Identification of fungi for sweet whey permeate utilization and eicosapentaenoic acid production

Summary Eighteen selected organisms of theEumycota division of the fungi kingdom were examined for eicosapentaenoic acid production and utilization of sweet whey permeate. The organisms belong to the subdivisionsMastigomycotina, Zygomycotina, Ascomycotina andDeuteromycotina. Seven organisms were initially identified as lactose utilizers (the predominant sugar in sweet whey permeate_ and eicosapentaenoic acid (EPA) producers. Utilization of lactose was demonstrated and EPA production was confirmed for four organisms, all of the subdivisionMastigomycotina. Growth studies showed thatP. ultimum had the best potential for future work.     

Effect of cultural conditions on production of eicosapentaenoic acid byPythium irregulare

Summary The effect of culture conditions upon lipid content and fatty acid composition of mycelia ofPythium irregulare was investigated with particular attention to increasing the yield of 5,8,11,14,17-eicosapentaenoic acid (205; –3) (EPA). All experiments were done by shake flask culture using a yeast extract + malt extract medium. The maximum growth rate was obtained at 25°C, but maximum EPA production was obtained at 12°C. The highest EPA production was 76.5 g EPA/ml 13 days fermentation at 12°C. Addition of glucose during fermentation increased the yield considerably. The highest yield was 112 g/ml, obtained at 13 days fermentation with spiking on day 11. Fermentation time could be shortened by initial incubation at 25°C for 2 days, followed by incubation at 12°C for 6 days. The culture also produced arachidonic acid and other -6 polyunsaturated fatty acids. EPA production was also obtained with lactose or sweet whey permeate, a by-product of cheese manufacture that contains lactose as the main carbohydrate.The mention of firm names or trade products does not imply that they are endorsed or recommended by the U.S. Department of Agriculture over other firms or similar products not mentioned.     

Statistical optimization of critical medium components for lipase production from Yarrowia lipolytica (MTCC 35)

Statistical optimization is an effective technique for the investigation of complex processes with minimal number of experimental runs. In this study, statistical approach was used to study the optimization of media components for lipase production from Yarrowia lipolytica MTCC 35. Mahua cake, glucose, MnCl₂ and KH₂PO₄ were screened to be the most significant variables among the nine medium variables that were tested to determine influence on lipase production by Plackett–Burman design. Central Composite Design was used for further optimization of these screened variables for enhanced lipase production. The determination coefficient (R²) value of 0.922 showed that the regression models adequately explain the data variation and represent the actual relationships between the variables and response. The optimum values of investigated variables for the maximum lipase production were 6.0% Mahua cake, 2.0% glucose, 0.2% MnCl₂ and 0.2% KH₂PO₄. The maximum lipase production (9.40 U mL^?1) was obtained under optimal condition.     

Docosapentaenoic acid (22:5n-3): A review of its biological effects

This article summarizes the current knowledge available on metabolism and the biological effects of n-3 docosapentaenoic acid (DPA). n-3 DPA has not been extensively studied because of the limited availability of the pure compound. n-3 DPA is an elongated metabolite of EPA and is an intermediary product between EPA and DHA. The literature on n-3 DPA is limited, however the available data suggests it has beneficial health effects. In vitro n-3 DPA is retro-converted back to EPA, however it does not appear to be readily metabolised to DHA. In vivo studies have shown limited conversion of n-3 DPA to DHA, mainly in liver, but in addition retro-conversion to EPA is evident in a number of tissues. n-3 DPA can be metabolised by lipoxygenase, in platelets, to form ll-hydroxy-7,9,13,16,19- and 14-hydroxy-7,10,12,16,19-DPA. It has also been reported that n-3 DPA is effective (more so than EPA and DHA) in inhibition of aggregation in platelets obtained from rabbit blood. In addition, there is evidence that n-3 DPA possesses 10-fold greater endothelial cell migration ability than EPA, which is important in wound-healing processes. An in vivo study has reported that n-3 DPA reduces the fatty acid synthase and malic enzyme activity levels in n-3 DPA-supplemented mice and these effects were stronger than the EPA-supplemented mice. Another recent in vivo study has reported that n-3 DPA may have a role in attenuating age-related decrease in spatial learning and long-term potentiation. However, more research remains to be done to further investigate the biological effects of this n-3 VLCPUFA.     

