WO2001076385A1

WO2001076385A1 - Method for the fractionation of oil and polar lipid-containing native raw materials using alcohol and centrifugation

Info

Publication number: WO2001076385A1
Application number: PCT/IB2001/000963
Authority: WO
Inventors: Steffen M. Hruschka; Stefan Kirchner; Jürgen RASSENHOVEL; Willi Witt; Bernd Best
Original assignee: Westfalia Separator Industry Gmbh
Priority date: 2000-04-12
Filing date: 2001-04-12
Publication date: 2001-10-18
Also published as: AU2001258705A1; PT1272049E; DK1272049T3; MXPA02010005A; AU783066B2; JP5461750B2; DE60140352D1; AU5249301A; EP1272048A2; CA2398053A1; EP1272049B1; ATE395835T1; EP1272048B1; DE60134128D1; ATE447332T1; WO2001076715A3; CN1422122A; WO2001076715A2; JP2003530448A; CN100403917C

Abstract

A process for the production of polar lipid-rich materials and preferably phospholipids. Preferably the polar lipid-rich materials are separated and recovered from native raw materials by extraction with water-soluble organic solvent and use of density separation to separate the resulting mixture.

Description

METHOD FOR THE FRACTIONATION OF OIL AND POLAR LIPID-CONTAINING NATIVE RAW MATERIALS USING ALCOHOL AND CENTRIFUGATION

FIELD OF THE INVENTION

The present invention relates to a process for the separation and recovery of polar lipid-rich fractions from mixtures such as native raw materials. Other fractions in the raw materials can also be recovered.

BACKGROUND OF THE INVENTION

Examples of polar lipids include phospholipids (e.g. phosphatidyl cholinc, phosphatidyl ethanolamine, phosphatidyl inositol, phosphatidyl serine, phosphatidylglycerol, diphosphatidylglycerols), cephalins, sphingolipids (sphingomyelins and glycosphingolipids), and glycoglycerolipids. Phospholipids are composed of the following major structural units: fatty acids, glycerol, phosphoric acid, amino alcohols, and carbohydrates. They are generally considered to be structural lipids, playing important roles in the structure of the membranes of plants, microbes and animals. Because of their chemical structure, polar lipids exhibit a bipolar nature, exhibiting solubility or partial solubility in both polar and non-polar solvents. The term polar lipid within the present description is not limited to natural polar lipids but also includes chemically modified polar lipids. Although the teπn oil has various meanings, as used herein, it will refer to the triacylglycerol fraction.

One of the important characteristics of polar lipids, and especially phospholipids, is that they commonly contain polyunsaturated fatty acids (PUFAs: fatty acids with 2 or more unsaturated bonds). In many plant, microbial and animal systems, they are especially enriched in the highly unsaturated fatty acids (HUFAs: fatty acids with 4 or more unsaturated bonds) of the omega-3 and omega-6 series. Although these highly unsaturated fatty acids are considered unstable in triacylglycerol form, they exhibit enhanced stability when incorporated in phospholipids. The primary sources of commercial PUFA-rich phospholipids are soybeans and canola seeds. These biomaterials do not contain any appreciable amounts of HUFAs unless they have been genetically modified. The phospholipids (commonly called lecithins) are routinely recovered from these oilseeds as a by-product of the vegetable oil extraction process. For example, in the production of soybean or canola oil, the beans (seeds) are first heat-treated and then crushed, ground, and/or flaked, followed by extraction with a non-polar solvent such as hexane. Hexane removes the triacylglycerol- rich fraction from the seeds together with a varying amount of polar lipids (lecithins). The extracted oil is then de-gummed (lecithin removal) either physically or chemically as a part of the normal oil refining process and the precipitated lecithins recovered. One disadvantage of this process is the use of the non-polar solvents such as hexane presents toxicity and flammability problems that must be dealt with.

