Immobilization of Candida rugosa lipase on hexagonal mesoporous silicas and selective esterification in nonaqueous medium

Candida rugosa lipases (CRLs) immobilized by physical adsorption, cross-linking and covalent binding methods on a MSU-H type mesoporous silica previously modified organically by different strategies, respectively, were examined as biocatalysts for esterification of conjugated linoleic acid (CLA) and ethanol in nonaqueous medium. MSU-H silica was modified by nonionic surfactant of triblock copolymer Pluronic P123, amino-functionalization and glutaraldehyde-grafting and confirmed by FT-IR analysis. Interaction mechanisms of CRLs and supports involve covalent and non-covalent interactions including electrostatic repulsion and hydrophobic interaction at pH 7. The immobilized CRLs containing surfactant were prepared by cross-linking via entrapping CRL aggregates inside the pores of silicas. The surfactant located inside the silicas could interfacially activate the immobilized CRLs and favored catalytic esterification. The biocatalyst containing 38 wt.% of surfactant afforded 1111.1 U/mg of specific activity about eight times higher than soluble CRL, and maximal 56.7% of total CLA esterification with 96.5% of 9c, 11t-CLA isomer esterification degree. The immobilized-CRL with 64.5 mg/g of loading amount of protein exhibited maximal hydrolytic activity of 2945.3 U/g-support for grafting glutaraldehyde. This derivative showed a high level of esterification activity and operational stability and remained 43.2–46.9% of total esterification for 32 h consecutive four runs.     

Synthesis and evaluation of novel azetidine analogs as potent inhibitors of vesicular [3H]dopamine uptake

Lobelane analogs that incorporate a central piperidine or pyrrolidine moiety have previously been reported by our group as potent inhibitors of VMAT2 function. Further central ring size reduction of the piperidine moiety in lobelane to a four-membered heterocyclic ring has been carried out in the current study to afford novel cis-and trans-azetidine analogs. These azetidine analogs (15a–15c and 22a–22c) potently inhibited [³H]dopamine (DA) uptake into isolated synaptic vesicles (K_i ? 66 nM). The cis-4-methoxy analog 22b was the most potent inhibitor (K_i = 24 nM), and was twofold more potent that either lobelane (2a, K_i = 45 nM) or norlobelane (2b, K_i = 43 nM). The trans-methylenedioxy analog, 15c (K_i = 31 nM), was equipotent with the cis-analog, 22b, in this assay. Thus, cis- and trans-azetidine analogs 22b and 15c represent potential leads in the discovery of new clinical candidates for the treatment of methamphetamine abuse.     

Esterification and transesterification reactions catalysed by addition of fermented solids to organic reaction media

We report for the first time both the production of the lipase of Burkholderia cepacia in solid-state fermentation and the biocatalysis of esterification and transesterification reactions through the direct addition of the lyophilised fermented solids to organic reaction media. B. cepacia produced a lipolytic activity equivalent to 108 U of pNPP-hydrolysing activity per gram of dry solids after 72 h growth on corn bran with 5% (v/w) commercial corn oil as the inducer. The fermented solid material was lyophilised and added directly to the reaction medium in esterification and transesterification reactions. A factorial design was used to study the effects on esterification of temperature, alcohol-to-acid molar ratio and amount of lipolytic activity added. All three variables affected the ester yield significantly, with the amount of enzyme being most important. A 94% ester yield was obtained at 18 h at 37 °C, with an alcohol-to-acid molar ratio of 5:1 and 60 U of added lipolytic activity. For the transesterification reaction, a factorial design was undertaken with the variables being the alcohol-to-acid molar ratio and the added lipolytic activity. Ester yields of over 95% were obtained after 120 h. Our results suggest that biocatalysis using direct addition of fermented solids to organic reaction medium should be further explored.     

