U.S. patent application number 10/072570 was filed with the patent office on 2003-06-12 for process for the preparation of one or more statins by frementation.
This patent application is currently assigned to Unilever Bestfoods North America, Division of Conopco, Inc.. Invention is credited to Ter Schure, Eelko Gerben, Trautwein, Elke A., Van Oorschot, Gijsbertus Johannes.
Application Number | 20030108657 10/072570 |
Document ID | / |
Family ID | 26076831 |
Filed Date | 2003-06-12 |
United States Patent
Application |
20030108657 |
Kind Code |
A1 |
Van Oorschot, Gijsbertus Johannes ;
et al. |
June 12, 2003 |
Process for the preparation of one or more statins by
frementation
Abstract
A process is described for the preparation of one or more
statins by fermentation, wherein a substrate is fermented with
statins producing fungus wherein the substrate comprises more than
20% by weight of soy ingredients. Further a food product is
described comprising an amount of one or more statins and an amount
of one or more compounds chosen from the group: polyunsaturated
fatty acids, phytosterols, proteins, peptides, dietary fibers,
polyphenols and saponins, wherein the food product has a Hue a*
value less than 20.
Inventors: |
Van Oorschot, Gijsbertus
Johannes; (Vlaardingen, NL) ; Ter Schure, Eelko
Gerben; (Vlaardingen, NL) ; Trautwein, Elke A.;
(Vlaardingen, NL) |
Correspondence
Address: |
UNILEVER
PATENT DEPARTMENT
45 RIVER ROAD
EDGEWATER
NJ
07020
US
|
Assignee: |
Unilever Bestfoods North America,
Division of Conopco, Inc.
|
Family ID: |
26076831 |
Appl. No.: |
10/072570 |
Filed: |
February 8, 2002 |
Current U.S.
Class: |
426/658 |
Current CPC
Class: |
A23C 9/1315 20130101;
A23L 13/65 20160801; A23L 11/50 20210101; A23L 33/105 20160801;
A23L 33/10 20160801; A23L 33/11 20160801; A61K 31/352 20130101;
A23L 7/126 20160801; A23D 7/0056 20130101; A23C 21/026 20130101;
A23G 9/38 20130101; A23V 2002/00 20130101; A23L 33/185 20160801;
A23L 13/46 20160801; C12P 17/06 20130101; A23L 13/424 20160801;
A61K 36/48 20130101; A61K 31/352 20130101; A61K 2300/00 20130101;
A61K 36/48 20130101; A61K 2300/00 20130101; A23V 2002/00 20130101;
A23V 2250/2116 20130101 |
Class at
Publication: |
426/658 |
International
Class: |
A23G 003/00 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 9, 2001 |
EP |
01200489.1 |
Feb 9, 2001 |
EP |
01200493.3 |
Claims
1. Process for the preparation of one or more statins by
fermentation, wherein a substrate is fermented with statins
producing fungus, characterized in that substrate comprises more
than 20% by weight of soy ingredients.
2. Process according to claim 1, wherein the substrate comprises
more than 50% by weight of soy ingredients.
3. Process according to claim 1, wherein the substrate comprises
more than 80% by weight of soy ingredients.
4. Process according to any one of claims 1, wherein the statins
producing fungus is a Monascus fungus.
5. Process according to any one of claims 1, wherein an amount of
oil being at least 5 wt. % oil is present in the substrate during
fermentation.
6. Process according to claim 5, wherein the oil is edible oil.
7. Process according to claim 5, wherein the oil is vegetable
edible oil.
8. Process according to any of claims 5, wherein the amount of oil
is at least 10 wt. %.
9. The Monascus strain F125 M1-4 deposited at the Centraal Bureau
voor Schimmelculturen on 14.11.2000 having number CBS 109070.
10. Food product comprising: a) an amount of one or more statins,
b) an amount of one or more compounds chosen from the group:
polyunsaturated fatty acids, phytosterols, proteins, peptides,
dietary fibers, polyphenols and saponins, wherein the food product
has a Hue a* value less than 20.
11. Food product according to claim 10, wherein the amount of one
or more statins is 5-500 mg/kg, comprising an amount of genistein
and genistin, wherein the amount of genistein is 10-99 wt. % of the
sum of the amounts of genistein and genistin.
12. Food product according to claim 11, wherein the amount of
genistein is 20-95 wt. % of the sum of the amounts of genistein and
genistin.
1. Food product according to claim 11, wherein the amount of
genistein is 20-80 wt. % of the sum of the amounts of genistein and
genistin.
13. Food product according to claim 10, wherein the amount of a) is
5-500 mg/kg and the amount of b) 1 wt. or higher.
14. Food product according to claim 14, wherein the amount of b) is
5 wt. % or higher.
15. Food product according any one of claims 10, wherein the food
product has a Hue a* value less than 10.
16. Food product according to claim 10, wherein the one or more
compounds chosen from the group: polyunsaturated fatty acids,
phytosterols, proteins, peptides, dietary fibers, polyphenols and
saponins are substantially derived from soybeans.
17. Food product according to claim 10, wherein the food product is
a margarine, dressing, sweet, bar meal replacer, breakfast cereal
or beverage.
