HK1101555B

HK1101555B - Composition comprising protein material and non-oxidizable fatty acid entities

Info

Publication number: HK1101555B
Application number: HK07109601.5A
Authority: HK
Inventors: 罗尔夫．贝格
Original assignee: 卑尔根技术转让公司
Priority date: 2004-07-19
Filing date: 2005-07-19
Publication date: 2013-10-11

Abstract

The publication number of the designated patent applied for is CN101010101A.

Description

Composition comprising proteinaceous material and non-oxidizable fatty acid entities

Technical Field

The use of a combination of non β -oxidizable fatty acid entities and proteinaceous substances has shown a surprising synergistic effect. The present invention relates to compositions prepared from a combination of a compound comprising non β -oxidizable fatty acid bodies and a proteinaceous material. Use of said composition for the preparation of a medicament or nutritional composition for the prevention and/or treatment of insulin resistance, obesity, diabetes, fatty liver, hypercholesterolemia, dyslipidemia, arteriosclerosis, coronary heart disease, thrombosis, stenosis, secondary stenosis, myocardial infarction, stroke, hypertension, endothelial dysfunction, procoagulant state, polycystic ovary syndrome, metabolic syndrome, cancer, inflammatory diseases and proliferative skin diseases. The composition may also be used as an additive to animal feed for conventional breeding of animals to generally affect their body composition, particularly fatty acid composition.

Technical Field

In earlier patent applications, the present inventors have described applications of the non β -oxidizable fatty acid analogues of the present invention in the treatment and prevention of obesity (NO 20005461), diabetes (NO 20005462), primary and secondary stenosis (NO 20005463), cancer (NO 20025930), proliferative skin diseases (NO 20031080), inflammatory and autoimmune diseases (NO 20032054). In other earlier patent applications, the inventors have described beneficial applications of the protein material of the invention, including single-cell protein material (NO 20033082), and fish protein hydrolysate (NO 20033078).

Disclosure of Invention

Surprisingly, the present inventors have now shown that the combined use of non β -oxidizable fatty acid entities with proteinaceous substances has a synergistic beneficial biological effect. The inventors show that the combination of non β -oxidizable fatty acid entities with proteinaceous substances reduces the concentration of plasma cholesterol, triglycerides and phospholipids and increases fatty acyl-coa oxidase activity. Furthermore, the inventors describe how non β -oxidizable fatty acid entities and protein material can be added directly to animal feed. The feed is digestible and has shown a surprising effect on the fatty acid composition of the animal. Based on these unexpected findings, it is therefore expected that the combination of non β -oxidizable fatty acid entities and proteinaceous substances will have an increased prophylactic and/or therapeutic effect on all diseases for which non β -oxidizable fatty acid entities are effective, compared to the effect of said fatty acid entities alone.

Detailed Description

The present invention relates to the use of a preparation comprising the following combination:

1) a proteinaceous substance; and

2) one or more compounds comprising a non-beta-oxidizable fatty acid entity represented by

(a) General formula R' -COO- (CH)₂)_2n+1-X-R', wherein X is a sulfur atom, a selenium atom, an oxygen atom, CH₂Radicals, SO radicals or SO₂A group; n is an integer of 0 to 11; and R 'is a linear or branched alkyl group, saturated or unsaturated, optionally substituted, wherein the backbone of R' contains from 13 to 23 carbon atoms and optionally one or more groups selected from the group consisting of oxygen, sulfur, selenium, oxygen, CH₂Radical, SO radical and SO₂Heterogroups of groups of bases (heterogroups); and R' is a hydrogen atom or an alkyl group containing 1 to 4 carbon atoms; and/or

(b) The general formula (I),

wherein R1, R2, and R3 represent

i) A hydrogen atom; or

ii) a group having the formula CO-R, wherein R is a linear or branched alkyl, saturated or unsaturated, optionally substituted, and the backbone of said R contains 1 to 25 carbon atoms; or

iii) has the formula CO- (CH)₂)_2n+1A group of-X-R', wherein X is a sulfur atom, a selenium atom, an oxygen atom, CH₂Radicals, SO radicals or SO₂A group; n is an integer of 0 to 11; and R 'is a linear or branched alkyl, saturated or unsaturated, optionally substituted, wherein the backbone of R' contains from 13 to 23 carbon atoms and optionally one or more groups selected from the group consisting of oxygen, sulfur, selenium, oxygen, CH₂Radical, SO radical and SO₂A hetero group of the group of radicals;

iv) is selected from the group consisting of-PO₃CH₂CHNH₃COOH (serine), PO₃CH₂CH₂NH₃(ethanolamine), PO₃CH₂CH₂N(CH₃)₃(Choline), PO₃CH₂CHOHCH₂OH (Glycerol) and PO₃(CHOH)₆(myo-inositol) group of entities;

wherein R1, R2, and R3 are each independently selected from i), ii), iii), or iv), but at least one of R1, R2, or R3 is defined by iii); and/or

(c) A compound of the general formula (II),

wherein A1, A2 and A3 are each independently selected from and represent an oxygen atom, a sulfur atom or a N-R4 group, wherein R4 is a hydrogen atom or a linear or branched alkyl group, saturated or unsaturated, optionally substituted, containing from 1 to 5 carbon atoms;

wherein R1, R2, and R3 represent

i) A hydrogen atom or a linear or branched alkyl, saturated or unsaturated, optionally substituted, group containing from 1 to 23 carbon atoms; or

iii) has the formula CO- (CH)₂)_2n+1A group of-X-R', wherein X is a sulfur atom, a selenium atom, an oxygen atom, CH₂Radicals, SO radicals or SO₂A group; n is an integer of 0 to 11; and R 'is a linear or branched alkyl, saturated or unsaturated, optionally substituted, wherein the backbone of R' contains from 13 to 23 carbon atoms and optionally one or more groups selected from the group consisting of oxygen, sulfur, selenium, oxygen, CH₂Radicals, SO radicals or SO₂A hetero group of the group of radicals;

A salt, prodrug or complex of a compound according to (a) - (c).

In a preferred embodiment of the compounds according to the invention, at least one of R1, R2 or R3 is alkyl.

In a preferred embodiment of the compounds according to the invention, at least one of R1, R2 or R3 is an alkene.

In a preferred embodiment of the compounds according to the invention, at least one of R1, R2 or R3 is an alkyne.

In a preferred embodiment of the compounds according to the invention, at least one of R1, R2 or R3 is tetradecylthioacetic acid.

In a preferred embodiment of the compounds according to the invention, at least one of R1, R2 or R3 is tetradecylselenoacetic acid (tetradecylselenoacetic acid).

A preferred embodiment of the compounds according to the invention are non β -oxidizable fatty acids.

In a preferred embodiment of the compounds according to the invention, X is a sulfur atom or a selenium atom.

Preferred embodiments of the compounds according to the invention are tetradecylthioacetic acid (TTA), tetradecylselenoacetic acid and 3-thio-15-heptadecyne.

In a preferred embodiment of the compounds according to the invention, n is 0 or 1.

In a preferred embodiment of the compound according to the invention, said compound is a phospholipid, wherein said phospholipid is selected from the group comprising phosphatidylserine, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylglycerol, diphosphatidylglycerol.

In a preferred embodiment of the compound according to the invention, said compound is triacylglycerol.

In a preferred embodiment of the compound according to the invention, said compound is a diacylglycerol.

In a preferred embodiment of the compound according to the invention, said compound is a monoacylglycerol.

In a preferred embodiment of the compounds according to the invention, said compounds are the Phosphatidylcholine (PC) derivative 1, 2-ditetradecylthioacetyl-sn-glycero-3-phosphorylcholine (1, 2-didecylthioacetyl-sn-glycero-3-phosphorylcholine).

In a preferred embodiment of the compound according to the invention, said compound is the Phosphatidylethanolamine (PE) derivative 1, 2-bistetradecylthioacetyl-sn-glycero-3-phosphoethanolamine (1, 2-didecylthioacetyl-sn-glycero-3-phosphoethanolamine).

Preferred embodiments of the compounds according to the invention are mono-, di-or triacylglycerides.

A preferred embodiment of the compounds according to the invention are triacylglycerides comprising tetradecylthioacetic acid (TTA).

In a preferred embodiment of the compounds according to formula (II), A1 and A3 both represent oxygen atoms, while A2 represents a sulfur atom or a N-R4 group in which R4 is a hydrogen atom or a linear or branched alkyl group, saturated or unsaturated, optionally substituted, containing from 1 to 5 carbon atoms.

The compounds according to the invention are analogues of natural compounds and are also recognized by the same system that handles natural compounds, including β -and in some cases ω -enzymes that oxidize natural long chain fatty acids. The analogs are different from their natural counterparts as they cannot be fully oxidized in this manner.