Vacuum level effects on knee contact force for unilateral transtibial amputees with elevated vacuum suspension

The elevated vacuum suspension system (EVSS) has demonstrated unique health benefits for amputees, but the effect of vacuum pressure values on knee contact force (KCF) is still unclear. The objective of this study was to investigate the effect of vacuum levels on KCF for unilateral transtibial amputees (UTA) using the EVSS. Three-dimensional gait was modeled for 9 UTA with five vacuum levels (0–20 inHg [67.73 kPa], 5 inHg [16.93 kPa] increments) and 9 non-amputees based on kinematic and ground reaction force data. The results showed that the vacuum level effects were significant for peak axial KCF, which had a relatively large value at 0 and 20 inHg (67.73 kPa). The intact limb exhibited a comparable peak axial KCF to the non-amputees at 15 inHg (50.79 kPa). At moderate vacuum levels (5 inHg [16.93 kPa] to 15 inHg [50.79 kPa]), co-contraction of quadriceps and hamstrings at peak axial KCF was similar for the intact limb, but was smaller for the residual limb comparing with the non-amputees. The intact limb showed a similar magnitude of quadriceps and hamstrings force at 15 inHg (50.79 kPa) to the non-amputees, but the muscle coordination patterns varied between the residual and intact limbs. These findings indicate that a proper vacuum level may partially compensate for the lack of ankle plantarflexor and reduce the knee loading. Of the tested vacuum levels, 15 inHg (50.79 kPa) appears most favorable, although additional analyses with more amputees are suggested to confirm these results prior to establishing clinical guidelines.     

Synthesis and anti-proliferative activity of aromatic substituted 5-((1-benzyl-1H-indol-3-yl)methylene)-1,3-dimethylpyrimidine-2,4,6(1H,3H,5H)-trione analogs against human tumor cell lines

Based on previous SAR studies on N-benzylindole and barbituric acid hybrid molecules, we have synthesized a series of aromatic substituted 5-((1-benzyl-1H-indol-3-yl)methylene)-1,3-dimethylpyrimidine-2,4,6(1H,3H,5H)-trione analogs (3a–i) and evaluated them for their in vitro growth inhibition and cytotoxicity against a panel of 60 human tumor cell lines. Compounds 3c, 3d, 3f and 3g were identified as highly potent anti-proliferative compounds against ovarian, renal and breast cancer cell lines with GI₅₀ values in low the nanomolar range. The 4-methoxy-N-benzyl analog (3d) was the most active compound with GI₅₀ values of 20 nM and 40 nM against OVCAR-5 ovarian cancer cells and MDA-MB-468 breast cancer cells, respectively. Two other analogs, 3c (the 4-methyl-N-benzyl analog) and 3g (the 4-fluoro-N-benzyl analog) exhibited equimolar potency against MDA-MB-468 cells GI₅₀ = 30 nM). Analog 3f (the 4-chloro-N-benzyl analog) exhibited a GI₅₀ value of 40 nM against renal cancer cell line A498. These results suggest that aromatic substituted N-benzylindole dimethylbarbituric acid hybrids may have potential for development as clinical candidates to treat a variety of solid tumors.     

Design,synthesis and aphicidal activity of N-terminal modified insect kinin analogs

The insect kinins are a class of multifunctional insect neuropeptides present in a diverse variety of insects. Insect kinin analogs showed multiple bioactivities, especially, the aphicidal activity. To find a biostable and bioactive insecticide candidate with simplified structure, a series of N-terminal modified insect kinin analogs was designed and synthesized based on the lead compound [Aib]-Phe-Phe-[Aib]-Trp-Gly-NH₂. Their aphicidal activity against the soybean aphid Aphis glycines was evaluated. The results showed that all the analogs maintained the aphicidal activity. In particular, the aphicidal activity of the pentapeptide analog X Phe-Phe-[Aib]-Trp-Gly-NH₂ (LC₅₀ = 0.045 mmol/L) was similar to the lead compound (LC₅₀ = 0.048 mmol/L). This indicated that the N-terminal protective group may not play an important role in the activity and the analogs structure could be simplified to pentapeptide analogs while retaining good aphicidal activity. The core pentapeptide analog X can be used as the lead compound for further chemical modifications to discover potential insecticides.     

