Recovery of c-phycocyanin from the cyanobacteriumSpirulina maxima

Spirulina biomass was separated into two fractions which may have various uses. A phycocyanin fraction may provide a food colourant and biomarkers, and a protein-rich leftover may be useful as aquaculture feed. Activated charcoal adsorption, ultrafiltration and spray drying were used effectively to produce a high quality colourant grade phycocyanin, while activated charcoal adsorption, ammonium sulphate precipitation, dialysis and chromatography were effective in preparing reagent grade phycocyanin.     

Box–Behnken analysis and storage of spray-dried collagenolytic proteases from Myceliophthora thermophila submerged bioprocess

Enzymes do not have long-term storage stability in soluble forms, thus drying methods could minimize the loss of enzymatic activity, the spray dryer removes water under high temperatures and little time. The aims of this study were to improve the stability of enzymatic extract from Myceliophthora thermophila for potential applications in industry and to evaluate the best conditions to remove the water by spray drying technique. The parameters were tested according to Box–Behnken and evaluated by analysis of variance (ANOVA), all the parameters measured were found to influence the final enzyme activity and spray drying process yield ranged from 38.65 to 63.75%. Enzyme powders showed increased storage stability than extract and maintained about 100% of collagenolytic activity after 180 days of storage at 30°C. The results showed that the microbial enzymes maintained activity during the spray drying process and were stable during long-term storage; these are promising characteristics for industrial applications.     

16S rRNA及rpoC1基因用于螺旋藻、节旋藻系统发育的比较北大核心CSCD

【目的】利用16S rRNA和rpoC1基因分子标记研究螺旋藻、节旋藻的系统发育关系,并对其区分能力进行比较。【方法】以84株螺旋藻、节旋藻为研究对象,对其进行16S rRNA、rpoC1基因序列的扩增、测序及分析,并对构建的系统发育树进行对比。【结果】rpoC1基因序列保守位点所占比例49.7%、平均G+C百分含量47.7%和序列相似度76%–100%明显低于16S rRNA基因序列的79.4%、55.6%和91%–100%,其变异程度高于16S rRNA基因;基于16S rRNA、rpoC1基因构建的系统发育NJ树拓扑结构基本一致,84株实验藻株分为2个属3个类群,其中仅F-351、F-904-2、F-1070和TJBC14-1藻株为螺旋藻,其余均为节旋藻;虽然2个基因都不能区分形态种和地理种,但rpoC1基因NJ树的置信度(100%)高于16S rRNA基因(99%),属内分群效果也明显优于16S rRNA基因。【结论】支持了螺旋藻、节旋藻为两个不同属的结论,且在属内分类时rpoC1基因比16S rRNA基因具有更高的区分度。     

Formulation development of the biocontrol agent Bacillus subtilis strain CPA-8 by spray-drying

Aims: To prepare commercially acceptable formulations of Bacillus subtilis CPA‐8 by spray‐drying with long storage life and retained efficacy to control peach and nectarine brown rot caused by Monilinia spp. Methods and Results: CPA‐8 24‐h‐ and 72‐h‐old cultures were spray dried using 10% skimmed milk, 10% skimmed milk plus 10% MgSO₄, 10% MgSO₄ and 20% MgSO₄ as carriers/protectants. All carriers/protectants gave good percentages of powder recovery (28–38%) and moisture content (7–13%). CPA‐8 survival varied considerably among spray‐dried 24‐h‐ and 72‐h‐old cultures. Seventy‐two hours culture spray dried formulations showed the highest survival (28–32%) with final concentration products of 1·6–3·3 × 10⁹ CFU g^?1, while viability of 24‐h‐old formulations was lower than 1%. Spray‐dried 72‐h‐old formulations were selected to subsequent evaluation. Rehydration of cells with water provided a good recovery of CPA‐8 dried cells, similar to other complex rehydration media tested. Spray‐dried formulations stored at 4 ± 1 and 20 ± 1°C showed good shelf life during 6 months, and viability was maintained or slightly decreased by 0·2–0·3‐log. CPA‐8 formulations after 4‐ and 6 months storage were effective in controlling brown rot caused by Monilinia spp. on nectarines and peaches resulting in a 90–100% reduction in disease incidence. Conclusions: Stable and effective formulations of biocontrol agent B. subtilis CPA‐8 could be obtained by spray‐drying. Significance and Impact of the Study: New shelf‐stable and effective formulations of a biocontrol agent have been obtained by spray‐drying to control brown rot on peach.     

