A PROCESS FOR REMOVING beta -CICLODEXTRIN FROM EGG YOLK OR EGG YOLK PLASMA

Abstract: The invention relates to a process for removing beta -cyclodextrin from egg yolk or egg yolk plasma using an alpha -amylase. This process is characterized in that the egg yolk or egg yolk plasma is treated with a maltogenic alpha -amylase originating from Bacillus stearothermophilus. The beta -cyclodextrin was incorporated into the egg yolk during a cholesterol extraction step. The invention further relates to egg yolk or egg yolk plasma obtained by the use of this process.

Description: Title: A process for removing β-cyclodextrin from egg yolk or egg yolk plasma. This invention relates to a process for removing β-cyclodextrin from egg yolk or egg yolk plasma using an α-amylase. In 1990, the World Health Organisation of the United Nations specified a standard for the maximum daily intake of cholesterol in its report 797 entitled "Diet, nutrition and the prevention of chronic diseases". The recommended maximum intake is 300 mg cholesterol per day per person. In the industrialized countries, the average cholesterol intake is approximately twice as high. Heretofore, a large number of procedures have been proposed to reduce cholesterol contents. These procedures can be divided into two groups: processes in which cholesterol is broken down and processes in which cholesterol is extracted. Examples of breakdown processes include degradation by microorganisms, conversion by means of enzymes, and chemical conversion. Cholesterol extraction can be carried out in the form of solvent extraction, by evaporation processes, and by adsorption to a fixed bed. It will be clear, however, that not all of these procedures can be applied to every cholesterol- containing foodstuff without any problem. It is generally known that eggs, and in particular egg yolks, contain a high percentage of cholesterol. For this reason, doctors advise patients who have a high cholesterol level in the blood to refrain from the consumption of eggs and egg products. EP-A-0 326 469 discloses a process for removing steroidal compounds from substances of biological origin, such as eggs and fats. If the biological substance is not present in liquid form, it is first liquefied. The liquid substance to be treated is contacted with β-cyclodextrin in an aqueous environment. β-Cyclodextrin is a cyclic oligomer consisting of seven -D- extrose units which are bound to each other via 1,4-α-glucosidic bonds. This cyclic oligomer has a hydrophobic central opening which makes it possible to bind particular lipophilic substances, such as cholesterol, into a complex. The complex formed can subsequently be removed from the treated substance, such as egg yolk, in a simple manner, for instance by means of centrifugation. What remains is a produc whose cholesterol content has been reduced by 70-95%. This extraction is highly selective when applied to diluted egg yolk. Cholesterol and free fatty acids are complexed, while useful lipophilic compounds such as vitamin and vitamin D remain behind in the product. A disadvantage arising in the removal of the β-cyclodextrin-cholesterol-complex from egg yolk is that approx. 0.5-2% of β-cyclodextrin remains behind in the egg yolk. The residual β-cyclodextrin does not fulfil any useful function and may therefore be regarded as ballast. It is endeavoured to have as few of such substances as possible present in foodstuffs. Accordingly, there is a need to reduce the β-cyclodextri content of egg yolk or egg yolk plasma to below 20 ppm, so that, foodstuff-technically, the substance involved can be merely qualified as a processing aid. EP-A-0 439 075 relates to a process for reducing β-cyclodextrin to glucose in egg yolk or egg yolk plasma. To that end, egg yolk or egg yolk plasma is mixed with an α-amylase selected from domestic hog pancreas amylase and α-amylases formed by or derived from Aspergillus niger r Asperαillus oryzae. Bacillus Dolvmvxa, Bacillus coag lans and Flavobacterium sp. Referring to the literature in the art, EP-A-0 439 075 mentions that cyclodextrins typically inhibit α- and β-amylas and are substantially resistant to these enzymes. A number of the α-amylases used in the process according to EP-A-0 439 07 were known to break down cyclodextrins only at a very low rate. However, if these