The crude lecithin extracted in the "de-gumming" process can contain up to about 33% oil (triacylglycerols). One preferred method for separating this oil from the crude lecithin is by extraction with acetone. The oil (triacylglycerols) is soluble in acetone and the lecithin is not. The acetone solution is separated from the precipitate (lecithin) by centrifugation and the precipitate dried under first a fluidized bed drier and then a vacuum drying oven to recover the residual acetone as the product is dried. Drying temperatures of 50-70°C are commonly used. The resulting dried lecithins contain approximately 2- 4% by weight of oil (triacylglycerols). Process temperatures above 70°C can lead to thermal decomposition of the phospholipids. However, even at temperatures below 70°C the presence of acetone leads to the formation of products that can impair the organoleptic quality of the phospholipids. These by-products can impart musty odors to the product and also a pungent aftertaste. To avoid use of non-polar solvents such as hexane and avoid the negative side effects of an acetone-based process, numerous processes have also been proposed involving the use of supercritical fluids, especially supercritical CO . For example, U.S. Patent No. 4,367,178 discloses the use of supercritical CO? to partially purify crude soy lecithin preparation by removing the oil from the preparation. German Patent Nos. DE-A 30 11 185 and DE-A 32 29 041 disclose methods for de-oiling crude lecithin with supercritical CO₂ and ethane respectively. Other supercritical processes have been proposed which include adding small amounts of hydrocarbons such as propane to the supercritical CO₂ to act as entraining agents. However, supercritical fluid extraction systems are very capital expensive and cannot be operated continuously. Further, extraction times are long and the biomaterials must be dried before extraction, and this increases the difficulties of stabilizing the resulting dry product with antioxidants. All of these factors make the supercritical process one of the most expensive options for extracting and recovering polar-lipid material or mixtures of these materials. As a result, alternative processes using extraction with liquid hydrocarbons at lower pressures have been described. For example U.S. Patent No. 2,548,434 describes a method for de-oiling oilseed materials and recovering crude lecithin using a liquid hydrocarbon at lower pressures (35-45 bars) but elevated temperatures (79° to 93°C). U.S. Patent No. 5,597,602 describes a similar process that operates at even lower pressures and temperatures. However, even with these improvements supercritical fluid extraction remains very expensive and is not currently used to produce phospholipids for food use on a large commercial scale.

The primary commercial source of HUFA-rich polar lipids is egg yolk. Two primary methods are used for the recovery of egg phospholipids on an industrial scale. Both require the drying of the egg yolk before extraction. In the first process the dried egg yolk powder is extracted first with acetone to remove the triacylglycerols. This is then followed by an extraction with pure alcohol to recover the phospholipids. In the second process, pure alcohol is used to extract an oil/lecithin fraction from the dried egg yolk. The oil/lecithin phase is then extracted with acetone to remove the triacylglycerols, leaving behind a lecithin fraction. Both of these methods require the use of acetone, which has the disadvantages discussed above.

Canadian Patent No. 1,335,054 describes a process for extracting fresh liquid egg yolk into protein, oil and lecithin fractions by the use of ethanol, elevated temperatures, filtration and low temperature crystallization with further filtration. The purity of the lecithin product is not disclosed. However one skilled in the art would expect that the lecithin fraction produced by this process would not be very pure. There would still be very significant amounts of oil associated with the lecithin because the chilling process would primarily remove the triglycerides containing saturated fatty acids. Those containing some unsaturated fatty acids would remain more soluble at low temperatures. Additionally, the filtration and the chilling/filtration processes employed in this method would be labor intensive and difficult to turn into a continuous process. In light of the current state of the art, there remains a need for an improved extraction technology for food-grade polar lipid products that is less expensive to operate and which protects the overall quality of the HUFAs in the polar lipid products. SUMMARY OF THE INVENTION

In accordance with the present invention, an improved process is provided for recovering polar lipids from native biomaterials, which does not involve the disadvantages of the prior art. One embodiment of the invention resides in a process for recovering polar lipids and/or polar lipid-containing mixtures from biomaterials using both high and low water-soluble organic solvent concentrations and centrifugation.

