Apparatus and method for obtaining glycoglycerolipids and glycosphingolipids from lipid phases
10239906 ยท 2019-03-26
Assignee
Inventors
Cpc classification
C07H15/04
CHEMISTRY; METALLURGY
International classification
C07H15/04
CHEMISTRY; METALLURGY
Abstract
The present invention relates to an apparatus and method for separating glycoglycerolipids and also glycoglycerolipids and glycosphingolipids from a lipid phase that contains glycoglycerolipids and acyl glycerides or glycoglycerolipids and glycosphingolipids and acyl glycerides, in mild conditions with no hydrolysis and while at the same time effectively depleting the lipid phase of said glycoglycerolipids, glycoglycerolipids and glycosphingolipids and their accompanying substances using an aqueous extraction process.
Claims
1. A method for the hydrolysis-poor separation of glycoglycerolipids from a lipoid phase which contains glycoglycerolipids and acylglycerides, comprising the steps: A1) providing a lipoid phase containing glycoglycerolipids and acylglycerides, B1) adding to the lipoid phase an aqueous phase containing anions of at least one salt which has a solubility of at least 30 g/l in water at 20 C. and which, upon dissociation in water, forms carbonate (CO.sub.3.sup.2), bicarbonate (HCO.sub.3.sup.), metasilicate (SiO.sub.3.sup.2), orthosilicate (SiO.sub.4.sup.4), disilicate (Si.sub.2O.sub.5.sup.2), trisilicate (Si.sub.3O.sub.7.sup.2), acetate (CH.sub.3COO.sup.), borate (BO.sub.3.sup.3), and/or tartrate (C.sub.4H.sub.4O.sub.6.sup.2); C1) mixing the lipoid phase and the aqueous phase; D1) separating the glycoglycerolipid-rich aqueous phase and obtaining a glycoglycerolipid-poor lipoid phase; and D2) recovering the glycoglycerolipids from the separated glycoglycerolipid-rich aqueous phase.
2. The method according to claim 1, comprising the following step E1) after step D1): E1) adding second aqueous phase containing at least one compound which has at least one amidino group and/or at least one guanidino group to the glycoglycerolipid-poor lipoid phase, followed by mixing the glycoglycerolipid-poor lipoid phase and the second aqueous phase and separating the second aqueous phase.
3. The method according to claim 1, wherein the glycoglycerolipids are lipophilic glycoglycerolipids having a lipophilicity index GL of 1.0GL6.0, wherein the lipophilicity index GL is calculated according to the following formula:
4. The method according to claim 1, wherein the glycoglycerolipids are glycosyldiacylglycerols, glycosylylalkylglycerols, and glycosyldialkylglycerols.
5. The method according to claim 1, wherein the glycoglycerolipid-rich aqueous phase do not contain any carboxylate, sulfate, sultanate, or phosphate group(s).
6. The method according to claim 1, wherein the lipoid phase in step A1) further comprises sterylglycosides which are separated in the aqueous phase in step D1).
7. The method according to claim 1, wherein in step B1) an aqueous phase which contains cations of a salt which has a solubility of at least 30 g/l in water at 20 C. and which, upon dissociation in water, forms Mg.sup.2+, Ca.sup.2+, Ti.sup.2+, Ti.sup.4+, Co.sup.2+, Co.sup.3+, Ni.sup.2+, Cu.sup.2+, Zn.sup.2+, Sn.sup.2+, or Sn.sup.4+ ions is added to the lipoid phase.
8. The method according to claim 1, wherein if phospholipids and/or fatty acids are contained in the lipoid phase, a phospholipid-free and/or fatty acid-free glycoglycerolipid-rich phase is obtained if the following step A2 or A2) is carried out after step A1) and before step B1): A2) adding water phase as aqueous phase to the lipoid, followed by mixing the lipoid phase and the aqueous phase and separating the aqueous phase, or A2) adding an aqueous carboxylic acid solution or an aqueous solution of an inorganic acid having a pH between 3.0 and 5.0 as aqueous phase to the lipoid phase, followed by mixing the lipoid phase and the aqueous phase and separating the aqueous phase.
9. The method according to claim 8, wherein the lipoid phase and the aqueous phase are intensively mixed in step C1) and/or A2) or A2).
10. The method according to claim 1, comprising the following step E1) after step D2): E1) adding an aqueous phase containing at least one compound which has at least one amidino group and/or at least one guanidino group to the lipoid glycoglycerolipid-poor phase, followed by mixing the lipoid glycoglycerolipid-poor phase and the aqueous phase and separating the aqueous phase.
11. A method for recovering hydrolysis-poor glycoglycerolipids and glycosphingolipids from a lipoid phase which contains glycoglycerolipids, glycosphingolipids, and acylglycerides, comprising the steps of: A1) providing a lipoid phase containing glycoglycerolipids, glycosphingolipids, and acylglycerides; B1) adding to the lipoid phase an aqueous phase containing anions of at least one salt which has a solubility of at least 30 g/l in water at 20 C. and which upon dissociation in water, forms carbonate (CO.sub.3.sup.2), bicarbonate (HCO.sub.3.sup.), metasilicate (SiO.sub.3.sup.2), orthosilicate (SiO.sub.4.sup.4), disilicate (Si.sub.2O.sub.5.sup.2), trisilicate (Si.sub.3O.sub.7.sup.2), acetate (CH.sub.3COO.sup.), borate (BO.sub.3.sup.3), Or tartrate (C.sub.4H.sub.4O.sub.6.sup.2); C1) mixing the lipoid phase and the aqueous phase; D1) separating the glycoglycerolipid-rich and/or glycosphingolipid-rich aqueous phase and obtaining a glycoglycerolipid-poor and/or glycosphingolipid-poor lipoid phase; and D2) recovering the glycoglycerolipids from the separated glycoglycerolipid-rich aqueous phase.
12. The method according to claim 11, wherein the glycosphingolipids are lipophilic glycosphingolipids having a lipophilicity index SL of 1.0SL7.0, wherein the lipophilicity index SL is calculated according to the following formula:
13. The method according to claim 11, wherein the glycoglycerolipids do not contain any carboxylate, sulfate, sulfonate, or phosphate group(s).
14. A method according to claim 11, wherein the lipoid phase in step A1) further comprises sterylglycosides which are separated in the aqueous phase in step D1).
15. The method according to claim 11, wherein in step Bib) an aqueous phase which contains cations of a salt which has a solubility of at least 30 g/l in water at 20 C. and which, upon dissociation in water, forms Mg.sup.2+, Ca.sup.2+, Ti.sup.2+, Ti.sup.4+, Co.sup.2+, Co.sup.3+, Ni.sup.2+, Cu.sup.2+, Zn.sup.2+, Sn.sup.2+, or Sn.sup.4+ ions is added to the lipoid phase.
Description
DESCRIPTION OF THE FIGURES
(1)
(2)
(3)
EXAMPLES
(4) Methods
(5) The efficiency of the inventive technique for separating a glycoglycerolipid-rich fraction from lipoid phases can be examined by various methods from the prior art.
