Sidhu, Gurcharn Singh (81 Cressy Road, North Ryde, NSW 2113, AU)
Rooney, Michael Laurence (120 Tambourine Bay Road, Lane Cove, NSW 2066, AU)
Oakenfull, David George (195 Foxvalley Road, Wahroonga, NSW 2076, AU)
Sidhu, Gurcharn Singh (81 Cressy Road, North Ryde, NSW 2113, AU)
Rooney, Michael Laurence (120 Tambourine Bay Road, Lane Cove, NSW 2066, AU)
|1.||1 to 6 and 8 wherein the complexing is carried out at a temperature below 18°C. • 10 A method according to claim 9 wherein the cholesterol containing components of the emulsion are presented in a form to ensure at least • 30% of the cholesterol is complexed within 20 minutes of contact between the cyclodextrin and cholesterol when the molar ratio of cyclodextrin to cholesterol is 2.|
|2.||11 A method according to anyone of the preceding claims wherein the emulsion comprises milk, , cream, egg yolk, egg yolk products, egg yolk plasma, whole yolk solutions or yolkalbumen mixtures.|
|3.||12 A method according to claim 11 wherein the emulsion comprises yolk, yolk plasma or yolkalbumen mixture and the emulsion is subjected to pretreatment to improve the extent and rate of complexing.|
|4.||13 A method according to claim 12 wherein the pretreatment comprises at' least one of the steps of dilution with water, dilution with milk, dissolution with salt solution, homogenisation, enzyme treatment, addition of lipid or combinations of these steps.|
|5.||14 A method according to claim 13 wherein the emulsion comprises a yolk^albumen mixture which is diluted with at least 40% by volume of milk prior to contact with the cyclodextrin.|
|6.||15 A method according to any one of the preceding claims wherein the precipitated cyclodextrincholesterol complex is subjected to a regeneration step whereby the choleεterol is removed from the complex to .provide free cyclodextrin.|
|7.||16 A method according to claim 15 wherein cyclodextrin is regenerated by extracting cholesterol with an appropriate solvent chosen from hexane, acetic acid or acetic acid/butanol mixture (l:v; v/v) .|
|8.||17 Products which have been treated in accordance with the method of any one of claims 1 to 16.|
This invention concerns a method for removing cholesterol from agueouε emulsions, especially from milk, dairy products and eggs.
It is widely accepted that serious health r ks attach to high plasma cholesterol levels. In Australia, coronary heart disease is responsible for more than 50,000 deaths every year, and death from coronary heart disease is twice as frequent as death from cancer. Dairy products, in particular, are perceived as contributing significantly to dietary cholesterol butterfat, for example, contains approximately 3mg cholesterol per gram. Egg yolk is high on any list of cholesterol-containing foods. An average hen's egg yolk is almost 2% cholesterol, ie 20 mg/g, or 5-8 times the concentration found in butterfat. Consequently there is considerable interest internationally in reducing the cholesterol level of dairy products.
The present invention is based on the recognition that cholesterol can be separated from aqueous emulsions by precipitation as a complex with a cyclodextrin.
Disclosure of Invention
This invention provides a method for removing cholesterol from aqueous emulsions which comprises:
i) contacting such an emulsion with a cyclodextrin to form a cyclodextrin-cholesterol complex; ii) causing the complex to precipitate; iii) separating the complex-precipitate.
The cyclodextrin may be added directly to the emulsion in powder form in which case ' the. emulsion acts as an aqueous solvent for the cyclodextrin or may be predissolved in water prior to addition to the emulsion.
I n instances where a material containing cholesterol is not in emulsion form it should be dispersed in a suitable liquid to form an emulsion prior to contacting with the cyclodextrin. However materials already in such form, such as milk or cream, may be contacted directly with the cyclodextrin without any pre- reatment.
The term "emulsion" as used herein is intended to include micellar solutions or cholesterol-containing fat associated with protein as in lipoproteins.
In a typical process according to this invention an aqueous emulsion containing cholesterol is shaken at 20 C to to 60 C with an aqueous solution of β-cyclodextrin . A cyclodextrin-cholesterol complex is formed which precipitates from solution on cooling to 20 , prefei-ably below 5 C. The precipitate is removed, for example, by centrifugation.
Surprisingly it has been found that the cyclodextrin used in the method of the invention can work quite effectively at lower temperatures even though the conventional thinking would suggest that higher temperatures should be required to obtain a satisfactory degree of complex formation. The cholesterol containing fats in many biological materials are solid at low temperature. It is therefore to be expected that the solid fats will reduce the rate of adsorption of cholesterol by the
cyclodextrin in comparison with the liquid form of the fats at higher temperatures. It is believed that the size of cholesterol containing globules of fats in the emulsions is a factor in ensuring that a satisfactory removal rate is realised. When the fat globules are very small, as in the case of milk, the cholesterol tends to accumulate at the surface of the globules with the result that it can readily transfer to the cyclodextrin even though the fat globules are solid.