Inhibitory effect of conjugated eicosapentaenoic acid on human DNA topoisomerases I and II

DNA topoisomerases (topos) and DNA polymerases (pols) are involved in many aspects of DNA metabolism such as replication reactions. We reported previously that long chain unsaturated fatty acids such as polyunsaturated fatty acids (PUFA) (i.e., eicosapentaenoic acid (EPA) and docosahexanoic acid (DHA)) inhibited the activities of eukaryotic pols in vitro. In the present study, we found that PUFA also inhibited human topos I and II activities, and the inhibitory effect of conjugated fatty acids converted from EPA and DHA (cEPA and cDHA) on pols and topos was stronger than that of normal EPA and DHA. cEPA and cDHA inhibited the activities of mammalian pols and human topos, but did not affect the activities of plant and prokaryotic pols or other DNA metabolic enzymes tested. cEPA was a stronger inhibitor than cDHA with IC(50) values for mammalian pols and human topos of 11.0-31.8 and 0.5-2.5 microM, respectively. Therefore, the inhibitory effect of cEPA on topos was stronger than that on pols. Preincubation analysis suggested that cEPA directly bound both topos I and II, but did not bind or interact with substrate DNA. This is the first report that conjugated PUFA such as cEPA act as inhibitors of pols and topos. The results support the therapeutic potential of cEPA as a leading anti-cancer compound that poisons pols and topos.     

Enhanced heterologous production of eicosapentaenoic acid in Escherichia coli cells that co-express eicosapentaenoic acid biosynthesis pfa genes and foreign DNA fragments including a high-performance catalase gene, vktA

Cellular eicosapentaenoic acid (EPA) makes up approximately 3% of total fatty acids in Escherichia coli DH5α, a strain that carries EPA biosynthesis genes (pEPAΔ1). EPA was increased to 12% of total fatty acids when the host cell co-expressed the vector pGBM3::sa1(vktA), which carried the high-performance catalase gene, vktA. Where this vector was co-expressed, the transformant accumulated a large amount of VktA protein. However, the EPA production of cells carrying the vector, that included the insert lacking almost the entire vktA gene, was approximately 6%. This suggests that the retention of a large DNA insert in the vector and the accumulation of the resulting protein, but not the catalytic activity of VktA catalase, would potentially be able to increase the content of EPA.     

Effects of eicosapentaenoic acid and docosahexaenoic acid on anxiety-like behavior in socially isolated rats

Abstract

The effects of fish oil for improving mental health have been reported. The present study was undertaken to compare the effects of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on anxiety-like behavior using a rat model. Experimental diets enriched in EPA or DHA as glycerides were prepared. Rats were exposed to social isolation stress and fed the experimental diet for 14 days. The results of behavioral tests revealed that rats fed the EPA-enriched diet exhibited less anxiety-like behavior than rats fed the control or DHA-enriched diets. Furthermore, EPA suppressed anxiety-like behavior only in socially isolated rats. The increase in EPA contents in the brain phospholipid fraction by feeding EPA-enriched diet was more significant than that of DHA by feeding DHA-enriched diet. These results suggest that dietary EPA is more anxiolytic than DHA in rats exposed to social isolation stress and is effective in increasing EPA content in brain membranes.     

