WO2006011702A1

WO2006011702A1 - Cholesterol lowering supplement and low cholesterol egg produced by using the same

Info

Publication number: WO2006011702A1
Application number: PCT/KR2004/003247
Authority: WO
Inventors: Jeong Hak Kim; Hyeon Jin Kim; Seong Tshool Hong
Original assignee: Jinis Biopharmaceuticals Co
Current assignee: Jinis Biopharmaceuticals Co
Priority date: 2004-07-30
Filing date: 2004-12-10
Publication date: 2006-02-02
Anticipated expiration: 2007-01-30
Also published as: KR100637762B1; EP1778218A1; BRPI0418928A; JP2008508259A; EP1778218A4; US20090181150A1; KR20060011578A; CN1993122B; CN1993122A

Abstract

This invention provides compositions and methods for producing low cholesterol poultry eggs using hypocholesterolemic compounds, cholesterol lowering supplements, and feeds therefrom. Preferably, this invention provides microbial statins without methyl group in C6, as shown in formula V, in order to produce low cholesterol poultry eggs. The invention allows producing low cholesterol poultry eggs without concomitant reduction of egg production by providing microbial statins, compactin and pravastatin as well as their derivatives, as cholesterol lowering components. This invention also provides methods for producing low cholesterol poultry eggs without huge increase of production cost since the effective amount of cholesterol lowering composition containing microbial statin costs 1/120 - ¼ of that of synthetic statin.

Description

CHOLESTEROLLOWERINGSUPPLEMENTANDLOWCHOLESTEROL EGG PRODUCEDBYUSINGTHESAME

Technical Field

This invention relates to compositions and methods for producing

low cholesterol poultry eggs using hypocholesterolemic compounds,

cholesterol lowering supplements, and feeds therefrom.

Background Art

Cholesterol is a kind of lipid in every animal product. It is an

essential constituent of cell membranes in human and serves as a starting

material for the synthesis of important biological compounds such as steroid

hormones and bile acids. However, it is important to maintain the

suggested level of serum cholesterol since exceed amount of cholesterol

can be harmful.

Specifically, high serum cholesterol levels are termed

hypercholesterolemia (more than 200 mg/dL of blood cholesterol), which is

a common chronic disease found in 52% of adults in the world.

Hypercholesterolemia is a major risk factor for arteriosclerosis, which leads

inter alia to myocardial infarction, angina pectoris, hypertension, and stroke.

Currently, coronary artery disease is the leading cause of human mortality in

the United States and in many other developed countries although it was

the forth cause of death in 1900s. It is generally accepted that high levels of cholesterol in the human diet due to more frequent use of animal

products such as eggs, meats, and dairy products (milk and buter) can

result in a rise in serum cholesterol and thereby increases the risk of

cardiovascular diseases.

However, the consumption of cholesterol-rich animal products

increases every year and it seems very difficult to limit the intake of animal

products significantly. Therefore, research and development efforts have

been directed to low cholesterol foods that can provide both basic nutrition

and may prevent coronary heart disease.

Chicken eggs are an excellent foodstuff from a nutritional standpoint

due to their composition of high-quality protein, saturated fatty acids, mono-

and polyunsaturated fatty acids, minerals, and vitamins. However, in

addition to these essential dietary components, eggs contain about 200 mg

of cholesterol per egg and have been considered a major source of dietary

cholesterol. Because of recent understanding of the association between

total plasma cholesterol levels and the incidence of coronary heart disease

(CHD), it is surmised that an increased amount of dietary cholesterol may

increase risk of CHD (Weggemans et ai, 2001). Growing numbers of

health-conscious consumers exclude eggs from their diets in an effort to

limit daily cholesterol consumption to 300 mg/day as recommended by the

American Heart Association (National Institutes of Health Consensus

Development Panel, 1985). Hence there has been a steady decrease of

per capita egg consumption in developed countries. In the US, increasing public concern over dietary cholesterol is reflected in annual per capita egg

consumption, which has declined from 303 to 256 during the past 35 years

(US Department of Agriculture, 2002). Eggs with reduced cholesterol

content may be an attractive, highly nutritious food for health-conscious

consumers and a lucrative product for egg producers. This invention

provides compositions and methods for producing low cholesterol poultry

eggs using hypocholesterolemic compounds, cholesterol lowering feed

supplements, and feeds therefrom.

