HK1015819B - Vegetable oil enzymatic degumming process by means of aspergillus phospholipase - Google Patents

Description

Enzymatic process for degumming vegetable oils with aspergillus phospholipase

Technical Field

The present invention relates to a process step of degumming in the preparation of edible oils, wherein vegetable oils-oil hydratable phospholipids have been removed as far as possible, preferably by prior water degumming-by enzymatic treatment of non-hydratable phospholipids to such an extent that they can undergo physical refining. This method is cost-effective and environmentally friendly.

Prior Art

The accepted refining processes for producing high quality edible oils typically include the process steps of degumming, deacidification, and decolorization and deodorization. In particular, much effort has recently been made to develop more efficient and less costly degumming schemes. The aim is to degum the oil to such an extent that it can subsequently be deacidified by distillation. The latter distillation deacidification process has a great advantage over the conventional, neutralization deacidification process, with no waste material being produced. However, it is carried out with the proviso that the phospholipid content is low, for example less than 15ppm, preferably less than 10ppm, of phospholipids in the oil. Ideally the phospholipid content is < 5 ppm.

The gum material of vegetable oils consists mainly of a mixture of phospholipids, the amount and composition of which depends on the method of preparation of the oil fruit and oil. The vast majority of phospholipids can be separated from their micellar solution in crude vegetable oil by hydration and used for lecithin recovery. Herein, it is referred to as wet degumming. A small portion of the phospholipids is not hydrated and remains in the oil. The chemical nature of this unhydrated phospholipid (NHP) has not been fully elucidated. It is known from tests that more than 50% of these consist of calcium and magnesium salts of phosphatidic acid (cf. Hermann Purdun, vegetable lecithin, chemical industry Press, H.Ziolkowsky KG, Osugueger, page 1988,181).

The aim of the usual industrial degumming process is to remove as much as possible the unhydrated phospholipids from the oil. The methods commonly used today are the "superscour" and "single degumming" methods by Wunie Afier, the "Total degumming (" TOP ") method by Van der M Tai, the" Alcon "method by Yuchi, and the" UF-method "by Clay mechanical industries, Inc. In these processes, conventional water degumming is mostly indispensable or a preceding stage thereof in order to remove hydratable phospholipids.

Typically, all of these degumming processes use purely mechanical or physicochemical methods, which are not always ideally suited for all oil qualities. These processes are very high in terms of equipment and energy consumption and do not guarantee the low phosphorus content necessary for deacidification by distillation.

As an effective principle, acid treatment is used as part of these degumming processes. It is known that strong acid agents are suitable for the post-degumming of oils that have been pre-degummed with water (see Pardun, Loc, cit, pp.185-189 or US-a 4698185). Citric acid is preferably used here.

An efficient enzymatic degumming process is described for the first time in European patent application 0513709. One of the edible oils is degummed with water and one of phospholipase (A)₂,A₁The aqueous solution of B) is emulsified and then separated from this aqueous phase. After this process, the oil contains less than 5ppm phosphorus and is suitable for subsequent deacidification by distillation. The important process parameters are the emulsification of the aqueous phase containing the enzyme into droplets of < 10 μm, the addition of citrate to the aqueous solution, the temperature being 50 to 70 ℃ and the pH-value preferably being 4 to 6. This pH-adjustment in acid is unexpected, since all known phospholipases have a pH-optimum at pH 8. The enzymatic degumming process used by luer company in the edible oil industry is the "Enzymax-process".

In DE-A4339556, a further variant of this process describes the reuse of enzymes, in which the used aqueous phase containing the contaminated pulp is dissolved by adding surfactants or solubilizers and the solution free of contaminated pulp is recovered as far as possible, which contains at least 10% of the used enzyme.

In the "Enzymax-method", the advantageous effect of citric acid can be used in order to degum as much as possible, in particular by a citric acid treatment arranged before or after the enzyme treatment. The simultaneous use of citric acid and enzymes is not possible.

