Solvent engineering applied to lipase-catalyzed glycerolysis of triolein

Solvent engineering was applied to lipase-catalyzed glycerolysis of triolein for the selective synthesis of monoolein and diolein. The effect of different binary mixtures of n-hexane and 2-methyl-2-butanol (2M2B) on the selective production of mono- or diacylglyceride was established. Conditions for high selectivity toward monoolein synthesis were enhanced from 10.6 mol% in pure n-hexane to 64 mol% in 2M2B. On the contrary, the highest production of diolein, corresponding to 62 mol%, was achieved in n-hexane. Concerning triolein conversion, the best results were obtained in 100% 2M2B, with a conversion of 75%. The effect of the n-hexane/2M2B ratio on diolein regioisomer production during triolein glycerolysis was also evaluated. Two different profiles of diolein regioisomers were observed as a function of solvent composition: Although the production of the 1,2-diolein isomer was favored as the proportion of 2M2B in n-hexane was increased, the 1,3-isomer was preferentially synthesized in reactions where n-hexane was the predominant solvent. When 100% n-hexane was used as a solvent, 1,3-diolein comprised 72 mol% of the total diolein population (58 mM). On the contrary, when the reaction was carried out in 100% 2M2B, the total concentration of diolein was lower (21 mM) but the 1,2-diolein regioisomer was preferentially formed (89%). These results were explained as a consequence of the different extents of hydrolysis-synthesis reactions involved in the glycerolysis process, which are strongly dependent on solvent mixtures and water concentration. Finally, some advantages of the use of binary mixtures of solvents compared with other strategies applied to glycerolysis reactions are discussed.     

Quantitative determination of Tri-, Di-,monooleins and free oleic acid by the thin layer chromatography-flame lonization detector system using internal standards and boric acid impregnated chromarod

The separation conditions for hydrolysates of triglycerides by lipase and their quantitative determination are discussed for a thin layer chromatography-flame ionization detector system utilizing internal standards. The complete separation of glyceride hydrolysis mixtures (triolein 1,3-diolein, 1,2-diolein, 1-monoolein and oleic acid) was achieved on a 3% boric acid-impregnated Chromarod S-II by development with benzene/chloroform/acetic acid (70∶30∶2, v/v/v) (mobile phase A) or hexane/ ether/acetic acid (70∶30∶1, v/v/v) (mobile phase B). Mobile phase B had an advantage over mobile phase A in terms of free space to add internal standards for simultaneous quantitation and was employed.p-Hydroxybenzoic acid andp-carboethoxy benzyl alcohol, which appeared between 1,2-diolein and 1-moloolein, were adopted as the internal standards. The calibration curves relating internal standards to each glyceride were all approximated by the equations Y=aX^b giving high correlations. The method was applied to hydrolysis of triolein by pancreatic lipase. Part of this investigation was reported at the annual meeting of JOCS in Tokyo, November 1982.     

Quantitative Glycerid-Bestimmung mittels Dünnschicht-Chromatographie und Densitometrie

Quantitative Determination of Glycerides by Thin-Layer Chromatography and Densitometry The quantitative determination of 1- and 2-mono-, 1,3- and 1,2-diolein and triolein in a mixture of oleic acid glycerides by thin-layer chromatography and densitometry is described. Low standard deviations are achieved by maintaining the Rf-values in the thin-layer chromatography around 0.1 and by charring the substance by the action of sulfuryl chloride vapour. The influence of other acidic components in glyceride molecule on the analytical results has been investigated.     

The behavior of glyceride-fatty acid mixtures in bile salt solution: Studies by gel filtration

Bile salt lipid emulsions were prepared which simulated the emulsified oil-micellar phase system of the small intestinal content during fat digestion. Application of such emulsions to gel columns prepared and eluted with 6 mM sodium taurodeoxycholate separated an emulsion phase and a micellar phase. The distribution of lipid solutes into the two phases under these conditions was measured. Micellar dimensions were larger as lipid concentrations were increased. Inclusion of multiple lipid classes resulted in larger micellar particles. Monoglyceride and fatty acids were eluted completely in the micellar phase under these conditions. Minimal measurable amounts of triolein were recovered in micellar solution. This was confirmed by extraction, chromatographic separation and quantitative analysis. As diolein concentration was increased, less was recovered in the micellar phase. When monoglyceride was added, more diolein entered the micellar phase. Addition of triglyceride enhanced the distribution of diolein into the emulsion phase.     