(6) Viscometry
(7) It has been shown that the separation of the glycolipid fraction significantly reduces the viscosity of the purified lipoid phase. Therefore, a reduction in the viscosity of the purified lipoid phase of at least 10%, more preferably at least 20%, and most preferably at least 30% as compared to the viscosity of the raw lipoid phases considered to be a result according to the invention.
(8) Alkaline Earth Metal and Metal Salt Binding Capacity
(9) Alkaline earth metal ions and metal ions virtually do not distribute in an apolar lipid phase. However, sugar residues of glycoglycerolipids and glycosphingolipids are capable of fixating such ions via hydrogen bonds, which is why many lipoid phases, such as vegetable oils, are contaminated with these ions. The binding capacity of a glycoglycerolipid-containing lipoid phase for such ions can therefore be used to estimate the content of sugar compounds. The binding capacity for alkaline earth metal ions as well as metal ions is preferably reduced by 80%, more preferably by >90% and most preferably by >95% with the methods according to the invention.
(10) The physico-chemical properties of the separated glycoglycerolipid-containing fraction can be investigated by established methods such as, HLB chromatography, tensiometry, and determination of the critical micellar concentration (CMC).
(11) Qualitative detection of glycoglycerolipids and glycosphingolipids can be performed by methods such as atomic emission spectroscopy and thin-layer chromatography (TLC). By means of the latter, separation into different compound classes is possible with subsequent differentiation of the sugar residues present. A narrow and sharp delineation of the bands indicates a high uniformity of the compounds present therein, whereas a broadening and unspecific limitation of the bands indicates a heterogeneity of the compounds and, in particular, of the sugar residues, and thus this criterion is suitable for the detection of a hydrolysis.
ABBREVIATIONS
(12) FFA: free fatty acids
(13) ppm: parts per million
(14) na: not applicable/not tested
(15) nd: not investigated/determined
(16) rpm: revolutions per minute
Example 1
(17) Recovery of a Glycoglycerolipid Lipid Fraction after Degumming of a Press Oil
(18) For testing whether after the degumming of a press oil or separation of fatty acids which still remain in the lipoid phase by means of an aqueous solution containing guanidino compounds, a crude oil of a screw pressing of stored jatropha nuts obtained at a temperature of 60 C. with the following key values: total phosphorus content 248 ppm, FFA 1.8 wt %, calcium 70 ppm, is refined according to the following scheme: a) 3% addition of a 0.5-1.0 molar NaOH solution b) 3% addition of a 75 wt % phosphoric acid solution a1) refined oil after step a) 0.3% addition of a 0.6 molar arginine solution b1) refined oil after step b) 0.3% addition of a 0.6 molar arginine solution The refined oils obtained after the above-described steps were then further treated with the following steps: c) 5% addition of a 10% sodium metasilicate solution d) 4% addition of a 15% potassium carbonate solution
(19) For each experiment, 200 ml was used for each refining step. The aqueous solutions were admixed by homogenization with an Ultrathurrax at 24,000 rpm for 3 minutes that was carried out by cycling movement of the container at room temperature. The resulting emulsions were centrifuged in a beaker centrifuge at 3,800 rpm for 5 minutes. Then upper phases were separated by decanting or withdrawing the phase.
(20) In the oil phases, contents of phosphorus, free fatty acids, and calcium were determined, and the quantity of dry mass of compounds that were contained in the aqueous phases was determined after steps C) and C). For the latter, the water was removed by vacuum drying. Furthermore, the water-binding capacity of the previously treated oils A1) and A1) and of the glycoglycerolipid-poor lipoid phases obtained after performing steps: A1)+C); A1)+D); B1)+C); B1)+D), according to the invention, were investigated by admixture of deionized water (1 ml to 50 ml) with an Ultrathurrax (20,000 rpm for 2 minutes) to the obtained oil phases and then the mixture was separated with a centrifuge at 4,000 rpm. The water content of the oils was determined by the Karl Fischer method. The dried organic materials from the separated water phases were dissolved in chloroform, followed by centrifugation at 5,000 rpm for 5 minutes. Then, the solvent phase was withdrawn and the organic matter was dried by means of a vacuum evaporator. Each of 20 mg of the obtained dry matter was dissolved in 1 L of deionized water. Samples therefrom were used for determination of the surface tension by means of a tensiometer (K 100, Krss, Germany).
(21) Results:
(22) The degumming procedures resulted in a substantial removal of hydratable phospholipids, free fatty acids, and a marked reduction of alkaline earth metal ions. Significant amounts of lipophilic organic compounds were separated into the aqueous medium by treatment with solutions of the salt compounds according to the invention which were admixed with an intensive mixing procedure; those compounds could then be removed. The amounts of the obtained dry substance were considerably greater than the calculated sum of the residual amounts of phospholipids, fatty acids, and alkaline earth metals which had also been separated. From the resulting dry mass, glycerolipids can be converted into an organic solvent and recovered therefrom. The obtained glycoglycerolipids exhibited extremely good surfactant properties in water.
Example 2
(23) The oily fraction after sedimentation of the press liquid of the Accocromia palm fruit with the key values: phosphorus content 128 ppm, FFA 2.6 wt %, calcium 48.8 mg/kg were examined with regard to separable glycoglycerolipid lipid fractions.
(24) For this purpose, 200 ml of the lipoid phase were pretreated by the following methods:
(25) a) addition of 5% of a low-ion water stirring with a propeller stirrer at 2,500 rpm while heating at 50 C. for 90 minutes. b) addition of 3% of a citric acid solution which is homogenized by means of an Ultrathurrax for 5 minutes at 24,000 rpm while heating the emulsion to about 50 C.
The lipoid phases that have been refined with the above-described steps which were obtained after centrifugation were further treated by each of the following steps: c) addition of 8% of a 15 wt % copper acetate solution, d) addition of 4% of a 20 wt % sodium hydrogen carbonate solution.
The aqueous phases of c) and d) are homogenized with the lipoid phases by means of an Ultrathurrax for 5 minutes at 24,000 rpm while heating the emulsions to about 50 C. The resulting emulsions were centrifuged in a beaker centrifuge at 4,000 rpm for 5 minutes. Subsequently phase separation was carried out by decanting or withdrawing the lipoid phases.
(26) The treated lipoid phases of refining steps c) and d) were reprocessed by the identical cleaning step as previously carried out and designated as c1) and d1). Thereafter the obtained lipoid phase c1) was treated according to process d) and the obtained lipoid phase d1) was treated according to process c).
(27) The content of phosphorus, free fatty acids, and calcium was determined in the lipoid phases, and for the aqueous phases of refining steps c) and d) the amount of the organic mass after drying were determined; further for the latter samples the HLB value was determined. Determination of the HLB value was carried out with an Asahipak GF-310 HQ multiple solvent GPC column. Here, ionic and nonionic surfactants can be differentiated and classified according to their HLB value.
(28) Thin layer chromatography was performed with silica gel G plates. Separation was carried out using a mixture of chloroform/acetone/water (30/60/2). These were developed using a naphthylenediamine reagent, whereby sugar residues of the glycerolipids can be color-coded.
