U.S. patent application number 11/070208 was filed with the patent office on 2005-07-07 for stanol composition and the use thereof.
This patent application is currently assigned to Raisio Benecol Ltd.. Invention is credited to Gylling, Helena, Miettinen, Tatu, Palmu, Tapio, Wester, Ingmar.
Application Number | 20050147729 11/070208 |
Document ID | / |
Family ID | 26160196 |
Filed Date | 2005-07-07 |
United States Patent
Application |
20050147729 |
Kind Code |
A1 |
Wester, Ingmar ; et
al. |
July 7, 2005 |
Stanol composition and the use thereof
Abstract
A stanol composition containing in addition to sitostanol as the
main component, also a substantial amount of at least 10%
campestanol has been found to effectively lower serum cholesterol
levels when incorporated in edible commodities. Upon esterification
the composition is especially useful in edible fats and oils and in
fat-containing foods.
Inventors: |
Wester, Ingmar; (Raisio,
FI) ; Palmu, Tapio; (Raisio, FI) ; Miettinen,
Tatu; (Espoo, FI) ; Gylling, Helena;
(Helsinki, FI) |
Correspondence
Address: |
ARENT FOX PLLC
1050 CONNECTICUT AVENUE, N.W.
SUITE 400
WASHINGTON
DC
20036
US
|
Assignee: |
Raisio Benecol Ltd.
|
Family ID: |
26160196 |
Appl. No.: |
11/070208 |
Filed: |
March 3, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11070208 |
Mar 3, 2005 |
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10223633 |
Aug 20, 2002 |
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10223633 |
Aug 20, 2002 |
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09051080 |
Jul 15, 1998 |
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Current U.S.
Class: |
426/611 |
Current CPC
Class: |
A61K 31/575 20130101;
A61P 3/00 20180101; A23D 7/013 20130101; A61K 31/575 20130101; A61K
2300/00 20130101; A23L 33/11 20160801 |
Class at
Publication: |
426/611 |
International
Class: |
A23D 007/00 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 9, 1996 |
FI |
963126 |
Claims
1. A composition, comprising a stanol mixture comprising a
sitostanol component selected from the group consisting of
sitostanol, a sitostanol fatty acid ester, and a combination
thereof; and a campestanol component comprising a campestanol fatty
acid ester, wherein the campestanol fatty acid ester is present in
an amount of 20 to 40 weight % of the stanol mixture, calculated as
free stanols.
2. The composition of claim 1, wherein the sitostanol fatty acid
ester is present in the stanol mixture.
3. The composition of claim 2, wherein the campestanol fatty acid
ester is present in an amount of 25 to 35 weight % of the stanol
mixture.
4. The composition of claim 2, wherein the campestanol fatty acid
ester is present in an amount of about 30 weight % of the stanol
mixture.
5. The composition of claim 1, wherein the stanol mixture is
derived from plant sterols.
6. The composition of claim 5, wherein the stanol mixture is
derived from vegetable oil sterols.
7. The composition of claim 5, wherein the stanol mixture is
derived from corn, soybean or rapeseed sterols.
8. A food composition, comprising a nutritional substance and a
blood cholesterol lowering effective amount of the stanol mixture
of claim 1.
9. The food composition of claim 8, wherein the nutritional
substance is selected from the group consisting of cooking oil,
margarine, butter, mayonnaise, salad dressing, shortening and
cheese.
10. A food composition, comprising a nutritional substance and a
blood serum cholesterol lowering effective amount of the stanol
mixture of claim 2.
11. The food composition of claim 10, wherein the nutritional
substance is selected from the group consisting of cooking oil,
margarine, butter, mayonnaise, salad dressing, shortening and
cheese.
12. The food composition of claim 10, wherein the campestanol fatty
acid ester is present in an amount of 25 to 35 weight % of the
stanol mixture.
13. The food composition of claim 10, wherein the campestanol fatty
acid ester is present in an amount of about 30 weight % of the
stanol mixture.
14. A method for lowering the cholesterol level in blood serum of a
human subject, comprising administering to the subject a blood
serum cholesterol level reducing effective amount of the stanol
mixture of claim 1.
15. A method for lowering the cholesterol level in blood serum of a
human subject, comprising administering to the subject a blood
serum cholesterol level reducing effective amount of the stanol
mixture of claim 2.
16. The method of claim 15, wherein the stanol mixture is
administered orally.
17. The method of claim 15, wherein the stanol mixture is
administered as a component of a food composition.
18. The method of claim 17, wherein the food composition comprises
a nutritional substance selected from the group consisting of
cooking oil, margarine, butter, mayonnaise, salad dressing,
shortening and cheese.
19. The method of claim 15, wherein the stanol mixture is
administered at a daily dose of about 3 grams per day.
20. The composition of claim 1, wherein the sitostanol fatty acid
ester, if present, and the campestanol fatty acid ester are
synthetically produced.
21. The composition of claim 1, wherein the sitostanol fatty acid
ester, if present, and the campestanol fatty acid ester are
prepared by a method comprising the step of esterifying free
sitostanol, free campestanol, or both free sitostanol and free
campestanol.