The effect of level of forage and oil supplement on biohydrogenation intermediates and bacteria in continuous cultures

The objective of this study was to investigate how level of forage and oils in ruminant animals’ diet affect selected strains of ruminal bacteria believed to be involved in biohydrogenation (BH). Four continuous culture fermenters were used in 4 × 4 Latin square design with a 2 × 2 factorial arrangement over four consecutive periods of 10 days each. The experimental diets used in this study were: high forage diet (700:300 g/kg (DM basis) forage to concentrate; HFC), high forage with oil supplement (HFO), high forage diet (300:700 g/kg (DM basis) forage to concentrate; LFC), and high forage with oil supplement (HFO). The oil supplement was a blend of fish oil (FO) and soybean oil (SBO) added at 10 and 20 g/kg DM, respectively. Acetate concentration was greater (P<0.01) with the high forage diets whereas propionate concentration was greater (P<0.02) with the low forage diets and both decreased (P<0.05) with oil supplementation. The concentrations of t11 C18:1 (vaccenic acid, VA) and c9t11 conjugated linoleic acid (CLA) were greater (P<0.01) with the high than the low forage diets and concentrations increased (P<0.01) with oil supplementation particularly when added with the high forage diet. The concentrations of t10 C18:1 and t10c12 CLA were greater (P<0.01) with the low than the high forage diets and concentrations increased (P<0.01) with oil supplementation particularly when added with the low forage diet. The DNA abundance of Butyrivibrio fibrisolvens, Ruminococcus albus, Ruminococcus flavefaciens, Anaerovibrio lipolytica and Butyrivibrio proteoclasticum were greater (P<0.03) with the high than the low forage diets. Oil supplementation reduced (P<0.05) the DNA abundance only for R. flavefaciens, B. fibrisolvens and R. albus especially when added with the high forage diet. Results from this study suggest that the greater trans fatty acids (FA) production seen with the high forage diets may be related to greater activity of B. fibrisolvens, R. flavefaciens and R. albus, and B. proteoclasticum appears to play a minor role in the production of C18:0 from trans C18:1.     

Effects of dietary conjugated linoleic acid on metabolic status,BW and expression of genes related to lipid metabolism in adipose tissue of dairy cows during peripartum

Conjugated linoleic acid (CLA) dietary supplementation reduces milk fat content and yield, but its effects on lipid metabolism and energy status remain controversial. The objective of this study was to investigate the effects of dietary CLA on adipose tissue (AT) mRNA abundance of genes related to lipid metabolism, plasma indicators of metabolic status, body condition score (BCS) and BW changes in dairy cows. Sixteen multiparous Holstein cows (3.2 ± 1.4 lactations, 615 ± 15 kg BW) were randomly assigned to treatments: 1) CLA; rumen-protected CLA (75 g/d) or 2) Control; equivalent amount of rumen inert fatty acid (FA) as the previous diet (78 g/d), from − 20.2 ± 3.2 (mean ± SEM) to 21 d relative to calving (d 0). Subcutaneous AT was biopsied from the tail-head region at d 21 to determine the mRNA abundance of genes related to lipid metabolism. Blood samples were collected at − 20.2 ± 3.2, 0, 7, 14 and 21 d relative to calving to determine plasma non-esterified fatty acids (NEFA), beta-hydroxybutyrate (BHBA), insulin and glucose. Conjugated linoleic acid decreased milk fat yield and milk fat content by 15 and 16%, respectively. Cows fed CLA had lower plasma NEFA and BHBA and greater glucose and insulin concentrations (P < 0.05). Mean BCS at 21 d postpartum was greater (P < 0.01; 2.89 vs 2.25), and BCS loss from the day of enrollment to 21 d postpartum was reduced (P < 0.01; − 0.13 vs − 0.64) in the CLA group. The expression of acylcoenzyme A oxidase, carnitine palmitoyltransferase 1A, hormone-sensitive lipase, β2 adrenergic receptor and acetyl-CoA carboxylase was downregulated by CLA supplementation, whereas the expression of sterol regulatory element binding protein, lipoprotein lipase and peroxisome proliferator-activated receptor gamma was upregulated (P < 0.01). In summary, CLA-supplemented cows showed signs of better metabolic status and less severe fat mobilization. Moreover, CLA increased mRNA abundance of genes related to lipogenesis and decreased mRNA abundance of genes related to FA oxidation and lipolysis in the AT of dairy cows during early lactation.     

