USE OF TALL OIL FATTY ACID IN BINDING TOXINS

FIELD OF THE INVENTION

The invention relates to use of a tall oil fatty acid and/or a tall oil fatty acid which is modi- fied by saponification in binding toxins.

BACKGROUND OF THE INVENTION

Toxins are poisonous substances produced within living cells or organisms. Toxins such as myco- toxins are a chemically variable group of secondary metabolites of fungi, which can be found in grains and other feedstuffs even in the absence of any visible fungal growth. High temperature and air humidity dur ^¬ ing the storage increase the likelihood of fungal growth, but mycotoxin contamination can also occur already in the field. Visible appearance or smell of grains or silage does not indicate the presence or ab ^¬ sence of mycotoxin contamination. Effects of toxins such as mycotoxins to farm animals are very variable, and range from increased mortality to decreased fer ^¬ tility and performance. Mycotoxins may also disturb the immune system of animals and make them more sus ^¬ ceptible to diseases.

Due to the chemical variability of mycotox- ins, analysis of all feedlots for even the most common mycotoxins would be too expensive. Therefore mycotoxin adsorbents are often used to give extra insurance against mycotoxin contamination in feeds. Mycotoxin adsorbents are substances that are itself not digested or absorbed by the animal. They are assumed to bind toxins during the passage through the alimentary canal. Thus, instead of being absorbed by the animals, the toxins get eventually voided via feces.

Toxin binders can also adsorb other types of toxins, like bacterial toxins or secondary metabolites of plants from the digestive tract. Activated carbon (charcoal) , which is a fine-granulated powder with a porous microstructure, is an efficient toxin binder. It is a recommended general toxin binder in various poisonings. However, charcoal also binds vitamins and minerals, which makes it unsuitable for continuous use in feeds. Silicate clays are also used as adsorbents in feeds .

Fractional distillation of crude tall oil, obtained as a by-product of the Kraft process of wood pulp manufacture, produces distilled tall oil (DTO) which typically comprises over 10% resin acids and less than 90% fatty acids. Further refinement of dis ^¬ tilled tall oil produces the tall oil fatty acid (TOFA) , which is available in a variety of composi- tions differing in the fatty acids and resin acids content. Because the TOFA is an inexpensive source of fatty acids, it has previously been used in animal nu ^¬ trition as an energy source. For instance, GB 955316 discloses the use of alkali metal salts of tall oil fatty acids to improve weight gain and nitrogen reten ^¬ tion in ruminant animals.

PURPOSE OF THE INVENTION

The purpose of the invention is to provide a new type of absorbent for use in binding toxins.

SUMMARY

Use of a tall oil fatty acid and/or a tall oil fatty acid which is modified by saponification ac- cording to the present invention is characterized by what is presented in claim 1.

DETAILED DESCRIPTION OF THE INVENTION FIG la Reduction of free ochratoxin A from the aqueous medium by the saponified TOFA. FIG lb Reduction of free zearalenone from the aqueous medium by the saponified TOFA.

FIG 2a The relative amount of ochratoxin A in the intestinal tissue of rat as a response to the saponified TOFA.

FIG 2b The relative amount of zearalenone in the intestinal tissue of rat as a response to the saponified TOFA.

The present invention is based on the reali- zation that the tall oil fatty acid and the modified tall oil fatty acid can be used in binding toxins.

The term "tall oil fatty acid" or "TOFA" should be understood as referring to a composition ob ^¬ tained by distillation of crude tall oil and further refinement of distilled tall oil. The TOFA or the TOFA which is modified by saponification typically compris ^¬ es 90-98% (w/w) fatty acids. Further, the TOFA or the TOFA which is modified by saponification may comprise 1-10% (w/w) resin acids.

Resin acids are known to have antimicrobial, including antibacterial, properties. The present in ^¬ ventors have found that resin acids of TOFA are not absorbable, and they have potential in toxin binding.

