Process for production of fatty acids, fatty acid esters and sterolesters from soapstock

FIELD OF INVENTION

This invention relates to processes which produces alkyl fatty acids, alkyl fatty acid esters and sterolesters via enzymatic catalysis using as feed soapstock waste generated by the vegetable oil refineries during the alkali refining process to produce edible oils. The combination of this technology with feedstock availability offers an economic and competitive approach to produce solvents, plastisicer from dimerised acids, biodiesel or raw material for the chemical industry. Additionally a new source of sterolesters is available for the food industry. Converting byproducts from renewable sources into more value added products using biotechnology is another real case of contribution from the chemical industry using more environmental friendly practices.

STATE OF THE ART

For each metric ton of alkali refined vegetable produced in the world approximately 30kg of soapstock is generated. There is a high potential source of raw material since vegetable oil production is growing, specially soybean in Brazil.

Soapstock waste has been used mostly as animal feed, raw material for soap makers, and feed stock for fatty acid production. The existent patents and commercial processes to make fatty acids from soapstock always refers to hydrolysis and acidification steps using strong acids such as sulfuric or hydrochloridic acids, producing a mixture of fatty acids, inorganic salts, water, and other small components such as glycerin, phospholipides. Due to the nature of this complex mixture separation of the crude fatty acids layer representing the organic phase from the aqueous phase is difficult demanding most of the time steps such as water washing, settling out, centrifuging, and filtration to separate the other components from the fatty acids. Some novelty has been introduced lately, for instance, the use of potassium soaps which generates lower viscosity feedstock, one of the biggest problem with sodium soaps, as described in the US patent 20030236422. Another patent disclosing procedure to make fluid soapstock is described in the US patent 5,156,879.

The invention is directed to a method for treatment of soapstock obtained by alkali refining of fats to provide a fluid, uniform, pumpable animal feed product. In the method, a raw i

soapstock is provided. The soapstock is pretreated by adding a strong, soluble base to the soapstock.

The US patent 6,475,758 disclose the use of an endogenic bacteria to acidulate soapstock. It is advantageously acidified by fermentation of endogenous soapstock nutrients and added nutrients under controlled conditions using acidogenic bacteria. The nutrients may include carbohydrate, nitrogen, phosphorous, sulfur from defined or undefined sources. The acidification reaction avoids the use of strong acids for the treatment of soapstock, minimizes wastewater contamination with salts and produces potentially valuable by-products including lactic acid, acetic acid, glyceric acid and nutrient rich microorganisms. All the above mentioned processes end up with a dark color crude fatty acids having residual moisture and other small components. Drying and distillation steps usually are necessary to produce commercial fatty acids to be sold in the marketplace or to use it as esterification feed because impurities is known to lower esterification reaction speed.

Usually, alkaline soapstock is converted into free fatty acids by treatment with strong acids. Afterwards, the fatty acids and water/salts are separated.

The process described in WO 2006/050589 to produce fatty acid esters from soapstock deals with enzymatic esterification with Lipase of the free fatty acids after acidification of the alkaline soapstock with strong acids. The conversion of triglycerides to fatty acids is not complete and the separation is difficult due to mono-/di-/triglycerides and phospholipids that act as emulsifiers. It was an object of the invention to optimize the conversion of Glycerides to fatty acid and to increase the yield of free fatty acids of the soapstock. It was also an object of the invention to minimize the emulsifing effects of the mixture to improve separation steps.

DESCRIPTION OF THE INVENTION

The subject of the invention is a process for production of fatty acids directly from any soapstock generated in the alkali refining process comprising of a) adding a lipase directly to the alkaline soapstock to facilitate hydrolysis of mono-,di- and triglycerides without prior neutralization, b) neutralizing and splitting the soaps with strong acids until reaching pH 1-6, preferably pH 4-6 c) separating the fatty acid phase from the aqueous by settling and/or centrifugation

This soapstocks typically contains 10-60% water, 35-85% of fatty derivatives including partial glycerides and phospholipids.

