Production and Characterization of DHA and GLA-Enriched Structured Lipid from Palm Olein for Infant Formula Use

Palm olein was modified via lipase-catalyzed acidolysis reaction to obtain fatty acid composition and positional distribution similar to human milk fat. In the reaction, a free fatty acid mix containing 23.23 % docosahexaenoic (DHA), 31.42 % gamma-linolenic (GLA), and 15.12 % palmitic acid was employed. The DHA and GLA were incorporated into the structured lipid (SL) product to improve its nutritional value. Response surface methodology (RSM) was used to investigate the effects of reaction time and substrate mole ratio (palm olein to a free fatty acid mix) on the amount of palmitic acid at the sn-2 position of SL triacyglycerols (TAG), and on the total DHA and GLA incorporation. Gram-scale production of SL was performed using the conditions predicted by RSM to maximize the content of palmitic acid at the sn-2 position. Verification of the predictions from RSM confirmed its practical utility. The resulting SL had 35.11 % palmitic acid at the sn-2 position, with 3.75 % DHA and 5.03 % GLA. Differential scanning calorimetry and HPLC analyses of the TAG revealed changes in their polymorphic profiles and TAG molecular species of SL compared to palm olein. The SL from this study can potentially be used in infant formula formulations.     

Lipase-Catalysed Enrichment of γ-Linolenic Acid from Evening Primrose Oil in a Solvent-Free System

The enrichment of γ-linolenic acid (GLA) was carried out in a solvent-free system by lipase-catalysed esterification of free fatty acids from evening primrose oil (EPO-FA) and 1-butanol (BtOH). The lipase employed to conduct this study was a free preparation of Candida rugosa. Variables evaluated were: substrate molar ratio (1:4, 1:6, 1:8, 1:10 and 1:12, EPO-FA:BtOH), temperature (10, 20, 30, 40, 50 and 60 °C), and enzyme loading (5, 10, 15 and 20 %, based on the total weight of substrates). GLA was highly enriched in the non-esterified fatty acid fraction since C. rugosa showed very low selectivity for this fatty acid. We were able to increase the content of GLA to ca. 70 wt.% under the following optimal conditions: 30 °C, 10 % enzyme loading and a 1:10 molar ratio (EPO-FA:BtOH), after 24 h. An additional set of experiments was conducted whereby the amount of water was controlled by addition of molecular sieves to the reaction mixture. The latter experiments produced a higher GLA concentrate (83.74 wt.%), under the optimal conditions described above and by adding 10 % molecular sieves (based on the total weight of substrates) after 36 h.     

Effects of Different Oil Sources and Residues on Biomass and Metabolite Production by Yarrowia lipolytica YB 423-12

Yarrowia lipolytica is known to have the ability to assimilate hydrophobic substrates like triglycerides, fats, and oils, and to produce single-cell oils, lipases, and organic acids. The aim of the present study was to investigate the effects of different oil sources (borage, canola, sesame, Echium, and trout oils) and oil industry residues (olive pomace oil, hazelnut oil press cake, and sunflower seed oil cake) on the growth, lipid accumulation, and lipase and citric acid production by Y. lipolytica YB 423-12. The maximum biomass and lipid accumulation were observed with linseed oil. Among the tested oil sources and oil industry residues, hazelnut oil press cake was the best medium for lipase production. The Y. lipolytica YB 423-12 strain produced 12.32 ± 1.54 U/mL (lipase activity) of lipase on hazelnut oil press cake medium supplemented with glucose. The best substrate for citric acid production was found to be borage oil, with an output of 5.34 ± 0.94 g/L. The biotechnological production of valuable metabolites such as single-cell oil, lipase, and citric acid could be achieved by using these wastes and low-cost substrates with this strain. Furthermore, the cost of the bio-process could also be significantly reduced by the utilization of various low-cost raw materials, residues, wastes, and renewable resources as substrates for this yeast.     

