesis and hydrolysis of fructans, the influence of different stresses (water, temperature, oxygen and others) on these process are discussed.     

Labeling of fructans in winter wheat stems

Fructans synthesized from newly formed assimilates accumulate in wheat stems as nonstructural carbohydrates. Experiments performed tested the hypothesis that the fructose moiety from translocated sucrose is used preferentially in biosynthesis of these fructans. Results indicated: (a) a large percentage of labeled sucrose was translocated and unloaded in an unaltered state; and (b) sucrose contributed its fructose moiety to fructan synthesis in stems.     

Microbial fructosyltransferases and the role of fructans

Microbial fructosyltransferases are polymerases that are involved in microbial fructan (levan, inulin and fructo-oligosaccharide) biosynthesis. Structurally, microbial fructosyltransferase proteins share the catalytic domain of glycoside hydrolases 68 family and are grouped in seven phylogenetically related clusters. Fructosyltransferase-encoding genes are organized in operons or in clusters associated with other genes related to carbohydrate metabolism or fructosyltransferase secretion. Fructosyltransferase gene expression is mainly regulated by two-component systems or phosphorelay mechanisms that respond to sucrose availability or other environmental signals. Microbial fructans are involved in conferring resistance to environmental stress such as water deprivation, nutrient assimilation, biofilm formation, and as virulence factors in colonization. As a result of the biological and industrial importance of fructans, fructosyltransferases have been the subject of extensive research, conducted to improve their enzymatic activity or to elucidate their biological role in nature.     

Fermentation of fructans by epiphytic lactic acid bacteria

A total of 712 strains of lactic acid bacteria isolated from forage grasses were studied for their ability to ferment fructans of phlein- as well as inulin-type. Only 16 strains utilized phlein and eight of these also fermented inulin. They were identified as Lactobacillus paracasei subsp. paracasei, Lact. plantarum, Lact. brevis and Pediococcus pentosaceus . In the species Lact. paracasei subsp. paracasei , all strains gave positive results, whereas the other positive strains possessed unique properties within their own species. In all but two cases (strains of the species Lact. plantarum ), the phlein was more intensively fermented than the inulin, as indicated by a lower pH and a higher lactic acid concentration. On the basis of the outcome of this study it seems worthwhile to inoculate grasses of low sugar content before ensiling with an active strain that can ferment fructans.     

Plant fructans stabilize phosphatidylcholine liposomes during freeze-drying.

Fructans have been implicated as protective agents in the drought and freezing tolerance of many plant species. A direct proof of their ability to stabilize biological structures under stress conditions, however, is still lacking. Here we show that inulins (linear fructose polymers) isolated from chicory roots and dahlia tubers stabilize egg phosphatidylcholine large unilamellar vesicles during freeze-drying, while another polysaccharide, hydroxyethyl starch, was completely ineffective. Liposome stability was assessed after rehydration by measuring retention of the soluble fluorescent dye carboxyfluorescein and bilayer fusion. Inulin was an especially effective stabilizer in combination with glucose. Analysis by HPLC showed that the commercial inulin preparations used in our study contained no low molecular mass sugars that could be responsible for the observed stabilizing effect of the fructans. Fourier transform infrared spectroscopy showed a reduction of the gel to liquid-crystalline phase transition temperature of dry egg PtdCho by more than 20 degrees C in the presence of inulin. A direct interaction of inulin with the phospholipid in the dry state was also indicated by dramatic differences in the phosphate asymmetric stretch region of the infrared spectrum between samples with and without the polysaccharide.     

Structure of fructans from excised leaves of New Zealand flax

The accumulation of total water-soluble carbohydrate, and specifically sucrose and fructan, by excised leaves of Phormium tenax and P. cookianum (family Phormiaceae J. G. Agardh, order Asparagales) was investigated. Total water-soluble carbohydrate content of excised leaves of P. tenax and P. cookianum increased during 48 h of continuous illumination at an average rate of 1.3 and 0.9 mg g(-1) fresh weight leaf per hour, respectively. The sucrose content of excised leaves increased throughout the experimental period. The fructan content of excised leaves of P. tenax increased slightly throughout the experimental period, whilst that of P. cookianum was variable and showed no overall change. Chemical and spectroscopic analysis of the fructans obtained from the two Phormium species showed that they were similar to each other and contained mostly 1-linked and terminal fructofuranosyl (Fruf) residues, together with smaller amounts of 6-linked Fruf, 1,6-branched Fruf, terminal and 6-linked glucopyranosyl residues. Separation of the fructans by thin-layer and high-performance anion-exchange chromatography revealed the presence of a complex mixture of fructo-oligosaccharides and higher molecular weight fructan. The branched structure of the fructans isolated from excised leaves of Phormium resembles that of fructans and fructo-oligosaccharides isolated from some related species within the order Asparagales (Agave vera cruz, Cordyline australis and Urginea maritima), but is distinct from the linear structure of fructans from others (Allium cepa and Asparagus officinalis). The structural heterogeniety of fructans within both the order Asparagales and superorder Liliiflorae may be a useful chemotaxonomic aid.     