In previous art of U.S. Pat. No. 6,177,121 , it was shown that low

cholesterol eggs were produced by administration of purified lovastatin,

simvastatin, or atorvastatin (Elkin et al., J. Nutr. 1999:129:1010-1019, Elkin

et al., J. Agric. Food Chem. 2003:51 :3473-3481). Although the cholesterol-

lowering effect was found to be satisfactory when the egg-laying chickens

are fed 0.03~0.06% of simvastatin or atorvastatin for 5 weeks, use of these

methods is impractical due to the high price of effective amounts of statins,

and consequently unmarketable production cost of low cholesterol eggs. In

addition, oral administration of simvastatin and atorvastatin decrease egg

production, increasing production cost of low cholesterol eggs again.

In the same art, lovastatin was shown to reduce egg cholesterol

level ineffectively, showing only 3-6% reduction at its high dose. Therefore,

it is very difficult to commercialize low cholesterol eggs using lovastatin,

simvastatin, or atorvastatin as key components of cholesterol lowering feed

supplements. Statins are cholesterol lowering agents by inhibition of the

biosynthesis of cholesterol restraining 3-Hydroxy-3-MethylglutaryI coenzyme

A reductase(HMG-CoA reductase). Cholesterol is synthesized by multi-step

biosynthesis starting from acetyl-CoA in humans and livestock, warm¬

blooded animals. The key rate-limiting step in cholesterol biosynthesis is

to convert 3-hydroxy-3-methylglutaryl-coenzyme A to mevalonic acid by the

key enzyme known as 3-hydroxy-3-methylglutaryl-coenzyme A reductase

(HMG-CoA reductase, Formula 1).

Formula 1.

Acetyl CoA 3-Hydroxy-3~Methy!g)utaryl CoA

HMG-CoA reductase

MevaSonate

Cholesterol Statin inhibits HMG-CoA reductase due to its structural similarity to

mevalonic acid, a substrate of HMG-CoA reductase. Several statins have

been developed as cholesterol lowering agents for the treatment of

hypercholesterolemia. These include mevastatin (disclosed in U.S. Pat.

No. 3,983,140), lovastatin (disclosed in U.S. Pat. No. 4,231 ,938),

pravastatin (disclosed in U.S. Pat. No. 4,346,227), simvastatin (disclosed in

U.S. Pat. Nos. 4,444,784 and 4,450,171), fluvastatin (disclosed in U.S. Pat.

No. 4,739,073), atorvastatin (disclosed in U.S. Pat. No. 5,273,995), and

cerivastatin (disclosed in U.S. Pat. No. 5,502,199).

Among above statins, compactin (mevastatin), lovastatin, and

pravastatin are produced by microbial culture while other statins are

synthesized. Microbial statins are classified by R group at C[6] as compactin

(R:-H), lovastatin (R.-CH3), and pravastatin (R:-OH). Statins can be

lactone structure, acid forms (formula V) or salt.

(V)

Compactin, having a hydrogen atom in C6 in the formula V, has

been isolated in the culture medium of Penicillium (U.S. Pat. No.

3,983,140). Thereafter, several compactin-producing strains were reported

in various species, including P. citrinum, P. brevicompactum, P. cyclopium,

P. adametzioides, Trichoderma viridae, Aspergillus terreus, Gliocladium sp.

(U.S. Pat. No. 3,983,140; 4,049,495; 4,137,322; 5,691 ,173; Korean Pat. No.

832801; 832329; 10-0378640). Unfortunately, the development of

compactin as hypocholesterolemic drugs was discontinued after clinical

trials showing serious toxicity of compactin. Then, other compounds found

to be structurally related to compactin have been isolated and studied for

hypocholesterolemic activity. These derivatives are 3-hydroxy compactin, 6-

hydroxy compactin, 8a-hydroxy compactin, 4a, 5-dihydrocompactic acid, 5'-

phosphocompactic acid, and ML-236A (Chakravarti et al., 2004, Appl.

Microbial. Biotechnol. 64:618-624).

