Technical Field

This invention relates to the feed supplement containing whole fruits and/or treated fruits of Milk Thistle (Silybum marianum (L.) GAERTNER) in a mixture with other plant materials or chemically treated oil from the fruits of Milk Thistle for use in feeding animals.

Background Art

The fruits of Milk Thistle contain several substances of which the pharmacologically most important are substances of the flavonol-lignan type, in aggregate named flavonolignans or also flavolignans. For their hepatoprotective, antioxidative, galactogenic, and vascular- beneficial effects, these substances are, according to the state of the art, utilized as efficient phytopharmacons in classical (see e.g. published International Application WO 2000/078325, European patent EP 1 774 963 Bl, and Russian patents RU 2 291 706 and RU 2 274 400), and in alternative medicine (see e.g. Czech patents CZ 293 046 and CZ 296 773, and Czech disclosed application PV 2004-905), and in cosmetic preparations (see e.g. disclosed Japan application JP 2006282568 and disclosed application DE 102005063063). According to the state of the art, however, use of flavonolignans is not limited to human area. Extracts containing flavonolignans are also fed in the form of feeding mixes or batches, for instance, to farm animals, in particular to cattle and poultry, with an objective to protect health and improve utility value of these animals and/or increase quality of products obtained from them - as it can be found e.g. in the publication Tedesco D. et al. J. Dairy Sci. 87(7), 2239-2247, 2004; Tedesco D. et al. J. Vet. Med. A Physiol. Pathol. Clin. Med.. 51(2), 85-89, 2004; Shiavohe A. et al. Journal of Animal Physiology and Animal Nutrition 9J_, 256-262, 2007, and also in French patent FR 2 719 451.

If animals were fed with the fruits of Milk Thistle these were, in particular, parts of the fruits after extraction of active substances (flavonolignans), as mentioned e.g. in Russian patent RU 2 099 076 where cows were fed with extracted cake after its previous extraction with aqueous ethanol. These experiments with feeding the fruits of Milk Thistle after previous extraction of flavonolignans or feeding the separated inner parts of the fruits (endosperm) containing flavonolignans just in very low concentration were also carried out elsewhere in the world with logical stress on the nutritional energy utilization only (Frankiewicz A. et al. Ann. Anim. Sci., 3 (1), 81-87, 2003) in feeding pigs or horses (Stopyra A. et al. Annates Universitas Mariae Curie-Sklodowska Lublin - Polonia, 61(1 1), 95-102, 2006) or calves (Potkanski A. et al. Rozcniki Akademii Rolniczej w Poznaniu, 209, 85-93, 1991). The fruits of Milk Thistle fed in this way, however, represent predominantly just a source of energy for animals the basis of which is fat (fatty oil), protein component, and starch contained in the fruits. Absolute amount and mutual ratio of these components vary depending on the said methods of processing the Milk Thistle fruits; however, the amount of bioactive flavonolignans received by the organism of an animal in this way into its digestive tract is generally low. Moreover, due to an intact matrix of plant cells and very low solubility of flavonolignans in stomach and intestinal fluids, bioavailability of these substances is quite limited; hence, for reaching their effective concentration in blood, considerable feeding batch s are required of which just a tiny part of flavonolignans is transferred into the internal environment of the organism.

