Response surface methodology applied to optimization of distilled monoglycerides production

This work demonstrates that response surface methodology (RSM) is a powerful tool for the optimization of the production of distilled MG. Experiments with a centrifugal molecular distillator having an evaporation area of 0.0046 m² were carried out using RMS to identify operating conditions that can lead to higher MG purity. The independent variables studied were the evaporator temperature (TEV) and the volumetric feed flow rate (Q). The experimental range was from 100 to 300°C for TEV and between 5 and 15 mL/min for Q. High-performance size exclusion chromatography was used to evaluate TG, DG, MG, FFA, and glycerol (GL) compositions. Results were presented as MG concentration surfaces. Starting from a material with 10.8% of TG, 37.7% of DG, 43.6% of MG, and 7.2% of GL, the maximum MG, purity in the distillate stream with just one distillation step was 82.6% at a TEV equal to 250°C and Q equal to 5 mL/min. At these conditions, the MG recovery was 61%. A strategy was developed to obtain distilled MG with 96.3% purity.     

Preparation of Diacylglycerol-Enriched Oil from Free Fatty Acids Using Lecitase Ultra-Catalyzed Esterification

Production of diacylglycerol-enriched oil by esterification of free fatty acids (FFA) with glycerol (GLY) using phospholipase A1 (Lecitase Ultra) was investigated in this work. The variables including reaction time (2–10 h), water content (2–14 wt%, FFA and GLY mass), enzyme load (10–120 U/g, FFA and GLY mass), reaction temperature (30–70 °C) and mole ratio of GLY to FFA (0.5–2.5) were studied. The optimum conditions obtained were as follows: reaction temperature 40 °C, water content 8 wt%, reaction time 6 h, molar ratio of GLY to FFA 2.0, and an enzyme load of 80 U/g. Under these conditions, the esterification efficiency (EE) of free fatty acids was 74.8%. The compositions of the FFA and acylglycerols of the upper oil layer (crude diacylglycerol) of the reaction mixture were determined using a high temperature gas chromatograph (GC). The crude diacylglycerol from the selected conditions was molecularly distilled at 170 °C evaporator temperatures to produce a diacylglycerol-enrich oil (DEO) with a purity of 83.1% and a yield of 42.7%.     

Conversion of oils to monoglycerides by glycerolysis in supercritical carbon dioxide media

Glycerolysis of soybean oil was conducted in a supercritical carbon dioxide (SC-CO₂) atmosphere to produce monoglycerides (MG) in a stirred autoclave at 150–250°C, over a pressure range of 20.7–62.1 MPa, at glycerol/oil molar ratios between 15–25, and water concentrations of 0–8% (wt% of glycerol). MG, di-, triglyceride, and free fatty acid (FFA) composition of the reaction mixture as a function of time was analyzed by supercritical fluid chromatography. Glycerolysis did not occur at 150°C but proceeded to a limited extent at 200°C within 4 h reaction time; however, it did proceed rapidly at 250°C. At 250°C, MG formation decreased significantly (P<0.05) with pressure and increased with glycerol/oil ratio and water concentration. A maximum MG content of 49.2% was achieved at 250°C, 20.7 MPa, a glycerol/oil ratio of 25 and 4% water after 4 h. These conditions also resulted in the formation of 14% FFA. Conversions of other oils (peanut, corn, canola, and cottonseed) were also attempted. Soybean and cottonseed oil yielded the highest and lowest conversion to MG, respectively. Conducting this industrially important reaction in SC-CO₂ atmosphere offered numerous advantages, compared to conventional alkalicatalyzed glycerolysis, including elimination of the alkali catalyst, production of a lighter color and less odor, and ease of separation of the CO₂ from the reaction products.     

