Increased stress tolerance of Bifidobacterium longum and Lactococcus lactis produced during continuous mixed-strain immobilized-cell fermentation

AIMS: The effect of immobilization and long-term continuous culture was studied on probiotic and technological characteristics of lactic acid and probiotic bacteria. METHODS AND RESULTS: A continuous culture in a two-stage system was carried out for 17 days at different temperatures ranging from 32 to 37 degrees C, with a first reactor containing Bifidobacterium longum ATCC 15707 and Lactococcus lactis subsp. lactis biovar. diacetylactis MD immobilized separately in gel beads, and a second reactor operated with free cells released from the first reactor. The tolerance of free cells from both strains produced in the effluent medium of both reactors to hydrogen peroxide, simulated gastric and intestinal juices, antibiotics and nisin, and freeze-drying markedly increased with culture time and was generally higher after 6 days than that of stationary-phase cells produced during free-cell batch fermentations. The reversibility of the acquired tolerance of B. longum, but not L. diacetylactis, to antibiotics was shown during successive free-cell batch cultures. CONCLUSIONS: Free cells produced from continuous immobilized-cell culture exhibited altered physiology and increased tolerance to various chemical and physico-chemical stresses. SIGNIFICANCE AND IMPACT OF THE STUDY: Continuous culture with immobilized cells could be used to produce probiotic and lactic acid bacteria with enhanced technological and probiotic characteristics.     

Growth of yeasts, lactic and acetic acid bacteria in palm wine during tapping and fermentation from felled oil palm (Elaeis guineensis) in Ghana

AIM: To investigate the microbiological and biochemical changes which occur in palm wine during the tapping of felled oil palm trees. METHODS AND RESUlts: Microbiological and biochemical contents of palm wine were determined during the tapping of felled oil palm trees for 5 weeks and also during the storage. Saccharomyces cerevisiae dominated the yeast biota and was the only species isolated in the mature samples. Lactobacillus plantarum and Leuconostoc mesenteroides were the dominated lactic acid bacteria, whilst acetic acid bacteria were isolated only after the third day when levels of alcohol had become substantial. The pH, lactic and acetic acid concentrations during the tapping were among 3.5-4.0%, 0.1-0.3% and 0.2-0.4% respectively, whilst the alcohol contents of samples collected within the day were between 1.4% and 2.82%; palm wine which had accumulated over night, 3.24% to 4.75%; and palm wine held for 24 h, over 7.0%. CONCLUSION: Accumulation of alcohol in palm wine occurs in three stages during the tapping and marketing with the concurrent lactic and acetic acid fermentation taking place as well. SIGNIFICANCE AND IMPACT OF THE STUDY: Yeasts, lactic and acetic acid bacteria are all important in the fermentation of palm wine and influence the composition of the product.     

Temporary storage compounds and sucrose-starch metabolism in seed coats during pea seed development (Pisum sativum)

Quantitative data for growth, carbohydrate, protein and free amino acid nitrogen content of pea ( Pisum sativum L. cv. Finale) seed coat were obtained during the main stage of seed development. These data allowed us to define the role of the seed coat storage compounds. High amounts of arginine were measured in the seed coat and this amino acid is hypothesized to be synthesized de novo in the seed coat cells. Starch appeared to be stored in a specific parenchyma layer of the seed coat. Starch storage was shown to occur from phloem-unloaded sucrose and high activities of some enzymes of sucrose-starch metabolism (sucrose synthase, EC 2.4.1.13 and ADP glucose pyrophosphorylase, EC 2.7.7.27) were measured. The contribution of seed storage compounds is discussed in terms of buffering embryo nutrition. The sink strength of the young pea seed may be located within the seed coat.     

