Isolation, characterization and structural determination of a unique type of arabinogalactan from an immunostimulatory extract of Chlorella pyrenoidosa

An arabinogalactan was isolated from a hot water extract of freeze-dried cells of the green microalga, Chlorella pyrenoidosa. This hot water extract is a proprietary immunomodulator, with the trademark Respondintrade mark (ONC-107). The arabinogalactan was recovered from the ethanol-soluble fraction of the supernatant resulting from a process that involved controlled ethanol precipitation followed by size exclusion chromatography on Sephadex G-100, then Cetavlon precipitation. Sugar analyses, GC-MS data for (S)-2-octyl glycosides, and 1D and 2D NMR experiments established unambiguously that the repeating unit was -->2)-alpha-L-Araf-(1-->3)-[alpha-L-Araf-(1-->4)]-beta-D-Galp-(1-->. This structure does not fit into any of the known classes of arabinogalactans. SEC/MALS experiments gave a molecular mass for the arabinogalactan isolated as 47 +/- 4 kDa but the original structure was probably larger.     

定向驯化对异养蛋白核小球藻硒的耐受性及富集能力的影响

硒(Se)是生物体必不可少的微量元素,硒缺乏会导致人产生克山病、大骨节病等疾病,缺硒也会给畜牧业带来巨大损失。目前的补硒产品存在硒含量和生物利用度低、安全性差等问题,而通过小球藻培养可获得生物利用度高、安全性好的有机硒,因此是非常有应用前景的补硒产品。首先,为了获得对硒的耐受性和富集能力更强的藻种,研究通过定向驯化的方式逐步提高培养基中Na₂SeO₃浓度来驯化蛋白核小球藻,并对驯化时间和驯化过程中Na₂SeO₃的浓度梯度进行了优化。结果表明,驯化后的藻种对硒的耐受性和富集能力明显提高。在5L发酵罐中,驯化后的藻株可以耐受40mg/L的Na₂SeO₃,胞内有机硒合成速率提高了175.6%。之后,在5L发酵罐中进一步优化了硒的补加方式,在异养培养过程中分批补料添加40mg/LNa₂SeO₃时,最终获得的蛋白核小球藻细胞干重达106.4g/L,有机硒含量为1227mg/kg,有机硒合成速率为1.36mg/(L·h)。研究结果与已有蛋白核小球藻异养富硒文献报道的最高细胞密度75g/L和最高有机硒含量560mg/kg相比分别提高了41.9%和119.1%。上述结果表明,通过定向驯化的方法,可大大提高蛋白核小球藻对硒的耐受性和富集能力。     

淡水小球藻分离培养及其对水体辛硫磷的降解功能研究

为了研究微藻对水体有机磷农药的降解和积累效果,从自然水体中富集纯化得到一株蛋白核小球藻(Chlorella pyrenoidosa),以该藻细胞为研究对象,以水产养殖中常用的有机磷杀虫剂辛硫磷为实验药物,通过高效液相色谱法研究了蛋白核小球藻对辛硫磷的富集和降解效果。结果显示,富集纯化藻细胞通过显微镜检与分子生物学检测鉴定为蛋白核小球藻,命名为MH-1。急毒性试验表明,辛硫磷浓度在高于2.0μg/mL时才会对MH-1生长产生显著抑制作用,表现在叶绿素含量与生物量显著下降。在2.0μg/mL及以下浓度辛硫磷存在的水体中,MH-1具有降解辛硫磷的功能,在0.2μg/mL和2.0μg/mL浓度组中,5 d内的辛硫磷净降解率分别为49.66%和39.61%,日平均降解速率分别为0.02μg/mL和0.38μg/mL。MH-1对辛硫磷也有一定的富集能力,72 h内MH-1对辛硫磷的富集呈现先升高后降低的趋势,120 h时MH-1对0.2μg/mL和2.0μg/mL的辛硫磷富集量分别为0.16 mg/g FW(Fresh weight, FW)和0.25 mg/g FW。结果表明,MH-1对辛硫磷具...     

