WO2007011222A2 - Cholesterol-lowering food additive - Google Patents

Cholesterol-lowering food additive Download PDF

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Publication number
WO2007011222A2
WO2007011222A2 PCT/NL2006/050180 NL2006050180W WO2007011222A2 WO 2007011222 A2 WO2007011222 A2 WO 2007011222A2 NL 2006050180 W NL2006050180 W NL 2006050180W WO 2007011222 A2 WO2007011222 A2 WO 2007011222A2
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WIPO (PCT)
Prior art keywords
polysaccharide
units
branching
links
cholesterol
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PCT/NL2006/050180
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French (fr)
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WO2007011222A3 (en
Inventor
Gerritdina Hendrika Van Geel-Schutten
Josepus Jan Emeis
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Nederlandse Organisatie Voor Toegepast-Natuurwetenschappelijk Onderzoek Tno
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Publication of WO2007011222A2 publication Critical patent/WO2007011222A2/en
Publication of WO2007011222A3 publication Critical patent/WO2007011222A3/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y204/00—Glycosyltransferases (2.4)
    • C12Y204/01—Hexosyltransferases (2.4.1)
    • C12Y204/01001—Phosphorylase (2.4.1.1)
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
    • A23L29/20—Foods or foodstuffs containing additives; Preparation or treatment thereof containing gelling or thickening agents
    • A23L29/269—Foods or foodstuffs containing additives; Preparation or treatment thereof containing gelling or thickening agents of microbial origin, e.g. xanthan or dextran
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
    • A23L29/20—Foods or foodstuffs containing additives; Preparation or treatment thereof containing gelling or thickening agents
    • A23L29/269—Foods or foodstuffs containing additives; Preparation or treatment thereof containing gelling or thickening agents of microbial origin, e.g. xanthan or dextran
    • A23L29/274—Pullulan
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
    • A23L29/20—Foods or foodstuffs containing additives; Preparation or treatment thereof containing gelling or thickening agents
    • A23L29/275—Foods or foodstuffs containing additives; Preparation or treatment thereof containing gelling or thickening agents of animal origin, e.g. chitin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y204/00—Glycosyltransferases (2.4)
    • C12Y204/01—Hexosyltransferases (2.4.1)
    • C12Y204/01018—1,4-Alpha-glucan branching enzyme (2.4.1.18), i.e. glucan branching enzyme
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2400/00—Lactic or propionic acid bacteria
    • A23V2400/11—Lactobacillus
    • A23V2400/173—Reuteri

Definitions

  • the present invention is in the field of nutritional health products.
  • the invention is concerned with the use of certain high molecular weight polysaccharides as cholesterol-lowering agents and for use in food products such as bakery products and dairy products and other food products.
  • Cardiovascular diseases are among the most important diseases and are the prime cause of death in the Western world. Increased blood levels of cholesterol and triglyceride imply an increased risk in the generation of cardiovascular diseases. Hence, many research efforts have been dedicated to the development of food ingredients and food products capable of lowering the blood cholesterol levels.
  • ⁇ -glucans such as oat ⁇ -glucans ( ⁇ -1, 3/1,4) or microbial ⁇ -glucans ( ⁇ - 1,3/1, 6) for lowering blood cholesterol levels is well known, see e.g. JP-A 6-135839.
  • WO 02/082929 discloses a food product containing dietary fibres, such as ⁇ -glucans from oat or inulin, and plant sterols for lowering serum total and/or LDL cholesterol levels.
  • EP-A 1060673 (WO 00/70964) describes the use of dextrans ( ⁇ -l,6-glucans) e.g. from Leuconostoc mesenteroides for selectively increasing the production of propionate in the gastro-intestinal tract of a mammal and for decreasing blood cholesterol and blood triglyceride levels in a mammal.
  • JP-A 59-137414 describes a drug containing elsinan (a glucan having ⁇ l,3- ⁇ l,4- ⁇ l,4 recurring units) for preventing arteriosclerosis.
  • the object of the invention is to provide a food ingredient enabling the preparation of food products exhibiting improved health characteristics, especially as to cholesterol lowering and prevention and treatment of hypercholesteroleaemia.
  • the blood cholesterol levels can be effectively decreased by supplying a food product containing a polysaccharide which is characterised by a molecular weight of at least 10 4 Dalton and an anhydroglucose content of at least 80%.
  • the invention is concerned with a method for decreasing blood cholesterol levels in a mammal, the method comprising enterally administering to a mammal a nutritional composition which contains such a polysaccharide.