The compounds according to the invention may be non-beta-oxidizable fatty acid analogues, such as R' CCO- (CH)₂)_2n+1-X-R' is as defined in. However, the compound may also be a more complex structure derived from one or more of the non β -oxidizable fatty acid analogues, as represented by general formula (I) or (II). These compounds are analogs of naturally occurring mono-, di-, and triacylglycerols or phospholipids, including phosphatidylserine, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylglycerol, and diphosphatidylglycerol. The compounds may also contain substitutions in the glycerol backbone as shown in formula (II). The substitution of oxygen is accomplished by replacing oxygen with a sulfur or nitrogen containing group. This can block hydrolysis prior to intestinal absorption, thus increasing the bioavailability of these compounds.

The above complex structures derived from one or more of said non β -oxidizable fatty acid entities have their effect because the fatty acid analogues they comprise cannot be fully β -oxidized. The complex structure may have the effect of an intact structure, and the effect of naturally occurring degradation products comprising the fatty acid analogues. Because the compounds cannot be fully beta oxidized, they will accumulate and this triggers an increase in beta oxidation of naturally occurring fatty acids. Many of these effects of the compounds according to the invention are due to this increase in beta oxidation.

During beta oxidation, a fatty acid is enzymatically oxidatively cleaved between carbons 2 and 3 (counting from the carboxyl terminus of the fatty acid), which results in the removal of two carbon atoms as acetic acid on one side of the oxidation site. This step is then repeated for the fatty acid shortened by two carbon atoms and repeated again until the fatty acid is fully oxidized. Beta oxidation is the usual way of catabolizing most fatty acids in the body. Blocking of beta oxidation with the compounds according to the invention is achieved by inserting a non-oxidizable group in the X position of the general formula according to the invention. Since the mechanism of beta oxidation is well known, X is defined as S, O, SO₂、CH₂Or Se. Any person skilled in the art will, without any inventive step, consider that all of these compounds can block beta oxidation in the same way.

Furthermore, the compound may comprise more than one block, i.e. R' may optionally comprise, in addition to X, one or more atoms selected from the group comprising an oxygen atom, a sulfur atom, a selenium atom, an oxygen atom, CH₂Radical, SO radical and SO₂Hetero groups of the group of groups. As an example, two or three sulfur can be inserted as X to induce changes in fatty acid degradation and thus modulated effects. The polysulfide atoms can also adjust polarity and stability to some extent. From a pharmacological point of view it is often desirable to be able to provide a wide range of compounds, rather than just a single compound, in order to avoid or counteract resistance problems.

In addition to the type of X, its location is also a problem. By how many CH there are₂The group placed between X and the carboxyl terminus of the fatty acid to define the distance of X from the carboxyl terminus of the fatty acidPer (CH)₂)_2n+1Where n is an integer from 0 to 11. Thus, that is to say, there are an odd number of CHs₂A group; the position of X relative to the carboxyl group therefore ultimately blocks beta oxidation. The selection range for n includes all variants of fatty acid analogues having the desired biological effect. Since in theory beta oxidation can act on an infinitely long molecule, n can thus be infinite, but in practice this is not the case. Fatty acids that normally undergo beta oxidation are typically 14-24 carbon atoms long, and thus this length is optimal for performing enzyme-catalyzed beta oxidation. The ranges for n and R' are thus specified such that the fatty acid entities will cover this range. (likewise, for analogs of the naturally occurring compound, option II) of formulae (I) and (II) and defining R has from 1 to 25 carbon groups, and option I) of formula (II) defines an alkyl group having from 1 to 23 carbon atoms). The total number of carbon atoms in the fatty acid backbone is preferably from 8 to 30, most preferably from 12 to 26. This size range is also ideal for uptake and transport of the fatty acid entities of the invention across the cell membrane.

Although all fatty acid analogues with a beta oxidation blocker X at odd positions away from the carboxy terminus block beta oxidation, the extent of their biological effect may vary. This is due to the difference in biological degradation time of different compounds. The inventors have conducted experiments to show the effect of moving X away from the carboxyl end of the fatty acid. In these experiments, the activity of mitochondrial beta oxidation of fatty acid analogues in the liver (nmol/min/mg/protein) was determined using sulfur at the 3, 5 and 7 positions relative to the carboxy terminus. The activity was 0.81 for the third position sulfur, 0.61 for the fifth position sulfur, 0.58 for the seventh position sulfur, and 0.47 for the non-beta oxidation blocking control palmitic acid. As expected, this shows that fatty acid analogues with different blocking positions do block beta oxidation and the effect is thereby reduced as the blocking position is further away from the carboxy terminus, as it requires a longer beta oxidation to reach the blocking position, so that more fatty acid analogues are subsequently degraded. However, since the drop from the third to fifth position is large, but the drop from the fifth to seventh position is small, it is reasonable to assume that this drop will continue to be small moving along the chain, and thus (compared to the control) it will actually be before the effect is seen at all.

Thus, as the compounds of the present invention, there are reasons to include fatty acid entities represented by general formulae (I) and (II) and other compounds (which comprise the fatty acid analogs) that block β oxidation at various distances from the carboxyl terminus of the analogs, since the compounds of the present invention in fact block β oxidation, even though their effects can be modulated. This regulation is after all different under a number of different (bearying) conditions; in different tissues, for many different doses (bearingdosage), and by modifying the fatty acid analogues so that it cannot be easily degraded, as will be described below. Thus, in the formula, all distances of the beta oxidation blocker from the carboxy terminus of the biologically relevant fatty acid analog are reasonably included.

Although fatty acid entities with a block at the X position as described are unable to undergo beta oxidation, they can still undergo omega oxidation. This is a generally rare and slower biological process that oxidizes fatty acids not from the carboxyl terminus, but from the methyl/hydrophobic head group, which is referred to herein as R'. In this pathway, the carbon atom at the omega terminus of fatty acids is hydroxylated by a member of the cytochrome P450 enzyme family. This hydroxylated fatty acid is subsequently converted into an aldehyde by an alcohol dehydrogenase, and the aldehyde is subsequently converted into a carboxyl group by an aldehyde dehydrogenase. Thus, the final product of this pathway is a dicarboxylic fatty acid, which can be further degraded from the omega terminus by omega oxidation.

Omega oxidation is considered to be the major degradation pathway of the fatty acid entities with blockade at the X position. Thus, an experiment to alter R' to block omega oxidation was performed by introducing a triple bond at the methyl terminus of the fatty acid entity. This resulted in the fatty acid analogue 3-thio-15-heptadecyne (heptadecene), which when tested showed the expected results: the in vivo degradation time is substantially increased. This is very important for the use of fatty acid entities in pharmaceutical preparations, since it may increase the effect of β -oxidizable fatty acid entities by further slowing their degradation.

On the other hand, because with the blocking of β oxidation, one can base knowledge on how ω oxidation occurs, one routinely finds other fatty acid entities that can block ω oxidation in exactly the same way. For example, a double bond will have exactly the same effect as a triple bond, and thus it is included in the definition of a methyl/hydrophobic head group terminus of a molecule, which is referred to herein as R', and which may be saturated or unsaturated. Branches can also block oxidation, whereby R' is defined as linear or branched.

In order to block omega oxidation by inserting a substituent in R ', said R' may be substituted in one or several positions with a substituent selected from the group consisting of oxygen, sulfur, selenium, oxygen, CH₂Radical, SO radical and SO₂Hetero groups in the group of groups. R' may also be selected from fluorine, chlorine, hydroxyl, C₁-C₄Alkoxy radical, C₁-C₄Alkylthio radical, C₂-C₅Acyloxy or C₁-C₄One or more compounds of the group of alkyl groups.

The compounds according to the invention are therefore either fatty acids which are similar to natural fatty acids but which cannot be beta-oxidised, or natural lipids comprising said fatty acid analogues. In vivo, fatty acid entities show a strong preference for incorporation into phospholipids. In some cases, it is actually advantageous to mimic properties and incorporate the fatty acid entities into natural lipids such as mono-, di-, and triglycerides and phospholipids. This alters the absorption of the compound (when the fatty acid is compared to a fatty acid incorporating a larger lipid structure) and may increase bioavailability or stability.

As an example, the complex may be prepared by including a fatty acid that cannot be β -oxidized in triacylglycerol. Such compounds are included in formulas (I) and (II). If such triacylglycerols are taken orally, for example in animal feed products, it may be transported like any triacylglycerols from the small intestine in chylomicrons and from the liver in blood in lipoproteins, stored in adipose tissue or utilized by muscle, heart or liver by hydrolyzing triacylglycerols to glycerol and 3 free fatty acids. In this regard, the free fatty acid is the parent compound of the present invention and is no longer a complex.

Other possible glycerophospholipids for fatty acids of the invention include, but are not limited to, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, and phosphatidylglycerol.