Soapstocks usually has 10 - 60% of water coming from alkali neutralization, and most refineries add extra water to make the soaps pumpable, the remaining part is composed by fatty acid soaps itself, 0,1 - 2% sterols, presence of mono-, di- and triglycerides and also low level of phospholipides. Some feedstock should also contain proteins coming from the extraction process which would end up as a solid material in the process. In a preferred embodiment the invention deals with a process where the soapstocks from alkali refining is selected from the group consisting of soybean, sunflower, rice, corn, coconut, palm kernel, rapeseed or cotton.

The advantage of this process is the addition of the Lipase without any pretreatment of the alkaline soapstock to enrich the free fatty acids in the soaps before esterification. This reduces the alkali concentration and manages the waste water. The process in a preferred embodiment is characterized in that an alkaline detergent Lipase is used for hydrolysis of glycerides in the soapstock.

The residual mono-/di-/triglycerides in the soapstock are hydrolysed by addition of an alkaline active lipase at pH > 8. Principally all known lipases are suitable for the hydrolysis of glycerides. The possible Lipases to be used in the process are produced by an organism selected from the group consisting of Aspergillus niger, Aspergillus orγzea, Bacillus species, Candida albicans, Candida antarctica, Candida cylindracea, Candida glabrata, Candida maltosa, Candida parapsilosis, Candida lipolytica, Candida tropicalis, Candida viswanathii, Chromobacterium viscosum, Geotrichum candidum, Issatchenkia orientalis (Candida krusei), Kluyveromyces marxianus (C. kefyr, C. pseudotropicalis), Mucor javanicus, Penicilium camenberti, Penicilium roqueforti, Pichia guilliermondii (Candida guilliermondii), Porcine pancreas, Pseudomonas cepacia, Pseudomonas fluorescens, Rhizomucor miehei, Rhizopus arrhizus, Rhizopus oryzae, Rhizopus niveus, Rhizopus javanicus and Thermomyces lanugenosus and mixtures thereof each in the form of free liquid, bulk or immobilisied, preferred free liquid or bulk. It is also preferred that the alkaline detergent Lipase is a Lipase of Thermomyces lanugenosus or from Pseudomonas alcaligenes. Most preferable from Thermomyces lanugenosus. The Lipases e.g. from Thermomyces or from Pseudomonas alcaligenes are optimized to high activity at alkaline pH, e.g. for washing applications. The lipase from Thermomyces is preferably used in a concentration of 0.1 % at

temperatures of 30 ⁰ C. Typical concentrations could be 1 ppm to 3% at temperatures from 10- 60°C. The lipase can be used in free or immobilized form, preferably it is used in the free form.

The invention also relates in a second embodiment to a process for production of fatty acids directly from any soapstock generated in the alkali refining process comprising of i) neutralizing and splitting the soaps with strong acids until reaching pH 1-6, preferably pH 4-6, ii) adding a Lipase to facilitate mono-/di-/triglyceride hydrolysis, iii) separating the fatty acid phase from the aqueous by settling and / or centrifugation.

In this variation of the process the hydrolysis of the glycerides occurs preferably by addition of a non-regioselective Lipase after neutralization of the alkaline soapstock.

The processes consists of hydrolysis of the mono-/di-/triglycerides either by addition of an alkaline Lipase directly to the soapstock before neutralization or in the second embodiment in addition of a non-regioselective Lipase after neutralizing and prior to separation. This second embodiment of the invention deals with a process where the soapstocks from alkali refining is also selected from the group consisting of soybean, sunflower, rice, corn, coconut, palm kernel, rapeseed or cotton and where the acids used to split the soaps are also strong acids like sulfuric acid or hydrochloride acids and the preferred pH is pH 1 -6 most preferable pH 5.

The residual mono-/di-/triglycerides in the soapstock are hydrolysed by addition of a non- regioselective lipase at ph bεlow 8. Principally all known Lipases are suitable tor the hydrolysis of glycerides. The possible Lipases to be used in the process are produced by an organism selected from the group listed before at the first embodiment of the invention and mixtures thereof. However, the lipases from Candida rugosa (cylindracea), Chromobacteήum or Geotήchum are preferred and well suited for the hydrolysis of triglycerides in this process, because they do not exhibit a regioselectivity. The lipase from Candida rugosa is used in a concentration of 0.02 % at temperatures of 30°-45°C. Typical concentrations for each Lipase could be 1 ppm to 1% at temperatures from 10-70 ⁰ C. The lipase can be used in free or immobilized form, preferably it is used in the free form.

