AU630446B2

AU630446B2 - Cholesterol removal from eggs, dairy products and other aqueous emulsions

Info

Publication number: AU630446B2
Application number: AU55112/90A
Authority: AU
Inventors: David George Oakenfull; Michael Laurence Rooney; Gursharan Singh Sidhu
Original assignee: Commonwealth Scientific and Industrial Research Organization CSIRO
Current assignee: Commonwealth Scientific and Industrial Research Organization CSIRO
Priority date: 1989-05-19
Filing date: 1990-05-17
Publication date: 1992-10-29
Anticipated expiration: 2010-05-17
Also published as: AU5511290A

Description

P/00/01i1 2EV5/91 Regulation 3.2 AkUSTRALIA Patents Act 1990

ORIGINAL

COMPLETE SPECIFICATION STANDARD PATENT ,,Cholesterol Removal from Invention Title: Eggs, Dairy Products and other Aqueous Emulsions" The following statement is a full description of this invention, including the best methodi of performing it known to me:- QC5. Ho qY~- I. 1. I I I I. 1 1 la This invention concerns a method for removing cholesterol from aqueous emulsions, especially from milk, dairy products and eggs.

It is widely accepted 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 frequent as death from cancer. Dairy products, in particular, are perceived as contributing significantly to dietary cholesterol butterfat, for exam:ple, 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.

.o The present invention is based on the recognition that o cholesterol can be separated from aqueous emulsions by precipitation as a complex with a cyclodextrin.

a a a 0This invention provides a method for removing cholesterol from a aqueous emulsions which comprises: 0 i) contacting such an emulsion with a cyclodextrin to form 0.0 a a cyclodextrin-cholesterol complex; o ii) causing the complex to precipitate by cooling; 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.

In instances where a material containing cholesterol is not in ~emulsion form it should be dispersed in a suitable liquid to -2form 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-treatment.

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 o< 60 0 C with an aqueous solution of B-cyclodextrin A cyclodextrin-cholesterol complex is formed which precipitates from solution on cooling to 200, preferably below 5 0

C.

The precipitate is removed, for example, by centrifugation.

O I 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 S, 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 I 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 3 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 preferably 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 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.

a; More specifically the method of the invention when applied to such egg components or products comprises steps such as the following: subjecting yolk, yolk plasma or egg white and yolk °mixture to a pretreatment procedure to improve the extent and rate of complexing, suitable procedures being i; dilution with water, dilution and dissolution with milk or salt solution, homogenisation, enzyme treatment, and addition of lipid or combinations of these steps; ii) treating the yolk preparation with cyclodextrin; iii) separating the precipitated cholesterol-cyclodextrin complex, typically by centrifugation.

-4 When the invention is applied to a yolk-albumen mixture it is preferred that the mixture be diluted to the extent of 40% by volume of milk prior to contact with cyclodextrin.

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 thereof can be S responsible for up to a three-fold increase in the amount of cholesterol removed from egg yolk on treatment with ij| 4 cyclodextrin.

.4, "C C Desirably the concentration of cyclodextrin use in treatments .i relating to eggs according to this invention should not be less than about 2% Cholesterol removal diminishes I rapidly when lower concentrations are used, on the other hand cholesterol removal may be substantially enhanced at higher concentrations.

In a preferred aspect of the invention the precipitated cyclodextrin-cholesterol complex may be subjected to a 4t regeneration step whereby the cholesterol is removed from the complex to provide free cyclodextrin. The cyclodextrin may be regenerated by extracting cholesterol with an appropriate solvent chosen from hexane, acetic acid or acetic acid/butanol mixture v/v).

MMMMM

4a Primarily, the term cyclodextri~nin tbi~s specification means B-cyclodextrin, but i t i S to b De uniderstood -to include acyclodextrin, cyclodextrins mnodC1i fi.ed(. to promote their cholesterol attracting .and r etaii. ng properties, and.

substituted cyclodextrins, which are solublIe or insoluble in water and are capable of forming a complex precipitate with cholesterol.