Presence of Apolipoprotein C-III Attenuates Apolipoprotein E-Mediated Cellular Uptake of Cholesterol-Containing Lipid Particles by HepG2 Cells

Apolipoprotein C-III (apoC-III) decreases the apolipoprotein E (apoE)-mediated uptake of lipoprotein remnants by the liver, and a high plasma concentration of apoC-III in VLDL is associated with hypertriglyceridemia and the risk of coronary heart disease. In this study, we prepared lipid emulsions containing triolein, phosphatidylcholine and cholesterol as model particles of lipoproteins, and examined the roles of apoC-III in apoE-mediated uptake of emulsions by HepG2 cells. Cholesterol in emulsion particles enhanced the apoE-mediated uptake via heparan sulfate proteoglycan and LDL receptor-related protein pathways. The amount of apoE bound to emulsion particles was increased by the presence of cholesterol at the particle surface, whereas cholesterol had no effect on the binding amount of apoC-III. Surface cholesterol alleviated the inhibitory effect of apoC-III on apoE incorporation into the emulsion surface. However, ApoC-III almost completely inhibited the apoE-mediated uptake of cholesterol-containing emulsions despite sufficient binding of apoE to emulsions. These findings suggest that apoC-III attenuates the binding of apoE to the lipoprotein surface and apoE-mediated cellular uptake of lipoprotein remnants. Furthermore, cholesterol may affect these functions of apoC-III and apoE involved in the clearance of lipoprotein remnants.     

Triolein-phosphatidylcholine-cholesterol emulsions as substrates for lipoprotein and hepatic lipases

Lipolysis of emulsified glycerol tri[9,10-³H]oleate by lipoprotein lipase purified from bovine milk (E.C. 3.1.1.34) and by hepatic lipase purified from rat liver perfusate was studied as a function of the phosphatidylcholine molecular species and the cholesterol content of the emulsions. Overall, the activities of the two enzymes were similar on a molar basis. Lipoprotein lipase initial lipolysis rates also were comparable for emulsions made with egg phosphatidylcholine or with saturated (dimyristoyl, dipalmitoyl and distearoyl) phosphatidylcholines when cholesterol was low. Increasing the cholesterol content of the emulsion from 2–3 mole percent to 7–14 mole percent reduced triolein lipolysis by lipoprotein lipase in emulsions made with saturated phosphatidylcholines. Rat hepatic lipase was more sensitive to increased cholesterol in emulsions made with saturated phosphatidylcholines than was lipoprotein lipase. The ability to maintain triolein lipolysis during longer incubations differed strikingly among the emulsions and for the two enzymes. Lymph chylomicrons were better substrates for both enzymes than any of the emulsions.     

Lipase-catalyzed production of wax esters

The lipase (triacylglycerol acylhydrolase, E.C. 3.1.1.3) catalyzed synthesis of wax esters has been investigated via two different approaches. All studies were performed using an immobilized 1,3-specific lipase [Lipozyme from Novo Industries (Montréal, Québec, Canada)]. The first approach involves reacting stoichiometric amounts of a fatty acid and stearyl alcohol in the presence of lipase. The medium is solvent-free, which allows for high substrate concentrations (1.55 M) and use of 5% (w/w) Lipozyme. In this reaction, maximum wax ester synthesis was found to be dependent upon the efficient removal of the water produced by the reaction. Under optimal conditions, yields of 100% were routinely reached after only 2 hr. The medium was then exclusively composed of the wax and the enzyme, no purification was required. The second method involves alcoholysis of a triglyceride, in this case triolein, with stearyl alcohol to produce 1,2-diolein, 2-monoolein and the wax ester of oleic acid. Again, no organic solvent was used. The wax ester yield was found to be directly dependent upon the alcohol concentration that was used to modulate the outcome of the reaction towards either the wax or the partial glycerides. The process was applied to the synthesis of waxes from high erucic acid rapeseed oil.     