  • the invention is further concerned with a food composition containing such a polysaccharide.
  • the polysaccharide to be used according to the invention preferably has a minimum branching percentage of 6%, more preferably at least 7%, even more preferably at least 8%, most preferably at least 10%.
  • the branching percentage may be as high as e.g. 30%, although percentages higher than 24% do not have additional advantages, the preferred upper level being 20%.
  • the branching percentage of the polysaccharide which can also be referred to as degree of branching, is defined as the proportion of branching units, i.e. anhydroglycose (monose) units being linked to three neighbouring anhydroglycose units.
  • the branching percentage i.e.
  • the proportion of branching units can be calculated by known methods including spectroscopic methods such as 1 H-NMR or 13 C-NMR spectroscopy and/or chromatographic methods either on the polysaccharide as such or after selective hydrolysis using iso-amylase followed by a coulometric titration with iodine, or after chemical preparation such as methylation followed by hydrolysis (see e.g. Van Geel-Schutten, et ah, Appl Environ. Microbiol. (1999) 65, 3008-3014).
  • spectroscopic methods such as 1 H-NMR or 13 C-NMR spectroscopy and/or chromatographic methods either on the polysaccharide as such or after selective hydrolysis using iso-amylase followed by a coulometric titration with iodine, or after chemical preparation such as methylation followed by hydrolysis (see e.g. Van Geel-Schutten, et ah, Appl Environ
  • the remainder of the anhydroglycose units are either chain units, i.e. units linked to two neighbouring units, or terminal units, i.e. units linked to a single neighbouring unit. Generally 46-88%, in particular 60-84% of the anhydroglycose units are chain units and 6-30%, in particular 8-24% are terminal units. In the polysaccharide, the proportion of branching units is close to the proportion of terminal units, as the total number of terminal units will normally equal the number of branching units plus two, and for high-molecular polysaccharides these numbers will practically become equal.
  • the degree of branching can also be estimated as the percentage of branching units plus the percentage of terminal units, divided by two.
  • the poly- saccharide to be used according to the invention can have a degree of pluriform linking, i.e. the links between the anhydroglycose (monose) units should be of at least two (preferably two) types including ⁇ -1,6 links, for example cc- 1,4 and ⁇ -1,6 or ⁇ -1,3 and CC- 1,6, wherein the two linking types should each have a frequency of at least 6%, more preferably at least 10%.
  • the links between the anhydroglucose units of the polysaccharide of the invention are of a least two different types.
  • the degree of second-type (minority-type) linking is at least 6%, more preferably at least 8% or even at least 10%.
  • An optional third or further type may be present in any percentage.
  • at least 6% of the links between the anhydroglucose units are CC- 1,6 links, and preferably at least 6%are CC- 1,4 links, which cc-1,6 and CC- 1,4 links may be either to chain units or to branching units or both.
  • the polysaccharide is in particular a glucan, i.e. comprising at least 80%, more preferably at least 90%, most preferably at least 96% of anhydroglucose units, calculated on the total number of anhydroglycose (any monose) units.
  • the polysaccharide can essentially be a homopolysaccharide.
  • the glucan is an cc- glucan, i.e. having the majority of anhydroglucose units linked through their cc- anomeric position. The remainder of the units, if any, can e.g.
  • the cc-glucan should contain cc-1,6 links, but the proportion of 1,6-linked units should not exceed 90%, especially should not exceed 80%, i.e. the glucan is not a dextran-type glucan.
  • the polysaccharide has an cc- 1,6-linked anhydroglucose content of 5-70%, more preferably of 15-50%.
  • the remainder of the cc-linked units may be 1,2-, 1,3- and/or 1,4-linked units, and branching units such as 1,2,6- 1,3,6- and/or especially 1,4,6-linked units.
  • Glucans having a substantial proportion of cc-l,4-units were found to be very suitable.
  • the polysaccharide has an cc- 1,4-linked anhydroglucose content of 15-75%, especially of 25-70%.
  • such a preferred polysaccharide has an cc- 1,4,6-linked anhydroglucose content of 6-24%, especially 8-20%.
  • the polysaccharide has an cc-l,3-linked anhydroglucose content of 10-60%, especially of 15-40%.
  • the molecular weight of the polysaccharide may typically be above 10 4 Da, up to e.g. 10 s , i.e. having a degree of polymerisation (DP) of about 60 up to about 10 6 .
