HK1139011B - A product based on conjugated linoleic acid and a method for the manufacture thereof - Google Patents

Description

Product based on conjugated linoleic acid and preparation method thereof

Technical Field

The invention relates to a product based on conjugated linoleic acid (hereinafter abbreviated as CLA) or a derivative thereof, having the characteristics described in the preamble of the main claim. The invention also relates to a method for preparing said product.

Background

The collective term CLA is intended to describe a mixture of geometric and positional isomers of conjugated dienoic acids derived from linoleic acid in the form of Free Fatty Acids (FFA) and their respective salts or other derivatives, especially esters.

CLA is found in nature in the milk and meat of ruminants and is an intermediate compound formed during the biohydrogenation of some of the polyunsaturated fatty acids contained in the diets of these animals, especially linoleic and alpha-linolenic acids. Some of these intermediate compounds are not fully hydrogenated and accumulate in the mammary gland.

CLA isomers are the major fatty component in ruminant milk, and are cis-9, trans-11, the mechanism by which they are endogenously synthesized has been proposed (Mahfouz et al, 1980, Pollard et al, 1980, Griinari et al, 2000).

Recent studies over 30 years have shown that this isomer, as well as the trans-10, cis-12 isomer, is involved in a number of physiological and metabolic functions, which has led to an increasing interest in CLA and its involved biochemical mechanisms.

In particular, among the many potential applications of CLA, the following have been proposed: i) inhibiting carcinogenesis; ii) improving immune function; iii) reducing inflammation; iv) reducing dissimilarity in immune stimulation; v) reducing asthma in an animal model; vi) reduced atherosclerosis (reduced LDL (low density lipoprotein) concentration and LDL: HDL (high density lipoprotein) ratio); vii) reducing body fat accumulation and increasing lean body mass; viii) promoting growth of rodent pups; ix) relief of diabetic symptoms in some experimental models; x) reducing hypertension.

Not all of the above physiological effects can be attributed to both of the above isomers (but so is the inhibition of breast carcinogenesis); in some cases, the effect is determined by only one of the two substances (e.g., trans-10, cis-12 is the only isomer responsible for reducing body fat mass, while the isomers cis-9, trans-11 improve growth and feeding efficiency in rodent pups), in other cases the effects of the two isomers appear to be a balance of opposite effects.

In view of the above potential benefits, there is a need to increase the availability of CLA by producing milk enriched with this compound from ruminants and preparing CLA-based products for direct administration to humans, for example in the form of food supplements or additives used in normal food production. In the first case, one of the possible alternatives is to supplement the animal feed with synthetic CLA.

It is also known to synthesize CLA in the laboratory, for example from vegetable oils, such as safflower oil or sunflower oil; the product obtained is generally a mixture of CLA isomers in various forms, for example in the form of methyl esters or Free Fatty Acids (FFA), which can be used in the human and animal diet.

However, CLA has a great disadvantage in terms of stability.

In fact, CLA is very susceptible to reaction with oxygen and other oxidizing agents, such as choline chloride or some minerals, and in particular in the presence of light or metals (such as copper and iron), to degrade rapidly, thus losing its activity. Their poor resistance to oxidation processes makes them particularly unstable and much more difficult to handle than the common polyunsaturated fatty acids.

Therefore, CLA must be properly protected from the external environment (during the period of storage from preparation to use, which is likely to be very long) and from the gastric or upper gastrointestinal environment (during use).

Moreover, the high instability of CLA also imposes considerable limitations on its preparation process, making it protective, and in fact, the process which requires high temperatures for a considerable time may lead to its rapid degradation.

The currently known methods of protecting CLA are to use CLA in the form of a calcium salt or ester, or to encapsulate molecules in a casein matrix treated with formaldehyde, even microencapsulated in cyclodextrins.

However, while it is desirable in the market place to provide the stability characteristics for CLA, it has not been achieved. Thus, there remains a need in the art to have available CLA-based products whose CLA content, and in particular the resistance to oxidation, remains substantially unchanged, even over a long period of several years, without the need for the use of antioxidants or storage under an inert atmosphere, while being capable of remaining stable in the gastrointestinal tract of animals and humans.

