Dependence of fructooligosaccharide content on activity of fructooligosaccharide-metabolizing enzymes in yacon (Smallanthus sonchifolius) tuberous roots during storage

ABSTRACT:  Tuberous roots of yacon ( Smallanthus sonchifolius ) accumulate about 10%, on a fresh weight basis, of inulin-type fructooligosacharides (FOSs), known as a food ingredient with various healthy benefits. However, we have a great difficulty to ensure these benefits because FOSs with a lower degree of polymerization (DP) decreased remarkably, and fructose increased when the tuberous roots were stored after harvesting even under previously recommended storage conditions of low temperature with high humidity. In the present study, to elucidate the involvement of FOS-metabolizing enzymes in FOS reduction during storage at 90% relative humidity and 8°C, we extracted a crude protein from yacon tuberous roots and measured the activities of invertase (β-fructofuranosidase, EC 3.2.1.26), sucrose:sucrose 1-fructosyltransferase (1-SST, EC 2.4.1.99), fructan:fructan 1-fructosyltransferase (1-FFT, EC 2.4.1.100), and fructan 1-exohydrolase (1-FEH, EC 3.2.1.80). The enzyme activities acting on sucrose, both invertase and 1-SST, were weakened after storage for a month. In addition, the activity of 1-FEH acting on short FOSs such as 1-kestose (GF₂) and 1-nystose (GF₃) was higher than that of 1-FFT. These results suggest that the continuous decline in FOSs of low DP during storage was dependent mainly on the 1-FEH activity. On the other hand, FOSs with a DP of ≥ 9 only slightly decreased in stored yacon tuberous roots during storage, though distinct 1-FEH activity was observed in vitro toward a high-DP inulin-type substrate, indicating that highly polymerized FOSs content was unlikely to be closely connected with the 1-FEH activity.     

Variation of Fructooligosaccharides and their Metabolizing Enzymes in Onion Bulb (Allium cepa L. cv. Tenshin) During Long-term Storage

ABSTRACT: The objective of this study was to assess the status of fructooligosaccharides (FOS) in onion bulbs ( Allium cepa L. cv. Tenshin) and their metabolizing-enzymes—1-fructoexohydrolase (1-FEH), 1-kestose hydrolyzing enzyme (1-KH), fructan:fructan 1^F-fructosyltransferase (1-FFT) and fructan:fructan 6^G-fructosyltransferase (6G-FFT)—during storage at 15°C. Fructose varies slightly, whereas 1-kestose peaked after 6 wk and then decreased progressively during the last 18 wk of storage. Lower degree of polymerization (DP) 3 to 6) FOS, higher (DP 7 to 12) FOS, total FOS, and total carbohydrates showed similar and close patterns during 24 wk. They varied slightly at the beginning of the storage period; afterward they decreased progressively and regularly during the last 20 wk of storage. 1-FEH and 1-KH activities were low but peaked abruptly after 12 and 16 wk, respectively, after which they decreased to levels higher (1-FEH) or similar (1-KH) to those observed at the beginning of the storage. Surprisingly, 1-FFT activity showed similar pattern to the variation of 1-KH hydrolyzing activity; on the other hand, 6G-FFT, although higher, was stable during 16 wk but decreased after that. The results allowed us to associate FOS to the dormancy and sprouting states, and the peaks of the degrading enzymes were shown to signal the release of dormancy of onion bulb.     