Another esterification found in vivo which can be readily used to prepare complexes of the compounds of the invention would be to prepare alcohols or polyols corresponding to the fatty acids, for example sphingolipid derivatives such as ceramides or sphingomyelins can be prepared by preparing the corresponding amino alcohols. Like glycerophospholipids, such complexes are very insoluble in water and less hydrophilic. These kinds of hydrophobic complexes of the present invention will pass through biological membranes more easily.

Other possible polar complexes of the invention may be, but are not limited to, lysophospholipids, phosphatidic acids, alkoxy compounds, glyceryl carbohydrates, gangliosides (gangliosides), and cerebrosides.

Although there may be large structural differences between the different compounds of the invention comprising non β -oxidizable fatty acid entities, it is expected that the biological functions of all these compounds are very similar, since they are all able to block β -oxidation in the same way. The inability of the fatty acid entities to be beta-oxidized (and in some cases omega-oxidized) causes the accumulation of these analogs in the mitochondria, which induces beta-oxidation of the native fatty acids in vivo, leading to many biological effects of compounds comprising the fatty acid entities of the invention. (Berge RK et al (2002) Curr Opin Lipidol13 (3): 295-

The beta oxidation pathway of fatty acids is the major pathway of fat metabolism. The initiation and rate-limiting reactions are carried out by acyl-CoA oxidase enzymes in the peroxisomes of the liver. acyl-CoA oxidase catalyzes the dehydrogenation of acyl-CoA thioesters to the corresponding trans-2-enoyl-CoA. The inventors have previously applied fatty acid analogues according to formula (I): tetradecylthioacetic Acid (TTA) was used to examine various biological effects of these fatty acids. In the present invention, its effect on acyl-CoA oxidase, and the effect of protein substances are examined separately or in combination.

The specific proteinaceous material examined herein is a fermented soy protein material. We also performed tests on single cell protein material and fish protein hydrolysates. Although these materials are complex and contain not only proteins, it is believed that the protein portion thereof acts as an active ingredient, enhancing the beneficial effects of the non- β oxidizable fatty acids of the present invention. Based on the results of fermenting soy protein material disclosed herein, we expect similar results for single-cell protein material and fish protein hydrolysate.

When the acyl-CoA oxidase activity was examined, TTA alone showed a great increase in the activity compared to the negative control. While fermented soy protein material alone has little activity. However, when TTA is used with fermented soy protein material, the activity of acyl-CoA oxidase is more than two-fold compared to the activity of TTA alone. This enhancement of the effect of TTA as an acyl-CoA oxidase activator by fermenting soy protein material was completely unexpected. It cannot of course be interpreted as the additive effect of TTA plus the fermented soy protein material; the unexpected synergistic effect is actually too strong.

In the present invention, the effect of non β -oxidizable fatty acid entities on phospholipid levels, as well as the effect of fermented soy protein material alone or in combination with TTA, was also examined. TTA did decrease phospholipid levels when compared to controls, whereas fermented soy protein material actually increased phospholipid levels slightly. However, when TTA is used with fermented soy protein material, it is surprising that phospholipid levels are reduced to a greater extent than TTA alone. This enhancement of the effect of TTA as a plasma phospholipid lowering agent by fermentation of soy protein material was completely unexpected. In contrast to acyl-CoA oxidase activity, it is also not interpreted as the additive effect of TTA plus the fermented soy protein material.

In the present invention, the effect of non β -oxidizable fatty acid entities on plasma cholesterol levels, as well as the effect of fermented soy protein material alone or in combination with TTA, was also examined. The effect of fish oil alone, with TTA, with fermented soy protein, or with TTA and fermented soy protein together was also examined. TTA alone showed a very significant reduction in plasma cholesterol levels, and fermented soy protein material or fish oil alone also showed cholesterol lowering effects. The fermented soy protein material and fish oil together also showed a stronger cholesterol lowering effect than each of them alone. The cholesterol lowering effect is surprisingly greater than that of TTA alone when fish oil or fermented soy protein material is added to TTA. When all three components: the cholesterol lowering effect was strongest when TTA, fish oil and fermented soy protein material were added simultaneously. This synergistic effect between TTA, fish oil and fermented soy protein material was completely unexpected.

TTA has been shown to reduce plasma triglyceride levels by increasing mitochondrial mass and stimulating mitochondrial beta-oxidation of normal saturated and unsaturated fatty acids to ketone bodies (Froyland Let al (1997) J Lipid Res 38: 1851-. In the present invention, it was found that this effect is further unexpectedly enhanced by the addition of a fermented soy protein material. In these experiments, the results of fermenting soy protein were very significant and unexpected. As expected, TTA did reduce triglyceride levels. Fermented soy protein alone actually increased triglyceride levels by 30% when compared to the control, but it still enhanced the triglyceride lowering effect of TTA by 50%. These synergistic effects are also highly unexpected.

In the present invention, the effect of feeding Atlantic salmon (Atlantic salmon) with a feed comprising non- β -oxidizable fatty acid analogs, oil, common feed components, and a fermented soybean protein material was examined. In example 2.1, the fish feed consisted of coating normal feed pellets with fish oil comprising TTA and fermented soy protein material. This feed was then used in example 2.2 as a food source for Atlantic salmon, and the presence of TTA had a beneficial effect on the so produced fish compared to feeding the fish with a corresponding feed without TTA (examples 2.3 and 2.4).

The common feed particles used mainly comprise fish meal, some wheat and vitamin and mineral additives. The oil used to encapsulate the particles was derived from capelin (capelin) in the sea, and varying amounts of TTA were mixed therein. Table 1 describes the formulation and chemical composition of the food. This is a common feed, well suited for the species tested (Atlantic salmon in this example), which shows beneficial effects by the addition of TTA. As previously shown in the present application, administration of TTA with a protein has additional beneficial effects compared to TTA alone. The fact that this normal feed contains high amounts of fat and protein and low amounts of carbohydrates may increase the beneficial effect of TTA compared to administering TTA alone, or more carbohydrates in the food.

In example 2.4, the effect of a specific protein material, a fermented soy protein material, was determined. The fermented soy protein material is produced by fermentation of soy. It comprises modified and unmodified soy proteins and isoflavones, as well as other soy components. A preferred embodiment of the present invention uses the fermented soy protein material Gendaxin。

Table 2 describes the fatty acid composition of the diets. There was only a small difference in the fatty acid composition of the diets (all containing close to 100% fish oil), the percentage of n-3 Fatty Acids (FA) being almost equal. However, food supplemented with TTA resulted in substantial changes in the percentage of n-3 fatty acid composition of Phospholipids (PL), Triacylglycerols (TAG) and Free Fatty Acids (FFA) in the gills, heart, and liver of Atlantic salmon. Administration of TTA during 8 cycles also resulted in a reduction in the percentage of saturated FAs in almost all lipid fractions. In the gill and core the percentage of n-3 FA, especially DHA, increased as seen in example 2.3.

Atlantic salmon fed a diet containing TTA grew at a slower rate than fish fed a control diet. In fish fed with TTA supplemented food, body lipid levels were significantly lower than in fish fed with control food.

Feeding the feed according to the invention has a health benefit to the fish themselves. Old fish, like humans, can suffer from arteriosclerosis and cause health problems, and lipid lowering will have a beneficial effect on this.

Generally, the lean meat obtained by the method of the invention is considered to be beneficial for feeding most animal species for consumption. Whereby a reduction of the total lipid level effect is advantageous per se. Furthermore, the specific modification of the fatty acid composition is particularly positive. It is now widely recognized that consuming less saturated fatty acids is healthy, and increasing n-3 consumption has been associated with many health benefits, such as from reducing the chances of onset of heart disease to anti-inflammatory effects and even smarter infants.

Other animal products obtained from animals fed the feed of the present invention may also have beneficial effects. The fish oil thus obtained has, as an example, a favourable nutritional composition when compared to oil from fish fed with commercial food. Other products, such as fish skin, may also have visible beneficial effects as the overall body composition improves.

The level of fatty acids in the blood is generally determined by the relative rates of lipolysis and esterification in adipose tissue and fatty acid absorption in muscle. In muscle, fatty acids inhibit glucose uptake and oxidation. Increased levels of fatty acids and triacylglycerols in blood and muscle are therefore associated with obesity and insulin resistance, as well as decreased activity to metabolize glucose (Olefsky JM (2000) J Clin Invest 106: 467-. We have shown that fatty acid oxidation and reduction of plasma fatty acid concentration is stimulated by non β -oxidizable fatty acid entities and proteinaceous matter, or optionally also comprising an oil component. It is therefore contemplated that the compositions of the present invention may be used for the prevention and treatment of insulin resistance and diseases caused thereby (Shulman GI (2000) J Clin Invest106 (2): 171-. TTA has been found to completely prevent high fat diet-induced insulin resistance and obesity in obese rats and to reduce obesity, hyperglycemia and insulin sensitivity (Madsen M et al (2002) J Lipid Res43 (5): 742-50). As the inventors have found unexpected synergistic results when TTA and proteinaceous matter are used together, and optionally also including oils, without being limited to any particular theory on why this result is shown, we now expect that such a combination will be more effective in treating these conditions. We also expect that fish and proteinaceous matter, and optionally also oil, will potentiate the role of TTA in the treatment of related diseases and conditions including hypertension, increased lipid and cholesterol levels, endothelial dysfunction, procoagulant state, polycystic ovary syndrome, and metabolic syndrome.