  • DP degree of polymerisation
  • smaller molecules e.g. down to a DP of about 10 are also suitable. It may be advantageous that the polysaccharide is relatively water-soluble.
  • the polysaccharide can be of any origin, such as plants, animals, microorganisms, and it can even be a (semi)synthetic product.
  • the polysaccharide is derived from a non-pathogenic organism, in particular a nonpathogenic, especially food-grade micro-organism, such as food-grade bacteria, yeasts and fungi.
  • Suitable micro-organisms are lactic acid bacteria and related bacteria, including the genera Lactobacillus, Leuconostoc, Lactococcus, Pediococcus, Propioni- bacterium and the like.
  • Lactobacillus strains for example Lb. reuteri, Lb.
  • the polysaccharides can be produced by (micro)organisms in a known way, e.g. by feeding the micro-organism a suitable carbohydrate source, such as sucrose, maltose, glucose, lactose, raffinose, etc., depending on the particular strain.
  • a suitable carbohydrate source such as sucrose, maltose, glucose, lactose, raffinose, etc., depending on the particular strain.
  • the polysaccharides are usually excreted by the micro-organism and can then simply be separated, if necessary, from the culture medium or production medium and from the cells by conventional means such as centrifugation, filtration, concentration, chromatography, etc.
  • the polysaccharides thus recovered may be fractionated where appropriate to remove short-chain analogues.
  • the polysaccharides may be used as such, or with (a part of) the production medium or even together with the producing microorganisms.
  • the polysaccharides may also be produced de novo by resting cells or by using isolated or immobilised glucan-producing enzymes (glucosyltransferases). Smaller molecules can be obtained by microbial synthesis or by partial hydrolysis of larger molecules.
  • the highly branched polysaccharides can be derived by modification of commonly available glucans - like starch, amylopectin, glycogen, pullulan or alternan - having a low degree of branching, using suitable micro-organisms or branching enzymes (EC 2.4.1.18) capable of producing the desired degree of branching.
  • suitable branching enzymes are ⁇ (l,4)-D-glucan: ⁇ (l,4)-glucan 6-glycosyltransferases and ⁇ (l,4)-D-glucan: orthophosphate ⁇ -glycosyltransferases (EC 2.4.1.1).
  • highly branched glucans by de novo synthesis from glucose or (phosphorylated) glucose derivatives using a combination of a glucan- synthesising enzyme and a branching enzyme, or by synthesis from sucrose using a combination of an amylosucrase and a branching enzyme.
  • highly branched ⁇ (l,4)(l,6)-glucans can be obtained from sucrose by the combined action of an amylosucrase and an ⁇ (l,4)-D-glucan: ⁇ (l,4)-glucan 6-glycosyltransferase, optionally expressed in modified crops.
  • a degree of branching up to 35% can be obtained as described in EP-A 1117802 (WO 00/22140, PlantTec).
  • the polysaccharide is not chemically modified, minor levels of substitution, e.g. by carboxyl, alkyl (such as methyl), hydroxyalkyl (such as hydroxyethyl), acyl (such as acetyl), phosphate, sulphate may be present, e.g. up to an average degree of substitution of 0.2, in particular not exceeding a degree of substitution of 0.1 (substituent per anhydroglycose unit).
  • the polysaccharides to be used according to the invention preferably have a reduced digestibility, i.e.
  • the digestibility (expressed as percentage glucose release in jejunum and ileum e.g. in the TNO gastro-intestinal model for the small intestine: Minekus et al, Alternatives to Laboratory Animals (ATLA) 23: 197-209, 1995) is less than 30% compared to native starch.
  • the polysaccharides have nutritional fibre characteristics.
  • An example of an excellent polysaccharide for use according to the invention is reuteran, a branched CC-1, 4/1, 6-glucan produced by some Lactobacillus reuteri strains such as strain LB 121 (see: van Geel-Schutten, G.H. et al, Appl. Microbiol. Biotechnol. (1998) 50, 697-703).
  • This glucan has a molecular weight of about 3,5 MDa and is highly branched with a structure consisting of a terminal, 4-substituted, 6-substituted, and 4,6-di-substituted ⁇ -glucose in a molar ratio 1.1 : 2.7 : 1.5 : 1.0; i.e. it has a degree of branching of around 15%.
  • the glucan and the enzyme producing it are described in WO 01/90372.
  • Another suitable glucan is glycogen, synthesised by animals, including mammals; glycogen usually has a degree of branching of 8-8.5%, while amylopectin, which is not be used according to the invention, usually has a degree of branching of 3.5-5%.