Removal of divalent nickel from aqueous solution using blue-green marine algae: adsorption modeling and applicability of various isotherm models

Adsorption of Ni(II) onto blue-green marine algae (BGMA) is investigated under batch condition. Under optimum experimental conditions, the initial Ni(II) metal ion concentration is varied from 25 to 250 ppm and the maximum adsorption capacity of BGMA is found to be 42.056 mg/g. The optimum pH, biomass loading, and an agitation rate on maximum removal of Cu(II) ion are found to be 6, 2 g, and 120 rpm, respectively. 24 h of contact time is allowed to achieve equilibrium condition. All the experiments are carried out at room temperature. The equilibrium experimental data infer that the isotherm is L-shaped. It is the indication of no strong competition between solvent and Ni(II) to occupy the active sites of BGMA. Also, it indicates that the BGMA has a limited sorption capacity for adsorption of Ni(II). The experimental data are tested with various isotherm models; subsequently, the mechanism of adsorption is identified and the characteristic parameters for process design are established. Fritz–Schlunder-V isotherm model is highly significant in establishing the mechanism of adsorption of Ni(II) under the conditions employed in this investigation followed by Freundlich. The q_max of 41.89 mg/g obtained by this model indicates its relevance more precisely with experimental data.     

Identification and characterization of the n‐6 fatty acid‐producing Mucor rouxii native isolate CFR‐G15

In zygomycetes fungi, many Mucor spp. have been known to produce γ‐linolenic acid (GLA) in their biomass. Among 250 soil samples screened, 20 Mucor isolates showed GLA in their mycelial mass under normal cultivation conditions. Sudan Black B was used for screening their qualitative oleaginesity. Among the representative isolates, Mucor sp. CFR‐G15, when grown in a fat‐producing medium, showed a maximum lipid content of 30 ± 1.32% in its mycelia and 14.42 ± 0.74% GLA. By using gene‐specific primers, the 18S rRNA gene and the Δ⁶ DES gene were amplified by PCR technique. The nucleotide sequences of the 18S rRNA and Δ⁶ DES genes exhibited >98% homology with M. rouxii ATCC 24905 (accession nos. AF117923 and AF296076, respectively), suggesting taxonomic identity. The native isolate M. rouxii CFR‐G15 reported in this study was found to be promising for the development of an economical process in the industrial production of GLA.     

Effect of Processing Parameters on Flocculation of Picochlorum oklahomensis

The flocculation process is commonly used to separate suspended solids from water. The microalgae strain, Picochlorum oklahomensis (PO), was investigated for its flocculation characteristics. Efficiencies of biopolymer addition, pH adjustment and electroflocculation for biomass recovery from the culture medium were examined. Flocculation efficiency of PO increased sharply above pH 11 and reached 97 % at pH 13. Chitosan was more effective in flocculating PO cells than sodium alginate and cationic starch. A generalized linear mixed model using a beta distribution for response was utilized for optimization of the chitosan flocculation process variables. Biomass: chitosan ratio, pH and settling time (ST) were the independent variables. There were significant 3-way interactions among the variables. The highest PO flocculation efficiency, 98.4 %, was obtained at biomass: chitosan ratio of 2.78, pH 9 and ST of 12 h. The electroflocculation efficiency improved with increasing current, operation time (OT) and ST. The highest electroflocculation efficiency, 99.74 %, was obtained under the following conditions; 0.8 A (ampere) and 15 min and 12 h, OT and ST, respectively. This study demonstrated that pH adjustment, chitosan addition and electroflocculation were all technically viable methods to flocculate PO cells. However, selection of the most suitable technique and the optimum treatment conditions needs to be based not only on the application of algal biomass, but also on an economic feasibility study.     

Restricted-Range Boraginaceae Species Constitute Potential Sources of Valuable Fatty Acids

The aim of this work was to establish the richness in γ-linolenic acid (GLA, 18:3n-6) and stearidonic acid (SDA, 18:4n-3) of the seed oil of several restricted-range Boraginaceae species, in a search for new valuable oils as advantageous alternatives to the commercially available sources of both polyunsaturated fatty acids. To this end, seeds of selected Boraginaceae species were collected and analyzed. The highest GLA contents (% total fatty acids) were found in the seed oils of Symphytum caucasicum M.Bieb. (22.9 %), Anchusa undulata subsp. undulata (Ten.) Cout. (22.0 %), Anchusa puechii Valdés (20.0 %), Glandora nitida Thomas (19.2 %), Echium pininana Webb & Berth. (17.1 %) and Pentaglottis sempervirens (L.) L. H. Bailey (17.0 %). With regard to SDA, the highest percentage was found in the seed oil of Echium cantabricum (M. Laínz) Fdez. Casas & M. Laínz (14.7 %), followed by Lappula patula (Lehm.) Asch ex Gürke (13.6 %). It is noticeable that several GLA-enriched species stand under a great threat of extinction, thus revealing the importance of the preservation of the natural ecosystems for endangered species.     