In a further preferred embodiment of both processes according to the invention the phospholipids are hydrolysed by addition of a phospholipase. The hydrolysis of glycerides

and phospholipids leads to a higher yield of free fatty acids and the separation of fatty acid and water phase is improved. The fatty acids are separated and purified by distillation and used for e.g. dimerization.

The phospholipids in the soapstock are optionally hydrolysed by addition of 1 ppm to 1 % of Phospholipase before or after neutralization step b) resp. step i) to facilitate the hydrolysis of the lecithin. Principally all known phospholipases are suitable for the hydrolysis of phospholipids. The possible phospholipases to be used in the process are also produced by an organism selected from the group listed before for the lipases at the first embodiment of the invention and also mixtures thereof. For the process Phospholipase from Thermomyces lanuginosus is preferred. The reaction is performed at 50-60 ⁰ C at pH 5-6. Typical ranges are from pH 3-7 and temperature from 20-70°C. The splitting of the phospholipids results in a higher yield of free fatty acid material due to generation of free fatty acids from the phospholipids and due to a facilitated separation without emulsifying phospholipids in the reaction mixture. The Phospholipase may be used in free form or in immobilized form, whereby free form is preferred. Synergistic effects may be achieved by using enzymes with different specifications.

The free fatty acid phase is separated from the water phase by means of settling or centrifugation. The water phase contains glycerol, lyso-lecithin and salts from the neutralization reaction.

After separation step, the fatty acid material could optionally be distilled. In a further preferred embodiment of the invention the distillation step is carried out by batch or continuous operation preferable by a thin film or wiped film evaporator and that a continuous distillation is operated at 180 ⁰ C - 260 ⁰ C at 1-10 mmhg pressure, preferable 220 ⁰ C at 3 mmhg.

These two processes according to the invention will make possible the use of soapstock in more added value applications, other than conventional animal feed and soap makers. The hydrolysis by alkaline lipase as in the first process or non-regioselective lipase as in the second embodiment of the invention can be performed in the presence of high amount of water and presence of other components other than fatty material. The final yield of fatty acids is improved. By using combinations of lipases and phospholipases, the process can be easily adapted to different raw materials and quality demands of the product. Another advantage of both processes is the easy separation of the aqueous phase and other impurities

increases dramatically. The process yield is higher compared to fatty acid generation without the use of enzymes. As a consequence less waste is generated in these processes.

Another subject matter of the invention is the use of the free fatty acids produced according the processes pursuant to the invention for enzymatic or chemical synthesis of Cl to C6 alcanol esters and / or for chemical dimerisation of the fatty acids. The preferable alcanol is a linear or branched Cl to C6 alcanol, preferred Cl Methanol or C2 Ethanol using batch or continuous technique. Also preferred is the use of said free fatty acids as raw material for the production of surfactants, as raw material for fabric softener, as raw material for conjugation, as raw material for technical ester production or as raw material for fatty acid ethoxylate production. Additionally the free fatty acids can be applied directly as foam inhibitors or boosters.

As a consequence this results in a next subject matter of the invention. This next subject matter is a process for obtaining sterolesters and/or fatty acid esters characterized in that the fatty acids obtained according to the processes for production of fatty acids directly from any soapstock as aforementioned described are x) esterifϊed with Cl to C6 alcanol with a Lipase that is selective for fatty acids and does not transesterify the sterolesters xx) the fatty acid esters are separated from the sterolesters by distillation

The resulting free fatty acids from the processes to produce fatty acids according to the invention are converted into C1-C6 alkyl esters, preferably methyl or ethyl esters by enzymatic conversion. A Lipase and the Cl to C6 alcanol are added. Principally all known Lipases are suitable for the synthesis of esters. The possible Lipases to be used in the process are produced by an organism selected from the group listed before at the first embodiment of the invention and mixtures thereof. However, it is known that some Lipases as e.g. Candida rugose types are also capable of esterifying sterols. As Lipases, preferably a Lipase from Thermomyces or alternatively from Candida Antarctica type B can be used. The Lipase from Thermomyces is added in concentrations of lppm to 5 %, usually 0.1 % is used. Temperature range from 15- 70°C, depending on type of enzyme. A 1 to 50 % alcanol concentration is used for esterification; typically 5 to 15 % are used. The conversion works also in the presence of

water. Typically 60-95 % conversion is obtained. The lipase may be used in free form or in immobilized form. An additional advantage of Thermomyces and Candida Antarctica type B lipase is the high activity in low-water environment even in the free form. Addition of the enzyme in the free form is preferred.

