Genome-scale metabolic modeling of Thraustochytrium sp. RT2316-16: Effects of nutrients on metabolism.

Marine thraustochytrids produce metabolically important lipids such as the long-chain omega-3 polyunsaturated fatty acids, carotenoids, and sterols. The growth and lipid production in thraustochytrids depends on the composition of the culture medium that often contains yeast extract as a source of amino acids. This work discusses the effects of individual amino acids provided in the culture medium as the only source of nitrogen, on the production of biomass and lipids by the thraustochytrid Thraustochytrium sp. RT2316-16. A reconstructed metabolic network based on the annotated genome of RT2316-16 in combination with flux balance analysis was used to explain the observed growth and consumption of the nutrients. The culture kinetic parameters estimated from the experimental data were used to constrain the flux via the nutrient consumption rates and the specific growth rate of the triacylglycerol-free biomass in the genome-scale metabolic model (GEM) to predict the specific rate of ATP production for cell maintenance. A relationship was identified between the specific rate of ATP production for maintenance and the specific rate of glucose consumption. The GEM and the derived relationship for the production of ATP for maintenance were used in linear optimization problems, to successfully predict the specific growth rate of RT2316-16 in different experimental conditions.

Coenzyme Q in Thraustochytrium sp. RT2316-16: Effect of the Medium Composition.

Coenzyme Q (CoQ; ubiquinone) is an essential component of the respiratory chain. It is also a potent antioxidant that prevents oxidative damage to DNA, biological membranes, and lipoproteins. CoQ comprises a six-carbon ring with polar substituents that interact with electron acceptors and donors, and a hydrophobic polyisoprenoid chain that allows for its localization in cellular membranes. Human CoQ has 10 isoprenoid units (CoQ10) within the polyisoprenoid chain. Few microorganisms produce CoQ10. This work shows that Thraustochytrium sp. RT2316-16 produces CoQ10 and CoQ9. The CoQ10 content in RT2316-16 depended strongly on the composition of the growth medium and the age of the culture, whereas the CoQ9 content was less variable probably because it served a different function in the cell. Adding p-hydroxybenzoic acid to the culture media positively influenced the CoQ10 content of the cell. The absence of some B vitamins and p-aminobenzoic acid in the culture medium negatively affected the growth of RT2316-16, but reduced the decline in CoQ10 that otherwise occurred during growth. The highest content of CoQ9 and CoQ10 in the biomass were 855 µg g-1 and 10 mg g-1, respectively. The results presented here suggest that the thraustochytrid RT2316-16 can be a potential vehicle for producing CoQ10. Metabolic signals that trigger the synthesis of CoQ10 in RT2316-16 need to be determined for optimizing culture conditions.

Cellulose Synthase in Atacama Cyanobacteria and Bioethanol Production from Their Exopolysaccharides.

Cyanobacteria produce exopolysaccharides (EPSs) as an adaptative mechanism against ultraviolet radiation and desiccation. Cellulose is present in the extracellular polymeric substance in some cyanobacteria genera and it has been proposed as a raw material for biofuel production. The goal of this work was to evaluate the cellulose presence in EPS of Atacama cyanobacteria strains and its use as an alternative and innovative biological source to produce bioethanol. The presence of cellulose was evaluated using techniques of molecular biology, bioinformatics, and electronic microscopy. The conserved motif D,D,D,35QXXRW, characteristic of processive ß-glycosyltransferase in all cellulose-producing organisms, was identified in the genome of the LLA-10 strain. This is evidence that cellulose synthase in the LLA-10 strain is a functional enzyme. EPS from Atacama cyanobacteria was hydrolyzed by ß-glucosidases (cellobiase and cellulase) and the released glucose was yeast-fermented to ethanol. Ethanol production reached 172.69 ± 0.02 mg ethanol/g EPS after 48 h of incubation. These results are the first step in the evaluation of EPS produced by native cyanobacteria isolated from northern Chile for future biotechnological applications such as the production of bioethanol.

Production of Carotenoids and Phospholipids by Thraustochytrium sp. in Batch and Repeated-Batch Culture.