The peroxisome proliferator-activated receptor (PPAR) family are pleiotropic modulators of cellular functions such as cell proliferation, differentiation and lipid homeostasis (Ye JM et al (2001) Diabetes 50: 411-417). The PPAR family includes three subtypes: PPAR α, PPAR β and PPAR γ. TTA is a potent ligand for PPAR α (Forman BM, Chen J, Evans RM (1997) ProcNatl Acad Sci 94: 4312-4317; Gottllicher M et al (1993) Biochem Pharmacol 46: 2177-2184; Berge RK et al (1999) Biochem J343 (1): 191-197), and also activates PPAR β and PPAR γ (Raspe E et al (1999) J Lipid Res 40: 2099-2110). TTA acts as a PPAR α activator, stimulating its catabolism by increasing cellular uptake of fatty acids. Lowering plasma triglyceride levels with TTA causes a shift in cellular metabolism of the liver to PPAR α -regulated fatty acid catabolism in the mitochondria (GrafHJ et al (2003) Jbiol Chem 278 (33): 30525-33). The effect of TTA on plasma triacylglycerols is guided by PPAR α activation, as demonstrated by the abrogation of this effect in PPAR α knockout mice, while fish oil reduces plasma triacylglycerol levels even in knockout mice (Dallongeville J et al (2001) J Biol Chem 276: 4634-.

The addition of dietary n-3 polyunsaturated fatty acids such as those found in fish oil stimulates acyl-CoA oxidase activity of liver peroxisomes and thereby stimulates fatty acid oxidation in the liver and to a lesser extent in skeletal muscle (Ukropec J et al (2003) Lipids 38 (10): 1023-9). Fish oil-enriched diets have been shown to increase the activity and mRNA levels of liver mitochondria and peroxisomal fatty acid oxidase lines (Hong DD et al (2003) Biochim Biophys Acta: Mol cell biol Lipids 1635 (1): 29-36). Fish oil caused an increase in peroxisomal acyl CoA oxidase abundance in rat liver, but not in rat muscle, and the authors proposed the hypothesis that this was due to the n-3 fatty acids avoiding fat-induced insulin resistance by acting as PPAR α ligands to cause liver (but not intramuscular) peroxisome proliferation. The expression of the PPAR α gene was unchanged. (Neschen S et al (2002) Am J Physiol Endocrinol Metab 282: E395-E401)

As can be seen in the above paragraphs, the biochemical details of how TTA, proteinaceous matter and optionally oil exactly affect fat metabolism are unclear. This effect may or may not be through the same pathway, e.g., both TTA and oil may act as ligands for PPAR α, or may act independently of PPAR α. If they act through the same pathway, TTA cannot be expected to be fortified by the oil, since TTA is a strong PPAR α activator, which is expected to fully saturate PPAR α activation. Thus, even if the additive effect of the two effects were to be obtained when TTA was combined with the oil, it would be unexpected. Even less is known about how proteins affect beta-oxidation or other aspects of fat metabolism. The effect of using both proteinaceous material and TTA cannot therefore be predicted. However, it would be highly surprising to obtain a synergistic effect far exceeding the additive effect, as seen from TTA and fermented soy protein material in all assays of the present invention. Beta-oxidizable fatty acid entities have many roles and we do not know how they occur, but based on the unexpected results of the present invention and without being bound to any particular theory we expect that they are all enhanced by proteinaceous matter and optionally oil.

PPAR ligands affect the proliferation of a variety of cancer cell lines. In particular TTA has been found to reduce proliferation of many Cancer cell lines (Berge K et al (2001) Cardigenesis 22: 1474-. This reduction is associated with a reduction in triacylglycerol levels (Tronstad KJ et al (2001) Biochem Pharmacol 61: 639-. Since fermented soy protein improves the ability of TTA to reduce triacylglycerols, it is highly likely that it will also improve the anti-proliferative effects of TTA, such that it improves the ability of TTA to prevent and treat cancer. TTA may be used in the prevention and/or treatment of cancer, including inhibition of: primary and secondary tumors, tumor growth, invasion of the primary tumor into connective tissue and formation of secondary tumors (NO 20025930).

PPAR agonists generally modulate the inflammatory response. TTA regulates the inflammatory response by inhibiting the release of the inflammatory cytokine interleukin-2 and by inhibiting PHA-stimulated proliferation of peripheral monocytes (Aukrust P et al (2003) Eur J Clin Invest 33 (5): 436-33). Modulation of cytokines by TTA can be PPAR-mediated or by altering levels of prostaglandins or by modifying lipid-mediated signaling, the latter also being the proposed mechanism of action for polyunsaturated fatty acids, as found in oils. Now that the present inventors have found unexpected results of the present invention, they expect that proteinaceous substances and optionally oils in combination with non β -oxidizable fatty acid entities will potentiate the effects of the fatty acid entities on inflammatory diseases, including immune-mediated disorders such as rheumatoid arthritis, systemic vasculitis, systemic lupus erythematosus, systemic sclerosis, dermatomyositis, polymyositis, various autoimmune endocrine diseases (e.g., thyroiditis and adrenalitis), various immune-mediated neurological diseases (e.g., multiple sclerosis and myasthenia gravis), various cardiovascular diseases (e.g., myocarditis, congestive heart failure, arteriosclerosis and stable and unstable angina, and wegener's granulomatosis), inflammatory bowel disease, crohn's disease, nonspecific colitis, pancreatitis, nephritis, biliary obstruction/fibrosis of the liver, And acute and chronic allograft rejection after organ transplantation, as well as proliferative skin diseases such as psoriasis, atopic dermatitis, non-specific dermatitis, primary irritant contact dermatitis, allergic contact dermatitis, ichthyosis lamellar, epidermolytic hyperkeratosis, pre-malignant sun-induced keratoses (pre-malignant sun-induced keratoses), and seborrheic dermatitis, and diseases with an inflammatory component such as alzheimer's disease or impaired/improvable cognitive function.

Drawings

FIG. 1 shows that fermented soy protein material potentiates the increase in fatty acyl-CoA activity of TTA.

Figure 2 shows that the fermented soy protein material potentiates the phospholipid-lowering effect of TTA.

Figure 3 shows the cholesterol lowering effect of fermented soy protein material and fish oil fortified TTA.

Figure 4 shows that the fermented soy protein material potentiates the triacylglycerol lowering effect of TTA.

Definitions as used in this application

Animal(s) production

In this context, the term "animal" includes mammals such as humans and farm (agricultural) animals, especially economically important animals such as poultry, cattle, sheep, goats and pig mammals, especially those that produce products suitable for human consumption such as meat, eggs and milk. In addition, the term also includes fish and shellfish, such as salmon, cod, tilapia, clams, oysters, lobsters or crabs. The term also includes domestic animals such as dogs and cats.

Animal feed

The term "animal feed" means the food of an animal (as defined above). Animal feed typically contains appropriate amounts of fats, proteins, carbohydrates, vitamins and minerals necessary to maintain the intended animal recipient alive, and may contain additional components for improving taste, texture, color, odor, stability, shelf life, etc., or antibiotics or other components added for the benefit of animal health. The animal feed is preferably, but not necessarily, dry matter, most preferably particulate matter. The term "animal feed" is also meant to include nutritional compositions, veterinary compositions, and/or functional food products for animal consumption.

Meat

The word "meat (eat)" means meat (flesh) from any animal as defined above. Thus, protein-containing meat (flesh) from mammals, birds, fish and shellfish are also referred to as meat (meat). The term "meat product" means any product produced from meat as defined above.

Vegetable oil and/or fish oil

These include all oils derived from plants and marine organisms including, but not limited to, fatty or fixed oils (fixed oils) and essential or volatile oils, and any combination thereof. They need not be in liquid form. The sunflower oil used in the present invention is actually oil from sunflower seeds and not the flower itself.

Fish oil

This term includes all oils derived from the ocean.

Nutritional composition

This term is meant to include any ingestible substance including, but not limited to, nutritional supplements for human and animal consumption, functional foods, herbal supplements (herbal supplements), and the like. The term also includes food products for human consumption and animal feed wherein the composition of the invention is an additive, not a major ingredient. This relates in particular to animal feeds, any of which may be supplemented with the composition of the invention in order to obtain its biological effect.