A continuous process for the glycerolysis of soybean oil

A continuous process for the glycerolysis of soybean oil with pure and crude glycerol, the co-product from the transesterification of soybean oil, was investigated in a pilot plant. The process was equipped with a static and a high-shear mixer. The experimental studies explored the effects of variations in mixing intensity, temperature, reactant flow rates, and reactant stoichiometry on the formation of MG and DG. The developed process resulted in high conversion of TG to MG. The most favorable conditions were 230°C, 40 mL/min total flow, 25 min of reaction time, 2.5∶1 molar ratio of glycerol/soybean oil, and 3600 rpm for the reactions involving crude glycerol where the concentrations of MG and DG in the product were about 56 and 36 wt%, respectively. Under similar conditions, glycerolysis of pure glycerol resulted in 58% MG and 33% DG. In general, higher temperatures and mixing intensities favored the conversion of TG to MG and DG. Reaction temperature had a greater influence on the extent of the reaction than mixing. The formation of MG approached equilibrium for nearly all cases under investigation.     

Lipase-catalyzed glycerolysis of olive oil in organic solvent medium: Optimization using response surface methodology

Enzymatic glycerolysis of olive oil for mono- (MG) and diglycerides (DG) synthesis was investigated. Several pure organic solvents and co-solvent mixtures were screened in a batch reaction system. The yields of MG and DG in co-solvent mixtures exceeded those of the corresponding pure organic solvents. Batch reaction conditions of the glycerolysis reaction, the lipase amount, the glycerol to oil molar ratio, the reaction time, and temperature, were studied. In these systems, the high content of reaction products, especially MG (55.8 wt%) and DG (16.4wt%) was achieved at 40 °C temperature and 0.025 g of lipase with relatively low glycerol to oil molar ratio (2: 1) within 4 h of reaction time in isopropanol/tert-butanol (1: 3) solvent mixture. Glycerolysis reaction was optimized with the assistance of response surface methodology (RSM). Optimal condition for reaction conversion was recommended as lipase amount 0.025 g, glycerol to oil molar ratio 2: 1, reaction time 4 h and temperature 40 °C.     

脂肪酶促乌桕脂组分甘油解及其反应机理研究

Glycerolysis of Chinese vegetable tallow （CVT） fraction was investigated using a 1,3-specific lipase from Rhizopus arrhizus as catalyst. Based upon a binary gradient HPLC with an evaporative light-scattering detector （ELSD）, the contents of free fatty acids （FFA）, monoglycerides （MG）, diglycerides（DG） and triglycerides （TG） with their positional isomers during the glycerolysis were determined. The effects of water content and the ratio of glycerol to oil on the product distribution of glycerolysis were studied. Under the optimum reactant conditions： 250 units lipase per gram oil at 37℃ with 1：2 molar ratio of oil to glycerol in a solvent-free system, after 24 h reaction, the product consisted of 7.2% TG, 25.6% MG, 56.1% DG and 4.9% FFA （all by mass）. Furthermore, the mechanism of glycerolysis was discussed in detail.     

The distribution of lipids in the germ,endosperm, pericarp and tip cap of amylomaize,LG-11 hybrid maize and waxy maize

The quantitative distribution of 23 acyl lipid classes and unsaponifiable matter in kernels of amylomaize, LG-11 hybrid maize and waxy maize is described. LG-11 and waxy maize were normal (oil content) varieties, containing 4.9% and 5.1% lipid, respectively, while amylomaize (9.3% lipid) was a high oil variety. The distribution of kernel lipids was 76–83% in germ, 1–2% in pericarp, 1% in tip cap, 1–11% in starch, and 13–15% in aleurone plus the nonstarch fraction of the starchy endosperm. Germ contained 39–47% lipid, which was nostly triglyceride (TG), with some steryl esters (SE) and diglycerides (DG), and small amounts of glycolipids (GL) and phospholipids (PL). Aleurone lipids appeared to be TG with some free fatty acids (FFA) and SE. The other nonstarch lipids in starchy endosperm were FFA with very small amounts of SE, DG, GL and PL. The starches had a little surface lipid (FFA) and true (internal) starch lipid (FFA, lyso-PL) in quantities roughly related to amylose content (amylomaize =ca. 73% amylose, 1.0% lipid; LG-11=23% amylose, 0.7% lipid; waxy maize =<5% amylose, 0.2% lipid). Pericarp lipids (0.8–2.5%) were mainly unsaponifiable matter, the acyl lipids being TG, SE, DG and FFA. Tip cap lipids (2.5–2.9%) had more TG, GL and PL than pericarp lipids, but were otherwise similar. Pericarp lipids and endosperm nonstarch lipids appeared to have suffered extensive degradation at some time during kernel development or after harvesting, while lipids in starch, germ and tip cap were evidently unaffected. FFA and lyso-PL are regarded as normal components of maize starch (rather than degradation products) and may occur as amylose inclusion complexes.     