Novozym 435-catalyzed 1,3-diacylglycerol preparation via esterification in t-butanol system

1,3-Diacylglycerol (1,3-DAG) oil has beneficial effects on suppressing the accumulation of body fat and preventing the increase of body weight. So, more and more attention has been paid to enzyme-mediated 1,3-DAG production in recent years due to its mild reaction condition and safe products. In this work, t-butanol was adopted as the reaction medium for lipase-catalyzed esterification for 1,3-DAG preparation. In t-butanol system, the harmful effects on lipase caused by glycerol could be eliminated completely, so the high enzymatic activity was maintained and the stability of the lipase could be improved significantly. Under the optimum conditions (60 °C, 1.00 g Novozym 435, 2.5:1 molar ratio of oleic acid to glycerol (10.0 g oleic acid and 1.3 g glycerol) and 6.0 g t-butanol), 1,3-DAG concentration of 40% was achieved and Novozym 435 can be used 100 times. A simplified model based on Ping-Pong Bi-Bi with substrate competitive inhibition by glycerol was found to fit the initial rate data and the kinetics parameters were evaluated by nonlinear regression analysis.     

Product-controlled steady-state kinetics between cytochrome aa3 from Rhodobacter sphaeroides and equine ferrocytochrome c analyzed by a novel spectrophotometric approach

Cytochrome c oxidase (CcO) catalyzes the reduction of molecular oxygen to water using ferrocytochrome c (cyt c^2 +) as the electron donor. In this study, the oxidation of horse cyt c^2 + by CcO from Rhodobacter sphaeroides, was monitored using stopped-flow spectrophotometry. A novel analytic procedure was applied in which the spectra were deconvoluted into the reduced and oxidized forms of cyt c by a least-squares fitting method, yielding the reaction rates at various concentrations of cyt c^2 + and cyt c^3 +. This allowed an analysis of the effects of cyt c^3 + on the steady-state kinetics between CcO and cyt c^2 +. The results show that cyt c^3 + exhibits product inhibition by two mechanisms: competition with cyt c^2 + at the catalytic site and, in addition, an interaction at a second site which further modulates the reaction of cyt c^2 + at the catalytic site. These results are generally consistent with previous reports, indicating the reliability of the new procedure. We also find that a 6 × His-tag at the C-terminus of the subunit II of CcO affects the binding of cyt c at both sites. The approach presented here should be generally useful in spectrophotometric studies of complex enzyme kinetics. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).     

Influence of organic systems on milk fatty acid profile and CLA in goats

The effect of organic system on the fatty acid profile of milk and CLA content was evaluated using 30 pregnant pluriparous goats, divided into two homogeneous groups (S and O) of 15 goats each. Group S was housed in a stable and received alfalfa hay as forage, while group O was raised according EC Regulation 834/2007 and led to pasture. After the kids weaning, goats were milked twice a day for 5 months. Daily milk yield was recorded and, monthly, representative milk samples from the two daily milkings were analysed for chemical and fatty acid profile. Average milk yield did not differ statistically between the groups. The goats of the O group had significantly higher fat content in milk than those of group S (65.9 g/day vs. 54.3 g/day, P < 0.01). Among milk fatty acids, organic system significantly affected the percentages of C18:1 c9, C18:1 t11, linoleic acid, alpha-linolenic acid, monounsaturated fatty acid and polyunsaturated fatty acid. Organic system highly influenced the c9 t11 conjugated linoleic acid (CLA) (0.810 g/100 g of fat vs. 0.542 g/100 g of fat, for groups O and S, respectively, P < 0.01), t10 c12 CLA (0.041 g/100 g of fat vs. 0.024 g/100 g of fat, for groups O and S, respectively, P < 0.01) and ∑CLA (0.87 g/100 g of fat vs. 0.58 g/100 g of fat for groups O and S, respectively, P < 0.01) concentrations of milk.     