Optimization of low-cost drying methods to minimize lipid peroxidation in <Emphasis Type="Italic">Spirulina platensis</Emphasis> grown in the Philippines

Three low-cost drying methods (sun, solar, and draft oven) were optimized to produce Spirulina powder of optimal quality. Optimization in the pre-dehydration stage included the use of two antioxidants, α-tocopherol and tertiary-butyl hydroquinone (TBHQ), and two blanching methods, microwave and water bath, to inactivate enzymes. The efficiency of the pre-dehydration treatments at minimizing lipid peroxidation were evaluated in terms of the product’s oxidative stability using the thiobarbituric acid (TBA) test. The sample with the lowest TBA reactive substance (TBARS) value was considered the most stable. TBHQ was found to be significantly better than α-tocopherol in minimizing lipid peroxidation in blanched samples while α-tocopherol was better than TBHQ in unblanched samples. Microwave blanching exerted a greater stabilizing effect than water bath blanching. The combined effect of TBHQ and microwave blanching was found to be the most effective pre-dehydration treatment for minimizing lipid peroxidation in drying Spirulina. Among the three low-cost optimized drying methods, sun-drying produced a dried product with the lowest TBARS value (0.472 mg malondialdehyde.kg⁻¹), which was closest to that of the spray-dried (control) sample (0.434 mg MDA.kg⁻¹). Draft oven and solar drying produced dried products with the same average TBARS value (0.56 mg MDA.kg⁻¹). Sun-drying, when optimized, produced a dried product that was almost as stable as the spray-dried product. Presented at the 6th Meeting of the Asian Pacific Society of Applied Phycology, Manila, Philippines     

Enhancing phagocytic activity of hemocytes and disease resistance in the prawn Penaeus merguiensis by feeding Spirulina platensis

Exposing the prawn Penaeus merguiensis to the bacteria Vibrio harveyi and Escherichia coli for an hour or feeding the prawns with Spirulina (Arthrospira) platensis (0.3% w/w feed) enhanced the phagocytic activity of their hemocytes. Improvement of the phagocytic activity was primarily through the activation of the hemocytes. The activated phagocytic hemocytes had a higher capacity to engulf foreign agents, such as bacteria, and a higher rate of phagocytosis. The phagocytic enhancement effect peaked on the fourth day of feeding with Spirulina. In the in vitro study, the granular cells from prawns took 45–60 min to complete the process of degranulation. Pre-exposure to Salmonella typhimurium and Bacillus subtilis did not result in enhancement of phagocytic activity of hemocytes. Only 10% prawns fed with Spirulina died in the first 14 days when challenged by V. harveyi at a concentration of 1 × 10⁴CFUs mL^–1, while all control prawns (basal feed without Spirulina) died within 14 days.     

Carbon dioxide fixation by Chlorella kessleri, C. vulgaris, Scenedesmus obliquus and Spirulina sp. cultivated in flasks and vertical tubular photobioreactors

CO₂ at different concentrations were added to cultures of the eukaryotic microalgae, Chlorella kessleri, C. vulgaris and Scenedesmus obliquus, and the prokaryotic cyanobacterium, Spirulina sp., growing in flasks and in a photobioreactor. In each case, the best kinetics and carbon fixation rate were with a vertical tubular photobioreactor. Overall, Spirulina sp. had the highest rates. Spirulina sp., Sc. obliquus and C. vulgaris could grow with up to 18% CO₂.     