It is known from JP-A-2-153997 to treat crude or previously degummed oil with an enzyme having phospholipase-A-activity. This prior art teaches that phospholipids are changed by adding phospholipase A so that it can be removed by an absorbent such as activated charcoal or bleaching earth. Thus in examples 1 and 2,3 of the examples were treated with bleaching earth addition combined into an enzyme. In example 3, the addition of bleaching earth is eliminated instead of bleaching earth, in particular a large amount of enzyme (2000-. An emulsion of oil in water now appears. It gives no indication of the distribution of the oil in the aqueous phase containing the enzyme, the adjustment of the pH, the co-use of citrate, and the reuse of the enzyme.

In JP-A-249593, ｃA similar enzymatic treatment of oil is described, however the aim is not degumming of oil but rather recovery of lysolecithin. It is then superfluous to adjust the pH value alone.

Also in the process of EP-A0328789, the conversion of soybean oil lecithin into lysolecithin by phospholipase is involved to prepare mayonnaise-like products.

EP-A0622446 describes an enzymatic process for degumming oils and fats, comprising a plurality of process steps. After the treatment with phospholipase, the enzyme solution was centrifuged off, and the remaining oil was washed with water at pH3-6 and finally treated with bleaching earth. This is characterized in that a large amount of water, i.e., 30 to 200% by weight, based on the oil used, is used not only in the enzyme treatment but also in the water washing step. Also here an emulsion of oil in water is produced. This situation means increased consumption of the apparatus, since large volumes of liquid have to be moved, and higher energy costs and costs for waste disposal. No description is given for the pH adjustment of the aqueous enzyme solution.

In the case of phospholipases, it is a particular problem to produce the necessary amount of enzyme to operate an industrial enzymatic process. The number of available uses is limited. Phospholipase A₁Commercially available, phospholipid B is only available in laboratory quantities; the source is an extract from rat liver or a culture of Streptomyces. Phospholipase A₂From snake venom, scorpion venom and peak venom. All these sources are not suitable for the production of industrially required quantities of enzyme. Current phospholipase A₂The only industrial preparation is from porcine pancreas. The worldwide collection of suitable glands is limited and never reproducible. In addition, phospholipase is only a minor by-product of the extraction process. The main product being trypsin, especially trypsinLactase, and porcine insulin. Even if the prevailing (hardly increasable) phospholipase A is estimated as the enzyme described in EP-A0513709₂The trade volume of (a) is sufficient only for not more than 2 to 3 oil mills.

There is therefore a need for a source from which the enzyme can be produced in unlimited amounts. According to the prior art, any number of enzymes are industrially available from microorganisms, for example from fungi or bacteria. For phospholipase A₁、A₂And B, no microorganism has been known at present which can produce the enzyme in a sufficient yield. Isolation of phospholipase A from Rhizopus arrhizus, Escherichia coli and Bacillus megaterium₁Phospholipase B was isolated from specific strains of Penicillium and Streptomyces. Surprisingly, there is no suggestion in the literature that phospholipase A enzymes₁、A₂Or B can be isolated from Aspergillus.

In contrast, lysophospholipase is known from EP0219269, which is obtained from Aspergillus niger. Lysophospholipases-referred to herein as phospholipases L₁Or L₂Has a different property compared to the previously extracted phospholipases in the sense that it is capable of cleaving monoacyl phospholipids such as lysolecithin. Pure lysophospholipase finds application in a completely different food technology area, namely in the filtration of wheat starch with improved yields.

Object of the Invention

It is an object of the present invention to provide an environmentally friendly and inexpensive process for reducing the content of phosphorus-containing components in vegetable oils to such an extent that the oils can be further processed by distillative deacidification, that is to say to a phosphorus content of less than 15ppm, preferably less than 10ppm, most preferably less than 5 ppm. These requirements can be achieved by enzymatic methods.

At the same time, there is a need for a microbial source that allows unlimited amounts of enzymes to produce phospholipase enzymes. According to the prior art, only enzymes having the property of cleaving acyl groups are sufficient, i.e.phospholipasesA₁,A₂And B. The use of an enzyme-producing microorganism, which has been employed in food technology for a long time and is therefore without doubt, has the advantage that it should not be underestimated. Examples here are different yeast strains such as Kluyveromyces cerivisiae, Bacillus hyphae such as B.subtilis or Aspergillus strains such as Aspergillus niger strains or Aspergillus oryzae strains.