  • suitable polysaccharides including an ⁇ - 1,3/1, 6 glucan produced by L. reuteri strain 180 was deposited as LMG P- 18389 at the BCCM/LMG, are described in WO 03/008618.
  • non-branched polysaccharides include a glucan having both ⁇ - 1,4 and CC-1, 6 links (about 2:1) such as pullulan, produced by Aureobasidium pullulans and constituted by CC- 1,6 linked blocks of three CC-1,4 linked anhydroglucose (maltotriose) units, and similarly with both ⁇ -1,3 and CC- 1,6 links (alternating, about 1:1) such as alternan, produced by certain strains of Leuconostoc mesenteroid.es..
  • a food ingredient according to the invention typically contains at least 1 wt.% of a polysaccharide as defined above, in particular between 3 and 30 wt.%, and at least one further component selected from carbohydrates, lipids, proteins, vitamins, minerals, other (non- ⁇ -glucan) fibres (e.g. inulin), emulsifiers, and other food additives.
  • the ingredient may also comprise other cholesterol-lowering agents such as long-chain polyunsaturated fatty acids (as such or in lipids), sphingolipids (ceramides, glycol- sphingo lipids, sphingomyelins), phytosterols (sterols and stanols), ⁇ -glucans, etc.
  • the weight ratio between the polysaccharides of the invention and other cholesterol- lowering agents is between 19:1 and 1:19, more preferably between 9:1 and 1 :4, most preferably between 4: 1 and 1 :2.
  • the branched and/or pluriformly linked polysaccharide to be used according to the invention can be incorporated in various food products, including complete foods, such as clinical foods, and food supplements.
  • the food products can also have the form of bakery products, dairy products, snacks, bars, etc., sport drinks and other beverages and the like.
  • the amount of polysaccharide of the invention to be incorporated in the food products depends on the nature of food product and the level at which it is typically consumed.
  • the amount of the polysaccharide can be between 5 mg and 2,5 g, preferably between 25 and 1000 mg per 100 kcal, or similar amounts per 100 ml in case of a complete liquid food.
  • the levels may even be higher, e.g. between 20 mg and 1O g, especially between 10 mg and 4 g per 100 ml. Alternatively, these amounts can be present per 100 g, also in case of non-liquid food products.
  • the branched polysaccharide to be used according to the invention can be administered in the form of a pharmaceutical composition, such as tablets, drinks, sachets, syrups, powders, containing the polysaccharide together with conventional excipients, and optional further ingredients.
  • the amount of polysaccharide can then be between 0.1 and 20 g, especially between 1 and 1O g, for single or multiple (e.g. 2-4 per day) daily dosage units.
  • the invention also pertains to the use of the polysaccharides as defined above for lowering blood cholesterol levels and/or blood triglyceride levels, or for preventing or treating hypercholesterolaemia or cardiovascular diseases, by administering the poly- saccharides in an amount of between 0.1 and 50 g, preferably between 0.5 and 20 g, most preferably between 1 and 5 g per day, either as such or as a part of a nutritional or pharmaceutical composition.
  • Example 1 Effect ofreuteran in transgenic APOE*3Leiden mice fed a "Western-type" diet
  • mice were used in which the human gene for the so-called Leiden mutation of apolipoprotein E3 (APOE*3Leiden transgenic mice) has been incorporated by transgenesis. Because of this transgenic change, these so-called APOE*3Leiden mice have a humanised lipoprotein profile, and are extremely suitable for studying the effect of compounds on lipoprotein metabolism (Van Vlijmen et al. 1996 J Clin. Invest.
  • mice Two groups of APOE*3Leiden mice were fed with a standard diet ("Western"), one without supplementation and the other with supplementation of reuteran. The plasma lipids were monitored for two weeks from the start of the experiment.
  • the composition of the Western diet (control diet) is as follows:
  • the reuteran-containing diet is obtained by adding 10% reuteran to the Western diet, and compensating for reduced cholesterol (by adding 0.025 % cholesterol), without compensation for the other components of the diet.
  • mice were allowed to feed on the diet ad libitum for 15 days.
  • the plasma concentration of cholesterol was measured at the start and at the end of the experiment and expressed in mmol/1.
  • the results are summarised in the table below.