enzymes were added to egg yolk treate with β-cyclodextrins, these cyclodextrins were broken down relatively rapidly and completely. According to this known process, a diluted aqueous egg yolk or egg yolk plasma solution is adjusted to a pH of between 4.5 and 6 with an edible acid, for instance citric acid. Subsequently, one of the specific α-amylases mentioned in EP-A-0 439 075 is added to the diluted egg yolk or egg yolk plasma. The α-amylase is incubated at a temperature of between 5° and 65°C for 2-50 hours. The α-amylase is added in an amount of 10-500 FAU per gram of β-cyclodextrin to be removed. A FAU (Fungal α-Amylase Unit) is a measure for the activity of an α-amylase originating from a fungus, and this measure is used by Νovo Industri A/S, Copenhagen, Denmark, for instance for the enzyme Fungamyl®. More particularly, 1 FAU is the amount of α-amylase originating from a fungus which breaks down 5.25 g starch in 1 hour under standard conditions. These standard conditions are: starch in the form of liquid starch, an incubation time of 7-20 minutes, 37°C and pH 4.7. It is not advisable to use an amount of enzyme in excess of the 10-500 FAU specified, because this does not give any better activity. Although, according to EP-A-0 439 075, efforts are made to reduce the β-cyclodextrin content to below 100 ppm, such efforts are only successful, as appears from the Examples, if Fungamyl ® (α- amylase from A. orizae. marketed by Νovo Industri A/S, Copenhagen, Denmark) is used. If other aiπylases are used, more than 800 ppm of β-cyclodextrin remains behind in the treated solution. It is further noted that the European patent application mentioned does not indicate any relation which makes it possible to relate the FAU unit to α-amylases which do not originate from fungi, such as the α-amylases from Bacillus sp., Flavobacterium sp and domestic hog pancreas amylase. It has now been found that β-cyclodextrin can also be broken down with a maltogenic α-amylase originating from Bacillus stearothermophilus. The present invention relates to a process for removing β-cyclodextrin from egg yolk or egg yolk plasma using an α-amylase, this process being characterized in that the egg yolk or egg yolk plasma iε treated with a maltogenic α-amylas originating from Bacillus stearothermophilus. The use of this maltogenic α-amylase now makes it possible to break down β-cyclodextrin substantially completel in a short time under conditions which are favourable for egg yolk. Depending on the reaction conditions, the reaction time will be, for instance, between 10 minutes and 2 hours. By the term "favourable for egg yolk" is meant that the functional properties of the egg yolk or egg yolk plasma are not adversely affected. It is known that egg yolk is a very delicate product. It can only be pasteurized at a temperature of 63°C at a maximum for 6 minutes. hen the pasteurization takes more time or is carried out at a higher temperature, th functional properties of the egg yolk components are affected. It is further known that if the pH is reduced to below 5.8 at an increased temperature of approximately 40°C or more, thickening arises due to coagulation of certain components. With the enzyme that is used according to the present invention, the treatment can be carried out in the temperatur range of 45°C to 63°C and at a pH ranging between 5.8 and 7.0. If a temperature lower than 45°C is used, the breakdown of β-cyclodextrin is still possible but takes more than 2 hours. Preferably, the temperature varies between 55°C and 63°C, because at these temperatures the egg yolk product can be pasteurized at the same time. In contrast with the α-amylases which are used according to EP-A-0 439 075, the α-amylase fro B. stearothermophilus is not inactivated at pasteurization temperatures. The preferred pH range is between 6.0 and 6.8. If the β-cyclodextrin degradation is combined with a pasteurization of the egg yolk product, the α-amylase can be added to the egg yolk product before heating for pasteurization begins. During the careful heating of the egg yolk product to a temperature of approximately 60°C, the enzyme used can break down the β-cyclodextrin. After being maintained at 60°C for a short time, the product can subsequently be cooled. Thus, a pasteurized egg yolk product is obtained, in which