Description
TECHNICAL FIELD
[0001] The present invention relates to process for the preparation
of one or more statins by fermentation. The invention further
relates to food product comprising one or more statins.
BACKGROUND OF THE INVENTION
[0002] Statins are compounds that are known to have a lowering
effect on levels of low-density lipoprotein cholesterol
(LDL-choloesterol) in the human blood. Elevated LDL-cholesterol
levels (hypercholesterolemia) is directly related to increased risk
of coronary heart disease. Statins inhibit the
hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase, the
rate-determining step in the cholesterol biosynthesis.
[0003] Scientific research has confirmed the healthy properties of
statins especially with respect to LDL blood-cholesterol and
triglyceride levels lowering activities, both in animals and in
humans (Li et al., Nutrition Research 18, 71-81 (1998); Heber et
al., Am. J. Clin. Nutr. 69, 231-236 (1999)).
[0004] Early reports of the effect of statins were made in 1979.
The Japanese scientist Endo isolated a metabolite from Monascus
that reduced artificially induced hyperlipoproteinemia in rats
(Endo, J. Antibiotics 32, 852-854, (1979)). These metabolites are
known as monacolins. Monacolin is identical to the cholesterol
lowering pharmaceutical lovastatin. Lovastatin is sold by Merck co.
under the tradename Mevacor. A derivative of lovastatin,
simvastatin, is sold as a cholesterol-lowering drug under the name
of Zocor. Other derivatives of lovastatin e.g. pravastatin, and
mevastatin, are also sold as lipid lowering drugs against
hypercholesterolemia. Monascus-extracts are sold in capsules in
Japan as the dietary product Monacolin. The usual dose of the above
statins is 20 mg/day, which results in at least 20% blood
LDL-cholesterol lowering.
[0005] The production of statins is also reported in fermentation
using fungi other than the above-mentioned Monascus species. It has
been shown that statins can be produced by a variety of filamentous
fungi, including Monascus, Aspergillus, Penicillium, Pleurotus,
Pythium, Hypomyces, Paelicilomyces, Eupenicillium, and
Doratomyces.
[0006] The preparation and purification of the statins used in
pharmaceutical preparations involves many process-steps, in which
ingredients are used that are not commonly used in the food
industry. The many process steps increase costs compared to
processes having less process steps. For these reasons the statins
prepared for pharmaceutical use are not used in the foods
industry.
[0007] As a food product, rice fermented with a red Monascus fungus
(red rice) has been known and used for hundreds of years in China.
Red rice was used and still is used in wine making, as a
food-colouring agent and as drug in traditional Chinese medicine.
We have found that most red rice available on the market contains
no statins or statins in very low amounts. The Food and Drug
Administration has concluded that red yeast rice available in the
market does not contain significant amounts of lovastatin (FDA,
Docket No. 97-0441, Final Decision).
[0008] WO 99/23996 describes a composition for treating elevated
serum cholesterol and/or triglycerides comprising a red rice
product containing at least 0.05% lovastatin by weight.
[0009] Red rice powder capsules are sold as dietary supplements
under the name of Cholestin by the firm Pharmanex. Pharmanex also
sells a Cholestin bar containing red yeast rice (Monascus purperus
went).
[0010] Red rice has an intensive red colour. Whereas the intensive
red colour of red rice is an advantage when it is used as colouring
agent, it is a disadvantage when it is used in food products. Due
to the intense red colour of red-rice products, the foods prepared
from red rice are coloured, depending on the amount of red-rice
product added to the food product yellow, orange or red. The higher
the amount of red rice added to the food, the more intense is the
red colour of the food product. In the known food products a
relatively large amount of red rice has to be added in order to add
enough statins. This results in a red colour of the products that
cannot be avoided.
[0011] In some food products the red rice colouring is undesirable.
In particular in the western world, consumers are reluctant to use
products of which the colour has changed from that they are used
to. For example spreads, including margarine, butter, low fat
spreads or salad oils are considered unacceptable by customers,
when the colour of such a product is orange or red. However, at the
same time these type of products have been found by us to be
excellent vehicles of the daily intake of amounts of statins
sufficient to obtain a blood LDL-cholesterol lowering effect.
SUMMARY OF THE INVENTION
[0012] It is an object of the invention to provide a food product
that has no undesired colouring due to the addition of statins. A
further object of the invention is to increase the health effects
of the known food products comprising statin. Another object is to
provide a process for the preparation of a food product comprising
statin, which involves less process steps than in the preparation
of statins as a pharmaceutical drug. Another object is to provide
such a process, which avoids the use of ingredients or process aids
that are not commonly used in the food industry.
[0013] One or more of these objects are attained by a process for
the preparation of one or more statins by fermentation, wherein a
substrate is fermented with statins producing fungus, characterized
in that substrate comprises more than 20% by weight of soy
ingredients.
[0014] Further one or more of the above objects are attained by a
food product comprising:
[0015] a) an amount of one or more statins
[0016] b) an amount of one or more compounds chosen from the
group:
[0017] polyunsaturated fatty acids, phytosterols, proteins,
peptides, dietary fibers, including soluble fibers, polyphenols and
saponins, wherein the food product has a Hue a value of less than
20, preferably less than 20, most preferably less than 0.