(29) Results:
(30) The lipoid phase of palm kernel peel material has a high content of nontriglyceride contaminants which consist largely of glycerol glycerides and sterols and contain only a small proportion of phospholipids. These accompanying substances cause turbidity and high viscosity of the oily phase. The residual values for phosphorus, free fatty acids, and alkaline earth metal ions were already significantly reduced by aqueous or acid degumming, but the lipoid phases remained highly viscous. An intensive mixing process of the aqueous solutions containing compounds according to the invention with the lipoid phase resulted in considerable formation of emulsions which further increased viscosity. However, further homogenization using a rotor-stator mixer enabled liquification of those emulsions which could be separated by centrifugation into a slightly turbid lipoid phase and a whitish semisolid mass. The amount of organic matter removed from the lipoid phase as achieved in the first separation step was largely independent of the previously performed degumming process and the salt dissolved in the aqueous phases according to the invention. It was found that even with a second separation, relevant amounts of oil contaminants could be separated. Then, in a further refining step using an intensive mixing process with the aqueous solutions of the salts according to the invention, virtually no additional accompanying substances could be separated. The total amounts of solids separated were far above the calculated sum of phospholipids, fatty acids, and metal ions separated. By thin layer chromatography, on the one hand, co-separation of relevant quantities of triacylglycerols could be excluded; on the other hand, digalactosyl- and monogalactosyldiglycerides as well as sterylglycosides could be detected. In the surfactant analysis, the presence of ionic surfactants in discrete amounts with an HLB value of 13 as well as a clear detection of nonionic surfactants with an average HLB value of 8 and 9 were found for the separated phase of c) and d).
Example 3
(31) Linseed press cake was placed in an aqueous solution with addition of 5% isopropanol and was homogenized with an immersion blender. The slurry was then stirred at 50 C. for 30 minutes. This was followed by the addition of a 3-fold amount of petroleum ether. After further homogenization, a centrifugal phase separation was carried out. The separated organic phase (OP1) was reduced to half its initial volume by means of a vacuum evaporator. This was followed by the addition of 5 vol % of a methanol/water mixture (95/5) to OP1, mixing of the solution, and subsequent centrifugal phase separation. The lightly turbid alcohol-water phase was pipetted off. The remaining organic phase (OP2) is fractionated and the following volume fractions and concentrations of the substances according to the invention are provided to the resulting fractions:
(32) A) potassium carbonate, B) sodium dihydrogen carbonate, C) mixture of sodium and potassium metasilicate, D) calcium acetate, E) aluminum acetate tartrate, F) sodium borate with concentrations of 3%, 10%, and 15% and a volumetric addition of 2%, 4%, and 8%, respectively.
(33) To 100 ml each of the organic phases (OP2), the solutions according to the invention were added and homogenized with an Ultrathurrax at 24,000 rpm for 30 seconds. After 3 minutes standing time, phase separation was performed at 4,000 rpm over a period of 10 minutes. The supernatant (organic phase, OP3) was then separated. Each of the corresponding aqueous phases (WP3) was intensively mixed with 50 ml of n-heptane, followed by phase separation as described above. The resulting aqueous phase (WP4) was dried with a vacuum evaporator and the dry matter was weighed. Analyses of the content of phosphorus (atomic absorption analysis) and nitrogen (Kjeldahl's method) of the dry mass were carried out for the investigations with a volume addition of 10% from all investigated substances. Thin layer chromatography according to Example 2 was prepared using the dry substance from each batch.
(34) Results:
(35) The mixing of the organic phases (OP2) with the aqueous salt solutions A)-F) according to the invention resulted in considerable emulsion formation. Phase separation could be carried out by centrifugation obtaining a creamy colored semisolid (at 2 vol %) to a viscous (at 15 vol %) aqueous phases (WP3) and a slightly to moderately turbid organic phase (OP3). After extraction of di- and triacylglycerides by means of an n-heptane extraction, WP3 was dried by vacuum drying, resulting in a brownish sticky, highly viscous residue. Only of small amounts of nitrogen-containing compounds (e.g. sphingolipids or proteins) or phosphate-containing compounds (e.g. phospholipids) were in there. In thin layer chromatography, bands corresponding to digalactosyl- and monogalactosyldiglycerides and sterylglycosides were present in all samples. It thus has been shown that a selective separation of glycoglycerolipids and glycosphingolipids from a lipoid phase of a phytoextraction is possible with the method according to the invention.
Example 4
(36) Rice bran from a standard process of rice processing with an oil content of 18% and a water content of 35% was stored at 8 C. after obtaining until lipid extraction. This starting material was mixed with 10% water and stirred at 30 C. for 2 hours. The mixture was extracted twice with n-hexane at 50 C. The aqueous phase (WP1) was concentrated to a highly viscous residue by means of a vacuum evaporator. Each of 100 g of the highly viscous mass was mixed with 300 ml of a mixture of chloroform and acetone (80:20), and the organic phase (OP2) was separated by means of centrifugal phase separation. Samples of 150 ml of OP2 were each mixed with either 20 ml aqueous solutions of a) sodium carbonate, b) sodium orthosilicate, c) copper acetate (Cu(OAc).sub.2), d) potassium tartrate or e) potassium borate, each at concentrations of 15%, and homogenized with an Ultrathurrax at 20,000 rpm for 20 s. After a standing period of 60 seconds, phase separation was carried out by a centrifuge at 5,000 rpm for 10 minutes. The respective organic phases (OP3) were separated and the respective aqueous phases (WP3), which had a highly viscous to semisolid consistency, were homogenized. From the homogenized aqueous phases (WP3), a 1 ml sample was separated, the remainder was dried by means of a vacuum evaporator and the quantity of substance which subsequently remained was weighed. The separated sample was hydrolyzed with sodium methoxide dissolved in methanol and then fractionated by means of silica gel chromatography. The fraction of glycosylceramides was dissolved in pyridine and MTPA-C1 (-methoxy--trifluoromethylphenylacetic acid chloride) was added at 0 C. The solution was stirred and concentrated over 24 hours at room temperature. After further purification by means of silica gel column chromatography with hexane/ethyl acetate (1:1) as eluent, a white solid was obtained after evaporation of the eluent. Using ESI-TOF-MS (electrospray time-of-flight mass spectrometry), sugar esters (m/z 1195.52 [M+H]+) were detected. The organic phases (OP3) were completely evaporated and the resulting solids were hydrolyzed and processed by the same method as described above.
(37) Results:
(38) Aqueous extraction of OP2, organic matter with quantities of a) 8.4 g, b) 11.7 g, c) 10.2 g, d) 9.9 g, and e) 10.1 g could be separated. In the separated organic matter which has been obtained from WP3 sugar compounds could be detected after hydrolysis, so that the presence of various sugar-containing lipid compounds (glycoglycerolipids, glycosphingolipids) can be assumed. In the organic phases (OP3), practically no sugar compounds could be detected after the hydrolysis, so that separation of sugar-containing compounds by means of the aqueous extraction process according to the invention is largely complete.
Example 5
(39) Examination on the extraction and recovery of glycoglycerolipids and glycosphingolipids from lipoid plant extracts and for their use as a baking aid.
