Rubisco small subunit, chlorophyll a/b-binding protein and sucrose : fructan-6-fructosyl transferase gene expression and sugar status in single barley leaf cells in situ. Cell type specificity and induction by light

Chungui Lu, Olga A. Koroleva, John F. Farrar, Joe Gallagher, Chris J. Pollock, and A. Deri Tomos (2002). Rubisco small subunit, chlorophyll a/b-binding protein and sucrose : fructan-6-fructosyl transferase gene expression and sugar status in single barley leaf cells in situ. Cell type specificity and induction by light. Plant Physiology, 130 (3) pp.1335-1348 Sponsorship: BBSRC RAE2008

Minimising colonic fermentation of high fructan foods : using food processing techniques to reduce levels of fructans in onion and garlic

Background: Irritable Bowel Syndrome (IBS) is a chronic functional disorder of the bowel, affecting up to 15% of Australian adults. Dietary triggers need to be identified and controlled. Researchers have shown that short chain carbohydrates, fructans (high in onion and garlic) play a major role in triggering IBS symptoms. Current dietary management aims to limit the intake of fructans in the diet. Another approach may be to use simple food processing to reduce fructans in foods.

Objective: To investigate if pickling will reduce fructan levels in garlic and shallots, and if pickled garlic and shallots reduce colonic fermentation, and  abdominal symptoms in human volunteers.

Design: Fructan levels of the garlic and shallots were measured using the Megazyme fructan assay. 18 volunteers (13 healthy and 5 IBS) participated in a single blinded, randomised cross over study. Subjects were randomly assigned to receive a breakfast (potato and salmon patty) that was either high (unprocessed) or low (processed/pickled) in garlic and shallots. Breath hydrogen was measured every hour over a ten hour period, and abdominal symptoms were assessed using validated questionnaires.

Outcomes: Pickling over a 12 day period significantly reduced fructan levels in both garlic (p=.0.00) and shallots (p=0.00). Consumption of the low fructan breakfast resulted in significantly lower breath hydrogen (p=0.05), abdominal pain (p=0.032), and wind (p=0.04).

Conclusion: Pickling results in significantly lowered fructan levels in problem foods- shallots and garlic, and lowered colonic fermentation and abdominal symptoms in both healthy and IBS volunteers. This study provides another dietary strategy for dietetic counselling of patients with IBS.

Cloning of Sucrose:Sucrose 1-Fructosyltransferase from Onion and Synthesis of Structurally Defined Fructan Molecules from Sucrose1

Sucrose (Suc):Suc 1-fructosyltransferase (1-SST) is the key enzyme in plant fructan biosynthesis, since it catalyzes de novo fructan synthesis from Suc. We have cloned 1-SST from onion (Allium cepa) by screening a cDNA library using acid invertase from tulip (Tulipa gesneriana) as a probe. Expression assays in tobacco (Nicotiana plumbaginifolia) protoplasts showed the formation of 1-kestose from Suc. In addition, an onion acid invertase clone was isolated from the same cDNA library. Protein extracts of tobacco protoplasts transformed with this clone showed extensive Suc-hydrolyzing activity. Conditions that induced fructan accumulation in onion leaves also induced 1-SST mRNA accumulation, whereas the acid invertase mRNA level decreased. Structurally different fructan molecules could be produced from Suc by a combined incubation of protein extract of protoplasts transformed with 1-SST and protein extract of protoplasts transformed with either the onion fructan:fructan 6G-fructosyltransferase or the barley Suc:fructan 6-fructosyltransferase.

Apoplastic Sugars, Fructans, Fructan Exohydrolase, and Invertase in Winter Oat: Responses to Second-Phase Cold Hardening

Changes in apoplastic carbohydrate concentrations and activities of carbohydrate-degrading enzymes were determined in crown tissues of oat (Avena sativa L., cv Wintok) during cold hardening. During second-phase hardening (−3°C for 3 d) levels of fructan, sucrose, glucose, and fructose in the apoplast increased significantly above that in nonhardened and first-phase-hardened plants. The extent of the increase in apoplastic fructan during second-phase hardening varied with the degree of fructan polymerization (DP) (e.g. DP3 and DP4 increased to a greater extent than DP7 and DP > 7). Activities of invertase and fructan exohydrolase in the crown apoplast increased approximately 4-fold over nonhardened and first-phase-hardened plants. Apoplastic fluid extracted from nonhardened, first-phase-hardened, and second-phase-hardened crown tissues had low levels, of symplastic contamination, as determined by malate dehydrogenase activity. The significance of these results in relation to increases in freezing tolerance from second-phase hardening is discussed.