Selective distribution of saturated fatty acids into the monoglyceride fraction during enzymatic glycerolysis

Four triglyceride fats and oils (beef tallow, lard, rapeseed oil and soybean oil) were reacted with glycerol while using lipase as the catalyst. For all fats examined, at reaction temperatures above the critical temperature (T_c), the fatty acid compositions of the monoglyceride (MG) and diglyceride (DG) fractions and of the original fat were similar. A relatively low yield of MG was obtained (20–30 wt%). When the reaction was carried out with beef tallow or lard at a temperature below the T_c (40°C), the concentration of saturated fatty acids in the MG fraction was 2 to 4 times greater than that in the DG fraction. Correspondingly, the concentration of unsaturated fatty acids in the DG fraction was more than two times greater than that in the MG fraction. At 5°C, a similar trend was observed for rapeseed oil and soybean oil. Direct analysis of partial glycerides during glycerolysis by high-temperature gas-liquid chromatography showed that below T_c the content of C16 MG increased relatively more than C18 MG. C36 DG and C54 TG were apparently resistant to glycerolysis. Preferential distribution of saturated fatty acids into the MG fraction was accompanied by a high yield of monoglyceride (45–70 wt%) and solidification of the reaction mixture. It is concluded that during glycerolysis below T_c, preferential crystallization occurs for MGs that contain a saturated fatty acid.     

Glycerolysis of Crude Methyl Esters with Crude Glycerol from Biodiesel Production

Glycerolysis of crude fatty acid methyl esters (FAME) with crude glycerol derived from biodiesel production was performed. The reaction was accomplished at temperatures ranging between 160 and 200 °C and molar ratios of FAME to glycerol ranging between 1.5 and 3.0. Increasing the temperature improved the formation rate of monoglycerides (MG) and diglycerides (DG). However, increasing both the temperature and the molar ratio of glycerol to FAME diminished the formation of MG. Best results (43 % MG and 26 % DG in 10 min) were obtained at 200 °C using the lowest concentration of glycerol. The effects of soap and NaOH present in crude glycerol were controlled by carrying out the reaction with pure glycerol. In comparison with NaOH-catalyzed reactions, soap-catalyzed reactions resulted in a slower formation rate of products. However, soap-catalyzed reactions were less prone to secondary reactions, affording maximum yields of MG and DG, which were higher than those obtained with NaOH-catalyzed reactions at 180 and 200 °C.     

Solid phase enzymatic glycerolysis of beef tallow resulting in a high yield of monoglyceride

A mixture of mono-, di- and triglycerides was obtained when beef tallow was reacted with glycerol using lipase enzyme as a catalyst. The reaction was carried out batchwise in a small vessel with agitation by magnetic stirring. The yield of monoglyceride (MG) was greatly influenced by the reaction temperature—at higher temperatures (48–50°C) a yield of approximately 30% MG was obtained, while at lower temperatures (38–46°C) a yield of approximately 70% MG was obtained. A sharp transition was observed between the high and low yield equilibrium states. The temperature at which this transition occurred is called the critical temperature (T_c) and was found to be 46°C in the case of tallow. During the course of the reaction, when approximately 40% MG had been synthesized, the reaction mixture became solid but the reaction continued until approximately 70% MG had been synthesized. A yield of 70% MG also was obtained with tallow at 42°C when a glycerol/tallow mole ratio ranging from 1.5 to 2.5 was used. The free fatty acid content at equilibrium depended on the water concentration in the glycerol phase and varied from 0.5% to 11.0% when the water content ranged from 0.6% to 12.5%. Above 8% water content, the yield of MG was reduced. Of the commercially available lipases that were investigated, lipase fromPseudomonas fluorescens orChromobacterium viscosum resulted in the highest yield of MG.     