[0018] Preferably the amount of a) is 5-100 mg/kg and the amount of
b) 1 wt. or higher. More preferably the amount of b) is 5 wt. % or
higher.
[0019] We have found that when the substrate for the Monascus
fermentation is soybeans and/or soybean ingredients, the red
colouring of the fermented product as in red rice fermentation is
avoided, i.e. a non-coloured or only slightly coloured fermentation
product is obtained. Further we have found that compounds having a
positive health effect, which are present in soybeans are also
present in the fermented product. These compounds include, but are
not limited to polyunsaturated fatty acids, phytosterols, proteins,
peptides, dietary fibers including soluble fibers, polyphenols and
saponins. As a result of the presence of these compounds in the
fermentation product, the food product according to the invention
has increased health effects compared to the known food products
comprising statin.
DETAILED DESCRIPTION OF THE INVENTION
[0020] The following definitions will be used.
[0021] Statins are defined as substances having the structural
formula, presented in formula (1). 1
[0022] In this structural formula, R1 and R2 can be any group.
Preferred statins are those given in table 1.
1TABLE 1 Preferred statins according to formula (1) R1 R2 monakolin
K CH3 2 monakolin L CH3 H monakolin J CH3 OH monakolin X CH3 3
monakolin M CH3 4 compactin (ML-236B) H 5 ML-236-A H OH NL-236-C H
H
[0023] Polyphenols herein are polyphenols having plant origin.
These include flavenoids, which include isoflavones. The
polyphenols include isoflavones, stilbenes, lignans, coumestans and
resorcyclic acid lactones. Examples of isoflavones are genistein,
daidzein, equol, glycitein, biochanin A, coumestrol, maitaresinol,
formononetin, O-desmethylengolesin, enterolactone and enterodiol.
Preferred isoflavones according to the invention are genistein and
daidzein and glycitein, which are present in soybeans.
[0024] Saponins are herein derived as .beta.-D-glucopyranosiduronic
acid derivates. Examples of saponins are Soya sapogenol A,B,C,D and
E, Soyasaponin I, II and III, as described in Lebensmittel Lexikon,
B.Behr's Verlag GmbH & Co. Hamburg, Bd.2, L-Z -3, 1993, pages
550-552.
[0025] Polyunsaturated fatty acid esters are defined as fatty acid
esters having more than one unsaturated group in the fatty acid
chain. Examples of polyunsaturated fatty acid esters are linoleic
acid esters, linolenic acid esters, arachidonic acid esters.
[0026] Dietary fibers are herein a collective term for a variety of
plant substances, that are resistant to digestion by the human
gastrointestinal enzymes. Depending on their solubility, dietary
fibers can be classified into insoluble (cellulose, some
hemicelluloses, lignins), and soluble (remainder of the
hemicelluloses, gums, mucilages. Soybean cotyledon fibers comprise
both soluble and insoluble dietary fibers.
[0027] Phytosterols are herein defined as sterol compounds produced
by plants, which are structually very similar to cholesterol except
that they contain some substitutions at the C24 position on the
sterol side chain. The phytosterols include 4-desmethylsterol,
4-monomethylsterols, 4,4'-dimethylsterols and mixtures thereof.
Examples of such phytosterols are .beta.-sitosterol, campesterol,
stigmasterol. The term phytosterols herein also includes
phytostanols, the saturated equivalents of phytosterols.
[0028] Polyphenols, polyunsaturated fatty acids, phytosterols,
proteins, peptides, dietary fibers, and saponins will hereinafter
collectively be referred to as soy actives.
[0029] Unless otherwise indicated, the amounts given will be
expressed, in wt. % or weight parts per million (ppm), mg/kg or
g/kg, relative to the total weight of the food product, unless
otherwise indicated.
[0030] The amounts of statins given herein are the sum of the
amounts of individual statins, as e.g. determined by
chromatography, unless otherwise indicated.
[0031] The substrate is herein defined as total of compounds in the
fermentation medium, without the solvent, for instance without
water, in case a liquid, water based fermentation medium is used.
In case no solvent is present the substrate equals the fermentation
medium.
[0032] The protein amounts given herein are the sum of the amounts
of individual proteins, unless otherwise indicated.
[0033] The amounts of soy actives are expressed as the sum (wt. %
or ppm) of polyunsaturated fatty acids, phytosterols, proteins,
peptides, dietary fibers including soluble fibers, polyphenols and
saponins.
[0034] The food product has a Hue a* value of less than 20,
preferably less than 20, most preferably less than 0. The Hue a*
value is determined as described hereinafter in the examples.
[0035] Preferably a food product according to the invention does
not include products especially suitable for the feeding of animals
(feed).
[0036] Several food products may be prepared according to the
invention, for example, spreads, soups, noodles, ice-cream, sauces,
dressing, snacks, cereals, beverages, bread, biscuits, other bakery
products, sweets, bars, chocolate, chewing gum, dairy products,
dietetic products e.g. slimming products or meal replacers etc.
[0037] The statins and soy actives are present in the food product
in an amount sufficient to obtain a blood LDL-cholesterol lowering
effect if the food product is used according to the common needs of
the consumer.