Treatment of

In the pharmaceutical use in relation to the present invention, the term "treatment" means reducing the severity of the disease.

Prevention of

The term "prevention" means the prevention of a given disease, i.e. the administration of a composition of the invention before the onset of the condition. This means that the compounds of the invention can be used as prophylactic or as an ingredient of a nutritional composition to prevent the onset or risk of a given disease.

Fermentation of

The organic matter is decomposed by microorganisms or enzymes, which includes hydrolysis.

Hydrolysis

Enzymatic or chemical breakdown of complex molecules into simpler units by chemical reaction with water.

Single cell protein material (SCP)

SCP is a material containing unicellular microorganisms. The microorganisms may be, inter alia, fungi, yeasts and bacteria. The SCP material contains a high proportion of protein.

Enzyme-treated Fish Protein Hydrolysate (FPH)

The FPH material is a protein hydrolysate produced from enzyme treated fish material. The FPH material contains a high proportion of proteins and peptides.

Fermented soy protein material

The fermented soy protein material is produced from fermented soy. It includes modified and unmodified soy proteins and isoflavones, as well as other soy components.

Nutrient composition

The term is meant to include any ingestible substance including, but not limited to, nutritional supplements, functional foods, herbal supplements, and the like for human and animal consumption. The term also includes food products for human consumption and animal feed wherein the composition of the invention is an additive, not a major ingredient. This relates in particular to animal feeds, any of which may be supplemented with the composition of the invention in order to obtain its biological effect.

Administration of the Compounds of the invention

As a medicament, the compositions of the invention may be administered directly to an animal by any suitable technique, including parenterally, intranasally, orally, or by absorption through the skin. They may be administered locally or systemically. The specific route of administration of each agent will depend, for example, on the medical history of the human or animal recipient.

Examples of parenteral administration include subcutaneous, intramuscular, intravenous, intraarterial, and intraperitoneal administration.

As a general proposition, a total pharmaceutically effective amount of each non β -oxidizable fatty acid entity administered parenterally will preferably be about 1 mg/kg/day to 200 mg/kg/day of the patient's body weight for a human, although as noted above, this will depend to a large extent on therapeutic judgment. A dosage of 5-50 mg/kg/day is most preferred. A dosage of 5-500 mg/kg/day of fermented soy protein material or other protein material is preferred, and a dosage of 50-300 mg/kg/day is most preferred. A1-300 mg/kg/day dose of fish oil or other oil is preferred, and a 10-150 mg/kg/day dose of fish oil or other oil is most preferred.

If administered continuously, each compound of the invention is typically injected 1-4 times per day or given as a continuous subcutaneous infusion, for example with a minipump. Intravenous bag solutions (intravenousbag solution) may also be used. The key factor in selecting an appropriate dosage is the result obtained, as determined by a reduction in total body weight or fat to lean mass ratio, or by other criteria measuring the control or prevention of obesity or obesity-related conditions, as deemed appropriate by the physician.

For parenteral administration, in one embodiment, the compounds of the invention are typically formulated as follows: each ingredient in the desired purity is combined in a unit dose injectable form (solution, suspension, or emulsion), and a pharmaceutically acceptable carrier, i.e., one that is non-toxic to the recipient at the dosages and concentrations administered and is compatible with the other ingredients of the formulation.

In general, the formulations are prepared by uniformly and intimately bringing into contact each of the compounds of the invention with liquid carriers or finely divided solid carriers or both. Subsequently, if necessary, the product is shaped into the desired dosage form. Preferably, the carrier is a parenteral carrier, more preferably a solution that is isotonic with the blood of the recipient. Examples of such carrier vehicles (carrier vehicles) include water, saline, ringer's solution, and dextrose solution. Non-aqueous vehicles such as fixed oils and ethyl oleate may also be used herein, as well as liposomes.

The carrier may suitably contain minor amounts of additives such as substances which enhance isotonicity and chemical stability. These substances are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, succinate, acetic acid, and other organic acids or salts thereof; antioxidants such as ascorbic acid; an immunoglobulin; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamic acid, aspartic acid, or arginine; monosaccharides, disaccharides, and other carbohydrates including cellulose or its derivatives, glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; counterions such as sodium; and/or a non-ionic surfactant such as a polysorbate, poloxamer (poloxamer), or PEG.

For oral pharmaceutical compositions, such carrier materials may be used, as for example, water, gelatin, gums, lactose, starch, magnesium stearate, talc, oils, polyalkylene glycols, petroleum jelly and the like. These pharmaceutical preparations may be in unit dosage form and may additionally contain other therapeutically valuable substances or conventional pharmaceutical adjuvants such as preservatives, stabilizers, emulsifiers, buffers and the like. The pharmaceutical preparations may be in conventional liquid forms, such as tablets, capsules, dragees, ampoules and the like, in conventional dosage forms, such as dry ampoules, and as suppositories and the like.

In addition to the compounds of the invention, i.e. beta-oxidizable fatty acid analogues and proteinaceous matter, or beta-oxidizable fatty acid analogues and proteinaceous matter and oil, can be used in nutritional preparations, as defined earlier, wherein preferably the dosage of non-beta-oxidizable fatty acid analogues is as described for the drug or less, while the amount of proteinaceous matter and oil is preferably suitable for the preparation of food and feed substances. As part of a nutritional composition, especially an animal feed, the oil and protein material may be an essential part of the feed and thus have nutritional value and fortify the non β -oxidizable fatty acid analogues. The fish oil may comprise up to all of the fat in the nutritional composition, while the fermented soy protein material may comprise up to all of the protein in the nutritional composition. In animal feed, the amount of non β -oxidizable fatty acid analogues can be up to 10 times that of the product for human consumption, that is to say up to 2 kg/mg/day of the animal's body weight. Such animal feed may be used for routine feeding of animals. The fermented soy protein material is particularly useful as a functional protein in food products, particularly when it is used as a substitute for natural plasma in animal feed and pet food. The animal feed composition may also include additional ingredients such as fats, sugars, salts, flavoring agents, minerals, and the like. The product can then be made into chunks that resemble chunks of natural meat in appearance and texture. The product of the invention has the further advantage that it is easily formulated to contain essential nutrients, is easily digestible by animals and is very palatable to animals.

Experimental part

Details of the preparation of non β -oxidizable fatty acid entities according to the present invention are disclosed in applicant's earlier norwegian patent application nos. 20005461, 20005462, 20005463 and 20024114. These documents also describe toxicity studies of TTA. Details of the preparation of mono-, di-, and triglycerides and nitrogenous lipids according to the present invention are disclosed in U.S. patent application No.10/484,350. Details of the preparation of phospholipids comprising serine, ethanolamine, choline, glycerol, and inositol according to the present invention are disclosed in applicant's earlier norwegian patent application No. 200045562.

The experimental results presented below have revealed that the proteinaceous material and/or oil substantially potentiates the biological effects of the non- β oxidizable fatty acid analogues.

Example 1 biological Effect of the composition according to the invention in rats

1.1 preparation of Fish Protein Hydrolysate (FPH)

Starting material

After the fish is sliced, FPH is produced from fish meat residues on salmon skeletons. Headless skeletons from freshly cut Atlantic Salmon (Salmon Salar, L.) slices were obtained directly from the production line and frozen at-20. + -. 2 ℃. Within one week, the frozen matrix was used for the enzymatic hydrolysis process.

Hydrolysis

By Protamex^TMThe enzymatic hydrolysis is carried out at a pH of about 6.5 and a temperature of 55. + -. 2 ℃. Protamex^TM(E.C.3.4.21.62/3.4.24.28) is a subtilisin complex from Novozymes AS (Bagsvaerd, Denmark) and meets the purity requirements for food grade enzymes. The ratio of salmon skeleton to water was 1.14. The ratio of enzyme to substrate 11.1 AU/kg crude protein was used in the hydrolysis. After 60 minutes of enzyme treatment, the temperature was raised to 98 ℃, which was reached after 105 minutes.

Purification of

The large bones are retained in the hydrolysis tank while the hydrolysate is filtered through a screen to remove small bones. Thereafter the insoluble fraction was removed in a two-phase separator (Westfalia, Germany, SC.35-26-177, 15kW, 7200rpm), and the remaining mixture was subsequently separated in a three-phase separator (Westfalia, Germany, SB-7-36- +76, 4kW, 8520rpm) into salmon oil, an emulsion fraction and a water fraction. The aqueous portion was concentrated (Nitroatomier, Denmark, Falling Film Evaporator, Ff100)And passed through an ultrafiltration membrane (ultra-membrane) having a nominal molecular weight limit of 100,000 (PCImembran systems, UK, PF100, 2,65 m)²) Filtration, and finally spray drying of the ultrafiltration membrane filtered fraction (UF) (Niro Atomizer, Denmark, P-63 tower, T)_in＝200℃，T_out＝84℃)。

End product

The UF fraction is called Fish Protein Hydrolysate (FPH). On a dry weight basis, the FPH material contains about 83% protein, 10% ash, and about 2% lipids. Further characteristics of FPH can be found in the applicant's previous application NO 20033078. The synthesis of FPH is given as an example, but is not intended to exemplify the synthesis of all protein materials or even fish protein hydrolysates of formula (I).