In this context, the term "toxin" should be understood as referring to any poisonous substance produced within living cells or organisms. Toxins are products of plants, animals, microorganisms, for exam ^¬ ple bacteria, viruses, fungi, rickettsiae, protozoa, etc. In this context, the term "mycotoxin" should be understood as referring to a toxic secondary metabo ^¬ lite produced by fungi, such as yeast and mould. The most common mycotoxins in grains or silage are for ex ^¬ ample aflatoxins, zearalenone, ochratoxin A, deoxyni- valenol, fumonisin and T-2 toxin. The toxins will vary depending on environmental factors.

In this context, the term "animal" should be understood as referring to all kinds of different ani- mals, such as monogastric animals, ruminants, fur ani ^¬ mals, pets and aquaculture. Non-limiting examples of different animals, including offspring, are cows, beef cattle, pigs, poultry, sheep, goats, horses, foxes, dogs, cats and fish.

In one embodiment of the present invention, the tall oil fatty acid which is modified by saponifi ^¬ cation comprises 1-10% (w/w) of resin acids.

In one embodiment of the present invention, the tall oil fatty acid which is modified by saponifi ^¬ cation comprises 2-9 % (w/w) resin acids.

In one embodiment of the present invention, the tall oil fatty acid which is modified by saponifi ^¬ cation comprises 5-9% (w/w) resin acids.

In one embodiment of the present invention, the tall oil fatty acid comprises 1-10% (w/w) of resin acids .

In one embodiment of the present invention, the tall oil fatty acid comprises 2-9 % (w/w) resin acids.

In one embodiment of the present invention, the tall oil fatty acid comprises 5-9% (w/w) resin ac ^¬ ids .

In this context, the term "resin acids" should be understood as referring to a complex mixture of various acidic compounds comprised by tall oil which share the same basic skeleton including a three- fused ring. The exact composition of the resin acids present in the TOFA varies e.g. according to the spe- cies of the trees the TOFA is obtained from and the processing conditions under which it is manufactured. Resin acids typically include compounds such as abiet- ic acid, dehydroabietic acid, levopimaric acid, neoab- ietic acid, pimaric acid and isopimaric acid, only to mention a few . In one embodiment of the present invention, the tall oil fatty acid which is modified by saponifi ^¬ cation comprises 90-98% (w/w) of fatty acids.

In one embodiment of the present invention, the tall oil fatty acid comprises 90-98% (w/w) of fat ^¬ ty acids .

Various processes for the saponification of the TOFA using e.g. NaOH or CaOH are known to a person skilled in the art.

In one embodiment of the present invention, the TOFA which is modified by saponification, the TOFA soap, is dried. The modified TOFA can be dried by spray drying, drum drying or by any other known suitable drying method.

The tall oil fatty acid or the tall oil fatty acid which is modified by saponification can be used as a feed additive which is effective in binding tox ^¬ ins .

In one embodiment of the present invention, the tall oil fatty acid which is modified by saponifi ^¬ cation is used together with silicate carrier.

In this context, the term "feed additive" should be understood as referring to a composition that may be added to a feed or used as such in the feeding of animals. The feed additive may comprise different active ingredients. The feed additive may be added in the feed in a concentration of 0.1 - 5 kg//ton of dry weight, preferably 0.2 - 3 kg/ton, most preferably 0.5 - 2 kg/ton of the dry weight of the to- tal amount of the feed. The TOFA or the TOFA which is modified by saponification or the feed additive com ^¬ prising the TOFA or the TOFA which is modified by saponification according to the invention may be added to the feed or feed additive as such, or it may in general be further processed as desired. Further, it may be added to the feed or feed additive, or it may be administered to an animal separately (i.e. not as a part of any feed composition) .

In this context, the term "feed composition" or "feed" should be understood as referring to the to- tal feed composition of an animal diet or to a part thereof, including e.g. supplemental feed, premixes and other feed compositions. The feed may comprise different active ingredients.

In one embodiment of the present invention, the feed additive comprises the TOFA which is modified by saponification and which is absorbed into a carrier material suitable for the feed composition such as sugarbeet pulp.

In one embodiment of the present invention, the feed additive comprises the TOFA which is absorbed into a carrier material suitable for the feed composi ^¬ tion such as sugarbeet pulp.