Disclosure of Invention

The problem underlying the present invention is to provide CLA-based products and methods for their preparation, which are structurally and functionally designed to overcome the limitations discussed above in connection with the cited references.

The present invention solves the above mentioned problems by means of a product and a method as described in the appended claims.

The product produced by the present invention is of the encapsulated type, comprising an inner core in which the CLA is substantially concentrated, and a coating which completely surrounds the inner core, covers and protects it.

The final product may have any suitable shape or size but is preferably in the form of granules having a size of 0.15 to 2 mm, at least 80% of the product having a particle size of less than 0.8 mm.

The granular product is preferably prepared by a microencapsulation process using spray cooling techniques as described in detail below.

The CLA used is preferably a synthetic derivative in the form of a free fatty acid and/or a methyl ester. It is in the form of oily droplets in which the content of the two main CLA isomers, cis-9, trans-11 and trans-10, cis-12, is as high as possible, preferably at least 50% by weight. These two isomers are usually present in almost equivalent amounts.

The content of the two main isomers of CLA present in the CLA synthetic oil will be described hereinafter by the term "CLA content".

The CLA content used in animal feed and human food may vary depending on the raw material (sunflower oil or safflower oil) used for its preparation and may be, for example, 60% or 80%.

The CLA used in the preparation of the products according to the invention has a peroxide number which is as low as possible (determined according to Italian standard NGD C35-1976), preferably less than 10, and even more preferably less than 3.

The peroxide value of CLA is a parameter indicative of the degree of degradation of CLA; the higher the peroxide number, the greater the degree of degradation of the CLA.

It has been found that the peroxide value of the CLA starting material used in the preparation of the product according to the invention is sufficiently low that the product retains its properties over a long period of time, but that if the starting material is already highly oxidized, its degradation process will continue, albeit at a very low degradation rate, even if a lipid matrix coating is present.

Similarly, the CLA used as starting material must be negative in the Kreis test (Italian standard NGD C56-1979) for identifying any aldehydes produced by the CLA degradation process, and the para-anisidine value should be as low as possible.

In the first step of the process for the preparation of the product according to the invention, the CLA, which is liquid at room temperature, is completely adsorbed onto the solid support. The solid support is preferably inorganic to withstand degradation over a longer period of time.

In order to minimize the time required to complete the step, it is carried out at a temperature of about 60-70 ℃ with a high-speed stirrer. Under these conditions, the desired effect is usually achieved in a few minutes.

In particular, the preferred solid support is based on silica, which is in the form of a powder having an average particle size of 10 to 80 microns, preferably 15 to 20 microns.

The silica used is preferably a synthetic derivative, substantially free of metals, in order to avoid triggering the oxidation reaction of the CLA and possible contamination of the CLA.

It is important to emphasize that, in addition to adsorbing the CLA, the silica also imparts to the mixture produced in the subsequent processing step a suitable consistency to promote its accurate formation into the final granular product after entering the spray cooling chamber.

Likewise, in order to adjust the consistency of the mixture entering the spray chamber, other mineral agents, such as calcium carbonate or calcium sulphate dihydrate, may optionally be used in addition to silica.

The amount of silica used is sufficient to achieve complete adsorption of the CLA, generally between 33% and 55% with respect to the CLA.

After completion of the first step of the process, a free-flowing powdery material is obtained, constituting the core of the final product.

Immediately after the first step, a second step is carried out in which the resulting powdered material is mixed with a lipid matrix which will form a coating covering and protecting the inner core.

According to a first aspect of the invention, the lipid matrix comprises at least 80% by weight of glycerides of saturated fatty acids having 16, 18, 20 and 22 carbon atoms (abbreviated as C16, C18, C20 and C22).

The term "saturated" is not to be understood in an absolute sense, but is intended to mean fatty acids having a degree of saturation of at least 99%.

As shown in the tests carried out by the applicant reported below, it is particularly important that the fatty acids present in the matrix of the invention are substantially in the form of glycerides and not free acids. For this purpose, the percentage of free acids in the lipid matrix must be less than 10%, preferably less than 1%.