Isomerization of positions of double bonds in molecules of polyunsaturated fatty acids, such as linoleic acid, contained in oil of Milk Thistle, can result in their so-called conjugated arrangement in which double and single bonds alternate in the fatty acid chain. This type of fatty acids plays an important role in metabolism of mammals as it participates in mechanisms regulating the amount and composition of body fats, stimulation of the immune system, and protection of the organism against cardiovascular and tumour diseases (Belury M. A.: Annu. Rev. Nutr. 22, 505-531, 2002; Singh M. et al.: Cancer Res. 54, 1212-1215, 1994). Important sources of conjugated fatty acids in human nutrition are foods of animal origin where these substances are mainly contained in the fatty tissue of animals and in milk. Concentration of conjugated fatty acids in foods of animal origin can be increased by feeding food with an increased content of conjugated fatty acids ^' to particular farm animals (Griinari J. M. and Bauman D.E.: AOCS Press, Champaign, 49, 180, 1999). Suitable feed supplements are obtained by synthetic method from plant oils containing polyunsaturated fatty acids (e.g. American patent US 6 015 833). Disadvantage of feeding thus obtained additives in a feedstuff is, in particular, their form of viscous oil to paste mass that, particularly in case of feeding small animals, make problem with intake of thus fortified food as well as with correctly and precisely batch d amount of conjugated fatty acids. Moreover, in such cases, it is quite difficult to ensure chemical stability of reactive conjugated fatty acids for a sufficiently long period, concerning, in particular, processes of their oxidative degradation (Brimberg U. I. and Kamal-Eldin A.: Eur. J. Lipid Sci. Technol. 105, 17-22, 2003). Disclosure of the Invention

The said shortcomings are solved by the feed supplement based on Milk Thistle according to this invention that provides stable mixtures of the Milk Thistle fruits with oil from the Milk Thistle seeds in which positions of double bonds in molecules of polyunsaturated fatty acids were transformed into conjugated positions, suitable for feeding animals, such as, in particular, gravid and lactating females of cattle, horses, camels, sheep, goats, dogs, and cats; this leads to protection of their organism against oxidative stress, protection of their liver tissue (liver parenchyma), increased utility value of farm animals, in particular milk and meat production of cattle, sheep and goats, improved quality of products obtained from farm animals, concerning particularly nutritional aspects, and improved physical condition and performance of sports animals, especially racing, show-jumping and draught horses, and also racing camels, stimulation of the immune system of animals and protection of their organism against tumour diseases.

The subject of this invention is the feed supplement particularly suitable for feeding animals that contains whole and/or treated fruits of Milk Thistle (Silybum marianum (L.) GAERTNER) and 5 % to 15 % by weight of oil from the seeds of Milk Thistle treated by conjugation of double bonds, relatively to weight of the fruits of Milk Thistle.

Solution according to this invention comprises also the method of producing this feed supplement, wherein this method includes the following steps:

a) treatment of the fruits of Milk Thistle, in particular, removing of their outer layers by peeling and/or abrading, if necessary;

b) pressing of the fruits under pressure of 40 to 60 MPa, wherein 10 % to 20 % of fatty oil is separated which consists of more than 90 % by weight of esters of higher fatty acids of chain length Cj ₄ - C ₂₂ with tribasic alcohol, and a tough pressed mass - the cake - is formed;

c) crushing and sieving of the cake to specified distribution of particle size; and

d) spreading of oil from seeds of Milk Thistle treated by conjugation of double bonds to the resulting cake.

Principal problem of utilizing active substances from the fruits of Milk Thistle is their low bioavailability. If, however, the fruits are disrupted by a suitable method, disintegration of cell walls can be ensured, which enables subsequent easier releasing of the substances into the outer environment, e.g. in the digestive tract of the organism. If this method of mechanical treatment is carried out under high pressures and low temperatures, the disintegration of cell walls can be accompanied by compacting of the material, which results in high specific weight of the material and formation of a pressed cake of small specific surface. In this cake, all its components are enclosed and protected against impacts of external environment similarly to the case of intact fruits. In the disclosed method of processing, a part of fat (oil) is separated from the pressed material; its total content in the final cake can thus be purposefully reduced to a required value the weight of which ranges between one third and two thirds of the original content.

The compacting can preferably be carried out by means of pressing on various types of presses; here, the most advantageous, concerning required properties of the resulting material, are screw presses. They can be used for compacting both the whole fruits of Milk Thistle and the fruits after previous treatment consisting e.g. in previous removing of outer layers of the fruits by peeling and/or abrading. In the process of pressing, the oil from disintegrated endosperm cells, where the oil is mainly contained, is uniformly sucked into the whole material. Thus, this material - cake - functions as a solid carrier saturated with the lipophilic component - the residual oil. Therefore, it can be preferably utilized as a base for spreading other lipophilic components, e.g. treated plant oils. With suitable ratios between the carrier and the spread material, the resulting mixtures are solid loose matters suitable for direct use as a feed or as additives to various forms of feeding mixes. In spreading the lipophilic substances on the fruits of Milk Thistle treated by pressing, the added liquid phase in a suitable type of homogenization device, e.g. a conical rotating screw homogenizer, is uniformly dispersed on surface of the solid carrier, subsequently partially sucked into the carrier, and dissolved in its own lipophilic component - oil. This process provides for saturating the carrier with the spread lipophilic material.