In one embodiment of the present invention, the feed additive comprises the TOFA which is modified by saponification and which is dried.

The feed composition can comprise the TOFA which is modified by saponification or the feed addi ^¬ tive comprising the TOFA which is modified by saponification according to the invention in an amount of 0.01 - 0.5 % (w/w) , preferably 0.02 - 0.3 % (w/w) , most preferably 0.05 - 0.2 % (w/w) of the dry weight of the total amount of the feed.

In one embodiment of the present invention, the feed composition comprises the TOFA or the feed additive comprising the TOFA in an amount of 0.01 - 0.5 % (w/w), preferably 0.02 - 0.3 % (w/w), most pref ^¬ erably 0.05 - 0.2 % (w/w) of the dry weight of the to ^¬ tal amount of the feed.

The modified tall oil fatty acid according to the invention is produced by saponification. The method comprises the steps of adding a base to an aqueous TOFA solution and heating the mixture. The mixture is stirred during the heating step. The mixture is heated at a temperature of 80 - 120 °C, preferably at 85 - 95 °C, for a period of 1 - 3 hours, preferably for 2 hours .

Any base suitable for saponification, such as an alkali metal hydroxide, can be used as the base. Normally, the base that is used is a sodium or potas ^¬ sium hydroxide.

In one embodiment of the present invention, the method of producing a modified tall oil fatty acid further comprises a step of drying. The drying can be carried out by spray drying, drum drying or by any other known drying method.

In one embodiment of the present invention, the TOFA which is modified by saponification is administered to an animal in an effective amount.

In one embodiment of the present invention, the TOFA is administered to an animal in an effective amount .

The present invention has a number of ad ^¬ vantages. The TOFA is a readily available, natural, low-cost and environmentally friendly material. Fur ^¬ ther, it is non-toxic and well tolerated. The inven ^¬ tion is effective in absorbing toxins. Subsequently, other benefits of the invention are e.g. improved ani ^¬ mal health and productivity.

The embodiments of the invention described hereinbefore may be used in any combination with each other. Several of the embodiments may be combined to- gether to form a further embodiment of the invention. A product, a method or a use, to which the invention is related, may comprise at least one of the embodi ^¬ ments of the invention described hereinbefore. EXAMPLES

In the following, the present invention will be described in more detail. EXAMPLE 1.

Test A: Toxin adsorption into solid phase in vitro

The capacity of a test product to remove tox ^¬ ins from aqueous medium was measured in this test. An efficient toxin adsorbent should be able to bind the toxin in all compartments of the digestive tract, to inhibit the toxin from getting absorbed by the animal. To evaluate the efficacy of the binder in the acidic stomach, the test was run at pH value 2.5 (50 mM gly- cine-HCl buffer) .

The test product was a saponified Tall Oil Fatty Acid (TOFA) product which contains 8.5~6 resin acids. The saponified TOFA was manufactured by adding 140 mg of NaOH (sodium hydroxide) to 1 gram of TOFA, adding enough water to adjust the total dry matter (TOFA) percentage of the mixture to 18-20%, heating the mixture to + 90 °C, keeping the temperature at + 90 °C for 120 minutes, during which time the mixture was gently stirred at 15 min intervals. The product tested was the saponified TOFA (8.5%) with or without silicate carrier.

The test A was conducted with two toxins

Ochratoxin A (OTA) and Zearalenone (ZEA) , at pH-value 2.5, three test substance levels 0,5, 1 and 2 kg/ton and four replicate samples per treatment. Control treatment was replicated 8 times.

Mycotoxins OTA and ZEA were available as 3H- labeled pure compounds, and radioactivity, measured by liquid scintillation counting, was used for their quantification in the samples.

