CA2071217A1

CA2071217A1 - Cholesterol reduction

Info

Publication number: CA2071217A1
Application number: CA002071217A
Authority: CA
Inventors: David George Oakenfull; Gurcharn Singh Sidhu; Michael Laurence Rooney
Original assignee: Individual
Current assignee: Commonwealth Scientific and Industrial Research Organization CSIRO
Priority date: 1990-01-23
Filing date: 1990-10-12
Publication date: 1991-07-24
Also published as: JPH05505516A; EP0607120A1; WO1991011114A1; EP0607120A4; NZ235649A

Abstract

Cholesterol is removed from aqueous emulsions of dairy or egg based products by contacting the aqueous emulsions with a cyclodextrin and forming a cyclodextrin-cholesterol complex generally at a temperature in the range 20 ·C to 60 ·C and causing the complex to precipitate from solution by chilling below 5 ·C. The precipitated complex is removed and the cyclodextrin is regenerated by extraction of cholesterol with a solvent chosen from hexane, acetic acid or acetic/butanol mixtures.

Description

WO 91tll]14 PCT/AU90/0~89 CHOLESTEROL REDUCTION

Technical Field This invention concerns a method for removi]lg cllolesterol from aqueous emulsions, especially from milk, ~3airy prod~lcts and eggs.

Backqround Art It is widely acce~ted that serious health risks 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 frequellt 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 removin~ cholesterol from aqueous emulsions which comprises: -' `,' ' WO91/11114 PCT/AU9-/0~-9 2071217 -2- ~
i, contactingsuc~lallemulsiol~wit]lacyclo~extri~lto form a cyclodextrin-cholesterol complex;
ii) causing the complex to precipitate;
iii) separating the complex-precipitate. t ~ ~
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The cyclodextrin may be added directly to the emulsiol1 in ~ 5 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.
, In instances where a material containing cholesterol is not in emulsion form it should be dispersed in a sl1itable liquid to form an emulsion prior to contacting Wit]1 the cyclodextrin.
However materials already in such form, s~ch as milk or cream, may be contacted directly with the cyclodextrin without any pre-treatment.

The term "emulsion" as used herein is il~tended 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 20C to to 60C with an aqueous solution of B-cyclodextrin . A
cyclodextrin-cholesterol complex is formed which precipitates from solution on cooling to 20, preferably below ~C.
The precipitate is removed, for example, by centl-ifugation.

- Surprisingly it has been ound that the cyclodextri11 used in the method of the invention can work qllite effectively at lower temperatures even though the conventiona] thinking would suggest that higher temperatures should be required to obtain a satisfactory degree of complex formatioll. 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 t11e W O 91/11114 PC~r/AU90/00489 ~ ` 2~712~7 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 emulcions is a factor in ensuring that a satisfactory removal rate is realised. Wilell the fat globules are very small, as in the 05 case of milk, the cholesterol tends to accum~late at the surface of the globules with t~e result t]lat it can readily transfer to the cyclodextrin even though the fat globules are solid.
, The low temperature capability of a preferled method of the invention is particularly useful in relation to biological materials, such as with milk, cream or egas 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 18C. More preferably a temperature range of 0-8C 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 concentration of cholesterol in egg yolk, or egg yolk products such as yolk plasma, whole yolk solutions and egg white and yolk.

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WO 91/11114 PCr/AU90/00489 -4- ~r 2 ~ 7 More specifically the method of the invention whell applied to such egg components or products comprises steps such as the following: .

i) preparing yolk, yolk plasma or egg white and yolk 05 mixture for maximum contact with cyclodextrin, suitable procedures being dilution with milk, water or salt solution, homogenisatioll, enzyme treatment, and addition of lipid;

10 ii) treating the yolk preparation with cyclodextrin;

iii) separating the . precipitated cholesterol-cyclodextrin complex, typically b~
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 inventiol- 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 thereof can be responsible for up to a three-fold increase in the amount of cholesterol removed from egg yolk on treatment with cycladextrin.
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Desirably the concentration of cyclodextrin use in treatments relating to eggs according to this inventioJl should not be ~ ,;,-.. . " ,. . : ,' - : , -. : ,: . - . : - : ~ .
- . -, - : : -.! 5 2~71217 less than about 2% (w/v). Cholesterol removal diminishes rapidly when lowe~- concentrations are used, on the other hand cholesterol removal may be substantially enhanced at higher concentrations.