Effect of a granulovirus on mortality and dispersal of potato tuber worm (Lepidoptera: Gelechiidae) in refrigerated storage warehouse conditions

Potato tuber worm (PTW), Phthorimaea operculella (Zeller), is a world-wide pest of potato. In rustic stores, PTW larvae can infest 100% of stored tubers. Treatment of tubers in rustic stores with the PTW granulovirus (PoGV) has been demonstrated to protect stored tubers. This is the first study to show the effects of PoGV for protection of tubers stored in refrigerated warehouse conditions. Tubers were treated by dipping in aqueous suspensions of PoGV or water. An estimated 0.0819 larval equivalents of virus or 1.88×10⁹ viral occlusion bodies were deposited on each kilogram of tubers. They were held at 16°C for 11 days before lowering the temperature by 0.5°C per day until 10°C was reached. The tubers were stored at this temperature for 53 days. Mean numbers of infested tubers at the end of the assay was affected by both pre-infestation rate and virus treatment. Mean numbers of infested tubers in the control treatment was 3 tubers per chamber higher than in the virus treatment providing strong evidence that PoGV controlled larvae and minimized spread into un-infested tubers. Of the larvae that were retrieved in virus-treated infested tubers, the mean mortality was 87% compared to 37% in controls.     

Seasonal variation in nitrogen storage reserves in the roots of leafy spurge (Euphorbia esula) and responses to decapitation and defoliation

Seasonal fluctuations of carbohydrates and nitrogenous components in the roots of the noxious perennial leafy spurge ( Euphorbia esula L.) are strongly associated with overwintering strategy, Amino acids and distinct soluble proteins accumulate during fall and remain at elevated levels throughout winter. The formation of carbohydrate reserves in roots was not significantly affected by decapitation or selective defoliation; however, maximum amino acid and soluble protein contents were markedly reduced. In particular, the accumulation pattern of a 26 kDa protein was altered. This protein may play a role in plant conditioning and regenerative potential.     

Importance of dormancy and sink strength in sprouting of onions (Allium cepa) during storage

In onion ( Allium cepa L.) postponement of sprouting is necessary to achieve long term storage. We studied the factors determining sprouting during dry storage at 16°C. The period to visible sprouting depends on the length of the dormancy period, if present, and on the growth rate of the sprout. In the three cultivars tested, sprouts were initiated within 2 weeks after harvest indicating the absence of a real dormancy period. Sprout length increased linearly during storage. The mitotic activity of the apex decreased before harvest, was low at the transition from scale to leaf formation, and increased again when the sprout was initiated. From a few weeks before harvest, the initially high fructan content of the scales decreased, leading to a large increase in fructose. The sprout always contained enough carbohydrates for growth (between 50 and 60 mg g⁻¹ dry weight, of which 30% was fructan). The activity of sucrose synthase (EC 2.4.1.13) increased as the sprout grew, indicating an increase in sink strength. Invertase (EC 3.2.1.26) was absent in all bulb organs, during the various developmental stages. Although carbohydrates and enzymes were available for fast sprouting, sprout growth was still linear instead of exponential during dry storage at temperatures favorable for growth (16°C). The relative importance of factors determining sprouting are discussed.     

The effect of long-term preservation of bacterial cells immobilized in poly(vinyl alcohol) cryogel on their viability and biosynthesis of target metabolites

The effect of cell storage at ?18°C for 18–24 months on reproductive capacity was investigated for various microorganisms (gram-positive and gram-negative bacteria, yeasts, and filamentous fungi) immobilized in poly(vinyl alcohol) cryogel. To examine the viability of immobilized cells after defrosting, the bioluminescent method of intracellular ATP determination was used. A high level of metabolic activity of immobilized cells after various periods of storage was recorded for Streptomyces anulatus, Rhizopus oryzae, and Escherichia coli, which are producers of the antibiotic aurantin, L(+)-lactic acid, and the recombinant enzyme organophosphate hydrolase, respectively. It was shown that the initial concentration of immobilized cells in cryogel granules plays an important role in the survival of Str. anulatus and Pseudomonas putida after 1.5 years of storage. It was found that, after slow defrosting in the storage medium at 5°C for 18 h of immobilized cells of the yeast Saccharomyces cerevisiae that had been stored for nine months, the number of reproductive cells increased due to the formation of ascospores.     