Purification, cloning, and functional expression of sucrose:fructan 6-fructosyltransferase, a key enzyme of fructan synthesis in barley.

Fructans play an important role in assimilate partitioning and possibly in stress tolerance in many plant families. Sucrose:fructan 6-fructosyltransferase (6-SFT), an enzyme catalyzing the formation and extension of beta-2,6-linked fructans typical of grasses, was purified from barley (Hordeum vulgare L.). It occurred in two closely similar isoforms with indistinguishable catalytic properties, both consisting of two subunits with apparent masses of 49 and 23 kDa. Oligonucleotides, designed according to the sequences of tryptic peptides from the large subunit, were used to amplify corresponding sequences from barley cDNA. The main fragment generated was cloned and used to screen a barley cDNA expression library. The longest cDNA obtained was transiently expressed in Nicotiana plumbaginifolia protoplasts and shown to encode a functional 6-SFT. The deduced amino acid sequence of the cDNA comprises both subunits of 6-SFT. It has high similarity to plant invertases and other beta-fructosyl hydrolases but only little to bacterial fructosyltransferases catalyzing the same type of reaction as 6-SFT.

羊草cDNA文库的构建及果聚糖水解酶的分离与鉴定

羊草(Leymus chinensis(Trin.) Tzvel.)又称碱草，隶属禾本科赖草属，是欧亚大陆草原区东部草甸草原及干旱草原上的重要建群种之一。作为一种兼具重要经济价值和生态价值的优良牧草，羊草受到了广泛的关注。但长期以来，对羊草的研究主要集中在生态学、生殖生物学方面，在分子生物学方面知之甚少。为了保存羊草基因资源，并在基因水平上研究羊草生物代谢的调控机理，本研究采用羊草根、叶片混合后做为材料，构建cDNA文库，并对文库中部分基因序列进行了分析。同时从中克隆获得羊草果聚糖水解酶全长基因并对这些基因进行了深入的生物信息学分析、转化毕赤酵母研究初步确定其功能，为深入了解羊草代谢的分子机制提供理论依据。主要结果如下： 1. 成功地构建了羊草根、叶混合cDNA文库，原始文库滴度达到4×106 pfu/ml，扩增文库滴度接近1011 pfu/ml ，重组率达97% 。PCR检测插入片段，均在0.5 kb到3 kb之间，l kb以上占68%。从文库中检测到了TC、γ-TMT、FEH基因，文库覆盖度达到要求且为PCR筛选文库提供了可能。 2. 随机挑取经检测过的597个单克隆进行测序，去除插入片段小于和污染序列后，获得了584条高质量的序列。所有584条EST序列与NCBI的核酸数据库比对时，有30.99%的EST序列与己知序列有很大的同源性：而与蛋白质数据库进行比对时，有61.27%的EST序列与已知序列有很大的同源性。核酸比对中，有32.87%的序列为未知功能新基因，而蛋白质比对结果只有11.27%的序列为未知功能新基因。其中获得5条全长基因。 3. 文库中测序得到果聚糖水解酶（FEH）片段，依据其核酸、蛋白序列，以羊草根茎为材料，结合果聚糖水解酶基因的保守序列设计引物，通过 RACE 方法，获得羊草果聚糖水解酶基因 Lc 1-FEH 的全长序列（2040bp），包含一个 1803bp 的开放阅读框，采用生物信息学方法对该基因编码蛋白质进行功能分析，该基因编码的氨基酸序列具有明显的果聚糖水解酶类蛋白特征(NDPNG，FRDP 和[WEC (V/P)D] 结构域)，其分子量为 66.8kD，等电点 pI 为 5.49，是一种酸性蛋白质。同源性分析结果表明Lc1-FEH与单子叶植物小麦、大麦和黑麦草细胞壁类酵素酶同源性最高，分别为89％、87％ 和72％。运用实时定量方法对Lc 1-FEH表达量在羊草发育各时期及不同逆境处理下进行测定，结果发现，幼苗中以叶中表达量最低，根茎中表达较高，成苗中花梗中的表达量最高；Lc1-FEH在转录水平明显受碱、ABA、SA及低温胁迫诱导，随着胁迫时间延长，表达量迅速增加，到达到最大值，之后表达水平逐渐降低，在盐、干旱的诱导下的表达量迅速降低。 4. 羊草果聚糖水解酶Lc1-FEH基因在毕赤酵母中的高效表达 将pMD-Lc1-FEH 质粒经双酶切后构建果聚糖酵母表达载体 pPICZα- Lc1-FEH 。将重组表达载体线性化后电击转化毕赤酵母Pichia pastoris X33，经抗生素 Zeocin 和酵母 PCR 筛选获得高效表达酵母工程菌。毕赤酵母表达的果聚糖水解酶蛋白经SDS-PAGE 分析表明Lc1-FEH表观分子量为 67 kD 左右。其最适反应 pH 值为 5.5，在 pH 值为 4.5~6.5 的范围内能保持较高的酶活力；表达Lc1-FEH的最适反应温度为 30 ℃；在温度在 20～30℃度范围内有较高的酶活。 Lc 1-FEH能够水解含有β-2，1糖苷键类型果聚糖：蔗果四糖、菊粉、6-蔗果三糖；而对β-2，6糖苷键类型果聚糖：6-蔗果三糖、新蔗果三糖、细菌类果聚糖及蔗糖基本不具水解活性。