(40) Cold pressed fruit pulp from kernels of the Acrocomia palm with an oil content of approx. 70% was diluted 1:1 with a mixture of acetone, dichloromethane, and hexane (ratio 1:1:5) and mixed well mechanically. Thereafter, two extraction steps were carried out with an aqueous 0.4 molar arginine solution at a volume addition of 4% in each. Phase separation was achieved by centrifugation. The lipoid phase was first mixed twice with a 10% sodium carbonate solution with a volume addition of 4% and the mixture was stirred; while the first mixing procedure was performed with an stirrer at 500 rpm and 15 minutes, the mixing procedure in the second extraction was carried out with an Ultrathurrax at 18,000 rpm for 5 minutes using an aqueous solution with identical volume and concentration. Phase separation was carried out by centrifugation at 5,000 rpm over 15 minutes. Each of the aqueous phases of the two subsequent separations consisted of a white viscous emulsion. The emulsions were combined and then freeze-dried. Subsequently, the lyophilized mass was dissolved in a mixture of chloroform and methanol (5:1), and forwarded to preparative column chromatography (silica gel matrix 60). Elution was performed with acetone/chloroform (5:1). The eluate was dried by means of a vacuum evaporator. A dry mass of 56 g was obtained; the initial quantity was 800 g.
(41) With the fraction thus obtained, containing glycoglycerolipids and glycosphingolipids, small-sized baking experiments were carried out according to a standard procedure: dough was prepared using 10 g flour, 7% fresh yeast, 2% of NaCl, 1% sucrose, 0.002% ascorbic acid, and water in which 30 mg of the mass containing glycoglycerolipids and glycosphingolipids had been dissolved by mixing (1200 rpm at 20 C. for 1 minute). The prepared samples were allowed to rise at 30 C. for 40 minutes, and were then baked at 185 C. for 10 minutes. Then the volume of the baked material was determined. For comparison, baking tests were carried out under identical conditions without addition of the mass containing glycoglycerolipids and glycosphingolipids and with addition of 0.3% of a pure lecithin powder (Jean-Puetz, Germany), which had been dissolved in water.
(42) Results:
(43) The liquid obtained by pressing the palm kernel fruit from palm trees of the genus Acrocomia is a lipoid phase in which a high proportion of glycoglycerolipids and glycosphingolipids, waxes, free fatty acids and fibers are present. Mixing with water results in formation of stable emulsions which cannot be broken by physical measures. It has been found that the separation of the free fatty acids is possible by means of an aqueous arginine solution while simultaneous dissolving of other lipoid substances in organic solvents and that subsequently a fraction containing glycoglycerolipids and glycosphingolipids can be separated off by aqueous extraction according to the invention. During the first addition of the solution with an intensive mixer, strong emulsion formation resulted; here subsequently performed phase separation had a poor result. When the aqueous solution was initially admixed by stirring with a propeller mixer, whereby input of air was avoided, there was also a marked formation of aggregates; however, phase separation was possible. After the first depletion of the lipoid phase, repeated admixture of the salt solution by means of an intensive mixer resulted in formation of an emulsion also, separation by means of centrifugal phase separation into a clear oil phase and a water phase containing solids was possible. The obtained aqueous fractions were combined, from this fraction a mixture of glycoglycerolipids and glycosphingolipids was obtained after preparative purification that was readily dissolvable in water. In the baking tests, a significant increase in the volume of the baking product was observed compared to baking results without addition of the mass containing glycoglycerolipids and glycosphingolipids (+300%) and compared to the result with lecithin added (+120%).
Example 6
(44) Investigations of the effect of process parameters on the extraction efficiency and hydrolysis stability of fractions containing glycoglycerolipids and glycosphingolipids
(45) Camelina oil, obtained by means of a screw press at 50 C., was intensively stirred with 3% deionized water for 1 hour at 45 C. Subsequently, the water phase was separated by means of a separator. To each 200 ml of the water-degummed oil (oil1), 4 vol % of an aqueous solution of potassium hydrogen carbonate, anhydrous sodium metasilicate, and calcium acetate (in each case 15 wt %) was added and homogenized with an Ultrathurrax for 5 minutes at 25,000 rpm. Immediately thereafter, centrifugation was performed at 5,000 rpm for 10 minutes, separating the oil phase (oil2) and the aqueous phase (WP1), from which oil-associated residues were removed by layering hexane and subsequent centrifugation, and then removing the organic phase. The thus obtained aqueous phase (WP2) had a highly viscous consistency. The WP2 was shaken and divided in 2 equal volume fractions; one of which was subjected to vacuum drying, then the dry weight was determined. Thin layer chromatographic studies were carried out from a sample of the degummed oil (oil1) and of the oil phase (oil2) obtained after the aqueous extraction. The degummed oil (oil1) showed distinct and sharply defined bands corresponding to monogalactosyldigigcerides, digalactosyldiglycerides, and glycosphingolipids. There were virtually no bands visible in the TLC of the oil phases (oil 2), which were treated with the above-mentioned aqueous solutions. The second volume fraction of WP 2 was intensively shaken immediately after preparation with a solvent mixture (chloroform/methanol/acetic acid, 90/8/2) and the organic phases (OP 1) were removed. The solvents of the obtained organic phases were removed by vacuum drying and the dry substance, which remained from the organic phase (OP1), was dissolved in the solvent mixture to subsequently perform thin layer chromatography. The chromatographic analyses were carried out in order to detect bands for glycoglycerolipids and glycosphingolipids and in respect of the width of the respective bands. Then aliquots of 100 ml each of oil phase 2 (oil 2) were homogenized with each of the previously used salt solutions by means of an Ultrathurrax for 5 minutes. Subsequently, phase separation was performed and the quantity of dry matter of organic compounds in the water phases was determined as described above.
(46) Various test modifications using 100 ml aliquots of the oil were performed using aqueous solutions of the above-mentioned salts according to the invention. Variations of the process temperatures (35, 55, and 75 C.) and the intensity of the mixing procedure after addition of the aqueous solutions with the above-mentioned salts according to the invention were performed. Furthermore, mixing devices differing in the achievable mixing intensities thereof were used: A) Ultrathurrax 25,000 rpm, B) Propeller mixer 2500 rpm, C) Ultrasonic. The mixing procedures were carried out for 5 minutes under continuous temperature control.
(47) In other investigations the mixing process was performed with mixer B) and C) for a duration of 10 and 20 minutes.
(48) Results:
(49) The intensive introduction of the aqueous solution with the substances according to the invention which was established in preliminary studies was applied in corresponding reference investigations, and it could be shown that an almost complete separation of glycoglycerolipids and glycosphingolipids was enabled. Moreover, thin layer chromatography results of the fractions obtained from the aqueous phases (WP2) resulted in identification of bands that correspond to glycoglycerolipids and glycosphingolipids, and which exhibited sharp boundaries, i.e., no relevant hydrolysis had taken place.
(50) The amounts of the separated dry matter obtained after a mixing procedure with a propeller mixer were markedly lower than those obtained by an intensive mixing procedure. In the same way, the amount of glycoglycerolipids and glycosphingolipids found in the oil treated in this way was also clearly discernible. By a renewed treatment by admixture of the respective salt solutions by means of an intensive mixer, further organic material was extracted; the quantities of the dry matter of both extractions were comparable to that obtained from an extraction by using an intensive mixer only.