Abstract

Nutritional and pharmaceutical compositions having cholesterol- lowering activities can be produced by incorporating at least 1 wt.% of a polysaccharide which is characterised by a molecular weight of at least 104 Dalton, an α-anhydroglucose content of at least 80% and a branching percentage of at least 6%. The corresponding glucan preferably has α-1,4 and α-1,6 bonds. The polysaccharide can be produced by a lactic acid bacterium, in particular a Lactobacillus strain.

Description

Cholesterol-lowering food additive
[0001] The present invention is in the field of nutritional health products. In particular, the invention is concerned with the use of certain high molecular weight polysaccharides as cholesterol-lowering agents and for use in food products such as bakery products and dairy products and other food products.
Background of the invention
[0002] Cardiovascular diseases are among the most important diseases and are the prime cause of death in the Western world. Increased blood levels of cholesterol and triglyceride imply an increased risk in the generation of cardiovascular diseases. Hence, many research efforts have been dedicated to the development of food ingredients and food products capable of lowering the blood cholesterol levels.
[0003] The use of β-glucans, such as oat β-glucans (β-1, 3/1,4) or microbial β-glucans (β- 1,3/1, 6) for lowering blood cholesterol levels is well known, see e.g. JP-A 6-135839. WO 02/082929 discloses a food product containing dietary fibres, such as β-glucans from oat or inulin, and plant sterols for lowering serum total and/or LDL cholesterol levels.
[0004] EP-A 1060673 (WO 00/70964) describes the use of dextrans (α-l,6-glucans) e.g. from Leuconostoc mesenteroides for selectively increasing the production of propionate in the gastro-intestinal tract of a mammal and for decreasing blood cholesterol and blood triglyceride levels in a mammal. JP-A 59-137414 describes a drug containing elsinan (a glucan having αl,3-αl,4-αl,4 recurring units) for preventing arteriosclerosis.
[0005] The object of the invention is to provide a food ingredient enabling the preparation of food products exhibiting improved health characteristics, especially as to cholesterol lowering and prevention and treatment of hypercholesteroleaemia.
Description of the invention
[0006] It was found that the blood cholesterol levels can be effectively decreased by supplying a food product containing a polysaccharide which is characterised by a molecular weight of at least 104 Dalton and an anhydroglucose content of at least 80%. Thus, the invention is concerned with a method for decreasing blood cholesterol levels in a mammal, the method comprising enterally administering to a mammal a nutritional composition which contains such a polysaccharide. The invention is further concerned with a food composition containing such a polysaccharide.
[0007] The polysaccharide to be used according to the invention preferably has a minimum branching percentage of 6%, more preferably at least 7%, even more preferably at least 8%, most preferably at least 10%. The branching percentage may be as high as e.g. 30%, although percentages higher than 24% do not have additional advantages, the preferred upper level being 20%. The branching percentage of the polysaccharide, which can also be referred to as degree of branching, is defined as the proportion of branching units, i.e. anhydroglycose (monose) units being linked to three neighbouring anhydroglycose units. The branching percentage, i.e. the proportion of branching units, can be calculated by known methods including spectroscopic methods such as 1H-NMR or 13C-NMR spectroscopy and/or chromatographic methods either on the polysaccharide as such or after selective hydrolysis using iso-amylase followed by a coulometric titration with iodine, or after chemical preparation such as methylation followed by hydrolysis (see e.g. Van Geel-Schutten, et ah, Appl Environ. Microbiol. (1999) 65, 3008-3014). Mixtures of highly branched polysaccharides and polysaccharides having a lower degree of branching (e.g. between 3 and 6 %) can also be used, while it is preferred that the average degree of branching of the mixture is above 6%. [0008] The remainder of the anhydroglycose units are either chain units, i.e. units linked to two neighbouring units, or terminal units, i.e. units linked to a single neighbouring unit. Generally 46-88%, in particular 60-84% of the anhydroglycose units are chain units and 6-30%, in particular 8-24% are terminal units. In the polysaccharide, the proportion of branching units is close to the proportion of terminal units, as the total number of terminal units will normally equal the number of branching units plus two, and for high-molecular polysaccharides these numbers will practically become equal. Thus, the degree of branching can also be estimated as the percentage of branching units plus the percentage