Comparison of bio- and autocatalytic esterification of oils using mono- and diglycerides

The purpose of this study was to investigate enzymatic and autocatalytic esterification of FFA in rice bran oil (RBO), palm oil (PO), and palm kernel oil (PKO), using MG and DG as esterifying agents. The reactions were carried out at low pressure (4–6 mm Hg) either in the absence of any added catalyst at high temperature (210–230°C) or in the presence of Mucor miehei lipase at low temperature (60°C). The reactions were carried out using different concentrations of MG, and the optimal FFA/MG ratio and time were 2∶1 (molar) and 6 h, respectively, in both auto- and enzyme-catalyzed processes. With DG as the esterifying agent in the autocatalytic process, the optimal temperature was 220°C, and the optimal FFA/DG ratio was 1∶1.25. For both MG and DG, the enzymatic process was more effective in reducing FFA and produced more favorable levels of unsaponifiable matter and color in the final product. The PV of the final products were also lower (1.8–2.9 mequiv/kg) by using the enzymatic process. To produce edible-grade oil, a single deodorization step would be required after enzymatic esterification; whereas, alkali refining, bleaching, and deodorization would be required after autocatalytic treatment.     

Reactions of olive oil and glycerol over immobilized lipases

The reaction of olive oil and glycerol over immobilized lipases was studied. For oil samples with free fatty acid (FFA) contents larger than 2%, FFA esterification and glycerolysis took place simultaneously, but the esterification reaction was faster than glycerolysis. Similar product distributions were obtained for glycerol/oil mole ratios of 3:1 and 6:1. Therefore, an excess of glycerol does not result in a significant increase in monoglyceride yield within the experimental range tested. The main reaction product at 80°C was diglyceride. No increase in monoglyceride yield was observed by lowering the reaction temperature to 10°C.     

<Emphasis Type="Italic">In situ</Emphasis> alkaline transesterification: An effective method for the production of fatty acid esters from vegetable oils

The production of simple alkyl FA esters by direct alkali-catalyzed in situ transesterification of the acylglycerols (AG) in soybeans was examined. Initial experiments demonstrated that the lipid in commercially produced soy flakes was readily transesterified during agitation at 60°C in sealed containers of alcoholic NaOH. Methyl, ethyl, and isopropyl alcohols readily participated in the reaction, suggesting that the phenomenon is a general one. Statistical experimental design methods and response surface regression analysis were used to optimize reaction conditions, using methanol as alcohol. At 60°C, the highest yields of methyl ester with minimal contamination by FFA and AG were predicted at a molar ratio of methanol/AG/NaOH of 226∶1∶1.6 with an approximately 8-h incubation. An increase in the amount of methanol, coupled with a reduced alkali concentration, also gave high ester yields with low FFA and AG contamination. The reaction also proceeded well at 23°C (room temperature), giving higher predicted ester yields than at 60°C. At room temperature, maximal esterification was predicted at a molar ratio of 543∶1∶2.0 for methanol/AG/NaOH, again in 8 h. Of the lipid in soy flakes, 95% was removed under such conditions. The amount of FAME recovered after in situ transesterification corresponded to 84% of this solubilized lipid. Given the 95% removal of lipid from the soy flakes and an 84% efficiency of conversion of this solubilized lipid to FAME, one calculates an overall transesterification efficiency of 80%. The FAME fraction contained only 0.72% (mass basis) FFA and no AG. Of the glycerol released by transesterification, 93% was located in the alcoholic ester phase and 75 was on the post-transesterification flakes.     