Spirulina enhances the viability of Lactobacillus rhamnosus E/N after freeze-drying in a protective medium of sucrose and lactulose

Aims: Response surface methodology (RSM) was used to optimize a protective medium for enhancing the viability of Lactobacillus rhamnosus E/N cells during lyophilization. Methods and Results: Spirulina, sucrose and lactulose were selected, on the basis of a Plackett‐Burman factorial design, as important protectants having the following protective effects on cell viability: 102·025, 36·885 and ?34·42, respectively. A full‐factorial central composite design was applied to determine optimal levels of three used agents. Conclusion: The optimal protective medium composition was determined to be: Spirulina 1·304% (w/v), lactulose 5·48% (w/v), and sucrose 13·04% (w/v) (Polish Patent P‐393189). The predictive value of cell viability in this medium was 89·619%, and experimental viability obtained during freeze‐drying was 87·5%. Significance and Impact of the Study: In this study, Spirulina was used for the first time as the protective agent in freeze‐drying medium, significantly increasing lactobacilli viability and giving synbiotic character of the final product.     

螺旋藻-壳寡糖生物被膜抑制剂的制备、表征及活性评价

病原菌形成的生物被膜严重威胁人类健康,显著增强了病原菌的耐药性,针对生物被膜的特效药物亟待研究。从虾、蟹壳等中提取得到的壳寡糖是一种天然碱性寡糖,具有良好的杀菌效果,但其对生物被膜的抑制作用仍有待提高。螺旋藻（Spirulina,SP）是一种表面带负电荷的微藻,其与壳寡糖形成的复合物可能发挥协同增效杀灭生物被膜深处病原菌的作用。针对提升壳寡糖的抑生物被膜作用,本研究首先通过浊度法筛选得到了杀菌效果显著的壳寡糖,并通过静电吸附作用将壳寡糖与螺旋藻结合,完成螺旋藻@壳寡糖（Spriulina@Chitooligosaccharides,SP@COS）复合物的制备。通过测定zeta电位、粒径和荧光标记等方法表征了壳寡糖和螺旋藻的结合情况,紫外-可见吸收光谱（ultraviolet-visible absorbance spectroscopy,UV-Vis）结果显示出螺旋藻对壳寡糖的包封率达90%,负载率达16%。制备的SP@COS对细菌、真菌生物被膜都有明显的增效抑制作用,且这种抑制效果主要是通过深入生物被膜内部、破坏细胞结构所实现。这些结果显示了螺旋藻-壳寡糖复合物具备作为生物被膜抑制剂的潜力,为提高壳寡糖的抑生物被膜作用、解决病原菌的危害提供了理论基础与新的思路。     

Influence of freeze-drying and spray-drying preservation methods on survivability rate of different types of protectants encapsulated Lactobacillus acidophilus FTDC 3081

ABSTRACT

The aims of this study were to compare the effectiveness of different drying methods and to investigate the effects of adding a series of individual protectant such as skim milk, sucrose, maltodextrin, and corn starch for preserving Lactobacillus acidophilus FTDC 3081 cells during spray and freeze-drying and storage at different temperatures. Results showed a remarkable high survival rate of 70–80% immediately after spray- and freeze-drying in which the cell viability retained at the range of 10⁹ to 10¹⁰ CFU/mL. After a month of storage, maltodextrin showed higher protective ability on both spray- and freeze-dried cells as compared to other protective agents at 4°C, 25°C, and 40°C. A complete loss in viability of spray-dried L. acidophilus FTDC 3081 was observed after a month at 40°C in the absence of protective agent.     

Functional Expression of <Emphasis Type="Italic">Spirulina</Emphasis>-Δ<Superscript>6</Superscript> Desaturase Gene in Yeast, <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Spirulina-acyl-lipid desaturases are membrane-bound enzymes found in thylakoid and plasma membranes. These enzymes carry out the fatty acid desaturation process of Spirulina to yield γ-linolenic acid (GLA) as the final desaturation product. In this study, Spirulina-Δ⁶ desaturase encoded by the desD gene was heterologously expressed and characterized in Saccharomyces cerevisiae. We then conducted site-directed mutagenesis of the histidine residues in the three histidine boxes to determine the role of these amino acid residues in the enzyme function. Our results showed that while four mutants showed complete loss of Δ⁶-desaturase activity and two mutants showed only trace of the activity, the enzyme activity could be partially restored by chemical rescue using exogenously provided imidazole. This study reveals that the histidine residues (which have imidazole as their functional group) in the conserved clusters play a critical role in Δ⁶-desaturase activity, possibly by providing a di-iron catalytic center. In our previous study, this enzyme was expressed in Escherichia coli. The results reveal that the enzyme can function only in the presence of an exogenous cofactor, ferredoxin, provided in vitro. This evidence suggests that baker’s yeast has a cofactor that can complement ferredoxin, thought to act as an electron donor for the Δ⁶ desaturation in cyanobacteria, including Spirulina. The electron donor of the Spirulina-Δ⁶ desaturation in yeast is more likely to be cytochrome b₅, which is absent in E. coli. This means that the enzyme expressed in S. cerevisiae can catalyze the biosynthesis of the product, GLA, in vivo.     