Eicosapentaenoic acid in the treatment of schizophrenia and depression: rationale and preliminary double-blind clinical trial results

It has been hypothesised that polyunsaturated fatty acids (PUFA) play an important role in the aetiology of schizophrenia and depression. Evidence supporting this hypothesis for schizophrenia includes abnormal brain phospholipid turnover shown by 31P Magnetic Resonance Spectroscopy, increased levels of phospholipase A2, reduced niacin skin flush response, abnormal electroretinogram, and reduced cell membrane levels of n-3 and n-6 PUFA. In depression, there is strong epidemiological evidence that fish consumption reduces risk of becoming depressed and evidence that cell membrane levels of n-3 PUFA are reduced. Four out of five placebo-controlled double- blind trials of eicosapentaenoic acid (EPA) in the treatment of schizophrenia have given positive findings. In depression, two placebo-controlled trials have shown a strong therapeutic effect of ethyl-EPA added to existing medication. The mode of action of EPA is currently not known, but recent evidence suggests that arachidonic acid (AA) if of particular importance in schizophrenia and that clinical improvement in schizophrenic patients using EPA treatment correlates with changes in AA.     

Optimisation of culture conditions for production of eicosapentaenoic acid byMortierella elongate NRRL 5513

Summary WhenMortierella elongata NRRL 5513 was cultured in shake flasks at 25°C, mycelial growth reached a stationary phase at 48 h but maximum eicosapentaenoic acid (EPA) production was observed at 6 days. When incubated at 11°C, EPA production also continued to rise during the stationary phase of growth, reaching a maximum after 10 days. An initial culture pH of 6.1 was found to be optimum for EPA production. The effect of temperature on EPA production was dependent on medium constituents. In glucose and linseed oil supplemented media, optimum temperature for EPA production was 11 and 15°C respectively. A maximum EPA yield of 0.61 g/l was obtained in linseed oil (2%), yeast extract (0.5%) supplemented basal medium. Maximum EPA content as a percentage of lipids (15.12%) was observed when the latter medium was supplemented with 0.25% urea.     

Identification of a cDNA encoding a novel C18-Δ polyunsaturated fatty acid-specific elongating activity from the docosahexaenoic acid (DHA)-producing microalga, Isochrysis galbana

Isochrysis galbana, a marine prymnesiophyte microalga, is rich in long chain polyunsaturated fatty acids such as docosahexaenoic acid (C22:6n-3, Δ^{4,7,10,13,16,19}). We used a polymerase chain reaction-based strategy to isolate a cDNA, designated IgASE1, encoding a polyunsaturated fatty acid-elongating activity from I. galbana. The coding region of 263 amino acids predicts a protein of 30 kDa that shares only limited homology to animal and fungal proteins with elongating activity. Functional analysis of IgASE1, by expression in Saccharomyces cerevisiae, was used to determine its activity and substrate specificity. Transformed yeast cells specifically elongated the C18-Δ⁹ polyunsaturated fatty acids, linoleic acid (C18:2n-6, Δ^9,12) and -linolenic acid (C18:3n-3, Δ^9,12,15), to eicosadienoic acid (C20:2n-6, Δ^11,14) and eicosatrienoic acid (C20:3n-3, Δ^11,14,17), respectively. To our knowledge this is the first time such an elongating activity has been functionally characterised. The results also suggest that a major route for eicosapentaenoic acid (C20:5n-3, Δ^5,8,11,14,17) and docosahexaenoic acid syntheses in I. galbana may involve a Δ⁸ desaturation pathway.     

Concentration of docosahexaenoic and eicosapentaenoic acids by enzymatic alcoholysis with different acyl-acceptors

The aim of this work was to produce docosahexaenoic (DHA) and eicosapentaenoic acid (EPA) enriched acylglycerols by alcoholysis of tuna and sardine oils, respectively, using isobutanol and 1-butanol as acyl-acceptors. The alcoholysis reactions were catalyzed by lipases Lipozyme® TL IM from Thermomyces lanuginosus and lipase QLG® from Alcaligenes sp., because these lipases have shown selectivity towards DHA and EPA, respectively. Studies were made to determine the influence of reaction time, alcohol/oil molar ratio, lipase amount and temperature. In the optimized conditions for the alcoholysis of tuna and sardine oils catalyzed by Lipozyme TL IM and lipase QLG, respectively, the DHA and EPA contents were trebled (from 22 to 69% for DHA, and from 19 to 61% for EPA). The stability of both lipases was also determined. Although Lipozyme TL IM is much more stable in isobutanol than in ethanol, with the former the conversion attained after four reaction cycles was about 40% of the initial conversion. In similar conditions, the conversion obtained with lipase QLG was about 88% of the initial conversion. In addition, the separation of DHA enriched acylglycerols and isobutyl esters from an alcoholysis reaction was studied by liquid–liquid fractionation using the ethanol–water–hexane biphasic system. The DHA enriched acylglycerols obtained were 97.6% pure (64.4% DHA).     