The glycerides are preferably in the form of triglycerides.

It is also preferred that the lipid matrix of the invention has a content of C18 saturated fatty acids of greater than 85% based on the total amount of saturated fatty acids constituting the glycerides.

This property is entirely unexpected given the protective action of the lipid matrix and therefore of the coating with CLA, which is much greater than that of the matrices in which the other fatty acids mentioned above predominate.

The melting point of the lipid matrix is thus between 60 and 75 deg.C, preferably between 65 and 68 deg.C.

The lipid matrix is first melted and then mixed with the powdery material obtained by adsorption of CLA on silica. Mixing may optionally be carried out in the presence of a suitable emulsifier to facilitate homogeneous dispersion of the silica powder in the lipid matrix.

The ratio of lipid matrix (triglyceride + emulsifier) to CLA depends on the type of product (in particular the size) that needs to be obtained. In the preferred embodiments described herein, the ratio is generally from 1.3 to 1.5, preferably 1.4.

Mixing is carried out for about 5 to 20 minutes, preferably about 10 minutes, to obtain a homogeneous mixture (although, more precisely, the resulting system is more preferably defined as a homogeneous suspension of the solid powder in the lipid matrix).

The mixture is then immediately injected under high pressure through a nozzle of suitable shape into a refrigerated spray chamber, the temperature of which is maintained between-2 ℃ and-12 ℃, so that the lipid matrix can solidify according to known methods (spray cooling technique) during the short time that the particles of the mixture remain in the gas phase.

A solid, granular product is thus obtained, comprising an inner core formed by solid-phase carrier particles adsorbing CLA and a coating formed by a lipid matrix for covering and protecting the inner core.

After spraying, the product is collected on a conveyor belt and forced to leave the cooling chamber at a temperature of less than 25 ℃ while the product is still inside the cooling chamber.

To prevent agglomeration of the granular product, an anti-agglomeration agent is sprayed, said anti-agglomeration agent consisting, for example, of silica with a particle size of 75-80 microns, in a proportion of about 0.5-2% of the product.

The particle size depends on the supply pressure and the nozzle shape, but the product can also be sieved, if necessary, to meet the requirements in terms of the desired size.

With the specific preparation method and matrix used, the resulting coating continuously and uniformly encapsulates the conjugated linoleic acid core adsorbed on the silica. This prevents exposure of the CLA to oxygen in the environment, light and oxidants present in formulations for human and/or animal use during storage prior to use. Furthermore, when used in the feed of ruminants, this may also prevent or reduce microbial biohydrogenation occurring in the rumen. The resulting microencapsulated product can be used in the preparation of medicaments and food supplements, which in turn can be used in the diet of humans and animals, depending on the dosage.

Another important advantage achieved by the process of the invention is that the time during which the CLA is exposed to the atmosphere is very limited, of the order of 20 minutes. This allows operation at normal atmospheric pressure.

Preparation examples of the products according to the invention

Example 1

34 g of CLA oil (content 60%) were introduced into a jacketed mixer, heated to 70 ℃ and adsorbed by 14.4 g of silica with 5 g of calcium carbonate added, giving a free-flowing powdery material.

The lipid matrix, comprising 43.4 g triglycerides of C16, C18, C20 and C22 saturated fatty acids (wherein the C18 content is 85%), and 3 g emulsifiers, was heated to 70 ℃, added to the powdered material and stirred for about 10 minutes to obtain a homogeneous suspension.

The resulting mixture is then placed in a cooling chamber maintained at a temperature of about-10 ℃ and sprayed with a nozzle suitable for the desired particle size to obtain granules having an inner core based on CLA adsorbed on silica and a lipid matrix coating the inner core.

About 0.9 grams of silica was dispersed onto the microcapsules removed from the cooling chamber, all sieved to define the size of the final product (80% less than 800 microns).

Example 2

A sample of the second product was prepared in the same manner as in the above example, except that the triglyceride contained about 45% C18 saturated fatty acids and about 60% C16 saturated fatty acids.