22. A food composition, comprising a nutritional substance and a
blood serum cholesterol lowering effective amount of the stanol
mixture of claim 3.
23. The composition of claim 22, wherein the stanol mixture is
administered as a component of a food composition.
24. A food composition, comprising a nutritional substance and a
blood serum cholesterol lowering effective amount of the stanol
mixture of claim 21.
25. The method of claim 14, wherein the stanol mixture is
administered orally.
26. The method of claim 14, wherein the stanol mixture is
administered as a component of a food composition.
27. A food composition, comprising a nutritional substance and a
blood serum cholesterol lowering effective amount of the stanol
mixture of claim 6.
28. A method for lowering the cholesterol level in blood serum of a
human subject, comprising administering to the subject a blood
serum cholesterol level reducing effective amount of the stanol
mixture of claim 6.
29. The method of claim 28, wherein the stanol mixture is
administered orally.
30. The method of claim 28, wherein the stanol mixture is
administered as a component of a food composition.
31. A method for reducing the absorption of cholesterol from the
intestines into the bloodstream of a human subject, comprising
orally administering to the subject a cholesterol absorption
reducing effective amount of the stanol mixture of claim 1.
32. The method of claim 31, wherein the stanol mixture is
administered as a component of a food composition.
33. A method for reducing the absorption of cholesterol from the
intestines into the bloodstream of a human subject, comprising
orally administering to the subject a cholesterol absorption
reducing effective amount of the stanol mixture of claim 2.
34. The method of claim 33, wherein the stanol mixture is
administered as a component of a food composition.
35. A method for reducing the absorption of cholesterol from the
intestines into the bloodstream of a human subject, comprising
orally administering to the subject a cholesterol absorption
reducing effective amount of the stanol mixture of claim 6.
36. The method of claim 35, wherein the stanol mixture is
administered as a component of a food composition.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to a sitostanol containing
composition of plant stanols especially for use as a serum
cholesterol level lowering substance. The invention also relates to
the corresponding esterified form of such a composition which
advantagously can be used in edible oils and fats and in
fat-containing foods.
BACKGROUND OF THE INVENTION
[0002] Plant sterols are essential components of all plants. Their
functions in plants resemble the functions of cholesterol in
mammals. The most abundant plant sterols in the flora are
.beta.-sitosterol, campesterol and stigmasterol. The chemical
structure of these plant sterols is very similar to that of
cholesterol the differences occuring in the side chain of the
backbone of the molecule. For example, compared to cholesterol, the
side chain of sitosterol contains an additional ethyl group and the
side chain of campesterol an additional methyl group.
[0003] Since 1950's plant sterols have been known to effectively
reduce the serum cholesterol levels. Even when administered in
relatively small doses (a few grams a day) they reduce the
absorbability of both biliary and dietary cholesterol effectively
and thus lower the serum total and LDL-cholesterol levels (12, 28,
see also 27, 32). The mechanism by which the restriction of
cholesterol absorption happens is still not known in detail, but it
is assumed that plant sterols displace cholesterol from the
micellar phase and thereby prevent its absorption. In practically
all of the early studies, sitosterol or its hydrogenated form
sitostanol has been the main plant sterol of interest. However, the
sterol composition of the tested preparations has not always been
well documented, and the sterol preparations used in most studies
have also contained different amounts of other sterols.
[0004] Plant sterols have been considered as a safe way of lowering
serum cholesterol levels, since they are natural components of
vegetable fats and oils. Additionally, their absorption from the
intestine of healthy subjects is limited, and the limited amounts
absorbed are excreted from the body in the bile. The absorbtion
rate of the plant sterols varies between individuals and between
the different plant sterols, but for healthy humans usually less
than 5% of the plant sterols are absorbed from the digestive tract
(27). However, up to 10% of dietary campesterol has been shown to
be absorbed (20).
[0005] In few rare diseases such as sitosterolemia plant sterols
are absorbed exceptionally efficiently, and also the elimination
from the body via the biliary route is impaired. Serum levels of
sitosterol, campesterol and also their saturated forms sitostanol
and campestanol are highly elevated. The elevated levels of the
saturated stanols are most probably due to their more effective
endogenous synthesis rather than a more effective absorption (10,
27). If untreated, sitosterolemia leads already at young age to
xanthomatosis and coronary heart disease. For people with this
disease, an administration of unsaturated plant sterols in amounts
greater than normally present in foods may lead to hazardous health
effects.
[0006] Lees and Lees (25) tested the effects of three different
sitosterol preparations on plasma lipid and lipoprotein
concentrations. One of the preparations was Cytellin, a commercial
preparation (Eli Lilly Co., USA) that contained 60-65% sitosterol
and 3540% other sterols, mainly campesterol. An average dose of 18
g/day divided in three doses resulted in a 10.5% average fall in
plasma total cholesterol and a 15% fall in LDL-cholesterol.
