Conversion of acid oil by-produced in vegetable oil refining to biodiesel fuel by immobilized Candida antarctica lipase

Acid oil, which is a by-product in vegetable oil refining, mainly contains free fatty acids (FFAs) and acylglycerols, and is a candidate of materials for production of biodiesel fuel. A mixture (acid oil model) of refined FFAs and vegetable oil was recently reported to be converted to fatty acid methyl esters (FAMEs) at >98% conversion by a two-step reaction system comprising methyl esterification of FFAs and methanolysis of acylglycerols using immobilized Candida antarctica lipase. The two-step system was thus applied to conversion of acid oil by-produced in vegetable oil refining to biodiesel fuel. Under similar conditions that were determined by using acid oil model, however, the lipase was unstable and was not durable for repeated use. The inactivation of the lipase was successfully avoided by addition of excess amounts of methanol (MeOH) in the first-step reaction, and by addition of vegetable oil and glycerol in the second-step reaction. Hence, the first-step reaction was conducted by shaking a mixture of 66 wt% acid oil (77.9 wt% FFAs, 10.8 wt% acylglycerols) and 34 wt% MeOH with 1 wt% immobilized lipase, to convert FFAs to their methyl esters. The second-step reaction was performed by shaking a mixture of 52.3 wt% dehydrated first-step product (79.7 wt% FAMEs, 9.7 wt% acylglycerols), 42.2 wt% rapeseed oil, and 5.5 wt% MeOH using 6 wt% immobilized lipase in the presence of additional 10 wt% glycerol, to convert acylglycerols to FAMEs. The resulting product was composed of 91.1 wt% FAMEs, 0.6 wt% FFAs, 0.8 wt% triacylglycerols, 2.3 wt% diacylglycerols, and 5.2 wt% other compounds. Even though each step of reaction was repeated every 24 h by transferring the immobilized lipase to the fresh substrate mixture, the composition was maintained for >100 cycles.     

A two steps enzymatic procedure to obtain sterol esters,tocopherols and fatty acid ethyl esters from soybean oil deodorizer distillate

Soybean oil deodorizer distillate (SODD) was enzymatically modified to obtain a product mixture comprised mainly of sterol esters, tocopherols, and fatty acid ethyl esters. Firstly, the original SODD was mixed with oleic acid to reduce its melting point from 65–70 to 30–35 °C and also to produce a reaction mixture with a ratio of free fatty acids (FFA) to sterols close to 2 to improve the progress of sterols esterification. Two enzymatic steps were used in order to separate sterols esterification and ethyl esterification in time and space. The first enzymatic step (in the presence of Candida rugosa lipase) allowed to efficiently transform more than 90% of the original sterols in a short period of time (5 h). The second enzymatic step (in the presence of Novozym 435) converted more than 95% of the FFA in less than 3 h. In addition, the stability of both biocatalysts has been evaluated and both bioprocesses have been scaled-up reutilizing the same batch of lipase up to 8 and 3 times for the first and the second enzymatic step, respectively. The final product obtained is intended to be used as starting material for the purification of sterol esters, tocopherols, and fatty acid ethyl esters via supercritical fluid extraction.     

Fermentation of soybean oil deodorizer distillate with Candida tropicalis to concentrate phytosterols and to produce sterols-rich yeast cells

Phytosterols have been recovered from the deodorizer distillate produced in the final deodorization step of vegetable oil refining by various processes. The deodorizer distillate contains mainly free fatty acids (FFAs), phytosterols, and tocopherols. The presence of FFAs hinders recovery of phytosterols. In this study, fermentation of soybean oil deodorizer distillate (SODD) with Candida tropicalis 1253 was carried out. FFAs were utilized as carbon source and converted into cellular components as the yeast cells grew. Phytosterols concentration in SODD increased from 15.2 to 28.43 % after fermentation. No significant loss of phytosterols was observed during the process. Microbial fermentation of SODD is a potential approach to concentrate phytosterols before the recovery of phytosterols from SODD. During SODD fermentation, sterols-rich yeast cells were produced and the content of total sterols was as high as 6.96 %, but its major sterol was not ergosterol, which is the major sterol encountered in Saccharomyces cerevisiae. Except ergosterol, other sterols synthesized in the cells need to be identified.     