Absence of turnover and futile cycling of sucrose in leaves of Lolium temulentum L.: implications for metabolic compartmentation

Cairns, A. J., Gallagher, J. A. (2004). Absence of turnover and futile cycling of sucrose in leaves of Lolium temulentum L.: implications for metabolic compartmentation. Planta, 219 (5), 836-846. Sponsorship: BBSRC RAE2008

Cloning and characterization of a putative fructosyltransferase and two putative invertase genes from the temperate grass Lolium temulentum L.

J. A. Gallagher, A. J. Cairns and C. J. Pollock (2004). Cloning and characterization of a putative fructosyltransferase and two putative invertase genes from the temperate grass Lolium temulentum L. Journal of Experimental Botany, 55 (397) pp.557-569 Sponsorship: BBSRC RAE2008

Carbohydrate preference, acid tolerance and bile tolerance in five strains of Bifidobacterium

Aims: To assess the suitability of bifidobacteria for inclusion in synbiotic products on the basis of carbohydrate preference, acid and bile tolerance. Methods and Results: Five strains of Bifidobacterium were analysed for their carbohydrate preference from 12 substrates. Maximum growth rates were used to compare substrate preferences. Galacto-oligosaccharides and isomalto-oligosaccharides were well utilized by all the test species. Most bacteria tested could also utilize at least one type of fructan molecule. To determine transit tolerance of potentially probiotic bifidobacteria, acid and bile resistance was tested. A wide range acid resistance was found. Bile tolerance also varied. Conclusions: GOS and IMO were generally well utilized by the tested species. Other substrates were used to different degrees by the different species. Most bifidobacteria are poorly resistant to strongly acidic conditions with the exception of Bifidobacterium lactis Bb12. Bile tolerances were widely variable and it was shown that caution should be exercised when using colorimetric methods to assess bile tolerance. Significance and Impact of Study: The study allows the comparison of the properties of bifidobacteria, allowing a cost effective screen for the best species for use in synbiotic products to allow better survival and efficacy.

The effect of the daily intake of inulin on fasting lipid, insulin and glucose concentrations in middle-aged men and women

The present study was carried out to examine the effect of the daily intake of 10 g inulin on fasting blood lipid, glucose and insulin levels in healthy middle-aged men and women with moderately raised total plasma cholesterol (TC) and triacylglycerol (TAG) levels. This study was a doubleblind randomized placebo-controlled parallel study in which fifty-four middle-aged subjects received either inulin or placebo for a period of 8 weeks. Fasting blood samples were collected before the supplementation period (baseline samples 1 and 2, separated by 1 week) and at weeks 4 and 8, with a follow-up at week 12. Compared with baseline values, insulin concentrations were significantly lower at 4 weeks (P,0×01) in the inulin group. There was a trend for TAG values, compared with baseline, to be lower in the inulin group at 8 weeks (P,0×08) returning to baseline concentrations at week 12. On comparison of the inulin and placebo groups, the fasting TAG responses over the 8-week test period were shown to be significantly different (P,0×05, repeated measures ANOVA), which was largely due to lower plasma TAG levels in the inulin group at week 8. The percentage change in TAG levels in the inulin group during the 8-week study was shown to correlate with the initial TAG level of the subjects (rs -0×499, P = 0×004). We therefore conclude that the daily addition of 10 g inulin to the diet significantly reduced fasting insulin concentrations during the 8-week test period and resulted in lower plasma TAG levels, particularly in subjects in whom fasting TAG levels were greater than 1×5 mmol/l. These data support findings from animal studies that fructans influence the formation and/or degradation of TAG-rich lipoprotein particles, and the insulin data are also consistent with recent studies showing attenuation of insulin levels in fructan-treated rats.