Pravastatin, having a hydroxyl group in C6 in the formula V, has

been shown to have hypocholesterolemic effect. Unlike compactin,

however, pravastatin is not toxic and has been developed as prescription

drugs for hypercholesterolemia patients. Currently, pharmaceutical

pravastatin is produced by enzymatic bioconversion process in which

hydrogen group in C6 of compactin is converted to hydroxyl group by

microorganisms (U.S. Pat No. 4,346,227). In previous arts, several species

in the genera of Streptomyces, carbophilus, S. roseochromogenus subsp.,

Nocardia and Actinomadura were reported to convert compactin to pravastatin by bioconversion process (U.S. Pat No. 5,942,423; 5,179,013;

4,537,859; 4,448,979; 4,346,227; Canadian Pat. No. 1 ,150,170; 1 ,186,647;

Korean Pat. No. 10-0414334; 10-0180706; Serizawa et al., 1983, J.

Antibiotics 36: 887-891).

Disclosure

Technical Problem

The invention provides economic and effective methods for

producing low cholesterol eggs, having lower than naturally-occurring levels

of cholesterol.

Technical Solution

The invention provides cholesterol lowering composition effective in

reducing the cholesterol content in poultry eggs with a little amount of

supplementation without reduction of egg production. The invention differs

with previous arts in which supplementation of atorvastatin or simvastatin

caused reduction of egg production which resulted in a rise of production

cost. Statins without methyl group in C6 of formula V, however, reduce egg

cholesterol at 1/10 to 1/2 amounts of atorvastatin, simvastatin, and

lovastatin for the same effects.

The invention provides poultry cholesterol lowering feed

supplements, cholesterol lowering feeds, and produced low cholesterol

eggs products therefrom as well as the methods. The present invention may be better understood with reference to

the accompanying examples that are intended for purposes of illustration

only and should not be construed to limit the scope of the invention, as

defined by the claims appended hereto.

Best Mode

As used herein, the following terms have the following meanings:

"Poultry" is intended to mean any domesticated poultry raised for

human consumption, including chicken, quail, duck, goose, ostrich and

turkey.

"Egg" is intended to mean any egg products for human consumption

from domesticated poultry, including chicken, quail, duck, goose, ostrich and

turkey.

"Low cholesterol egg products" is intended to encompass egg

products with reduced cholesterol content compared to eggs produced by

conventional husbandry methods.

This invention provides compositions for reducing the cholesterol

content in poultry eggs. In one aspect of the invention, the composition

comprises cholesterol lowering components selected from the group

consisting of: compactin and its derivatives of the formula I, compactin and

its derivatives of the formula II, a pharmaceutically acceptable salt of said

compactin and its derivatives, and an ester of said compactin and its derivatives. The compactin is characterized as statins having only hydrogen

groups at C6 of formula I or II.

( I )

(H)

Compactin is called as mevastatin or ML236B and includes lactone

structure (formula I), free acid structure (formula II), salt and esters

therefrom. The compactin derivatives are any statins with a hydrogen group

in C6 of formula V₁ including 3-hydroxy compactin, 6- hydroxy compactin,

8a-hydroxy compactin, 4a, 5-dihydrocompactic acid, 5'-phosphocompactic

acid, ML-236A. Compactin derivatives are also microbial inhibitors of cholesterol biosynthesis but are not limited thereto. Compactin producing

strains include Streptomyces roseochromogenus for 3-hydroxy compactin,

Mucor hiemalis for 6-hydroxy compactin, Schizophyllum commune for 8a-

hydroxy compactin, Penicillum citrinum for 4a, 5-dihydrocompactic acid,

Carcinella muscae for 5'-phosphocompactic acid, and Emericella unguis for

ML-236A.

In another embodiment of the invention, the composition for reducing

the cholesterol content in eggs, comprises cholesterol lowering components

selected from the group consisting of: pravastatin and its derivatives of the

formula III, pravastatin and its derivatives of the formula IV, a

pharmaceutically acceptable salt of said pravastatin and its derivatives and

an ester of said pravastatin and its derivatives. The pravastatin is

characterized as statins having a hydroxy! group at C6 of formula III or IV.

(in)

(IV)

Pravastatin, called as eptastatin, mezalotin, or pravachol, is

produced by bioconversion from microorganism. Pravastatin in this art

includes lactone structure (formula III), free acid structure (formula IV), salt

and esters therefrom. The pravastatin derivatives are any statins with a

hydroxy! group in C6 of formula V.

This invention also provides poultry feed supplements and feed

containing the said cholesterol lowering compositions.

This invention also provides a method of producing low cholesterol

eggs comprising administering to poultry a composition containing

cholesterol lowering components selected from the group consisting of:

compactin and its derivatives of the formula I₁ compactin and its derivatives

of the formula II, a pharmaceutically acceptable salt of said compactin and

its derivatives, and an ester of said compactin and its derivatives.