However, when only traces of plant sterols including campesterol
are normally detected in plasma (10, 33), the plasma concentration
of campesterols varied from 4 to 21 mg/dl in the subjects tested by
Lees and Lees (25). In the discussion the authors stated very
strongly that since the atherogenicity of campesterol is unknown,
the use of a sitosterol preparation with a relatively high
campesterol content like the Cytellin preparation used in their
study cannot be recommended.
[0007] Further, Lees et al. (26) studied the efficacy of plant
sterols from soybean oil and tall oil in lowering the blood
cholesterol level. They used two different physical forms of each
plant sterol, namely a suspension and a powder. The soy sterol
consisted of 60-65% sitosterol and 35% campesterol, and a daily
dose of an average 18 g of sterols per day (range 9-24 g) was given
in three equal doses. A tall oil sterol preparation with only about
5% campesterol was used in this study. A daily dose of 3 grams of
both tall oil sterol preparations (powder and suspension) was
tested. Additionally, a dose of 6 grams of the tall oil sterol
suspension was tested.
[0008] Soy sterol in both physical forms and tall oil sterol in
powder form reduced the plasma cholesterol content by on average
12% (26). However, the relatively high absorbability of campesterol
that has already been shown earlier, was observed also in this
study. In the 5 patients tested the plasma campesterol levels
ranged from 5 to 21 mg/dl (mean 16 mg/dl). Thus again, even if the
cholesterol-lowering effect of soy sterol was proved to be
significant, the authors did not recommend its use as a
cholesterol-lowering agent. On the contrary, they recommended that
pharmaceutical plant sterol preparations should contain a minimum
of campesterol and a maximum of sitosterol. Based on the two
studies cited above, it can be concluded that the use of vegetable
oil based sterols such as soy sterol are strongly not
recommendable.
[0009] Saturated plant sterols such as sitostanol and campestanol
are present in most vegetable oils only in trace amounts. However,
tall oil sterols contain 10-15% of sitostanol, the saturated form
of sitosterol. Sitostanol can also be made by hydrogenation of the
double bond in sitosterol. In the latest studies made with both
experimental animals and humans, sitostanol has been proven to be
more effective as a cholesterol-lowering agent than sitosterol (8,
16, 17, 18, 19, 36).
[0010] An additional advantage of sitostanol is that it is
virtually unabsorbable. Several studies (e.g. 9, 16, 17, 21) have
shown that sitostanol is practically unabsorbable while small
amounts (<5%) of its unsaturated form sitosterol (33) can be
absorbed. Similarily, in an in vitro study Amstrong and Carey (6)
also showed that cholestanol, a saturated form of cholesterol, was
more hydrophobic and less absorbable than cholesterol.
[0011] When sitostanol is made by hydrogenation of the most usual
plant sterol sources, also another saturated plant sterol, namely
campestanol, is formed from campesterol. Until recently, relatively
little has been known about the absorbability and the possible
hypocholesterolemic effect of this stanol. Based on the data cited
above stating that saturated sterols are less absorbable than their
unsaturated forms, it could be hypothesized that campestanol might
be virtually unabsorbable.
[0012] To study the absorbability of different plant sterols
Heinemann et al. (20) compared the intestinal absorption of
cholesterol with campesterol, sitosterol, stigmasterol and also low
concentrations of sitostanol and campestanol in humans by means of
intestinal perfusion technique. The results showed that the
absorption rate of the differed plant sterols varied between
different plant sterols being on average 4.2% for sitosterol, 4.8%
for stigmasterol, 9.6% for campesterol and 12.5% for campestanol.
Large variation between the absorption efficacy in the ten male
subjects was detected.
[0013] Thus, according to Heinemann et al. (20) campestanol was
found to be more efficiently absorbed than its unsaturated form
campesterol. This is against the assumption based on studies cited
earlier that showed that the saturated sterols (sitostanol,
cholestanol) would be less absorbable than the unsaturated ones
(sitosterol, cholesterol). The reason for this remains unclarified.
Heinemann et al. (20) speculated, though, that the reason for this
conflicting result might be that the study of Amstrong and Carey
(6) was made with in vitro conditions and that the theory of the
hydrophobicity being a major factor in micellar binding and/or
absorption might not be relevant in in vivo conditions. However,
this speculation does not explain the fact that several studies
that have shown the poorer absorbability of sitostanol compared to
that of sitosterol have been made under in vivo conditions. Thus
the results of Heinemann et al. (20) that conflict with previous
results remained unexplained by the authors.
[0014] Sugano et al. (34) studied the hypocholesterolemic activity
of corn sterols (composition: 31% campesterol, 4% stigmasterol and
65% sitosterol) and corn stanols (composition: 31% campestanol and
69% sitostanol) obtained by hydrogenation of a corn oil sterol
mixture. Two experiments were carried out in rats. Both the sterol
and the stanol showed hypocholesterolemic effects at the level of
0.5-1% of the diet when cholesterol (1% in the diet) was ingested.
In the first experiment no significant difference was sen in the
hypocholesterolemic effect of phytosterols and phytostanols.