the presence of β-cyclodextrin can no longer be demonstrated. For egg yolk, the conditions specified are much milder than the conditions described in EP-A-0 439 075, where, as specified, treatment times are much longer (2-50 hours) at a temperature of up to 65°C and at a considerably more acidic pH of between 4.5 and 6. For the reasons outlined, the process according to the present invention has great advantages over the process according to EP-A-0 439 075. In a preferred embodiment, in the process according to the present invention, the maltogenic α-amylase used is Maltogenase ® . Maltogenase is the maltogenic α-amylase from Bacillus stearothermophilus which has been expressed and produced by a Bacillus subtilis strain. This product iε marketed by Νovo Industri A/S, Copenhagen, Denmark. Maltogenase is officially approved for use in foodstuffs. Using the process according to the present invention, the β-cyclodextrin is broken down into glucose and maltose. The presence of these sugars in an egg product does not cause any problems with respect to official food regulations. More in particular, the heat-stable maltogenic α-amylase hydrolyseε the l,4-α-glucosidic compounds in cyclodextrin. As stated above, β-cyclodextrin can be rapidly broken dow by the maltogenic α-amylase from B. stearothermophi ..IF. under favourable conditions for egg yolk. The breakdown occurs rapidly, notwithstanding the fact that the optimum pH for this enzyme is 5.3. β-Cyclodextrin is broken down completely within 2 hours if more than 60 MANU α-amylase according to the invention is used. A MAΝU (Maltogenic Amylase Νovo Unit) is the amount of enzyme that breaks down l μmol maltotriose per minute under standard conditions. These standard conditions are: a substrate concentration of 10 mg/ml, a temperature of 37°C, a pH of 5.0 and an incubation time of 30 minutes. In contrast with an obεervation to the contrary in EP-A-0 439 075, better, more rapid conversion is achieved if a larger amount of α-amylase iε uεed. In most caseε, however, a conversion time of 2 hours will not be objectionable. Accordingly, it will suffice to use an amount of about 60 MAΝ per ml substrate. The maltogenic α-amylase which is uεed in accordance wit the present invention, and Maltogenase in particular, are approved for use in the food induεtry. Maltogenaεe iε difficult to remove from the substrate or to inactivate. Due to the thermostability of this enzyme, it is imposεible to inactivate it by increasing the temperature without adverεely affecting the quality of the egg yolk. Nor iε thiε enzyme inactivated after prolonged incubation at pH 8.3 or after a treatment with hydrogen peroxide. It did prove to be possible to remove Maltogenase with modified starch, for instance Perfectamyl ® , AVEBE B.A., Foxhol, the Netherlandε. If it is desired that as much enzyme as poεεible be removed from the treated product, the enzyme can be bound to a suitable support, for instance Eupergit®C. Eupergit ® C is supplied by Rohm Pharma GmbH, Darmstadt, Germany. This material consists of oxirane-acrylic beads having an average diameter of 150 μm. Other suitable εupports are Sepharose ® (agarose) , Sephadex ® (dextrose) , celluloεe, εtarch and supportε baεed on glaεε and εilicone. However, becauεe α-amylaεe is partly inactivated by immobilization on the support, a considerably larger amount of enzyme iε required to break down all of the β-cyclodextrin within 2 hours. Thiε iε not a problem, however, becauεe the εupported enzyme iε reuεable. During incubation, less than 1% of the α-amylase is released from the support. It iε further obεerved that EP-A-0 415 287 diεcloεeε a proceεε in which β-cyclodextrin is used as a solid adsorption agent for the removal of cholesterol. This process, however, is complicated. The use of a supported enzyme for removing β-cyclodextrin is not suggested in that application. Finally, the present invention relates to low-choleεtero egg yolk or egg yolk plasma obtained by the use of the proces according to the present invention. Substantially no β- cyclodextrin is present in this product anymore. This product can be used in the same way aε egg yolk having a normal choleεterol content. The preεent invention will now be illuεtrated in and by the following Exampleε. Exam le i To 3 ml 20 mM phosphate buffer, pH 6.4, with 18 wt.