Preferably, poultry animals are fed cholesterol lowering supplements

of the invention, said compactin and its derivatives independently or by mixing with the conventional animal feed for the final amount of 0.0001 -

3% by weight. A preferred feeding schedule is to feed the animal at least

once a day with the supplemented feed for a period of more than 5 days

duration although the minimum duration and feeding amount to produce low

cholesterol eggs can be adjusted depending on the poultry species.

This invention also provides a method of producing low cholesterol

eggs comprising administering to poultry a composition containing

cholesterol lowering components selected from the group consisting of:

pravastatin and its derivatives of the formula III, pravastatin and its

derivatives of the formula IV, a pharmaceutically acceptable salt of said

pravastatin and its derivatives, and an ester of said pravastatin and its

derivatives.

Preferably, poultry animals are fed cholesterol lowering supplements

of the invention, pravastatin and its derivatives of the formula (III), formula

(IV), salt and ester form, independently or by mixing with the conventional

animal feed for the final amount of 0.0001 - 3% by weight.

This invention also provides derivatives of compactin and pravastatin

which can be any intermediates obtained during hypocholesterolemic

microbial culture and subsequent purification process of microbial statins.

Compactin, pravastatin, and their derivatives are not synthetic

statins but microbial statins which are products of microbial culture. Thus,

manufacturing of compactin and pravastatin for pharmaceutical purpose starts with the culture of microorganisms followed by purification of statins

from the culture media, containing several statin-derived intermediates.

Manufacturing of pravastatin, for example, starts with the culture of

C-6 hydroxylation microorganisms, such as Streptomyces. Next, compactin

was added into the Streptomyces culture to initiate bioconversion of

compactin to pravastatin via C-6 hydroxylation. After bioconversion reaction,

the culture broth was recovered by centrifugation of culture for column

chromatography to yield pure pravastatin. The culture broth for column

chromatography usually contains pravastatin as well as unused compactin

and other derivatives due to low conversion rate of about 40-70% and

incomplete hydroxylation. Streptomyces cell precipitates after removal of

broth also contains compactin, pravastatin, and other derivatives in small

amounts. In addition, prewashing solution, washing solution and pass-

through during column purification also contains compactin, pravastatin, and

other derivatives in small amounts due to incomplete recovery of statin.

These statin-containing intermediates from manufacturing process of

pharmaceutical drugs can be applied directly to animal although its use for

human is limited without further purification. Therefore, side products from

manufacturing pharmaceutical statins can be used as cholesterol lowering

supplements to produce low cholesterol eggs at economic price.

Preferably, these cholesterol lowering supplements can be supplied as solid

form after lyophilization. As used herein, "industrial intermediates or by-products" is intended

to encompass any products obtained from industrial microbial culture

containing hypocholesterolemic compounds, including every intermediates

from each production steps, such as microbial culture, statin-containing

broth, microbial cell precipitates, washed solution from preparative column,

solution from column elution, etc.

The present invention may be better understood with reference to

the accompanying examples that are intended for purposes of illustration

only and should not be construed to limit the scope of the invention, as

defined by the claims appended hereto.

Example 1

Low choSesterol eggs by compactin (1) Administration of cholesterol lowering supplements

Healthy ISA brown hens, 45-week old, were assigned randomly to

each dietary group (control group, 8 birds; experimental group, 6 birds each).

Each bird was placed in an individual cage in an environmentally controlled

room (25⁰C, 50% relative humidity, 16L8D). The hens were allowed for 2

weeks in which to adapt to the feed with no additives and the housing.

Control birds were fed a commercial diet based on corn and soybean meal

(Table 1), while birds in experimental groups were fed diets supplemented

with 0.003 or 0.03% of compactin (Sigma Aldrich Korea) for 6 weeks. Feed and water were provided ad libitum throughout the experiment. Feed

consumption, egg production and egg weight were recorded daily. Egg

production was expressed as percent hen day production; (100 x number of

eggs laid) / (number of hens x days). Table !