However, in the second experiment, at the same dietary levels the
phytostanols showed considerably greater ability to lower the
plasma cholesterol concentration tham did the phytosterols
(statistically significant at p<0.02). Moreover, rats fed the
1.0% stanol diet had plasma cholesterol levels significantly lower
(p<0,02) than that of the animals fed the diet free of
cholesterol. This was not observed in rats fed the 1.0% sterol
diet.
[0015] Sugano et al. (34) did not study the difference in
hypocholesterolemic effect between stanol mixtures with a high
content of sitostanol and a low content of campestanol (tall oil
sterol based) and stanol mixtures with a substantially higher level
of campestanol (vegetable oil sterol based). They compared the
hypocholesterolemic effect of an unsaturated sterol mixture with
the corresponding saturated stanol mixture. Later studies made by
this research group have been focused on the cholesterol lowering
effect of sitostanol specificly and compared to sitosterol (21, 22,
23, 35). In fact, in a later publication (23) they refer to the
phytostanol study mentioned above (34) mentioning only the
hypocholesterolemic effect of .beta.-sitostanol compared to
.beta.-sitosterol without discussing any hypocholesterolemic effect
of saturated sterols (including campestanol) compared to
unsaturated sterols. In the later studies mentioned above sterol
mixtures with the typical composition of hydrogenated tall oil
sterols with a high content of sitostanol (>90%) have been
used.
[0016] Miettinen and Vanhanen (30) have shown that sitostanol in
fatty acid ester form is more effective than free sitostanol in
lowering serum cholesterol levels. Later studies have also shown
that the use of sitostanol esters as a part of a daily diet is an
effective way of reducing serum total and LDL-cholesterol
concentrations (13, 14, 15, 31, 37, 38). The benefit of using
stanol esters instead of free stanol is also that the stanol esters
are fat-soluble and can therefore easily be incorporated into a
wide variety of foods without changing the taste, flavor or
physical behavior of the final product. The method for the
preparation of sitostanol fatty acid esters and the use of
fat-soluble stanol esters in foods have been disclosed in U.S. Pat.
No. 5,502,045 (2), hereby incorporated by reference. Straub (3)
suggests the use of saturated stanols (sitostanol, clionastanol,
22,23-dihydrobrassicastanol, campestanol and mixtures thereof in a
method for making a food additive composition where stanols are
mixed with an edible solubility agent, an effective amount of a
suitable antioxidant and an effective amount of a suitable
dispersant. These food additives are intended to reduce cholesterol
absorption from foods and beverages which contain cholesterol, e.g.
meat, eggs and dairy products. However, in this patent no data
showing either any clinical effects or the absorbtion of dietary
sterols is presented.
[0017] Eugster et al. (1) teach the use of small amounts of
sterols, their fatty acid esters and glucosides for the treatment
of tumors. The methods of preparation proposed by Eugster et al.
involve hazardous chemical reagents like N,N'-carbonyl-diimidazole,
thionyl chloride and solvents like tetrahydrofuran, benzen,
chloroform or dimethylformamide. Eugster et al. comment on the
possible use of these substances as dietary foods and as food
additives, but do not present any data on hypocholesterolemic
effects or make any claims covering such use. From the disclosure
of Eugster et al. it is hard to get a clear picture of how the end
product is purified to yield a pure enough sterol ester in large
amounts enough to be used as a food component. The only purifying
processes referred to are thin layer chromatography and high
performance liquid chromathoghaphy. This being the case, the
preparation method referred to in the patent by Eugster et al. is
limited to small amounts only.
[0018] The U.S. Pat. No. 3,751,569 (4) discloses the addition of
plant sterol fatty acid esters to cooking oil with the objective of
lowering the serum cholesterol levels in man. The patent proposes,
for use in the esterification of free sterols, a method which in no
case fullfills the requirement for preparation of a food-grade
product. According to the patent, the esterification is carried out
between a free sterol and a fatty acid anhydride, with perchloric
acid acting as a catalyst. The catalyst and reagent used cannot be
accepted in food processes. In addition, the patent relates to the
fatty acid esters of only native plant sterols. The method proposed
in the German patent DE 22 48 921 (5) for the esterification of
sterols present in oils and fats by a chemical interesterification
technique fullfills the criteria of food processes. In this patent,
free sterol and an excess of fatty acid esters are added to a
mixture of oil or fat, whereafter the entire fat blend is
interesterified by a commonly known interesterification technique.
In the resulting fat blend virtually all free sterols have been
converted to fatty acid esters. The purpose of this is to protect
free sterols in vegetable and animal oils against possible changes
during processing.
[0019] Earlier data shows that campesterol, one of the major plant
sterols, is absorbed relatively efficiently. Therefore it has been
recommended that only plant sterol mixtures with a minimum content
of campesterol should be used. This has in practice lead to the use
of sterol mixtures such as tall oil sterols with a high content of
sitosterol.
[0020] Most work on stanols has covered sitostanol only. The study
of Heinemann et al. (20) showing that campestanol, the saturated
form of campesterol, is more readily absorbed than campesterol or
sitosterol (12.5%, 9.6% and 4.2% respectively) has lead to a
"consensus" that saturated sterol mixtures with "elevated" levels
of campestanol are unsafe due to the absorption of campestanol. A
clear evidence of this is that all clinical studies covering the
use of stanols (sitostanol) have been based on sterol mixtures with
a high level of sitostanol and a low level of campestanol.
