Title of Invention

"ENZYMATIC MUCILAGE REMOVAL PROCESS"

Abstract

The invention relates to the mucilage-removal process step in the manufacture of edible oils, in which vegetable or animal oils from which the hydratable phosphatides have, preferably, been largely removed by a prior aqueous mucilage-removal, are freed from non-hydratable by an enzyme treatment so that they may be physically refined. The enzyme used is a phospholipase from an aspergillus strain. The process is gentle, cost saving and environmentally friendly.

Full Text

Enzymatic Mucilage Removal Process The invention relates to a mucilage remova process step in the preparation of edible oils, in which vegetable oils from which hydratable phosphatides have, preferably, been largely removed by a prior aqueous mucilage-removal, are freed from non-hydratable phosphatides by an enzyme treatment so that they may be subjected to physical refining. The process is gentle, cost-saving and environmentally friendly. The known refining processes for preparing high-quality edible oils normally include the steps of mucilage-removal and de-acidification, as well as bleaching and de-odourisation. Efforts have recently been made to make the mucilage-removal process more efficient and economical. The aim of this process is to remove sufficient mucilage from the oil so that it may subsequently be de-acidified by distillation. This distillation de-acidification process has the great advantage over conventional processes of acid-removal which involve neutralisation in that there is no waste. One of the requirements, however, when carrying out this process, is a low amount of phosphatides in the oil, for example, a phosphorus content of less than 15 ppm, preferably of less than 10 ppm. Amounts of phosphorus of The mucilage in vegetable oils is mainly a mixture of phosphatides, the amount and composition of which depends on the oil seed and the way the oil is recovered. Most of the phosphatides may be separated from their micelle solutions in the crude oils through hydration, and then used for obtaining lecithin. This is called wet mucilage removal. A small amount of the phosphatides, however, cannot be hydrated and remains in the oil. The chemical characteristics of these "non hydratable" phosphatides (NHP) have not been fully explained. Investigations have shown that the phosphatides comprise more than 50% of calcium and magnesium salts of phosphatide acids (see Hermann Pardun, Die Pflanzenlecithine, Verlag fur chem. Industrie H. Ziolkowsky KG, Augsburg, 1988, Page 181) . Conventional industrial mucilage-removal processes have the object of extracting the largest possible amount of hydratable phosphatides from the oil. The processes normally used currently include the "super degumming process" and the "unidegumming process" of Unilever, the "Total degumming ("TOP") process" of Vandemoortele, the "Alcon-process" of Lurgi and the "UF-process" of Krupp Maschinentechnik GmbH. The classical aqueous mucilage-removal step for the extraction of hydratable phosphatides is frequently integrated in these processes or carried out beforehand. A typical feature of all these mucilage-removal processes is that mechanical or physico-chemical methods are exclusively used, which are not always optimally suitable for all the oil grades. Furthermore, these processes are costly in terms of equipment and energy, and do not ensure that the low phosphorus content required for a subsequent de-acidification by distillation is produced. Some of these mucilage-removal processes use an acid treatment as an effective method. It is known that strongly acidic agents are suitable for subsequent mucilage-removal in oils treated beforehand in an aqueous mucilage-removal step (cf. Pardun, loc. cit., Page 185-189 or US-A 4 698 185). Preferably, citric acid is used for this. European Patent Application No.0 513 709 discloses, for the first time, an effective enzymatic process for mucilage-removal. Here, an edible oil previously freed from mucilage by water is emulsified with an aqueous solution of phospholipase (A2, Alf B) and then separated from this aqueous phase. When this process has been carried out, the oil contains less than 5 ppm of phosphorus and it is suitable for subsequent de-acidification by distillation. Important process parameters include an emulsification of the enzyme-containing aqueous phase to form droplets DE-A 43 39 556 discloses the reuse of the enzyme as a further variant of this process. The enzyme is removed from an already used slime-containing aqueous phase by adding tensides or solubilisers and recovered as a largely slime-free solution which comprises at least 10% of the enzyme used. The "EnzyMax-process" makes it