Steryl ester synthesis,storage and hydrolysis: A contribution to sterol homeostasis

Sterols are essential lipids of all eukaryotic cells, appearing either as free sterols or steryl esters. Besides other regulatory mechanisms, esterification of sterols and hydrolysis of steryl esters serve to buffer both an excess and a lack of free sterols. In this review, the esterification process, the storage of steryl esters and their mobilization will be described. Several model organisms are discussed but the focus was set on mammals and the yeast Saccharomyces cerevisiae. The contribution of imbalanced cholesterol homeostasis to several human diseases, namely Wolman disease, cholesteryl ester storage disease, atherosclerosis and Alzheimer's disease, Niemann-Pick type C and Tangier disease is described.     

Biodiesel production from rice bran by a two-step in-situ process

The production of fatty acid methyl esters (FAMEs) by a two-step in-situ transesterification from two kinds of rice bran was investigated in this study. The method included an in-situ acid-catalyzed esterification followed by an in-situ base-catalyzed transesterification. Free fatty acids (FFAs) level was reduced to less than 1% for both rice bran A (initial FFAs content = 3%) and rice bran B (initial FFAs content = 30%) in the first step under the following conditions: 10 g rice bran, methanol to rice bran ratio 15 mL/g, H₂SO₄ to rice bran mass ratio 0.18, 60 °C reaction temperature, 600 rpm stirring rate, 15 min reaction time. The organic phase of the first step product was collected and subjected to a second step reaction by adding 8 mL of 5 N NaOH solution and allowing to react for 60 and 30 min for rice bran A and rice bran B, respectively. FAMEs yields of 96.8% and 97.4% were obtained for rice bran A and rice bran B, respectively, after this two-step in-situ reaction.     

Exploring a new,soluble lipase for FAMEs production in water-containing systems using crude soybean oil as a feedstock

Performance of a new lipase from Novozymes (Callera Trans L) was studied for fatty acid methylesters (FAMEs) production. In order to reduce the costs of the industrial enzymatic biodiesel production process, the enzyme was used in its soluble form instead of the common immobilized preparations. Cost reduction was also achieved by using crude (non-degummed) soybean oil as a cheaper raw material. The effect of water content during Callera Trans L-catalyzed FAMEs production was explored from evaluation of free fatty acids (FFAs), tri- di or monoacylglycerides (TAGs, DAGs, MAGs) variation during 24 h reaction. An excellent 96% FAMEs release was achieved when low (3–5%) water concentrations were used in the conversion of crude soybean oil. Time course HPLC analysis of the reaction products suggests that the soluble enzyme proceeds through a mechanism of methylester formation based on a first hydrolysis step that releases FFAs, DAGs or MAGs, followed by esterification of FFAs with methanol for FAMEs production.     

The incorporation of mevalonate-[2-14C] into the sterol fractions of Calendula officinalis

The incorporation of mevalonate-[2-¹⁴C] into the free sterols, steryl esters, steryl glucosides, acylated steryl glucosides and water-soluble complexes was investigated and the sterols of each fraction were separated into stanols, Δ⁷ sterols, Δ⁵ sterols, stigmasterol, clerosterol and methylene-cholesterol. The stanols and Δ⁷ sterols were more strongly labelled in the steryl esters than in the free sterols. The Δ⁵ sterols and stigmasterol were more intensively labelled in the free sterols than in the steryl esters. All sterol types were more labelled in the steryl glycosides than in the acylated steryl glucosides. Stanols were probably formed from Δ⁷ or Δ⁵ precursors.     

Metabolic interconversion of free sterols and steryl esters in Saccharomyces cerevisiae.

The interconversion of free and esterified sterols was followed radioisotopically with [U-14C]acetate and [methyl-14C]methionine. In pulse-chase experiments, radioactivity first appeared mainly in unesterified sterols in exponential-phase cells. Within one generation time, the label equilibrated between the free and esterified sterol pools and subsequently accumulated in steryl esters in stationary-phase cells. When the sterol pools were prelabeled by growing cells aerobically to the stationary phase and the cells were diluted into unlabeled medium, the prelabeled steryl esters returned to the free sterol form under several conditions. (i) During aerobic growth, the prelabeled sterols decreased from 80% to 45% esters in the early exponential phase and then returned to 80% esters as the culture reached the stationary phase. (ii) Under anaerobic conditions, the percentage of prelabeled steryl esters declined continuously. When growth stopped, only 15% of the sterols remained esterified. (iii) In the presence of an inhibitor of sterol biosynthesis, which causes accumulation of a precursor to ergosterol, prelabeled sterols decreased to 40% steryl esters while the precursor was found preferentially in the esterified form. These results indicate that the bulk of the free sterol and steryl ester pools are freely interconvertible, with the steryl esters serving as a supply of free sterols. Furthermore, there is an active cellular control over what types of sterol are found in the free and esterified sterol pools.     