Molecular cloning and characterization of Polygalacturonase-Inhibiting Protein and Cinnamoyl-Coa Reductase genes and their association with fruit storage conditions in blueberry (Vaccinium corymbosum)

Blueberry is a widely grown and easily perishable fruit crop. An efficient post-harvest handling is critical, and for that purpose gene technology methods have been part of ongoing programmes to improve crops with high food values such as blueberry. Here we report the isolation, cloning, characterization and differential expression levels of two cDNAs encoding Polygalacturonase-Inhibitor Protein (PGIP) and Cinnamoyl-Coa Reductase (CCR) from blueberry fruits in relation to various storage conditions. The open reading frame of PGIP and CCR encodes a polypeptide of 329 and 347 amino acids, respectively. To assess changes in the expression of blueberry PGIP and CCR after harvest, a storage trial was initiated. The northern blots hybridization showed a clear differential expression level of PGIP and CCR between freshly harvested and stored fruits as well as between fruits stored under various storage conditions. Although the prospects of exploiting such a strategy for crop improvement are limited, the results provide further insight into the control of the quality over the storage period at the molecular level.     

浙江早稻谷中稻水象甲的存活研究

研究了早稻收获和储藏期间稻水象甲Lissorhoptrus oryzophilus Kuschel一代成虫的数量动态及其存活时间与温湿度的关系。早稻收割时尚未迁离稻田的象甲中,仅3～13%的个体随脱落操作落入稻谷中,且这些个体经太阳曝晒2天后存活率不足1%;收割时残留于稻草中的虫量为稻谷中的5.1～12.3倍。储藏1个月的稻谷中所有成虫均死亡,但储藏相同时间的秕谷和稻草中存活成虫分别达到250头/50 kg秕谷和17头/50 kg稻草。成虫在≥90% RH下的存活时间显著长于35～75%RH下,在玻璃管中的存活时间显著长于稻谷中。在稻谷中,温度对成虫存活时间的影响不明显,但在玻璃管中,20℃（恒温）下的存活时间显著长于27.0～33.2℃（室温）下。利用生存分析的指数模型分析了成虫存活与湿度、温度和存放基质的关系,表明在75～98% RH之间,湿度越低则成虫死亡风险越大,较高温度（27.0～33.2℃）和处于稻谷中其死亡风险亦高。这些结果为开展稻谷中稻水象甲的检疫提供了依据。     

The influence of nitrogen availability on carbon and nitrogen storage in the biennial Cirsium vulgare (Savi) Ten. I. Storage capacity in relation to resource acquisition, allocation and recycling

Plants of Cirsium vulgare (Savi) Ten. were cultivated under five different nitrogen regimes in order to investigate the effects of nitrogen supply on the storage processes in a biennial species during its first year of growth. External N supply increased total biomass production without changing the relationship between ‘productive plant compartments’ (i.e. shoot plus fine roots) and ‘storage plant compartments’ (i.e. structural root dry weight, which is defined as the difference between tap root biomass and the amount of stored carbohydrates and N compounds). The amount of carbohydrates and N compounds stored per unit of structural tap root dry weight was not affected by external N availability during the season, because high rates of N supply increased the concentration of N compounds whilst decreasing the carbohydrate concentration, and low rates of N supply had the opposite effect. Mobilization of N from senescing leaves was not related to the N status of the plants. The relationship between nitrogen compounds stored in the tap root and the maximum amount of nitrogen in leaves was an increasing function with increasing nitrogen supply. We conclude that the allocation between vegetative plant growth and the growth of storage structures over a wide range of N availability seems to follow predictions from optimum allocation theory, whereas N storage responds in a rather plastic way to N availability.     

Formation of oleosomes (storage lipid bodies) during embryogenesis and their breakdown during seedling development in cotyledons of Sinapis alba L.

