Method for producing egg-yolk of decreased cholesterol content

Elevated human serum cholesterol is a risk factor for arteriosclerosis and coronary heart disease.

In many pathologic cases, lowering cholesterol intake can restore normal serum cholesterol levels. On this basis, efforts by the food industry are aimed at significantly reducing the amount of cholesterol and cholesterol esters in fat-rich foods of animal origin.

This is a major challenge for the far-reaching preservation of the organoleptic and nutritional properties of foods.

A number of methods for isolating cholesterol and cholesterol esters have already been reported, but many of these methods are not suitable for reducing the cholesterol content of food due to chemical changes in essential constituents of the starting material (such as proteins, triglycerides, etc.).

A relatively gentle process, which has become known only recently, is for the removal of cholesterol or cholesterol ester by high pressure extraction with carbon dioxide. Krukonis, V .: Supercritical Fluid Processing, International Symposium on Supercritical Fluids, Nice, 1988 /.

Although this process is distinguished by the physiological tolerance of the extraction agent (carbon dioxide), work under high pressure is technically quite complicated. In addition, cholesterol and cholesterol esters cannot be selectively removed under gentle conditions by this process, since triglycerides are also co-extracted.

While it is fundamentally possible to improve selectivity by increasing the temperature, this has a detrimental effect on the cholesterol and cholesterol ester uptake capacity of the carbon dioxide and the quality of the product obtained.

EP-A-0 326 469 discloses the separation of cholesterol derivatives from egg yolk using β-cyclodextrin. The disadvantage of this process is the long reaction time and the relatively small decrease in cholesterol content in egg yolk.

It is an object of the present invention to provide a process for the removal of cholesterol and / or cholesterol esters from egg yolk which is free of the above-mentioned disadvantages of the prior art process and allows for a highly selective reduction of these materials under mild conditions and with little equipment.

According to the invention, this object is solved by:

(a) dilute egg yolks with water or brine,

b) selectively complexing the cholesterol and cholesterol esters in the diluted egg yolk with 3-cyclodextrin,

c) separating the β-cyclodextrin bound to the cholesterol and / or cholesterol esters from the diluted egg yolk;

d) removing the added water from the yolk,

e) enzymatically digesting the amount of β-cyclodextrin in egg yolk using α-amylase and / or CTGase;

f) recovering the β-cyclodextrin from the β-cyclodextrin complex by treating with water and / or an alcohol and optionally recycling it to process step b).

Surprisingly, it has been found that in this way, a low total cholesterol, good organoleptic product is obtained. In addition, the resulting cholesterol-reduced egg yolk contains practically no β-cyclodextrin. It was not foreseeable that we would achieve this.

In the process of the invention, in step a), the egg yolk is diluted by the addition of water or aqueous saline. The amount of water used can vary within wide limits, but for reasons of economy it has proven advantageous to add 10-400% by weight, preferably 100-300% by weight, of egg yolk starting weight. Upon addition of water, the granule fraction precipitates in the form of a solid and can be very easily separated from the liquid egg yolk plasma by conventional solid / liquid separation techniques such as centrifugation. In this case, egg yolk plasma is used instead of egg yolk for the remainder of the process.

Instead of water, you can add an aqueous saline solution to the egg yolk. In this embodiment, the separation of the egg yolk emulsion into granular fraction and egg yolk plasma is prevented. The saline solution is preferably 5-20% sodium chloride solution or 1-10% ammonium bicarbonate solution to achieve the desired degree of dilution.

In a subsequent step b) of the process of the invention, cholesterol and cholesterol derivatives are then removed from the diluted egg yolk or egg yolk plasma by complexing with β-cyclodextrin; this allows for particularly selective binding of cholesterol.

The amount of cyclodextrin may vary within wide limits, but preferably 3-40% by weight of β-cyclodextrin, based on the dry weight of the egg yolk.

Depending on the amount of β-cyclodextrin used, complexing with β-cyclodextrin, which can be accomplished by known methods, such as simple mixing or mixing, may remove about 60-99% of the cholesterol and cholesterol esters, while the remainder of the egg yolk components. they remain in the liquid phase. Prior dilution of egg yolk with water allows very gentle and complete removal of cholesterol with β-cyclodextrin at lower temperatures, such as 4-20 ° C. Complex formation is particularly advantageously carried out by stirring the mixture at 4-10 ° C.

In step c) of the process of the invention, the β-cyclodextrin coupled to cholestine and / or cholestine esters is separated from the liquid egg yolk phase. In principle, conventional methods for separating solids and liquids may be used. Centrifugation is preferably used in the process of the invention for rapid and complete separation. Other separation methods, such as filtration, are likewise well applicable.

After separation of the β-cyclodextrin complexes, the addition salts can be removed from the egg yolk in a known manner, if the presence of these salts is not desired for certain uses. In the case of ammonium bicarbonate, removal is very easy._r · · · · ·

6 by allowing the compound to evaporate by heating the egg yolk mixture to 40-80 ° C. The distillation is preferably carried out in vacuo at 55-70 ° C. If sodium chloride is to be removed from the egg yolk, it is preferably accomplished by dialysis, electrodialysis, cross-flow ultrafiltration or other methods.