If the used lipophilic spread material is the chemically treated oil from the fruits of Milk Thistle in which double bonds in molecules of polyunsaturated fatty acids, in particular linoleic acid, were rearranged into conjugated positions by chemical reaction, and the oil is re- esterified from the original form of acylglycerols to the form of ethyl esters, this combination of materials shows two principal beneficial properties: 1) High compatibility of residual oil in the cake of the fruits of Milk Thistle used as a carrier with the spread chemically treated oil, particularly concerning physical-chemical properties (complete mutual miscibility, chemical structural affinity, etc.)

2) Protection of the added chemically treated oil against degradation by oxidative processes, as it is dissolved in the matrix of natural oil showing higher reactivity against oxidative agents, particularly air oxygen; it thus functions as an antioxidant. In this way, the added chemically treated oil from the fruits of Milk Thistle is efficiently protected against degradation and its beneficial biological properties can be fully utilized.

The obtained mixtures of the pressed fruits of Milk Thistle with spread chemically treated oil from the fruits of Milk Thistle according to this invention can be preferably stabilized by minimizing access of oxygen to the mixture. This can be achieved e.g. by expelling oxygen from the mixture by flushing with a suitable inert gas (nitrogen, argon, carbon dioxide) and subsequent keeping of the material in an environment with inert atmosphere created by the above mentioned gases. Another method of protecting the product according to this invention lies in sucking-off of gases and thus also oxygen from the mixture using vacuum with subsequent keeping of the product in evacuated containers. In this way, the final materials can be kept under prescribed conditions (temperature max. 30°C, original closed containers without access of light) for up to three years without any noticeable decrease in concentration of determinable biologically active substances.

Influence of the prepared feeding mixes on conditions of animals has been verified on the bellow given specific examples by carried out clinical experiments. These experiments were thematically focused on safety of feeding and monitoring, in particular, of biochemical parameters of tested animals in comparison with reference groups, their utility value in case of farm animals, and overall physical condition in case of other animals.

Best Mode for Carrying Out of the Invention

Example 1 Preparation of the feed supplement from the fruits of Milk Thistle and ethyl esters of conjugated fatty acids

Provided batch (5,000 kg) of the fruits of Milk Thistle (loss on drying 7.21 % by weight; content of flavonolignans 3.17 % by weight) was pressed on a press with multiple-run screw (type: "Shark" PL 200 LCPV7, manufacturer: Farmet, spol. s r. o, Ceska Skalice) under pressure 50 MPa; 4.150 kg of the pressed Product 1 and 655 kg of the fatty oil was obtained. Product 1 was crushed and sieved on a crushing machine (type: PS- 12, manufacturer Haiva, spol. s r. o., Tasovice) fitted with a sieve of mesh size 12 mm at its outlet. Flow rate of the material through the crusher was 6,000 kg.h ^"1 . In total, 4.140 kg of the crushed and sieved cake (Product 2) was obtained.

A part of Product 2 (500 kg) and 26.3 kg of oil from the seeds of Milk Thistle treated by conjugation of double bonds and transferred to the form of ethyl esters (content of ethyl esters of conjugated linoleic acid 48 % by weight) were processed by mixing on a conical rotary screw homogenizer (type: HV.3000.1, manufacturer ERGON, n.p., Kromefiz) with the screw speed 1.0 s ^"1 and the arm speed 0.033 s ^"1 for 60 min. In total, 526 kg of the product (Product 3) was obtained.