1.2 preparation of Single Cell Protein (SCP) Material

Starting material

In a loop fermenter in ammonium/mineral salts medium (AMS) at 25 deg.C and pH6.5 for 0.15h^-1The dilution ratio (c) microbial cultures comprising all of the Methiococcus capsulatus (Bath), Ralstonia sp., Brevibacillus agri and Aneurinibacillus sp commercially available from Norferm Denmark AS, Odense, Denmark were produced by continuous aerobic fermentation of natural gas. AMS medium contained the following ingredients per liter: 10mg NH₃、75g H₃PO₄·2H₂O、380mg MgSO₄·7H₂O、100mgCaCl₂·2H₂O、200mg K₂SO₄、75mg FeSO₄·7H₂O、1.0mg CuSO₄·5H₂O、0.96mg ZnSO₄·7H₂O、120μg CoCl₂·6H₂O、48μg MnCl₂·4H₂O、36μgH₃BO₃、24μgNiCl₂·6H₂O and 1.20. mu.g NaMoO₄·2H₂O。

Production of

The fermenter was filled with water which had been heat sterilized at 125 ℃ for 10 seconds. The addition of different nutrients is adjusted according to their consumption. Continuous fermentation was carried out with 2-3% biomass (dry weight basis).

Single cell material was harvested continuously and centrifuged at 3000rpm in an industrial continuous centrifuge followed by ultrafiltration with membranes having an exclusion size of 100,000 daltons. The resulting product was then sterilized in a heat exchanger at 130 ℃ for about 90 seconds.

Further features of SSP can be found in the applicant's prior application NO 20033082. The synthesis of SSP is given as an example, but is not intended to illustrate the synthesis of all proteinaceous material or even single-cell proteinaceous material of formula (I).

1.3 fermenting Soy protein Material

Fermented soy protein material is produced by fermentation of soybeans. It includes modified and unmodified soy proteins and isoflavones, as well as other soy components. A preferred embodiment of the present invention uses the fermented soy protein material GendaxinIt is commercially available from Aximed, Bergen, Norway. Giving GendaxinAs an example, but not intended to be an illustration of all proteinaceous matter or even fermented soy protein matter of formula (I).

Chemical product

Chemicals are obtained from common commercial sources and are reagent grade. Carboxymethylcellulose (CMC) was used as a control (negative). Fish oil was obtained commercially from hordador.

Animal(s) production

Male Wistar rats weighing 250-358g were purchased from AnLab Ltd. (Prahg, the check Republic.) and kept indoors in iron cages at a temperature of 22+/-1 ℃ and light controlled (light from 7. early to 7. late). There is no restriction on food and water intake. Three rats were placed in each cage. Weight gain and food intake were monitored daily.

Food product

Rats were fed with standard Chow ST1 diet (from Velaz, Prahg, The CheckRepublic).

Treatment of

Male Wistar rats were allowed to acclimate to the new environment before the experiment began. They were then treated daily by gavage for 10 consecutive days. CMC was used as a vehicle and negative control. 4 rats were counted per treatment group. The TTA-treated group was given 150mg/kg body weight/day dissolved in CMC or oil. The fish oil treatment group was given 3mL (about 2.5g) per kg body weight per day. The amount of the fermented soybean protein material-treated group was 0.45g/kg body weight/day. CMC was used as a vehicle and negative control. The day after the final treatment rats were sacrificed.

Sacrifice and tissue acquisition

Subcutaneous injection of Hypnorm^TM(fentanyl citrate 0.315mg/ml and fluanidone 10mg/ml, Janssen Animal Health) and Dormicum(midazolam 5mg/ml, F. Hoffmann-La Roche) in a 1: 1 mixture. Blood was drawn directly from the heart with a heparin-rinsed syringe. The livers were then immediately excised, weighed and divided into two portions, which were immediately cooled on ice or frozen in liquid nitrogen, respectively. Plasma and tissues were preserved at-80 ℃ until analysis. The protocol was approved by the Norwegian national institute of Biological Experiments with Living Animals (Norwegian StateBoard of Biological Experiments).

Preparation of hepatocyte fractions

Liver from rats were individually homogenized in ice-cold sucrose solution (0.25mol/L sucrose in 10mmol/L HEPES buffer pH7.4 and 1 mmol/LEDTA) using a Potter-Elverhjem homogenizer. The liver was sub-cellularly fractionated as described previously (Berge RK et al (1984) Eur J Biochem 141: 637-44). This operation was carried out at 0-4 ℃ and the components were stored at-80 ℃. Proteins were analyzed using the BioRad protein assay kit with bovine serum albumin as standard.

Enzyme assay

The measurement of fatty acyl-CoA oxidase activity in peroxisomal liver fractions was performed as described previously (Small GM, Burdett K, Connock MJ (1985) Biochem J227: 205-10). Results are given as fatty acyl-CoA oxidase activity per total protein, minus baseline activity (control activity), and data presented in figure 1 are normalized to TTA activity.

Lipid analysis

Plasma and liver lipids were measured enzymatically on a Technicon Axon system (Miles, Tarrytown, NY) using a triglyceride kit from Bayer, Total cholesterol (Bayer, Tarrytown, NY), and a PAP150 kit for choline-containing phospholipids from biomerilux. The results are given as per total protein and the data presented in FIGS. 2-4 are normalized to the activity of the positive control (no TTA or oil added; i.e., "normal" levels). The results are given as per total protein and the data presented in FIGS. 2-4 are normalized to the activity of the positive control (no TTA or oil added; i.e., "normal" levels).

Example 2

Biological effects of the composition according to the invention in Atlantic salmon

2.1 Experimental protocol including preparation of Fish feed

Experimental food based on fish meal was provided by EWOS and contained 0.01% Y₂O₃As an inert marker for digestibility determination (3mm particles). Table 1 shows the formulations and chemical compositions of the three foods. All three foods were produced from one feed mixture. Different foods were obtained by coating conventional feed pellets with different oils and mixtures. Food or fish oil (hair)Capelin oil (control), fish oil with 0.5% TTA added (0.5% TTA), or fish oil with 1.5% TTA added (1.5% TTA).

Table 1: food formulation and chemical composition

Food: fish oil (control), fish oil with 0.5% TTA added (0.5% TTA), fish oil with 1.5% TTA added (1.5% TTA)

^aFish oil from hair scales, Norsildmel, Norway.

^bAsta，BASF，lucanthin red.

^cCanta，lucanthin pink.

The fatty acid composition of the diet clearly reflects the fatty acid composition of the fish oil used (capelin oil) (table 2). Lepidopodium oil contains relatively high levels of monounsaturated Fatty Acids (FA) and is also rich in long-chain n-3 FA, 20:5 n-3(EPA) and 22:6 n-3 (DHA). However, the feed contains significant amounts of fish meal, which contains n-3 FA, ensuring that the level of these FAs in the diet is higher than in the added oil.

In addition to the above diets, the same diet but with 0.5% Gendaxin and 0% or 0.9% TTA (based on total dry weight of the feed) was prepared.

Table 2: fatty acid composition of foods

Comparison: fish oil, 0.5% TTA: fish oil with 0.5% TTA added, 1.5% TTA: fish oil with 1.5% TTA was added. The amount of each fatty acid is given as a percentage of the total fatty acids.

2.2: atlantic salmon fed with TTA-containing feedFish, device and Experimental design

In AKVAFORSK Research Station, Sunndalsra, Norway, performed the assay. An average starting weight of about 86g of Atlantic Salmon (Salmon salar) was placed in 15 column-cone cans (0.85m diameter) with 40 fish per can. The tank was filled with seawater thermostatted at 12 ℃. The fish were acclimated and fed with commercial feed for 2 weeks prior to the start of the trial. The growth test consisted of an 8 week period.

The diet was as described in table 2 above, either with fish oil (capelin oil) (control), with 0.5% TTA added fish oil (0.5% TTA), or with 1.5% TTA added fish oil (1.5% TTA). Three foods were randomly distributed into triple duplicate pots. Electrically-driven disc feeders (Akvaprodukter AS, Sunndals)ra) dispensing the feed. This tank is designed so that waste feed can be collected from the effluent with a wire cage. The waste feed is collected and this allows the weight consumed by the feed to be calculated.

The food containing Gendaxin was used in the isolated test, but the test design was the same as described above.