(51) Chromatographic bands corresponding to glycoglycerolipids and glycosphingolipids were present in all samples of the dry masses obtained by the extractions with the above-mentioned salt solutions. There was no broadening of the boundaries of the chromatographic bands for the investigations that were performed with durations <5 minutes, indicating the absence of hydrolysis products. A longer duration of the mixing process with the propeller mixer increased the amounts of extractable organic matter to an amount comparable with that obtained by an intensive mixing process. While at a mixing temperature of 35 C., only a treatment time of more than 10 minutes caused slight signs of a hydrolysis of the glycoglycerolipids and glycosphingolipids, hydrolysis-induced widening of the bands of the glycoglycerolipids and glycosphingolipids could be seen already after 5 minutes at higher treatment temperatures which increased significantly when mixing was performed longer at these temperatures.
Example 7
(52) Grape press residues were microbially decomposed in a fermentation tank under continuous percolation conditions and addition of carboxylic acids. After 7 days, a sample of 1 liter was taken and intensively mixed with 1 liter of biodiesel (C8 to C18-methyl ester). The mixture was centrifuged and the heavily turbid organic phase (OP1) was removed. Then 100 ml of the organic phase (ON) was mixed with 5 ml of magnesium hydrogen carbonate or potassium acetate (10% strength aqueous solution) at 30 C. using a propeller mixer at 1,000 rpm for 7 minutes. This was followed by centrifugation at 4,000 rpm for 10 minutes. The resulting organic phase (OP2) was only slightly turbid; the aqueous phase (WP2) consists of a cream-colored semisolid mass. After carefully decanting the organic phase (OP2), the aqueous phase (WP2) was added to 100 ml of chloroform and mixed (OP3). Then 2 ml of 0.1 molar HCl (in deionized water) were added to WP2 and intensely mixed. The resulting mixture was centrifuged and the aqueous phase (WP3) pipetted off. Thereafter, 2 ml of a methanol-water mixture (80:20) were admixed and mixture was then centrifuged. After pipetting off the slightly turbid methanol phase, the solvent of the organic phase (OP4) was removed by means of a vacuum evaporator and the dry substance was weighed. A 5 g sample thereof was separated and dissolved with a mixture of chloroform and methanol (90:10) and subsequently applied to a thin layer chromatography plate (Macherey-Nagel, Germany); for the development a mixture of chloroform:methanol:water (70:28:2) was used as eluent.
(53) Development and dyeing of the plates was performed with anisaldehyde reagent (Sigma, Germany) at 200 C. For reference, concentrates from vegetable mono- and diglycoglycerolipids were applied separately. The cleaved sugar-containing compounds can be classified based on the color reaction: glycoglycerolipidsgreen/blue-green, glycosphingolipidsblue, glycerophospholipidsgray or violet, hydrolyzed glycoglycerophospholipidsintensely red or violet.
(54) Results:
(55) From a lipoid substance mixture which was converted into an organic phase, relevant amounts of the glycoglycerolipids and glycosphingolipids dissolved therein were separated into an aqueous phase (with magnesium carbonate 8 g and with potassium acetate 7 g) by means of the aqueous extraction process according to the invention. By means of thin layer chromatography, the presence of ceramides, sphingolipids, and glycosphingolipids as well as glycoglycerolipids could be identified, whereby monoglycosylglycero- and diglycosylglycerolipids could be detected for the latter. Only bands suggesting glycophospholipids were shown.
(56) The CMC (critical micelle formation concentration) was 55 mg/l for both extracts. The determined surface tension was 28.1 mN/m.
Example 8: Single Steps
(57) For the steps described in the following, 200 kg of lipoid phase (cold-pressed camelina oil) were used.
Example 8A: Step A2) Treatment with Citric Acid Solution
(58) The lipoid phase in the feed tank is heated to 60 C. and then 0.1 wt % of citric acid (33 wt %, at room temperature) is added and the mixture is intensively stirred for 30 seconds and then stirred for 10 minutes at about 100 to 150 rpm. Then, 0.3 wt % of water is added.
(59) The mixture of lipoid phase and dilute citric acid is then pumped into the separator and the aqueous phase is separated from the oily phase at a capacity of 200 l/h. For further processing, the oily phase is transferred to a further receiving tank (receiving tank 2).
Example 8B: Step A2) Treatment with Water
(60) The lipoid phase in the feed tank is heated to 65 C. and then 3 wt % of water (at room temperature) is added and stirred intensively for 30 seconds and then stirred for 10 minutes at about 100 to 150 rpm.
(61) The mixture of lipophilic phase and water is then pumped into the separation separator and the aqueous phase is separated from the oily phase at a capacity of 200 l/h. For further processing, the oily phase is transferred to a further master tank (master tank 2).
Example 8C: Step B1) Treatment with Sodium Bicarbonate/Sodium Acetate Solution
(62) The lipoid phase is brought to a process temperature of 45 C. to 50 C. and a sufficient volume of 8% sodium hydrogen carbonate solution/sodium acetate solution is added. Subsequently, a fraction (A)) is intensively stirred for 30 seconds by means of a Ystral mixer and then stirred normally for 10 minutes. A second fraction is homogenized with the intensive mixer (B)) according to the invention for 2 minutes.
(63) The mixture of A) is then pumped into a standard separation separator and the aqueous phase is separated from the oily phase at a capacity of 200 l/h. The oily phase is transferred to a master tank for further processing.
(64) The mixture of B) is then pumped into the separation separator according to
Example 8D: Step E1) Treatment with Arginine Solution
(65) The lipoid phase is brought to a process temperature of 40 C. to 45 C., and a volume of 0.6 M arginine solution is added, in such a way that 1.5 moles of arginine per mole of free fatty acids are present. The mixture is then carefully stirred for 10 minutes.
(66) The mixture is then pumped into the separation separator and the aqueous phase is separated from the oily phase at a capacity of 200 l/h. The oily phase is collected.
(67) Results:
(68) The oily phases had a significant turbidity after the steps A2 and a slight turbidity after A2) and had a concentrations of: FFA 0.31%, iron 0.23 ppm, phosphorus 34 ppm and a content of H.sub.2O of 0.55% after A2), and of: FFA 0.42%, iron 0.15 ppm, phosphorus 19.6 ppm, and a content of H.sub.2O of 0.30% after A2).
(69) The intensive mixing with a standard mixer in the course of process step B1) resulted in a significant emulsion formation, which was associated with an increase in viscosity. An introduction into a standard separating separator could only be made possible by increasing the temperature in the receiving container to 60 C. The aqueous phase obtained with the standard separating separator has a slightly yellowish color and had a cream-like consistency. The dry matter obtained from the aqueous phases of the separation according to process step A2) amounted to 2.3 kg and of the separation according to process step A2) 2.1 kg. It was found that about 20% of the anhydrous residue corresponded to triacylglycerides. The oily phases were markedly turbid.