Kinetic study on enzymatic esterification of tuna fish oil fatty acids with butanol

Docosahexaenoic acid (DHA) is an important polyunsatured fatty acid (PUFA) which can be purified from tuna fish oil fatty acids by selective enzymatic esterification. The present paper investigates the kinetic study for selective esterification of tuna fish oil fatty acids with butanol catalyzed by Rhizopus oryzae lipase (ROL) in biphasic solvent system. Under the most suitable reaction conditions, 76.2% esterification was achieved in 24 h. Different kinetic models for esterification given by Segel [1], Oliveira et al. [2], Gogoi et al. [3], and Kraai et al. [4] were tested for fitting the esterification data and the model given by Oliveira et al. [2] was found to be most suitable. The model given by Prazeres et al. [5] for hydrolysis was also tested for esterification and the model with second order product inhibition was found to provide better match between the predicted and experimental values than that of model by Oliveira et al. [2]. The kinetic model was fitted using MATLAB^® to determine the best kinetic parameters. The average value of kinetic constants using the model given by Prazeres et al. were estimated as K_m = 23.6 μmoles FFA/ml, K_i1 = 4.6 × 10⁻⁵ μmoles FFA/mg enzyme h, K_i2 = 0.0062 μmoles FFA/mg enzyme h and K₂ = 149.5 μmoles FFA/mg enzyme h.     

Effects of dietary sources of vegetable fats on performance of dairy ewes and conjugated linoleic acid (CLA) in milk

Two experiments were carried out to study the effects of supplementing the ration of lactating ewes with vegetable fats (sunflower oil, SO or hydrogenated palm oil, HPO; HIDROPALM^®) on diet digestibility, milk yield and milk composition, and on the concentration of the conjugated linoleic acid (CLA) C18:2 cis-9 trans-11 and C18:1 trans-11 (vaccenic acid, VA) and other main fatty acids in milk fat. Treatments involved a control diet, without added oil, and 2 diets supplemented with either 12 g/kg SO or 12 g/kg HPO on a dry matter (DM) basis. In the first experiment, 6 non-pregnant, non-lactating Lacaune ewes were used following a 3 × 3 replicated Latin Square design. Addition of vegetable fat supplement to the diet increased digestibility of DM, organic matter (OM) and crude protein (CP), but did not affect that of the ether extract (EE), neutral detergent fibre (NDF) or acid detergent fibre (ADF). In the second experiment, 60 Lacaune dairy ewes mid-way through lactation (120 ± 12 days in milk, 0.98 ± 0.03 kg/day average milk yield) were divided into three equal-sized groups each of which was assigned to one of the three experimental diets for 4 weeks. Compared with the control treatment, supplementation with HPO increased milk yield and energy-corrected milk. But neither vegetable fat supplement modified percentages of fat and protein in milk. Supplementation with HPO increased C14:1, C16:1 and C16:0 content and reduced C18:0 and C18:1 cis-9 content in milk fat. Supplementation with SO increased the VA content in milk fat by 36% and that of cis-9 trans-11 CLA by 29% in comparison with the control diet. Supplementation with HPO led to milk fat with 15% more cis-9 trans-11 CLA than control milk. In conclusion, adding a moderate dose of HPO or SO to the diets increased CLA concentration in milk fat. Nevertheless, supplementation with SO was more effective than HPO in increasing CLA concentration in milk fat and reducing the atherogenicity index, improving milk quality from the human health standpoint.     