Enhancement of proliferation and differentiation in bone marrow hematopoietic cells by Spirulina (Arthrospira) platensis in mice

This study evaluates whether Spirulina, including its components such as phycocyanin, enhances or sustains immune functions by promoting immune competent-cell proliferation or differentiation. The effects of Spirulina of a hot-water extract (SpHW), phycocyanin (Phyc), and cell-wall component extract (SpCW) on proliferation of bone marrow cells and induction of colony-forming activity in mice were investigated. The Spirulina extracts, SpHW, Phyc, and SpCW, enhanced proliferation of bone-marrow cells and induced colony-forming activity in the spleen-cell culture supernatant. Granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin-3 (IL-3) were detected in the culture supernatant of the spleen cells stimulated with the Spirulina extracts. Bone marrow-cell colony formation in soft-agar assay was also significantly induced by the blood samples and the culture supernatants of the spleen and Peyer's patch cells of the mice which ingested Spirulina extracts orally for 5 weeks in in vivo study. Ratios of neutrophils and lymphocytes in the peripheral blood and bone marrow, consequently, increased in the mice. Spirulina may have potential therapeutic benefits for improvement of weakened immune functions caused by, for example, the use of anticancer drugs.     

高沥水性钝顶螺旋藻新品系的选育及超微结构与RAPD分析

[目的]选育高沥水性的螺旋藻新品系,显著降低藻粉生产中干燥的能耗。[方法]以用于工厂化培植的钝顶螺旋藻ZJU0115为出发品系,用组织匀浆-离心沉降法制得其原生质球,并先以0.6%EMS处理30 min再用2.4 kGy的~(60)Coγ射线辐照,经含0.02%黄原胶(xanthan gum)的Zarrouk's培养液筛选、藻丝单体分离培养、藻泥持水率和胞外多糖(EPS)等检测及生产培植试验。[结果]获得了一株产量、蛋白质和多糖含量与ZJU0115相当,而藻泥持水率和EPS含量分别下降5.9%和29.7%的突变体,命名为ZJU0115(HD)。超微结构与随机扩增多态性DNA标记(random amplified polymorphic DNA,RAPD)分析结果显示,与其亲本ZJU0115相比,ZJU0115(HD)藻丝表面更光滑,可能为酸性多糖的乳白状粘附物也更少;ZJU0115(HD)细胞内的多磷酸盐颗粒显著变小且呈弥散状;基因组DNA在随机引物S90的扩增产物中显示出多态性差异。[结论]ZJU0115(HD)在工厂化培植中生产性状好、高沥水性能稳定,藻泥的干燥能耗降低了近50%,它的育成与应用,有助于推进当前螺旋藻产业迈向高效、节能、绿色、环保发展的新阶段。     

Germination-associated events and the desiccation sensitivity of recalcitrant seeds — a study on three unrelated species

The storage behaviour of recalcitrant seeds was assessed using three diverse species: a gymnosperm, Araucaria angustifolia (Bert.) O. Kuntze; a herbaceous monocotyledon, Scadoxus membranaceus (Bak.) Friis Nordal; and a woody dicotyledon, Landolphia kirkii Dyer. Seeds were stored under conditions of high relative humidities that maintained seed moisture content and under low relative humidities that caused drying. At regular intervals moisture content was determined, germinability assessed and the ultrastructure of radicle meristem cells examined. Under storage at high relative humidity, seed moisture content was maintained at the original level and subcellular germination events were initiated in the short-term. Such seeds showed enhanced rates of germination when removed from storage and planted. Long-term storage under these conditions resulted in the initiation of subcellular damage which intensified with time and ultimately resulted in the loss of viability. The rate at which germination events proceeded varied among the three species, and could be directly correlated with the period of viability retention under humid storage conditions. Storage under desiccating conditions resulted in subcellular damage and rapid loss of viability. The rate at which the seeds dried varied among the three species. The proportion of water loss tolerated by the different species before loss of viability, correlated with the rate of drying. The storage behaviour of the seeds of these three species is discussed in terms of a previously described model.     

