Alterations in allocation and composition of lipid classes in Euonymus fruits and seeds.

The spindle tree (Euonymus europaeus L.) is a much-branched deciduous shrub or small tree. Its fruit capsules contain seeds with remarkably high content of oil interesting for industry, especially the 3-acetyl-1,2-diacyl-sn-glycerols (AcDAG) synthesized by a specific acetyl-CoA diacylglycerol acetyltransferase. The distribution and amount of individual triacylglycerols (TAG) and especially acetyl-triacylglycerols (AcDAG) in Euonymus fruit have previously been assigned to specific tissues. Using anatomical and microscopical observations, we studied the fruit morphology, and for the first time, we identified a more detailed allocation of oil bodies in individual tissue structures. Thin layer chromatography separation of extracts from immature and mature fruits confirmed TAG and AcDAG as the most abundant lipid classes in both endosperm and embryo, followed by fatty acids and polar lipids. The abundance of fatty acids was further studied in the TAG and AcDAG fractions using gas chromatography. Data revealed particular FAs in both fractions allocated in tissue-specific manner and/or as indicators of maturation of E. europaeus seeds. While the abundance of cis-vaccenic-, linoleic as well as α-linolenic acids in the AcDAG structures generally drop with maturation in both embryo and endosperm, content of oleic acid increases. Abundance of cis-vaccenic acid in TAG was recorded in immature endosperm. For embryo, the abundance of stearic acid in AcDAG and oleic acid in TAG fraction was distinctive. Deeper understanding of underlying metabolic processes will be essential for targeted metabolic engineering and/or application for oilseed crops.

Effect of fungal solid-state fermented product enriched with gamma-linolenic acid and ß-carotene on blood biochemistry and immunology of broiler chickens.

The aim of this study was to investigate the effect of the addition of fungal solid-state fer-mented product (FP) enriched with gamma-linolenic acid (GLA) and ß-carotene to feed on the haematological and immunological parameters of broiler chickens. Eighty 1-day-old COBB 500 broiler chickens were divided into two groups. The control group was fed with basic diets and chickens of the experimental group received 10% addition of FP, while the amount of basic diet was reduced. FP was produced during a solid-state fermentation (SSF) process using Umbellopsis isabellina CCF2412 as a producer of GLA and ß-carotene. After 38 days of feeding, blood sam-ples were collected and analyzed. Lower total and LDL-cholesterol values were measured in blood samples of the experimental animals (p⟨0.05). However, the triacylglycerol content was higher in the experimental group (p⟨0.05). Significantly higher levels of hematocrit and hemo-globin, and lower eosinophil and basophil content in the experimental group were recorded (p⟨0.05). The experimental group showed higher numbers of B lymphocytes and greater phago-cytic capacity (p⟨0.05). The results indicate that a fermented product produced by SSF, using the fungal strain Umbellopsis isabellina, is a good source of GLA and ß-carotene, which can influence the biochemical, hematological and immunological parameters of broiler chickens.

Effect of fungal gamma-linolenic acid and beta-carotene containing prefermented feed on immunity and gut of broiler chicken.

Gamma-linolenic acid (GLA) is a fatty acid from the ω-6 family. It is able to deliver a wide range of health benefits arising from its anti-inflammatory effects. An insufficient supply of GLA from agricultural and animal sources resulted in the development of a fermentation technique using lower filamentous fungi, which have the ability to accumulate high concentrations of GLA and beta-carotene during solid-state fermentation of cereals. The goal of this study was to observe the influence of the addition of prefermented cereal product, containing high amounts of GLA and beta-carotene, into the feed of broiler chickens on their immune status, and also the number of lactic acid bacteria and enterobacteria in gut content, which has never been studied before. Immunostimulation in the GLA group was manifested by a significant increase in the oxidative burst of phagocytes, CD4+CD8- lymphocytes in blood, and the CD4: CD8 ratio. Upregulation of gene expression for IgA in the GLA group indicates that the B-lymphocytes were stimulated at a local gut level. In the caecum, increased mRNA expression for mucin-2 and insulin-like growth factor was observed in the GLA group, which could contribute mainly to the protection of the intestinal mucosa and to better growth and regeneration of skeletal muscles. Improved immune activation and protection of the intestinal mucosa were subsequently reflected in a change of the microbial composition in gut contents; a significant reduction of enterobacteria occurred after GLA administration. We can conclude that prefermented cereals containing fungal GLA and beta-carotene represent a low-cost supplement for broiler diet having a beneficial health effect.