Fed-batch fermentation of Tuber melanosporum for the hyperproduction of mycelia and bioactive Tuber polysaccharides

For the first time, a fed-batch fermentation process of Tuber melanosporum was developed for the efficient production of bioactive mycelia and Tuber polysaccharides. Each 1.67 g/L of peptone and 8.33 g/L of yeast extract were added on day 3, 6, and 9, respectively, and sucrose was fed to maintain its concentration around 35–5 g/L when its residual level decreased to 10–5 g/L. Then, the maximal biomass, the production of extracellular polysaccharides (EPS) and intracellular polysaccharides (IPS) reached 53.72 ± 2.57 g DW/L, 7.09 ± 0.62 and 4.43 ± 0.21 g/L, respectively. Compared with the batch culture conducted in the enriched medium, the biomass, the production of EPS and IPS were enhanced by 55.8%, 222.3% and 103.2%, respectively. Not only the cell density but also the production of EPS and IPS were the highest ever reported in truffle fermentation, and the biomass was also the highest as ever reported in mushroom fermentation.     

Enzyme assisted extraction of polysaccharides from the fruit of Cornus officinalis

Process of enzyme assisted extraction (EAE) of polysaccharides from Cornus officinalis was optimized by response surface methodology (RSM). The influence of four different factors on the yield of C. officinalis polysaccharides (COP) was studied. Results showed that the optimal conditions were compound enzyme amount of 2.15%, extraction pH of 4.2, extraction temperature of 55 °C and extraction time of 97 min. Under these conditions, the COP yield was 9.29 ± 0.31%, which was well in agreement with the value predicted by the model. The three methods, EAE, hot water extraction (HWE), ultrasound-assisted extraction (UAE) for extracting COP by RSM were further compared. Results showed that EAE had the largest yield of polysaccharides with lower equipment cost.     

Amoebae and algae can prolong the survival of Campylobacter species in co-culture

Several species of free-living amoebae can cause disease in humans. However, in addition to the direct pathogenicity of e.g. Acanthamoebae and Naegleria species, they are recognized as environmental hosts, indirectly involved in the epidemiology of many pathogenic bacteria. Although several studies have demonstrated intracellular survival of many different bacteria in these species, the extent of such interactions as well as the implications for the epidemiology of the bacterial species involved, are largely unknown and probably underestimated. In this study, we evaluated eight different unicellular eukaryotic organisms, for their potential to serve as environmental hosts for Campylobacter species. These organisms include four amoebozoas (Acanthamoeba polyphaga, Acanthamoeba castellanii, Acanthamoeba rhysodes and Hartmanella vermiformis), one alveolate (Tetrahymena pyriformis), one stramenopile (Dinobryon sertularia), one eugoenozoa (Euglena gracilis) and one heterolobosea (Naegleria americana). Campylobacter spp. including Campylobacter jejuni, Campylobacter coli and Campylobacter lari are the most common cause of gastroenteritis in the western world. Survival and replication of these three species as well as Campylobacter hyointestinalis were assessed in co-cultures with the eukaryotic organisms. Campylobacter spp. generally survived longer in co-cultures, compared to when incubated in the corresponding growth media. The eukaryotic species that best promoted bacterial survival was the golden algae D. sertularia. Three species of amoebozoas, of the genus Acanthamoeba promoted both prolonged survival and replication of Campylobacter spp. The high abundance in lakes, ponds and water distribution networks of these organisms indicate that they might have a role in the epidemiology of campylobacteriosis, possibly contributing to survival and dissemination of these intestinal pathogens to humans and other animals. The results suggest that not only C. jejuni, but a variety of Campylobacter spp. can interact with different eukaryotic unicellular organisms.     

Differential lipid and fatty acid profiles of photoautotrophic and heterotrophic Chlorella zofingiensis: Assessment of algal oils for biodiesel production

The objective of this study was to document and compare the lipid class and fatty acid composition of the green microalga Chlorella zofingiensis cultivated under photoautotrophic and heterotrophic conditions. Compared with photoautotrophic cells, a 900% increase in lipid yield was achieved in heterotrophic cells fed with 30 g L⁻¹ of glucose. Furthermore heterotrophic cells accumulated predominantly neutral lipids (NL) that accounted for 79.5% of total lipids with 88.7% being triacylglycerol (TAG); whereas photoautotrophic cells contained mainly the membrane lipids glycolipids (GL) and phospholipids (PL). Together with the much higher content of oleic acid (C18:1) (35.2% of total fatty acids), oils from heterotrophic C. zofingiensis appear to be more feasible for biodiesel production. Our study highlights the possibility of using heterotrophic algae for producing high quality biodiesel.     