[0021] It is an established fact from many studies (e.g. 8, 17, 18,
19, 23, 36), that sitostanol, the saturated form of sitosterol, is
more effective than the corresponding unsaturated sitosterol in
reducing the blood cholesterol level. Furthermore saturated sterols
are absorbed in very limited amounts, which make the use of
saturated sterols a safe mean of reducing cholesterol on a
population bases. Of the unsaturated sterols especially campesterol
is absorbed in amounts high enough to call for strong
recommendations against the use of sterol mixtures with eleveted
levels of campesterol (eg. vegetable oil based sterol mixtures)
(25, 26).
[0022] Accordingly there has been a strong prejudice against using
campestanol in any substantial amounts as a substance to be added
to foods and this has seriously limited the spectrum of phytosterol
containing raw materials to such containing a relatively minor
amount of campesterol and its saturated form, campestanol.
BRIEF DESCRIPTION OF THE INVENTION
[0023] This invention relates to plant stanol compositions
containing sitostanol as a main component but with substantial
amounts of campestanol, either in free form or esterified as fatty
acid esters for lowering the level of blood serum cholesterol.
[0024] The invention further relates to the use of stanol
compositions containing sitostanol as the main component but also
substantial amounts of campestanol, or fatty acid esters thereof in
edible commodities as a dietary component for lowering blood serum
cholesterol levels.
[0025] The object of the present invention is to broaden the
spectrum of plant raw materials useful in the preparation of
substances for edible commodities, especially edible oils and fats
and fat-containing foods intended to control cholesterol levels in
blood serum. The invention enables using as raw materials for these
purposes plant oils and fats containing in addition to sitosterol
also a substantial amount of campesterol.
[0026] Suitable raw materials for use in the preparation of the
compositions of the present invention are e.g. corn, soybean and
rapeseed but also other plants with a phytosterol composition high
in campesterol may be used.
[0027] The novel composition of the present invention, and
especially its esterified form, may be incorporated in food
substances such as cooking oils, margarines, butter, mayonnaise,
salad dressings, shortenings, cheeses (including unripened and
ripened cheeses) and other fat-containing foods.
[0028] The composition of the present invention can also be
consumed as such.
DETAILED DESCRIPTION OF THE INVENTION
[0029] According to the present invention, the plant stanol
composition contains, in addition to its main component,
sitostanol, also a substantial amount of at least 10%
campestanol.
[0030] The compostion preferably contains as much as from 20% to
40% and most preferably from 25% to 35%, e.g. about 30% campestanol
or its fatty acid ester when the composition has been esterified to
make it lipophilic.
[0031] Throughout this specification all percentages are given by
weight, unless otherwise specified. In this specification the
bracketed numbers refer to publications listed in the appended List
of References.
[0032] Data obtained surprisingly and against prevailing prejudice
shows that a hydrogenated stanol mixture containing sitostanol as
the main component but with substantial amounts of campestanol is
at least as effective as a stanol mixture containing over 90%
sitostanol and a low level of campestanol, indicating that
campestanol is at least as effective in reducing the absorption of
cholesterol as sitostanol. Moreover, data from sterol analysis of
blood serum clearly shows that campestanol remains virtually
unabsorbed, with blood serum contents being about 40% smaller than
that of sitostanol. Thus a stanol mixture containing sito-stanol as
a major component but with substantial amounts of campestanol must
be regarded as at least as safe as a conventional tall sterol based
stanol mixture. This data is in striking contrast to current
opinion regarding the efficacy and safety of stanol mixtures with
elevated amounts of campestanol (see 20, 27, 34).
[0033] The U.S. Pat. No. 5,502,045 (2) showed that fatty acid
esters of sitostanol are more effective in reducing the blood
cholesterol level than the free sitostanol. Later studies have
clearly confirmed the cholesterol lowering effect of a margarine
containing fat soluble sitostanol fatty acid esters (e.g. 31).
[0034] The use of stanol fatty acid esters instead of free stanols
is crucial for a broad use of these in various fat containing food
products because only the stanol fatty acid esters are soluble in
edible oils and fats in amounts high enough to reach levels
effective in reducing the absorption of both dietary and biliary
cholesterol from the digestive tract.
[0035] The solubility of stanol esters in edible oils and fats is
as high as 35-40%, whereas the solubility of free sterols in edible
oils and fats is limited to a maximum of 2 per cent by weight only
at the temperature of 21.degree. C. (24). Higher amounts could be
incorporated by using different surfactancts, solubilizing or
dispersing agents, but even the use of these substances does not
ensure fat solubility. The use of the above substances is usually
restricted or even prohibited by law. Furthermore free sterols at a
level of 1% will affect the physical properties of the fat or oil,
causing changes in the structure and physical behaviour of the
product. This is not the case when stanol fatty acid esters are
used since the physical properties of the fat mixture can easily be
modified by altering the fatty acid composition of the mixture.