Endophytic Streptomyces sp. AC35, a producer of bioactive isoflavone aglycones and antimycins.

In this research, a microbial endophytic strain obtained from the rhizosphere of the conifer Taxus baccata and designated as Streptomyces sp. AC35 (FJ001754.1 Streptomyces, GenBank) was investigated. High 16S rDNA gene sequence similarity suggests that this strain is closely related to S. odorifer. The major fatty acid profile of intracellular lipids was also carried out to further identify this strain. Atomic force microscopy and scanning acoustic microscopy were used to image our strain. Its major excreted substances were extracted, evaluated for antimicrobial activity, purified, and identified by ultraviolet-visible spectroscopy (UV-vis), liquid chromatography-mass spectrometry (LC-MS/MS) and nuclear magnetic resonance as the bioactive isoflavone aglycones-daidzein, glycitein and genistein. Batch cultivation, performed under different pH conditions, revealed enhanced production of antimycin components when the pH was stable at 7.0. Antimycins were detected by HPLC and identified by UV-vis and LC-MS/MS combined with the multiple reaction monitoring. Our results demonstrate that Streptomyces sp. AC35 might be used as a potential source of effective, pharmaceutically active compounds.

Substrates enriched by the fungus Cunninghamella echinulata: an in vitro study of nutrient composition, sheep rumen fermentation and lipid metabolism.

AIMS: Enrichment of wheat bran (WB), corn meal (CM) and barley flakes (BF) with the oleaginous fungus Cunninghamella echinulata (CE) might lead to effective use of these by-products in ruminant nutrition. We examined their effects on rumen fermentation and lipid metabolism. METHODS AND RESULTS: WB, CM and BF substrates without or with brewer's grains (WBG, CMG, BFG) and enriched with CE were incubated with meadow hay (MH, 500 : 500, w/w) in rumen fluid in vitro for 24 h. The dry matter of the CE-enriched substrates increased (by 2-4%); however, digestibility decreased (P < 0·01). Adverse effects of CE-enriched substrates on the rumen ciliate population were observed. Little effect on the rumen eubacterial population was detected by the 16S-polymerase chain reaction/denaturizing gradient gel electrophoresis method. The increase in γ-linolenic acid output in the MH + BFGCE diet (800 : 200, w/w) was accompanied by an increase in rumen biohydrogenation of polyunsaturated fatty acids. CONCLUSION: The diet substrates enriched with the fungus CE were less digestible than the untreated cereal substrates; no appreciable positive effect was observed on rumen fermentation patterns or the eubacterial and ciliate populations. SIGNIFICANCE AND IMPACT OF THE STUDY: The in vitro study showed that adding CE-enriched substrates to ruminant diets is not effective for improving rumen fermentation.

Lower omega-3 index is a marker of increased propensity of hypertensive rat heart to malignant arrhythmias.

Polyunsaturated omega-3 fatty acids (omega-3 PUFA) are important components of cell membrane affecting its function and their deficiency is deleterious to health. We have previously shown that spontaneously hypertensive rats (SHR) are prone to life-threatening arrhythmias that are reduced by omega-3 PUFA intake. Purpose of this study was to explore plasma and red blood cells (RBC) profile of omega-3 and omega-6 PUFA as well as to determine omega-3 index, a risk factor for sudden cardiac death, in aged SHR and the effect of omega-3 PUFA intake. Male and female 12-month-old SHR and age-matched Wistar rats fed with omega-3 PUFA (200 mg/kg BW/day/2 month) were compared with untreated rats. Composition of omega-3 PUFA: alpha linolenic acid, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) as well as omega-6 PUFA: linoleic acid and arachidonic acid was analyzed by gas chromatography. Results showed sex- and strain-related differences of basal omega-3 and omega-6 PUFA levels in plasma and RBC as well as in response to omega-3 PUFA intake. Comparing to Wistar rats omega-3 index, expressed as a percentage of EPA+DHA of total fatty acids, was lower in SHR and it increased due to consumption of omega-3 PUFA. Findings support our hypothesis that lower omega-3 index may be also a marker of increased propensity of the hypertensive rat heart to malignant arrhythmias.

Use of several waste substrates for carotenoid-rich yeast biomass production.