A rapid method for the isolation of eicosapentaenoic acid-producing marine bacteria

Bacterial production of long chain polyunsaturated fatty acids (LC-PUFAs) is a promising biotechnological approach for the mass production of these valuable compounds, but extensive screening is currently needed to select a strain that meets industrial requirements.A method was developed for the rapid screening and isolation of eicosapentaenoic acid (EPA)-producing marine bacteria from mixed cultures using the dye 2,3,5-triphenyltetrazolium chloride (TTC). The method was first validated using two bacteria from the Shewanella genus, S. gelidimarina (known to contain EPA) and S. fidelis (known not to contain EPA), and subsequently applied to a range of bacterial samples collected from seven randomly selected New Zealand fish species.By incorporating TTC in both solid and liquid state fermentation treatments, a clear association between the reduction of TTC to the red-coloured triphenyl formazan (TF) and the presence of EPA within Gram-negative bacteria was confirmed. Incubation in 0.1% w/v TTC was optimal for colour response and cell growth in agar plates and liquid cultures. Bacteria that produce EPA reduced TTC to TF, but a number of non-EPA-producing bacteria also showed this capacity. By conducting a subsequent Gram staining, all EPA-producing strains were revealed to be G (−) rod bacteria while the non-producing ones were all G (+) cocci. The fatty acid methyl esters of the isolated bacteria that reduced TTC to TF were analysed using gas chromatography-mass spectrometry and the content of EPA was confirmed by gas chromatography.From a pool of 2.0 × 10⁸ CFU/ml, this method allowed the rapid isolation of 16 bacteria capable of producing EPA. This new approach significantly reduces the number of samples submitted for GC analysis and therefore the time, effort and cost of screening and isolating strains of EPA-producing marine bacteria.     

Improvement of arachidonic acid and eicosapentaenoic acid production by increasing the copy number of the genes encoding fatty acid desaturase and elongase into <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Genes encoding Δ6 desaturase, Δ6 fatty acid elongase, and Δ5 desaturase from the alga, Phaeodactylum tricornutum, were co-expressed in Pichia pastoris to produce arachidonic acid (ARA; 20:4 Δ^{5, 8, 11, 14}) and eicosapentaenoic acid (EPA; 20:5 Δ^{5, 8, 11, 14, 17}). A panel of Pichia clones carrying progressively increasing copies of the heterologous gene expression cassette was created using an in vitro multimerization approach. ARA and EPA accumulated up to 0.3 and 0.1% of total fatty acids, respectively, in the recombinant P. pastoris carrying with double copies of these three heterologous genes, as compared to 0.1 and 0.05%, respectively, in the recombinant P. pastoris with single copy. Yun-Tao Li and Mao-Teng Li contributed equally to this work.     

Screening of diatoms for heterotrophic eicosapentaenoic acid production

Nine strains of diatoms (representing four species) were screened for their ability to produce eicosapentaenoic acid (EPA) when cultured heterotrophically on glucose. Four strains were able to produce EPA heterotrophically using glucose as its carbon and energy source. Of the four,Nitzschia laevis was the best EPA producer, yielding 0.017 g g^–1 dry cell weight.N. laevis was the only species tested which synthesised more EPA heterotrophically than photosynthetically. This study shows thatN. laevis is a potential source of EPA production using heterotrophic culture conditions with glucose as the carbon and energy substrate.Author for correspondence     

Changes in biochemical and hemocyte parameters of the Pacific oysters Crassostrea gigas fed T-Iso supplemented with lipid emulsions rich in eicosapentaenoic acid