(2) Egg laying performance

The effects of 0.003% or 0.03% compactin on the laying performance

of 45-week old ISA brown hens were investigated (Table 2). Egg weight

was maintained at more than 60 grams after 6 weeks of oral administrations

of compactin at both doses. Feeding of 0.003% or 0.03% compactin did not

cause any difference in egg production compared to the control group

without administration of compactin. Maintenance of laying performance

with compactin feeding is of contrast to the previous art in which atorvastatin

reduced egg production down to 70% after feeding for 5-week. Table 2

(3) Egg cholesterol analysis

For egg cholesterol analysis, eggs from each bird were collected and

egg yolk was separated, weighed, and sampled for analysis. Lipids from

eggs were extracted by the method of Folch et al. (1957). Briefly, one

gram of sample was saponified with 3 ml_ of 33% KOH and incubated with

30 ml_ of 95% methanol in a 65⁰C water bath for 90 min. After

saponification, 10 mL hexane and 3 mL water were added, followed by

vigorous shaking for 10 min. 5α-cholestan (Sigma C-8300) was used as an

internal standard. Cholesterol content was determined by a gas

chromatography (Shimadzu, Japan) using column VB-1 (30 m x 0.25

mm x 0.25 μ m, VICI Inc.) with a split ratio of 100:1 and nitrogen as

carrier gas for a column flow rate of 0.54 mL/min. Injector, column, and

detector temperatures were 275⁰C, 290⁰C, and 340⁰C, respectively. Table

3 shows the effect of compactin on egg cholesterol. The average control

yolk weight was 17.2 g. Yolk weight showed a slight decrease with

compactin administration compared to the control. Administration of

compactin caused the significant reduction in yolk weight, compared to the

control with 12.8 mg cholesterol per gram of yolk. When expressed as total yolk cholesterol content, percentage changes compared to that of

control were 16% and 29% at 0.003% and 0.03% compactin, respectively.

It should be noted that low cholesterol egg can be produced by

administration of compactin less than 0.03%, indicating compactin is 2 or 3

times more effective than atorvastatin in producing low cholesterol eggs.

Table 3

Example 2

Low cholesterol eggs by pravastatin

Egg production and yolk cholesterol analysis was performed with

0.003 and 0.03% pravastatin administered group with the same procedure

as described in Example 1 above. Hen performance was investigated by

measuring egg weight and egg production as in Table 4. Pravastatin group

had maintained above 80% of egg production rate after 6-week

administration, same as control. This is in contrast with previous art in which

atorvastatin resulted in decrease of egg production rate more than 15%

compared to control. Table 4

Table 5 shows the effect of pravastatin on egg cholesterol.

Administration of pravastatin caused reduction in both yolk weight and

cholesterol concentration compared to control, resulting decrease of total

cholesterol content. Administration of 0.03% pravastatin produce eggs with

24% less cholesterol compared to control.

Table 5

Example 3

Low cholesterol eggs by industrial products containing statin.

Streptomyces cell precipitates and cell culture broth after compactin

bioconversion were used as cholesterol lowering supplements respectively.

First, compactin bioconversion was done as following. Single colony of

streptomyces carbophilus was inoculated into 100ml R2YE in 500ml erlenmyer flask and incubated at 27 ⁰C at 200 rpm for 3 days. After

incubation, 10 ml of seed culture was added to 100 ml of conversion media

(glucose 2.0%, corn steep liquor 0.2%, K₂HPO₄ 0.15%, yeast extract 0.1 %,

MgSO₄JH₂O 0.15%, ZnSO₄7H₂O 0.001%, NH₄NO₃ 0.1%, peptone 0.1%,

pH 7.0) in 500 ml flask and incubated at 27⁰C at 200 rpm for 3 days. Then,

filter sterilized compactin salt was added into culture upto 0.1% weight of

culture and incubated for another 4 days. After biovonversion reaction,

culture media was analyzed by TLC to confirm the presence of compactin

and pravastatin. This culture was freeze dried for 24 hours and then used as

cholesterol lowering supplements. Also Streptomyces cells were obtained

from bioconversion reaction by centrifugation at 3,000 X g. Streptomyces

cell precipitates recovered as pellets was dried at 60 ⁰C and used as

cholesterol lowering supplements. Streptomyces cell precipitates as well as

bioconversion reaction broth was dried and used as cholesterol lowering

supplements as shown in Example 1. Egg production and cholesterol

amount were measured afterward.