[0036] It is obvious that stanol fatty acid esters easily can be
incorporated to other foods than margarines and spreads as
described in this invention. The U.S. Pat. No. 5,502,045 (2) gives
further examples of possible use. It is, however, obvious to those
skilled in the art that stanol fatty acid esters can be added to a
wide variety of foods, especially fat-containing foods.
[0037] Many methods for preparing fatty acid esters of sterols have
been proposed. The drawbacks of these methods are that almost all
of them use reagents, which cannot be accepted in the production of
a product intended to be used as a macronutrient in foods. The use
of toxic reagents like thionyl chloride or anhydride derivatives of
fatty acids is common.
[0038] The preferred method of preparing stanol fatty acid esters
of sterols is described in the U.S. Pat. No. 5,502,045 (2, hereby
incorporated by reference). This procedure is based on the
interesterification process used widely by the edible fat and oil
industry. This esterification process deviates advantageously from
previous methods in that no other substances than the free stanol,
a fatty acid ester or a fatty acid ester mixture and a
interesterification catalyst like sodium ethylate are used. One
important feature of the method is that one of the reactants, the
fatty acid ester is used in excess and functions as a solvent,
solubilizing the stanol under the conditions used (vacuum 5-15
mmHg). The reaction gives a mixture of fatty acid esters and stanol
fatty acid esters. The stanol fatty acid ester can easily be
concentrated into almost pure stanol fatty acid esters by vacuum
destillation, which removes the excess of fatty acid esters.
Alternatively the blend can be added as such to the final fat blend
before the deodorizing step is carried out.
[0039] Stanols are found in small amounts in nature eg. in wheat,
rye, corn and tritricale and can thus be found in small amounts
(11, 14) in the daily food. Stanols can easily be produced by
hydrogenation of natural sterol mixtures. Only tall sterol mixtures
with high enough purity (sterol content >98%) to be used as such
for food use were commercially available in early 1996. Plant
sterols with substantial amounts of campesterol such as vegetable
oil based sterol mixtures can e.g. be obtained as a by-product of
tocopherol production from vegetable oil distillates. The obtained
plant sterols can be converted into stanols by prior known
hydrogenation techniques such as that based on the use of Pd/C
catalyst in organic solvents (7, hereby incorporated by reference).
It is obvious for those skilled in the art that a wide variety of
Pd catalysts and solvents can be used to carry out the
hydrogenation, which when done under optimized conditions leaves
only small amounts of unsaturated sterols unconverted while the
formation of the typical dehydroxylated by-products stanes and
stenes remains at a low level (<1.5%).
[0040] The instant invention compares the hypocholestelolemic
effect of a stanol mixture containing a high level of sitostanol
that is generally regarded by experts in the field to be the safest
and most effective plant sterol in reducing cholesterol absorption
and thereby serum cholesterol levels with a stanol mixture
containing a substantial amount of campestanol. In this
specification, for the first time, hypocholesterolemic effects of
vegetable oil based stanols in humans have been reported. This
invention is the first to show that a stanol mixture with a
substantial amount of campestanol (over 10% and preferably about
30%) is at least as effective as stanol mixtures with high levels
of sitostanol. Furthermore, the results of the present study
clearly indicate that campestanol on the contrary to what has been
reported by Heinemann et al. (20) is virtually unabsorbed.
[0041] Clinical Studies
[0042] To study the hypocholesterolemic effects of vegetable oil
stanol ester and tall oil stanol ester margarines a 5-week double
blind cross over study with a 2 weeks wash-out period was designed.
The test arrangement of the study was as follows:
[0043] Test Arrangement of the Intervention Study.
[0044] Numbers 1-6 indicate the blood samples collected at the home
diet (1, 2), after the first intervention period (3, 4) and after
the second intervention period (5, 6). VS=vegetable oil based
stanol ester margarine, TS=tall oil based stanol ester
margarine.
1 1
[0045] Twenty-four voluntary, free-living, healthy women with a
moderately elevated cholesterol level (average 6.12.+-.0.16 mmol/l)
consumed about 25 g per day (a 250 g tub/10 days) of the test
margarines as a part of the daily diet in a random order. Serum
lipids (total cholesterol, LDL-cholesterol, HDL-cholesterol and
tri-glycerides) and serum sterol contents were measured at the home
diet and at the end of each test period. Blood samples were taken
twice, one week apart at the home diet and by the end of each test
margarine perios. The obtained serum lipid values are shown in
Table 1 below.
2TABLE 1 Serum lipid concentrations (mmol/l, mean .+-. SE) during
the home diet and after the five-week treatment with vegetable oil
stanol ester margarine (VS) and tall oil stanol ester margarine
(TS), (n = 24). Home diet VS TS Total cholesterol 6.12 .+-. 0.16
5.77 .+-. 0.18* 5.95 .+-. 0.23 LDL-cholesterol 4.03 .+-. 0.15 3.60
.+-. 0.17* 3.76 .+-. 0.19* HDL-cholesterol 1.54 .+-. 0.09 1.62 .+-.