The degree of polymerization of inulin-like fructans affects cecal mucin and immunoglobulin A in rats

ABSTRACT:  Cecal amounts of mucin and immunoglobulin A (IgA) were examined through the cecal fermentation pattern in Wistar (WS) or Sprague–Dawley (SD) rats fed inulin-type fructans differing in their degree of polymerization (DP). The animals were fed a control diet or a diet containing one of the fructans with an average DP of 4, 8, 16, or 23, at 60 g/kg diet for 10 d. Cecal fermentation products substantially differed between WS and SD rats fed DP8 fructan, with short-chain fatty acids (SCFAs) as the major organic acids in the former but lactate predominating in the latter. Cecal fermentability of fructans in both strains generally decreased with increasing DP of fructans, and this was especially manifest in reduction of the amounts of lactate in DP16 and 23. In WS rats, cecal mucin and IgA were greater in all fructan groups than in the control group. In SD rats, cecal mucin was greater only in the DP8, 16, and 23 groups as compared to the control group, while IgA was greater in the DP4 and 8 groups. In both strains, cecal mucin correlated with the sum of cecal SCFAs, but not with lactate, succinate, or total organic acids. In contrast, only cecal lactate correlated with cecal IgA in both strains. The present study shows that the different fermentation patterns of fructans affect cecal mucin and IgA; mucin is likely to respond to cecal SCFA production, whereas IgA increases when fermentation occurs rapidly and lactate is a major fermentation product.     

Expression of the 1Ax1 transgene in an elite Chinese wheat variety and its effect on functional properties

BACKGROUND: The transfer of transgenes from ‘model’ wheat genotypes into elite wheat cultivars using conventional plant breeding is an alternative strategy for improving the dough quality of wheat. Thus a cross was made between a popular Chinese elite wheat cultivar of the Yangzi River down‐central area that expresses high‐molecular‐weight glutenin subunit (HMW‐GS): 1Bx7 + 1By8 plus 1Dx2 + 1Dy12, and a model transgenic wheat line B102‐1‐2 which over‐expresses HMW‐GS 1Ax1 in an L88‐31 genetic background that includes HMW‐GS 1Bx17 + By18. RESULTS: F₁ to F₆ generations of crosses between B102‐1‐2 (paternal) and Emai (maternal) were analysed for their HMW‐GS compositions, allowing the selection of pure F₆ lines over‐expressing HMS‐GS 1Ax1 in the presence of the endogenous HMW‐GS: 1Bx7 + 1By8 with 1Dx2 + 1Dy12, and 1Bx17 + 1By18 with 1Dx2 + 1Dy12. Analysis of the F₆ lines showed changes in the extensograph parameters, with increases in peak area, resistance to extension, and extensibility and peak resistance to extension, similar to those observed in the transgenic paternal line B102‐1‐2. CONCLUSION: This work showed that the expression levels of the 1Ax1 transgene and the effects on dough properties were similar in the transgenic parental line B102‐1‐2 and the F₆ progeny generated from a cross with an elite Chinese cultivar Emai. The feasibility of using transgenic lines expressing HMW‐GS subunits in conventional breeding programmes was demonstrated. Copyright © 2009 Society of Chemical Industry     

Hot air-assisted radio frequency heating effects on wheat and corn seeds: Quality change and fungi inhibition

Hot air-assisted radio frequency (RF) heating has been proposed and studied as an alternative disinfestation method for grain seeds. In this study, hot air-assisted RF heating effects on fungi inhibition and its influence on physiological (seed vigor, germination rate) and biochemical (color, enzyme activity) qualities of wheat and corn seeds were further investigated. Results showed that high intensity hot air-assisted RF treatment (70 °C, 10 min) reduced seed vigor and germination rate for both wheat and corn seeds, but relatively mild intensity treatment (65 °C, 10 min) showed no significant influence on color and germination rate, and even enhanced enzyme activity (Superoxide dismutase-SOD, catalase-CAT and peroxidase-POD). Moreover, hot air-assisted RF treatment (65 °C, 10 min) reduced Aspergillus flavus by 2 and 3 log-scale (CUF/g) in wheat and corn seeds with the moisture content of 12.0%, and 3 and 4 log-scale for wheat and corn seeds when moisture content increased to 15.0%, respectively. These results indicated higher moisture content (a_w) of grain seeds would benefit fungi inhibition for hot air-assisted RF treatment, and with proper control of treatment conditions, hot air-assisted RF heating has the potential to inhibit fungi and maintain physiological and biochemical quality of grain seeds.     