The aim of this study was to assess the effect of dietary eicosapentaenoic acid (20:5n-3) on hemocyte parameters such as hemocyte concentration, phagocytosis, and non-stimulated reactive oxygen species (ROS) production in Pacific oysters Crassostrea gigas, as well on proximate biochemical and fatty acid compositions. One-year-old oysters (C. gigas) were fed T-Isochrysis aff. galbana (T-Iso), which is low in 20:5n-3, either alone or with supplements of a lipid emulsion rich in 20:5n-3 at 1%, 10% or 50% (dry weight of the algal ration) for up to 7 weeks. Changes in gill fatty acid composition demonstrated that the lipid emulsion was well ingested by oysters during the dietary conditioning. Biochemical analysis indicated that oysters fed supplements of 50% and, to a lesser extent, 10% lipid emulsions had a higher total lipid content compared with oysters fed other diets, suggesting a more advanced reproductive status for the oysters fed high doses of lipid emulsion. Moreover, some oysters in these two treatment groups spawned during the last three weeks of the seven-week feeding experiment. Lipid supplements had a significant influence on hemocyte concentration, phagocytic index and non-stimulated hemocyte ROS production. After 4 weeks, highest hemocyte concentrations were found in oysters fed on a supplement of 50% lipid emulsion compared with those fed on other diets but the hemocytes derived from these oysters had the lowest short-term phagocytic index. After 7 weeks of dietary conditioning, the ROS production in non-stimulated hemocytes of oysters fed 10% and 50% lipid emulsion declined. These results suggested that 20:5n-3, and perhaps its eicosanoid metabolites, affected oyster hemocyte functions; however, the reproductive status of oysters may also have interfered with the 20:5n-3 dietary effect.     

Crypthecodinium cohnii with emphasis on DHA production: a review

Docosahexaenoic acid (DHA) is a polyunsaturated fatty acid (PUFA) that belongs to the ω-3 group. In recent years, DHA has attracted much attention because of its recognized beneficial effect on human health. At present, fish oil is the major source of DHA, but it may be produced by microorganisms with additional benefits. Marine microorganisms may contain large amounts of DHA and are considered a potential source of this important fatty acid. Some of these organisms can be grown heterotrophically on organic substrates without light, offering the possibility of greatly increasing microalgal cell concentration under controlled and monitored conditions, resulting in a very high quality product. Among the heterotrophic marine dinoflagellates, Crypthecodinium cohnii has been identified as a prolific producer of DHA. The organism is extraordinary in that it produces no other PUFAs than DHA in its cell lipid in any significant amount, which makes the DHA purification process very attractive, particularly for pharmaceutical and nutraceutical applications. This paper reviews recent advances in the biotechnological production of DHA by C. cohnii.     

Development of a semidefined growth medium for Pedobacter cryoconitis BG5 using statistical experimental design

Pedobacter cryoconitis BG5 are psychrophiles isolated from the cold environment and capable of proliferating and growing well at low temperature regime. Their cellular products have found a broad spectrum of applications, including in food, medicine, and bioremediation. Therefore, it is imperative to develop a high-cell density cultivation strategy coupled with optimized growth medium for P. cryoconitis BG5. To date, there has been no published report on the design and optimization of growth medium for P. cryoconitis, hence the objective of this research project. A preliminary screening of four commercially available media, namely tryptic soy broth, R2A, Luria Bertani broth, and nutrient broth, was conducted to formulate the basal medium. Based on the preliminary screening, tryptone, glucose, NaCl, and K₂HPO₄ along with three additional nutrients (yeast extract, MgSO₄, and NH₄Cl) were identified to form the basal medium which was further analyzed by Plackett–Burman experimental design. Central composite experimental design using response surface methodology was adopted to optimize tryptone, yeast extract, and NH₄Cl concentrations in the formulated growth medium. Statistical data analysis showed a high regression factor of 0.84 with a predicted optimum optical (600?nm) cell density of 7.5 using 23.7?g/L of tryptone, 8.8?g/L of yeast extract, and 0.7?g/L of NH₄Cl. The optimized medium for P. cryoconitis BG5 was tested, and the observed optical density was 7.8. The cost-effectiveness of the optimized medium was determined as 6.25 unit prices per gram of cell produced in a 250-ml Erlenmeyer flask.     