The carotenogenic thraustochytrid Thraustochytrium sp. RT2316-16 was grown in batch and repeated-batch cultures using different feeds containing glucose, or glycerol, and yeast extract, for the production of lipids, phospholipids and carotenoids. RT2316-16 produced canthaxanthin, astaxanthin and ß-carotene. The effects of biotin, ascorbic acid, light and temperature were evaluated in some of the experiments. In 2-day-old batch cultures, the combined mass percentage of eicosapentaenoic acid and docosahexaenoic acid in total lipids was between 16.5% (glycerol-based medium in the dark; biomass concentration = 4.2 ± 1.1 g L-1) and 42.6% (glucose-based medium under light; biomass concentration = 3.3 ± 0.1 g L-1), decreasing to 3.8% and 6.1%, respectively, after day 4. In repeated-batch cultures, the total lipids in the biomass increased after glucose or glycerol was fed alone, whereas the total carotenoids (168 ± 7 µg g-1 dry weight (DW)) and phospholipids in the biomass increased after feeding with yeast extract. The biomass with the highest content of phospholipids (28.7 ± 4.3 mg g-1 DW) was obtained using a feed medium formulated with glycerol, yeast extract and ascorbic acid. Glycerol was the best carbon source for the production of a biomass enriched with total lipids (467 ± 45 mg g-1 DW). The composition of carotenoids depended strongly on the composition of the feed. Repeated-batch cultures fed with yeast extract contained canthaxanthin as the main carotenoid, whereas in the cultures fed only with glucose, the biomass contained mainly ß-carotene.

Pathogens and predators impacting commercial production of microalgae and cyanobacteria.

Production of phytoplankton (microalgae and cyanobacteria) in commercial raceway ponds and other systems is adversely impacted by phytoplankton pathogens, including bacteria, fungi and viruses. In addition, cultures are susceptible to productivity loss, or crash, through grazing by contaminating zooplankton such as protozoa, rotifers and copepods. Productivity loss and product contamination are also caused by otherwise innocuous invading phytoplankton that consume resources in competition with the species being cultured. This review is focused on phytoplankton competitors, pathogens and grazers of significance in commercial culture of microalgae and cyanobacteria. Detection and identification of these biological contaminants are discussed. Operational protocols for minimizing contamination, and methods of managing it, are discussed.

Nitrogen Sources Affect the Long-Chain Polyunsaturated Fatty Acids Content in Thraustochytrium sp. RT2316-16.

The psychrophilic marine microorganism Thraustochytrium sp. RT2316-16 can produce carotenoids as well as lipids containing the omega-3 polyunsaturated fatty acids (PUFA) eicosapentaenoic acid and docosahexaenoic acid. This work reports on the effects of the composition of the culture medium, including certain amino acids, on growth and lipid synthesis by RT2316-16. Compared with the culture on glutamate, the use of lysine, alanine, or serine, increased the content of the omega-3 PUFA in total lipids. In the media that contained yeast extract, glutamate, and glucose, lipid accumulation occurred when organic ammonium was exhausted earlier than glucose. In contrast, lipid mobilization was promoted if glucose was exhausted while organic ammonium (supplied by yeast extract and glutamate) remained in the medium. The total content of carotenoids in the lipid-free biomass decreased during the first 12 to 24 h of culture, simultaneously with a decrease in the total lipid content of the biomass. The experimental data suggested a possible interrelationship between the metabolism of carotenoids and lipids. A high content of omega-3 PUFA in the total lipids could be obtained by growing the thraustochytrid in a medium with a low glucose concentration (6 g L-1) and a high concentration of organic nitrogen (yeast extract 12 g L-1; glutamate 1.06 g L-1), after glucose was exhausted. These observations may guide the development of a strategy to enhance omega-3 PUFA in the biomass.

Antarctic Thraustochytrids as Sources of Carotenoids and High-Value Fatty Acids.

Eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and carotenoids are needed as human dietary supplements and are essential components in commercial feeds for the production of aquacultured seafood. Microorganisms such as thraustochytrids are potential natural sources of these compounds. This research reports on the lipid and carotenoid production capacity of thraustochytrids that were isolated from coastal waters of Antarctica. Of the 22 isolates, 21 produced lipids containing EPA+DHA, and the amount of these fatty acids exceeded 20% of the total fatty acids in 12 isolates. Ten isolates were shown to produce carotenoids (27.4-63.9 µg/g dry biomass). The isolate RT2316-16, identified as Thraustochytrium sp., was the best producer of biomass (7.2 g/L in five days) rich in carotenoids (63.9 µg/g) and, therefore, became the focus of this investigation. The main carotenoids in RT2316-16 were ß-carotene and canthaxanthin. The content of EPA+DHA in the total lipids (34 ± 3% w/w in dry biomass) depended on the stage of growth of RT2316-16. Lipid and carotenoid content of the biomass and its concentration could be enhanced by modifying the composition of the culture medium. The estimated genome size of RT2316-16 was 44 Mb. Of the 5656 genes predicted from the genome, 4559 were annotated. These included genes of most of the enzymes in the elongation and desaturation pathway of synthesis of ω-3 polyunsaturated fatty acids. Carotenoid precursors in RT2316-16 were synthesized through the mevalonate pathway. A ß-carotene synthase gene, with a different domain organization compared to the gene in other thraustochytrids, explained the carotenoid profile of RT2316-16.

Macrocystis pyrifera Extract Residual as Nutrient Source for the Production of Sophorolipids Compounds by Marine Yeast Rhodotorula rubra.

Seaweed processing generates liquid fraction residual that could be used as a low-cost nutrient source for microbial production of metabolites. The Rhodotorula strain is able to produce antimicrobial compounds known as sophorolipids. Our aim was to evaluate sophorolipid production, with antibacterial activity, by marine Rhodotorula rubra using liquid fraction residual (LFR) from the brown seaweed Macrocystis pyrifera as the nutrient source. LFR having a composition of 32% w/w carbohydrate, 1% w/w lipids, 15% w/w protein and 52% w/w ash. The best culture condition for sophorolipid production was LFR 40% v/v, without yeast extract, artificial seawater 80% v/v at 15 °C by 3 growth days, with the antibacterial activity of 24.4 ± 3.1 % on Escherichia coli and 21.1 ± 3.8 % on Staphylococcus aureus. It was possible to identify mono-acetylated acidic and methyl ester acidic sophorolipid. These compounds possess potential as pathogen controllers for application in the food industry.

Temperature Differentially Affects Gene Expression in Antarctic Thraustochytrid Oblongichytrium sp. RT2316-13.

Oblongichytrium RT2316-13 synthesizes lipids rich in eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). The content of these fatty acids in the total lipids depended on growth temperature. Sequencing technology was used in this work to examine the thraustochytrid's response to a decrease in growth temperature from 15 °C to 5 °C. Around 4% (2944) of the genes were differentially expressed (DE) and only a few of the DE genes (533 upregulated; 206 downregulated) had significant matches to those in the SwissProt database. Most of the annotated DE genes were related to cell membrane composition (fatty acids, sterols, phosphatidylinositol), the membrane enzymes linked to cell energetics, and membrane structure (cytoskeletal proteins and enzymes). In RT2316-13, the synthesis of long-chain polyunsaturated fatty acids occurred through ω3- and ω6-pathways. Enzymes of the alternative pathways (Δ8-desaturase and Δ9-elongase) were also expressed. The upregulation of the genes coding for a Δ5-desaturase and a Δ5-elongase involved in the synthesis of EPA and DHA, explained the enrichment of total lipid with these two long-chain fatty acids at the low temperature. This molecular response has the potential to be used for producing microbial lipids with a fatty acids profile similar to that of fish oils.

Dynamic flux balance analysis of biomass and lipid production by Antarctic thraustochytrid Oblongichytrium sp. RT2316-13.

Production of biomass and lipids in batch cultures of the Antarctic thraustochytrid Oblongichytrium sp. RT2316-13, is reported. The microorganism proved capable of producing nearly 67% docosahexaenoic acid (DHA) and 15% eicosapentaenoic acid (EPA) in its total lipid fraction. Biomass with a maximum total lipid content of 33.5% (wt/wt) could be produced at 15°C in batch culture using a medium containing glucose (20 g/L), yeast extract (10.5 g/L), and other minor components. A lower culture temperature (5°C) reduced biomass and lipid productivities compared to culture at 15°C, but enhanced the DHA and EPA content of the lipids by 6.4- and 3.3-fold, respectively. Both a simple minimally structured mathematical model and a more complex genome-scale metabolic model (GEM) allowed the fermentation profiles in batch cultures to be satisfactorily simulated, but the GEM provided much greater insight in the biochemical and physiological phenomena underlying the observed behavior. Unlike the simpler model, the GEM could be interrogated for the possible effects of various external factors such as oxygen supply, on the expected outcomes. In silico predictions of oxygen effects were consistent with literature observations for DHA producing thraustochytrids.