After separation of the β-cyclodextrin complexes from the largely cholesterol-free diluted egg yolk or egg yolk plasma and optionally, after removal of the salts, the egg yolk (plasma) is concentrated in step d) until it returns to its original solids content. This step can be carried out by conventional methods such as vacuum evaporation or membrane technique very gently and without difficulty.

In step e) of the process according to the invention, the residual amount of cyclodextrin present in the egg yolk, which is usually 0.1 to 1.0% by weight, is decomposed using α-amylase and / or CTGase. Preferably, α-amylase produced by Aspergillus niger, Aspergillus oryzae, Bacillus polymyxa, Bacillus coagulans and Flavobacteria, domestic pancreatic amylase, or α-amylase derived therefrom are used. Of the CTGases (cyclodextrin transglycosylases = EC 2.4.1.19), they are particularly suitable for the Bacillus group (e.g., Bacillus macerans, Bacillus megaterium, Bacillus stearothermophils, Bacillus circulans and Bacillus ohbensis), the Klebsiella group (e.g. for example variant) and alkalophilic bacteria such as CTGases produced by or derived from Nos. 38-2 and 17-1. These α-amylases and CTGases allow the synthesis of α-amylases and CTGases.

- Practically complete degradation of the 7 β-cyclodextrins. Prior to adding the enzymes to the egg yolk, it is advisable to adjust the pH of the egg yolk to the optimum pH of the respective enzyme, which can be done, for example, with standard food grade acids such as citric acid. The amount of enzyme required is essentially dependent on the content of the starting? -Cyclodextrin in the yolk and, in the case of α-amylase, is generally 10-500 FAU per g of β-cyclodextrin to be removed.

(1 FAU / unit of fungal α-amylase / under standard conditions / substrate: soluble starch, incubation time: 7-20 minutes, temperature: 37 ° C, pH: 4.7 / 1 hour decomposes 5.26 g starch) . In the case of CTGases, this amount is preferably 0.5 to 20 U per gram of β-cyclodextrin to be removed (1 U is the amount of substrate that is converted per minute). We can work with larger amounts of enzyme, but this quickly becomes uneconomic as it does not produce a better effect. Treatment conditions, such as temperature and reaction time, can be varied within wide limits, but temperatures of 5 to 65 ° C have been particularly preferred, with a treatment time of 0.5 to 50 hours being typical. In a preferred embodiment, a mixture of α-amylase and CTGase is used.

Enzymatic degradation of cyclodextrin can also be performed in the diluted egg yolk phase, i.e. prior to the concentration step; so the order of steps d) and e) can be reversed. However, this is not beneficial.

If cholesterol removal is performed in egg yolk plasma, after enzymatic treatment, the separated granule fraction can be resuspended in egg yolk plasma.

• » · · · · * 4 ·« • ·«··«·4 • 4 ·4 4

- 8 The virtually cholesterol-free egg yolk may be further processed depending on the intended use. Thus, an appropriate amount of egg whites may be added to the egg yolk to produce whole eggs with reduced cholesterol content.

In step f) of the process of the invention, the β-cyclodextrin complexed with cholesterol and / or cholesterol esters is optionally purified and then regenerated. This optional purification step is carried out by washing with an aqueous or aqueous saline solution such as sodium chloride or ammonium bicarbonate. The amount of water used can be varied within a relatively wide range. In general, however, a 1-5 fold amount based on the weight of the β-cyclodextrin complex is sufficient to separate the β-cyclodextrin complexes from any contaminants such as proteins. In a preferred embodiment, the β-cyclodextrin-cholesterol complex is mixed with 1-2 times the amount of water and the solid (β-cyclodextrin complex) is separated, for example by centrifugation. The substantially protein-containing wash water may optionally be reused to dilute the egg yolk (step a).

After optional washing, the β-cyclodextrin complex is treated with water and / or alcohol to remove β-cyclodextrin from cholesterol (cholesterol derivatives) and any fats that may also be present. The temperature of the water or alcohol is preferably 40-100 ° C to destabilize the β-cyclodextrin complexes and simultaneously dissolve the released β-cyclodextrin in water. As the alcohol, a C 1-4 alcohol, preferably ethanol, is used as the alcohol. · · · · · · · · · · · · · · · · · · · · · · · · · · ·

- 9 to apply.

The resulting mixture of fatty cholesterol, optionally after alcohol recovery, can be used directly as a raw material in the cosmetic industry or further processed to recover cholesterol.

Preferably, the β-cyclodextrin purified with water or alcohol, optionally in several steps, is dissolved in water and, after evaporation of the aqueous solution or water, is reused in powder form for complexing the cholesterol derivatives in step b).