possible to use the advantageous effect of citric acid for extensive mucilage removal, i.e. by a citric acid treatment before or after the enzyme treatment. The simultaneous use of citric acid and enzymes is not possible. The treatment of crude or previously de-slimed oil by means of an enzyme which exhibits phospholipase-A-activity is known from jP-A-2-153997. This document teaches that the use of phospholipase A changes phosphatides in such a way that they may be easily removed by adsorbents such as Aktivton or bleaching clay. In Example 1 and 2 of three examples for carrying out the enzyme treatment, the latter is combined with a bleaching earth treatment. Example 3 dispenses with the use of bleaching earth. Instead, especially large amounts of enzyme (2,000 to 20,000 units) in large amounts of water (100 to 1,000 wt.% based on the oil) are used. Here, an oil-in-water emulsion is obtained but there is no teaching for distributing the oil in the enzyme-containing water phase, for adjusting the pH value, for the simultaneous use of citrate and the reuse of the enzyme. JP-A-2-49593 discloses a similar enzyme treatment of oils which, however, does not aim to remove the mucilage in oil but to produce lysolecithin. The setting of special pH values is not necessary here. The process disclosed in EP-A 0 328 789 also deals with the transformation of lecithins in soya oil by phospholipase A to form lysolecithin for producing mayonnaise-type products. EP-A 0 622 446 describes an enzymatic process comprising several steps for the removal of mucilage in oil and fat. After the treatment with phospholipase, the enzyme solution is centrifuged off, the remaining oil is washed with water of pH 3-6 and finally treated with bleaching earth. The characterising feature here is that both in the enzyme treatment and in the washing step large amounts of water are required, i.e. 30 to 200 wt.% based on the oil used. Again, oil-in water emulsions are produced. This process requires more costly equipment, as large volumes of fluid have to be moved, as well as resulting in higher energy and waste-removal costs. pH values of the aqueous enzyme solution are not disclosed. The provision of the required amount of enzyme for carrying out a large-scale industrial enzymatic process is a specific problem in the case of phospholipases as the available amount is limited. Phospholipase A: is not commercially available and phospholipase B is only available in laboratory amounts; sources are extracts from rat livers or from Streptomyces cultures. Phospholipase A2 occurs in the poison of snakes, scorpions and bees. None of these sources are suitable for producing industrially relevant enzyme amounts. At present the only industrial process for preparing phospholipase A2 is the extraction from swine pancreatic glands. However, the availability of suitable glands worldwide is greatly limited and, at any rate, cannot be increased as required. Furthermore, phospholipase is only a less important by-product in the extraction process. The main products are pancreatic proteases, especially trypsin, as well as porcine insulin. It is estimated that at present the commercially available volume of phospholipase A2, which can hardly be further increased, is only sufficient for 2 to 3 oil mills at the most, even if the enzyme were reused in the process as described in EP-A 0 513 709. There is therefore a need for a source which provides the enzyme in unlimited quantities. According to the prior art, industrial enzymes are obtained from microorganisms such as fungi or bacteria, in any amount required. However, no microorganism is known at present which produces phospholipase Alf A2 or B enzymes in sufficient yield. In isolated cases, phospholipase Ax was obtained from Rhizopus arrhizus, Escherichia coli and Bacillus megaterium and phospholipase B from Penicillium notatum and Streptomyces strains. Surprisingly, no mention is made in the literature that phospholipase A1( A2 or B may be isolated from Aspergillus. However, lysophospholipases obtained from Aspergillus niger are known from EP 0 219 269. Lysophospholipases, called phospholipase Lx or L2 here, have a different specificity compared to the above phospholipases in that they are capable of separating only monoacyl phosphatides such as lysolecithin. Pure lysophospholipases are used in an entirely different field of foodstuff technology, i.e. for improving the yield in wheat-starch filtration. There is still a need for a gentle, environmentally friendly and economical process for reducing the amount of phosphorus-containing ingredients in vegetable oils up to a degree which enables further treatment of the oil through de-acidification by distillation, that