Enzymatic synthesis of symmetrical 1,3-diacylglycerols by direct esterification of glycerol in solvent-free system

1,3-Diacylglycerols were synthesized by direct esterification of glycerol with free fatty acids in a solvent-free system. Free fatty acids with relatively low melting points (<45°C) such as unsaturated and medium-chain saturated fatty acids were used. With stoichiometric ratios of the reactants and water removal by evaporation at 3 mm Hg vacuum applied at 1 h and thereafter, the maximal 1,3-diacylglycerol content in the reaction mixture was: 84.6% for 1,3-dicaprylin, 84.4% for 1,3-dicaprin, 74.3% for 1,3-dilinolein, 71.7% for 1,3-dieicosapentaenoin, 67.4% for 1,3-dilaurin, and 61.1% for 1,3-diolein. Some of the system’s parameters (temperature, water removal, and molar ratio of the reactants) were optimized for the production of 1,3-dicaprylin, and the maximal yield reached 98%. The product was used for the chemical synthesis of 1,3-dicapryloyl-2-eicosapentaenoylglycerol. The yield after purification was 42%, and the purity of the triacylglycerol was 98% (both 1,3-dicapryloyl-2-eicosapentaenoylglycerol and 1,2-dicapryloyl-3-eicosapentaenoylglycerol included) by gas chromatographic analysis, of which 90% was the desired structured triacylglycerol (1,3-dicapryloyl-2-eicosapentaenoylglycerol) as determined by silver ion high-performance liquid chromatographic analysis.     

In vitro and in vivo effects of exogenous lipids on the enzymatic hydrolysis of rat bile phospholipids

The addition of total phospholipids, phosphatidylcholines, triglycerides, cholesterol or glycerol to incubation media containing rat pancreatic juice and bile labeled with [9,10³H₂] oleic acid (90% of the radioactivity present as phospholipids) had no effect on the hydrolysis of bile endogenous phospholipids. The introduction of 2 or 10 mg of phosphatidylcholines and 0.5 ml of bile (≈ 1.5 mg of phospholipids)into the rat upper duodenum decreased the rate of absorption of rative bile phospholipids. It was not followed by an increase of free fatty acids released from biliary phospholipids in the intestinal lumen. The introduction of bile (0.5 ml) and small amounts of triolein (1.4–3.5 mg) into the duodenum had little effect on the rate of hydrolysis and absorption of native bile phospholipids, but caused a reduced absorption of the free fatty acids released or those coming from initial nonphosphorus biliary lipids. The introduction of bile (0.5 ml) and large amounts of triolein (30 mg) into the duodenum increased the rates of hydrolysis and absorption of endogenous bile phospholipids. These observations suggest that luminal lipid components can modify the organization of luminal micelles and, consequently, the action of the pancreatic phospholipase A₂ and the absorption of bile lipids.     

Factors affecting droplet size of sodium caseinate‐stabilized O/W emulsions containing β‐carotene

This work was initiated to prepare an oil‐in‐water (O/W) emulsion containing β‐carotene by microfluidization. The β‐carotene was dissolved in triolein and microfluidized with an aqueous phase containing sodium caseinate (SC) as the emulsifier. Microfluization at 140 MPa resulted in O/W emulsions with a mean droplet diameter of ca. 120 nm, which was further confirmed by transmission electron microscopy analysis. The influences of SC concentration and microfluidization parameters on the droplet size of the emulsions were studied. The results showed that the mean droplet diameter decreased significantly (p <0.05) from 310 to 93 nm with the increase in SC concentration from 0.1 to 2 wt‐%. However, a further increase in SC concentration did not much change the droplet diameter, although the polydispersity of the emulsions was slightly improved. The droplet diameter of the emulsions was found to decrease from 200 to 120 nm with increasing microfluidization pressure, with narrower droplet size distribution. The storage study showed that the emulsions were physically stable for about 2 weeks at 4 °C in the dark. The results provide a better understanding of the performance of SC in stabilizing the O/W emulsions.     