Enrichment of carotenoids in flaxseed (Linum usitatissimum) by metabolic engineering with introduction of bacterial phytoene synthase gene crtB

Linseed flax (Linum usitatissimum L.) is an industrially important oil crop, which includes large amounts of alpha-linolenic acid (18:3) and lignan in its seed oil. We report here the metabolic engineering of flax plants to increase carotenoid amount in seeds. Agrobacterium-mediated transformation of flax was performed to express the phytoene synthase gene (crtB) derived from the soil bacterium Pantoea ananatis (formerly called Erwinia uredovora 20D3) under the control of the cauliflower mosaic virus (CaMV) 35S constitutive promoter or the Arabidopsis thaliana fatty acid elongase 1 gene (FAE1) seed-specific promoter. As a result, eight transgenic flax plants were generated. They formed orange seeds (embryos), in which phytoene, alpha-carotene, and beta-carotene were newly accumulated in addition to increased amounts of lutein, while untransformed flax plants formed light-yellow seeds, in which only lutein was detected. Interestingly, despite the control of the CaMV 35S promoter, the expression of crtB was not observed in the leaves but in the seeds in the transgenic flax plants. Total carotenoid amounts in these seeds were 65.4-156.3 microg/g fresh weight, which corresponded to 7.8- to 18.6-fold increase, compared with those of untransformed controls. These results suggest that the flux of phytoene synthesis from geranylgeranyl diphosphate was first promoted by the expressed crtB gene product (CrtB), and then phytoene was consecutively decomposed to the downstream metabolites alpha-carotene, beta-carotene, and lutein, as catalyzed by endogenous carotenoid biosynthetic enzymes in seeds. The transgenic flaxseeds enriched with the carotenoids could be valuable as nutritional sources for human health.     

Study of total fructan and fructooligosaccharide content in different onion tissues

The objective of this work was to determine the fructan and fructooligosaccharide (FOS) content of different onion tissues in order to evaluate the potential use of onion by‐products from the food industry as a source of FOS and fructans. Assays with two methods were carried out to optimise the extraction procedure. The main FOS, namely kestose (GF₂), nystose (GF₃) and fructofuranosylnystose (GF₄), were measured directly using standard sugars. The method for total fructans was based on enzymatic treatment (Novozym 230) of ethanolic/aqueous extract followed by determination of released fructose and glucose by HPLC. Data showed a clear predominance of GF₂ in every onion tissue and no occurrence of highly polymerised fructans. The tissues richest in fructans were the fleshy layers, so that the outer two fleshy layers turn out to be the best onion by‐product as a possible fructan source. © 2000 Society of Chemical Industry     

Comparison of the water-soluble carbohydrate composition and fructan structures of Agave tequilana plants of different ages

The composition of water-soluble carbohydrates from Agave tequilana plants of 2, 4 and 6½ years were compared by HPLC, HPAEC-PAD, MALDI-TOF-MS and GC–MS. The plants of 2 years exhibited the highest levels of free monosaccharide and low molecular weight fructans (DP 3–DP 6) with potential application as prebiotics. A maximum of fructan polymerisation was achieved at 4 years with mean DP from 3 to 30, then it was decreased at 6½ years with mean DP from 4 to 24. The linkage analysis showed an increase and decrease in the branching degree from 2 to 6½ years with a maximum at 4 years, correlated to changes in t-β-d-Fruf linkages with increased and decreased synthesis of (2→1) and (2→6)-β-d-Fruf as well as 1,6-di-β-d-Fruf linkages.     