is, to amounts of phosphorus of less than 15 ppm, preferably less than 10 ppm, more particularly less than 5 ppm. These requirements may be fulfilled by an enzymatic process. There is therefore also a need for a microbial source which makes it possible to produce the enzyme phospholipase in an unlimited quantity. According to the prior art, only enzymes with an acyl-cleaving specificity may be used, namely phospolipases Alf A2 and B. In the process of the invention, it is highly advantageous if an enzyme-producing microorganism is used which has been employed in the foodstuffs industry for a long time and is therefore safe. Examples of this include various yeast strains such as Kluyveromyces cerivisiae, Bacillus strains such as B. subtilis or Aspergillus strains such as A. niger or oryzae. It would also be advantageous to combine the effect of two known processes, i.e. the acid treatment and the enzyme treatment, into one step in the mucilage-removal process. Finally, of the smallest possible amounts of enzyme and acid in order to obtain a particularly economical process would also be advantageous. The invention provides a process for reducing the content of phosphorus-containing ingredients in vegetable oils through the enzymatic degradation of the oils using Aspergillus acyl-cleaving phospholipase enzymes. The preparation of industrial enzymes through the cultivation of Aspergillus strains is an important and highly developed field of biotechnology. Enzymes from Aspergillus niger are, for example, widely used in starch manufacture (amyloglucosidases), in fruit juice manufacture (pectinases) and in the manufacture of baked goods (xylanases). Enzymes from Aspergillus, especially A. niger, have been known in the foodstuffs industry for a long time and are regarded as being safe. Phospholipases Al7 A2 or B from this source are not known. The use in the mucilage-removal process of phospholipases derived from this source is an important feature of this invention. The search for phospholipase-containing strains was preceded by the standard screening method of improving the strain by mutation and selection to achieve a higher phospholipase activity. The specificity of the phospholipase obtained from this source may be varied and complex. In contrast to the prior art phospholipase A2 from the pancreas which, by definition, only separates the acyl group at the C2 atom of a phospholipid molecule, most phospholipases from Aspergillus contain various acyl separating specificities simultaneously. Apart from an A! and A2 specificity, a lysophospholipase activity has also been found. The known enzyme specificities have the E.G. numbers 3.1.1.32 (A^ , 3.1.1.4 (A2) and 3.1.1.5. Lysophospholipase (EC number 3.1.1.5) is also called phospholipase B. According to the literature, however, it is unclear whether a distinction has to be made between lysophospholipase and phospholipase B (cf. Pardun, loc.cit., p. 140). The feature common to both of them is that they are capable of further cleaving lysolecithin, i.e. up to glycerophosphorylcholine. Phospholipase B is also able to affect lecithin. As the phospholipase according to the invention has both specificities, namely with respect to both substrates lecithin and lysolecithin, it could also be called phospholipase B. Pure lysophospholipases from Aspergillus which are able to cleave lysolecithin but not lecithin appear to be ineffective in this mucilage-removal process, especially under acidic reaction conditions. The same is also true for the non-acyl cleaving phospholipases C and D. Thus, the specificity in respect of lecithin i.e. phospholipase Al and/or A2 activity which are difficult to distinguish analytically, is an important feature of the enzyme according to the invention. The simultaneous presence of these various specificities may be a reason for the advantageous effect of the enzyme according to the invention. Though this enzyme is in most cases a single enzyme, its effect is similar to that of an enzyme complex. When using the enzyme according to the invention, its degree of purification is not greatly significant. The fermentation fluid itself, which is the resultant enzyme rich product obtained after an ultra filtration, or the resultant precipitate i.e. the enzyme protein, may, for example, also be used. It is certainly within the scope of the invention that instead of using an enzyme obtained from a conventional Aspergillus strain, an enzyme from a strain which has undergone genetic engineering is used. Genetic engineering now offers a great number of possibilities for cloning the gene required for forming phospholipase, from Aspergillus, and to express it in a high yield in a suitable host strain. Suitable