In accordance with an embodiment of the present invention, a process for fractionation of an oil-, polar lipid-, and protein-containing mixture is provided. The process includes the steps of adding a high concentration of water-soluble organic solvent to the mixture, separating protein from the mixture by subjecting the mixture to density separation, e.g., using gravity or centrifugal force, to form a protein-rich fraction and a polar lipid/oil-rich fraction, reducing of the concentration of water-soluble organic solvent in the polar lipid/oil-rich fraction, and subjecting this fraction to density separation, e.g., using gravity or centrifugal force, to form a polar lipid-rich fraction and an oil-rich fraction.

In accordance with another embodiment of the present invention, a process for recovering polar lipid from a polar lipid-containing mixture employing the use of a water- soluble organic solvent, wherein the relatively high solubility of polar lipid in a water- soluble organic solvent, in which the water-soluble organic solvent comprises greater than 68 percent by weight of the aqueous solution, followed by process steps which utilize water-soluble organic solvent concentrations of from about 5 to about 35% by weight, are employed to assist in the recovery.

In accordance with another embodiment of the present invention, a process for fractionation of an oil-, polar lipid-, and protein-containing mixture is provided. The process includes the steps of adding a high concentration water-soluble organic solvent to the oil-, polar lipid-, and protein-containing mixture, and separating protein from the mixture to form a protein-rich fraction and a polar lipid/oil-rich fraction. As used herein, the term "high concentration water-soluble organic solvent" will mean greater than 68 percent organic solvent, preferably greater than 80 percent organic solvent, more preferably greater than 90 percent, more preferably from about 80 percent to about 95 percent organic solvent.

Preferably, the process steps are conducted under oxygen-reduced atmospheres that can include use of inert or non-reactive gases (e.g. nitrogen, carbon dioxide, argon, etc), use of solvent vapors, use of a partial or full vacuum, or any combination of the above.

BRIEF DESCRIPTION OF THE FIGURES The present invention may be more readily understood by reference to the following figures, wherein

FIG. 1 is a graphical representation of the solubility of phospholipids, a form of polar lipids, as a function of alcohol concentration.

FIG. 2 is a graphical representation of a phospholipid extraction process (as an example of a polar lipid extraction process) based on a high concentration of alcohol.

DETAILED DESCRIPTION OF THE INVENTION Because of their bipolar nature, polar lipids (including phospholipids) are of significant commercial interest as wetting and emulsifying agents. These properties may also help make HUFAs in the phospholipids more bioavailable, in addition to enhancing their stability. These properties make phospholipids ideal forms of ingredients for use in nutritional supplements, food, infant formula and pharmaceutical applications.

We have unexpectedly found that polar lipids are soluble not only in high water- soluble organic solvent concentrations (e.g., at water-soluble organic solvent concentrations greater than about 68% w/w) but also in low water-soluble organic solvent concentrations (less than about 35% water-soluble organic solvent w/w) (FIG. 1). As used herein, water-soluble organic solvent concentration means the weight percentage of water-soluble organic solvent in an aqueous solution. The aqueous solution includes added water and water present in the materials. For the purpose of this invention, phospholipids are described as "soluble" if they do not settle or separate from the continuous phase (sometimes also called supernatant or light phase) when subjected to centrifugation by equipment described in this invention. In the water-soluble organic solvent concentration range from about 35% w/w to about 68% w/w water-soluble organic solvent, polar lipids exhibit significantly lower solubility. The present invention exploits this property of polar lipids (enhanced solubility/dispersibility at low water- soluble organic solvent concentrations), which can then be exploited in several ways (along with the high solubility of phospholipids in high water-soluble organic solvent concentrations) to develop processes for inexpensively extracting and recovering polar lipids, and especially phospholipids, from native biomaterials.