The low temperature capability of a preferred method of the invention is particularly useful in relation to biological materials, such as with milk, cream or eggs which can spoil if they are not chilled.
Thus in a preferred aspect of the invention the temperature at which the complexation is carried out is below 18 C. More preferably a temperature range of 0-8 C is appropriate. Where such low complexation temperatures are used it is preferred that the fats in the emulsion be of sufficiently small size to ensure that at least 30% of the cholesterol is reacted to form a complex precipitate within 20 minutes of being mixed with the cyclodextrin. -
In order to ensure a high level of removal, the molar ratio of cyclodextrin to cholesterol should not be lower than 0.5, more preferably it should exceed 2. The ratio may be such as to ensure removal of at least 15% of the cholesterol from the emulsion, more preferably 40%.
The invention is suitable for decreasing the concentrat on of cholesterol in egg yolk, or egg yolk products such as yolk plasma, whole yolk solutions and egg white and yolk.
More specifically the method of the invention when applied to such egg components or products comprises steps such as the . following:
i) preparing yolk, yolk plasma or egg white and yolk mixture for maximum contact with cyclodextrin, suitable procedures- being dilution with milk, water or salt solution, homogenisation, enzyme treatment, and addition of lipid;
ϋ) treating the yolk preparation with cyclodextrin;
iii) separating the precipitated cholesterol-cyclodextrin complex, typically by centrifugation.
An optional step following (iii) is the regeneration of a whole yolk product by addition of the yolk granules and concentration, or by removal of salt and concentration.
The diluted egg yolk or liquid whole egg is brought to the original concentration by removal of water and/or salt using a semi-permeable membrane. If necessary the granules-free ' 'yolk is re-mixed with yolk granules, the purpose being to reconstitute the original " composition of liquid whole egg.
In a particularly preferred embodiment of the invention the yolk or yolk product is exposed to the action of the cyclodextrin in " the presence of egg white. We have found that, surprisingly, egg white or a component thei-eof can be responsible for up to a three-fold increase in the amount of cholesterol removed from egg yolk on treatment with cyclodextrin.
Desirably the concentration of cyclodextrin use in treatments relating to eggs according to this invention should not be
less than about 2% (w/v). Cholesterol removal diminishes rapidly when lower concentrations are used, on the other hand cholesterol removal may be substantially enhanced at higher concentrations.
Primarily, the term cyclodextrin in thi specification means β-cyclodextrin, but it is to ' be understood to include α cyclodextrin, cyclodextrinε modified to promote their cholesterol attracting and retaining properties, and substituted cyclodextrins, which are soluble or insoluble in water and are capable of forming a complex precipitate with cholesterol .
Best Modes for Carrying Out the Invention
The invention will now be described with reference to the following examples:
Extraction of Cholesterol from an Oi1-in-Water Emulsion Cholesterol, labelled with 14C was emulsified with oleic acid, onoolein and taurocholic acid in phosphate buffer (pH
7.0). This emulsion was shaken with β-cyclodextrin in powder form (0.1-2% w/v) at temperatures ranging from 20 to 60 C and subsequently cooled to below 5 C. The cyclodextrin-cholesterol complex was removed by centrifugation. In a typical experiment, the adsorbent removed 85% of the cholesterol from the emulsion (As shown in
Table 1) .
TABLE 1 - Removal of cholesterol from an emulsion by β-cyclodextrin
Activity of Labelled Cholesterol
Untreated Emulsion 7049
After exposure to the cyclodextrin 1095
10 EXAMPLE 2
Extraction of Cholesterol from Milk Milk was shaken with powdered β-cyclodextrin (1% w/v) for one hour at 40 C. On cooling in ice followed by centrifugation, the adsorbent was found to have removed 70% of the cholesterol from the fat (as
15 shown in Table 2) .
TABLE 2 Removal of cholesterol from milk by β-cyclodextrin.
Concentration of Molar ratio cholesterol in of CP:CH*
Untreated milk 3.20 30
After exposure to the cyclodextrin 0.96
_.. * Molar ratio of β-cyclodextrin to cholesterol
Extraction of Cholesterol from Cream Cream containing 18% fat was shaken with powdered β-cyclodextrin (2% w/v) for one
30 , j_ Λ „ hour at 40o C„. O_n cool,i.ng a.n i.ce f ÷ ol ,l ■,owedj br_y cent * .ri -f ÷ ugatJ.i-on, the cyclodextrin was found to have removed 79% of the cholesterol from the fat (as shown in Table 3).