host strains include, for example, Aspergillus strains but also other fungal strains and even bacterial strains. Depending on the degree of purification, the amounts of enzyme used may be between 0.0001 and 1% based on the oil to be de-slimed. The extremely low activity of the enzymes according to the invention which is required for carrying out the mucilage-removal is unexpected but advantageous. In the prior art pancreatic phospholipase A2 activities of about 1,000 lecitase units (LU) per litre of oil were used (see Example 1 in EP-A 0 513 709) . With the enzymes from Aspergillus according to the invention, under the above conditions, activities of only 5 to 50 LU per litre of oil are sufficient. The lysophospholipase activity found in purified Aspergillus phospholipases is even higher than the phospholipase A2 activity i.e. by 1 to 100 times. The amounts to be used, therefore, are 5 to 5,000 lysolecitase units (LLU) per litre of oil, preferably 50 to 1,000 LLU. These activity data are in respect of preparation mixtures which are cleared from mucilage in batches. When the enzyme is reused, the required enzyme addition based on 1 litre of oil is significantly lower, for example l/5th to l/10th of the above values. Because of these low enzyme amounts, reuse of the enzyme can be dispensed with. It is also possible to mix the Aspergillus phospholipase, according to the invention, with other acyl-cleaving phospholipases, in a targeted manner, for example with further phospholipases from aspergillus or with phospholipase A2 from the pancreas. In the latter case, a pH value must be set which is close to both pH optima i.e. about pH 3 to 5. Surprisingly, further objectives can also be achieved by the use of phospholipase from Aspergillus. Thus, it is possible to use the enzyme in a citric acid solution, thereby combining the action of the enzyme with that of the citric acid. The use of an enzyme in relatively concentrated citric acid solutions is unusual. Few enzymes are known in enzymology which are stable at such low pH values and which even exhibit optimal activity at such values. One of the few examples of such-a spectrum of characteristics include the pepsin of the digestive tract. The enzyme is dissolved in a 1 to 20% citric acid solution. Here, pH values of pH Instead of citric acid, it is also possible to use lactic acid, acetic acid, formic acid or phosphoric acid as well as other inorganic or organic acids. Flavouring acids such as citric acid, however, are preferred. It must be noted that acid solutions alone, i.e. without using enzymes, do not produce sufficiently low phosphorus contents, especially with oils previously freed from mucilage. The pH optimum of 2 to 3 which has been found in the process according to the invention, does not coincide with the pH optimum, that is pH = 8, found in conventional analytical processes. Here, egg yolk is emulsified as a substrate in the enzyme solution at 40°C, and the activity is determined as a function of the pH value. The reason for the surprisingly low pH process optimum could be the particular conditions of the phase boundary where the pH value measured is possibly higher than that of the aqueous phase (bulk phase). The enzyme is thoroughly mixed with oil in the acidic aqueous solution. It should be endeavoured to keep the volume of the aqueous phase as low as possible compared to the oil phase, so that only the smallest possible volumes need to be handled during the subsequent separation. As a rule, volumes Since the phospholipase affects lecithin, the use of oils which contain high amounts of lecithin, such as crude soya oil, in the process according to the invention would not be suitable. Starting products, therefore, are oils previously substantially freed from mucilage, especially in water, which normally have a phosphorus content between 50 and 300 ppm. It is only in exceptional cases that oils previously freed from mucilage have higher phosphorus contents and hardly ever more than 500 ppm of phosphorus. Oils of varying quality may be processed in the same plant. It is also possible to simultaneously use de-slimed oils as well as pressure oils or extraction oils, namely in a mixture with previously de-slimed oils. By way of exception, the phosphorus may then be above 500 ppm. A prior drying of the oil may be dispensed with. Both phases, i.e. the oil phase and the enzyme-containing water phase, must be thoroughly mixed with one another to be able to act. A simple stirring is not sufficient. Satisfactory distribution in the oil is ensured if a small amount of water (0.5 to 5 wt.