of terminal units, divided by two. [0009] Instead of, or in addition to a minimum branching percentage, the poly- saccharide to be used according to the invention can have a degree of pluriform linking, i.e. the links between the anhydroglycose (monose) units should be of at least two (preferably two) types including α-1,6 links, for example cc- 1,4 and α-1,6 or α-1,3 and CC- 1,6, wherein the two linking types should each have a frequency of at least 6%, more preferably at least 10%. Thus, it is preferred that the links between the anhydroglucose units of the polysaccharide of the invention are of a least two different types. The degree of second-type (minority-type) linking, be it branching or not, is at least 6%, more preferably at least 8% or even at least 10%. An optional third or further type may be present in any percentage. Thus, in the polysaccharide to be used according to the invention, at least 6% of the links between the anhydroglucose units are CC- 1,6 links, and preferably at least 6%are CC- 1,4 links, which cc-1,6 and CC- 1,4 links may be either to chain units or to branching units or both. [0010] The polysaccharide is in particular a glucan, i.e. comprising at least 80%, more preferably at least 90%, most preferably at least 96% of anhydroglucose units, calculated on the total number of anhydroglycose (any monose) units. Thus, the polysaccharide can essentially be a homopolysaccharide. Preferably, the glucan is an cc- glucan, i.e. having the majority of anhydroglucose units linked through their cc- anomeric position. The remainder of the units, if any, can e.g. be of the type of β-linked glucose, mannose, rhamnose, galactose, fructose, xylose, or the like. The presence of low levels of non-saccharidic residues, such as lipid (lipopolysaccharides), peptidic (proteoglycans) or other residues, is not preferred, but is not detrimental up to a level of about 20% (by weight of the total average molecule). [0011] The cc-glucan should contain cc-1,6 links, but the proportion of 1,6-linked units should not exceed 90%, especially should not exceed 80%, i.e. the glucan is not a dextran-type glucan. Preferably, the polysaccharide has an cc- 1,6-linked anhydroglucose content of 5-70%, more preferably of 15-50%. The remainder of the cc-linked units may be 1,2-, 1,3- and/or 1,4-linked units, and branching units such as 1,2,6- 1,3,6- and/or especially 1,4,6-linked units. Glucans having a substantial proportion of cc-l,4-units were found to be very suitable. Thus, in a preferred embodiment, the polysaccharide has an cc- 1,4-linked anhydroglucose content of 15-75%, especially of 25-70%. In particular, such a preferred polysaccharide has an cc- 1,4,6-linked anhydroglucose content of 6-24%, especially 8-20%. In another preferred embodiment, the polysaccharide has an cc-l,3-linked anhydroglucose content of 10-60%, especially of 15-40%. [0012] The molecular weight of the polysaccharide may typically be above 104 Da, up to e.g. 10s , i.e. having a degree of polymerisation (DP) of about 60 up to about 106. However, smaller molecules, e.g. down to a DP of about 10, are also suitable. It may be advantageous that the polysaccharide is relatively water-soluble. [0013] The polysaccharide can be of any origin, such as plants, animals, microorganisms, and it can even be a (semi)synthetic product. However, it is preferred that the polysaccharide is derived from a non-pathogenic organism, in particular a nonpathogenic, especially food-grade micro-organism, such as food-grade bacteria, yeasts and fungi. Suitable micro-organisms are lactic acid bacteria and related bacteria, including the genera Lactobacillus, Leuconostoc, Lactococcus, Pediococcus, Propioni- bacterium and the like. Especially preferred are Lactobacillus strains, for example Lb. reuteri, Lb. plantarum, Lb. fermentum, Lb. parabuchneri and Lb. sakei. Another suitable micro-organism is Aureobasidium pullulans. [0014] The polysaccharides can be produced by (micro)organisms in a known way, e.g. by feeding the micro-organism a suitable carbohydrate source, such as sucrose, maltose, glucose, lactose, raffinose, etc., depending on the particular strain. The polysaccharides are usually excreted by the micro-organism and can then simply be separated, if necessary, from the culture medium or production medium and from the cells by conventional means such as centrifugation, filtration, concentration, chromatography, etc.. The polysaccharides thus recovered may be fractionated where appropriate to remove short-chain analogues. Alternatively, the polysaccharides may be used as such, or with (a part of) the production medium or even together with the producing microorganisms. The polysaccharides may also be produced de novo by resting cells or by using isolated or immobilised glucan-producing enzymes (glucosyltransferases). Smaller molecules can be obtained by microbial synthesis or by partial hydrolysis of larger molecules.