Sugars in crop plants

We review current knowledge of the most abundant sugars, sucrose, maltose, glucose and fructose, in the world's major crop plants. The sucrose-accumulating crops, sugar beet and sugar cane, are included, but the main focus of the review is potato and the major cereal crops. The production of sucrose in photosynthesis and the inter-relationships of sucrose, glucose, fructose and other metabolites in primary carbon metabolism are described, as well as the synthesis of starch, fructan and cell wall polysaccharides and the breakdown of starch to produce maltose. The importance of sugars as hormone-like signalling molecules is discussed, including the role of another sugar, trehalose, and the trehalose biosynthetic pathway. The Maillard reaction, which occurs between reducing sugars and amino acids during thermal processing, is described because of its importance for colour and flavour in cooked foods. This reaction also leads to the formation of potentially harmful compounds, such as acrylamide, and is attracting increasing attention as food producers and regulators seek to reduce the levels of acrylamide in cooked food. Genetic and environmental factors affecting sugar concentrations are described.

An exploratory study into the putative prebiotic activity of fructans isolated from Agave angustifolia and the associated anticancer activity

Linear Inulin type fructan (ITF) prebiotics have a putative role in the prevention of colorectal cancer, whereas relatively little is known about branched fructans. This study aims to investigate the fermentation properties and potential prebiotic activity of branched fructans derived from Agave angustifolia Haw, using the Simulator of Human Intestinal Microbial Ecosystem (SHIME) model. The proximal, transverse and distal vessels were used to investigate fructan fermentation throughout the colon and to assess the alterations of the microbial composition and fermentation metabolites (short chain fatty acids and ammonia). The influence on bioactivity of the fermentation supernatant was assessed by MTT, Comet and transepithelial electrical resistance (TER), respectively. Addition of Agave fructan to the SHIME model significantly increased (P<0.05), bifidobacteria populations (proximal and transverse), SCFA concentrations (proximal, transverse and distal) and decreased ammonia concentrations in the distal vessel. Furthermore, the fermentation supernatant significantly (P<0.05) increased the TER of a Caco-2 cell monolayer (%) and decreased fluorescein-based paracellular flux, suggesting enhanced barrier function and reduced epithelial barrier permeability (proximal and distal vessel). While cytotoxicity and genotoxicity remained unaltered in response to the presence of Agave fructans. To conclude, branched Agave fructans show indications of prebiotic activity, particularly in relation to colon health by exerting a positive influence on gut barrier function, an important aspect of colon carcinogenesis.

A randomised, double- blind, cross-over study investigating the prebiotic effect of agave fructans in healthy human subjects

This placebo-controlled, randomised, double-blind, cross-over human feeding study aimed to determine the prebiotic effect of agave fructans. A total of thirty-eight volunteers completed this trial. The treatment consisted of 3 weeks' supplementation with 5 g/d of prebiotic agave fructan (Predilife) or equivalent placebo (maltodextrin), followed by a 2-week washout period following which subjects were crossed over to alternate the treatment arm for 3 weeks followed by a 2-week washout. Faecal samples were collected at baseline, on the last day of treatment (days 22 and 58) and washout (days 36 and 72), respectively. Changes in faecal bacterial populations, SCFA and secretory IgA were assessed using fluorescent in situ hybridisation, GC and ELISA, respectively. Bowel movements, stool consistencies, abdominal comfort and mood changes were evaluated by a recorded daily questionnaire. In parallel, the effect of agave fructans on different regions of the colon using a three-stage continuous culture simulator was studied. Predilife significantly increased faecal bifidobacteria (log10 9·6 (sd 0·4)) and lactobacilli (log10 7·7 (sd 0·8)) compared with placebo (log10 9·2 (sd 0·4); P = 0·00) (log10 7·4 (sd 0·7); P = 0·000), respectively. No change was observed for other bacterial groups tested, SCFA, secretory IgA, and PGE2 concentrations between the treatment and placebo. Denaturing gradient gel electrophoresis analysis indicated that bacterial communities were randomly dispersed and no significant differences were observed between Predilife and placebo treatments. The in vitro models showed similar increases in bifidobacterial and lactobacilli populations to that observed with the in vivo trial. To conclude, agave fructans are well tolerated in healthy human subjects and increased bifidobacteria and lactobacilli numbers in vitro and in vivo but did not influence other products of fermentation