Native biomaterials that are rich in HUFA-containing polar lipids include fish, crustaceans, microbes, eggs, brain tissue, milk, meat and plant material including oilseeds. As used herein, the terms fish, crustaceans, microbes, eggs, brain tissue, milk, meat and plant material including oilseeds will include genetically modified versions thereof. The content of phospholipids in these materials is generally low, usually ranging from 0.1% to about 4% by wet weight. As a result large amounts of raw materials need to be processed to recover these phospholipids. Because of the high costs of prior extraction techniques, phospholipids and especially HUFA-enriched phospholipids were very expensive and therefore restricted to use in the infant formula, pharmaceutical and cosmetic industries. One of the advantages of the present invention is that it provides for the extraction of polar lipids, and in particular phospholipids, in a cost-effective manner.

A polar lipid recovery process utilizing high concentrations of water-soluble organic solvent in a polar lipid/oil concentration step followed by the use of low water- soluble organic solvent concentrations in a step recovering the polar lipids from the oil phase is outlined in FIG. 2. Liquid egg yolk is used as the polar-lipid rich biomaterial in this example. It is understood, however, that other polar lipid-containing biomaterials (e.g. fish, crustaceans, microbes, brain tissue, milk, meat and plant material including oilseeds) could also be processed in a similar manner with minor modification to the process.

In the first step of the process 12, the material is dried, if necessary. For a more efficient recovery of the protein, the material is optionally subjected to size reduction 14. A water-soluble organic solvent (e.g., alcohol) is added 14. The concentration of water- soluble organic solvent in the water-soluble organic solvent/water solution is at least about 68% w/w, preferably at least about 80% w/w, preferably at least about 90% w/w, more preferably from about 80 to about 95% w/w, more preferably from about 85 to about 95% w/w, and more preferably from about 90 to about 95% w/w. The more moisture that is present in the material, the greater the amount and/or the higher the concentration of the water-soluble organic solvent that will be needed to achieve the desired concentration when mixed with the material. In other words, if the material is relatively dry, less water-soluble organic solvent and/or lower concentration water- soluble organic solvent can be employed. On the other hand, if the material is relatively wet, more water-soluble organic solvent and/or higher concentration water-soluble organic solvent must be employed.

The denatured protein 20 is then separated by density separation 18. Since proteins are not soluble in high concentrations of water-soluble organic solvent, they precipitate (while the polar lipids and oil dissolves in the high concentration water-soluble organic solvent) and the precipitated proteins 20 are separated from the polar lipid/oil- enriched fraction 22 by density separation 18, e.g., using gravity or centrifugal force. Using egg yolk as an example, this results in two fractions being recovered: (1) a fraction with approximately 60-95% oil (as % dry weight) and about 5-40% dry weight as polar lipids; and (2) a protein fraction, preferably with more than 90% of the protein of the egg yolk.

If it is desired to separate the polar lipid from the oil, the oil/polar lipid fraction 22 is mixed 26 with water 24 to a final concentration of water-soluble organic solvent in water of from about 5 to about 35% w/w, preferably from about 20 to about 35% w/w, more preferably from about 25 to about 30% w/w. A less desirable alternative would be to dry the oil/polar lipid fraction 22 and then add a water-soluble organic solvent, and water as necessary, to achieve the desired concentration of water-soluble organic solvent. The polar lipid is then separated from the oil by means of density separation 28. A polar lipid-enriched fraction 30 and an oil-enriched fraction 32 are formed. Further processing can be performed on the polar lipid-enriched fraction 30 and/or oil-enriched fraction 32 as desired or necessary. For example, counter-current washing/centrifugation or crosscurrent washing/separation of the oil and polar lipid products can be employed to improve the purity of the products and economics of the overall process.

In an alternative embodiment, the drying step can be eliminated. For example, instead of drying a material such as eggs, wet eggs can be used. The process is similar to that described above, however, the drying step is eliminated. As a result, a larger amount and/or higher concentration of water-soluble organic solvent is employed to precipitate the protein.