Volatile compounds in heated oleic acid-esterified propoxylated glycerol

Static headspace and capillary gas chromatography, in combination with infrared and mass spectrometry, were used to collect, separate, identify, and quantitate the oxidative and thermal decomposition products in two heated model compound triacylglycerols, oleic acid-esterified propoxylated glycerol (EPG-08 oleate) and triolein. The EPG-08 oleate and triolein were each heated in a deep-fat fryer at 192±8°C for 12 h each day until the oil contained ≥20% polymeric material. Most of the volatile compounds identified in heated triolein and EPG-08 oleate were associated with the oxidative and thermal decomposition of oleates, such as short-chain alkanes, aldehydes, and alcohols. The major volatile compounds (concentration >20 ppm) in heated EPG-08 oleate and triolein were heptane, octane, heptanal, octanal, trans-2-decenal, nonanal, and trans-2-undecenal. However, a few minor volatile compounds, not previously found in heated fats and oils, were identified in the heated EPG-08 oleate samples. These compounds included 1,2-propanediol, 1-hydroxy-2-propanone (hydroxyacetone), 1-acetoxy-2-propanone (acetoxyacetone), 2-ethyl-4-methyl-1,3-dioxolane, 2,2,4-trimethyl-1,3-dioxolane, 2-heptyl-4-methyl-1,3-dioxolane, and 4-methyl-2-octyl-1,3-dioxolane. Alkyl-substituted dioxolanes have been found in heated methyl linoleate.     

Evidence for diglycerides as attractants in an ant-seed interaction

The chemical basis of an ant-seed interaction was investigated for the antAphaenogaster rudis and the ant-dispersed violetViola odorata. A laboratory behavioral bioassay was developed to chemically identify the attractant responsible for the interaction. The ant attractant, localized in the elaiosome, was classified as a lipid by both field and laboratory bioassays. Assays of partially purified lipid extracts revealed that the principal attractant may be a diglyceride. Gas-liquid chromatography analysis of the hydrolyzed diglyceride fraction revealed oleic acid as the major fatty acid present, suggesting that either 1,2- or 1,3-diolein may be the attractant. Structure-activity correlations for lipid standards demonstrated a clear preference for the diglyceride 1,2-diolein. The data also suggest that ricinoleic acid is not the lipid eliciting the ant response toViola odorata, as had been previously suggested.     

Enzymatic interesterification of triolein with tripalmitin in canola lecithin-hexane reverse micelles

Lipase-catalyzed interesterification of tripalmitin with triolein in canola lecithin-hexane reverse micelles allowed for the successful modification of triolein and tripalmitin to yield a fat of intermediate properties between the two initial substrates. Acetone-insoluble canola lecithin (AIL) reverse micelles containingRhizopus arrhizus lipase in buffer, or plain 0.1M sodium phosphate buffer of pH 7.0, formed readily in hexane. Both had an average Stokes’ radius of approximately 40Å, as determined by quasielastic light-scattering determinations. The reverse micelle system was stable and did not form higher-order micelle oligomers or aggregates. Biotransformation of the triglycerides was performed at 47°C in a 50-mM AIL-hexane reverse micelle system containing 50% (w/w) oil at a water-to-surfactant ratio (w_o) of 5.5. Dynamic (oscillatory) mechanical analysis indicated that the crystallization temperature of the fat dropped from 47.7 to 37.5°C as judged by the storage (G′) and loss (G″) modulusvs. temperature profiles after 48 h of reaction. Differential scanning calorimetric studies showed that the melting point of the fat dropped from 61 to 57°C after 48 h of reaction. Triglyceride analysis of the fat mixture by gas-liquid chromatography (GLC) indicated that, after 48 h of reaction, the tripalmitin content dropped from 34.5 to 29% (w/w), the triolein content dropped from 64.5 to 52.1% (w/w) and the 1-oleyl-2,3-dipalmitin content reached 7.5% (w/w) while the 1-palmitoyl-2,3-diolein content reached 7.2% (w/w). 1,2-Dipalmitoyldiglyceride and 1,2-dioleyldiglyceride contents reached 1.6 and 2.4% (w/w), respectively, after 48 h. Free fatty acid analysis of the fat mixture by GLC revealed that the free palmitic acid content increased from 0.28 to 2.4% (w/w) while the free oleic acid content increased from 1.4 to 5.4% (w/w) in the initial 24 h, after which the levels remained constant. The relatively high initial free fatty acid content of the mixture was due to free fatty acids present in the canola lecithin and not in the oils. This enzymatic interesterification protocol utilizes, for the first time, an organic solvent commonly used in food processing operations and a food-grade and inexpensive surfactant that readily forms reverse micelles and yields a modified fat with improved rheological properties for use as an edible plastic fat.     