The invention will. now be described( wiLhi reference to the following examples: 000000 ,0 0 00 0 00 0 0 0 00 00 0 000 0 00 5 EXAMPLE 1 Extraction of Cholesterol from an Oil-in-Water Emulsion 14 Cholesterol, labelled with 14C was emulsified with oleic acid, monoolein and taurocholic acid in phosphate buffer (pH This emulsion was shaken with B-cyclodextrin in powder form W/v) at temperatures ranging from 200 to 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 STable 1).

TABLE 1 Removal of cholesterol from an emulsion by o o B-cyclodextrin 9o9 Activity of Labelled Cholesterol

(DPM)

o, Untreated Emulsion 7049 After exposure to the cyclodextrin 1095 EXAMPLE 2 Extraction of Cholesterol from Milk Milk was shaken with powdered B-cyclodextrin w/v) for one hour at 400C. On cooling in ice followed by centrifugation, the adsorbent was found to have removed 70% of the cholesterol from the fat (as shown in Table 2).

TABLE 2 Removal of cholesterol from milk by B-cyclodextrin.

I i 6 Concentration of Molar ratio cholesterol in of CP:CH* milk (mg/g fat) Untreated milk 3.20 After exposure to the cyclodextrin 0.96 Molar ratio of B-cyclodextrin to cholesterol EXAMPLE 3 Extraction of Cholesterol from Cream Cream containing 18% fat was shaken with powdered B-cyclodextrin w/v) for one hour at 4 0 C. 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).

TABLE 3 Removal of cholesterol from cream by B-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 EXAMPLE 4 Removal of cholesterol from egg yolk plasna The yolk from White Leghorn eggs was diluted 1:1 with isotonic saline (0.16 M NaCI) and the mixture centrigued at 20,000 rpm (50,000 g) for 30 min at 10 0 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

I

-~ii f 7 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 0 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 Concentration w/v) Cholesterol Concentration in Yolk Plasma (mg/ml) Molar Ratio

CD:CH

Cholesterol left removed I t I I *tr I lIt 1 1 I II: I I

III

Dilution 1+1 0 1 2 0 1.4 5.40 4.87 4.13 6.66 5.80 5.83 4.73 0.7 1.6 0.47 1.4 90.2 76.5 9.8 23.5 87.0 13.0 81.0 19.0 Dilution 1+3 2.62 1.62 4.2 61.5 38.5 it

II

EXAMPLE Removal of cholesterol from whole volk 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

I

8 Cyclodextrin Concentration w/v) Cholesterol Concentration in Yolk Plasma (mg/ml) Cholesterol left removed Dilution 1+2.25 5.25

I-

EXAMPLE 6 Removal of cholesterol from liquid whole egg 64 t Ir Ir

PI

4 46 64B 6 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 TABLE 6 TREATMENT OF WHOLE EGG PULP 64' Cyclodextrin Concentration w/v) Cholesterol Molar Concentration ratio in Yolk Plasma of (mg/ml)

CD:CH

Cholesterol left removed I II 6e 4 S I 666111 I I Australorp Dilution 1+3 1.13 Leghorn Dilution 1+3 1.54 Australorp Dilution 1+2 2.41 Leghorn Dilution 1+4 (NaC1) 6.1 5 (water) 4.4 14 86 (NaCI) 2.8 37 63 (NaC1) 1.40 4.9 11 89 I 9 -9- Labelled milk (LM) was prepared by evaporating 4 ml of a chloroform solution containing 0.25 \iCi 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 0 C for min. 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 4 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.

EXAMPLE 7 44 Labelled milk (LM) was mixed with B-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 Table demonstrates the improved efficiency at low temperatures.) TABLE 7 Removal of cholesterol from milk by B-cyclodextrin.

t I _i~ 10 Cyclodextrin w/v) Contact Temp Cholesterol time (min.)