This recovery or recycling of O-cyclodextrin is one of the major economic advantages of the process of the invention. In addition, an egg yolk with a residual cyclodextrin content of less than 20 ppm is obtained with a total cholesterol content reduction of about 80-95%. Based on such a good reduction of cholesterol and β-cyclodextrin content, with further advantages such as low equipment requirements and good organoleptic properties of the resulting egg yolk, the method of the present invention is particularly suitable for industrial application.

The following non-limiting examples illustrate the present invention.

First pounds of egg yolk (dry matter 950 g) with a cholesterol content of 1.2% are mixed with 2 kg of 10% w / w aqueous sodium chloride solution.

The egg yolk-salt mixture was then mixed with 280 g of β-cyclodextrin and stirred for 60 minutes at 5 ° C. The cholesterol-containing β-cyclodextrin is then separated from the egg yolk phase by centrifugation.

The diluted egg yolk phase is then subjected to cross-flow ultrafiltration, whereby the moisture content is adjusted to its original value and the sodium chloride content is reduced to about half. In the next step, the sodium chloride content is reduced to about 0.3 wt% by electrodialysis.

Finally, egg yolk is treated with Fungamyl 800 enzyme preparation (1000 FAU / kg material) for 45 minutes at pH 5.5 and incubated at 50 ° C with a concentration of about 0.5% w / w in 3-cyclodextrin. below ppm.

Yield 0.12% of total cholesterol, which corresponds to a 90% reduction compared to untreated egg yolk.

The separated β-cyclodextrin-cholesterol complex

350 g, which contains trapped fats and proteins, is mixed with 400 ml of water and the solid is separated by centrifugation from the liquid phase. The proteinaceous aqueous solution is hereinafter used to dilute the egg yolk. The solids-separated 0-cyclodextrin-cholesterol complex was washed in several steps with nine times 98% ethanol. The ethanol phase is then separated by centrifugation from the solid β-cyclodextrin. The β-cyclodextrin is then dissolved in hot water and filtered to remove insoluble constituents. From this solution finally 198 g of pure β-cyclodextrin

- We win 11_z which is used repeatedly to complex the cholesterol.

Second EXAMPLE 1 kg of egg yolk (solids content 950 g) with a cholesterol content of 1.2% is mixed with 2 kg of a 4% solution of ammonium bicarbonate.

The egg yolk-salt mixture is then mixed with 300 g / 3-cyclodextrin and stirred for 50 minutes at 5 ° C. The complex-linked β-cyclodextrin is then separated from the egg yolk phase by centrifugation.

The diluted egg yolk phase is then concentrated in vacuo at 65 ° C while removing the ammonium carbonate and adjusting the moisture content to its original value.

Finally, the egg yolk is adjusted to pH 6.0 with citric acid and mixed with Funamyl 800 (300 FAU / kg). After incubation for 45 minutes at 50 ° C, 50 U of a CTGase from Bacillus macerans (EC 2.4.1.19) is added and incubated for another 45 minutes at 50 ° C.

The product yields an egg yolk content of 0.1% total cholesterol, which corresponds to a 92% reduction compared to untreated egg yolk. No residual β-cyclodextrin could be detected in the product (limit of detection 20 ppm).

The separated / 3-cyclodextrin-cholesterol complex

350 g, which contains trapped fats and proteins, is mixed with 3800 g of water and cooked for 30 minutes. The solution is then liberated by filtration to remove the undissolved solution · · · · · · · · · · · · · · · · · · · · · · · ·

- 12 ingredients. From this solution, 205 g of pure β-cyclodextrin are finally recovered and reused to complex the cholesterol.

Example 3 kg of egg yolk (solids content 950 g) with a total cholesterol content of 1.2% was mixed with distilled water in a 1: 3 weight ratio and centrifuged for 15 minutes at 4 ° C.

6.3 kg of the plasma fraction thus obtained were decanted from the granule fraction and mixed thoroughly with 238 g of β-cyclodextrin for 60 minutes at 4 ° C. The complex-bound cyclodextrin is then separated from the liquid plasma phase by centrifugation.

The plasma phase is incubated with Fungamyl 800 enzyme preparation (400 FAU / kg material) and a CTGase from Bacillus macerans (10 U / kg starting material) for 120 minutes at pH 5.5 and 50 ° C.

The plasma fraction so treated is then subjected to vacuum evaporation at 50 ° C, whereby the moisture content is adjusted to its original value. The separated granule fraction is resuspended in concentrated egg yolk plasma and the whole mass is mixed with egg protein in a weight ratio of 1: 2. The product obtained is a whole egg with a cholesterol content of 0.07%.

350 g of the separated β-cyclodextrin complex is mixed with 700 g of water and the solids are centrifuged to remove water-soluble impurities such as · · · ······················································

- 13 proteins. The β-cyclodextrin complex is then cooked three times with 900 g of ethanol for 60 minutes. The solids separated by filtration were dissolved in boiling water, filtered to remove insoluble residues, and the resulting solution of [beta] -cyclode.extrin was concentrated and crystallized to give 160 g of pure β-cyclodextrin.