Remobilization of fructans in Phippsia algida during rapid inflorescence development

Plants of Phippsia algida (Sol.) R. Br. were cultivated in short days (SD; 8 h summer daylight) and in long days (LD; 8 h summer daylight + 16 h low irradiance extension of 5 μmol m⁻² s⁻¹) at 9, 15, and 21°C. In this plant, inflorescence primordia are initiated in both LD and SD, but LD are required for heading and inflorescence development (Heide, O.M.; Physiol. Plant. 85: 606–610. 1992). Total dry matter production was slightly increased by LD over SD at 9°C, while it was little affected by daylength at 15 and 21°C. Phippsia algida contained mainly fructans with a low degree of polymerization, largely of the kestose series. After 29 to 42 days (depending on the temperatature) of photoperiodic treatment, fructans constituted 15–20 percent of dry mass of SD-grown plants compared with only 2–3 percent of dry mass for LD-grown flowering plants. There was no difference due to photoperiod in levels of mono- and disaccharides. Shifting the SD-grown plants to LD conditions resulted in rapid inflorescence development, accompanied by a parallel rapid decrease in the fructan level, while the level of mono- and disaccharides remained constant. The results show that fructans are important as storage carbohydrates in the late snow-bed species P. algida that normally requires several growing seasons for completing its life cycle. Exhaustion of this storage pool during the extremely fast flower and fruit development constitutes an essential part of the plants adaption to a very short growing season.     

In vitro comparison of the prebiotic effects of two inulin-type fructans

Faecal cultures were used to compare the prebiotic effects of a new fructan containing high solubility inulin (HSI) and of a well-established prebiotic containing oligofructose (OF) with a negative control (CT). Changes in the intestinal microbiota, pH, ammonia, volatile organic acids and lactic acid were monitored during incubation. Molecular techniques for microbial enumeration indicated that both HSI and OF led to a significant increase in bifidobacteria (P< or =0.05) and lactobacilli (P< or =0.05) compared to the control. Significant changes in the pH and levels of ammonia with both inulin-type fructans were observed, as well as higher levels of acetic, lactic and formic acids (P< or =0.05). The fermentative metabolism appeared to be faster on OF than on HSI. Both OF and HSI showed clear prebiotic effects, but had differences in fermentation kinetics because of to the different degree of polymerization (DP). This study provides proof for the prebiotic effectiveness of HSI, and shows that inulin-type fructans with higher DP might have a prolonged bifidogenic effect, thus could extend the saccharolytic metabolism and low pH to the distal parts of the colon.     

水分和腐植酸对燕麦果聚糖代谢的影响

探讨水分和腐植酸(HA)对燕麦不同器官非结构性碳水化合物(NSC)积累与分配的影响,进一步明确水分和HA对燕麦糖代谢和粒重形成的作用机制,可为旱作地区燕麦的推广种植提供理论指导和技术支撑。试验以‘蒙农大燕1号'和‘内燕5号'两个燕麦品种为材料,分别在旱作(无灌溉)和有限灌溉(拔节期和抽穗期每次灌水60 mm)两个水分条件下喷施HA与清水(CK),研究燕麦开花后不同时期NSC组分在茎、叶、穗中的动态变化以及叶片中碳代谢相关酶活性的变化。结果表明: 两个燕麦品种茎、叶、穗中的NSC组分含量均随开花后时间的延长先升高后降低,且两品种各器官中的NSC组分含量大致相同;与CK相比,在灌水条件下喷施HA后蒙农大燕1号穗部的果聚糖含量提升幅度明显大于旱作条件;喷施HA后蒙农大燕1号叶片中果聚糖外水解酶和转化酶活性分别显著提高了27.1%和30.6%,单穗粒重显著提高了55.9%,且与旱作条件下相比提高幅度更大;蒙农大燕1号籽粒千粒重和单穗粒重与叶片果聚糖含量呈显著正相关关系。综上,水分和腐植酸协同作用可以有效调节燕麦果聚糖的积累及主要代谢酶活性,从而提高千粒重和单穗粒重,促进产量形成。     

An improved method for quantitative analysis of total fructans in plant tissues

Current methods for measuring fructan levels in plant tissues are time-consuming and costly. They often involve multiple or sequential extractions, enzymatic or acid hydrolysis of fructan polymers, and multiple HPLC runs to quantify fructan-derived hexoses. Here we describe a new method that requires a single extraction step, followed by selective precipitation of fructans by acetone, acid hydrolysis of the precipitate, and a short (10 min) HPLC run to complete the procedure. We used perennial ryegrass samples to show that the new method has similar sensitivity, but better reproducibility, than a more complex method that is widely used. We have used the new method to study developmentally related changes in fructan levels in glasshouse-grown perennial ryegrass plants.     