DPM

Initial Final Molar Ratio removed of

CD:CH

2.0 2.0 1188 1188 1188 1188 1188 1188 1188 1952 1952 1952 1952 442 353 441 352 287 240 224 326 179 190 139 62.5 70.3 62.9 70.4 S(0 4 43 00O I 4~ 4 U 4r( 0 444 77.1 81.8 82.1 83.3 90.8 90.3 92.9 EXAMPLE 8 Labelled scrambled egg mix (SEM) was mixed with 4 1 4 11

SII

B-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 determined from the loss of radioactivity. The results are shown in Table 8.

TABLE 8 Removal of cholesterol from scrambled egg mix by B-cyclodextrin.

i 11 Cyclodextrin Contact Temp.

time C) DPM Cholesterol Molar Initial Final removed Ratio of 0

CD:CH

w/v) (min.) 719 719 719 719 719 719 719 719 92.9 95.5 96.5 95.6 95.5 94.4 93.3 93.3 4.3 4.3 4.3 4.3 4.3 4.3 4.3 4.3 0 0 a 00 Or I 00o 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 cooled to 0°C, centrifuged and the cholesterol removed by determined from the loss of radioactivity. The results are shown in Table 8.

TABLE 9. Removal of cholesterol from egg low density lipoprotein (6mg cholesterol/ml) by 8-cyclodextrin.

12 Cyclodextrin w/v) Contact Temp. DPM Molar Cholesterol time Initial Final Ratio removed (min.) of

CD:CH

rl t 4..

2.a 2.7 2.

2.7 2.a 2.7 2.7 2.7 2.7 3391 3391 3391 3433 3433 3433 1192 1192 1192 1192 1192 1192 1192 1192 2921 2893 2811 3083 2914 3000 427 321 433 280 359 363 280 280 0.85 0.85 0.85 0.85 0.85 0.85 4.6 4.6 4.6 4.6 4.6 4.6 4.6 4.6 14.0 15.0 17.0 10.0 15.0 13.0 64.2 73.1 63.7 76.5 69.1 69.5 76.5 76.5 LDL solution was sodium chloride diluted 3-fold with aqueous 0.5 M I 4 I 44t Reconstituted liquid whole egg products prepared by methods according to this invention could be used to substitute for conventional liquid or dried whole egg in various food applications. Eggs are an extremely nutritious food that are at present underused, partly because of fears about 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.

EXAMPLE Cream (201) containing 20% fat was treated with cyclodextrin w/v) to remove cholesterol. Butter was prepared from it

L__

13 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 TABLE 10. Removal of cholesterol from butter by B-cyclodextrin Cholesterol Content (mg/g fat) Cholesterol Removed 4r *r 4 *4 4r Untreated cream 3.2 Treated cream 0.42 *4 4 1 4*41 ItI I I Ie I 4 1 1 411 1

Claims

2. A method according to claim 1 wherein the molar ratio of cyclodextrin to cholesterol in the emulsion is at least
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 1 cyclodextrin. 4 S 5. A method according to claim 4 wherein at least 40% of the S cholesterol is removed from the emulsion. S6. A method according to any one of the preceding claims wherein the cyclodextrin is in solid form and is added to the emulsion.
7. A method according to any one of the preceding claims 0 wherein the complexing is carried out at a temperature in C the range 20°C to 600C.
8. A method according to any one of the preceding claims wherein cyclodextrin-complex is precipitated from solution by chilling below 5 0 C. 15
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. A method according to claim 9 wherein the cholesterol containing components of the emulsion are presented under conditions 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 or 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 I comprises at least one of the steps of dilution with water, dilution and dissolution with milk or 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 'i 4cyclodextrin. 4 A method according to any one of the preceding claims wherein the precipitated cyclodextrin-cholesterol complex is subjected to a regeneration step whereby the U t cholesterol is removed from the complex to provide free cyclodextrin. -I i Pi;: i"i:d r I i i 16
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 v/v).
17. Products which have been treated in accordance with the method of any one of claims 1 to 16.
18. A method according hereinbefore described. to claim 1 substantially Dated this day of August 1992 COMMONWEALTH ORGANISATION SCIENTIFIC AND INDUSTRIAL RESEARCH 4I a 02 a~ *La a a, 0 3 lia 4 *l 2 00 3 4 42 art ~.1 td§