Effects of Regioselectivity and Lipid Class Specificity of Lipases on Transesterification,Exemplified by Biodiesel Production

Lipase-catalyzed ethanolysis of triolein was studied as a model for biodiesel production. Four lipases were immobilized on porous polypropylene, and ethanolysis reactions were carried out in methyl t-butyl ether. The reaction products were analyzed using gas chromatography. Three of the four lipases studied were efficient in the conversion of triolein to 2-monoolein, but slow in the final step of producing glycerol. However, Candida antarctica lipase B was slow in the conversion of triolein, but more efficient in the subsequent two steps than the other lipases. The 1,3-selectivity of the lipases was less pronounced for the monooleins than for triolein. Silica gel was investigated as a catalyst for acyl migration, showing an increase in biodiesel yield with three of the lipases, but a reduction in yield when C. antarctica lipase B was used. The highest biodiesel yield (96 %) was obtained with a combination of Rhizopus arrhizus lipase and C. antarctica lipase B.     

Inhibitory effect of 1,3-diglycerides during adsorption of β-carotene onto attapulgite and sepiolite

The adsorption isotherms of β-carotene on attapulgite and sepiolite were measured in the presence of 1,3-diglyceride in n-hexane to elucidate the relative inhibitory power of the diglycerides, hydroxyl group, carbon number, and double bond for adsorption bleaching. The adsorption mode of β-carotene was the Langmuir type. The inhibition of β-carotene adsorption may be caused by the polarity of diglycerides and the formation of diglyceride micelles. The relative inhibitory power of 1,3-diolein, 1,3-distearin, and 1,3-dipalmitin toward β-carotene adsorption was 1.49, 1.40, and 0.99, respectively. Therefore, the relative inhibitory power due to the hydroxyl group and increase in the carbon atom of the fatty acid in glyceride was 0.41–0.49.     

改性明胶用盐絮凝对乳剂感光度的影响

本文研究了由改性明胶制备的乳剂用盐絮凝[(NH_4)_2SO_4]对乳剂感光度的影响。与用酸絮凝相比,对于中性法乳剂用盐絮凝感光度可提高80％,而氨法乳剂用盐絮凝感光度可提高30％左右。同时也研究了改性明胶以及絮凝时的pH值对使乳剂絮凝所需盐量的影响。     

Lipolysis of different oils using crude enzyme isolate from the intestinal tract of rainbow trout, <Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis>

Crude enzyme isolate was prepared from the intestine of rainbow trout. Positional specificity of the crude enzyme isolate was determined from both 1(3)- and 2-MAG products after in vitro lipolysis of radioactive-labeled triolein. The ratio of 2-MAG/1(3)-MAG was 2∶1, suggesting that the overall lipase specificity of the enzyme isolate from rainbow trout tended to be 1,3-specific; however, activity against the sn-2 position also was shown. In vitro lipolysis of four different unlabeled oils was performed with the crude enzyme isolate. The oils were: structured lipid [SL; containing the medium-chain FA (MCFA) 8∶0 in the sn-1,3 positions and long-chain FA (LCFA) in the sn-2 position], DAG oil (mainly 1,3-DAG), fish oil (FO), and triolein (TO). MCFA were rapidly hydrolyzed from the SL oil. LCFA including n−3 PUFA were, however, preserved in the sn-2 position and therefore found in higher amounts in 2-MAG of SL compared with 2-MAG of FO, DAG, and TO. Lipolysis of the DAG oil produced higher amounts of MAG than the TAG oils, and 1(3)-MAG mainly was observed after lipolysis of the DAG oil. The positional specificity determined and the results from the hydrolysis of the different oils suggest that n−3 very long-chain PUFA from structured oils may be used better by aquacultured fish than that from fish oils.     