Another unsolved object, which is not yet solved by the present invention, is to combine the effective actions of two known processes, namely the action of acid treatment and enzyme treatment, into one step in the degumming process.

Finally, it is an object of the present invention to be able to carry out the process of the invention particularly economically with as small a feed of enzymes and acids as possible.

Technical scheme

The object of the present invention is achieved by a method for reducing phosphorus-containing components contained in vegetable oils by enzymatic decomposition using an acyl-decomposing phospholipase, characterized in that an enzyme derived from an Aspergillus strain is used.

The preparation of industrial enzymes by cultivation of strains of aspergillus is an important highly developed field in biotechnology. Enzymes from the aspergillus niger bud are used in large amounts in the starch industry (amyloglucosidase), in the fruit juice industry (pectinase) and in the pasta industry (xylanase). Enzymes from Aspergillus, in particular from Aspergillus niger, have long been used in the food industry and are known to be harmless. Phospholipase A from such sources₁,A₂Or B is not known, the use of phospholipases of this origin in degumming processes is an important feature of the present invention. In the search for phospholipase-containing strains, the product VERON1 sold by the Applicant was found^91 or ROHAPECT^7104 there is sufficient phospholipase A₂-activity. In the strain improvement screening methods known per se, higher activity is manifested by mutations and increased selectivity for phospholipase activity (see: W.Gerhartz, "Industrial enzymes", VCH publishing Co., Ltd., 1990, p 35).

The properties of phospholipases obtained from such sources can be multifaceted and intricate. Different from the pancreatic phospholipase A known from the prior art₂This enzyme is defined as decomposing only C in phospholipid molecules₂Acyl groups on atoms, most phospholipases from the genus Aspergillus simultaneously contain different acyl-decomposing properties. Thus, except for A₁And A₂In addition to the properties, lysophospholipase properties were also found. The enzyme properties mentioned correspond to e.c. numbers 3.1.1.32,3.1.1.4 and 3.1.1.5. Lysophospholipase (EC-No. 3.1.1.5) is also known as phospholipase 3; however, it is unclear whether a distinction has to be made between lysophospholipase and phospholipase B, as characterized in the literature (see Pardun, Loc, cit, p.140). They have in any case in common that they can be broken down further to lysolecithin, but also up to glycerophosphocholine. Phospholipase B can also damage lecithin incidentally. Since the phospholipase of the present invention has two characteristics of not only the matrix lecithin but also the matrix lysolecithin, it can be referred to as phospholipase B. As far as the applicant is aware, pure lysophospholipases from Aspergillus, which are capable of decomposing lysolecithin first but not, are ineffective in the present degumming process, especially under acidic reaction conditions. This also applies to phospholipase C and D which do not decompose acyl groups.

Thus the properties of lecithin, i.e. phospholipase A₁And/or A₂The activity, which is analytically difficult to distinguish between the two, is an important feature of the enzyme of the invention. The simultaneous presence of such different properties may be the basis for the advantageous action of the enzymes of the invention. Although it must be called a single enzyme in most cases, it has the function of a complex enzyme.

When the enzyme of the invention is used, its purity is of no great significance. For example, the fermentation liquor itself, the enzyme-rich retentate obtained after ultrafiltration or the enzyme protein precipitated therefrom can be used.

It is within the scope of the present invention to use enzymes obtained from production strains modified by genetic techniques instead of enzymes obtained from conventional Aspergillus production strains. During this period the genetic technology offers a number of possibilities, and the genes necessary for the production of phospholipase are cloned from Aspergillus and propagated in high yields in a suitable host strain (exprimieren). As host strains, for example, also Aspergillus strains can be considered, but also other strains of fungi and even bacterial strains.

The amount of enzyme used corresponds to the purity and can be between 0.0001 and 1%, based on the oil to be degummed.