自养和兼养条件下蛋白核小球藻昼夜节律的响应

【目的】研究自养和兼养两种培养方式对蛋白核小球藻(Chlorella pyrenoidosa)生长、细胞分裂和生化组分积累的影响,探讨人工培养蛋白核小球藻的昼夜节律响应机制和优化技术。【方法】小球藻自养培养采用BG11培养基,兼养培养基在BG11培养基中添加4种不同浓度(1、5、10、20 g/L)的葡萄糖,培养周期为10 d。血球板计数法测定藻细胞浓度,干重法测定藻细胞生物量。显微观察藻细胞大小和分裂情况。脂染色法测定小球藻总脂的含量,藻细胞的叶绿素、蛋白和淀粉分别采用甲醇、氢氧化钠、硝酸钙浸提后通过紫外分光光度法定量测定。【结果】葡萄糖兼养培养对蛋白核小球藻具有显著的促生长效应,最适浓度为10 g/L。10 d收获时,兼养组(10 g/L葡萄糖)藻细胞浓度和干重分别是自养组的2.57倍和6.73倍。分析一昼夜中的藻细胞增殖规律可知,第2天和第5天时自养组中增殖的新生子细胞约有76.00%在黑暗期分裂产生,而兼养组中第2天和第5天光照期的新细胞增殖量占比分别达到40.90%和67.50%。一昼夜内藻细胞大小的迁移动态监测表明,第2天自养组藻细胞的体积变化静息期为8 h,兼养组只有4 h;第5天两组藻细胞大小迁移动态的昼夜节律明显,但兼养组黑暗结束后较大细胞(D6μm)占比显著高于自养组。第8天时,兼养组藻细胞已处于稳定期,总脂和蛋白含量均显著高于自养组,藻细胞总脂和色素含量在一昼夜中相对稳定,但蛋白和淀粉含量分别在光照8 h和12 h左右达到峰值。从第2天开始,对兼养组细胞每天进行2 h光延长,收获时藻细胞浓度和干重分别比对照组提高13%和11%。【结论】葡萄糖兼养培养能大幅提高蛋白核小球藻的生物量。蛋白核小球藻生长增殖与生化组分积累均受昼夜节律调控,自养条件下藻细胞以光照期生长黑暗期增殖为主。兼养培养提高藻细胞生物量的机制在于缩短藻细胞生长静息期,在昼夜节律中加速藻细胞生长并显著提高通过细胞周期检查点的细胞比例,光照期效应尤其明显。藻细胞蛋白和淀粉含量昼夜节律明显,最佳收获时间分别在光照8 h和12 h后。     

Isolation and structural characterization of a polysaccharide FCAP1 from the fruit of Cornus officinalis

A water-soluble polysaccharide, FCAP1, was isolated from an alkaline extract from the fruits of Cornus officinalis. Its molecular weight was 34.5 kDa. Monosaccharide composition analysis revealed that it was composed of fucose, arabinose, xylose, mannose, glucose, and galactose in a molar ratio of 0.29:0.19:1.74:1:3.30:1.10. On the basis of partial acid hydrolysis and methylation analysis, FCAP1 was shown to be a highly branched polysaccharide with a backbone of β-(1→4)-linked-glucose partially substituted at the O-6 position with xylopyranose residues. The branches were composed of (1→3)-linked-Ara, (1→4)-linked-Man, (1→4,6)-linked-Man, (1→4)-linked-Glc, and (1→2)-linked-Gal. Arabinose, fucose, and galactose were located at the terminal of the branches. The structure was further elucidated by a specific enzymatic degradation with an endo-β-(1→4)-glucanase and MALDI-TOF-MS analysis. Oligosaccharides generated from FCAP1 indicated that FCAP1 contained XXXG-type and XXG-type xyloglucan fragments.     

Isolation and characterization of a heteropolysaccharide from the corm of Amorphophallus campanulatus

A water-soluble polysaccharide isolated from the aqueous extract of the corm of Amorphophallus campanulatus was found to contain d-galactose, d-glucose, 4-O-acyl-d-methyl galacturonate, and l-arabinose in a molar ratio 2:1:1:1. Structural investigation of the polysaccharide was carried out using acid hydrolysis, methylation analysis, periodate oxidation study, and NMR studies (¹H, ¹³C, DQF-COSY, TOCSY, NOESY, ROESY, HMQC, and HMBC). On the basis of the above-mentioned experiments the structure of the repeating unit of the polysaccharide was established as:This molecule showed splenocyte activation.     