Thermoxidation of substrate models and their behavior during hydrolysis by porcine pancreatic lipase

The behavior of thermoxidized triacylglycerols during hydrolysis catalyzed by porcine pancreatic lipase was evaluated using nonpolar triacylglycerols isolated from palm olein (NPTPO), triolein, and sn-1,3 diolein substrates. Substrates were thermoxidized at 180°C for 1 to 4 h. Owing to formation of polymers and dimers of triacylglycerols, the molecular weight of the thermoxidized substrates increased. After 1 h heating, the concentration of polymers and dimers was similar for the sn-1-3 diolein and triolein samples but higher in NPTPO samples. Conjugated double bonds were formed in all samples, and α,β-unsaturated carbonyl compounds developed through allylic oxidations. These caused increased ultraviolet absorbance at 232 nm. The hydrolysis of heated and unheated samples by the lipase can be described by a Michaelian equation. The enzyme showed a higher apparent V _max and K _M with heated sn-1,3 diolein and triolein than with their unheated counterparts. This was due to the generation of polar compounds which acted as emulsifiers and which favored the formation of an oil/water microemulsion. This behavior was not observed in NPTPO, where heating decreased the apparent V _max and K _M over the first 2 h. Later, a tendency to increase these values was observed. The results could be explained by a balance between concentration of surfactants and of natural emulsifiers in the thermoxidized samples.     

Solubility in and affinity for the bile salt micelle of plant sterols are important determinants of their intestinal absorption in rats

Intestinal absorption of various plant sterols was investigated in thoracic duct-cannulated normal rats. Lymphatic recovery was the highest in campesterol, intermediate in brassicasterol and sitosterol, and the lowest in stigmasterol and sitostanol. Higher solubility in the bile salt micelle was observed in sitosterol, campesterol, and sitostanol than in brassicasterol and stigmasterol. The solubility of the latter two sterols was extremely low. When the affinity of plant sterols for the bile salt micelle was compared in an in vitro model system, which assessed sterol transfer from the micellar to the oil phase, the transfer rate was the highest in brassicasterol, intermediate in campesterol and stigmasterol, and lowest in sitosterol and sitostanol. Although no significant correlations between lymphatic recovery of plant sterols and their micellar solubility or transfer rate from the bile salt micelle were observed, highly positive correlation was obtained between the lymphatic recovery and the multiplication value of the micellar solubility and the transfer rate. These observations strongly suggest that both solubility in and affinity for the bile salt micelle of plant sterols are important determinants of their intestinal absorption in rats.     

Distribution among tissues of intravenously administered sucrose octaoleate

The distribution of [¹⁴C]oleate label in rat tissues in the 6 hr after intravenous administration of sucrose octa-[¹⁴C]oleate (7.5 mg; SuO₈) was compared with that observed after administration of [¹⁴C]triolein. The [¹⁴C]-oleate label, whether injected as triolein emulsion, or as chylomicrons obtained from donor animals, rapidly cleared from the serum; only 10% or less remained in the serum 15 min after injection. Labeled SuO₈ disappeared less rapidly from the serum; about one-third of the dose was present after 15 min, and after 120 min 14% remained. In the liver, there was an initial greater accumulation of fatty acid label when and emulsion of either triolein or SuO₈ was given rather than the chylomicrons. The octaester continued to accumulate in the liver throughout the 6 hr of study, and 78% of the initial dose was present at that time. By contrast, although one-third of the triolein, as of SuO₈, was found in the liver shortly after injection, levels subsequently decreased; at 6 hr, 12% of the label remained associated with that organ. A small portion, up to 8% of the acid label, whether administered as chylomicrons or as a triolein emulsion, was found in the epididymal fat pads. Smaller amounts, usually 1% or less, of the [¹⁴C]oleate label were found in fat pads following the injection of labeled SuO₈. In a separate study, the levels of acid label in the liver and spleen were monitored for 21 days following the intravenous administration of [¹⁴C]SuO₈. There was an initial accumulation of approximately half of the injected lipid label in the liver and one-quarter in the spleen. By day 21, the level in the liver had decreased to one-third of that administered, while the level in spleen remained at one-quarter.