A great advantage and unexpected effect of the enzymes used according to the invention is their activity necessary for degumming, which activity needs to be very small. When phospholipase A from pancreas was used in the prior art₂When using Lecithinase Units (LU) with an activity of about 1000 lecithinase units per liter of oil (see example 1 in EP-A0513709), it is sufficient to use the enzyme of the invention from Aspergillus under the above-mentioned conditions with an activity of only 5 to 50LU per liter of oil. With a corresponding extension of the reaction time, even use amounts of less than 5LU per liter of oil can be used to bottom out. Lysophospholipase activity found in purified Aspergillus phospholipase was even higher than phospholipase A₂Activity, more precisely about 1 to 100 times. This results in the use of amounts of 5 to 5000 lysolecithinase-units (LLU) per liter of oil, preferably 50 to 1000 LLU. This activity data applies to the batch degummed material. The amount of enzyme added necessary for reuse of the enzyme is significantly lower on a 1 liter oil basis, for example 1/5 to 1/10 of the above values. This small amount of enzyme even allows to abandon the reuse of the enzyme.

The Aspergillus phospholipase of the invention can be mixed in a targeted manner with other acyl-decomposing phospholipases, for example with another phospholipase from Aspergillus or with phospholipase A from the pancreas₂. In the latter case, however, the pH must be adjusted, the two pH optima being close, that is to say about pH3 to 5.

The use of phospholipases from the genus Aspergillus surprisingly also solves the otherwise proposed task. It is thus possible to put the enzyme into a citric acid solution, combining the action of the enzyme with that of the citric acid. It is not unusual to use enzymes in relatively concentrated citric acid solutions. Some enzymes are hardly known in enzymology, are stable at such low pH values and even have an optimum action. One of the few examples of such a profile is pepsin in the digestive tract. The enzyme is soluble in 1-20% citric acid. At the same time, the pH is adjusted to a value of pH < 4, preferably to a value of pH < 3. For example, a 5% citric acid solution, a pH of about 2.3 is obtained.

Lactic acid, acetic acid, formic acid, phosphoric acid, and other inorganic and organic acids may also be used in place of citric acid. But preferably is a food acid, especially citric acid. It should be borne in mind that a sufficiently low phosphorus content is not obtained with the acid solution alone, that is to say without the addition of enzymes, in particular for oils which have been degummed beforehand.

In the process of the invention, the pH optimum is adjusted to a value of from 2 to 3, which is not in harmony with the pH optimum obtained by conventional methods. The latter is at pH =8, where egg yolk is used as a base, emulsified in an enzyme solution at 40 ℃, and the activity is pH-dependent. One can account for this unexpectedly low pH-process optimum with specific phase interface conditions, where there may be a higher pH-value than measured in the aqueous phase (bulk phase).

The enzyme is intimately mixed with the oil in this acidic aqueous solution. In order to keep the volume as small as possible during the subsequent separation, efforts are made to keep the water phase as small as possible compared to the oil phase. In general, a volume of < 10% (based on the oil phase) is sufficient, preferably < 5%. In each case a water-in-oil emulsion was produced.

The oil phase to be treated may be soybean oil, sunflower oil or rapeseed oil. The former is the most important oil product. Other vegetable oils, such as soy bean oil, coconut oil or palm oil, and animal oils (animal oils contain interfering phospholipids), can also be treated according to the present invention.

Because phospholipases attack lecithin, oils high in lecithin, such as crude soybean oil, are of little interest for use in the inventive process. The feedstock is therefore a pre-degummed, in particular water-degummed, oil which is generally characterized by a phosphorus content of between 50 and 300 ppm. With exceptions, the pre-degummed oil has a high phosphorus content, almost no more than 500ppm phosphorus at a time. Oil of fluctuating quality can be processed with the same equipment. The partially degummed oil, as well as the pressed or extracted oil, may also be additionally invested, specifically mixed with the previously degummed oil. The phosphorus content can likewise be above 500 ppm. Pre-drying of the oil is not necessary.

It is possible that the enzyme is active and it is necessary to intimately mix the two phases, i.e. the oil phase and the aqueous phase containing the enzyme, with each other. Pure stirring is absolutely insufficient.