Optimization of Lipase-Catalyzed Fractionation of Two Conjugated Linoleic Acid (CLA) Isomers

The separation of two isomers of conjugated linoleic acid is highly significant since each exhibits different biochemical properties. The aim of this study was to investigate and optimize several factors affecting the esterification of l-menthol with the c9,t11-CLA isomer in an organic solvent-free system using lipase from Candida rugosa (Lipase AY-30). D-optimal design with 5 factors and 3 levels were employed to evaluate the effects of synthesis parameters; reaction time (8–24 h), temperature (30–50 °C), enzyme content (2–20 U/ml), substrate molar ratio of conjugated linoleic acid oil to l-menthol (2:1–1:2) and pH (6–8) on esterification of c9,t11-CLA with l-menthol. Based on the analysis of the residual amount of c9,t11-CLA in the free fatty acid fraction after just one-step esterification, the optimum synthesis conditions were as follows: reaction time 23.12 h, temperature 32.65 °C, enzyme amount 135.40 U, molar ratio of CLA oil to l-menthol at 1:1.7 and pH at 7.7; the lowest purity of c9,t11-CLA in free fatty acid fraction based on the total content of c9,t11 and t10,c12-CLA isomers was 8.6 %.     

pH-/temperature-sensitive carboxymethyl chitosan/poly(N-isopropylacrylamide-co-methacrylic acid) IPN: preparation,characterization and sustained release of riboflavin

Dual crosslinked pH-/temperature-sensitive interpenetrating polymer networks (IPN) were prepared by free-radical copolymerization of N-isopropylacrylamide and methylacrylic acid (MAA) using N,N′-methylenebisacrylamide as a crosslinker in carboxymethyl chitosan (which was crosslinked by Ca²⁺) aqueous solution. Scanning electron microscopy was used to observe the morphologies of the IPN at different pH values and temperatures. The effects of MAA content and environmental pH on the “pH-/temperature-induced” phase transition behavior of the IPN hydrogels were investigated. The phase transition temperature was adjusted to 37 °C by changing the MAA content. The effects of drug-loaded content, crosslinking density, environmental pH, and temperature on the drug release behavior of the drug-loaded IPN hydrogel were also explored. Based on results, the hydrogel possessed pH/temperature sensitivity. The swelling ratio and phase translation temperature of the hydrogel were lower at lower pH. These values were lowest at pH 3.0. The release behavior of riboflavin was dependent on preparation condition, environmental pH, and temperature. Drug cumulative release was only 6 % at pH 1.8 for 2 h. The drug cumulative release was 13 % before the drug-loaded hydrogel reached the position with pH 6.8. The drug release rate was higher at lower temperature. Therefore, dual-crosslinked hydrogel holds much potential as a drug site-specific carrier.     

Optimization of Rhamnolipid Production by <Emphasis Type="Italic">P. aeruginosa</Emphasis> Isolate P6

Rhamnolipids are interesting microbial surfactants having great industrial importance. However, the main obstacles towards an economic production of rhamnolipids are low productivity and high raw-material costs. Therefore, this study aimed at optimization of the culture media as well as culture conditions using response surface methodology for maximum rhamnolipid production by Pseudomonas aeruginosa isolate P6, a promising rhamnolipid-producing isolate. The optimum medium for maximum rhamnolipid production was found to be a mineral salts medium with glycerol 2 % v/v as the carbon source. The optimum cultivation conditions using response surface methodology were found to be an incubation temperature of 30 °C, an agitation rate of 250 rpm, an inoculum size of 5 % v/v and unlike most studies, an initial pH of 7.5. The resulting model predicted data points that corresponded well to the experimental values. Optimization resulted in a threefold increase in rhamnolipid production reaching 7.54 g/L. The data are potentially useful for further industrial exploitation of rhamnolipid production by the studied isolate.     