05 Primarily, the term cyclodextrin in this s~ecificatio1l means B-cyclodextrin, but it is to be understood to include n cyclodextrin, cyclodextrins modified to promote their cholesterol attracting and retainin~ properties, and substituted cyclodextrins, which are soluble Ol insoluble in water and are capable of forming a complex precipitate with cholesterol.

Best Modes for CarrYina Out the Invention The invention will now be described Wit]l reference to the following examples:

~ from an Oil-in-Water EmulS10 n Cholesterol, labelled with l4C was emulsified with oleic acid, monoolein and taurocholic acid in phosphate buffer (pH
7.0). This emulsion was shaken Wit}l B-cyclodextrin in powder form (0.1-2% w/v) at temperatures ranging from 20 to 60C and subsequently cooled to below 5C. The cyclodextrin-cholesterol complex was removed by centrifugation. In a typical experiment, the adsorbent removed 85% of the cholesterol from the emulsion (~s shown in Table l).

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WO9l/11l14 PCT/AU90/0~89 2~7~17 -6- ~ I
i TABLE 1 - Removal of cholesterol from an em~llsion by B-cyclodextrin -.
Activity of Labelled Cholesterol 05 (DPM) . . .
Untreated Emulsion 7049 .
After exposure to the cyciodextrin 1095 ~xtraction of Cholesterol from Milk Milk was shaken with powdered ~-cyclodextrin (1% w/v) for one hour at 40C. On :~
cooling in ice followed by centrifu~ation, the adsorbellt 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 B-cyclodextrin.
_ ........... ._ Concentration of Molar ratio -.
cholesterol in of CP:CH* ~ ~
milk (mg/g fat) Untreated milk 3.20 After exposure to the cyclodextrin 0.96 25 * Molar ratio of ~-cyclodextrin to cholesterol Extraction of Cholesterol from Cream Cream containing 18%
fat was shaken with powdered ~-cyclodextrin (2% w/v) or one :
30 hour at 40OC On cooling in ice followed by centrifugation, . ~.
the cyclodextrin was found to have removed 79% of the cholesterol from the fat (as shown in Table 3).

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TABLE 3 Removal of cholesterol from cream by - ~-cyclodextrin.
Concentration Molar ratio of cholestelol of CD:CH
in cream (mg/g fat) 05 __ _ _ _ _ _ _.
Untreated cream 3.10 1 After exposure to the cyclodextrin 0.65 _ _ ... . _ .. _ . .

Removal of cholesterol from egg Yolk plasma The yolk from White Leghorn eggs was diluted 1:1 with isotonic saline (0.16 M NaC1) and the mixture centrigued at 20,000 rpm 15 (50,000 g) for 30 min at 10C. The yolk gl-anules (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 20 temperatures ranging from 10-50C). Cyclodextrin was then centrigued off at low speed. Cholesterol was determined itl the solution by a chemical method.
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- . : . . , . : . -WO91/11114 PCT/AU9OtO0489 c~ ~ 7 ~ 8-Cyclodextrin Cholesterol MolarCholesterol Concentration Concentration Ratio in Yolk Plasma CD:CH
(% w/v) ~mg/ml) % left % removed _ . _ Dilution 1~1 0 5.40 1 4.87 0.7 90.29,8 2 4.13 1.6 76.523.5 ~-, 0 6.66 1.4 5.80 0.47 87.013.0 0 5.83 - :.
2 4.73 1.4 81.019.0 Dilution 1~3 2.62 2 1.62 4.2 61.538.5 . . . _ EXAMPLE 5 ,~-Removal of cholesterol from whole yolk 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 25 for Example 4.