Electron microscopic and biochemical investigations of developing embryonic mustard cotyledons provided no evidence for the widely accepted hypothesis that oleosomes of fat-storing tissues originate from the endoplasmic reticulum and are surrounded by a unit- or half-unit membrane. In contrast, it was found that the first lipid droplets appear (about 12–14 d after pollination) in the ground cytoplasm near the surface of plastids. Subsequently these nascent lipid droplets, which lack any detectable boundary structure at this stage, become encircled by a cisterna of rough endoplasmic reticulum. At the same time an osmiophilic coat of about 3 nm thickness becomes detectable at the lipid/water interface. In the cotyledon cells of germinating seedlings a centrifugally moving front of fat degradation moves from the central vacuoles(s) towards the cell periphery, leaving behind collapsed coats of oleosomes which are depleted of their lipid contents (saccules). Although saccules appear tripartite in cross section, they are structurally different from endoplasmic reticulum membranes. The oleosome coats can be isolated from oleosome preparations by extracting lipids with organic solvents. The coat material is insoluble in detergents like Triton X-100 or deoxycholate and shows a tripartite, lamellar structure (similar to collapsed saccules) under the electron microscope. Upon dissolution with dodecylsulfate, polyacrylamide gel electrophoresis revealed a polypeptide composition (9 major bands) which is qualitatively different from that of the endoplasmic reticulum membrane. Also the buoyant densities of defatted oleosome coats and defatted endoplasmic reticulum membranes are very different. It is concluded that oleosome lipids accumulate in the ground cytoplasm and are bounded by a lamellar structure originating de novo from proteinaceous elements synthesized by specific regions of the endoplasmic reticulum.Abbreviation ER endoplasmic reticulum     

顽拗性黄皮胚脱水过程中核酸、蛋白质代谢的变化

黄皮种子发育晚期,胚内核酸、蛋白质合成能力增强,而花生胚的核酸、蛋白质合成能力在发育晚期则呈下降趋势。黄皮胚的发育在达到生理成熟后维持着活跃的生理代谢并转入萌发状态;而花生胚的代谢活性逐步降低并转入生理静止状态。脱水处理引起生理成熟期黄皮胚核酸、蛋白质合成能力急剧下降,核酸水解酶活性增强。不同程度脱水的黄皮胚吸胀２４ｈ,核酸、蛋白质的合成能力随脱水程度的加深而降低;生物大分子代谢能力的变化是顽拗性     

Changes in amino acid content of an algal feed species (Navicula sp.) and their effect on growth and survival of juvenile abalone (Haliotis rubra)

The growth and survival of juvenile Haliotis rubra, when fed with the diatom Navicula sp. cultured in f/2 medium containing combined nitrogen at 24.71 mg NO₃-N L^–1 (high), 12.35 mg NO₃-N L^–1 (standard) or 2.47 mg NO₃-N L^–1 (low), were compared in a 33-day trial. The alga in the low nitrogen medium contained 37% less total amino acid than that in the high and standard nitrogen media. There was a slightly greater reduction in essential amino acids (40%) compared to non-essential amino acids (35%). Juvenile abalone feeding on Navicula grown in medium with low nitrate and lower total amino acid content grew more slowly than when fed on the same species grown in standard or higher nitrogen medium with a higher amino acid content. The growth rate of juveniles was highest (43 m d^–1) in the high nitrate treatment followed (40 m d^–1) by the standard nitrate treatment and lowest (31 m d^–1) in the low nitrate treatment. The survival of the juveniles was also effected by the diet. Survival was better in the high and standard nitrogen media (88%) than the low nitrogen medium (75%). The results suggest that in order to achieve uniformity in nutritional quality of diatoms and good growth of abalone juveniles in commercial abalone nurseries, the nitrogen concentration in tanks should be monitored and additional nitrate added to provide an optimum concentration of between 2 and 12 mg NO₃-N L^–1.     