(70) Under the application of the intensive mixer according to the invention, as shown in
(71) The following values are determined in the chemical analysis:
(72) B1) with standard mixer/separator: FFA 0.21%, H.sub.2O content 0.65%, iron 0.15 ppm, phosphorus 20 ppm after A2), and FFA 0.22%, H.sub.2O content 0.52%, iron 0.1 ppm, phosphorus 15 ppm after A2);
(73) B1) with mixer/separator according to
(74) The process step E1) was carried out with the lipid phase obtained from the process B1) with the mixer/separator system according to
(75) The following values are determined in the chemical analysis:
(76) Lipoid phase from process step A2): FFA 0.13%, H.sub.2O content 0.25%, iron content 0.1 ppm, phosphorus content 5 ppm;
(77) Lipoid phase from process step A2): FFA 0.12%, H.sub.2O content 0.20%, iron 0.1 ppm, phosphorus 3 ppm.
(78) All percentages (%) made herein are by weight (wt %) unless otherwise specified in the respective specification.
Example 9: Two-Stage Methods
Example 9A: Steps B1) and E1)
(79) 130 kg of rapeseed oil (FFA content 1.40%, H.sub.2O content 0.17%, iron content 0.44 ppm, phosphorus content 65.0 ppm) are filled into the master tank (master tank 1).
(80) Subsequently, the crude oil in the receiving tank 1 is brought to a process temperature of 45 C. and mixed with 3.9 kg of 10% sodium metasilicate solution. The mixture is then intensively stirred for 30 seconds by means of a Ystral mixer and then stirred normally for 10 minutes.
(81) The resulting mixture is then pumped into the separation separator and the aqueous phase A is separated from the oily phase A at a capacity of 200 l/h. The aqueous phase A is collected and stored until further use. The oily phase A is transferred back into the master tank 1 for further processing. 125 ml of oily phase A were used for chemical analysis (FFA content 0.10%, H.sub.2O content 0.15%).
(82) The oily phase A is brought to a process temperature of 40 to 45 C. and a volume of 0.6 M arginine solution is added in such a way that 1.5 moles of arginine per mole of free fatty acids are present and an introduction of air was avoided during addition. The mixture is then carefully stirred for 10 minutes. The resulting mixture is then pumped into the separation separator and the aqueous phase B is separated from the oily phase B at a capacity of 200 l/h. The aqueous phase B and the oily phase B are collected separately. 125 ml of oily phase B were used for chemical analysis (FFA content 0.1%, H.sub.2O content 0.2%).
Example 9B: Steps B1) and E1)
(83) 200 to 350 kg of rapeseed oil (FFA content 0.42%, H.sub.2O content 0.03%, iron content 0.42 ppm, phosphorus content 66.6 ppm) are filled into the master tank (master tank 1).
(84) The crude oil is brought to a process temperature of 45 to 50 C. and a sufficient volume of 8% sodium hydrogen carbonate solution is added. The mixture is then intensively stirred for 30 seconds by means of a Ystral mixer and then stirred normally for 10 minutes. The resulting mixture is then pumped into a separation separator and the aqueous phase A is separated from the oily phase A at a capacity of 200 l/h. The aqueous phase A is collected and stored until further use. The oily phase A is transferred back into the master tank 1 for further processing. 125 ml of oily phase A were used for chemical analysis (FFA content 0.31%, H.sub.2O content 0.30%, iron content 0.15 ppm, phosphorus content 19.6 ppm).
(85) The oily phase A is brought to a process temperature of 40 to 45 C. and a volume of 0.6 M arginine solution is added in such way that 1.5 moles of arginine per mole of free fatty acids are present and an introduction of air was avoided during addition. The mixture is then carefully stirred for 10 minutes. The resulting mixture is then pumped into the separation separator and the aqueous phase B is separated from the oily phase B at a capacity of 200 l/h. The aqueous phase B and the oily phase C are collected separately. 125 ml of oily phase B were used for chemical analysis (FFA content 0.13%, H.sub.2O content 0.41%, iron content 0.09 ppm, phosphorus content 12.8 ppm).
Example 9C: Steps B1) and E1)
(86) 200 to 350 kg of rapeseed oil (FFA content 0.42%, H.sub.2O content 0.01%, iron content 0.42 ppm, phosphorus content 67.9 ppm) are filled into the master tank (master tank 1).
(87) The crude oil is brought to a process temperature of 45 to 50 C. and a sufficient volume of 8% strength sodium acetate solution is added. The mixture is then intensively stirred for 30 seconds by means of a Ystral mixer and then stirred normally for 10 minutes. The resulting mixture is then pumped into the separation separator and the aqueous phase A is separated from the oily phase A at a capacity of 200 l/h. The aqueous phase A is collected and stored until further use. The oily phase A is transferred back into the master tank 1 for further processing. 125 ml of oily phase A were used for chemical analysis (FFA content 0.42%, H.sub.2O content 0.55%, iron content 0.23 ppm, phosphorus content 34 ppm).
(88) The oily phase A is brought to a process temperature of 40 C. to 45 C. and a volume of 0.6 M arginine solution is added in such a way that 1.5 moles of arginine per mole of free fatty acids are present and an introduction of air was avoided during addition. The mixture is then carefully stirred for 10 minutes. The resulting mixture is then pumped into the separation separator and the aqueous phase B is separated from the oily phase B at a power of 200 l/h. The aqueous phase B and the oily phase B are collected separately. 125 ml of oily phase B were used for chemical analysis (FFA content 0.16%, H.sub.2O content 0.45%, iron content 0.1 ppm, phosphorus content 11.8 ppm).
Example 9D: Steps B1) and E1)
(89) 200 to 350 kg of rapeseed oil (FFA content 0.43%, H.sub.2O content 0.12%, iron content 1.15 ppm, phosphorus content 57.4 ppm) are filled into the master tank (master tank 1).
(90) The crude oil is brought to a process temperature of 45 to 50 C. and a sufficient volume of 8% strength sodium carbonate solution is added. The mixture is then intensively stirred for 30 seconds by means of a Ystral mixer and then stirred normally for 10 minutes. The resulting mixture is then pumped into the separation separator and the aqueous phase A is separated from the oily phase A at a capacity of 200 l/h. The aqueous phase A is collected and stored until further use. The oily phase A is transferred back into the master tank 1 for further processing. 125 ml of the oily phase A were used for chemical analysis (FFA content 0.26%, H.sub.2O content 0.25%, iron content 0.16 ppm, phosphorus content 18.75 Ppm).
(91) The oily phase A is brought to a process temperature of 40 to 45 C. and a volume of 0.6 M arginine solution is added in such a way that 1.5 moles of arginine per mole of free fatty acids are present and an introduction of air was avoided during addition. The mixture is then carefully stirred for 10 minutes. The resulting mixture is then pumped into the separation separator and the aqueous phase B is separated from the oily phase B at a capacity of 200 l/h. The aqueous phase B and the oily phase B are collected separately. 125 ml of oily phase B were used for chemical analysis (FFA content 0.11%, H.sub.2O content 0.32%, iron content 0.11 ppm, phosphorus content 9.0 ppm).