<Emphasis Type="Italic">Spirulina</Emphasis> (<Emphasis Type="Italic">Arthrospira</Emphasis>) industry in Inner Mongolia of China: current status and prospects

This paper outlines an investigation on current situation of Spirulina (Arthrospira) industry in Inner Mongolia, an internal region of China with temperate continental climate. More than 20 Spirulina plants have been established in Inner Mongolia since 2001, most of which are located at Wulan Town in the Ordos Plateau. By the end of 2009, the total annual production of Spirulina in the Ordos Plateau surpassed 700 t (dw), which account for ca. 80% of the total productivity of Inner Mongolia, and ca. 20% of China. Besides abundant solar radiation and enough freshwater favorable for Spirulina production, the three technical strategies contribute to the prosperity and success of Spirulina industry in the region: (1) reducing the cost or investment by overall advantages of rich local natural resources with low cost for Spirulina production, such as alkaline lakes, coal, electricity, and sandy land; (2) controlling the culture temperature and to avoid contamination by building plastic greenhouses on raceway ponds, (3) reducing investment by simplifying the construction of the ponds and the greenhouses. As the result, the growth period of Spirulina has been prolonged from about 120 to about 165 days, the cost of Spirulina has decreased by 25–30%, and the quality of products has been enhanced substantially. Inner Mongolia is expected to become the largest base for Spirulina production not only in China, but also in the world in the near future.     

Current knowledge on potential health benefits of Spirulina

Spirulina is a microscopic filamentous alga that is rich in proteins, vitamins, essential amino acids, minerals and essential fatty acids like γ-linolenic acid (GLA). It is produced commercially and sold as a food supplement in health food stores around the world. Up to very recently, the interest in Spirulina was mainly in its nutritive value. Currently, however, numerous people are looking into the possible therapeutic effects of Spirulina. Many pre-clinical studies and a few clinical studies suggest several therapeutic effects ranging from reduction of cholesterol and cancer to enhancing the immune system, increasing intestinal lactobacilli, reducing nephrotoxicity by heavy metals and drugs and radiation protection. This paper presents a critical review of some published and unpublished data on therapeutic effects of Spirulina.     

Metabolic flux distribution for γ-linolenic acid synthetic pathways in<Emphasis Type="Italic">Spirulina platensis</Emphasis>

Spirulina produces γ-linolenic acid (GLA), an important pharmaceutical substance, in a relatively low level compared with fungi and plants, prompting more research to improve its GLA yield. In this study, metabolic flux analysis was applied to determine the cellular metabolic flux distributions in the GLA synthetic pathways of twoSpirulina strains, wild type BP and a high-GLA producing mutant Z19/2. Simplified pathways involving the GLA synthesis ofS. platensis formulated comprise of photosynthesis, gluconeogenesis, the pentose phosphate pathway, the anaplerotic pathway, the tricarboxylic cycle, the GLA synthesis pathway, and the biomass synthesis pathway. A stoichiometric model reflecting these pathways contains 17 intermediates and 22 reactions. Three fluxes—the bicarbonate (C-source) uptake rate, the specific growth rate, and the GLA synthesis rate—were measured and the remaining fluxes were calculated using linear optimization. The calculation showed that the flux through the reaction converting acetyl-CoA into malonyl-CoA in the mutant strain was nearly three times higher than that in the wild-type strain. This finding implies that this reaction is rate controlling. This suggestion was supported by experiments, in which the stimulating factors for this reaction (NADPH and MgCl₂) were added into the culture medium, resulting in an increased GLA-synthesis rate in the wild type strain.     