An in vitro digestion study of encapsulated lactoferrin in rapeseed phospholipid-based liposomes.

Effectiveness of liposomes elaborated with rapeseed phospholipid (RP) extracted from a residue of oil processing, stigmasterol (ST) and/or hydrogenated phosphatidylcholine (HPC) for the encapsulation lactoferrin (LF) was studied; lipid membrane of liposomes was characterized (bilayer size, chain conformational order, lateral packing, lipid phase, and morphology) and the protection offered to the encapsulated LF during in vitro digestion was determined. Liposomes composed of RP+STLC(low concentration) showed spherical and irregular vesicles without perforations. Lamellar structure was organized in a liquid-ordered phase with a potential orthorhombic packing. Stability and size of the liposomes were more affected by gastric digestion than intestinal digestion; 67-80% of the initially encapsulated LF remained intact after gastric digestion whereas the percentage was reduced to 16-35% after intestinal digestion. Our results shows that liposomes elaborated with RP, properly combined with other lipids, can be a useful oral delivery system of molecules sensitive to digestive enzymes.

Antarctic thraustochytrids: Producers of long-chain omega-3 polyunsaturated fatty acids.

Thraustochytrids have been isolated from different aquatic systems; however, few studies have reported their occurrence in Antarctica. In this study, 13 strains close to strains belonging to the genera Oblongichytrium, Thraustochytrium, and Aurantiochytrium were isolated from seawater samples collected near the Antarctic Base Professor Julio Escudero (S 62°12'57' E 58°57'35″). Docosahexaenoic acid (DHA) was found in the total lipids of all the isolates; DHA content of the biomass (dry weight) varied between 3.3 and 33 mg/g under the growth conditions for isolation. Five of the Antarctic thraustochytrids were able to accumulate lipids at levels higher than 20% w/w. Two strains, RT2316-7 and RT2316-13, were selected to test the effect of the incubation temperature (at 5°C for 14 days and at 15°C for 5 days). Incubation temperature had little effect on the lipid content and biomass yield; however, its effect on the fatty acid composition was significant (p < .05). The low incubation temperature favored the accumulation of eicosapentaenoic acid (EPA), palmitic acid and stearic acid in the total lipids of RT2316-7. Percentage of EPA, DHA and the omega-6 fatty acid dihomo-γ-linolenic acid of total fatty acids of RT2316-13 was higher at the low incubation temperature. RT2316-13 accumulated the highest lipid content (30.0 ± 0.5%) with a carbon to nitrogen mass ratio equal to 16.9. On the contrary, lipid accumulation in RT2316-7 occurred at high concentration of the nitrogen sources (monosodium glutamate or yeast extract). The capability to accumulate lipids with a fatty acid profile that can be tuned through cultivation temperature make the Antarctic thraustochytrid RT2316-13 a candidate for the production of lipids with different uses.

Probiotics and prebiotics potential for the care of skin, female urogenital tract, and respiratory tract.

The prebiotics and probiotics market is constantly growing due to the positive effects of its consumption on human health, which extends beyond the digestive system. In addition, the synbiotic products market is also expanding due to the synergistic effects between pre- and probiotics that provide additional benefits to consumers. Pre- and probiotics are being evaluated for their effectiveness to treat and prevent infectious diseases in other parts of the human body where microbial communities exist. This review examines the scientific data related to the effects of pre- and probiotics on the treatment of diseases occurring in the skin, female urogenital tract, and respiratory tract. The evidence suggests that probiotics consumption can decrease the presence of eczema in children when their mothers have consumed probiotics during pregnancy and lactation. In women, probiotics consumption can effectively prevent recurrent urinary tract infections. The consumption of synbiotic products can reduce respiratory tract infections and their duration and severity. However, the outcomes of the meta-analyses are still limited and not sufficiently conclusive to support the use of probiotics to treat infectious diseases. This is largely a result of the limited number of studies, lack of standardization of the studies, and inconsistencies between the reported results. Therefore, it is advisable that future studies consider these shortcomings and include the evaluation of the combined use of pre- and probiotics.

Production of Lipids and Proteome Variation in a Chilean Thraustochytrium striatum Strain Cultured under Different Growth Conditions.