First and last sampling

The fish were fasted for 2 days prior to initial sampling. 6 fish per pot were anesthetized at the beginning and end of the experiment in MS-222 and their average weight and average length were determined. The 6 fish were sacrificed by hitting the head and the abdomen was cut open. Samples of liver, heart, gill and kidney were immediately frozen in liquid nitrogen and stored at-80 ℃. These samples were then used for analysis of fatty acid composition. Another 5 fish in each tank were anesthetized and sacrificed. These fish are used to determine the composition of the whole body.

The fish were not fasted until the final sampling. According to the method described by Austreng (Aquaculture, 197813: 265) 5 fish per tank were peeled open to collect a fecal sample. The fecal samples from each tank were pooled. Samples were stored at-20 ℃ before analysis.

A second gill arch was removed from the anaesthetised fish and rinsed in ice cold SEI buffer (150mM sucrose, 10nMEDTA, 50mM imidazole, ph7.3) and immediately frozen in liquid nitrogen. Gill tissue was stored at-80 ℃. The liver was homogenized in ice-cold sucrose medium.

Growth of

In all food groups, the average body weight of the fish increased almost 3-fold during the test period from 86g of the starting value to about 250g of the final value. From 1.8 for SGR in the control group to 0.5% for SGR in the TTA group and 1.7 for SGR in the 1.5% TTA group (table 3), SGR decreased with increasing TTA dose in the food. The conditioning factor (conditioning factor) was not significantly different between the food groups (table 3).

Table 3: effect of a diet comprising TTA and oil on feed intake and growth of Atlantic salmon

The values are mean. + -. SEM (n ═ 3)

CF (%): conditional coefficients, SGR: specific growth rate, TGC: thermal growth coefficient (Thermalgrowth coefficient), FER: feed efficiency ratio (wet weight gain/dry feed intake).

^abcThe difference between the mean values within a given row is significant (p ≦ 0.05), which is represented by a different superscript letter.

Feed intake and nutrient digestibility

In this test, there was only a small difference in digestibility (table 4). In all food groups, the digestibility of FA was high, more than 96% of the total amount of FA for fish fed the control food and 0.5% TTA food, and more than 90% for fish fed the 1.5% TTA food. Generally, the digestibility of saturated FA is lower than that of other FAs.

Table 4: digestibility of nutrients in Atlantic salmon

Protein, fat, energy and selected fatty acid controls in Atlantic salmon fed with a diet containing: fish oil, 0.5% TTA: fish oil with 0.5% TTA added, 1.5% TTA: fish oil data with 1.5% TTA addition are% mean. + -. SEM

The numerical values of different superscripts in the same row have significant differences; nd is not detected

2.3 biological Effect of TTA

Chemical product

Acetic acid, chloroform, petroleum ether and methanol are all from Merek (Darmstadt, Germany). Benzene was from Rathburn Chemicals ltd. (walker, Scotland), 2 ', 7' -dichlorofluorescein was from Sigma Chemical co. (st. louis, MO, USA). Methanol hydrochloride (Methanolic HCl) and 2, 2-dimethoxypropane were purchased from Supelco Inc (Bellfonte, PA, USA). Glass-based silica gel K6 plates were obtained from Whatman International Ltd (Maidstone, England).

Chemical analysis

The fish sampled at the beginning and end of the experiment were analyzed for dry matter, fat, protein, ash and energy. All food and fecal samples were analyzed for dry matter (dried to constant weight at 105 ℃), fat (by ethyl acetate extraction as described in NS 9402, 1994), protein (by Kjeltec autoanalyser-N × 6.25), starch, ash (by heating to constant weight at 550 ℃), energy and yttrium oxide (ICP-AES after wet ashing the samples). The energy content of food, fecal and whole fish samples was determined by adiabatic Bomb Calorimeter using a Parr 1271 Bomb Calorimeter.

Lipid extraction and fatty acid analysis

Total lipids were extracted from homogenized gills, liver and heart using the method described by Folch (J Biol Chem 1957226: 497-Asa 509). The chloroform-methanol phase of the gills was dried under nitrogen and dissolved in hexane. Phospholipid (PL), Triacylglycerol (TAG) and Free Fatty Acid (FFA) were separated by Thin Layer Chromatography (TLC) using a mixture of petroleum ether, diethyl ether and acetic acid (113: 20: 2 by volume) as the mobile phase. Lipids were visualized by methanol spray TLC plates with 0.2% (w/v)2 ', 7' -dichlorofluorescein and identified by comparison to known standards under UV light.

Spots corresponding to PL, FFA and TAG were scraped into glass tubes and subsequently trans-methylated with 2, 2-dimethoxypropane, methanol hydrochloride and benzene at room temperature overnight (trans-methylated) as described by Mason and Waller (Anal Chem 196436: 583). Substantially as RsjThe (Fish Physiol Biochem 199413: 119-132) separated these methyl esters by gas chromatography on a non-polar fused capillary column. Methyl esters of FA were separated in a gas chromatograph (Perkin-Elmer Auto system GC equipped with an injector, programmable split/no split injector) with a CP wax 52 column (25m long, 0.25mm inner diameter and 0.2 μm film thickness), a flame ionization detector and 1022 data system. The carrier gas was He and the temperature of the injector and detector was 280 ℃. The furnace temperature is 10 ℃ min^-1At a rate of 50 ℃ to 180 ℃ followed by 0.7 ℃ min^-1The rate of (2) increased to 240 ℃. The relative amount of each fatty acid present is determined by measuring the area under the peak of the corresponding fatty acid.

Computing

The Apparent Digestibility Coefficient (ADC) was calculated as described in Austreng (Aquaculture, 197813: 265-272). Condition Coefficient (CF), liver body index (HSI), specific growth index (SGR), and thermal unit growth coefficient (TGC) were calculated based on individual records of weight and length as follows:

SGR＝(e^{(lnw1-lnw 0/day)}-1)*100

TGC＝(W₁ ^1/3-W₀ ^1/3) 1000/(days x deg.C)

Wherein W₀Is the initial weight, W₁Is the final weight, and t days temperature.

CF 100W (fork length)^-3

HSI 100 liver weight W^-1

Statistical analysis

All data were subjected to one-way analysis of variance (ANOVA) and differences were ranked using the Duncan multiple range test. The significance level was set at 5%.

Body and liver composition

Fish fed 1.5% TTA food (9.6%) had lower body lipid levels than fish fed control food (10.6%) (table 5). Total liver lipid content was not statistically significantly different between fish fed the control food and fish fed the TTA food (table 6). The liver body index of fish fed 1.5% TTA food (1.2%) was significantly higher than the liver body index of fish fed control food (1.1%) (table 6).

Table 5: chemical composition of carcass based on wet weight%

Comparison: fish oil, 0.5% TTA: fish oil with 0.5% TTA added, 1.5% TTA: fish oil with 1.5% TTA was added.

^abThere is a significant difference between the mean values for the given row (p ≦ 0.05), which is represented by a different superscript letter.

Table 6: effect of a diet comprising TTA and oil on liver body index (HSI) and liver lipid content

The results are mean ± SEM (n ═ 3). There are significant differences in the values with different superscripts within the same row.

Fatty acid composition of liver, gill and heart

The fatty acid composition of PL, TAG and FFA of gill, liver and heart are shown in tables 7, 8 and 9. TTA was incorporated into the PL fractions of the gill (0.8%) and heart (0.7%) of atlantic salmon fed a 1.5% TTA diet. TTA was also incorporated into TG and FFA portions of gills (Table 7). Trace amount of TTA and its Delta⁹Desaturase products are integrated into liver lipids, however, TTA Δ is not recovered in cardiac and gill lipids⁹Desaturase products.

The percentage of n-3 FA in the liver, gill and heart also depends on the food fed to the fish. The percentage of EPA + DHA in all lipid fractions of gills and hearts was significantly higher for fish fed 1.5% TTA diet than for control fish. On the other hand, in the liver, TTA only results in a modest increase in the DHA percentage and a slight decrease in the EPA percentage. The percentage of palmitic acid (16: 0) and the total of all saturated FAs in the PL fraction of gills, heart and liver of fish fed 1.5% TTA diet was significantly lower than the percentage in fish fed control diet (tables 7, 8, 9). The total amount of monounsaturated FA in TG and FFA fractions of gills was significantly lower in fish fed the 1.5% TTA diet than in fish fed the control diet (table 8). In contrast, the percentage of total monounsaturated FA in the PL and TAG fractions of the liver was higher in fish fed more TTA doses.

2.4 composition and biologically acting chemical of fermented Soy protein Material according to the invention

Gendaxin was obtained from Aximed, Bergen, Norway. One GendaxinThe capsules contained 35mg of isoflavones, 10mg of Genistein (Genistein) and 15mg of Daidzein (Daidzein).