Revealing differentially expressed proteins in two morphological forms of <Emphasis Type="Italic">Spirulina platensis</Emphasis> by proteomic analysis

Spirulina is distinguished from other cyanobacteria by its spiral morphology; however, this cyanobacterium has frequently been observed with a linear morphology in laboratory and industrial conditions. In our laboratory conditions, the simultaneously presence of the linear and spiral forms has also been observed. In the present study, the two forms of S. platensis C1 were separated and grown as axenic cultures in order to study the proteins that were differentially expressed in the soluble and insoluble protein fractions of the spiral and the linear forms. Two dimensional-differential gel electrophoresis (2D-DIGE) was performed to separate differentially expressed proteins that were subsequently identified by mass spectrometry. The differentially expressed proteins suggested two points. First, the morphological change is possibly induced by various environmental stresses such as oxygen level, carbon dioxide level, nutrient availability, and light. Second, the change of cell-shape might be a result of the change in a cell shape determination mechanism. Thus, this study is the first to show evidence at the protein level that may explain this morphological transformation in Spirulina.     

Evaluation of drying methods on nutritional constituents and antioxidant activities of Chlorella vulgaris cultivated in an outdoor open raceway pond

Chlorella vulgaris is known for its protein, growth factor, and nutritional constituents. Chlorella vulgaris was cultivated in a 1000-L outdoor open raceway pond with a maximum volumetric productivity of 130 mg L^-1 day^-1. The harvested biomass was dried through different methods, viz., sun drying (30 °C), oven drying (60 °C), lyophilization (?110 °C), drum drying (120 °C), and spray drying (100–150 °C). The effect of the drying method on proximate composition, pigments (chlorophyll, carotenoids), bioactive compounds (total phenolic content, flavonoid content), vitamin B₁₂, antioxidant properties (ferric reducing antioxidant power, DPPH, and total antioxidant activity), and the color quality of C. vulgaris biomass was evaluated. Surface characterization by scanning electron microscopy (SEM) and functional group characterization through Fourier transform infrared spectroscopy were also performed. The biomass dried through lyophilization and sun drying retained maximum bioactive compounds and antioxidant activities. In contrast, drum drying resulted in a loss of nutrients, viz., protein (up to 44%), lipid (up to 41%), vitamin B₁₂ (up to 40%), total phenolic content (> 50%), total flavonoid content (> 50%), and antioxidant activity (> 50%). Oven drying led to a loss of 30% in total flavonoid content and 17% in ferric reducing antioxidant power. SEM showed the destruction of cell wall integrity in the drum-dried sample and porous structure in the spray-dried sample. This study suggests that drying methods affect the nutrients and bioactive compounds of C. vulgaris biomass, and therefore a drying method should be selected carefully depending on the end use of the biomass.

     

Role of light and photosynthesis on the acclimation process of the cyanobacteriumSpirulina platensis to salinity stress

The response ofSpirulina platensis cells to salinity stress was studied. Once adapted to the higher osmoticum, photosynthetic parameters such as the maximum rate of photosynthesis under saturating irradiance (P_max) and the initial slope of the P-I curve () are reduced by 15% and 25% in 0.5 M NaCl grown cells, respectively. Salt-adapted cells have a modified biochemical composition; reduced protein and chlorophyll content, and an increased level of carbohydrates. The reduction in the photosynthetic capacity of the salt-adaptedSpirulina cells reflects a lower ability to utilize light energy and results in an increase in the susceptibility of the stressed cells to photoinhibition. This conclusion is supported by the finding that cultures exposed to salt stress show not only a decrease in growth rate (), but lose the ability to respond to increased irradiance with an increase in growth. The use of variable fluorescence as a fast and reliable measurement to follow the changes in PSII of salt-stressesSpirulina cells enables following the early events of salinity shock. It indicates that as soon as the cells are exposed to salt, a protection mechanism is induced. This mechanism does not require any protein synthesis and may take place even in the dark, though at somewhat reduced effectiveness. The significance of the result in providing a better understanding of the interaction between two environmental stresses — light and salinity — and their application in the outdoor mass cultivation ofSpirulina are discussed.Author for correspondence