Rumen microbial responses in fermentation characteristics and production of CLA and methane to linoleic acid in associated with malate or fumarate

An in vitro incubation in batch was conducted to investigate the effect of propionate precursor (malate or fumarate) on fermentation characteristics, and production of CLA and methane by rumen microbes when incubated with linoleic acid (C_18:2). Sixty milligrams of C_18:2 alone (LA), 60 mg C_18:2 with 24 mM malic acid (M-LA), or 60 mg C_18:2 with 24 mM fumaric acid (F-LA) was added to 150 ml culture solution consisting of 75 ml strained rumen fluid and 75 ml McDougall's artificial saliva. Culture solution for incubation was also made without malate, fumarate, and C_18:2 (control). Two grams of feed consisting of 1.4 g concentrate and 0.6 g ground alfalfa (DM basis) was also added to the culture solution of each treatment. An in vitro incubation in batch was made anaerobically in a shaking incubator for up to 12 h at 39 °C.The pH of the culture solution was increased (P<0.0001) in M-LA or F-LA treatments from 3 h to 12 h compared with the control and LA treatments. At 12 h incubation, the concentration of total VFA in the culture solution was higher (P<0.01) in M-LA and F-LA than in control and LA treatments. Concentration of C₃ by M-LA and F-LA was increased at 3 h (P<0.01), 6 h (P<0.01) and 12 h (P<0.01) compared with control and LA. However, no difference in C₃ concentration was observed between control and LA, or between M-LA and F-LA. Accumulated total gas produced for up to 12 h incubation was increased (P<0.01) by M-LA or F-LA compared with the control. Accumulated total methane produced for up to 12 h incubation, however, was greatly reduced (P<0.01) by all the supplements compared with control, and its production from M-LA or F-LA was smaller than the LA. The M-LA or F-LA also increased (P<0.05–<0.001) the concentrations of cis9, trans11-CLA for all incubation times and trans10, cis12-CLA at 1 h (P<0.01), 3 h (P<0.05), and 12 h (P<0.05) incubation times compared with LA.It can be concluded that malate and fumarate, as propionate precursors, act as alternative electron sinks and may compete with CH₄ generation and bio-hydrogenation of C_18:2 in the utilization of metabolic H₂. The highest CLA concentration at the early incubation stage (1 h) was accompanied by reduced propionate proportion. Linoleic acid is also considered one of the potential alternatives to suppress CH₄ generation.     

Synthesized tyrosyl hydroxyphenylacetate,a novel antioxidant,anti-stress and antibacterial compound

New tyrosyl ester derivative, a naturally occurring phenol with interesting biological properties, has been synthesized in good yield by a direct esterification of tyrosol (Ty) with p-hydroxyphenylacetic acid (p-HPA) using Candida antarctica lipase as a catalyst. The response surface methodology was used to modulate the effects of the enzyme amount (10–50 mg), the tert-butanol/hexane (v/v) ratio (0.16–0.84), the temperature (35–55 °C) and the reaction time (15–45 h) on the tyrosyl hydroxyphenylacetate (Ty-HPA) conversion yield. Under the optimal predicted conditions (enzyme amount: 10 mg, solvents volume ratio 0.16, reaction temperature; 45 °C and 34 h of incubation), a high conversion yield of 79.33 ± 4% was reached. The obtained ester was purified and characterized by NMR, LC/MS and FT-IR methods. ABTS free radical quenching potency demonstrated that the esterified tyrosol (Ty-HPA) was more effective than the natural separated antioxidants: Ty and p-HPA. Furthermore, when used at a non-cytotoxic concentration (100 μM), tyrosyl ester showed significant effectiveness in preventing iron-induced oxidative stress in blood cells compared to the two separated compounds. The antibacterial activity of Ty, p-HPA, mixed solution of Ty + p-HPA and Ty-HPA was performed by determining the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) using a micro-well dilution method. Compared to the separated substrates, synthesized ester exhibits the most antibacterial effect mainly against Gram+ bacteria.     