Because of the simplicity of the equipment required in the process, the entire process can very easily be conducted under a reduced-oxygen atmosphere (e.g., nitrogen, a preferred embodiment of the process), further protecting any HUFAs in the polar lipids from oxidation. For example, a gas tight decanter can be used to separate protein from the mixture. A suitable decanter is model CA 226-28 Gas Tight available from Westfalia Separator Industry GmbH of Oelde Germany, which is capable of continuous separation of protein from suspensions with high protein solids content in a centrifugal field. A gas tight separator useful for separating polar lipids from oil is model SC 6-06-576 Gas Tight available from Westfalia Separator Industry GmbH of Oelde Germany, which is capable of continuous separation of polar lipids from oil in a centrifugal field.

The concentration of water-soluble organic solvent in the protein removal step is preferably greater than about 68% w/w, more preferably greater than about 70% w/w, more preferably greater than about 80% w/w, more preferably greater than about 90% w/w. In principle, it is believed that the higher the water-soluble organic solvent concentration, the stronger the protein contraction, but the more nonpolar the aqueous/water-soluble organic solvent phase, more polar lipids may be dissolved in the oil phase. The appropriate concentration and temperature must therefore be found, for example, by conducting a few preliminary experiments (centrifuge tests), for each raw material. The present invention, in various embodiments, includes components, methods, processes, systems and/or apparatus substantially as depicted and described herein, including various embodiments, subcombinations, and subsets thereof. Those of skill in the art will understand how to make and use the present invention after understanding the present disclosure. The present invention, in various embodiments, includes providing devices and processes in the absence of items not depicted and/or described herein or in various embodiments hereof, including in the absence of such items as may have been used in previous devices or processes, e.g., for improving performance, achieving ease and/or reducing cost of implementation.

The foregoing discussion of the invention has been presented for purposes of illustration and description. The foregoing is not intended to limit the invention to the form or forms disclosed herein. Although the description of the invention has included description of one or more embodiments and certain variations and modifications, other variations and modifications are within the scope of the invention, e.g., as may be within the skill and knowledge of those in the art, after understanding the present disclosure. It is intended to obtain rights which include alternative embodiments to the extent permitted, including alternate, interchangeable and/or equivalent structures, functions, ranges or steps to those claimed, whether or not such alternate, interchangeable and/or equivalent structures, functions, ranges or steps are disclosed herein, and without intending to publicly dedicate any patentable subject matter.

Claims

We claim:

1. A process for fractionation of an oil-, polar lipid-, and protein-containing mixture, comprising the steps:

(a) adding a water-soluble organic solvent to said mixture and separating protein from said mixture to form a protein-rich fraction and a polar lipid/oil-rich fraction;

(b) reducing the concentration of water-soluble organic solvent in said polar lipid/oil-rich fraction; and

(c) subjecting the water/water-soluble organic solvent and polar lipid/oil-rich fraction to density separation to form a polar lipid-rich fraction and an oil-rich fraction.

2. The process of Claim 1, wherein the separation of protein of step (a) comprises the steps:

(a) adding water-soluble organic solvent to said oil-, polar lipid-, and protein-containing mixture to obtain a water-soluble organic solvent concentration of a least about 68% w/w; and

(b) separating by density separation the resulting mixture into a protein-rich fraction and a polar lipid/oil-rich fraction

3. The process of Claim 1, wherein said oil-, polar lipid-, and protein- containing mixture is derived from eggs.

4. The process of Claim 1, wherein water-soluble organic solvent is recovered from the protein-rich fraction and the polar lipid/oil-rich fraction after the density separation.

5. The process of Claim 1, wherein said polar lipid/oil-rich fraction formed in step (a) comprises from about 5% to about 40% by weight polar lipid and from about

60% to about 95% by weight oil.

6. The process of Claim 1, wherein said protein-rich fraction formed in step (a) comprises from about 80% to about 95% by weight protein on a dry basis.