Lipid analysis

Plasma lipids were measured enzymatically on a Technicon Axon system (Miles, Tarrytown, NY) using a triglyceride kit from Bayer, Total cholesterol (Bayer, Tarrytown, NY), and a PAP150 kit for choline-containing phospholipids from biomerilux. The results are given in mmol/l and are shown in Table 10 below.

Table 10: total cholesterol, triglycerides and phospholipids of plasma

The above data clearly show that the addition of Gendaxin to fish feed has a positive effect on the fatty acid composition of salmon plasma. When compared to the control, cholesterol, triglyceride and phospholipid levels are all reduced if 0.25% Gendaxin is added to the fish feed. In addition, further addition of Gendaxin and TTA additionally improved the fatty acid composition of plasma.

Enzyme assay

Fatty acyl-CoA oxidase activity was measured in peroxisomal liver fractions as described previously (Small GM, Burdett K, Connock MJ (1985) Biochem J227: 205-10). The results are given as fatty acyl-CoA oxidase activity per total protein and are shown in table 11 below.

Table 11: beta oxidation of liver

	Beta oxidation
		Control 0.5% Gendaxin + 0.9% TTA	0.9401.501

The above data clearly show that addition of Gendaxin and TTA to fish feed has a positive effect on beta oxidation, as beta oxidation is highly increased.

Example 3

Consistent with the experimental protocol given in example 1, we have conducted feeding trials on male Wistar rats with the following feed composition:

30% fat

20% protein

5% of fiber

10% sucrose

3.5% AIN93G mineral mixture

1.0% AIN-93 vitamin mixture

And the rest is: starch

The fat component is 30% lard, or 2.5-5% lard is replaced by fish oil, or 0.15% lard is replaced by TTA. The proteinaceous matter is 20% milk protein (casein), or half of it is exchanged for fish protein or "Bioprotein".

Claims

1. Use of a preparation for the preparation of a pharmaceutical or nutraceutical composition for the prevention and/or treatment of obesity, diabetes, fatty liver, hypercholesterolemia, dyslipidemia, arteriosclerosis, coronary heart disease and proliferative skin diseases, said preparation comprising a combination of:

1) a protein material selected from the group consisting of fermented soy protein materials produced from fermented soy and including modified and unmodified soy protein and isoflavones and other soy components, and

2) tetradecylthioacetic acid or a salt thereof or a mono-, di-or triacylglyceride thereof.

2. Use according to claim 1, wherein the proliferative skin disease is selected from the group comprising: psoriasis, atopic dermatitis, non-specific dermatitis, primary irritant contact dermatitis, allergic contact dermatitis, ichthyosis, epidermolysis hyperkeratosis, pre-malignant sun-induced keratosis, and seborrheic dermatitis.

3. Use of an animal feed comprising a common feed component and a combination of:

4. Use according to claim 3 wherein the improvement in total lipid composition comprises a reduction in the total lipid level in the body.

5. Use according to claim 3 wherein the improvement in total lipid composition comprises a reduction in the total saturated fatty acid levels of the body.

6. Use according to claim 3 wherein the improvement in total lipid composition comprises an increase in the total n-3 fatty acid level of the body.

7. Use according to any one of claims 1 to 6, wherein the proteinaceous material is fermented.

8. Use according to claim 1, wherein the ester of tetradecylthioacetic acid is a triacylglycerol containing tetradecylthioacetic acid.

9. Use according to any one of claims 1 to 8, wherein the composition further comprises a vegetable oil and/or a fish oil.

10. Use according to claim 9, wherein the vegetable or fish oil comprises polyunsaturated fatty acids.

11. Use according to claim 10, wherein the vegetable oil is selected from the group comprising sunflower oil, soybean oil and olive oil.

12. Use according to any one of the preceding claims, wherein the composition is for administration to or feeding to an animal.

13. The use according to claim 12, wherein the animal is a human.

14. Use according to claim 12, wherein the animal is an agricultural animal.

15. Use according to claim 12, wherein the animal is poultry, cattle, sheep, goats or pigs.

16. Use according to claim 12, wherein the animal is a domestic or pet animal.

17. Use according to claim 12, wherein the animal is a dog or cat.

18. Use according to claim 12, wherein the animal is a fish or shellfish.

19. Use according to claim 12, wherein the animal is salmon, cod, tilapia, clams, oysters, lobsters or crabs.

20. Use according to any one of the preceding claims, wherein the tetradecylthioacetic acid or salt thereof, or mono-, di-or triacylglyceride thereof, comprises a daily dose of about 1 to 200mg/kg for human consumption and about 1 to 2000mg/kg for animal consumption.

21. Use according to claim 20, wherein the tetradecylthioacetic acid or salt thereof, or mono-, di-or triacylglyceride thereof comprises a daily dose for human consumption of 5-50 mg/kg.

22. Use according to claim 20, wherein the tetradecylthioacetic acid or salt thereof, or mono-, di-or triacylglyceride thereof comprises a daily dose of 5 to 500mg/kg for animal consumption.

23. Use according to any of the preceding claims, wherein the proteinaceous matter comprises a daily dose of about 5-500mg/kg for human consumption and a daily dose of from 5mg/kg up to the total daily protein consumption for animal consumption.

24. Use according to claim 23, wherein the proteinaceous matter comprises a daily dose for human consumption of 50-300 mg/kg.

25. Use according to claim 9, wherein the oil comprises a daily dose of about 1-300mg/kg for human consumption and a daily dose of from 1mg/kg up to the total daily fat consumption for animal consumption.

26. Use according to claim 25, wherein the oil comprises a daily dose for human consumption of from 10 to 150 mg/kg.

27. Use according to claim 3, wherein the animal feed may be a nutritional composition, a veterinary composition, and/or a functional food product.

28. A composition having a synergistic biological effect on increasing fatty acyl-coa oxidase activity and lowering cholesterol, triglyceride and phospholipid concentrations, characterized in that the composition comprises a combination of:

1) a protein material selected from the group consisting of fermented soy protein materials, which are fermented and/or hydrolyzed proteins, which are produced from fermented soy and include modified and unmodified soy protein and isoflavones and other soy components, and

29. The composition according to claim 28, wherein said composition comprises a daily dosage of about 1 to 200mg/kg of tetradecylthioacetic acid or salt thereof or mono-, di-or triacylglyceride thereof for human consumption and about 1 to 2000mg/kg of tetradecylthioacetic acid or salt thereof or mono-, di-or triacylglyceride thereof for animal consumption.

30. The composition according to claim 29, wherein the composition comprises a daily dose for human consumption of 5-50mg/kg of tetradecylthioacetic acid or salt thereof, or mono-, di-or triacylglyceride thereof.

31. The composition according to claim 29, wherein the composition comprises a daily dose for consumption by an animal of 5-500mg/kg of tetradecylthioacetic acid or salt thereof, or mono-, di-or triacylglyceride thereof.

32. The composition according to claim 28, wherein the composition further comprises a vegetable oil and/or a fish oil.

33. The composition according to claim 28, wherein said ester of tetradecylthioacetic acid is a triacylglyceride comprising tetradecylthioacetic acid.

34. The composition of claim 28, wherein the vegetable or fish oil comprises polyunsaturated fatty acids.

35. A composition according to claim 28, wherein said composition comprises a daily dosage of from about 5 to 500mg/kg of proteinaceous matter for human consumption and a daily dosage of from 5mg/kg up to the total daily protein consumption of proteinaceous matter for animal consumption.

36. A composition according to claim 35, wherein the composition comprises for human consumption a daily dose of 50-300mg/kg of proteinaceous matter.

37. A composition according to claim 28, wherein said composition comprises a daily dosage of from about 1 to 300mg/kg of oil for human consumption and a daily dosage of from 1mg/kg up to the total daily fat consumption of the oil for animal consumption.

38. The composition according to claim 37, wherein the composition comprises a daily dose for human consumption of 10-150mg/kg of oil.

39. The composition according to claim 28, wherein the composition is an animal feed further comprising common feed ingredients.

40. The composition according to claim 28, wherein the animal feed is fish feed.

41. The composition according to claim 28, wherein the fish feed is salmon feed.

42. A composition according to claim 28 wherein the common feed component comprises fish meal and/or fish oil.

43. Use of an animal feed in the preparation of a nutritional, veterinary and/or functional food product for the production of an animal based product with improved fatty acid composition, wherein the animal feed comprises common feed components and the following combinations and is used to feed an animal producing the product:

1) a protein material selected from the group consisting of fermented soy protein materials produced by fermented soy and including modified and unmodified soy protein and isoflavones and other soy components; and

44. The use according to claim 43, wherein the animal-based product is a meat product.

45. The use according to claim 43, wherein the animal-based product is an oil-based product.

46. The use according to claim 43, wherein the animal-based product is a skin-based product.