Trade-offs between reproductive and somatic (storage) investments in animals: a comparative test of the Van Noordwijk and De Jong model

Classical life-history theory predicts ‘trade-offs’ between reproductive and somatic investments. However, empirical studies have shown that intraspecific phenotypic correlations between these two resource investments are often positive or nonsignificant, rather than negative as predicted. The model of Van Noordwijk and De Jong (1986) was proposed to explain these unexpected results. According to their model, positive correlations between reproductive and somatic investments will result if individual variation in resource acquisition exceeds that of resource allocation, whereas negative correlations will result if individual variation in resource allocation exceeds that of resource acquisition. To test this model, I used body storage/condition as an index of somatic investment because it is usually strongly related to level of resource acquisition. I predicted that laboratory studies should more often show negative correlations between reproductive and somatic investments than field studies, because individual variation in resource acquisition is expected to be lower in controlled laboratory environments than in variable natural environments. A literature review revealed that correlations between somatic (storage) investment and reproductive investment (estimated as clutch/litter mass, number of offspring per clutch/litter, or number of clutches/litters) among conspecific breeding female animals are more often positive (15 species) or nonsignificant (17 species) than negative (6 species). Moreover, as expected, five of six negative correlations were observed in laboratory studies, whereas 13 of 15 positive correlations were observed in field studies. It is concluded that future empirical and theoretical work on life histories should consider individual variation in both resource acquisition and allocation and the interaction between the two. This revised version was published online in July 2006 with corrections to the Cover Date.     

氮肥对夏秋季茶树吸收根生物量和养分储量的影响

以12年生龙井43茶树为研究对象,在7月至翌年1月利用土钻法对连续5a施用不同氮肥处理后的茶树吸收根生物量和养分含量进行了研究。结果表明茶树吸收根生物量在0.34-0.72 mg/dm3之间,碳、氮、磷、钾和镁储量变异范围分别为12.6-25.2 mg/dm~3、4.55-11.2 mg/dm~3、0.47-1.19 mg/dm~3、1.31-4.05 mg/dm~3、0.30-1.19 mg/dm~3。茶树吸收根生物量和各养分含量随月份变化呈现双峰型,峰值分别在8月和翌年1月,而7月和11月生物量和养分储量均较低。与不施肥对照相比,施用氮肥影响茶树吸收根生物量,氮肥施用对茶树吸收根生物量的影响因氮肥施用时间而异。不同氮肥施用水平下茶树吸收根总碳浓度和总碳含量均不存在显著差异。受氮肥施用时间影响,施氮对茶树吸收根氮浓度的影响不同月份间存在差异,其中7月、8月和1月施氮处理下氮浓度较高,而9月、10月和11月不施氮处理下氮浓度较高。氮肥施用对各月份茶树吸收根氮养分储量均没有显著影响。氮肥施用降低了部分月份茶树吸收根磷、钾和镁的浓度和储量。施用中等用量的氮肥能缩小茶树吸收根夏秋季氮磷钾镁养分储量的月份间差异。     

稻株含氮量和密度对褐飞虱存活、发育和生殖特性的影响

对褐飞虱种群在不同含氮量稻株和若虫密度条件下的反应进行了研究。结果表明,若虫密度对褐飞虱存活的抑制作用随若虫密度提高而增强,但随寄主含氮量的增加而显著下降,表现为低含量的寄主植物可以增强对种群调节的负反馈作用。在低氮稻株上的饲养代数也明显影响若虫存活率,而在高氮稻株上饲养的不同代别褐飞虱之间则无显著差异。若虫率与稻株含氮量呈极显著的负相关,即在高若虫密度下寄主含氮量的增加可显著缩短褐飞虱若虫的发育历期。与高含氮量稻株上的褐飞虱种群相比,饲养在低含氮量稻株上的褐飞虱种群的若虫发育时间在高若虫密度下显著延长。在每盆40头褐飞虱若虫的密度下,成虫性比与稻株含氮量呈极显著的正相关,而在不同若虫密度下,随着若虫密度的增加雌性成虫比例显著下降。在每盆160头的若虫密度时低氮稻株上褐飞虱种群的性比低于0.3,显著低于在高氮稻株上的褐飞虱种群的性比0.85。在含氮量低的稻株上的雌成虫体重随若虫密度的增加极显著减少,连续取食第2代的雌成虫又比取食第1代时的轻。在所研究的所有若虫密度下,取食高含氮量稻株的褐飞虱种群的雌成虫寿命均为取食低含氮量稻株褐飞虱种群雌成虫寿命的3倍左右,差异极为显著。稻株含氮量和若虫密度对褐飞虱生殖力的作用最大,特别是在低氮稻株上若虫密度对褐飞虱生殖力的作用更为突出。在高含氮量稻株上的卵孵化率均随若虫密度的增加而有所下降,但在相同含氮量稻株上卵孵化率的差异均不显著。结果推测由于施用氮肥较多的水稻可以承受高密度的褐飞虱,提高了它们的迁出临界密度,减少了褐飞虱在克服逆境过程中的种群损失,从而造成更高的密度和更重的田间危害程度。     