Vanillin bioproduction from alkaline hydrolyzate of corn cob by Escherichia coli JM109/pBB1

Hydrolysis of corn cob performed for 6 h with 0.5 N NaOH at solid/liquid ratio of 0.084 g/g allowed obtaining a hydrolyzate containing 1171 ± 34 mg/l ferulic acid and 2156 ± 63 mg/l p-coumaric acid that was used as a medium for vanillin bioproduction by the engineered strain Escherichia coli JM109/pBB1. Aiming at maximizing vanillin bioproduction, the effects of medium heat sterilization, one-stage or two-stage pre-cultivation, adaptation of the microorganism to the hydrolyzate and inoculum biomass level were investigated. Biomass pre-cultivated once in unsterilized hydrolyzate was able to effectively convert ferulic and p-coumaric acids to a mixture of vanillin, vanillic acid and vanillyl alcohol provided with the typical vanilla flavor. At initial biomass concentration of 0.5 g_DM/l, maximum values of vanillin concentration (239 ± 15 mg/l), vanillin yield on consumed ferulic acid (0.66 ± 0.03 mol/mol) and vanillin volumetric productivity (10.9 ± 0.7 mg/lh) were obtained after 22 h.     

Enzymatic purification of dihomo-γ-linolenic acid from Mortierella single-cell oil

Purification of dihomo-γ-linolenic acid (20:3n−6; DGLA) from a single-cell oil containing 39 wt.% DGLA was attempted. The process comprised: (i) non-selective hydrolysis of the oil to prepare a mixture of free fatty acids (FFAs); (ii) urea adduct fractionation of the FFA mixture to remove saturated fatty acids; and (iii) repeated selective esterification of the resulting mixture with two kinds of lipases. In the first step, Candida rugosa lipase (Lipase-OF from Meito Sangyo Co. Ltd., Aichi, Japan) was the most effective for preparation of the FFAs from the oil; 99% hydrolysis was achieved by the reaction at 40 °C for 72 h. Urea adduct fractionation of the FFA mixture removed almost completely behenic and lignoceric acids, and the content of DGLA increased from 39 to 55 wt.%. The FFAs were esterified with 2 mol equivalent of lauryl alcohol (LauOH) using C. rugosa lipase (Lipase-AY from Amano Enzyme Inc., Aichi, Japan). In consequent, DGLA was enriched to 86 wt.% in the unesterified FFA fraction. To further increase the content of DGLA, the esterification was repeated using the same lipase. Accordingly, the content of DGLA increased to 91 wt.%, but the preparation was contaminated with 3.3 wt.% γ-linolenic acid. This contaminant was removed finally by selective esterification of the FFAs with 2 mol equivalent of LauOH using Pseudomonas aeruginosa lipase. A series of procedures purified DGLA to 95 wt.% in a yield of 51% of the initial content in the single-cell oil.     

Effect of dietary conjugated linoleic acid on serum levels of N2O5 and l-citrulline in goat kids

Forty new born male kids were allotted into two groups for the evaluation of the effects of dietary conjugated linoleic acid (CLA) on nitric oxide (NO) and l-citrulline levels in serum. The control group received no supplement CLA and the CLA group received 20 g/kg milk DM of CLA from birth to 1-month old. The kids were fed colostrum for 2 days and milk replacer from days 3 to 29. Blood samples (n = 40) were taken at 1, 8, 15, 22, and 29 days of age. N₂O₅ (nitrite + nitrate) concentration in serum ranged from 21.93 to 26.15 and 34.90 to 40.59 μmol in control and CLA kids. The l-citrulline values ranged from 0.30 to 0.40 and 15.54 to 19.81 μmol in control and CLA kids.     