Intracellular distribution and origin of sterols in Calendula officinalis leaves

In Calendula officinalis leaves 66% of all steryl forms are present in the ‘microsomal fraction’ (IV), 24% in the mitochondrial and Golgi membranes (III), 5% in the ‘chloroplast’ (II), 4% in the ‘cell wall and membrane’ (I) fraction and 1%. in the cytosol. Free sterols, their esters, glycosides and acylated glycosides are present in varying proportions in all cellular subtractions. Mevalonate-[2¹⁴C] labelling of sterols derived from various steryl forms showed that free sterols and all their derivatives, i.e. steryl esters and glucosides, are formed in fraction IV and are then translocated to other organelles. Fraction III is the main site of glycosylation of transported sterols as well as of acylation of steryl glycosides.     

The Neutral Lipids of Paramecium tetraurelia: Changes with Culture Age and the Detection of Steryl Esters in Ciliary Membranes1

Neutral lipids, particularly triglycerides, accounted for the major decrease in the total lipid content in Paramecium cells that occurs with culture age. Sterols, triglycerides, and steryl esters were the major classes of neutral lipids in cells and isolated cilia. Free as well as high concentrations of esterified sterols were detected in purified ciliary membrane preparations. Stigmasterol and 7-dehydrostigmasterol were the major components of both free and esterified sterols of cells and cilia; however, when cholesterol was present in the growth medium, it was desaturated to 7-dehydrocholesterol and incorporated into cellular and ciliary lipids. Free fatty acids from cells and triglycerides from cells and cilia were low in polyunsaturated fatty acids and reflected the composition of fatty acids in the culture medium. An exception was the reduced concentration of stearate in triglycerides from whole cells. Greater than 50% of triglyceride fatty acids from cilia were saturated. The fatty acid compositions of cellular triglycerides and ciliary steryl esters did not change with culture age, but those of cellular steryl esters and ciliary triglycerides did change. In comparison with phospholipids, these neutral lipid fatty acid compositional changes were smaller. The sensitivity of these stigmasterol-containing cells to polyene antibiotics indicated that they were killed by nystatin > filipin > amphotericin B. The unexpected finding of high concentrations of steryl esters in ciliary membrane preparations is discussed.     

Sterols in Germinating Seeds and Developing seedlings of Longleaf Pine, Pinus palustris

Sterols in germinating embryos and young seedlings of longleaf pine (Pinus palustris Mill.) were identified and quantities determined for different periods after germination. Sterol analyses were performed by gas-liquid chromatography (GLC) and verified by combination of GLC-mass spectrometry. Campesterol and β-sitosterol were two major sterols which accounted for most of the sterol composition while stigmasterol was present in very small amounts. No cholesterol was revealed by GLC-mass spectrometry although there was a minor peak appearing on the sterol gas-liquid chromatograms with a retention time close to that of authentic cholesterol. By fractionation, three different forms of sterols were obtained: steryl esters, steryl glycosides, and free sterols. The sterols were mainly found in the esterified fraction, while steryl glycosides and free sterols only made up a small portion of the total sterol value. The total sterol content in general increased during seedling development, and this increase reflected mainly a change in steryl esters. The low levels of both free and glycosidic sterols remained nearly unchanged throughout the experimental germination period.     