Example 3 (comparative example)

A third sample of product was prepared in the same manner as in example 1, with the difference that the matrix used was formed from free long-chain saturated fatty acids with a stearic acid (C18) content of 98%.

Product analysis

A first set of laboratory tests were performed on the samples prepared above to determine their stability over time with respect to environmental factors and different oxidants. Specifically, the various samples were packaged in paper bags with aluminum foil and internal PE and stored in a storage.

Stability was evaluated by measuring the CLA content and the peroxide value over time, which as mentioned above constitutes an index of the extent of degradation of the CLA and of the lipid matrix.

The preliminary test consists in evaluating the stability over time of the CLA adsorbed on silica without coating with a lipid matrix. The samples were stored in the dark for one week, at the end of which the CLA content was reduced from 19.8% to 12.9%, by 35%. This test shows that the presence of the coating is essential for the stability of the CLA.

This test shows that the sample of example 3, in which the matrix is formed substantially from C18 fatty acid rather than triglyceride, is clearly less stable on storage. In fact, the samples started to lose their initial content after several days of preparation, the CLA concentration was zero after 3 months (initial content 20.35%; content 0% after 3 months of storage), and the peroxide value increased from 3.2 to 50.

The product also shows a significant tendency to agglomerate due to the exothermic chemical reaction between the CLA and the matrix.

A slight improvement in stability was observed from the sample of example 2, with a loss of CLA content of about 84% after 6 months of storage, decreasing from the initial 19.98% to 3.19%. Moreover, CLA tends to escape the microcapsules through the coating.

Quite surprisingly, on the other hand, example 1 has excellent stability during the entire measurement process. In fact, after storage for up to 2 years, the measured CLA content was reduced by only 6%, from 20.0% to 18.8%.

A second set of experiments was performed in cows to examine the protective effect of CLA in the gastro-intestinal tract of ruminants.

The test was carried out using samples of CLA-based product obtained according to example 1 and according to example 3, given to the groups of cows in various doses over a period of 1 month.

After the treatment, the milk obtained from each group of animals was analyzed to determine its CLA methyl ester content and compared with samples of milk obtained from the untreated milk group and with samples of milk sold in commercial packages.

Each milk sample was then further analyzed to identify its acid profile by measuring the proportion of each of saturated, monounsaturated, and polyunsaturated fatty acids.

The type of sample administered to each group of animals, the dose administered and the results obtained by analyzing the milk samples are summarized in table 1 below.

Group of	1	2	3	4	5*
						Dosage of product (g/head/day)	20	50	50	0	-
Type of product	Example 1	Example 1	Example 3	-	-
						CLA (milligram/kilogram milk)	0.95	2.14	1.28	0	0
% saturated fatty acid	67.85	56.80	58.29	77.30	72.30
						% monounsaturated fatty acid	30.07	39.54	39.24	20.65	25.20
% polyunsaturated fatty acids	2.09	3.67	2.47	2.05	2.07

TABLE 1

Commercial milk samples

From this table, it is clear that CLA methyl ester is not present in the untreated animals, and its presence was confirmed because exogenous CLA was administered. It is clear from comparing group 2 and group 3 that the matrix based on triglycerides with 85% C18 fatty acids is much more effective in protecting CLA from ruminant biohydrogenation processes and enabling its absorption by the mammary gland than the matrix consisting of C18 free fatty acid.

Moreover, a comparison of groups 1 and 2 shows that the utilization of CLA is substantially proportional to the amount of CLA administered, regardless of the type of animal feed.

Another important effect shown by the above tests is that the administration of CLA according to the invention to cows advantageously improves the acid profile of the milk obtained, significantly reduces the proportion of saturated fatty acids in the milk, favours the proportion of mono-and polyunsaturated fatty acids and produces all the positive effects resulting therefrom.

The present invention thus solves the problems discussed above in connection with the prior art while providing a number of other advantages.