[0038] The skilled person will be able to adjust the percentage of
statins and soy actives in the food product to get the above
effect. The percentages will depend on the type of food product,
since the food products are used in different serving sizes.
Moreover the pattern in a food product is consumed (servings per
day and distribution over days) is dependent on the food product.
Data about serving sizes may be found in the list published by the
United States Food and Drug Administration (FDA) titled: "Reference
Amounts customarily consumed per Eating Occasion".
[0039] Preferably in the food products according to the invention,
the amount of statin is 5-500 mg/kg and the amount of soy actives
is 1 wt. or higher. More preferably the amount of soy actives is 5
wt. % or higher or 10 wt % or higher. Most preferably the amount of
soy actives is 20 wt. % or higher.
[0040] As an illustration table 2 indicates a number of products,
which may be prepared according to the invention, and a typical
serving size.
2 TABLE 2 Product Daily Serving Margarine 15 g Meat product 50 g
Dressing 30 g Sweet 10 g Bar 75 g Meal replacer drink 330 ml
Beverages 200 ml
[0041] Preferably the food product according to the invention
comprises statin and non-glycosylated isoflavone. In soy beans and
soy materials derived from soy, isoflavones are present
substantially in the glycosylated form. Typically about 5 wt. % of
the isoflavones is present in the non-glycosylated form. The most
important glycosylated isoflavones are genistin, daidzin and
glycetin. The non-glycosylated forms are respectively genistein,
daidzein and glycetein. Genistein, daidzein and glycetein have been
reported to have advantageous health effects, including estrogenic
and antioxidant properties.
[0042] We have found that due to the fermentation according to the
invention the glycosylated isoflavones are converted into the
corresponding non-glycosylated isoflavones, which are more
benificial. For instance, the amount of genistein and daidzein is
increased in the fermented soy compared to the non-fermented soy.
Surprisingly this advantageous conversion occurs simultaneously
with the production of statin.
[0043] The invention therefore further relates to a food product
wherein the amount of statin is 5-500 mg/kg, and an amount of
genistein and genistin, wherein the amount of genistein is 10-99
wt. %, preferably 15-99 wt. %, more preferably 20-95 wt. %, still
more preferably 20-90 wt. %, most preferably 20-80 wt. % of the sum
of the amounts of genistein and genistin.
[0044] The invention therefore further relates to a food product
wherein the amount of statin is 5-500 mg/kg, and an amount of
daidzein, wherein the amount of daidzein is 10-99 wt. %, preferably
15-99 wt. %, more preferably 20-95 wt. %, still more preferably
20-90 wt. %, most preferably 20-80 wt. % of the sum of the amounts
of daidzein and daidzin.
[0045] The absolute amounts of genistein and daidzein may, for each
food product, be adjusted by the skilled person to a desired level.
This may for instance be done by selection of the soy material to
be fermented from materials having a different isoflavone content,
by adjustment of the fermentation conditions, such as fermentation
time, and by selecting the amount of fermented soy added to the
food product. In such way the amount may be adjusted to a desired
daily intake of the isoflavones, that could be for instance 50-80
mg/day for genistein. The preferred absolute level of genistein in
the food products according to the invention, depends on the food
type, and may be 50 mg/kg or more, more preferably 100 mg/kg or
more, 200 mg/kg or more, 500 mg/kg or more and most preferably
200-5000 mg/kg. Also the absolute level of daidzein depends on the
food type, and may be 50 mg/kg or more, more preferably 100 mg/kg
or more, 200 mg/kg or more, 500 mg/kg or more and most preferably
200-5000 mg/kg.
[0046] Preferably the food product according to the invention is a
spread, meat product, sauce, such as soy sauce, vinegar, soup,
bakery good, beverage or bar. These products are preferred because
the way in which they are consumed results in a more constant
intake of statins and soy actives than in other food products. More
preferred food products according to the invention are a spread,
cereal bar, beverage or breakfast cereal.
[0047] The invention will now be further illustrated by the
description of suitable embodiments of the more preferred food
products. It belongs to the ability of the skilled person to use
the teaching provided therewith to prepare other products of the
invention.
[0048] Spreads
[0049] Typically, spreads according to the invention are oil in
water or water in oil emulsions, although also spreads, which are
substantially fat free, are covered. Spreads are to include
margarines and liquid cooking products. The spreads may be
spreadable and not pourable or may be pourable at the temperature
of use e.g. 2-10.degree. C. Fat levels may vary in a wide range
e.g. full fat margarines with 60-90 wt. % of fat, medium fat
margarines with 30-60 wt. % of fat, low fat products with 10-30 wt.
% of fat and very low or fat free margarines with 0 to 10 wt. % of
fat.
[0050] The fat in the margarine or other spread may be any edible
fat, often used are soybean oil, rapeseed oil, sunflower oil and
palm oil. Fats may be used as such or in modified form e.g.
hydrogenated, esterified, refined etc. Other suitable oils are well
known in the art and may be selected as desired.
[0051] The pH of a margarine or spread may advantageously be from
5.0 to 6.5, though other pH's are possible.
[0052] Examples of spreads other than margarines are cheese
spreads, sweet spreads, yoghurt spreads etc.
[0053] Optional further ingredients of spreads may be emulsifiers,
colourants, vitamins, preservatives, emulsifiers, gums, thickeners
etc. The balance of the product will normally be water.