Hydrolysis of steryl esters by a lipase (Lip 3) from Candida rugosa

A well-known lipase, Lip 3 of Candida rugosa, was purified to homogeneity from a commercial lipase preparation, using hydrophobic interaction and anion exchange chromatography. Lip 3, which has been reported to act on cholesteryl esters, was also found to be active on plant-derived steryl esters. Lip 3 had optimal activity at pH 5-7 and below 55 degrees C. It was able to hydrolyse steryl esters totally in a clear micellar aqueous solution. However, the action on a dispersed colloidal steryl ester solution was limited and only about half of the steryl esters were degraded. The degree of hydrolysis was not improved by addition of fresh enzyme. The composition of released fatty acids and sterols was, however, almost identical to that obtained by alkaline hydrolysis, showing that all the different steryl esters were hydrolysed equally and that none of the individual components were responsible for incomplete hydrolysis. Thus, it appeared that the physical state of the colloidal steryl ester dispersion limited the action of Lip 3. Wood resins contain both triglycerides and steryl esters among the hydrophobic components, which create problems in papermaking. The simultaneous enzymatic hydrolysis of triglycerides and steryl ester is therefore of considerable interest and Lip 3 is the first enzyme reported to act on both triglycerides and steryl esters.     

Sterols in hardening winter wheat

The main sterols in winter wheat crowns and roots were sitosterol and campesterol, with significant amounts of stigmasterol and traces of cholesterol. The main groups of sterol-containing lipids were free sterols, steryl glucosides, steryl esters and esterified steryl glucosides. Sterol analysis within each group showed little difference between them. Steryl esters were relatively rich in cholesterol and poor in stigmasterol. Free sterols were rich in stigmasterol. Low temperature caused an increase in sterol content but had little effect on sterol composition and sterol to lipid P ratio. There was some increase in steryl esters and some decrease in free sterols. Cholesterol and stigmasterol decreased in the steryl ester and free sterol fractions, respectively. There was little evidence for involvement of sterols in winter wheat frost hardening.     

Concentration gradients of free sterols,steryl esters and lipid phosphorus in the trunkwood of Scot's pine (Pinus sylvestris L.)

The amounts of free sterols, steryl esters and lipid phosphorus were determined in the sapwood and heartwood of mature, and in the outer and inner sapwood of young Pinus sylvestris trees. In the mature trees (up to 70 years old) the heartwood contains significantly higher amounts of free sterols than the sapwood. No radial gradient can be demonstrated in the amounts of steryl esters. Lipids extracted from the sapwood contain higher amounts of phosphorus than those from the heartwood. Stems of young Pinus sylvestris trees (up to 13 years old) show in the inner sapwood higher amounts of both free sterols and steryl esters than the peripheral younger wood zone. The inner sapwood of the young stems shows slightly higher amounts of lipid phosphorus than the outer sapwood. The results indicate that Pinus sylvestris accumulates both free sterols and steryl esters in the stems at a very early stage of the life cycle. Sterol accumulation in the innermost parts of the stems seems not to depend on heartwood formation.     

气相色谱法测定啤酒中的游离脂肪酸

气相色谱法测定啤酒中辛酸到二十二碳酸共１１种游离脂肪酸,采用多级溶剂萃取及薄层色谱纯化技术进行样品制备,并采充氮措施抑制脂肪酸的氧化产生,此方法有较好的重复性和回收率。     

Towards a commercially potential process: Enzymatic recovery of phytosterols from plant oil deodoriser distillates mixture

In order to examine the industrial potential to indirectly isolate phytosterols from deodoriser distillates (DODs), enzymatic transesterification of an industrial rapeseed and soybean oil DOD mixture with bioethanol was investigated using commercial lipases and a few newly immobilised preparations of lipases. The lipases from different sources and differing preparation forms were evaluated, in terms of thermostability, enzyme efficiency, and toleration of ethanol. Lipozyme 435 and Lipozyme NS-40044 TLL were found to be most effective biocatalysts in catalysing ethanolysis of glycerides and steryl esters from DODs. The optimum conditions are 10% enzyme load (wt% of DODs), ethanol/DODs of 3.0:1.0 (mol/mol), water content 0.125% (based on the weight of total mixture), and reaction at 30 °C for 5 h. The results demonstrated that >95% sterols can be recovered as free form (>85% sterol esters were liberated as free sterols within 4 h). With this process, the system was simplified as fatty acid ethyl esters and free sterol as major components, where free sterols can be recovered via solvent extraction or molecular distillation. Furthermore, a reuse study of enzyme in consecutive batch reactions demonstrated an excellent operation stability and reusability of Lipozyme 435 and Lipozyme NS-40044 TLL with the developed process. This work indicated that the industrially refined waste DODs can be directly subjected to an enzymatic process for high efficacy recovery of phytosterol without any pre-process, driven by robust lipase preparations.     