TABLE 3 Removal of cholesterol from cream by β-cyclodextrin.
Concentration Molar ratio of cholesterol of CD:CH in cream (mg/g fat)
Untreated cream 3.10
After exposure to the cyclodextrin 0.65 15
Removal of cholesterol from egg yolk plasma
The yolk from White Leghorn eggs was diluted 1:1 with isotonic saline (0.16 M NaCl) and the mixture centrigued at 20,000 rpm (50,000 g) for 30 min at 10°C. The yolk granules (about 12% of the yolk) sedimented and the supernatant solution (about 20% w/v) after dilution with water, was used for cholesterol removal by treating with the powdered cyclodextrin (concentration ranging from 0.5 to 3.5% w/v for 20 minutes at temperatures ranging from 10-50 C). Cyclodextrin was then centrigued off at low speed. Cholesterol was determined in the solution by a chemical method.
TABLE 4 - TREATMENT OF YOLK PLASMA
Cyclodextrin Cholesterol Molar " Cholesterol Concentration Concentration Ratio in Yolk Plasma CD:CH
(% w/v) (mg/ml) left % removed 5
0 5.40 1 4.87 0.7 90.2 9.-8 2 4.13 1.6 76.5 23.5 0
0 6.66 1. 5.80 0.47 87.0 13.0
0 5.83 2 4.73 1.4 81.0 19.0
Dilution 1+3 5
0 2.62 2 1.62 4.2 61.5 38.5
Removal of cholesterol from whole yolk 0
Yolk from White Leghorn hens' eggs (39.2 g) was diluted with 2M sodium chloride (13 ml) and stirred to dissolve the granules. The mixture was diluted with 0.5M sodium chloride and treated with powdered cyclodextrin at various dilutions as described $ for Example 4.
TABLE 5 ' - TREATMENT OF WHOLE YOLK
Cyclodextrin Cholesterol Cholesterol
(% w/v) in Yolk Plasma (mg/ml) % left % removed
Dilution 1+2.25 5.25 80 20
Removal of cholesterol from liquid whole egg
The yolks and whites of Australorp and Leghorn hens' eggs were mixed and beaten lightly. The mixture was then diluted with water or 0.5M sodium chloride and cholesterol extracted with cyclodextrin as for Examples 4 and 5.
TABLE 6 - TREATMENT OF WHOLE EGG PULP
Cyclodextrin Cholesterol Molar Cholesterol
Concentration Concentration ratio in Yolk Plasma of (% w/v) (mg/ml) CD:CH % left % removed
Dilution 1+3 (NaCl)
2 1.13 6.-1 5 . 95 Leghorn
Dilution 1+3 (water)
2 1.54 4.4 .14 86
Dilution 1+2 (NaCl) 2 2.41 2.8 37 63
Dilution 1+4 (NaCl)
2 1.40 4.9 11 89
Labelled milk (LM) was prepared by evaporating 4 ml of a chloroform solution containing 0.25 μCi ' of ^C-labelled cholesterol in a round bottomed flask containing 10 g of fine silica beads. Milk (50 ml) was added and mixed at 20 C for 30 in. using a rotary evaporator. Radioactive cholesterol readily exchanged with the cholesterol already in the milk.
Labelled scrambled egg mix (SEM) or labelled egg yolk low density lipoprotein (LDL) were prepared by introducing l ''C-labelled cholesterol by the same method as described for LM. The scrambled egg mix was prepared by combining the yolk and white from whole egg and mixing with an equal volume of full cream homogenised milk.
Labelled milk (LM) was mixed with β-cyclodextrin • and samples were maintained at different temperatures with different contact times. They were then cooled to 0 C, centrifuged and the cholesterol removed determined from the loss of radioactivity. The results are shown in Table 7. (This 5 Table demonstrates the improved efficiency at low temperatures.)
TABLE 7 Removal of cholesterol from milk by β-cyclodextrin.
Cyclodextrin Contact Temp DPM
(% w/v) time (°C) Initial Fin.
(min. ) (%) CD:CH
1 0 10 40 1188 1 0 30 40 1188
1.0 10 8 1188 1.0 30 8 1188
1. 10 4 1188 1. 20 4 1188 1. 30 4 1188
20 2 5 4 1952 2 10 4 1952 2, 15 4 1952 2, 20 4 1952
EXAMPLE 8 2.5
Labelled scrambled egg mix (SEM) was mixed with β-cyclodextrin and samples were maintained at different temperatures with different contact times. They were then cooled to 0 C, centrifuged and the cholesterol removed was 30 etermined from the loss of radioactivity. The results are shown in Table 8.
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