Synthesis of structured lipids by two enzymatic steps: Ethanolysis of fish oils and esterification of 2-monoacylglycerols

This paper studies the synthesis of structured triacylglycerols (STAGs), rich in polyunsaturated fatty acids (PUFAs) by a two-step enzymatic process: (i) alcoholysis of fish oils (cod liver and tuna oils) with ethanol to obtain 2-monoacylglycerols (2-MAGs), catalyzed by 1,3 specific lipases and (ii) esterification of these 2-MAGs with caprylic acid (CA, 8:0), also catalyzed by a 1,3 specific lipase, to produce STAGs of structure CA–PUFA–CA. As regards the alcoholysis reaction, three factors have been studied: the influence of the type of lipase used (lipase D from Rhizopus oryzae, immobilized on Accurel MP1000, and Novozym 435 from Candida antarctica), the operational mode of a stirred tank reactor (STR operating in discontinuous and continuous mode) and the intensity of treatment (IOT = lipase amount × reaction time/oil amount). Although higher 2-MAG yields were obtained with lipase D, Novozym 435 was selected due to its greater stability in the operational conditions. The highest 2-MAG yield (63%) was attained in the STR operating in discontinuous mode at an IOT of 1 g lipase × h g oil^?1 (at higher IOT the 2-MAGs were degraded to glycerol). This system was scaled up to 100 times the initial volume, achieving a similar yield (65%) at the same IOT. The 2-MAGs in the final alcoholysis reaction mixture were separated from ethyl esters by solvent extraction using solvents of low toxicity (ethanol and hexane); the 2-MAG recovery yield was over 90% and the purity was approximately 87–90%. Regarding the esterification of the 2-MAGs, the following factors were studied: the influence of the lipase type used, the presence or absence of solvent (hexane) and the reaction time or intensity of treatment (IOT = lipase amount × reaction time/2-MAG amount). Of the five lipases tested, the highest STAG percentages (over 90%) were attained with lipases D and DF, immobilized on Accurel MP1000. These STAGs contain 64% CA, of which 98% is at positions 1 and 3. Position 2 contains 5% CA and 45% PUFAs, which means that all the PUFAs that were located at position 2 in the original oil remain in that position in the final STAGs. The lipase D immobilized on Accurel MP1000 is stable in the operational conditions used in the esterification reaction. Finally the purification of STAGs was carried out by neutralization of free fatty acids with hydroethanolic solution of KOH and extraction of STAGs with hexane. By this method purity was over 95% and separation yields were about 80%.     

Seasonal variations in fatty acid composition of pasture forage plants and CLA content in ewe milk fat

The relations between fatty acids (FAs) composition of pasture forage plants and the content of conjugated linoleic acid (CLA) as a total content of cis-9, trans-11 + trans-7, cis-9 + trans-8, cis-10 CLA isomers in ewes’ milk fat during natural pasture season (April–September) were investigated. The extracts of ewes’ milk fat samples as well as the pasture samples were analyzed for fatty acid composition by capillary gas chromatography with flame ionization and mass spectrometric detection. α-Linolenic, linoleic, and palmitic acids were predominant in pasture plants, and their contents varied during pasture season. The most abundant and most varied fatty acid compound in pasture plants was α-linolenic acid. Its content significantly decreased from 62% to 39% (of total FA) (P < 0.001) from May to August, and subsequently it slightly (57%) increased from August to September (P < 0.05), compared with the beginning of pasture season. Similarly, the content of CLA in ewes’ milk fat decreased from 2.4% in May to 1.3% in August (P < 0.001), and subsequently it rose to 2.6% in September (P < 0.001). The α-linolenic/linoleic acid ratio in the pasture sample decreased from 4.36 in May to 1.97 in August (P < 0.001), and subsequently it increased to 3.14 in September (P < 0.001); thus, it reached the level approaching to that at the beginning of pasture season. The pasture seasonal variations in the ratio were directly proportional to the corresponding content of CLA and indirectly proportional to the ratio in ewes’ milk fat. The results suggest that the seasonal variations in CLA content in ewes’ milk fat are related primarily to the seasonal variation in α-linolenic acid content in grass lipids.