Lipid production by oleaginous Mucorales cultivated on renewable carbon sources

Mortierella isabellina and Cunninghamella echinulata were cultivated on glucose‐, pectin‐, starch‐ and lactose‐based media. Culture on glucose at two initial C/N ratios favored lipid synthesis in the media with increased C/N. Starch was an adequate substrate for both molds, but lipid (in g/g of biomass) was produced in lower quantities compared with the glucose trial. Pectin was inadequate for C. echinulata whereas growth of M. isabellina was satisfactory (8.4 g/L), followed by moderate lipid production. Growth of C. echinulata on lactose was negligible, while that of M. isabellina was notable (9.5 g/L) although lipid in biomass was only 0.36 g/g. Hydrolytic enzymes (α‐amylase, polygalacturonase and β‐galactosidase) activities of both strains seemed to be low enough to saturate their metabolic capabilities. This seemed a major cause for the lower amount of lipid accumulated during growth on complex media compared with that on glucose. Cellular fatty acids of M. isabellina were oleic, linoleic and palmitic acid, while γ‐linolenic acid (GLA) was produced in low quantities. In C. echinulata grown on glucose, lactose or starch, GLA concentration was notable at the beginning and end of culture. Growth on pectin at the first growth steps was accompanied by the production of saturated fatty acids, the amount of which decreased thereafter.     

Equilibrium,Thermodynamic and Kinetic Studies on Biosorption of Mercury from Aqueous Solution by Macrofungus (Lycoperdon perlatum) Biomass

The present research is to investigate the possibility of macrofungus Lycoperdon perlatum biomass, which is an easily available, renewable plant, low-cost, as a new biomass for the removal of mercury (Hg(II)) ions from aqueous solutions. The effects of various parameters like pH of solution, biomass concentration, contact time, and temperature were studied by the using the batch method. The Langmuir model adequately described the equilibrium data. The biosorption capacity of the biomass was found to be 107.4 mg · g^?1 at pH 6. The mean free energy value (10.9 kJ · mol^?1) obtained from the D–R model indicated that the biosorption of Hg(II) onto fungal biomass was taken place via chemical ion-exchange. Thermodynamic parameters showed that the biosorption of Hg(II) onto L. perlatum biomass was feasible, spontaneous, and exothermic in nature. The kinetic results showed that the biosorption of Hg(II) onto fungal biomass followed second-order kinetics. This work also shows that L. perlatum biomass can be an alternative to the expensive materials like ion exchange resins and activated carbon for the treatment of water and wastewater containing mercury ions due to its ability of selectivity and higher biosorption capacity and also being low cost material.     

Effect of Natural and Pretreated Soybean Hulls on Enzyme Production by Trichoderma reesei

The enzyme induction utility of soybean hulls (SBH), consisting in excess of 50 wt% non-starch polysaccharides (NSP) cellulose, hemicellulose, and pectin, was studied through cultivation of the carbohydrase-producing fungus Trichoderma reesei Rut C-30. Shake flask systems of T. reesei were grown in a medium consisting of defatted soybean flour as a nitrogen source and SBH, some of which were untreated and others pretreated by liquid hot water, alkaline, and supercritical carbon dioxide, as carbon source. Cellulase, xylanase, and polygalacturonase activities were measured for the systems, and the natural hull systems were found to yield optimum enzyme production. Controlled batch fermentation experiments were carried out to compare enzyme production resulting from media with Avicel^® (FMC BioPolymer, Philadelphia, PA, USA) versus natural SBH with and without soybean flour as the nitrogen source. Soybean hull fermentations were also performed at several pH levels to observe the effects on enzyme production. Soybean hulls induced comparable levels of cellulase, and higher levels of xylanase and polygalacturonase, than Avicel^®. With SBH, cellulase and xylanase production were enhanced at higher pH levels (6.0), and polygalacturonase was enhanced at lower pH levels (4.0–4.5). Enzyme production was largely unaffected by the presence of soybean flour as the nitrogen source.     