[0054] A typical size for an average serving of margarine or other
spreads is 15 grams. Preferred levels of statins in the margarine
or spread are: 20-500 mg/kg statin, more preferred ranges are 50 to
250 mg/kg statin.
[0055] Beverages
[0056] Preferred food products according to the invention are
beverages, for example tea, fruit juice, soft drinks,
meal-replacers, etc. Meal replacer drinks will be described in more
detail herein below. It will be apparent that similar levels and
compositions apply to other beverages comprising statins and soy
actives.
[0057] A typical serving size of a beverage is taken to be 200 ml.
Preferred levels of statins in a beverage are: 5-100 mg/kg, more
preferably 10-80 mg/kg.
[0058] Bars, including cereal bars
[0059] These products often comprise a matrix of edible material
wherein the statin and soy actives can be incorporated. For example
the matrix may be fat based (e.g. couverture or chocolate) or may
be based on bakery products (bread, dough, cookies etc). Preferably
the food product is a cereal bar, in which the matrix is based on
agglomerated cereal particles (rice, grain, nuts, raisins, fruit
particles).
[0060] The matrix material of a bar may be present in an amount of
60-95 wt. % of the weight of the bar, preferably 70-90 wt. % most
preferred 75-85 wt %.
[0061] Other ingredients in the cereal bar may be starch, sugar
(e.g. 0-10 wt %), sirups, honey, milk solids, salt (e.g. 0-5 wt. %)
calcium carbonate, vitamins, flavouring and colouring.
[0062] The ingredients are usually mixed and cooked (e.g. by
cooking-extruding) to produce the (cereal) bar.
[0063] A typical size for a bar could be from 20 to 200 g,
generally from 40 to 100 g. Preferred level in such products would
be: 25 to 500 mg/kg statin. More preferred range for this level is
50 to 300 mg/kg.
[0064] Further ingredients may be added to the product such as
flavouring materials, vitamins, minerals etc.
[0065] Preparation Of The Food Product
[0066] According to the invention a substrate, prepared from
soybeans and/or ingredients thereof is fermented with a statin
producing fungus and the fermentation product is used in the
preparation of a food product. These steps will be illustrated
below. In this illustration the statin producing fungus is
Monascus.
[0067] Fermentation is conducted in known way. The fermentation is
conducted in at least one fermentation vessel (fermenter) in which
a medium comprising soybeans and/or ingredients thereof is present.
The fermentation is started (inoculated) by adding a suspension of
spores of the Monascus fungus (inoculum), which has been prepared
by fermenting Monascus fungus on a separate medium. The
fermentation may be executed batchwise or as a continuous
process.
[0068] The fermentation involves the following steps, which are
executed in the given order:
[0069] a) Preparation of the medium for the inoculum and the medium
to be used in the fermenter
[0070] b) Sterilization of the media, fermenters and ancillary
equipment
[0071] c) Production of inoculum
[0072] d) Addition of the inoculum to the medium comprising
soybeans and/or ingredients thereof, in the fermenter.
[0073] e) Conducting the fermentation
[0074] f) Removal of the fermentation product from the
fermenter
[0075] The fermentation product is used in the preparation of the
food products according to the invention.
[0076] Optionally, before the fermentation product is used in the
preparation of the food products, the following additional process
steps may be executed:
[0077] g) Sterilization of the fermentation product
[0078] h) Drying of the fermentation product (or sterilized
fermentation product)
[0079] i) One or more separation steps, for instance extraction, to
separate statins and soy actives from Monascus biomass in the
fermentation product
[0080] The medium used in the fermenter may be solid or liquid.
Advantageously the medium is solid, most preferred the medium
substantially consists of crushed whole soybeans, which have been
soaked with water (e.g. 30 wt % water). In case the medium is
liquid, usually water is present as major constituent of the
medium.
[0081] Whole soybean are preferably used as a substrate for the
fermentation. Typical composition of whole soybeans is 42% wt %
protein, 20 wt % oil, 35% wt % total carbohydrates, 5 wt % ash and
5.5 wt % crude fiber (Kawamura, S., Tech. Bull. Faculty Agric.,
Kagawa Univ., 18, 117 (1967)).
[0082] Instead of whole soybeans, parts or ingredients of soybean
may be used in the medium for the fermenter, for instance soy
protein (including textured vegetable protein), soy milk,
soy-flakes etc. Care has to be taken that the medium contains
compounds that can provide a carbon source and a nitrogen source
for growth of the Monascus fungus.
[0083] The Monascus fungus used according to the invention may be
any Monascus fungus that produces statins. Preferably the fungus is
chosen from the group of Monascus ruber Most preferred is Monascus
ruber F125 M1-4.
[0084] Strains F125 and F125 M1-4 are deposited at the Centraal
Bureau voor Schimmelculturen (CBS) as no. CBS 109070 on 14.11.2000
and no. CBS 109269 on 23.01.2001.
[0085] These deposits were made under the provisions of the
Budapest Treaty on the International Recognition of the Deposit of
Microorganisms for the Purpose of Patent Procedure and the
Regulations thereunder (Budapest Treaty).