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l' ~ , . s 2~7~7 TABLE 5 - TREATMENT OF WHOLE YOL~

Cyclodextrin Cholesterol Cholesterol Concentration Concentration 05 (% w/v) in Yolk Plasma (mg/ml) % left ,~ Iemoved ?
_ _ _ Dilution 1+2.25 2 5.25 sn 20 . _ lO EXAMPLE 6 Remo~al of cholesterol from liquid whole eqg The yolks and whites of Australorp and Leghorn hens' eggs were mixed and beaten lightly. The mixture was then diluted with ~ :
15 water or 0.5M sodium chloride and cholesterol extracted with cyclodextrin as for Examples 4 and 5. :

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Cyclodextrin Cholesterol Molar Cholesterol Concentration Concentration ratio in Yolk Plasma of O (% w/v) (mg/ml) CD:CH % left % removed , Australorp Dilution 1+3 (NaCl) 2 1.13 6.1 5 . 95 Leghorn Dilution 1~3 (water) 2 1.54 4.4 14 85 Australorp Dilution 1~2 (NaC1) 15 2 2.41 2.8 37 63 Leghorn Dilution 1+4 ~NaC1) 2 1.40 4.9 11 89 -_ . . . _ Labelled milk (LM) was prepared by evaporating 4 ml of a chloroform solution containing 0.25 ~Ci of ~4C-labelled cholesterol in a round bottomed flask containing 10 g of fine silica beads. Milk (50 ml) was added and mixed at 20C for 25 30 min- using a rotary evaporator. Radioactive cholesterol readily exchanged with the cholesterol already in the milk.

Labelled scrambled egg mix ~SEM) or lakelled egg yolk l~w density lipoprotein (LDL) were prepared by introducing 30 I~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.

-` 2~712 ~
Labelled milk (LM) was mixed with B-cyclodextrin and samples were maintained at different temperatures with different contact times. They were then cooled to 0C, centrifuged and the cholesterol removed determined from the loss of radioactivity. The results are shown in Table 7. (This Table demonstrates the improved efficiency at low temperatures.) TABLE 7 Removal of cholesterol from milk by B-cyclodextrin.
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Cyclodextrin Contact Temp DPM __ Molar Cholesterol Ratio (% w/v) time (C) Initial Final removed of (min.) (%) CD:CH ~-.... .
1.0 lO 40 1188 442 62.5 30 1.0 30 40 1188 353 70.3 30 1.0 10 8 1188 441 62.9 30 1.0 30 8 1188 352 70.4 30 l.0 lO 4 1188 287 77.1 30 1.0 20 4 1188 240 ~1.8 30 1.0 30 4 1188 224 82.1 30 ~0 2,0 5 4 1952 326 83.3 60 2.0 10 4 1952 179 90.8 60 2.0 15 4 1952 190 90.3 60 2.0 20 4 1952 139 92.9 60 .. ....

Labelled scrambled egg mix (SEM) w~s mixed with B-cyclodextrin and samples were maintained at different temperatures with different contact times. They were then cooled to 0C, centrifuged and the cholesterol removed was 30determined from the loss of radioactivity. The results are shown in Table 8.

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2 ~ 7 ~ ~ ~ jl TABLE 8 Removal of cholesterol from scrambled egg mix :.
by B-cyclodextrin.

Cyclodextrin Contact Temp. DPM Cholesterol Molar time (C) Initial Final removed Ratio 05 (% w/v) (min.) (%) CD:CH

. . .. . _ _ .
4.0 5 40 719 5~ 92.9 4.3 4.0lO 40 719 32 95.5 4.3 4.015 40 719 25 96.5 4.3 10 4 0 30 40 71 36 95.6 4.3 4.0 5 lO 719 35 95.5 4.3 4.0lO lO 71a 41 94.4 4.3 4.015 10 719 48 93.3 4.3 4.030 lO 719 48 93.3 4.3 ~ ~-15 ExAMpLE 9 Labelled egg yolk low density lipoprotein (LDL) was mixed with B-cyclodextrin and samples were maintained at different -~ .
temperatures with different contact times. They were then 20 cooled to 0C, centrifuged and the cholesterol removed by determined from the loss of radioactivity. The results are '~ ' .