Cloning and optimization of induction conditions for mature PsaA (pneumococcal surface adhesin A) expression in Escherichia coli and recombinant protein stability during long-term storage

The gene corresponding to mature PsaA from Streptococcus pneumoniae serotype 14 was cloned into a plasmid with kanamycin resistance and without a purification tag in Escherichia coli to express high levels of the recombinant protein for large-scale production as a potential vaccine candidate or as a carrier for polysaccharide conjugation at Bio-Manguinhos/Fiocruz. The evaluation of induction conditions (IPTG concentration, temperature and time) in E. coli was accomplished by experimental design techniques to enhance the expression level of mature recombinant PsaA (rPsaA). The optimization of induction process conditions led us to perform the recombinant protein induction at 25°C for 16 h, with 0.1mM IPTG in Terrific Broth medium. At these conditions, the level of mature rPsaA expression obtained in E. coli BL21 (DE3) Star by pET28a induction with IPTG was in the range of 0.8 g/L of culture medium, with a 10-fold lower concentration of inducer than usually employed, which contributes to a less expensive process. Mature rPsaA expressed in E. coli BL21 (DE3) Star accounted for approximately 30-35% of the total protein. rPsaA purification by ion exchange allowed the production of high-purity recombinant protein without fusion tags. The results presented in this work confirm that the purified recombinant protein maintains its stability and integrity for long periods of time in various storage conditions (temperatures of 4 or -70°C using different cryoprotectors) and for at least 3 years at 4 or -70°C in PBS. The conformation of the stored protein was confirmed using circular dichroism. Mature rPsaA antigenicity was proven by anti-rPsaA mouse serum recognition through western blot analysis, and no protein degradation was detected after long periods of storage.     

Influence of initial organic N reserves and residual leaf area on growth, N uptake, N partitioning and N storage in alfalfa (Medicago sativa) during post-cutting regrowth

BACKGROUND AND AIMS: The influence of initial residual leaf area and initial N reserves on N uptake, final N distribution, and yield in alfalfa regrowing after cutting, were studied. METHODS: The effects of two levels of initial residual leaf area (plants cut to 15 cm, with (L+) or without (L-) their leaves) and two initial levels of N status [high N (HN) or low N (LN)] on growth, N uptake and N partitioning, allocation and storage after 29 d of post-cutting regrowth were analysed. KEY RESULTS: During most of the regrowth period (8-29 d after the initial harvest), HN and L+ plants had higher net N uptake rates than LN and L- plants, respectively, resulting in a greater final mineral N uptake for these treatments. However, the final partitioning of exogenous N to the regrowing shoots was the same for all treatments (67 % of total exogenous N on average). Final shoot growth, total plant N content, and N allocation to the different taproot N pools were significantly lower in plants with reduced initial leaf area and initial N reserve status. CONCLUSIONS: Although both initial residual leaf area and initial N reserves influenced alfalfa regrowth, the residual leaf area had a greater effect on final forage production and N composition in the taproot, whereas the N uptake rate and final total N content in plant were more affected by the initial N reserve status than by the residual leaf area. Moreover, N storage as proteins (especially as vegetative storage proteins, rather than nitrate or amino acids) in the taproot allowed nitrate uptake to occur at significant rates. This suggests that protein storage is not only a means of sequestering N in a tissue for further mobilization, utilization for growth or tissue maintenance, but may also indirectly influence both N acquisition and reduction capacities.