[0086] Availability of the deposited material is not to be
construed as a license to practice the invention in contravention
of the rights granted under the authority of any government in
accordance with its patent laws.
[0087] The medium will ordinarily be sterilized before
fermentation, e.g. by heat treatment, like pasteurization.
[0088] The medium in the fermenter may contain other substances,
which may aid the fermentation, for instance sugars, amino acids
and vitamins.
[0089] The fermentation may be carried out in a manner, which can
be determined by the skilled person on the basis of common general
knowledge of fermentation technology. As illustration preferred
embodiments are described hereunder.
[0090] The fermentation temperature may be important. The
temperature is preferably in the range of 10 to 37, .degree. C.
more preferably 20 to 30.degree. C. We have found that at
37.degree. C. and higher the production of statins decreases.
[0091] Preferably during fermentation the medium is aerated, e.g.
by stirring, shaking etc. Aeration may be carried out by blowing
air through the fermentation medium. Preferably the air is wholly
or partly saturated with water vapour in case solid state
fermentation is used. This avoids drying out of the fermentation
medium.
[0092] The relative levels of statins to soy actives will depend on
the fermentation time. The fermentation time is therefore dependent
on the desired amount of statins in the fermentation product.
Preferred fermentation time is 1-60 days, more preferably 1-50
days, still more preferably 15-40 most preferably 20-30 days.
[0093] Prior to addition to the food product, the fermentation
product may be subjected to a separation step, to separate statins
and soy actives from Monascus biomass in the fermentation product.
This separation may be done with known separation techniques, e.g.
filtration or centrifugation.
[0094] The fermentation product may also be extracted and extract
may be used in the preparation of the food product. Preferred
extraction agents are food-grade extraction agents. More preferred
extraction agent is ethanol. Most preferred extraction agent is
vegetable oil, e.g. soybean oil or sunflower oil.
[0095] The extraction may be done on the fermentation product.
Alternatively the Monascus biomass may be separated from the
fermentation product prior to extraction, e.g. by filtration. The
Monascus biomass may be separately extracted and the resulting
extract can also be used in the preparation of the food
product.
[0096] Extracts may be used as such in the preparation of food
products. Preferably extraction solvent may be removed from the
extracts, e.g. by evaporation of the extraction solvent.
[0097] Advantageously edible oil may be used as extractant, the
edible oil is preferably vegetable oil, such as for instance soy
bean oil or sunflower oil. When the fermentation product is
extracted with vegetable oil, it was found that the statin is
effectively extracted and an oil phase containing substantially all
statin is obtained. The resulting extract is very suitable to be
used directly as a food ingredient.
[0098] Most advantageously the extractant, e.g. vegetable oil is
added to the fermentation medium during fermentation. We have found
that in the presence of an extractant, the production of statins
during fermentation is considerably increased. It is possible to
increase the amount of statin produced by at least a factor 10,
more preferably at least a factor 40, compared to fermentation
without extractant, by the addition of vegetable oil during the
fermentation. Preferably the extractant should not interfere with
the fermentation, especially it should not be poisonous for the
statins producing fungus. The amount of oil is preferably at least
5 wt. % oil (w/w on substrate), more preferably more than 10 wt. %,
most preferably at least 20 wt. %, being present in the substrate
during fermentation. Preferably the oil is edible oil, more
preferably vegetable edible oil, such as for instance sunflower oil
or soybean oil. Though animal and vegetable fat may be used these
are less preferred for hearth health reasons.
[0099] The fermentation product (including extracts etc.) may
directly be added to food product ingredients in the process of
preparation of the food products according to the invention. It can
be added to the other ingredients of the food product composition,
or it may be added to part of the ingredients, before other
ingredients are added. If more than one phase is present in the
food product the fermentation product may be present in one or more
of these phases. Preferably the fermentation product will
substantially be present in an oil phase, if such oil phase is
present.
The invention will be further illustrated in the examples.
EXAMPLES
Example 1
[0100] A. Preparation of Monascus strain F125 M1-4
[0101] Monascus ruber strain F125 was cultivated in malt water
liquid medium at 30.degree. C. for 4 days. Of this culture, 1 ml
was used as an inoculum for a Hybond-N filter (Amersham, UK) placed
on a YE plate (4% glucose, 0.3% KH.sub.2PO.sub.4, 1.0% yeast
extract (Difco), 1.5% agar). After 3 days incubation at 30.degree.
C., the spores were harvested by washing the filters with 10 ml
physiological saline containing 0.1% Tween 80. The spores were
filtered 4 times through Mira cloth filters to obtain a hyphae free
spore suspension. This suspension was used for subsequent
mutagenesis.
[0102] The spores were diluted to a concentration of 10.sup.8
spore/ml then exposed to UV light at an intensity of 100
joules/m.sup.2. The mutagenised spores were plated on Potato
Dextrose Agar (Oxoid) and incubated for 3 weeks. One of the
resulting colonies, which had a lighter colour than the others was
selected and is herein defined as Monascus strain F125 M1-4.
[0103] B. Preparation of Monascus spores
[0104] Monascus f125M1-4 spores were prepared by harvesting
Monascus mycelium from a PDA (potato dextrose agar) (oxoid) slope
by washing with 5 ml physiological saline and incubating the
mycelium in 150 ml malt water (oxoid) for 4 days at 30.degree. C.