[0015] Alternatively, the highly branched polysaccharides can be derived by modification of commonly available glucans - like starch, amylopectin, glycogen, pullulan or alternan - having a low degree of branching, using suitable micro-organisms or branching enzymes (EC 2.4.1.18) capable of producing the desired degree of branching. Examples of suitable branching enzymes are α(l,4)-D-glucan: α(l,4)-glucan 6-glycosyltransferases and α(l,4)-D-glucan: orthophosphate α-glycosyltransferases (EC 2.4.1.1). [0016] It is also possible to produce the highly branched glucans by de novo synthesis from glucose or (phosphorylated) glucose derivatives using a combination of a glucan- synthesising enzyme and a branching enzyme, or by synthesis from sucrose using a combination of an amylosucrase and a branching enzyme. As an example, highly branched α(l,4)(l,6)-glucans can be obtained from sucrose by the combined action of an amylosucrase and an α(l,4)-D-glucan: α(l,4)-glucan 6-glycosyltransferase, optionally expressed in modified crops. A degree of branching up to 35% can be obtained as described in EP-A 1117802 (WO 00/22140, PlantTec). [0017] Although it is preferred that the polysaccharide is not chemically modified, minor levels of substitution, e.g. by carboxyl, alkyl (such as methyl), hydroxyalkyl (such as hydroxyethyl), acyl (such as acetyl), phosphate, sulphate may be present, e.g. up to an average degree of substitution of 0.2, in particular not exceeding a degree of substitution of 0.1 (substituent per anhydroglycose unit). [0018] The polysaccharides to be used according to the invention preferably have a reduced digestibility, i.e. increased resistance to degradation by intestinal enzymes, compared to e.g. starch. Preferably, the digestibility (expressed as percentage glucose release in jejunum and ileum e.g. in the TNO gastro-intestinal model for the small intestine: Minekus et al, Alternatives to Laboratory Animals (ATLA) 23: 197-209, 1995) is less than 30% compared to native starch. Thus, the polysaccharides have nutritional fibre characteristics.
[0019] An example of an excellent polysaccharide for use according to the invention is reuteran, a branched CC-1, 4/1, 6-glucan produced by some Lactobacillus reuteri strains such as strain LB 121 (see: van Geel-Schutten, G.H. et al, Appl. Microbiol. Biotechnol. (1998) 50, 697-703). This glucan has a molecular weight of about 3,5 MDa and is highly branched with a structure consisting of a terminal, 4-substituted, 6-substituted, and 4,6-di-substituted α-glucose in a molar ratio 1.1 : 2.7 : 1.5 : 1.0; i.e. it has a degree of branching of around 15%. The glucan and the enzyme producing it are described in WO 01/90372. [0020] Another suitable glucan is glycogen, synthesised by animals, including mammals; glycogen usually has a degree of branching of 8-8.5%, while amylopectin, which is not be used according to the invention, usually has a degree of branching of 3.5-5%. Further examples of suitable polysaccharides, including an α- 1,3/1, 6 glucan produced by L. reuteri strain 180 was deposited as LMG P- 18389 at the BCCM/LMG, are described in WO 03/008618.
[0021] Examples of suitable non-branched polysaccharides include a glucan having both α- 1,4 and CC-1, 6 links (about 2:1) such as pullulan, produced by Aureobasidium pullulans and constituted by CC- 1,6 linked blocks of three CC-1,4 linked anhydroglucose (maltotriose) units, and similarly with both α-1,3 and CC- 1,6 links (alternating, about 1:1) such as alternan, produced by certain strains of Leuconostoc mesenteroid.es.. [0022] A food ingredient according to the invention typically contains at least 1 wt.% of a polysaccharide as defined above, in particular between 3 and 30 wt.%, and at least one further component selected from carbohydrates, lipids, proteins, vitamins, minerals, other (non-α-glucan) fibres (e.g. inulin), emulsifiers, and other food additives. The ingredient may also comprise other cholesterol-lowering agents such as long-chain polyunsaturated fatty acids (as such or in lipids), sphingolipids (ceramides, glycol- sphingo lipids, sphingomyelins), phytosterols (sterols and stanols), β-glucans, etc. Preferably, the weight ratio between the polysaccharides of the invention and other cholesterol- lowering agents is between 19:1 and 1:19, more preferably between 9:1 and 1 :4, most preferably between 4: 1 and 1 :2.