Bio‐ and auto‐catalytic esterification of high acid mowrah fat and palm kernel oil

An alternative process for the deacidification of high acid palm kernel oil (PKO) and mowrah fat, MF, was investigated by autocatalytic and enzyme (Mucor miehei)‐catalyzed esterification of free fatty acids (FFA). In the process monoglyceride (MG) or glycerol was used as esterifying agent. The results of the autocatalytic esterification process were compared with that of bioesterification with respect to reduction of FFA. For the former process the optimum reaction temperature and oil to MG or glycerol proportion were established. The optimum reaction temperature for PKO free fatty acids with stoichiometric quantity of glycerol is 160—165 °C (at 10 mm Hg pressure (1.33 kPa)) and after 6 h the FFA content is reduced to 1.6%, w/w, (from 25.0%, w/w, original). However, if a stoichiometric amount of MG is used as an esterifying agent the optimum esterification temperature is found to be 195—200 °C (at 10 mm Hg pressure (1.33 kPa)), and after 8 h the FFA content is reduced to 3.4%, w/w. On the contrary, biorefining of PKO at 60 °C temperature and 10 mm Hg pressure (1.33 kPa) using optimum (40% excess) quantity of glycerol or 50% excess MG reduces the FFA level to 1.2% and 0.7%, w/w, respectively. Similar study on bio‐ and auto‐catalytic esterification of MF was also carried out and got comparable results. The final products were then characterized.     

Enzymatic glycerolysis of soybean oil

Enzymatic glycerolysis of soybean oil was studied. Of the nine lipases that were tested in the initial screening, Pseudomonas sp. resulted in the highest yield of monoglycerides. Lipase from Pseudomonas sp. was further studied for the influence of temperature, thermal stability, enzyme/oil ratio, and glycerol/oil ratio. A full factorial optimization approach was performed. The following conditions were tested over the specified ranges: temperature (30–70°C), thermal stability (30–70°C), enzyme/oil ratio (0.05–0.2 g enzyme/10 g oil), glycerol/oil ratio (1:1–3:1 glycerol/oil molar ratio) and 1 h reaction time. The stability of the enzyme at the reaction temperature was also incorporated as a separate variable. At temperatures above 40°C enzyme denaturation offset the higher activity. The optimal conditions were selected to be the basis for a continuous process: 40°C, a glycerol/oil molar ratio of 2:1, and an enzyme/oil ratio of 0.1 g enzyme/10 g oil. A definition for glycerolysis activity was adopted. The glycerolysis activity (1 GU) was defined as the amount of enzyme necessary to consume 1 μmol of substrate (glycerol and oil) per minute. This research is intended to explore the reaction parameters that are important in a continuous enzymatic glycerolysis process.     

High-yield enzymatic glycerolysis of fats and oils

Several triglyceride fats and oils were reacted with glycerol using lipase as catalyst. A batch system with magnetic stirring was used without the addition of any solvents or emulsifiers. In all cases a mixture of mono-, di- and triglycerides was obtained. However, the yield of monglyceride (MG) depended strongly on the reaction temperature: at higher temperatures approximately 30% MG was produced at equilibrium while at lower temperatures a yield of 65%–90% MG was obtained for most of the fats examined. The upper temperature limit below which a high MG yield could be attained was designated the critical temperature (T_c). The value of T_c depended on the fat type and was found to vary between 30°C and 46°C for naturally occurring hard fats. A high MG yield could not be obtained for fully hydrogenated tallow and lard under the conditions described here. Of the three liquid oils examined, rapeseed oil and olive oil had a T_c of 5°C and 10°C respectively whereas a high yield of MG could not be obtained with corn oil at 5°C or greater. The maximum yield of MG below T_c also depended on the fat type: the highest yields being obtained for olive oil (90%), palm stearin and milk fat (80%) and the lowest yield for palm oil (67%). In all cases a high yield of MG was accompanied by solidification of the reaction mixture. The effect of enzyme type on MG production was examined for palm oil and palm stearin and the effect of water concentration was examined for palm oil.     