The spores were harvested by filtration through a Mira cloth
filter.
[0105] C. Fermentation
[0106] C.1. Preparation Of An Inoculum
[0107] A shake flask containing soybeans is inoculated with
filamentous spores suspended in a physiological water solution
containing 0.1 wt % Tween 80 (polyoxyethylene sorbitan fatty acid
ester, available from ICI Specialty Chemicals.TM.). This shake
flask is incubated to let the mould grow. This resulted in a
Monascus spore suspension, which was adjusted by dilution to a
concentration of 1*10.sup.6 spores/ml.
[0108] C.2. Preparation Of Fermentation Medium
[0109] 1 kg of soybeans is soaked in tap water (50.degree. C.) for
30 minutes. After soaking the beans are rinsed with cold tap water.
Subsequently 50 g batches of the soaked beans (97% soybean) were
brought in to 300 ml Erlenmeyer flasks.
[0110] C.3. Fermentation
[0111] The shake flasks were each inoculated with 1 ml of the
prepared Monascus spore suspension and incubated for 30 days at
30.degree. C. A sample from the shake flask was taken every week to
monitor the statin production.
[0112] After fermentation, about 600 g soybeans remained.
[0113] D. Statin analysis of fermentation product
[0114] The fermentation product samples are extracted in a 50 ml
tube (Falcon) by adding 6 ml of a mixture of acetonitril, water and
phosphoric acid (1:1:0.05, v/v/v). The mixture is blended with an
Ultraturrax for 1 min. The mixture was then incubated at room
temperature on a rollerbank for over 24 hours. Hereafter the
samples were centrifuged and the supernatant liquid used for HPLC
analysis. Samples were separated using HPLC analysis on a Shimadzu
apparatus according to the method of Morovjan et al., J.
chromatogr. A 763 (1997) 165-172. The system consists of the
Shimadzu SCL-10A system controller, CTO-10AS column oven, LC-10AT
vp pump system, RID-10A refraction index detector, SPD-M10A diode
array detector and SIL-10AD autoinjector. For the chromatographic
determination of statins a Waters NovaPak C18 (150.times.3.9 mm
I.D., 4 .mu.m) column was used operating at 25.degree. C. The
eluent was acetonitril-0.1% phosphoric acid (50:50,v/v) solution
flowing at 1.5 ml/min. Runs were performed for 15 min. The
detection was performed using a diode array detector from 190 nm up
to 800 nm. The sum of the area of all peaks in the spectrum
belonging to statins is measured. Comparison to a standard
(Mevinolin, Sigma) allows the calculation of a statin content
(expressed in mg/kg analysed product).
[0115] The analysis results in a statin content of 1200 mg/kg
product.
[0116] E. Extraction of the fermentation product
[0117] The fermentation product was extracted with ethanol and
acetonitril. The soybean extracts contained 0.0545 g statin/kg
(ethanol extract) or 0.0978 g statin/kg (acetonitril extract) as
determined by HPLC, as described under D.
[0118] F. Colour analysis of extract
[0119] When colours are classified, they can be broken down into
the three primary elements. One is the Hue (colour) the other is
Value (brightness) and the third is Chroma (Saturation like vivid
colours or dull colours).
[0120] To enable anyone to tell anyone else exactly what colour
they are talking about a common numerical code is used. This
numerical code used is L*a*b*. When a colour is expressed in this
system, Value becomes L*, while Hue and Chroma are expressed as a*
and b* respectively. The L*a*b* was measured of different time
samples during fermentation. The supernatant of the samples was
filtered sterilized with a milipore 0.22 .mu.m filter. Of the clear
liquid L*a*b* was measured with a UV 1601 spectofotometer of
Shimatzu.
[0121] L*a*b* values of the soy extracts
[0122] Soy fermented with M. ruber M1-4
[0123] L*=90.2 ; a*=-4.8 ; b*=33.8
[0124] G. Preparation of a pourable margarine
[0125] An ethanol extract of the fermentation product was used for
further processing. Ethanol was removed by means of rotary
evaporator. The residue was used in a pourable margarine
composition. A pourable margarine composition without fermentation
product residue was used for comparison with respect to colour.
[0126] The composition of the pourable margarine is given in table
3.
3TABLE 3 Composition of the pourable margarine of example 1. Amount
Ingredient (wt. % on total) Sunflower oil 79.6 Rapeseed oil 1.95
hardened to a melting temperature of 70.degree. C. Lecithin (BOLEC
MT) 0.18 Lecithin (Cetinol) 0.2 Potassium sorbate 0.0125 Water 18.0
Statins 0.00011
[0127] The colour of the pourable margarine was not different from
a control with no statins extract, but 20 wt. % water.
Comparative Example A
[0128] Example 1, steps B to G were repeated, but instead of
Monascus strain F125 M1-4, a Monascus strain isolated from red rice
(commercial product from China) was used.
[0129] The L*a*b* values of the extracts were determined according
to example 1, step F. These values were:
[0130] L*=90.2 ; a*=56.1; b*=63.9
Comparative Example B
[0131] The procedure of example 1, steps B to G. was repeated,
however in step C.2., instead of 1 kg of soybeans, 1 kg of rice
(Parboiled rice, Oryza) was used. After fermentation about 500 g
rice remained.