Egg production and yolk cholesterol analysis was performed with

the same procedure as described in Example 1 above except 0.5 and 1% of

Streptomyces cell precipitates and bioconversion reaction broth were

administered instead of compactin. Hen performance was investigated by

measuring egg weight and egg production as in Table 6. Both

experimental groups showed usual hen performance similar to control group

while egg production rate is marginally increased compared to control. Table 6

Table 7 shows the data of egg cholesterol analysis. Egg cholesterol

amount as well as yolk weight and cholesterol concentration were reduced

in correlation with the amounts of supplements compared to the control.

Administration of dried broth material resulted in reduction of egg

cholesterol about 17% and 24% in 0.5% and 1% supplement group,

respectively. Administration of dried cell material resulted in reduction of egg

cholesterol about 13% and 17% in 0.5% and 1% supplement group,

respectively.

Table 7

Industrial Applicability

This invention provides a method for producing low cholesterol poultry

eggs without concomitant reduction of egg production. This invention allows

minimal increase of production costs, as low as 1/20 - 1/4 of that of

synthetic statins by providing compactin, pravastatin and their derivatives.

The invention also allows producing low cholesterol poultry eggs by

providing industrial products from microbial culture of statin-producing

microorganisms as cholesterol lowering supplements. This invention allows

providing low cholesterol poultry eggs with economic price, contributing

prevention of hypercholesterolemia by decreasing consumption of dietary

cholesterol.

Claims

1. A composition for reducing the cholesterol content in poultry eggs,

comprising cholesterol lowering components selected from the group

consisting of:

a. compactin and its derivatives of the formula I

b. compactin and its derivatives of the formula Il

c. a pharmaceutically acceptable salt of said compactin and its

derivatives

d. an ester of said compactin and its derivatives ( I )

(π)

2. The composition of claim 1 ,

wherein the cholesterol lowering components are any by-products,

intermediates, and/or isolates from the industrial preparation of microbial

cultures, comprising compactin and its derivatives of formula I or II.

3. A composition for reducing the cholesterol content in poultry eggs,

comprising cholesterol lowering components selected from the group

consisting of:

a. pravastatin and its derivatives of the formula III

b. pravastatin and its derivatives of the formula IV

c. a pharmaceutically acceptable salt of said pravastatin and its

derivatives

d. an ester of said pravastatin and its derivatives

(Ef)

(IV)

4. The composition of claim 3,

wherein the cholesterol lowering components are any by-products,

intermediates, and/or isolates from the industrial preparation of microbial

cultures, comprising pravastatin and its derivatives of formula III or IV.

5. Poultry feed supplements, comprising the composition of claim 1 or 2.

6. A poultry feed, comprising an oviparous vertebrate animal food and

the cholesterol lowering composition of claim 1 or 2.

7. Poultry feed supplements, comprising the composition of claim 3 or 4.

8. A poultry feed, comprising an oviparous vertebrate animal food and

the cholesterol lowering composition of claim 3 or 4.

9. A method of producing low cholesterol eggs comprising

administering to poultry a composition containing cholesterol lowering

components selected from the group consisting of:

a. compactin and its derivatives of the formula I

b. compactin and its derivatives of the formula Il c. a pharmaceutically acceptable salt of said compactin and its

derivatives

d. an ester of said compactin and its derivatives

( I )

( II )

10. The method of claim 9,

wherein the cholesterol lowering components are by-products,

intermediates, and/or isolates from the industrial preparation of microbial

cultures, comprising compactin and its derivatives of formula I or II.

11. The method of claim 9, wherein poultry feed is supplemented with

from 0.0001 - 3% by weight of the compactin and its derivatives and the

poultry is fed the feed at least once per day for more than 5 days.

12. Low cholesterol eggs produced in accordance with the method of

claim 9, 10 or 11.

13. A method of producing low cholesterol eggs comprising

administering to poultry a composition containing cholesterol lowering

components selected from the group consisting of:

a. pravastatin and its derivatives of the formula III

b. pravastatin and its derivatives of the formula IV

c. a pharmaceutically acceptable salt of said pravastatin and its

derivatives

d. an ester of said pravastatin and its derivatives

(IE)

(IV)

14. The method of claim 13,

wherein the cholesterol lowering components are by-products,

intermediates, and/or isolates from the industrial preparation of microbial

cultures, comprising pravastatin and its derivatives of formula III or IV.

15. The method of claim 13, wherein poultry feed is supplemented with

from 0.0001 - 3% by weight of the pravastatin and its derivatives and the

poultry is fed the feed at least once per day for more than 5 days.

16. Low cholesterol eggs produced in accordance with the method of

claim 13, 14 or 15.