(92) The following Table 3 describes the type and appearance of the reduced lipoid phase as well as of the aqueous phase after separation by centrifugation after step B1) and before step E1) in a series of tests with rape seed oil (FFA content 0.43%, H.sub.2O content 0, 12%, iron content 1.15 ppm, phosphorus content 57.4 ppm) according to the instructions above:
(93) TABLE-US-00005 Spinning test 1 min./20 C. Added Oil phase Substance conc. volume water-% FFA-% appearance Vol % heavy phase MS 10% 0.5% 0.28 0.65 turbid 6 (brown) 1.0%* 0.12 0.21 blank 10 (brown) pH = 13 3.0%* 0.07 0.07 sl. turbid 10 (nougat-brown) 5.0% 0.18 0.04 turbid 9 (nougat-brown) NC 10% ig 0.5% 0.24 0.56 turbid 7(brown) 1.0%* 0.14 0.36 blank 6 (brown) pH = 11 3.0%* 0.16 0.07 sl. turbid 8 (light-brown) 5.0% 0.18 0.05 turbid 9 (light-brown) NAc 10% ig 0.5% 0.39 0.96 turbid 0.3 (brown) 1.0% 0.12 0.60 blank 13 (brown) pH = 8.1 3.0%* 0.15 0.59 blank 6.5 (light-brown) 5.0% 0.15 0.59 sl. turbid 7.5 (light-brown) NHC 8% ig 0.5% 0.09 0.65 alm blank 15/thereof 0.6 water (brown) 1.0% 0.09 0.62 alm blank 15/thereof 0.6 water (brown) pH = 8.1 3.0%* 0.11 0.58 blank 8.5/thereof 0.6 water (brown) 5.0% 0.11 0.62 blank 7.5/thereof 2.5 water (brown) MS: Na metasilicate; NC: Na Carbonate; NAc: sodium acetate; NHC: Na bicarbonate FFA: free fatty acids; alm blank: almost blank; sl turbid: slightly turbid
Example 10 Three-Step Procedures
Example 10A: Steps A2) and B1) and E1)
(94) 130 kg of rapeseed oil (FFA content 1.40%, H.sub.2O content 0.17%, iron content 0.44 ppm, phosphorus content 65.0 ppm) are filled into the master tank (master tank 1).
(95) The lipoid phase in the feed tank 1 is then heated to 50 to 55 C., and 6 kg of water are then added and the mixture is intensively stirred for 30 seconds and then stirred for 10 minutes at about 100 to 150 rpm. The mixture of lipophilic phase and water is then pumped into the separation separator and the aqueous phase A is separated from the oily phase A at a capacity of 200 l/h. The aqueous phase A is collected and stored until further use. For further processing, the oily phase A is transferred into a further master tank (master tank 2). 125 ml of oily phase A were used for chemical analysis (FFA content 1.05%, H.sub.2O content 0.18%).
(96) 49 kg of the thus obtained oily phase A are brought to a process temperature of 40 to 45 C. and 1.5 kg of 10% sodium metasilicate solution is added. The mixture is then stirred intensively for 30 seconds using a Ystral mixer, free of air entry and afterwards 10 minutes stirred normally without entry of air.
(97) The resulting mixture is then pumped into the separation separator and the aqueous phase B is separated from the oily phase B at a power of 200 l/h. The aqueous phase B is collected and used to extract the separated glycoglycerolipids. The glycoglycerolipids were recovered from aqueous phase B by extraction with chloroform. 125 ml of oily phase B were used for chemical analysis (FFA content 0.13%, H.sub.2O content 0.2%).
(98) The oily phase B is brought to a process temperature of 40 C. to 45 C. and a volume of 0.6 M arginine solution is added in such a way that 1.5 mol of arginine per mole of free fatty acids are present and an introduction of air was avoided during addition. The mixture is then carefully stirred for 10 minutes. The resulting mixture is then pumped into the separation separator and the aqueous phase C is separated from the oily phase C at a capacity of 200 l/h. The aqueous phase C and the oily phase C are collected separately. 125 ml of the oily phase C was used for chemical analysis. The content of free fatty acids could be reduced to 0.14% by weight. In addition, the amount of potassium, phosphorus, iron and calcium was reduced to less than 5 ppm (K<5 ppm, P<5 ppm, Fe<5 ppm, Ca<5 ppm).
Example 10B: Steps A2) and B1) and E1)
(99) Approx. 200 kg of rapeseed oil (FFA content 0.5%, H.sub.2O content 0.04%, iron content 0.63 ppm, phosphorus content 74.8 ppm) are filled into the master tank (master tank 1).
(100) The lipoid phase is then heated to 40 to 60 C. in the receiver tank 1 and then 0.1% by weight of citric acid (33 wt %, to room temperature) is added and the mixture is intensively stirred for 30 seconds and then stirred for 10 minutes at about 100 C. at 150 rpm; 0.3% by weight of water is added then.
(101) The mixture of lipoid phase and dilute citric acid is then pumped into the separation separator and the aqueous phase A is then separated from the oily phase A at a capacity of 200 l/h. The aqueous phase A is collected and stored until further use. For further processing, the oily phase A is transferred into a further master tank (master tank 2). 125 ml of oily phase A were used for chemical analysis (FFA content 0.48%, H.sub.2O content 0.33%, iron content 0.13 ppm, phosphorus content 15.9 ppm).
(102) The oily phase A obtained in this way is brought to a process temperature of 40 to 45 C. and a sufficient volume of 8% sodium hydrogen carbonate solution is added so that a theoretical degree of neutralization of the free fatty acids of 90% is achieved. Subsequently, intensive mixing by means of a Ystral mixer for 30 seconds, without entry of air, and then 10 minutes normal stirring still without entry of air, that means without introduction of gas. The resulting mixture is then pumped into the separation separator and the aqueous phase B is separated from the oily phase B at a capacity of 200 l/h.
(103) The aqueous phase B is collected. In this, sterylglycosides were detected by means of DC. The oily phase B is transferred back into the master tank 1 for further processing. 125 ml of oily phase B were used for chemical analysis (FFA content 0.39%, H.sub.2O content 0.41%, iron content 0.06 ppm, phosphorus content 4.08 ppm).
(104) The oily phase B is brought to a process temperature of 40 to 45 C., and a volume of 0.6 M arginine solution is added in such a way that 1.5 mol of arginine per mole of free fatty acids are present and an introduction of air was avoided during addition. The mixture is then carefully stirred for 10 minutes. The resulting mixture is then pumped into the separation separator and the aqueous phase C is separated from the oily phase C at a capacity of 200 l/h. The aqueous phase C and the oily phase C are collected separately. 125 ml of the oily phase C was used for chemical analysis. The content of free fatty acids could be reduced to 0.15% by weight. In addition, the amount of potassium and calcium was reduced to less than 0.5 ppm (K<1 ppm, Ca<1 ppm) and the amount of phosphorus was reduced to 0.8 ppm and the amounts of iron were reduced to 0.02 ppm.
Example 10C: Steps A2) and B1) and E1)
(105) Approx. 250 kg rapeseed oil (FFA content 0.42%, H.sub.2O content 0.08%, iron content 0.43 ppm, phosphorus content 70 ppm) are filled into the receiving tank (receiving tank 1).
(106) Subsequently, the lipoid phase is heated to 50 to 55 C. in the receiving tank 1 and then 0.1% by weight of citric acid (33 wt %, to room temperature) is added and the mixture is intensively stirred for 30 seconds and then stirred for 10 minutes at about 100 C. at 150 rpm; 0.3% by weight of water is added then.