实时荧光聚合酶链式反应检测转基因小麦B73-6-1、B72-8-11b和B102-1-2品系

目的：建立实时荧光聚合酶链式反应检测转基因小麦B73-6-1、B72-8-11b和B102-1-2品系的鉴定方法。方 法：以转基因小麦B73-6-1、B72-8-11b和B102-1-2为研究对象,在转基因小麦外源片段与小麦染色体重组的边界序 列分别设计引物和探针,以其他多种转基因产品和非转基因小麦为对照进行特异性实验,以B73-6-1样品模拟制备 10 个含量梯度的添加样品进行灵敏度实验。结果：本研究设计的引物和探针具有很好的品系鉴定特异性,检测灵敏 度可达到0.01%（m/m）。结论：该方法特异性好、灵敏度高,可快速、准确地鉴定转基因小麦B73-6-1、B72-8-11b 和B102-1-2品系。     

RNAi沉默淀粉分支酶sbe3基因对水稻直链淀粉的影响

本研究利用RNAi技术,通过RT-PCR方法克隆出水稻淀粉分支酶sbe3基因片断,经两步组装法将sbe3基因片断分别正向、反向组装入pRNAi-ubi,成功构建sbe3基因RNAi转化载体pRNAi-ubi/sbe3。在农杆菌介导下,对粳稻中花11未成熟胚诱导的愈伤组织进行转化,通过PCR、Southern杂交鉴定获得一批转基因植株。半定量RT-PCR鉴定出转化苗T1代种子中sbe3基因表达被明显抑制,但只引起转基因水稻胚乳中直链淀粉含量少量的提高,说明采用RNAi仅沉默sbe3基因对水稻胚乳直链淀粉含量没有显著影响。     

A comparison of the non-starch carbohydrates in cereal grains

The carbohydrates soluble in 80% ethanol and in water at 100°C were extracted from two varieties of wheat, barley, oats, rye, triticale, rice and malt. The highest concentrations of sugars soluble in 80% ethanol were found in malt and rye with the lowest in rice. Barley contained the most raffinose and rye contained the most fructan. Rye had the highest levels of water-soluble and total pentosans and barley the highest levels of water-soluble and total (1→3), (1→4)-β-glucans. The total arabinose:xylose ratio varied from 1.2 in wheat and triticale to 0.3 in oats. All were very low in uronic acids.     

Effect of Germination,under Different Environmental Conditions,on Saponins,Phytic Acid and Tannins in Lentils (Lens culinaris)

The effect of germination conditions on some antinutrients of Lens culinaris var Magda 20 seeds were studied. The seeds were germinated at 20°C under variable conditions of time, water and light. Quantitative analyses of the soyasapogenols, inositol phosphates and tannins were carried out by capillary gas chromatography, high-performance liquid chromatography and spectrophotometric techniques respectively. Germinated seeds at day 6 contained higher levels soyasapogenol B than the controls, whereas in general the tannin content was reduced. Total phytic acid amounts did not decrease after 3 days of germination but was greatly reduced after 6 days. This work shows that the optimal conditions to reduce some antinutritional factors (tannins and phytic acid) in lentils were 6 days of seed germination in dark and with alternate watering. Therefore, germination conditions offer a good opportunity to improve the nutritional quality of lentils. © 1997 SCI.     

Changes in quantities of inositol phosphates during maturation and germination of legume seeds

 The changes in the quantities of inositol phosphates during the maturation and germination of pea, faba bean and lupin seeds were determined in two consecutive (1993 and 1994) years of differing weather conditions. Irrespective of the year, all seeds accumulated predominantly inositol hexaphosphate (IP₆). The weather conditions influenced the accumulation of inositol phosphates in maturing seeds, but they did not influence the total content. Gradual degradation of inositol phosphates occurred during seed germination. After 8 days of germination, IP₆ was degraded by some 80% in peas, 78% in faba beans and 42% in lupin seeds. The enzymic hydrolysis of higher forms of inositol phosphates (IP₆ and IP₅) in germinating seeds was assumed to yield inositol tetraphosphate (IP₄) and inositol triphosphate (IP₃), because the quantities of these compounds increased during seed germination. Received: 1 August 1997 / Revised version: 13 October 1997     