Carotenoids are industrially significant pigments produced in many bacteria, fungi, and plants. Carotenoid biosynthesis in yeasts is involved in stress response mechanisms. Thus, controlled physiological and nutrition stress can be used for enhanced pigment production. Huge commercial demand for natural carotenoids has focused attention on developing of suitable biotechnological techniques including use of liquid waste substrates as carbon and/or nitrogen source. In this work several red yeast strains (Sporobolomyces roseus, Rhodotorula glutinis, Rhodotorula mucilaginosa) were enrolled into a comparative screening study. To increase the yield of these pigments at improved biomass production, several types of exogenous as well as nutrition stress were tested. Each strain was cultivated at optimal growth conditions and in medium with modified carbon and nitrogen sources. Synthetic media with addition of complex substrates (e.g. yeast extract) and vitamin mixtures as well as some waste materials (whey, potato extract) were used as nutrient sources. Peroxide and salt stress were applied too. The production of carotene enriched biomass was carried out in flasks as well as in laboratory fermentor. The best production of biomass was obtained in inorganic medium with yeast extract. In optimal conditions tested strains differ only slightly in biomass production. All strains were able to use most of waste substrates. Biomass and pigment production was more different according to substrate type. In laboratory fermentor better producers of enriched biomass were both Rhodotorula strains. The highest yields were obtained in R. glutinis CCY 20-2-26 cells cultivated on whey medium (cca 45 g per liter of biomass enriched by 46 mg/L of beta-carotene) and in R. mucilaginosa CCY 20-7-31 grown on potato medium and 5% salt (cca 30 g per liter of biomass enriched by 56 mg/L of beta-carotene). Such dried carotenoid-enriched red yeast biomass could be directly used in feed industry as nutrition supplement.

Effects of prefermented cereal-derived substrates (ground barley and rye bran) enriched with fungal γ-linolenic acid on rumen fermentation parameters and lipid metabolism in vitro.

AIMS: To increase rumen output of γ-linolenic acid (GLA), we used two cereal-derived substrates, ground barley (GB) and rye bran (RB), enriched with fungal GLA as components of feed rations. We examined their effects on rumen fermentation patterns, lipid metabolism and the ciliated protozoan population in an artificial rumen. METHODS AND RESULTS: Four diets consisting of meadow hay (MH) plus unfermented (GB or RB) or prefermented (GB - TE or RB - TE) cereal-derived substrates were fermented in an artificial rumen with ovine rumen inoculum. The cereal-derived substrates were prefermented with the fungus Thamnidium elegans (TE) by fungal solid-state fermentation. The diets with TE increased the rumen input of dietary GLA (mg day(-1)) from 0 to 21 (GB - TE) or 26 (RB - TE). Both experimental diets increased the rumen output of GLA (P < 0.001). Adverse effects on the ciliate population were observed. Both diets also had an effect on the fatty acids profile. Fermentation patterns were also affected with MH + RB - TE. CONCLUSION: Cereal-derived substrates enriched with GLA effectively enhanced the output of GLA in artificial rumen. SIGNIFICANCE AND IMPACT OF THE STUDY: The ability of the fungal strain T. elegans to grow and utilize various agro-industrial substrates might be useful in developing potential new animal diets enriched in GLA.

Comparison of the extrapancreatic action of gamma-linolenic acid and n-3 PUFAs in the high fat diet-induced insulin resistance [corrected].

OBJECTIVE: The effect of dietary borage oil (rich in the gamma-linolenic acid [GLA]) on insulin sensitivity and lipid metabolism was compared with that of fish oil (rich in n-3 polyunsaturated fatty acids [PUFAs]) in high fat (HF) diet-induced insulin resistance (IR) of rats. METHODS: Male Wistar rats were fed ad libitum for 3 weeks a standard laboratory chow (Controls) or high fat diet consisting of 70-cal % fat. In addition, a group of rats was fed high fat (HF) diet where a part of saturated fat was replaced with fish oil as a source of n-3 PUFAs (HF+FO), or borage oil as a source of GLA (HF+GLA). In vivo insulin action was assessed by the euglycemic hyperinsulinemic clamp. Glucose, insulin, free fatty acids (FFA), triglycerides (Tg) and glycerol levels in blood and tissue depots were also measured. RESULTS: Increased levels of Tg, FFA and glycerol in circulation after HF diet were accompanied by their raised accumulation in insulin sensitive tissues. FO feeding lowered the concentration of all lipids in serum and prevented their accumulation in both tissues. On the other hand GLA supplementation into the high fat diet did not suppress increased levels of Tg, FFA and glycerol in circulation and tissue depots as well. FO feeding significantly reduced HF diet-induced in vivo IR, while GLA supplementation did not improve the in vivo insulin sensitivity in HF diet induced insulin resistance. CONCLUSIONS: 1. Substitution of FO into the high fat diet led to an improvement of in vivo insulin action; 2. this insulin sensitizing effect of FO was accompanied by a decrease of circulating Tg, FFA and glycerol levels in the postprandial state and by a lower lipid content in liver and skeletal muscle. 3. on the opposite, GLA treatment failed to improve in vivo insulin action; and 4. was associated with an adverse effect on lipid levels both in circulation and tissue depots.