Isolation of anti-Candida albicans compounds from Markhamia obtusifolia (Baker) Sprague (Bignoniaceae)

An increase in clinical cases of Candidiosis globally as well as fungal resistance to drugs prompted the search for novel anti-Candida albicans agents from plant sources. Leaf extracts of Markhamia obtusifolia were screened for activity against C. albicans in vitro. An acetone extract obtained following serial exhaustive extraction contained mainly the active components with at least four active zones on the bioautogram. Bioassay guided fractionation of this extract led to the isolation of three compounds which inhibited the growth of three C. albicans strains. Based on spectroscopy studies (NMR and MS), the compounds were identified as 3β-hydroxyurs-12-en-28-oic acid, ursolic acid (1) 3β, 19α-dihydroxyurs-12-en-28-oic acid, pomolic acid (2) and 2β, 3β, 19α -trihydroxy-urs-12-en-28-oic acid, 2-epi-tormentic acid (3). The most active compound was 3β, 19α-dihydroxy-12-ursen-28-oic acid (2) with a minimum inhibitory concentration (MIC) value of 12.5 µg/mL for C. albicans isolated from dog and 25.0 µg/mL for C. albicans from cat and ATCC 90028 at 24 h following incubation. However, at 48 h of incubation MICs were > 400 µg/mL for all the three compounds isolated. This study indicated that M. obtusifolia could be a potential source of active principles against C. albicans.     

Biological hydrogen production by the algal biomass Chlorella vulgaris MSU 01 strain isolated from pond sediment

Chlorella vulgaris MSU 01 strain isolated from the sediment of the pond is able to produce molecular hydrogen in a clean way. To relate the dynamic coupling between the cultural conditions and biological responses, an original lab scale set up has been developed for hydrogen production. Different sources like mannitol, glucose, alanine, citric acid, aspartic acid, l-alanine, l-cysteine, sodium succinate and sodium pyruvate were used for algal media optimization. Corn stalk, from 1 to 5 g/L was tested for the effective algal growth and hydrogen production. The cell concentration of 1.6-19 g/L dry cell weight (DCW) was found at the 10th day. The kinetic parameters involved in the hydrogen production at 4 g/L corn stalk using the algal inoculum (50 mL) in the bioreactor volume (500 mL) was found to be with the hydrogen production potential (P_s) of 7.784 mL and production yield of (P_r) 5.534 mL respectively. The growth profile of the algal biomass at the above mentioned condition expressed the logistic model with R² 0.9988. The final pH of the broth was increased from 7.0 to 8.5-8.7. The anaerobic fermentation by C. vulgaris MSU 01 strain involved in the conversion process of complex carbon source has increased the H₂ evolution rate and higher butyrate concentration in the fermentate.     

The effects of algae species and densities on the population growth of the marine rotifer, Colurella dicentra

Colurella dicentra clones isolated from bay water in the Mississippi Gulf Coast were cultured with artificial seawater. Experiments were conducted to determine the effects of six algae species (Nannochloropsis oculata, Tetraselmis chuii, Chaetoceros gracilis, Rhodomonas salina, Isochrysis galbana, and Prorocentrum micans), six C. gracilis densities, and six N. oculata densities (25,000, 50,000, 100,000, 250,000, 500,000, and 1,000,000 cells ml^− 1) on C. dicentra population growth. Algae type influenced rotifer production (p < 0.0001). C. gracilis treatment (9120 ± 3351SD) produced the highest number of rotifers followed by N. oculata (5760 ±2232SD). P. micans had the lowest number of rotifers, although not significantly different from numbers in T. chuii, R. salina, and I. galbana treatments (p > 0.05).The population growth rate (r) varied with algae species treatment. The highest values were recorded for C. gracilis treatment (0.22 to 0.26 d^− 1), followed by N. oculata (0.21 to 0.24 d^− 1), and the lowest for P. micans (− 0.19 to 0.14 d^− 1). C. gracilis and N. oculata densities had significant effects (p < 0.0001) on C. dicentra population growth. The highest rotifer production was recorded at a C. gracilis density of 100,000 cells ml^− 1, followed by 250,000 cells ml^− 1 and 50,000 cells ml^− 1. Algae densities of 500,000 cells ml^− 1 and above produced the lowest rotifer numbers. Population growth rate (r) varied with C. gracilis densities. The highest values were observed for C. gracilis concentrations of 100,000 cells ml^− 1 (0.17 to 0.19 d^− 1), and the lowest for concentrations of 500,000 cells ml^− 1 and above (− 0.19 to 0.09 d^− 1). The 100,000 cells ml^− 1N. oculata density gave the highest rotifer production followed by 50,000, 250,000, 25,000, and 500,000 cells ml^− 1. Algae densities of 1,000,000 cells ml^− 1 produced the lowest rotifer numbers. Population growth rate (r) varied with N. oculata densities, with the highest values obtained for algae densities of 100,000 cells ml^− 1 (0.35 to 0.40 d^− 1), and the lowest for concentrations of 1,000,000 cells ml^− 1 (0.05 to 0.012 d^− 1). This is the first report of C. dicentra in Mississippi Coastal waters, and perhaps the smallest marine rotifer species (93 by 49 μm) ever cultured successfully.     