Product 3 was sampled for pertinent analyses. Results of the analyses are shown in Tables 1 and 2.

Results of analyses of product of processing the fruits of Milk Thistle and ethyl esters of conjugated fatty acids

Results of sieve analysis of feed supplement from fruits of Milk Thistle and ethyl esters of conjugated fatty acids

Example 2 Preparation of feed supplement from fruits of Milk Thistle and ethyl esters of conjugated fatty acids

Provided batch (15,000 kg) of the fruits of Milk Thistle (loss on drying 7.21% by weight; content of flavonolignans 3.17 % by weight) underwent abrading on a peeling machine (type:„Ekonos" 3.22, manufacturer TMS Group, a.s., Pardubice) at weight flow-rate 800 kg.h ^"1 . At its outlet, the machine was fitted with a sieve of mesh size 1.2 x 20 mm. 2,200 kg of abraded outer layers of the fruits and 12,600 kg of the peeled fruits (Product 4) was obtained. A part of Product 1 (1000 kg) was pressed on a press with multiple-run screw (type: "Shark" PL 200 LCPV7, manufacturer: Farmet, spol. s r. o, Ceska Skalice) under pressure 60 MPa; 790 kg of the pressed Product 5 and 192 kg of the fatty oil 2 was obtained. Product 2 was crushed and sieved on a crushing machine (type: PS-12, manufacturer Haiva, spol. s r. o., Tasovice) fitted with a sieve of mesh size 12 mm at its outlet. Flow rate of the material through the crusher was 6,000 kg.h ^"1 . In total, 780 kg of the crushed and sieved cake (Product

6) was obtained.

A part of Product 6 (500 kg) and 55.6 kg of oil from the seeds of Milk Thistle transferred to the form of ethyl esters by conjugation of double bonds (content of ethyl esters of conjugated linoleic acid 53% by weight) were processed by mixing on a conical rotary screw homogenizer (type: HV.3000.1, manufacturer ERGON, n.p., Kromefiz) with the screw speed 1.0 s ^"1 and the arm speed 0.033 s ^"1 for 60 min. In total, 555 kg of the product (Product

7) was obtained.

Product 7 was sampled for pertinent analyses. Results of the analyses are shown in Tables 3 and 4.

Table 3: Results of analyses of product of processing the treated fruits of Milk Thistle and ethyl esters of conjugated fatty acids

Results of sieve analysis of feed supplement from the treated fruits of Milk Thistle and ethyl esters of conjugated fatty acids

Summary of results of clinic experiments

Experiment 1 Flock of pigs in stable breeding

The experiment was carried out with a flock of 40 pigs (cross-breeds Pietrain x Hampshire) in stable breeding fed with a feeding batch composed of maize, soy, dried lucerne fodder, and fodder concentrate. The flock was divided into two halves - experimental and reference under otherwise the same conditions of feeding and stabling. The experimental group was fed with the feeding mix enriched with an additive of the processed fruits of Milk Thistle Product 3 in the amount of 3 % (corresponding to 0.15 % of ethyl esters of conjugated fatty acids from Milk Thistle oil) of the feeding batch. The reference group received 0.15 % of the chemically non-treated Milk Thistle oil to the feeding mix. Animals in both groups were fed in the food amount ad libitum for 8 weeks before the slaughter.

After the slaughter, nutritional parameters of the products (fatty acid composition in saddle and inner lard, fat content in the saddle musculature) and parameters of utility value of animals (ratio of weight increase to food intake, slaughter weight of the animal relative to initial weight) were evaluated.

The results proved that the average composition of fatty acids in respective types of body fats of the group fed with the feed supplement according to this invention showed statistically significant increase in the content of conjugated fatty acids. This fact is particularly important regarding the definition of sources of conjugated fatty acids for the human organisms. Decrease in content of fat was also found in the saddle musculature of the experimental group by 6.3 %, which documents the function of conjugated fatty acids as a regulators of metabolism of body fats and their storage. Lower content of fats in the musculature is a positive fact in relation to human nutrition as it directly reduces fat intake in consumption of the given type of meat. Composition of fatty acids in respective body fats is shown in Table 5.