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W09l/11114 PCT/AU90/0~89 fi .~ - 1 3-20~121 7 shown in Table 8.
TABLE 9. Removal of cholesterol from egg low density lipoprotein (6mg cholesterol/ml) by ~-cyclodextrin.
Cyclodextrin Contact Temp. DPM Molar Cholesterol (% w/v) time (C) Initial Final Ratio removed (min.) of (%) 05 CD:CH

1.5 5 40 3391 2921 0.~ ~4.0 1.5 10 - 40 3391 2893 0.85 15.0 1.5 15 40 3391 2811 0.85 17.0 1.5 5 5 3433 3083 0.~5 10.0 10 1.5 lO 5 3433 2914 0.85 15.0 1.5 15 5 3433 3000 0.85 13.0 2.7a 5 40 1192 427 4.6 64.2 2.7a 10 40 1192 321 4.6 73.1 2 7a 15 40 1192 433 4 6 63.7 2.7a 30 40 1192 280 4.6 76.5 15 2.7a 5 lO 1192 359 4.6 69.1 2.7aa 10 10 1192 363 4.6 6~.5 ; -2.7 15 lO 1192 280 4.6 76.5 2.7a 30 10 1192 280 4.6 76.5 _ _ a LDL solution was diluted 3-fold with aqueous 0.5 M
sodium chloride. ?

Reconstituted liquid whole egg products prepared by methods according to this invention could be used to substitute for 25 conventional liquid or dried whole egg in various food applications. Eggs are an extremely nutritio~ls food that are at present underused, partly because of ears abff~lt their relatively high cholesterol content. A method, such as that described here, would benefit people on low-cholesterol diets.
It would also benefit primary producers by enhancing the demand for eggs by for example processors of liquid, dried, and frozen egg; and food manufacturers such as those making mayonnaise, ice creams, omelets; and in particular, bakeries and makers of cake mixes.-'2~ ~ C~
Cream (201) containing 20% fat was treated with cyclodextrin (2%; w/v) to remove cholesterol. Butter was prepared from it .
using a small laboratory churn. The colesterol in this butter was estimated using a standard chemical procedure and compared with the cholesterol content of butter prepared from untreated cream. The results are given in Table lO. ~ ~

TABLE 10. Removal of cholesterol from butter by ~, ~-cyclodextrin j:

Cholesterol Cholesterol Content Removed (mg/g fat) (%) . .
Untreated cream 3.2 Treated cream 0.42 87 :;

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Claims

C L A I M S

1 A method for removing cholesterol from aqueous emulsions comprising contacting the emulsion with a cyclodextrin to form a cyclodextrin-cholesterol complex, precipitating the complex and separating the precipitate from the emulsion.

2. A method according to claim 1 wherein the molar ratio of cyclodextrin to cholesterol in the emulsion is at least 0.5.

3. A method according to claim 2 wherein the molar ratio is at least 2.

4. A method according to claim 2 or claim 3 wherein the ratio and conditions of removal are such as to ensure removal of at least 15% of the cholesterol from the emulsion within 20 minutes of contact between the cholesterol and cyclodextrin.

5. A method according to claim 4 wherein at least 40% of the cholesterol is removed from the emulsion.

6. A method according to any one of the preceding claims wherein the cyclodextrin is added to the emulsion in solid form.

7. A method according to any one of the preceding claims wherein the complexing is carried out at a temperature in the range 20°C to 60°C.

8. A method according to any one of the preceding claims wherein cyclodextrin-complex is precipitated from solution by chilling below 5°C.

9. A method according to any one of the claims 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.

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 yolk-albumen mixtures.

12. A method according to claim 11 wherein the emulsion comprises yolk, yolk plasma or yolk-albumen mixture and the emulsion is subjected to pre-treatment to improve the extent and rate of complexing.

13. A method according to claim 12 wherein the pre-treatment 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.

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.

15. A method according to any one of the preceding claims wherein the precipitated cyclodextrin-cholesterol complex is subjected to a regeneration step whereby the cholesterol is removed from the complex to provide free cyclodextrin.

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 (1:v; v/v).

17. Products which have been treated in accordance with the method of any one of claims 1 to 16.