Cytotoxic dihydrobenzophenanthridine alkaloids from the roots of Macleaya microcarpa

Five dihydrobenzophenanthridine alkaloids, named as maclekarpine A-E, were isolated from the roots of Macleaya microcarpa, together with 10 known benzophenanthridine/dihydrobenzophenanthridine derivatives and a known amide. Their structures were determined on the basis of spectroscopic methods. The relative configuration of maclekarpine A was solved by X-ray single-crystal diffraction, which also contributed to the establishment of the relative configuration of maclekarpine B and C, as well as the assignment of the absolute configuration of maclekarpine A-C with aid of their CD curves. Cytotoxicity of the isolated compounds against five human tumor cell-lines was evaluated, with some having activity with IC₅₀ values ranging from 0.1 to 3.5 μM.     

Sulfated polysaccharides from Laminaria angustata: Structural features and in vitro antiviral activities

Sulfated polysaccharides potently inhibit the infectivity of herpes simplex virus (HSV) in cultured cells. In this study, we have analyzed sulfated xylogalactofucan and alginic acid containing fractions generated from Laminaria angustata, a marine alga. The xylogalactofucan that has apparent molecular mass of 56 ± 5 kDa and unusually low sulfate content contains, inter alia, 1,3-, 1,4- and 1,2-linked fucopyranosyl residues. The algin (molecular mass: 32 ± 5 kDa) contains gulo- (55.5%) and mannuronic (44.5%) acid residues. Introduction of sulfate groups enhanced the macromolecules capability to inhibit the infection of cells by HSV-1. The 50% inhibitory concentration (IC₅₀) values of these macromolecules against HSV-1 were in the range of 0.2-25 μg ml⁻¹ and they lacked cytotoxicity at concentrations up to 1000 μg ml⁻¹. The sulfate content appeared to be an important hallmark of anti-HSV-1 activity. Our results suggest the feasibility of inhibiting HSV attachment to cells by direct interaction of polysaccharides with viral particles.     

Physiology and cryosensitivity of coral endosymbiotic algae (Symbiodinium)

Coral throughout the world are under threat. To save coral via cryopreservation methods, the Symbiodinium algae that live within many coral cells must also be considered. Coral juvenile must often take up these important cells from their surrounding water and when adult coral bleach, they lose their endosymbiotic algae and will die if they are not regained. The focus of this paper was to understand some of the cryo-physiology of the endosymbiotic algae, Symbiodinium, living within three species of Hawaiian coral, Fungia scutaria, Porites compressa and Pocillopora damicornis in Kaneohe Bay, Hawaii. Although cryopreservation of algae is common, the successful cryopreservation of these important coral endosymbionts is not common, and these species are often maintained in live serial cultures within stock centers worldwide. Freshly-extracted Symbiodinium were exposed to cryobiologically appropriate physiological stresses and their viability assessed with a Pulse Amplitude Fluorometer. Stresses included sensitivity to chilling temperatures, osmotic stress, and toxic effects of various concentrations and types of cryoprotectants (i.e., dimethyl sulfoxide, propylene glycol, glycerol and methanol). To determine the water and cryoprotectant permeabilities of Symbiodinium, uptake of radio-labeled glycerol and heavy water (D₂O) were measured. The three different Symbiodinium subtypes studied demonstrated remarkable similarities in their morphology, sensitivity to cryoprotectants and permeability characteristics; however, they differed greatly in their sensitivity to hypo- and hyposmotic challenges and sensitivity to chilling, suggesting that standard slow freezing cryopreservation may not work well for all Symbiodinium. An appendix describes our H₂O:D₂O water exchange experiments and compares the diffusionally determined permeability with the two parameter model osmotic permeability.