The parameter of utility value of the daily weight increase of the animal to the amount of food intake showed slight increase in the experimental group compared with the reference group. Slaughter weight relative to initial weight was slightly lower in the experimental group in comparison with the reference group. These parameters are shown in Table 6.

Table 5: Composition of fatty acids

Relative increase in 336 4.9 10.5 % of experimental

group compared

with reference group

Inner lard

Reference group 0.10 36.84 0.60

Experimental group 0.52 37.45 0.65

Relative increase in 420 1.7 8.3

% of experimental

group compared

with reference group

Parameter of utility value of daily weight increase relative to food intake and ratio of slaughter weight of animal to initial weight

Experiment 2 Group of police dogs

The experiment was carried out with 10 individuals of police dogs of the breed German shepherd aged 3 - 5 years; the dogs were divided into two groups, reference and experimental, with 5 individuals in each. Both groups were fed with standard feeding granules for adult dogs (firm Delikan, Lipnik nad Becvou), wherein the experimental group received an extra batch of 20 g of Product 7 per dog and day for 30 days. The feed supplement was integrated by mixing into the feeding granules. Biochemical parameters of blood were tested with a stress on metabolism of parenchymatous organs (liver, kidney) with respect to protein, energy, mineral, and muscular metabolism. Moreover, clinical symptoms of the tested animals were subjectively evaluated: feed intake, activity - impact on performance and vitality, allergic reactions, welfare - well behaviour. Results of the tests documented improved clinical symptoms in the experimental group as shown in Table 7. From the monitored biochemical parameters, haematological profile was not adversely influenced; values of all monitored haematological parameters and mineral metabolism ranged within physiological values. On the other hand, parameters of liver metabolism (ALT, AST, GMT and bilirubin) were not improved in the experimental group in comparison with the reference group. Key results of the relevant monitored parameters are shown in Table 8.

The values indicate that the administered feed supplement according to this invention has a positive effect on all values of monitored liver enzymes (transaminases - ALT, AST, GMT) in the group of experimental individuals fed with the additive according to this invention compared with the reference group fed with the feeding batch without the additive according to this invention 30 days after feeding, which documents fast regeneration of liver cells in case of providing the feed supplement according to this invention. Lower loading of organism in case of the group fed with the feed supplement according to this invention is also documented by marked decrease in metabolites indicated by decreased concentration of bilirubin in the experimental group as compared with the reference group.

Table 7: Clinical symptoms of providing the feed supplement containing the processed fruits of Milk Thistle and ethyl esters of conjugated fatty acids according to this invention in comparison with common fodder of police dogs: scale 0 to 10 points (0 points = no effect, 10 points = effect in maximum intensity) - average values of both groups

Relevant biochemical parameters of liver enzymes in providing the feed supplement containing the processed fruits of Milk Thistle and ethyl esters of conjugated fatty acids according to this invention in comparison with common fodder of police dogs - average values of both groups

0th day ... day before the start of providing the feed supplement

Industrial Applicability

The feed supplements based on the processed fruits of Milk Thistle according to the invention are an important form of feed supplements for a wide group of animals. Due to safety of feeding and proved positive effects on health of animals, they can be used for preventative and curative feeding of animals with a positive effect on their productivity, in case of farm animals, and overall health conditions and performance, in case of sports and domestic animals. Long-term shelf life of the said feed supplements, together with a possibility to feed them to animals either directly or integrated into various types of feeding mixes and products for animal nutrition, ensure their universal applicability and enable active substances from the feed supplements to be administered to animals in an optimum batch and form.

The said method of producing the feed supplements based on the processed fruits of Milk Thistle transfers active substances from this fruits (flavonolignans) into forms showing their high availability from the digestive tract and transfer into the internal environment of the animal where their biological properties beneficial to the animal health can be subsequently asserted. The by-product of processing the fruits of Milk Thistle - fatty oil - can be, after chemical modification, re-integrated into the feed supplements where it represents a source of conjugated fatty acids necessary for correct function of the animal organism.