Claims (28)

1. A product based on conjugated linoleic acid, comprising an inner core in which the conjugated linoleic acid CLA is concentrated and a coating for covering and protecting the inner core, characterized in that the CLA is adsorbed on an inorganic solid support, the coating being formed by a lipid matrix comprising a fraction of glycerides of C16, C18, C20 and C22 saturated fatty acids of greater than 80% by weight, the glycerides being triglycerides of free acidity less than 10%.

2. The product of claim 1, wherein said free acidity is less than 1%.

3. The product of claim 1, wherein the C18 fatty acid moieties are equal to or greater than 85% relative to the total fatty acids contained in the lipid matrix.

4. The product of claim 1, wherein said CLA is adsorbed on a solid support in powdered form in said inner core.

5. The product of claim 4, wherein the solid support has an average particle size of 10 to 80 microns.

6. The product of claim 5, wherein the solid support has an average particle size of 15 to 20 microns.

7. The product of claim 4, wherein the solid support is based on metal-free silica.

8. The product of claim 5, wherein the solid support is based on metal-free silica.

9. The product according to claim 7, characterized in that, in addition to silica, minerals based on calcium carbonate or calcium sulphate dihydrate are present.

10. The product according to claim 8, characterized in that, in addition to silica, minerals based on calcium carbonate or calcium sulphate dihydrate are present.

11. The product of claim 1, wherein the lipid matrix comprises an emulsifier.

12. A product according to claim 1, wherein the CLA contained in the inner core is in the form of a methyl ester, free fatty acid or salt thereof, and contains a fraction of cis-9, trans-11 and trans-10, cis-12 isomers greater than 50% by weight.

13. The product of claim 12, wherein the CLA comprises about 60% by weight of the cis-9, trans-11 and trans-10, cis-12 isomer fractions.

14. The product of claim 12, wherein the CLA comprises about 80% by weight of the cis-9, trans-11 and trans-10, cis-12 isomer fractions.

15. The product of claim 1, wherein the product is in the form of particles having a particle size of 0.15 to 2 mm and at least 80% of the product is less than 800 microns.

16. The product according to claim 15, characterized in that the weight ratio between the lipid matrix and the CLA is comprised between 1.3 and 1.5.

17. A process for the preparation of a product based on Conjugated Linoleic Acid (CLA), comprising the step of coating an inner core in which the CLA is concentrated with a coating, the CLA being adsorbed entirely on a solid support in powdered form, before being coated with the coating, to provide a free-flowing powdered material, the powdered material obtained by adsorbing the CLA on a solid support being mixed with a lipid matrix at a temperature above the melting point of the matrix, the melting point being 60-75 ℃, to obtain a mixture formed from a solid suspension of a molten lipid matrix, the mixture being sprayed through a nozzle at high pressure into a cooled spray chamber maintained at a temperature of-2 ℃ to-12 ℃, to form a granulated solid product, wherein the CLA is adsorbed on an inorganic solid support, the coating being formed from a lipid matrix comprising more than 80% by weight of C16, Glycerides of C18, C20 and C22 saturated fatty acids, which are triglycerides with less than 10% free acidity.

18. The method of claim 17, wherein the free acidity is less than 1%.

19. The method of claim 17, wherein the C18 saturated fatty acid fraction is equal to or greater than 85% relative to the total fatty acids contained in the lipid matrix.

20. The method of claim 17, wherein said CLA has a peroxide number of less than 10.

21. The method of claim 17, wherein the solid support has an average particle size of 10 to 80 microns.

22. The method of claim 21, wherein the solid support has an average particle size of 15 to 20 microns.

23. The method of claim 17, wherein the solid support is based on metal-free silica.

24. The method of claim 17, wherein the adsorption is achieved by high speed mixing.

25. The method of claim 17, wherein the mixing of the adsorbed CLA with the lipid matrix lasts for 5-20 minutes.

26. The method of claim 17, wherein a silica-based powder is dispersed in the granular solid product as an anti-agglomeration agent.

27. Use of a product based on Conjugated Linoleic Acid (CLA) according to any one of claims 1 to 16 as a food supplement for human consumption.

28. Use of a product based on Conjugated Linoleic Acid (CLA) according to any one of claims 1 to 16 as a feed supplement for animals.