0.09* 1.63 .+-. 0.10* Triglycerides 1.22 .+-. 0.13 1.20 .+-. 0.11
1.26 .+-. 0.15 *p < 0.05 or less
[0046] Both test margarines resulted in favourable changes in serum
lipids. The reduction in LDL-cholesterol values and the increase in
HDL-cholesterol values were statistically significant (p<0.05 or
less). Furthermore, the vegetable oil based sterol ester resulted
also in a statistically significant reduction of total cholesterol.
The obtained reduction of total cholesterol and LDL-cholesterol was
higher with the vegetable oil based stanol ester margarine compared
to the tall oil based stanol ester margarine. No changes in
triglyceride levels were obtained. The serum lipid results obtained
indicate that a vegetable oil stanol ester margarine containing a
substantial amount of campestanol in its stanol fraction might be
even more effective than the tall oil stanol ester margarine. Tall
oil stanol ester margarine has in earlier studies (14, 15, 31)
shown effective hypocholesterolemic effects. Thus, based on the
cross-over design of this study, it can be concluded that vegetable
oil based stanols are showing at least as effective
hypocholesterolemic effects as tall oil based stanols.
[0047] Serum sterol concentrations were quantified with gas-liquid
cromatography according to a previously published method (29,
hereby incorporated by reference). The means of two measurements of
serum lipids from the blood samples taken at each period were
calculated. The data on mean serum plant sterol concentrations at
the home diet and after each test period and the mean changes
observed in these concentrations are presented in Tables 2 and 3
below.
3TABLE 2 Serum plant sterol concentrations (mean .+-. SE, .mu.g/dl)
during the home diet and after each intervention period (n = 24).
Vegetable oil based stanol ester margarine, TS = tall oil based
stanol ester margarine. Home diet VS TS Campestanol 47 .+-. 2 58
.+-. 3 47 .+-. 3 Sitostanol 94 .+-. 3 92 .+-. 5 96 .+-. 5
Campesterol 472 .+-. 37 337 .+-. 25 350 .+-. 28 Sitosterol 277 .+-.
17 198 .+-. 12 227 .+-. 15 *p < 0.05 or less
[0048]
4TABLE 3 Mean changes (.+-.SE) in the serum plant sterol
concentrations (.mu.g/dl), (n = 24). VS = Vegetable oil based
stanol ester margarine, TS = tall oil based stanol ester margarine,
HD = home diet. .DELTA.(VS-HD) .DELTA.(TS-HD) .DELTA.(VS-TS)
Campestanol 11 .+-. 2* 0 .+-. 2 11 .+-. 2* Sitostanal -2 .+-. 3 2
.+-. 4 -4 .+-. 4 Campesterol -134 .+-. 19* -122 .+-. 21* -12 .+-.
13 Sitosterol -80 .+-. 11* -51 .+-. 12* -29 .+-. 8* *p < 0.05 or
less
[0049] Both test margarines significantly lowered serum campesterol
and serum sitosterol levels. The serum concentration of campesterol
is known to reflect intestinal cholesterol absorption in humans
(29, 39). Thus, the lower the campesterol value, the lower the
percentage of intestinal cholesterol is absorbed.
[0050] Marked falls in serum campesterol levels (25-28%) during the
study periods indicates that both stanol ester margarines decreased
the intestinal absorption of cholesterol. Furthermore, no
differences in the serum sitostanol concentration could be seen
while mean serum campestanol concentration after the vegetable oil
stanol ester period was significantly higher than at the home diet
and after the tall oil stanol ester period. However, the absolute
concentration of campestanol was only about 63% of that of
sitostanol, which is generally regarded as virtually unabsorbable.
This low serum concentration of campestanol clearly indicates that
the absorption of campestanol is very limited, which is in conflict
with the results presented by Heinemann et al. (20). Thus, since
stanol mixtures containing high levels of sitostanol are regarded
as safe for human ingestion, stanol mixtures containing substantial
amounts of campestanol must be regarded as equally safe based on
the fact that campestanol is like sitostanol virtually
unabsorbable.
[0051] The preparation of the stanol ester composition of the
invention and the margarines used in the above clinical studies are
disclosed in detail in the following working examples:
EXAMPLE 1
Hydrogenation of Sterol Mixtures
[0052] A commercially available sterol mixture obtained from
vegetable oil distillate (composition: brassicasterol 2.7%,
campesterol 26.7%, stigmasterol 18.4% sitosterol 49.1% and
sitostanol 2.9% ) was hydrogenated in a pilot scale reactor (25 l).
26 g of a fibrous Pd catalyst (Smop-20; Pd content 10 weight-%,
Smoptech, Turku, Finland), 26 g distilled water for the activation
of the catalyst and 11.7 kg propanol was feed into the reactor. The
reactor was flushed with nitrogen and the activation of the
catalyst was carried out under hydrogen gas at a pressure of 1 bar
and at a temperature of 65.degree. C. for 30 min. After the
activation the blend was cooled to 40.degree. C., after which 1.3
kg of the sterol blend was added.