Differential effects of eicosapentaenoic acid and docosahexaenoic acid in promoting the differentiation of 3T3-L1 preadipocytes

The objective of this study was to determine the effects of enrichment with n-3 fatty acids, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), on the differentiation of 3T3-L1 preadipocytes. Enrichment with DHA but not EPA significantly increased the differentiation markers compared to control differentiated cells. DHA compared to EPA treatment led to a greater increase in adiponectin secretion and, conditioned media collected from DHA treated cells inhibited monocyte migration. Moreover, DHA treatment resulted in inhibition of pro-inflammatory signaling pathways. DHA treated cells predominantly accumulated DHA in phospholipids whereas EPA treatment led to accumulation of both EPA and its elongation product docosapentaenoic acid (DPA), an n-3 fatty acid. Of note, adding DPA to DHA inhibited DHA-induced differentiation. The differential effects of EPA and DHA on preadipocyte differentiation may be due, in part, to differences in their intracellular modification which could impact the type of n-3 fatty acids incorporated into the cells.     

Eicosapentaenoic acid modifies lipid composition in caveolae and induces translocation of endothelial nitric oxide synthase

Endothelial nitric oxide synthase (eNOS) plays a crucial role in the regulation of a variety of cardiovascular functions. Many studies have shown that dietary n-3 polyunsaturated fatty acids (PUFAs) have beneficial effects on coronary atherosclerosis. However, the mechanisms of n-3 PUFAs regulation in eNOS activation remain unknown. In the present study we investigated the effects of eicosapentaenoic acid (EPA, 20:5 n-3) on subcellular distribution of eNOS and lipid composition of caveolae. We demonstrated for the first time that EPA treatment profoundly altered lipid composition and fatty acyl substitutions of phospholipids in caveolae. We found that caveolin-1 was solely located in caveolae fractions in control cells, and EPA treatment displaced caveolin-1 from caveolae. eNOS was detected in the caveolin-enriched fractions and noncaveolae fractions in control cells. EPA treatment induced the translocation of eNOS from caveolae fractions to soluble fractions. P-eNOS was also distributed in both fractions. After EPA treatment, the level of p-eNOS in each fraction was increased but the distribution of which was unaffected. Moreover, the results of immunofluorescence confirmed that EPA could redistribute caveolin-1 and eNOS in plasma membrane. eNOS activity in HUVEC cells was increased after EPA treatment, which was in a dose dependent manner. And incubation with 50 microM EPA had the maximum effect on eNOS activity. Our results suggested that eNOS translocation was paralleled by a stimulated capacity for NO production in the cells. We found that total Akt and p-Akt were primarily presented in heavy membranes in control cells, and the relative level of p-Akt increased but the distribution did not change after EPA treatment. The distribution of CaM was slightly changed after EPA treatment. Our results indicated that n-3 PUFAs profoundly altered caveolae microenvironment, thereby modifying location and function of proteins in caveolae. EPA-induced alterations of lipid and proteins in caveolae may be an important mechanism in the pathophysiologic process of atherosclerosis.     

Statistical optimization of production conditions of alkaline pectin lyase from Bacillus cereus using response surface methodology

Alkaline pectin lyase finds applications in the degumming and retting of plant fibres, textile industry and pectic wastewater treatment where it degrades highly methylesterified pectin without prior action of any other pectinase. Response surface methodology (RSM) has been frequently utilized for the optimization of production process of industrially important enzymes from microbes. In the present work, fermentation conditions for the production of pectin lyase from Bacillus cereus were optimized using the factorial and central composite design of RSM. The cubic order polynomial regression model was found to be adequate and significant with a determination coefficient R² of 0.9505 (p?相似文献