Production of eicosapentaenoic acid bySaprolegnia sp. 28YTF-1

Saprolegnia sp. 28YTF-1, isolated from a freshwater sample, is a potent producer of 5,8,11,14,17-cis-eicosapentaenoic acid (EPA). The fungus used various kinds of carbon sources, such as starch, dextrin, sucrose, glucose, and olive oil for growth, and olive oil was the best carbon source for EPA production. The EPA content reached 17 mg/g dry mycelium (0.25 mg/L) when the fungus was grown in a medium that contained 2.5% olive oil and 0.5% yeast extract, at pH 6.0 and 28°C for 6 d with shaking. Accompanying production of arachidonic acid (AA; 3.2 mg/g dry mycelia, EPA/AA = 5.1) and other ω6 polyunsaturated fatty acids was low. Both EPA content and EPA/AA ratio increased in parallel by lowering growth temperature. Triglyceride was the major mycelial lipid (ca. 84%), but EPA comprised only 2.2% of the total fatty acids of this lipid. About 40% of the EPA produced was found in polar lipids, such as phosphatidylethanolamine (EPA content, 28.2%), phosphatidylcholine (13.6%), and phosphatidylserine (21.2%).     

Effect of Growth Temperature on the Physico-Chemical Characteristics of Fungal Oil

The fungus Fusarium oxysporum f. sp. lini (Bolley) has been found to be a potential source for biotechnological production of oil. When grown as static surface culture in the best synthetic basal medium containing 15% glucose and 0.4% ammonium nitrate as sole carbon and nitrogen sources, respectively, at optimum pH 5.9 and temperature 28°±1°C, it gives 0.4% biomass of high protein (23.7%) and oil (18.9%) content, within a period of 14 days. The oil appears to be of economic importance since it contains appreciable proportions of nutritionally significant unsaturated fatty acids and resembles edible palm oil in composition.     

Peach-palm (Bactris gasipaes Kunth.) waste as substrate for xylanase production by Trichoderma stromaticum AM7

This work investigated the xylanase production by fungi isolates from tropical agroforestry systems using peach-palm industrial waste as a substrate. Trichoderma stromaticum AM7 was the best xylanase producer and there was a 160% increase in xylanase activity after optimizing by the Box–Behnken statistical design. The optimization process demonstrated that the maximum activity occurred at 0.95% nitrogen concentration after 6 days of cultivation at 32°C, which achieved a yield of 1440?U?g^?1 of dry initial substrate. Analysis by scanning electron microscopy showed degradation of the fibers after cultivation. The optimum pH and temperature for xylanase activity were 5.0 and 50°C, respectively. The extensive degradation of the peach-palm waste and xylanase production by T. stromaticum AM7 was due to the combination of a good physicochemical composition of the culture medium and the characteristics of the selected fungus.     

Enzyme-Assisted Aqueous Extraction of Kalahari Melon Seed Oil: Optimization Using Response Surface Methodology

Enzymatic extraction of oil from Kalahari melon seeds was investigated and evaluated by response surface methodology (RSM). Two commercial protease enzyme products were used separately: Neutrase^® 0.8 L and Flavourzyme^® 1000 L from Novozymes (Bagsvaerd, Denmark). RSM was applied to model and optimize the reaction conditions namely concentration of enzyme (20–50 g kg^?1 of seed mass), initial pH of mixture (pH 5–9), incubation temperature (40–60 °C), and incubation time (12–36 h). Well fitting models were successfully established for both enzymes: Neutrase 0.8 L (R ² = 0.9410) and Flavourzyme 1000 L (R ² = 0.9574) through multiple linear regressions with backward elimination. Incubation time was the most significant reaction factor on oil yield for both enzymes. The optimal conditions for Neutrase 0.8 L were: an enzyme concentration of 25 g kg^?1, an initial pH of 7, a temperature at 58 °C and an incubation time of 31 h with constant shaking at 100 rpm. Centrifuging the mixture at 8,000g for 20 min separated the oil with a recovery of 68.58 ± 3.39%. The optimal conditions for Flavourzyme 1000 L were enzyme concentration of 21 g kg^?1, initial pH of 6, temperature at 50 °C and incubation time of 36 h. These optimum conditions yielded a 71.55 ± 1.28% oil recovery.     