Kinetic analysis of oil biosynthesis by an arachidonic acid-producing fungus, Mortierella alpina 1S-4.

Kinetic analysis of arachidonic acid (AA)-oil biosynthesis by Mortierella alpina 1S-4 growing under lipid-accumulating (LN medium) and non-lipid-accumulating (HN medium) conditions was investigated and compared with industrial AA fermentation. Various kinetic parameters of these cultivation processes demonstrate a characteristic pattern of the lipogenesis in this fungus, where growth phase, phase of oil accumulation and phase of AA synthesis are distinct from each other. The fungus utilizing LN medium synthesized 32.3 g fatty acid 100 g(-1) glucose on the 4th day of cultivation and reached the maximum daily fatty acid accumulation (expressed as differential specific rate qD(FA/B)) of 9.5%. Our results also indicate that a qD(FA/B) value of about 2.5% might be critical for lipid overproduction in M. alpina. AA was rapidly incorporated into triacylglycerols (90% of total AA) at the later cultivation phase and overall AA yield was directly related to the total yield of fatty acid.

Biosynthesis and regulation of microbial polyunsaturated fatty acid production.

Growing interest in polyunsaturated fatty acid (PUFA) applications in various fields coupled with their significance in health and dietary requirements has focused attention on the provision of suitable sources of these compounds. Isolation of highly efficient oleaginous microorganisms has led to the development of fermentation technologies as an alternative to agricultural and animal processes. Particularly active in PUFA synthesis are the Zygomycetes fungi and certain microalgae. Emphasis is placed on increasing the product value by employing new biotechnological strategies (e.g. mutation techniques, molecular engineering and biotransformations) which allow the regulation of microbial PUFA formation with satisfactory yield in order to be competitive with other sources. Comparative successes in fungal PUFA production demonstrate microbial potential to synthesize high-value oils and provide the main stimulus for their applications.

Characterization of the second form of NADH-cytochrome b5 reductase gene from arachidonic acid-producing fungus Mortierella alpina 1S-4.

The second type of cytochrome b5 reductase (Cb5R-II) gene was characterized in the arachidonic acid-producing fungus Mortierella alpina 1S-4. Its cDNA (897 bp) and predicted amino acid (298 aa) sequences show more than 70% similarity to the previously isolated first type of Cb5R. Highly conserved exon-intron organization suggests that the two genes evolved from the duplication of a common ancestral gene. Cb5R-II has a flavin-binding domain at its highly hydrophobic N-terminal and an NADH-binding domain at the C-terminal. In comparison with Cb5R genes from other sources, high homology (46-54%) was found for yeast and plant genes. Phylogenetic analysis revealed that microbial and plant Cb5R genes represent a gene family evolved from one prototype and are different from mammalian Cb5R genes.

Characterization of triacylglycerol biosynthesis in subcellular fractions of an oleaginous fungus, Mortierella ramanniana var. angulispora.