Enrichment of eicosapentaenoic acid and docosahexaenoic acid in saponified menhaden oil

Eicosapentaenoic acid (EPA, 20∶5n−3) and docosahexaenoic acid (DHA, 22∶6n−3) in free fatty acids (FFA) derived from saponified menhaden oil were concentrated by the solubility differences of FFA-salts in organic solvent. FFA-salts were formed by adding NaOH to a solution containing FFA. A Buchner funnel was used to separate solid phases from liquids containing FFA-salts. FFA that are rich in EPA and DHA can be recovered from the liquid phase by the addition of 12 N HCl. The effects of reaction time, the amount of NaOH, and solvent used on the concentration of EPA and DHA were systematically investigated. With a total volume of 112 mL, made up of 1.85% 15 N NaOH, 88.1% acetone, and 10.0% FFA, a reaction temperature of 30°C, and a reaction time of 1 h, the resulting liquid phase contained 65.4 wt% EPA and DHA, with a corresponding yield of 41.5%. By replacing the acetone with a mixture of 45% acetone and 55% acetonitrile and then storing the liquid phase at −70°C overnight, the content and yield of EPA and DHA in the final liquid phase were 61.4 wt% and 66.2%, respectively.     

Biodiesel from an alkaline transesterification reaction of soybean oil using ultrasonic mixing

The feasibility of using ultrasonic mixing to obtain biodiesel from soybean oil was established. The alkaline transesterification reaction was studied at three levels of temperature and four alcohol-to-oil ratios. Excellent yields were obtained for all conditions. For example, at 40°C with ultrasonic agitation and a molar ratio of 6∶1 methanol/oil, the conversion to FAME was greater than 99.4% after about 15 min. For a 6∶1 methanol/oil ratio and a 25 to 60°C temperature range, a pseudo second-order kinetic model was confirmed for the hydrolysis of DG and TG. Reaction rate constants were three to five times higher than those reported in the literature for, mechanical agitation. We suspect that the observed mass transfer and kinetic rate enhancements were due to the increase in interfacial area and activity of the microscopic and macroscopic bubbles formed when ultrasonic waves of 20 kHz were applied to a two-phase reaction system.     

Separation of oil constituents in organic solvents using polymeric membranes

Different types of commercial nonporous (reverse osmosis and gas separation) polymeric membranes were screened for their abilities to separate FFA, MG, DG, and TG from a lipase hydrolysate of high-oleic sunflower oil after diluting it with organic solvents (ethanol and hexane). Cellulose acetate (CA) (NIR-1698) membrane gave the largest difference in rejection between FFA and glycerides and high flux in oil/ethanol mixtures. In the hexane system, the values of permeate flux and rejection were generally lower than those in the ethanol system. The silicone-polyimide composite membrane (NTGS-2100) gave the highest flux and rejections of solutes (70.2% for FFA, 94.4% for TG) in oil/hexane mixtures. In the ethanol system with the CA membrane, TG had the highest rejection (98%) followed by DG (90%) and MG and FFA (50–70%) when the oil concentration was varied from 6.3 to 45.8%. A discontinuous diafiltration process (16 batches) using the CA membrane with ethanol changed the composition of the oil from 31∶28∶9∶32 TG/DG/MG/FFA to 65∶30∶1∶4. The results of this study showed that oil constituents can be separated in suitable solvents using appropriate nonporous membranes.     

Ethyl esters from the single-phase base-catalyzed ethanolysis of vegetable oils

The effects of alcohol/oil molar ratio, base concentration, and temperature on the single-phase base-catalyzed ethanolyses of sunflower and canola oils were determined. The use of tetrahydrofuran as co-solvent, as well as higher than usual alcohol/substrate molar ratios, prevented glycerol separation. This allowed each reaction to reach equilibrium rather than just steady-state conditions. High conversions of oil lowered the concentrations of MG and DG surfactants in the products, and thereby mitigated the formation of emulsions usually associated with ethanolysis reactions. An alcohol/oil molar ratio of 25∶1, together with the necessary amount of cosolvent, gave optimal results. At this molar ratio, despite equilibrium being achieved, ethanolysis, unlike methanolysis, did not quite produce biodiesel-standard material, the MG content being approximately 1.5 mass%. For methanolysis and 1-butanolysis, the corresponding values were 0.6 and 2.0 mass%, respectively. The use of 1.4 mass% KOH (equivalent to 1.0 mass% NaOH) led to ethanolysis equilibrium within 6–7 min at 23°C rather than 15 min when only 1.0 mass% was used. At 60°C, equilibrium was reached within only 2 min. Soybean and canola oils behaved the same.