% egg yolk powder, which contained approximately 2 wt.% β-cyclodextrin, waε added 0.5 ml of a 2 mM potassium phosphate buffer, pH 6.4, into which 60 MANU/ml Maltogenase ® had been incorporated. This mixture was incubated at 45°C and after 2 the enzyme reaction was terminated by heating the mixture at 100°C for 10 min. The β-cyclodextrin content of the treated egg yolk produc was determined using HPLC. The HPLC analysis was carried out with a Benson BCX4 cation exchanger in the calcium form (particle size 15-20 μm, column 250 x 9 mm) , with 100 ppm Ca-EDTA as mobile phase, a flow rate of 0.4 ml/min, a column temperature of 85°C and a sample size of 20 μl. The index of refraction of the eluted material was meaεured with a Jaεco 830-RI detector at 40°C and regiεtered with a SP4400 Chromjet integrator of Spectra Phyεicε at a paper rate of 2.5 mm/min. No β-cyclodextrin waε demonεtrated. Exam p le 2 The procedure according to Example 1 was repeated. Now, however, incubation occurred at 60°C and the enzyme reaction was ended after 30 min. β-Cyclodextrin was no longer demonstrated. Example 3 ' Maltogenase® was bound to Eupergit®C in accordance with the directions of the manufacturer. To that end, 73 ml of this enzyme, having a total enzyme content of 8.2 g/l, was dialysed at 4°C for 18 h against 2 volumes of 2 liters of 0.1 M potasεium monohydrogen phoεphate buffer, pH 7.5, and subεequently for 5 h againεt 2 volumes of 2 liters of l M potassium monohydrogen phosphate buffer, pH 7.5, with 0.05% p-hydroxybenzoic acid methyl ester. 17 g Eupergit ® C was added to the dialysed solution. This mixture was incubated at room temperature for 70 h. The solution was then filtered uεing a P2 glaεs filter. The Eupergit ® C was washed with 0.1 M potasεium monohydrogen phosphate buffer, pH 7.5. It waε found that 45% of the originally added protein was still present in the filtrate, i.e., per gram Eupergit ® C, 19 mg Maltogenase had been immobilized. 1.0 g Maltogenase-Eupergit was added to 6 ml 20 mM phosphate buffer, pH 6.4, with 18 wt.% egg yolk powder, which contained β-cyclodextrin. After incubation with revolving at 45°C for 2 h, the supported enzyme waε removed by centrifugation (5 min, 900 g) . 4 ml of the remaining egg yolk solution waε incubated at 100°C for 10 min in order to inactivate any enzyme preεent. It was found that all of the β-cyclodextrin in this solution had been broken down. The Maltogenase-Eupergit removed by centrifugation was washed 3 times with 8 ml phosphate buffer, pH 7.5. This washed material was added to 8 ml of a similar egg yolk powder solution and incubated again at 45°C for 2 h. After centrifugation, the cyclodextrin content was determined again. It was found that the activity of the Eupergit beads with Maltogenase thereon was high enough to break down all of the β-cyclodextrin.

Claims: CLAIMS 1. A process for removing β-cyclodextrin from egg yolk or eg yolk plasma uεing an α-amylase, characterized in that the egg yolk or egg yolk plasma is treated with a maltogenic α-amylase originating from Bacillus stearothermophilus. 2. A process according to claim l, characterized in that the maltogenic α-amylase used is Maltogenase ® . 3. A process according to claim l or 2, characterized in that it is carried out at a temperature of between 45 and 63°C. 4. A process according to claim 3, characterized in that the temperature is set between 55 and 63°C. 5. A procesε according to any one of the preceding claimε, characterized in that it is carried out at a pH ranging from 5.8 to 7.0. 6. A procesε according to claim 5, characterized in that the pH iε adjuεted to between 6.0 and 6.8. 7. A process according to any one of the preceding claims, characterized in that the α-amylase is immobilized on a support material. 8. A procesε according to claim 7, characterized in that the support material used is Eupergit®. 9. Low-choleεterol egg yolk or egg yolk plasma obtained by the use of the proceεε according to any one of claimε 1-8.

Inventors: ROS ALBERT JACOBUS, VAN PANHUIS GIJSBERTUS, ROS, ALBERT, JACOBUS, VAN PANHUIS, GIJSBERTUS
Applicants: NL IND EIPRODUKTEN BV, PANHUIS GIJSBERTUS VAN, ROS ALBERT JACOBUS, B.V. NEDERLANDSE INDUSTRIE VAN EIPRODUKTEN, ROS, ALBERT, JACOBUS, VAN PANHUIS, GIJSBERTUS
Application year: 1993
Main classification: A23L1
IPC codes: A23L1