(107) The mixture of lipoid phase and dilute citric acid is then pumped into the separation separator and the aqueous phase A is then separated from the oily phase A at a capacity of 200 l/h. The aqueous phase A is collected and stored until further use. For further processing, the oily phase A is transferred into a further master tank (master tank 2). 125 ml of oily phase A were used for chemical analysis (FFA content 0.4%, H.sub.2O content 0.30%, iron content 0.13 ppm, phosphorus content 17 ppm).
(108) The oily phase A obtained in this way is brought to a process temperature of 45 to 50 C. and a sufficient volume of 8% sodium acetate solution is added so that a theoretical degree of neutralization of the free fatty acids of 90% is achieved. Subsequently, the mixture is stirred intensively and preferably without entry of gas by means of a Ystral mixer for 30 seconds and then stirred for 10 minutes normally and preferably without entrance of gas. The resulting mixture is then pumped into the separator and the aqueous phase B is separated from the oily phase B at a capacity of 200 l/h.
(109) Sterylglycosides were detected in the aqueous phase B by means of TLC. The oily phase B is transferred back into the master tank 1 for further processing. 125 ml of oily phase B were used for chemical analysis (FFA content 0.37%, H.sub.2O content 0.40, iron content 0.07 ppm, phosphorus content 6 ppm).
(110) The oily phase B is brought to a process temperature of 40 to 45 C., and a volume of 0.6 M arginine solution is added in such a way that 1.5 mol of arginine per mole of free fatty acids are present and an introduction of air was avoided during addition. Subsequently, the mixture is stirred gently for 10 min. The resulting mixture is then pumped into the separation separator and the aqueous phase C is separated from the oily phase C at a capacity of 200 l/h. The aqueous phase C and the oily phase C are collected separately. 125 ml of the oily phase C was used for chemical analysis. The content of free fatty acids could be reduced to 0.12% by weight. In addition, the amount of potassium and calcium was reduced to below 5 ppm (K<5 ppm, Ca<5 ppm) and the amount of phosphorus was reduced to 1.1 ppm and the amounts of iron were reduced to 0.05 ppm.
Example 10D: Steps A2) and B1) and E1)
(111) Approx. 300 kg rapeseed oil (FFA content 0.47%, H.sub.2O content 0.04%, iron content 0.53 ppm, phosphorus content 85.1 ppm) are filled into the master tank (master tank 1).
(112) Subsequently, the lipoid phase is heated to about 70 C. in the receiver tank 1 and then 0.1% by weight of citric acid (33%, to room temperature) is added and the mixture is intensively stirred for 30 seconds and then 10 minutes at about 100 to 150 rpm; 0.3% by weight of water is added then.
(113) The mixture of lipoid phase and dilute citric acid is then pumped into the separation separator and the aqueous phase A is then separated from the oily phase A at a capacity of 200 l/h. The aqueous phase A is collected and stored until further use. For further processing, the oily phase A is transferred into a further master tank (master tank 2). 125 ml of oily phase A were used for chemical analysis (FFA content 0.46%, H.sub.2O content 0.53%, iron content 0.13 ppm, phosphorus content 16.2 ppm).
(114) The oily phase A obtained in this way is brought to a process temperature of 40 to 45 C. and a sufficient volume of 8% sodium carbonate solution is added so that a theoretical neutralization degree of the free fatty acids of 90% is achieved. The mixture is then stirred intensively and preferably without gassing by means of a Ystral mixer for 30 seconds and then stirred normally for 10 minutes, preferably without entry of air. The resulting mixture is then pumped into the separation separator and the aqueous phase B is separated from the oily phase B at a capacity of 200 l/h. The aqueous phase B is collected and used to extract the separated glycoglycerolipids. The glycoglycerolipids were recovered from aqueous phase B by extraction with chloroform. The oily phase B is transferred back into the master tank 1 for further processing. 125 ml of oily phase B were used for chemical analysis (FFA content 0.24%, H.sub.2O content 0.48%, iron content 0.03 ppm, phosphorus content 2.25 ppm).
(115) The oily phase B is brought to a process temperature of 40 to 45 C., and a volume of 0.6 M arginine solution is added in such a way that 1.5 mol of arginine per mole of free fatty acids are present and an introduction of air was avoided during addition. Subsequently, the mixture is stirred gently for 10 minutes and free of air introduction. The resulting mixture is then pumped into the separation separator free of air and thus the aqueous phase C is separated from the oily phase C at a capacity of 200 l/h. The aqueous phase C and the oily phase C are collected separately. 125 ml of the oily phase C was used for chemical analysis. The content of free fatty acids could be reduced to 0.10% by weight. In addition, the amount of potassium and calcium was reduced to below 0.4 ppm (K<0.4 ppm, Ca<0.5 ppm), and the amount of phosphorus was reduced to 0.8 ppm and the amounts of iron to 0.02 ppm.
Example 10E
(116) According to Example 10D), rapeseed oil was examined as a lipoid phase. The data are given in mg per kg lipoid phase except for FFA. For FFA, the data are in % by weight. Raw denotes the initial values of the lipoid phase. A2 ) mean the values after step A2). B1) are the values after step B1). E1) are the values after step E1).
(117) TABLE-US-00006 raw A2) B1) E1) FFA [%] 0.58 0.55 0.28 0.18 P [mg/kg] 96.18 10.04 1.20 0.712 Fe [mg/kg] 0.64 0.15 0.032 0.012 Ca [mg/kg] 48.71 2.74 0.506 0.473 Mg [mg/kg] 8.62 0.56 0.131 0.109 Cr [mg/kg] 0.016 0.009 0.009 0.007 Zn [mg/kg] 0.167 0.027 0.015 0.007 Mn [mg/kg] 0.136 0.015 0.004 0.001 P Fe Ca Mg Sample name H.sub.2O FFA % mg/kg mg/kg mg/kg mg/kg Crude - NC 0.05 0.54 78.32 0.53 33.04 5.70 NC- 0.53 0.48 16.57 0.15 1.78 0.28 Degumming = A2 NC Lipoids = B1) 0.49 0.25 2.21 0.15 0.32 0.07 NC Native 0.59 0.23 0.90 0.04 0.34 0.09 neutral = E1)
Example 10F
(118) According to Example 10C), rapeseed oil was examined as a lipoid phase. The data are given in mg per kg lipoid phase except for FFA. For FFA, the data are in % by weight. Raw denotes the initial values of the lipoid phase. A2) mean the values after step A2). B1) are the values after step B1). E1) are the values after step E1).
(119) TABLE-US-00007 raw A2) B1) E1) Mg [mg/kg] 8.62 0.511 0.125 0.093 Cr [mg/kg] 0.016 0.007 0.009 0.006 Mn [mg/kg] 0.136 0.016 0.004 0.002 P Fe Ca Mg Sample name H.sub.2O FFA % mg/kg mg/kg mg/kg mg/kg Crude-NAc- 0.05 0.43 52.52 0.60 31.33 5.43 means NAc- 0.26 0.43 12.49 0.17 1.85 0.40 Degumming = A2 NAc-Lipoids = B1 0.24 0.44 5.79 0.09 0.89 0.25 NAc-Native 0.37 0.13 0.80 0.00 0.21 0.07 neutral = E1