The obtained esters depend on the content of the free fatty acid material resulting from the alkaline soapstock. If soybean- or rapeseed oil was used as basis for the soapstock, esters of palmitinic acid, stearic acid, oleic acid, linoleic acid and/or linolenic acid in different concentrations are obtained. Surprisingly it was found that the pattern of the esters obtained from the soybean oil based soapstock are different from the fatty acid profile of soy. The esters contain less polyunsaturated fatty acids and therefore fit in contrast to esters obtained from soybean oil directly to the EU legislation for Biodiesel fuel EN 14214. Additionally the oxidation problems experienced with Biodiesel from rapeseed oil are attributed mainly to the linolenic acid content in rapeseed oil of around 10 %. Soybean oil has a similar content of linolenic acid. Surprisingly it was found that the linolenic acid content in the soapstock is significantly lower (3-4 %), wich results in a Biodiesel fuel with an enhanced oxidation stability. The residual free fatty acids after enzymatic esterification are preferably turned into their ionic form by addition of a mineral base like KOH, NaOH or Ca(OH) ₂ solution to prevent co- distillation with the esters. This results in a better separation of esters from residual fatty acids. Afterwards the mixture is separated in a distillation, preferred in a short path distillation. The sterolesters are enriched in the bottom phase, the methyl esters are enriched in the distillate. The new process for obtaining sterolesters and/or fatty acid esters consists of a selective esterification of the fatty acids with lipase while leaving the sterolesters unchanged. The so produced esters, which correspond to 70 - 95 % of the fatty acid residue are separated by distillation and sterolesters are enriched in the bottom fraction. The sterolesters can be further purified by e.g. crystallization, extraction or fractionated distillation. Sterolesters are the preferred form for human nutrition and not the free sterols. In a chemical process the sterolesters are transesterified at least partially into free sterols and fatty acid methyl esters.

A further preferred embodiment of the invention relates to the process for obtaining natural sterolesters and/or fatty acid esters whereby the Lipase is a Lipase of Thermomyces. Further preferred is this process if the unreacted fatty acids are transformed into their salts prior to destination of the fatty acid esters. As an advantage the esterification at low temperatures simplifies the process and equipments. This gives this process high flexibility allowing the use of existent plants with minor changes. For new plants capital investment is considerable lower. The esterification process according to the invention simplicity allows the existence of small ester production plants near to the oil refineries saving a lot of handling and transportation cost. The process generates concentrated sterolesters as high valued product. The isolated sterolesters are mainly esters from sterols selected from the group consisting of beta Sitosterol, Campesterol, Stigmasterol and mixtures thereof. In soybean oil for example the amounts of the sterols are 54 % beta Sitosterol, 18 % Campesterol, 15 % Stigmasterol and 5 % mixtures of other sterols.

As a consequence of the process for obtaining sterolesters and/or fatty acid esters, further subject matters of the invention are the use of the sterolesters obtained by said process for animal feed, food, health foods and as pharmaceutical agent for lowering cholesterol and/or as precursor for steroid synthesis. Usually chemical processes lead to the formation of free sterols, which have to be reesterified for use in food products. With this process natural sterolesters are obtained, which can be supplemented to food products directly after purification.

Another subject matter of the invention is the use of the fatty acid esters obtained by said process as solvent, for the production of fatty alcohols, as biofuel or Biodiesel, as plastisicer or for dimerization. Further embodied applications are the use of the methyl esters as raw material for fatty alcohol production or the use as raw material for the production of surfactants as e.g. sulfo esters or fabric softeners.