7. The process of Claim 1, wherein said oil-, polar lipid-, and protein- containing mixture further comprises cholesterol and a substantial amount of said cholesterol reports to said oil-rich fraction pursuant to the separation of step (c).

8. The process of Claim 1, wherein said water-soluble organic solvent in step (a) is present in a water-soluble organic solvent/water mixture in which said water-soluble organic solvent comprises from about 80% to about 95% by weight of said water-soluble organic solvent/water mixture.

9. The process of Claim 1, wherein said water-soluble organic solvent in step (c) is present in a water-soluble organic solvent/water mixture in which said water-soluble organic solvent comprises from about 5% to about 35% by weight of said water-soluble organic solvent/water mixture.

10. The process of Claim 1, wherein said water-soluble organic solvent is recovered by countercurrent washing, evaporation or drying.

1 1. The process of Claim 1 , wherein said polar lipid-rich fraction is dried to recover water-soluble organic solvent, washed with an water-soluble organic solvent/water mixture comprising greater than about 80% by weight water-soluble organic solvent in order to precipitate residual protein and further dried to recover the water-soluble organic solvent.

12. The process of Claim 11, wherein the addition of said water-soluble organic solvent results in the precipitation of at least some of said protein, which is recovered by density separation.

13. The process as claimed in Claims 1-12, wherein said water-soluble organic solvent comprises a polar solvent.

14. The process as claimed in Claims 1-13, wherein said water-soluble organic solvent comprises an alcohol.

15. The process as claimed in Claims 1-14, wherein said water-soluble organic solvent comprises a C_.-C₈ alcohol.

16. The process as claimed in Claims 1-15, wherein said water-soluble organic solvent comprises isopropanol, ethanol or mixtures thereof.

17. The process as claimed in Claims 1-16, wherein the pH during processing is from pH 4 to about pH 10.

18. The process as claimed in Claims 1-17, wherein said mixture is obtained from at least one of eggs, fish, crustaceans, microbes, brain tissue, milk, meat and plant material including oilseeds.

19. The process as claimed in Claims 1-18, wherein at least 60% of the polar lipids originally present in the mixture are recovered in a polar lipid-rich fraction.

20. The process as claimed in Claims 1-19, wherein at least 80% of the polar lipids originally present in the mixture are recovered in a polar lipid-rich fraction.

21. A process for recovering polar lipid from a polar lipid-containing mixture employing the use of a water-soluble organic solvent, wherein the relatively high solubility of polar lipid in an aqueous solution of the water-soluble organic solvent, in which the water-soluble organic solvent comprises more than 68 percent by weight of the aqueous solution, followed by employing the use of a water-soluble organic solvent, wherein the relatively high solubility of polar lipid in an aqueous solution of the water- soluble organic solvent, in which the water-soluble organic solvent comprises less than 35 percent by weight of the aqueous solution, are employed to assist in said recovery.

22. The process as claimed in Claim 21 , wherein said mixture is obtained from at least one of eggs, fish, crustaceans, microbes, brain tissue, milk, meat and plant material including oilseeds.

23. The process of any of Claims 1-22, wherein said polar lipid comprises a phospholipid.

24. The process of any of Claims 1-23, wherein at least a portion of said process is performed in an oxygen-reduced atmosphere.

25. The process as claimed in Claims 1-24, wherein said mixture is obtained from at least one of eggs, fish, crustaceans, microbes, brain tissue, milk, meat and plant material including oilseeds.

26. The process as claimed in Claim 1, wherein said steps of adding and subjecting are repeated at least once.

27. A process for fractionation of an undried oil-, polar lipid-, and protein- containing mixture, comprising the steps:

(b) separating by density separation the resulting mixture into a protein-rich fraction and a polar lipid/oil-rich fraction.

28. An oil-containing, polar lipid-containing or protein-containing product produced by any of the processes of Claims 1-27.