[0053] The propanol sterol mixture was heated under nitrogen
atmosphere to 65.degree. C., after which nitrogen was displaced by
hydrogen. After that a thorough flushing with hydrogen was done,
the hydrogenation reaction was carried out at a hydrogen pressure
of 1 bar. The normal conversion time is about 120 min. The
conversion can easily be monitored by taking aliquots, which are
analyzed by HPLC.
[0054] The hydrogen pressure was dropped and the reactor was
flushed with nitrogen. The fibrous catalyst was filtered off with
nitrogen pressure. The propanol stanol blend was left to
crystallize overnight at 10.degree. C. after which the stanol
crystals were vacuum filtered and the cake was washed with 0.5 kg
cold propanol. The obtained stanol mixture was dried at 60.degree.
C. in a vacuum cupboard. The yield was 75% and the composition of
the obtained stanol mixture was as follows according to capillary
GC analysis: campesterol 0.2%, campestanol 28.9%, stigmasterol
0.1%, sitosterol 0.2%, sitostanol 70.1%. It should be noted that
brassicasterol is hydrogenated into 24.beta.-methyl cholestanol, an
epimer of campestanol, but since these appear in the same peak with
ordinary capillary gas chromatographic procedures which is unable
to separate according to chirality, it is usually calculated as
campestanol. Based on the initial sterol mixture the content of
24.beta.-methyl cholestanol should be 2.7%.
EXAMPLE 2
Preparation of Stanol Fatty Acid Esters
[0055] A stanol fatty acid ester mixture was prepared on a pilot
scale. 6 kg stanols obtained by combining several batches obtained
by the hydrogenating procedure given in example 1 was dried
overnight at 60.degree. C. and esterified with a 8.6 kg low erucic
acid rapeseed oil methyl ester mixture. The sterol composition of
the stanol blends used was as follows: Campesterol 0.4%,
campestanol (+24.beta.-methyl cholestanol) 29.7%, stigmasterol
0.1%, sitosterol 0.4% and sitostanol 68.0%. The stanol content of
the blend was 98.2%. The esterification was carried out as
follows:
[0056] A mixture of stanols and low erucic rapeseed oil fatty acid
methyl ester was heated in a reactor vessel at 90-120.degree. C.
under a vacuum of 5-15 mmHg. After drying for 1 hour, 21 g
Na-ethylate was added and the reaction was continued for about 2
hours. The catalyst was destroyed by the addition of 30% water (by
weight) at 90.degree. C. After phase separation the water phase was
removed and a second washing was carried out. After the separation
of the water phase, the oily phase was vacuum dried at 95.degree.
C. with a stirring effect of 200 rpm. The stanol fatty acid mixture
was lightly bleached for 20 min. at 30 mmHg and a temperature of
110.degree. C. with 1.0% of bleahing earth (Tonsil Optimum FF,
Sudchemie, Germany) under a stirring effect of 200 rpm. The
bleaching earth was filtered off and the obtained mixture of fatty
acid methyl esters and stanol fatty acid esters can be added as
such to fat blends prior to deodorization or the excess of methyl
esters can be distilled off under vacuum. Accordingly the blend can
be deodorized to obtain a tasteless stanol fatty acid ester
mixture, which can be added as such to different food manufacturing
processes.
[0057] The conversion of the esterification process is normally
>99% measured by a fast HPLC method and the yield is in the
range of 95%.
EXAMPLE 3
Production of Margarines for the Clinical Studies
[0058] 80% margarines with tall oil stanol fatty acid esters and
vegetable oil based stanol fatty esters were produced on a
Gerstenberg & Agger 3.times.57 pilot scale perfector. Tall oil
stanol fatty acid esters were obtained from the normal production
of Benecol.RTM. margarine by Raision Margariini, Finland. A normal
trans fatty acid free fat blend (composition: 30% non-hydrogenated
interesterified vegetable fat and 70% liquid LEAR oil) to wich the
stanol fatty acid mixtures were added was used. The stanol content
of the final product was targeted to be 12 g/100 g product, which
would provide a daily intake of 3 g stanols at usage level of 25
g/day. The products were produced according to following
recipe:
5 Fat blend including the stanol fatty acid esters 80% Water 19%
Salt 0.5% Emulsifier, Dimodan BP Na-bicarbonate and citric acid as
pH-regulating agents .beta.-carotene as colouring agent
Flavours.
[0059] The obtained margarines were packed into 250 g polypropene
tubs, which were sealed by an aluminium foil. The taste and texture
of the products were equal to commercial margarines.
[0060] The stanol content of the tall oil stanol margarine was 12.7
g/100 g product and of the vegetable oil based stanol margarine
12.6 g/100 g product. The sterol composition of the two products
were as follows:
6 Tall oil based Vegetable oil based stanol margarine stanol
margarine Brassicasterol 0.3% 0.4% Campesterol 2.2% 2.4%
Campestanol 7.5% 27.6% Sitosterol 7.4% 4.2% Sitostanol 82.5% 63.8%
Others 0.1% 1.6%
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