STEROLIC BIOMARKERS IN MARINE PHYTOPLANKTON. II. FREE AND CONJUGATED STEROLS OF SEVEN SPECIES USED IN MARICULTURE

The sterols of seven unicellular algae widely used in mariculture and belonging to different classes (Prasinophyceae, Haptophyceae, Eustigmatophyceae, and Diatomophyceae) were examined for free and combined forms. Under standard culture conditions, all synthesized free sterols and combined forms (steryl esters, acyl steryl glycosides, and steryl glycosides). Free sterols were dominant in five of the species. In contrast, sterols were mostly esterified in the eustigmatophyte Nannochloropsis oculata (Droop) Hibberd and the diatom Thalassiosira pseudonana Hasle et Heimdal, whereas in another diatom, Chaetoceros calcitrans (Paulsen) Takano, glycosylated forms represented over 60% of total sterols. The pennate diatom Haslea ostrearia (Gaillon) Simonsen synthesized large amounts of steryl glycosides consisting mainly of an unusual sterol, 23,24-dimethylcholest-5-en-3β-ol, occurring in some dinoflagellates.     

Sterols in relation to ageing of seeds of Helianthus annuus and Cicer arietinum

Under accelerated ageing at high relative humidity and high temperature for 4 days germination and membrane permeability remained unaffected both in sunflower and chick pea seeds. However, the steryl glycoside concentration in the pooled leachate increased progressively with ageing. Total sterols, as well as steryl glycosides and free sterols of the seeds, increased with a concomitant decline in steryl esters under accelerated ageing. Pretreatment with the sterol biosynthesis inhibitor SK & F 7997A₃ prevented the increase of total sterols under accelerated ageing conditions but there were increases in the amounts of steryl glycosides and free sterols and a decrease in steryl ester after such treatment, therefore, indicating interconversions of the various sterol types. Accelerated ageing also caused increases in free amino acids and soluble carbohydrate. Low relative humidity-high temperature and high relative humidity-low temperature failed to produce such effects.     

Effect of Light on Mevalonic Acid Incorporation into the Phytosterols of Nicotiana tabacum L. Seedlings

Mevalonic acid-2-¹⁴C was readily incorporated into the free, esterified, and glycosidic sterol fractions of tobacco (Nicotiana tabacum L. var. Burley 21) seedlings. The time course of mevalonic acid-2-¹⁴C incorporation was different for the various individual sterols. Campesterol and sitosterol (group I) became radioactive as the free sterol and subsequently as the steryl ester. The reverse order was observed for cholesterol and stigmasterol (group II). Light stimulated the incorporation of mevalonic acid-2-¹⁴C into the group I free sterols and during the first 6 to 9 hours into the steryl esters of group II. The increase in specific radioactivity of the group II steryl esters was followed by a decline. Based on time course studies it is suggested that the group II steryl esters turn over rapidly and that light influences the rate of turnover.     

Cellular pregnenolone esterification by acyl-CoA:cholesterol acyltransferase

Pregnenolone (PREG) can be converted to PREG esters (PE) by the plasma enzyme lecithin: cholesterol acyltransferase (LCAT), and by other enzyme(s) with unknown identity. Acyl-CoA:cholesterol acyltransferase 1 and 2 (ACAT1 and ACAT2) convert various sterols to steryl esters; their activities are activated by cholesterol. PREG is a sterol-like molecule, with 3-β-hydroxy moiety at steroid ring A, but with much shorter side chain at steroid ring D. Here we show that without cholesterol, PREG is a poor ACAT substrate; with cholesterol, the V(max) for PREG esterification increases by 100-fold. The binding affinity of ACAT1 for PREG is 30-50-fold stronger than that for cholesterol; however, PREG is only a substrate but not an activator, while cholesterol is both a substrate and an activator. These results indicate that the sterol substrate site in ACAT1 does not involve significant sterol-phospholipid interaction, while the sterol activator site does. Studies utilizing small molecule ACAT inhibitors show that ACAT plays a key role in PREG esterification in various cell types examined. Mice lacking ACAT1 or ACAT2 do not have decreased PREG ester contents in adrenals, nor do they have altered levels of the three major secreted adrenal steroids in serum. Mice lacking LCAT have decreased levels of PREG esters in the adrenals. These results suggest LCAT along with ACAT1/ACAT2 contribute to control pregnenolone ester content in different cell types and tissues.