[0023] The branched and/or pluriformly linked polysaccharide to be used according to the invention can be incorporated in various food products, including complete foods, such as clinical foods, and food supplements. The food products can also have the form of bakery products, dairy products, snacks, bars, etc., sport drinks and other beverages and the like. The amount of polysaccharide of the invention to be incorporated in the food products depends on the nature of food product and the level at which it is typically consumed. For a complete food, such as a clinical food, the amount of the polysaccharide can be between 5 mg and 2,5 g, preferably between 25 and 1000 mg per 100 kcal, or similar amounts per 100 ml in case of a complete liquid food. In case of liquids or semi-liquids such as drinks, yoghurts the levels may even be higher, e.g. between 20 mg and 1O g, especially between 10 mg and 4 g per 100 ml. Alternatively, these amounts can be present per 100 g, also in case of non-liquid food products. [0024] Alternatively, the branched polysaccharide to be used according to the invention can be administered in the form of a pharmaceutical composition, such as tablets, drinks, sachets, syrups, powders, containing the polysaccharide together with conventional excipients, and optional further ingredients. The amount of polysaccharide can then be between 0.1 and 20 g, especially between 1 and 1O g, for single or multiple (e.g. 2-4 per day) daily dosage units.
[0025] The invention also pertains to the use of the polysaccharides as defined above for lowering blood cholesterol levels and/or blood triglyceride levels, or for preventing or treating hypercholesterolaemia or cardiovascular diseases, by administering the poly- saccharides in an amount of between 0.1 and 50 g, preferably between 0.5 and 20 g, most preferably between 1 and 5 g per day, either as such or as a part of a nutritional or pharmaceutical composition.
EXAMPLES
Example 1: Effect ofreuteran in transgenic APOE*3Leiden mice fed a "Western-type" diet
In this experiment mice were used in which the human gene for the so-called Leiden mutation of apolipoprotein E3 (APOE*3Leiden transgenic mice) has been incorporated by transgenesis. Because of this transgenic change, these so-called APOE*3Leiden mice have a humanised lipoprotein profile, and are extremely suitable for studying the effect of compounds on lipoprotein metabolism (Van Vlijmen et al. 1996 J Clin. Invest.
97:1184-1192; Van Vlijmen et al. 1998 Arzneimittelforschung 48: 396-402).
Two groups of APOE*3Leiden mice were fed with a standard diet ("Western"), one without supplementation and the other with supplementation of reuteran. The plasma lipids were monitored for two weeks from the start of the experiment.
The composition of the Western diet (control diet) is as follows:
Figure imgf000008_0001
The reuteran-containing diet is obtained by adding 10% reuteran to the Western diet, and compensating for reduced cholesterol (by adding 0.025 % cholesterol), without compensation for the other components of the diet.
The mice were allowed to feed on the diet ad libitum for 15 days. The plasma concentration of cholesterol was measured at the start and at the end of the experiment and expressed in mmol/1. The results are summarised in the table below. Standard diet Standard diet + 10% reuteran t = 0 t = 15 d t = 0 t = 15 d cholesterol level 14. 8 13.0 15.5 9.8 in mmol/1 (± sd) (± 2 •4) (± 1.2) (± 3.6) (± 1.7)
The results show that the presence of reuteran in the diet leads to a significant reduction of cholesterol levels in the plasma after 15 days. The triglyceride levels are affected to a lower extent.

Claims

Claims
1. Use of a polysaccharide for preparing a nutritional or pharmaceutical composition for lowering blood cholesterol levels, the polysaccharide being characterised by a molecular weight of at least 104 Dalton and an anhydroglucose content of at least 80%, and by the links between the anhydroglucose units being of a least two different types comprising α-1,6 links and CC- 1,4 links, each one accounting for at least 6% of the total number of links.
2. Use according to claim 1, wherein the polysaccharide has a branching percentage of between 6 and 24%.
3. Use according to claim 1 or 2, wherein the polysaccharide has an α-l,6-linked anhydroglucose content of 5-70%, preferably of 15-50%.
4. Use according to any one of claims 1-3, wherein the polysaccharide has an α-1,4- linked anhydroglucose content of 15-75%, preferably of 25-70%.
5. Use according to any one of claims 1-4, wherein the polysaccharide is produced by a lactic acid bacterium, in particular a Lactobacillus strain.
6. Use according to any one of claims 1-5, wherein the polysaccharide is administered at a level of between 0.5 and 20 g per day.
7. Use according to any one of claims 1-6, for preventing or treating cardiovascular diseases.
8. A nutritional or pharmaceutical composition containing a polysaccharide as defined in any one of claims 1-6 together with another cholesterol- lowering agent.
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US10568839B2 (en) 2011-01-11 2020-02-25 Capsugel Belgium Nv Hard capsules
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US11576870B2 (en) 2017-04-14 2023-02-14 Capsugel Belgium Nv Pullulan capsules
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