Triacylglycerol (TG) biosynthetic enzymes were characterized in subcellular fractions of an oleaginous fungus, Mortierella ramanniana var. angulispora. When the membrane or lipid body fraction of this fungus was incubated with [14C]oleoyl-CoA without adding exogenous acyl acceptors, radioactivity was incorporated predominantly into TG, indicating that diacylglycerol acyltransferase (DGAT) used endogenous diacylglycerol to incorporate [14C]oleoyl-CoA into TG. Adding glycerol 3-phosphate or lysophosphatidic acid increased radiolabeled phosphatidic acid (PA) in the membrane fraction, which reflected the presence of glycerol-3-phosphate acyltransferase (GPAT) and lysophosphatidic acid acyltransferase (LPAAT). Label accumulation did not occur in lysophosphatidic acid when glycerol 3-phosphate was added, suggesting that GPAT was rate-limiting in sequential acylation. In the lipid body fraction, adding lysophosphatidic acid similarly increased radiolabeled PA, whereas adding glycerol 3-phosphate caused much lower increase in radiolabeled PA. Quantitative assays for GPAT, LPAAT, phosphatidic acid phosphatase (PAP), and DGAT essentially confirmed the results obtained from [1-14C]oleoyl-CoA incorporation; LPAAT had the highest activity in the membrane and lipid body fractions, GPAT was significantly lower in the lipid body fraction, and DGAT was much higher in the lipid body fraction. GPAT and LPAAT in the membrane fraction had a strong preference toward oleoyl-CoA as a substrate over palmitoyl-CoA. Results indicate that TG biosynthetic enzymes had different subcellular distribution with the sequence of enrichment in the lipid body fraction, i.e., GPAT

Oleate 6-hydroxylase activity in the membrane fraction from an oleaginous fungus, Mortierella ramanniana var. angulispora.

Conversion of [1-14C]oleoyl-CoA (OA-CoA) in subcellular fractions of an oleaginous fungus, Mortierella ramanniana var. angulispora (IFO 8187) was investigated. The membrane fraction actively catalyzed the transformation of OA-CoA into one metabolite, which was identified as 6-hydroxy oleic acid (6-HOOA) by GC-MS analysis. The enzyme activity to form 6-HOOA was dependent on OA-CoA concentrations and increased linearly with incubation time and protein concentrations under appropriate experimental conditions. Since the enzyme activity required molecular oxygen and reduced pyridine nucleotides (NADH and NADPH), it was most probable that the formation of 6-HOOA was ascribed to direct hydroxylation to the C6 position, oleate 6-hydroxylase. 6-HOOA was formed from free oleic acid (OA) and OA-NH4 salt as substrate as well as OA-CoA, but not from OA covalently bound to phosphatidylcholine or triacylglycerol. Since CoA stimulated the hydroxylation of free OA and OA-NH4 salt but not that of OA-CoA, OA may be converted to OA-CoA and then hydroxylated. The enzyme activity was distinctly reduced by addition of lysophosphatidic acid, phosphatidic acid and dithiothreitol. Since hydroxy fatty acids are very minor components in this fungus, it may raise the possibility that the 6-hydroxylase activity works for other functions such as comprising one step for desaturation as well as forming the hydroxy fatty acids.

Effect of salt stress on fatty acid alterations in some strains of Dipodascopsis and Dipodascus spp.

The effect of salt stress (8% w/v NaCl) on fatty acid composition of eight strains of Dipodascus and Dipodascopsis spp. varied from being of slight influence only (Dipodascopsis uninucleata), to decreasing the content of 18:2 (D. reesii, D. tetrasperma and D. australiensis) and to decreasing both 18:1 and 18:2 (D. tothii and D. aggregatus) with a concomitant rise of 14:1 and 16:1. With the exception of D. aggregatus, NaCl inhibited lipid accumulation in all strains. Only trace amounts of fatty acids over C18 in chain length were found.

Formate dehydrogenase activity in methanol-utilizing yeasts.

The growth and activity of formate dehydrogenase (FDH) in five methanol-utilizing yeasts at various methanol concentrations were investigated. The parameters observed were inhibited at 4% methanol concentration in the medium. For Candida boidinii and Pichia trehalophila FDH activity was not found. The highest value was detected for Pichia lindneri (0.14 U/mg protein).

Selection of Rhizopus strains for L(+)-lactic acid and gamma-linolenic acid production.

The production of L(+)-lactic acid and formation of gamma-linolenic acid by 50 Rhizopus strains growing on saccharidic substrates were investigated. Formation of acids was observed on solid cultivation media but mainly during submerged fermentation. Strains with the highest selectivity of both L(+)-lactic acid production and gamma-linolenic acid formation were tested in a laboratory fermenter. The best producer was treated by UV irradiation to increase the fatty acid content in the biomass, especially that of gamma-linolenic acid. The conversion of 10% saccharidic substrate by this newly prepared strain Rhizopus arrhizus CCM 8109 results in more than 95% of theoretical yield of L(+)-lactic acid and permits a volume productivity of 0.4 g gamma-linolenic acid per liter.