Total lipids and docosahexaenoic acid (DHA) production by a Chilean isolated thraustochytrid were evaluated under different growth conditions in shake flasks. The analyzed strain was identified as Thraustochytrium striatum according to an 18S rRNA gene sequence analysis. The strain (T. striatum AL16) showed negligible growth in media prepared with artificial seawater at concentrations lower than 50% v/v and pH lower than 5. Maltose and starch were better carbon sources for growth than glucose. DHA content of the biomass grown with maltose (60 g L-1) was doubled by increasing the agitation rate from 150 to 250 rpm. The DHA (0.8-6%) and eicosapentaenoic acid (0.2-21%) content in the total lipids varied depending on culture conditions and culture age. Lipid and DHA concentration increased (up to 5 g L-1 and 66 mg L-1, respectively) by regularly feeding the culture with a concentrated starch solution. Carotenoid accumulation was detected in cells grown with maltose or starch. Contrasting conditions of starch and glucose cultures were selected for comparative proteomics. Total protein extracts were separated by two-dimensional gel electrophoresis; 25 spots were identified using ESI-MS/MS. A protein database (143,006 entries) for proteomic interrogation was generated using de novo assembling of Thraustochytrium sp. LLF1b - MMETSP0199_2 transcriptome; 18 proteins differentially expressed were identified. Three ATP synthases were differentially accumulated in cultures with glucose, whereas malate dehydrogenase was more abundant in cells cultured with starch.

Metabolic modelling and simulation of the light and dark metabolism of Chlamydomonas reinhardtii.

A metabolic network model of the green microalga Chlamydomonas reinhardtii was used to characterize photoautotrophic and heterotrophic (i.e. growth on stored compounds) growth under light and dark, respectively. The metabolic network comprised 2514 reactions distributed among nine intracellular compartments and the extracellular space. The metabolic network included all the key biochemical pathways for synthesis and metabolism of starch and triacylglycerols (TAGs). Under light and nitrogen limitation, the model simulated the accumulation of the energy-rich compounds (TAGs and starch) in the cell. In the dark, the model could simulate cell growth and maintenance on stored compounds. The model-predicted consumption rates of storage compounds (starch or TAGs) to enable growth in the dark, were found to be greater than the rates of synthesis under light. This implied utilization of the storage compounds for cell maintenance in the dark. Under constant illumination, the simulations of cell growth and intracellular starch content agreed closely with independent experimental data. In other simulations, compared with the case without photorespiration, light uptake rate increased 1.04-fold when the ratio of the rates of oxygenation and carboxylation (Rubisco) was 0.1. Although extensive experimental work exists on culture and physiology of microalgae, it does not allow quantitative predictions of the influence of dark metabolism on the productivity of metabolites to be made. This limitation is overcome using the present model. A metabolic network model of Chlamydomonas reinhardtii is shown to simulate growth and synthesis of energy-rich compounds (triacylglycerols and starch) under light. The same model also simulates dark growth and maintenance through consumption of the stored energy-rich compounds.

Effective Lactobacillus plantarum and Bifidobacterium infantis encapsulation with chia seed (Salvia hispanica L.) and flaxseed (Linum usitatissimum L.) mucilage and soluble protein by spray drying.

Mucilage (M) and soluble protein (SP) extracted from chia seed and flaxseed were used as encapsulating material for two probiotic bacteria: Bifidobacterium infantis and Lactobacillus plantarum by spray drying. Probiotic survival and viability after spray drying and during storage were evaluated. B. infantis and L. plantarum displayed high survival (⩾98%) after encapsulation with mixtures of maltodextrin (MD) combined with M and SP from flaxseed (MD:FM:FSP - 7.5:0.2:7.5%, w/w/w) and chia seed (MD:CM:CSP - 7.5:0.6:7.5%, w/w/w), respectively. These ternary blends protected the probiotics and enhanced their resistance to simulated gastric juice and bile solution. Probiotics encapsulated with the ternary blends incorporated in instant juice powder exhibited high viability (>9Log10CFU/g) after 45days refrigerated storage. Encapsulation with the ternary blends reduced particle size of the probiotic powders thereby offering additional functional benefits. Our results reveal that chia seed and flaxseed are excellent sources of probiotic encapsulating agents.

Production of eicosapentaenoic acid by Nannochloropsis oculata: Effects of carbon dioxide and glycerol.