Changes in quantities of inositol phosphates during maturation and germination of legume seeds

 The changes in the quantities of inositol phosphates during the maturation and germination of pea, faba bean and lupin seeds were determined in two consecutive (1993 and 1994) years of differing weather conditions. Irrespective of the year, all seeds accumulated predominantly inositol hexaphosphate (IP₆). The weather conditions influenced the accumulation of inositol phosphates in maturing seeds, but they did not influence the total content. Gradual degradation of inositol phosphates occurred during seed germination. After 8 days of germination, IP₆ was degraded by some 80% in peas, 78% in faba beans and 42% in lupin seeds. The enzymic hydrolysis of higher forms of inositol phosphates (IP₆ and IP₅) in germinating seeds was assumed to yield inositol tetraphosphate (IP₄) and inositol triphosphate (IP₃), because the quantities of these compounds increased during seed germination. Received: 1 August 1997 / Revised version: 13 October 1997     

油菜BnROP1同源基因沉默导致拟南芥雄性不育

基于油菜杂合双显性雄性核不育系和拟南芥全基因组芯片的表达谱分析结果,选择了一个油菜差异表达基因,其对应的拟南芥同源基因为At3G51300（AtROP1）,采用荧光定量PCR方法,对油菜中该基因（BnROP1）在根、茎、叶、花、雌蕊、雄蕊、角果中的表达模式进行了分析,在此基础上构建了AtROP1基因的microRNA干扰载体,通过农杆菌介导的拟南芥转化,获得12株转基因苗。T1转基因植株大多出现角果数量减少或不结实、无花粉、花药萎缩以及小孢子数量大大减少的表型。对转基因T2多个株系的荧光定量PCR分析说明,目标基因的干扰是有效的,靶基因的表达水平都显著下调。说明转基因植株的雄性不育表型和ROP1基因的表达沉默有关,ROP1基因不仅控制了花粉管的极性和伸长,还和小孢子的正常发育密切相关。     

Detection and prediction of Sitophilus oryzae infestations in triticale via visible and near-infrared spectral signatures

Triticale is a hybrid of wheat and rye grown for use as animal feed. In Florida, due to its soft coat, triticale is highly vulnerable to Sitophilus oryzae L. (rice weevil) and there is interest in development of methods to detect early-instar larvae so that infestations can be targeted before they become economically damaging. The objective of this study was to develop prediction models of the infestation degree for triticale seed infested with rice weevils of different growth stages. Spectral signatures were tested as a method to detect rice weevils in triticale seed. Groups of seeds at 11 different levels (degrees) of infestation, 0–62%, were obtained by combining different ratios of infested and uninfested seeds. A spectrophotometer was used to measure reflectance between 400 and 2500 nm wavelength for seeds that had been infested at different levels with six different growth stages from egg to adult. The reflectance data were analyzed by several generalized linear regression and classification methods. Different degrees of infestation were particularly well correlated with reflectances in the 400–409 nm range and other wavelengths up to 967 nm, although later growth stages could be detected more accurately than early infestation. Stepwise variable selection produced the lowest mean square differences and yielded a high R² value (0.988) for the 4th instars, pupae and adults inside the seed. Models were developed to predict the level of infestation in triticale by rice weevils of different growth stages. Overall, this study showed a great potential of using reflectance spectral signatures for detection of the level of infestation of triticale seed by rice weevils of different growth stages.     