Typoselectivity of Crude Geobacillus sp. T1 Lipase Fused with a Cellulose-Binding Domain and Its Use in the Synthesis of Structured Lipids

Typoselectivity of crude CBD-T1 lipase (Geobacillus sp. T1 lipase fused with a cellulose binding domain) was investigated. Multi-competitive reaction mixtures including a set of n-chain fatty acids (C8:0, C10:0, C12:0, C14:0, C18:1 n-9, C18:2 n-6 and C18:3 n-3) and tripalmitin-enriched triacylglycerols were studied in hexane. The crude CBD-T1 lipase discriminated strongly against C18:1 n-9 [competitive factor (α) = 0.23] and showed the highest preference for C8:0 (α = 1). Utilizing the catalytic properties of crude CBD-T1 lipase, acidolysis of soybean oil with C8:0 was selected as a model reaction to investigate the ability of the lipase to produce MLM-type (medium-long-medium) structured lipids. Several reaction parameters (added water amount, reaction temperature, substrate molar ratio and reaction time) examined for incorporating C8:0 into soybean oil, the optimum conditions were: 1:3 (soybean oil/C8:0) of molar ratio, 3 mL of hexane, 50 °C of temperature, 48 h of reaction time, 20 % of crude CBD-T1 lipase (w/w total substrates), and 7.5 % of water (w/w enzyme). Under these conditions, the incorporation of C8:0 was 29.6 mol%. The results suggest that crude CBD-T1 lipase, which showed different fatty acid specificity profiles, is a potential biocatalyst for the modification of fats and oils.     

Lipidomics Reveals Mitochondrial Membrane Remodeling Associated with Acute Thermoregulation in a Rodent with a Wide Thermoneutral Zone

Mongolian gerbils (Meriones unguiculatus) have high physiological flexibility in response to acute temperature changes, and have the widest thermoneutral zone (TNZ, 26.5–38.9 °C) reported among small mammals. At the upper critical temperature (T _uc, 38.9 °C), body temperatures of gerbils were significantly increased (39–41 °C) while metabolic rates were maintained at the basal level. In contrast, below the lower critical temperature (T _lc, 26.5 °C), metabolism was elevated and body temperature stable. Rapid changes in mitochondrial membrane lipidome were hypothesized to play an important role during acute thermoregulation of gerbils. Taking advantage of a recent lipidomic technique, we examined changes in the membrane phospholipids environment and free fatty acids (FFA) production in mitochondria between 38 and 27 °C (in the TNZ), and between 27 and 16 °C (below the TNZ). At 38 °C, acute heat stress elicited distinct remodeling in mitochondrial membrane lipidome which related to a potential decrease in mitochondrial respiration and membrane fluidity compared to 27 °C. At 16 °C, a sharply decreased unsaturation index and increased chain lengths were detected in mitochondrial FFA production both in muscle and brown adipose tissue. Our results suggest that mitochondrial membrane lipid remodeling may stabilize membrane function and activity of respiration related membrane protein to maintain a stable metabolic rate at T _uc, and improve heat production by decomposing less fluid fatty acid conjugates of membrane lipids under acute cold exposure. These data therefore imply an important role of membrane remodeling during acute thermoregulation in a non-hibernating endotherm.     

Simultaneous saccharification and fermentation of wheat bran flour into ethanol using coculture of amylotic Aspergillus niger and thermotolerant Kluyveromyces marxianus

Studies on simultaneous saccharification and fermentation (SSF) of wheat bran flour, a grain milling residue as the substrate using coculture method were carried out with strains of starch digesting Aspergillus niger and nonstarch digesting and sugar fermenting Kluyveromyces marxianus in batch fermentation. Experiments based on central composite design (CCD) were conducted to maximize the glucose yield and to study the effects of substrate concentration, pH, temperature, and enzyme concentration on percentage conversion of wheat bran flour starch to glucose by treatment with fungal α-amylase and the above parameters were optimized using response surface methodology (RSM). The optimum values of substrate concentration, pH, temperature, and enzyme concentration were found to be 200 g/L, 5.5, 65°C and 7.5 IU, respectively, in the starch saccharification step. The effects of pH, temperature and substrate concentration on ethanol concentration, biomass and reducing sugar concentration were also investigated. The optimum temperature and pH were found to be 30°C and 5.5, respectively. The wheat bran flour solution equivalent to 6% (w/V) initial starch concentration gave the highest ethanol concentration of 23.1 g/L after 48 h of fermentation at optimum conditions of pH and temperature. The growth kinetics was modeled using Monod model and Logistic model and product formation kinetics using Leudeking-Piret model. Simultaneous saccharificiation and fermentation of liquefied wheat bran starch to bioethanol was studied using coculture of amylolytic fungus A. niger and nonamylolytic sugar fermenting K. marxianus.