Biodiesel produced in this way is suitable for Biodiesel according to EN 14214 because the esters contain less polyunsaturated fatty acids and therefore fit in contrast to esters obtained from soybean oil directly to the EU legislation for Biodiesel fuel EN 14214.

The fatty acid esters according to the processes can be used as Biodiesel with an improved oxidation stability, as additive in Diesel or as 100 % fuel for Diesel engines.

EXAMPLES

Example 1 : To 50 g alkaline soapstock from soybean, 10 g soybean oil was added. 50 μl of Lipolase 100 EX was stirred at 30 ⁰ C. The triglyceride content was determined by gas chromatography and was 0 % after 44 hrs reaction time.

Example 2: 50 g neutralized soapstock from soybean, 6.25 g of methanol and 50 μl of Lipolase 100 EX was stirred at 30°C. The acid value of the reaction was determined. Starting acid value was 152, the acid value reached 15.0 after 30 hrs conversion. Methyl ester phase was separated by settling.

Example 3: 50 g soapstock was spiked with 5 g lecithin and reaction was started by addition of 0,05 g phospholipase. Hydrolysis of lecithin was determined by TLC. After 18 hrs reaction time, no lecithin could be detected in the soapstock mixture.

Example 4: To 177 kg of alkaline soapstock, 75 g of lipase was added and stirred at 40 ⁰ C for 1.5 hrs. The initial triglyceride content was 27.5 %. After hydrolysis, 3.2 % triglycerides could be determined by GPC. It was observed that the separation of oil and water phase was faster when the soapstock was treated with alkaline lipase.

Example 5: To 50 g neutralized soapstock from soybean, 5 g soybean oil was added. 5 mg of Lipase from Candida rngosa was added and the mixture was stirred at 45°C. The triglyceride content was determined by gas chromatography and was 0.5 % after 46 hrs reaction time.

Example 6: Acidified soapstock was adjusted to pH 4-5 with NaOH, 4 % water and 250 ppm phospholipase was added and stirred for 8 hrs at 55 ⁰ C. After this pre-treatment, enzymatic esterification with lipase and short chain alcohol was carried out according to example 2. The yield of the combined phospholipase and lipase treatment was 89.1 % after separation compared to 80.4 % without phospholipase treatment.

Example 7: Large scale alkaline enzymatic hydrolysis of soapstock and separation of oil and water phase. In a 85 m ³ tank enzymatic hydrolysis of the soapstock was performed by

addition of 0.1 % Lipolase. The initial acid value of 124 rose to 170 after a hydrolysis time of 2 hours at 40 °C.

Example 8: Distillation of fatty acid esters obtained from soapstock and analysis of the sterol content in the bottom fraction.

The soapstock has an average content of 2 % sterols of which 80 % are found in the form of sterolesters. A chemical esterification of the dried soapstock fatty acids was performed with zinc oxids as catalyst in a 20 m ³ reactor to an acid value of 5. In parallel an enzymatic esterification of the soapstock with Lipolase was done in a 100 m ³ tank to an acid value of 30.

The two esters where distilled and the bottom fractions were analyzed for their sterol content: In the distillation residue of the chemical esterification a total sterol content of 8.8% with a free sterol portion of 5.3 % was found corresponding to a sterolester content of 40 % in the sterol fraction. In the distillation residue of the enzymatical esterification a total sterol content of 4.6 % with a free sterol portion of 0.95 % was found corresponding to a sterolester content of 19 %. From these results it can be seen that in the enzymatic esterification the sterolesters are not transesterified to a significant amount while in the chemically catalyzed esterification a significant part of the sterolesters is transesterified.

Example 9: Analysis of the distillate and comparison against soybean and rapeseed fatty acid profile. The EU Biodiesel norm is adapted to the iodine value typically seen in rapeseed oil methyl esters with a maximum of 120.

Result: The esters obtained from the soapstock have a iodine value of 110, which is in specification of the EN 24124 (120 specification limit). Additionally the low percentage of

linolenic acid is positive because oxidation problems in Biodiesel are especially attributed to the highly oxidation sensitive linolenic acid content.

If not indicated in another way, all amounts are in weight% related to the overall weight.

I l