Good distribution of the enzyme in the oil is ensured if the enzyme is solubilized in a trace amount of water in an amount of about 0.5 to 5 weight-% (based on the oil) and in this form emulsified in the oil as droplets having a diameter (weight average) of less than 10 μm. Preferably droplets smaller than 1 micron. Vigorous stirring at shaft speeds above 100 cm/sec proved to be effective. This method can be replaced by tumbling the oil in the reactor by means of a centrifugal pump on the outside of the rib. The distribution of the aqueous phase containing the enzyme can also be made fine by the action of ultrasound, usually a dispersing machine such as Ultraturrax.

The reaction of the enzyme may be carried out at the interface between the oil phase and the aqueous phase. The aim of all such mixing measures is to create as large a surface as possible for the enzyme-containing aqueous phase. The addition of the surfactant enhances the uniform distribution of the aqueous phase. Thus, surfactants having an HLB-value above 9, such as sodium dodecyl sulfate, are added to the enzyme solution as the case may be, as described in EP-A0513709. A similarly effective method to improve emulsification is the addition of lysolecithin. The amount of addition may be from 0.001% to 1% based on the oil. In any case, for the inventive process, a highly dispersed water-in-oil emulsion is obtained.

The temperature of the enzymatic treatment is not critical. Temperatures between 20 and 80 ℃ are suitable. The latter allows only short-term use. In general, the temperature resistance of the phospholipase of the invention is superior. It is not damaged when a low pH is used. It is desirable to use temperatures of from 30 to 50 ℃.

The treatment time depends on the temperature and can be shortened with increased temperatures. Usually times of from 0.1 to 10, preferably from 1 to 5 hours are sufficient. The reaction is carried out in a degumming reactor which may be divided into several layers as described in DE-A4339556. Thus, in addition to a batch mode of operation, a continuous operation is also possible, wherein the reaction can also be carried out in different temperature zones. For example, it may be stopped at 40 ℃ for 3 hours, followed by incubation at 60 ℃ for 1 hour. The staged reaction process also offers the possibility of setting different pH values in the individual stages. The pH of the solution can thus be adjusted, for example, to 7 in the first stage and to 2.5 in the second stage with the addition of citric acid. However, in at least one stage, the pH of the enzyme solution must be below 4, preferably below 3, according to the invention. When the pH value is additionally adjusted below the limits according to the invention, a deterioration of the effect is observed. Thus citric acid is preferably added to the enzyme solution before it is mixed into the oil.

After the end of the enzymatic treatment, the enzymatic solution together with the cleavage products of the NHPs absorbed therein are separated (batchwise or continuously) from the oily phase, preferably by centrifugation. Since the enzymes have a high degree of stability and absorb only a small amount of decomposition products, which precipitate out as a sludge, the same aqueous enzyme phase can be used repeatedly. It is likewise possible to separate the enzyme from the dirty pulp in accordance with DE-A4339556, so that the enzyme solution largely freed of dirty pulp can be returned to service.

Oils containing less than 15ppm phosphorus are obtained by the degumming process according to the invention. The original aim was to have a phosphorus content of less than 10 ppm; ideally less than 5 ppm. Further processing of the oil with a phosphorus content of less than 10ppm is undoubtedly possible according to the distillative deacidification method. In the process of the invention, a range of other ions such as magnesium, calcium, zinc and iron are also largely removed from the oil. Owing to the low iron content achieved, mostly below 0.1ppm, the products have the ideal prerequisite for good oxidation stability during further processing and storage.

Examples

Example 1

500g of wet degummed soybean oil with a residual phosphorus content of 190ppm was heated to 40 ℃ in a round bottom flask. To this was added 26g of water in which 5g of citric acid and 0.19g of the powdered enzyme preparation had been dissolved. The enzyme preparation is derived from a production batch of an aspergillus niger fermentation and contains 60 phospholipase units (= lecithinase units LU) per gram. 1 Lecithase Unit (LU) is the amount of enzyme that releases 1 micromole of fatty acid from egg yolk within one minute at 40 ℃ pH = 8. The enzyme preparation may also be tested for lysophospholipase activity. 1001 lysophospholipase units (= lysolecithinase unit LLU)/per g were measured. 1 lysolecithin unit is the amount of enzyme that liberates 1 micromole of fatty acid per minute from the lysolecithin-emulsion at 55 ℃ and pH = 4.5. The enzyme preparation is dephosphorylated Lipase A, considering that the phospholipase content is not specifically purified₂It also contains a significantly high lysophospholipase activity and can be called phospholipase B.