Development of simple and efficient in Planta transformation method for wheat (Triticum aestivum L.) using Agrobacterium tumefaciens

Wheat (Triticum aestivum L. var. Shiranekomugi) seeds were soaked in water at 22 degrees C for 1 d. Thereafter, the embryo of the soaked seeds was inoculated with Agrobacterium tumefaciens by piercing a region of the embryonic apical meristem with a needle that had been dipped in an A. tumefaciens inoculum. The inoculated seeds were incubated at 22 degrees C for 2 d and sterilized by cefotaxime (Claforan) (1000 ppm water solution) treatment and then vernalized at 5 degrees C for 25 d. Finally, the seedlings were grown to maturation (T(0) plants) and allowed to pollinate naturally for seed setting (T(1) plants) in pots under nonsterile condition. To examine the transformation by various means, four different strains of A. tumefaciens were used for transformation. The following five lines of evidence proved the transformation: altered phenotype and its transmittance to the next generation, resistance of T(1) seed germination to geneticin or hygromycin B, the detection of a transgene in T(1) plants by PCR analysis and Southern hybridization and the rescue of the plasmid consisting of the integrated T-DNA and flanking wheat genome DNA from T(1) plants. The transformation efficiency of T(1) plants, which were transformed using different A. tumefaciens strains, was estimated to be 33% by PCR analysis, 75% by Southern hybridization and 40% by plasmid rescue.     

Evaluation of zinc and magnesium bioavailability from pea (Pisum sativum, L.) sprouts. Effect of illumination and different germination periods

The effect of sprouting pea seeds (Pisum sativum, L) for 2, 4 and 6 days, with and without light, on the content of zinc (Zn) and magnesium (Mg) and their nutritive utilisation by growing rats was studied. Soaking of pea seeds prior to germination caused a 49% reduction in Zn content followed by minor losses during germination. The content of Mg decreased by 6% as a result of the soaking process, and by 20–28% during germination. Sprouting for 2 and 4 days improved the bioavailability of Zn and Mg from pea seeds (from 32.2 to 88.6–108.0 μg retained Zn per day, and from 1.64 to 2.97–4.79 mg retained Mg per day in raw and sprouted pea flour diets, respectively), outweighing the reductions in Zn and Mg content because of seed soaking. The presence or absence of light during the germination process did not affect the results. We conclude that sprouting of peas for 4 days was the most effective treatment to improve the bioavailability of Zn and Mg in pea seeds.     

fps基因过量表达对烟叶类胡萝卜素生物合成的影响

为探索法呢基焦磷酸合酶(FPS)基因过量表达对烤烟叶片类胡萝卜素生物合成的影响,利用RT-PCR技术,比较了4个fps转基因株系(K4、K-6、K-17、K-35)及其非转基因对照中参与类胡萝卜素生物合成的关键酶基因的表达强度,并利用LC/MS技术,分析了叶片发育过程中类胡萝卜素及其各组分含量的变化规律.结果表明:外源fps在烟草中的过量表达,对烟叶类胡萝卜素合成相关基因Psy、Lycb皆有正向调节作用,但对Zds的表达影响不大;化学分析表明fps转基因烤烟株系中,总类胡萝卜素及其各组分的含量在叶片不同发育期均高于未转基因K326对照株系.说明外源fps基因在烟草中的过表达,对类胡萝卜素生物合成具有促进作用.     

Synbiotic potential of fresh cream cheese supplemented with inulin and Lactobacillus paracasei in co-culture with Streptococcus thermophilus

The influence of the addition of Lactobacillus paracasei and Streptococcus thermophilus on the fructan content at the beginning and at the end of storage at 4 ± 1 °C of a potentially synbiotic fresh cream cheese manufactured with inulin was investigated. Three cheese-making trials were prepared, all supplemented with a lactic culture of S. thermophilus (T1, T2 and T3). L. paracasei subsp. paracasei was added in T1 and T2. Inulin was added in T2 and the fructan content was measured after 1 and 21 days of storage. Samples of T2 possessed similar mean concentrations of fructans after 1 and 21 days of storage, 7.32% and 7.27%, respectively, and no significant difference was observed. These results indicated that the metabolism of starter and probiotic bacteria did not degrade the fructans present in those cheeses. Additionally, synbiotic cheeses possessed a fructan content higher than 7 g per 100 g, sufficient to confer prebiotic potential during the entire storage period of these products.