%, based on oil) is dissolved in the oil and emulsified in this form to form droplets of less than 10 micrometers in diameter (weight average value). Preferably, the droplets are smaller than 1 micrometer. Vigorous stirring at radial speeds of more than 100 cm/sec, has proved suitable. However, the oil may also be circulated in the reactor by means of an external centrifugal pump. The enzyme-containing water phase may also be finely distributed by means of ultrasound. Dispersing devices such as Ultraturrax are usual. The enzymatic reaction takes place at the boundary surface between the oil water phases. The aim of the mixing measures is to ensure the largest possible surface for the enzyme-containing water phase. The addition of tensides increases the distribution of the water phase. In some cases, therefore, the enzyme solution is mixed with tensides with HLB values of more than 9, such as an Na-dodecylsulphate, as described in EP-A 0 513 709. A similarly effective method for improving emulsification is the addition of lysolecithin. The amounts added may be about 0.001 to 1% based on the oil. In the process according to the invention, highly dispersed water-in-oil emulsions are always produced. The temperature of the enzyme treatment is not critical. Temperatures between 20 and 80°C are suitable, but the latter may only be used for a short time. The phospholipase according to the invention is noted, as a whole, for its high temperature resistance. It is also not affected by the low pH value used. Temperatures of 30 to 50°C are of optimum value. The duration of the treatment depends on the temperature and may be reduced as the temperature increases. Treatment times of 0.1 to 10, preferably 1 to 5 hours are normally sufficient. The reaction takes place in a mucilage-removal reactor which may be divided into stages, as disclosed in DE-A 43 39 556. In addition to batch operation in batches, a continuous operation is also possible. The reaction may also be carried out at various temperatures. It is thus possible to carry out the incubation for e.g. 3 hours at 40°C and then for 1 hour at 60°C. A reaction in stages also enables the adjustment of different pH values in the individual stages. In a first stage, for example, the pH value of the solution may be adjusted to 7 and in a second stage to 2.5 by adding citric acid. According to the invention, in at least one stage, however, the pH value of the enzyme solution must be below 4, preferably below 3. When the pH value was subsequently adjusted below that of the invention, a reduction in activity was found. Therefore, the citric acid is preferably added to the enzyme solution before the solution and the oil are mixed. When the enzyme treatment has finished, the enzyme solution including the NHP degradation products contained in the solution are separated from the oil phase, either in batches or continuously, preferably by centrifuging. As the enzymes have a high stability and the amount of degradation products taken up, which precipitate as slime, is low, the same aqueous enzyme phase may be reused several times. According to DE-A 43 39 556, it is also possible to separate the enzyme from the slime so that a largely slime-free enzyme solution may be used again. The mucilage-removal process according to the invention produces oils which contain less than 15 ppm of phosphorus. It is preferable to obtain phosphorus contents of less than 10 ppm, ideally of less than 5 ppm. With phosphorus contents below 10 ppm it is easily possible to further process the oil through deacidification by distillation. During the process according to the invention, a number of other ions such as magnesium, calcium, zinc and iron are also removed from the oil. Thanks to the resultant low iron content, which is mostly below 0.1 ppm, the product is highly oxidation resistant during further processing and storage. EXAMPLES The following Example 1 is a non-limiting illustration of the invention Example 1 500 g of soya oil from which the wet mucilage has been removed and which has a residual phosphorus content of 190 ppm is heated to 40°C in a round flask. 26 g of water in which 5 g of citric acid and 0.19 g of a powdered enzyme preparation have been dissolved is added to the oil. The enzyme preparation is derived from an aspergillus niger fermentation batch and comprises 60 phospholipase units (=Lecitase Units, LU) per g. 1 lecitase unit (LU) is the amount of enzyme which liberates 1 micromol of fatty acid from egg yolk in 1 minute at 40°C, pH = 8. The enzyme preparation was also examined for its lysophospholipase activity. 