The contents of the round-bottomed flask were vigorously dispersed using an external circulation pump. At this point the contents of the flask were dispersed about once per minute. Wherein the particle size of the aqueous phase is less than 1 μm. At 1 hour intervals, samples were taken several times and the phosphorus content was determined at the points where the following data were obtained:

time of day	0	2	4	6	20
						Phosphorus content ppm	190	81	27.9	5.4	4.2

This low phosphorus content of < 10ppm required for deacidification by subsequent distillation is obtained after 6 hours by the process according to the invention.

Comparative example 1

The procedure is as in example 1, except that instead of the enzyme preparation, a corresponding amount of whey protein, i.e. protein without enzyme, is added. After the same treatment time, the samples taken as above show: by the enzyme-free treatment, the phosphorus content is reduced by not less than 121 ppm. The addition of citric acid alone is therefore not sufficient. The oil obtained is not suitable for deacidification by distillation.

Time of day	0	2	4	6	20
						Phosphorus content ppm	190	152	128	123	121

Comparative example 2

The procedure is as in example 1, except that the phospholipase from the genus Mycoplasma is replaced by commercial pure lysophospholipase (G-enzyme, enzyme biosystems, USA 1172.LLU per G). It has no phospholipase A₂And (4) activity. After the same treatment time, the samples taken as above show: under the conditions specified above, the phosphorus content is reduced by not less than 85ppm by adding lysophospholipase alone. The distillation deacidification of the oil obtained is not suitable.

Time of day	0	2	4	6	20
						Phosphorus content ppm	190	138	124	119	85

Claims (17)

1. Method for reducing phosphorus-containing components contained in vegetable oils by enzymatic decomposition with acyl-cleaving phospholipases, characterized in that an enzyme from the genus Aspergillus is used which contains not only phospholipase A₂Activity, see EC-No. 3.1.1.4, and/or phospholipase A₁Activity, see EC-No. 3.1.1.32, but also lysophospholipase activity, see EC-No. 3.1.1.5; adjusting the pH of the enzyme solution used to a value of < 4; and the process is carried out at a temperature of from 20 to 80 ℃.

2. The process as claimed in claim 1, wherein the pH of the enzyme solution used is adjusted to < 3.

3. A process according to claim 1, characterized in that the pH is adjusted with citric acid, the enzyme being activated in the presence of citric acid.

4. A process according to any one of claims 1-3, characterized in that the process is carried out at a temperature of 30 to 50 ℃.

5. A method according to any one of claims 1-3, characterized in that the aqueous phase containing the enzyme in oil is emulsified into particles having a particle size below 10 μm.

6. A method according to any of claims 1-3, characterized in that at least partly pre-degummed oil is used.

7. A method according to claim 6, wherein said pre-degummed oil is a wet degummed oil.

8. A method according to claim 7, characterized in that the phosphorus content of the oil is reduced from not more than 500ppm to less than 15 ppm.

9. A method according to claim 8, characterized in that the phosphorus content of the oil is reduced to below 15ppm from 50 to 300 ppm.

10. A method according to claim 8 or 9, characterized in that the phosphorus content of the oil is reduced to below 10 ppm.

11. A method according to claim 10, characterized in that the phosphorus content of the oil is reduced to less than 5 ppm.

12. A method according to claim 8 or 9, characterized in that soybean oil is processed.

13. A method according to claim 8 or 9, characterized in that rapeseed oil or sunflower oil is processed.

14. A process according to any of claims 1-3, characterized in that after the reaction the aqueous solution of phospholipase is separated from the treated oil and reused.

15. A method according to any of claims 1-3, characterized in that it is carried out in a batch mode.

16. A method according to any one of claims 1-3, characterized in that it is carried out continuously.

17. A method according to any of claims 1-3, characterized in that the iron content of the oil is reduced at the same time as the phosphorus-containing component.