[0132] The L*a*b* values of the extracts were determined according
to example 1, step F. These values were
[0133] L*=80 a*=54; b*=49
Example 2
[0134] A spread having the composition as in table 4 was
prepared.
4TABLE 4 Composition of spread of example 2 Ingredient Amount (wt %
on total) Sunflower oil 69.295 Fat blend 10.5 Beta carotene 0.125
Monoglygceride (Hymono 0.08 8903) Water 13.33 Ethanol extract of
6.67 example 1C Total 100
[0135] The fat blend was a mixture of 65 wt. % mid fraction of palm
oil stearin and 35 wt % palm kernel oil.
[0136] The spread was prepared by premixing the ingredients in a
premix tank at 55.degree. C. in a premix tank and feeding the
premix to a Votator with two scraped surface heat exchangers
(A-units) operated at 800 rotations per minute (rpm) and one
crystallizer (C-unit) operated at 200 rpm. The configuration was
A-A-C.
[0137] The amount of statin in the spread was 74 mg/kg. The colour
of the spread was not different from a control with no statins
extract, but 20 wt. % water.
Example 3
[0138] Fermentation with oil addition
[0139] 10 litre reactors were filled with 8 litres substrate
(Glucose 20 9/1, glycerol 100 g/l, starch 20 g/l, NaNO3 2 g/l MgSO4
5 g/l) and set to an airflow of 6 volume change per minute (vvm). A
pre-culture was prepared in YPD at 30.degree. C. for two days and
used to inoculate the fermentor with a concentration of 1.times.10
7 spores/l Monascus ruber F125M1-4. The cultures were incubated at
25.degree. C. and 200 rpm for 3 weeks. After 22 days, 400 ml
soybean oil was added to the 10 litre reactor.
[0140] After 49 days the fermentation was stopped and the amount of
statins in the water and the oil phase were measured. The results
are shown in Table 5.
5TABLE 5 Amount of statin (mg/l) in the different phases after 49
day of fermentation. Water phase Oil phase Total Volume of the
phase 6.6 0.4 7 Statin (mg/l) 2.37 1420 83.4
[0141] At the end of the fermentation 97 wt % of the statins were
found in the oil phase. A big difference is seen between the statin
levels in the oil and the water phase. The supplementation of oil
increases the total amount of statin in the reactor. A control
reactor without oil supplementation contained 1.8 mg/l statins,
while the reactor with oil supplementation shows a total amount of
83.4 mg/l statins. The reactor with oil supplementation shows a 45
times higher statin content compared with the amount of statins in
the fermentation without oil.
[0142] Addition of soybean oil to solid state fermentations also
stimulated the production of statin.
Example 4
[0143] Steps A, B and D. were done according to example 1.
[0144] C. Fermentation
[0145] C.1. Preparation of an Inoculum
[0146] A shake flask containing dehulled soybeans is inoculated
with filamentous spores suspended in a physiological water solution
containing 0.1 wt % Tween 80 (polyoxyethylene sorbitan fatty acid
ester, available from ICI Specialty Chemicals.TM.). This shake
flask is incubated to let the mould grow. This results in a
Monascus spore suspension, which was adjusted by dilution to a
concentration of 1*10.sup.6 spores/ml.
[0147] C.2. Preparation of Fermentation Medium
[0148] 1 kg of dehulled soybeans were soaked in tap water
(50.degree. C.) for 30 minutes and subsequently air dried for two
hours. Subsequently 50 g batches of the dried dehulled beans were
brought in to 300 ml Erlenmeyer flasks.
[0149] C.3. Fermentation
[0150] The shake flasks were each inoculated with 1 ml of the
prepared Monascus spore suspension and incubated for 30 days at
30.degree. C. A sample from the shake flask was taken every week to
monitor the statin production.
[0151] After fermentation, about 600 g soybeans remained. The
analysis according to step D results in a statin content of 2800
mg/kg product.
[0152] E. Isoflavone analysis of fermentation product
[0153] The isoflavone concentration was measured according to the
HPLC method described in Franke A.A., et al. (1998): HPLC analysis
of isoflavonoids and other phenolic agents from foods and human
fluids; Proceed. Soc. Exp. Biol. Med; 217 (3), 274-280.
[0154] Two samples were tested. The first (comparative) sample was
taken from non-fermented fermentation medium, as prepared in step
C2 above. The second sample was of equal (solid) weight, but taken
from the fermentation product. The results of the isoflavone
measurement of these samples are given in table 3.
6TABLE 3 Isoflavone concentration in fermented and non- fermented
soy Isoflavone concentration (g/kg) Fermentation medium
Fermentation Isoflavone (unfermented) product Daidzin 1.107 0.304
Genistin 1.608 0.476 Daidzein 0.057 0.9 Genistein 0.085 0.494 Total
2.9 2.2
[0155] Table 3 shows that the amount of isoflavones in total in the
fermentation product is slightly decreased compared to the
non-fermented material, but surprisingly the amounts of genistein
and daidzein are increased. The fermentation product contains
substantial amounts of isoflavones in addition to the statin.
* * * * *