The experiment was conducted in silanized glass vials in 1 ml volume of buffer. In the test sys ^¬ tem, the bound radioactive toxin becomes removed from the liquid phase through co-pelleting with the insolu- ble components of the potential binder. The following procedure was used: 1. The test products were weighed into the vials, 2. 3H -labeled and intact mycotoxin was mixed with the buffers to get the final toxin con- centration of 10 μg/l, 3. 1 ml of the buffer-mycotoxin solution was added to the vials, 4. The vials were sealed and kept for 2 hours at 37 °C in constant slow shaking, 5. The vials were centrifuged for 10 min at 3000 x g 6. 50 μΐ of the supernatant was mixed with 150 μΐ of liquid scintillation cocktail (Optiphase) into wells of a 96-well microtiter plate and 7. The radioactivity of the samples was measured with a liq ^¬ uid scintillation counter for five minutes. Results

The results are illustrated in Figure la and lb. The saponified TOFA was able to bind OTA from the aqueous medium statistically significantly, and the binding was dependent on the concentration of the test product (Figure la) . The saponified TOFA adsorbed 25- 60% of the free OTA from the medium.

The saponified TOFA significantly decreased the amount of free ZEA even at the lowest dosages (p < 0.001; Figure lb) . The saponified TOFA removed approx- imately 30-60% of the free toxin.

In some cases the silicate carrier may be a beneficial addition to the combination of the saponified TOFA, in order to increase the range of toxins to which the product combination is effective.

EXAMPLE 2.

Test B: Inhibition of toxin uptake ex vivo This test studied the ability of the saponi ^¬ fied TOFA to inhibit the uptake of toxins Ochratoxin A (OTA) and Zearalenone (ZEA) into live intestinal tis ^¬ sue of a rat ex vivo.

The saponified TOFA comprising 8.5~6 resin ac ids was manufactured as described in Example 1. The saponified TOFA was tested with silicate carrier.

The test B was conducted with two toxins Ochratoxin A (OTA) and Zearalenone (ZEA) , two test substance levels 1 and 2 kg/ton and three replicate samples per treatment. Control treatment was replicat- ed 6 times.

The rats were not exposed to mycotoxins prior to the experiment.

The test was conducted as follows for the my ^¬ cotoxins: 1. The ileum of euthanized rat was immedi- ately removed, opened, emptied and rinsed with a phys ^¬ iological, buffered saline solution (128 mM NaCl, 4.7 mM KC1, 2.5 mM CaC12, 1.2 mM KH2P04, 2.6 mM MgS04, 2.0 mM NaHC03, pH 7.3), 2. The ileum was cut into trans ^¬ verse 2-mm slices and kept in the saline solution un- til the incubations. Peyer' s patches were removed, 3. The slices were immersed in the same basal saline so ^¬ lution amended with the radioactively labeled mycotox- in (10 μg/l), and with or without saponified TOFA, 4. After 5 min incubation at room temperature, the slices were quickly (2-3 seconds) rinsed with the same saline solution without the toxin or test substances, 5. Ex ^¬ cess saline was removed, 6. The slices were placed in pre-weighed 3-ml liquid scintillation vials, and re- weighed to calculate their mass, 7. 0.25 ml of tissue solvent was added to solubilize the tissue slices overnight at room temperature, 8. 2.5 ml of liquid scintillation cocktail was added to the vials after the slices were fully dissolved into the solubilizer, 9. The radioactivity of the slices was calculated with a liquid scintillation counter and 10. The radioactivity per unit mass in the treatments was compared against of the control treatment. Results

The results are illustrated in Figure 2a and 2b. The results were expressed in relation to the con ^¬ trol treatment without binders. Treatment values less than 100 % indicate inhibition of toxin uptake by the test product. The saponified TOFA decreased the uptake of OTA (p < 0.05 for the 1 kg/tn treatment) (Figure 2a) . The saponified TOFA dose-dependently decreased the uptake of ZEA (p < 0.01 for the 2 kg/tn treatment) (Figure 2b) . These results show that the saponified TOFA is an efficient agent for toxin binding in the ex vivo model with the presence of live intestinal tis ^¬ sue .

It is obvious to a person skilled in the art that, with the advancement of technology, the basic idea of the invention may be implemented in various ways. The invention and its embodiments are thus not limited to the examples described above; instead they may vary within the scope of the claims.