Plant fructans in stress environments: emerging concepts and future prospects

Plants are sessile and sensitive organisms known to possess various regulatory mechanisms for defending themselves under stress environments. Fructans are fructose-based polymers synthesized from sucrose by fructosyltransferases (FTs). They have been increasingly recognized as protective agents against abiotic stresses. Using model membranes, numerous in vitro studies have demonstrated that fructans can stabilize membranes by direct H-bonding to the phosphate and choline groups of membrane lipids, resulting in a reduced water outflow from the dry membranes. Inulin-type fructans are flexible random-coiled structures that can adopt many conformations, allowing them to insert deeply within the membranes. The devitrification temperature (T(g)) can be adjusted by their varying molecular weights. In addition, above T(g) their low crystallization rates ensure prolonged membrane protection. Supporting, in vivo studies with transgenic plants expressing FTs showed fructan accumulation and an associated improvement in freezing and/or chilling tolerance. The water-soluble nature of fructans may allow their rapid adaptation as cryoprotectants in order to give optimal membrane protection. One of the emerging concepts for delivering vacuolar fructans to the extracellular space for protecting the plasma membrane is vesicle-mediated, tonoplast-derived exocytosis. It should, however, be noted that natural stress tolerance is a very complex process that cannot be explained by the action of a single molecule or mechanism.     

Genetic engineering of rice capable of synthesizing fructans and enhancing chilling tolerance

Fructans are water-soluble fructose oligomers and polymers thatare based on sucrose, and have been implicated in protectingplants against water stress. Rice (Oryza sativa L.) is highlysensitive to chilling temperatures, and is not able to synthesizefructans. Two wheat fructan-synthesizing enzymes, sucrose:sucrose1-fructosyltransferase, encoded by wft2, or sucrose:fructan6-fructosyltransferase, encoded by wft1, were introduced intorice plants, and rice transformants that accumulate fructanswere successfully obtained. The mature leaf blades of transgenicrice lines with wft2 or wft1 accumulated 16.2 mg g^–1 FWof oligo- and polysaccharides mainly composed of inulin oligomersof more than DP7, and 3.7 mg g^–1 FW of oligo- and polysaccharides,mainly composed of phlein oligomers of more than DP15, respectively.The transgenic rice seedlings with wft2 accumulated significantlyhigher concentrations of oligo- and polysaccharides than non-transgenicrice seedlings, and exhibited enhanced chilling tolerance. Theoligo- and polysaccharide concentrations of seedlings expressingwft1 were obviously lower than those of lines expressing wft2,and no correlation between oligo- and polysaccharide concentrationsand chilling tolerance was detected in wft1-expressing ricelines. The results suggest that transgenic rice lines expressingwheat-derived fructosyltransferase genes accumulated large amountsof fructans in mature leaf blades and exhibited enhanced chillingtolerance at the seedling stage. This is the first report owingthat fructan accumulation enhanced tolerance to non-freezinglow temperatures. Key words: Chilling tolerance, fructan, fructosyltransferase, Oryza sativa, transgenic plant     

Structural diversity of fructans from members of the order Asparagales in New Zealand

The water-soluble carbohydrates (WSCs) extracted from the underground parts of Arthropodium cirratum, Astelia banksii, Bulbinella hookeri, Dianella nigra and Xeronema callistemon, and the flower stem of Phormium tenax have been investigated. Extracts of A. cirratum, B. hookeri, D. nigra and P. tenax contained 108.4, 28.1, 41.9 and 29.7 mg gFW(-1) WSCs, respectively. Thin-layer chromatography (TLC) showed that these species contained fructans. Extracts of A. banksii and X. callistemon each contained 11 mg gFW(-1) WSCs or less, and TLC detected only monosaccharides and sucrose. Reverse-phase (RP) HPLC and glycosyl linkage analysis showed that extracts of B. hookeri contained predominantly a linear series of fructans, with 1-linked and terminal fructofuranosyl (Fruf) residues and terminal glucopyranose (Glcp). RP-HPLC of extracts of A. cirratum, D. nigra and P. tenax showed a more complex pattern of oligosaccharides. Linkage analysis showed that these extracts contained fructans with 1-linked Fruf, 6-linked Fruf and 1,6-branched Fruf. Extracts of D. nigra and P. tenax contained 6-Glcp with only trace amounts of terminal Glcp, while A. cirratum contained mostly terminal Glcp with a trace of 6-Glcp. Differences in fructan and fructo-oligosaccharide structure between species shown in this study, together with published data, suggests that there are differences in the activities of the various fructosyltransferases responsible for biosynthesis of fructans in species within the Asparagales.