1,001 lysophospholipase units (=lysolecitase units, LLU) per g were measured. One lysolecitase unit is the amount of enzyme which liberates 1 micromol of fatty acid per minute from a lysolecithin emulsion at 55°C, pH=4.5. The enzyme preparation which was not specially purified of the amount of phospholipases also contains, in addition to phospholipase A2 a remarkably high activity of lysophospholipase and could be called phospholipase B. The contents of the round flask is intensively dispersed by means of an external centrifugal pump. In so doing, the contents of the flask is mixed about once per minute. The water phase is present in a particle size of below 1 µm. Samples are taken in 1 hour intervals and examined for the phosphorus content. The following values are found: (Table Removed) The low phosphorus content of Comparison Test 1 The process used is as described in Example 1. Instead of the enzyme preparation, however, a corresponding amount of lactic protein i.e. non-enzymatic protein, is used. The samples taken after the same treatment times, as described above, show that the phosphorus contents do not drop below 121 ppm when an enzyme-free treatment is carried out. Simply adding citric acid, therefore, is not sufficient. The resultant oil is not suitable for a deacidification by distillation. (Table Removed) Comparison Test 2 The process is carried out as in Example 1. Instead of an aspergillus phospholipase, however, a commercially available pure lysophospholipase (G-zyme) made by Enzyme Biosystems, USA, 1172 LLU pro g) is used. It does not exhibit phospholipase A2 activity. The samples taken, as described above, after the same treatment times, show that the phosphorus contents do not drop below 85 ppm under the conditions as specified, when only lysophospholipase is used. The resultant oil is not suitable for a deacidification by distillation. (Table Removed) We Claim 1. A process for reducing the phosphorus content in animal or vegetable oils containing undesired phosphatides which comprises subjecting the oil to enzymatic degradation using an acyl-cleaving phospholipase derived from Aspergillus. 2. A process according to claim 1, wherein the enzyme exhibits phospholipase A2 activity and/or phospholipase Ax activity, in addition to lysophospholipase activity. 3. A process according to claim 1 or 2, wherein degradation is carried out using an enzyme solution with a pH 4. A process according to claim 1 or 2, wherein degradation is carried out using an enzyme solution with a pH 5. A process according to claim 3 or claim 4, wherein, the pH value is adjusted by the addition of citric acid. 6. A process according to any one of claims 1 to 5, wherein the process is carried out at a temperature of 20 to 80°C. 7. A process according to any one of claims 1 to 6 wherein the process is carried out at a temperature of 30 to 50°C. 8. A process according to any one of claims 1 to 7, wherein the enzyme is added to the oil as an aqueous solution. 9. A process according to any one of claims 1 to 8, wherein an aqueous phase containing the enzyme is emulsified in the oil to form droplets with a diameter of less than 10 micrometers. 10. A process according to any one of claims 1 to 9, wherein an oil is used from which mucilage, more particularly wet slime, has at least partially been removed beforehand. 11. A process according to claim 10, characterised in that an initial phosphorus content of 50 to 500 ppm in the oil is reduced to less than 15 ppm. 12. A process according to claim 11 wherein the phosphorus content is reduced to below 10 ppm. 13. A process according to claim 12 wherein the phosphorus content is reduced to below 5 ppm. 14. A process according to any one of claims 1 to 13, wherein soya oil is processed. 15. A process according to any one of claims 1 to 13, wherein rapeseed oil or sunflower oil is processed. 16. A process according to any one of claims 1 to 15, wherein after the reaction the aqueous solution of the phospholipase is separated from the treated oil and reused. 17. A process according to any one of claims 1 to 15, which is carried out in batches. 18. A process according to any one of claims 1 to 15, that it is carried out continuously. 19. A process according to any one of claims 1 to 18, wherein the iron content of the oil is also reduced. 20. A process for reducing the phosphorus content in vegetable or animal oils substantially as hereinbefore described and with reference to Example 1.

Documents:

1638-del-1996-abstract.pdf

1638-del-1996-assignment.pdf

1638-del-1996-claims.pdf

1638-del-1996-correspondence-others.pdf

1638-del-1996-correspondence-po.pdf

1638-del-1996-description (complete).pdf

1638-del-1996-form-1.pdf

1638-del-1996-form-13.pdf

1638-del-1996-form-19.pdf

1638-del-1996-form-2.pdf

1638-del-1996-form-3.pdf

1638-del-1996-form-4.pdf

1638-del-1996-form-6.pdf

1638-del-1996-gpa.pdf

1638-del-1996-petition-137.pdf

1638-del-1996-petition-others.pdf