The marine microalga Nannochloropsis oculata is a potential source of eicosapentaenoic acid (EPA, C20:5n3) and carotenoids for use in functional foods and nutraceuticals. Mixotrophic culture of N. oculata using glycerol was examined as a possible way of increasing the biomass and metabolite productivity relative to a pure photoautotrophic culture in modified f/2 medium. The effect of CO2 supply was also tested. EPA production in semi-continuous culture with and without glycerol and CO2 was evaluated. The effects of glycerol supplementation and light/dark cycling on the production of the biomass and EPA are reported for cultures conducted at a constant pH controlled using CO2. Consumption of glycerol was small, but its effects were significant. Glycerol enhanced the lipid content of the biomass but reduced the chlorophyll a content. Mixotrophic cultivation favored the production of lipids with a high percentage of saturated fatty acids that are generally desired in oils for making biodiesel. EPA concentration (5.3±0.6 to 27.5±1.6mg EPA/L) in N. oculata cultures depended strongly on growth conditions. The highest EPA concentration occurred in non-aerated mixotrophic culture with intermittent CO2 supply without pH control. This EPA concentration (=27.5±1.6mg/L) was comparable to that obtained in semi-continuous culture without glycerol and pH control, and aerated with CO2 enriched air during the light period (=23.6±1.1mg/L).

High carotenoid bioaccessibility through linseed oil nanoemulsions with enhanced physical and oxidative stability.

Carotenoid (astaxanthin or lycopene) emulsions obtained by high pressure homogenization were investigated for their physical, oxidative and storage stability and biological fate on an in vitro digestion model of bioaccessibility. Emulsion stability evaluated at various processing environments (20-50°C, 2-10 pH, 0-500 mM NaCl, and 0-35 days storage at 25°C) depended on carotenoid and homogenization pressures (5, 10, 100 MPa). Trolox increased the oxidative stability of nanoemulsions (100 MPa) and acted synergistically with BHT in increasing the stability of lycopene nanoemulsion. Intestinal digestibility depended on homogenization pressures with the fastest release and lower amount of free fatty acids observed at 100 MPa. Carotenoid nanoemulsions (100 MPa) were partially (66%) digested and highly bioaccessible (>70%). Therefore, nanoemulsions provide an effective and stable system for efficient astaxanthin or lycopene delivery and bioavailability in foods, beverages, nutraceuticals and/or other agriproducts.

Oil bodies as a potential microencapsulation carrier for astaxanthin stabilisation and safe delivery.

Astaxanthin (AST) is a valued molecule because of its high antioxidant properties. However, AST is extremely sensitive to oxidation, causing the loss of its bioactive properties. The purposes of this study were to define conditions for microencapsulating AST in oil bodies (OB) from Brassica napus to enhance its oxidative stability, and to test the bioactivity of the microencapsulated AST (AST-M) in cells. Conditions for maximising microencapsulation efficiency (ME) were determined using the Response Surface Methodology, obtaining a high ME (>99%). OB loaded with AST showed a strong electrostatic repulsion in a wide range of pH and ionic strengths. It was found that AST-M exposed to air and light was more stable than free AST. In addition, the protective effect of AST against intracellular ROS production was positively influenced by microencapsulation in OB. These results suggest that OB offer a novel option for stabilising and delivering AST.

Seed oil bodies from Gevuina avellana and Madia sativa.

In this study, oil bodies (OBs) from Gevuina avellana (OBs-G) and Madia sativa (OBs-M) were isolated and characterized. Microscopic inspection revealed that the monolayer on OB-G was thinner compared to that on OB-M. Cytometric profiles regarding size, complexity, and staining for the two OB sources were similar. Fatty acid to protein mass ratio in both OBs was near 29, indicating high lipid enrichment. OBs-G and OBs-M showed a strong electrostatic repulsion over wide ranges of pH (5.5-9.5) and NaCl concentration (0-150 mM). Proteins displaying highly conserved sequences (steroleosins and aquaporins) in the plant kingdom were identified. The presence of oleosins was immunologically revealed using antibodies raised against Arabidopsis thaliana oleosins. OBs-G and OBs-M exhibited no significant cytotoxicity against the cells. This is the first report about the isolation and characterization of OBs-G and OBs-M, and this knowledge could be used for novel applications of these raw materials.