The invention relates to use of a tall oil fatty acid and feed supplement and feed composition comprising said tall oil fatty acid.

BACKGROUND OF THE INVENTION

Imbalances in microbial populations and growth of harmful bacteria in the digestive tract of animals can cause significant losses in animal growth and production. These imbalances manifest themselves as intestinal disorders such as diarrhea. While micro ^¬ bial infections of animals have been prevented by the use of e.g. antibiotics and other agents that prevent the growth of microorganisms, stricter regulations on their use are expected. Generally, there is an in ^¬ creasing demand for ingredients for use in animal feeding that can modulate the microbial population in the animal digestive tract but which are readily available, well tolerated and environmentally friend- ly.

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 tall oil fatty acid (TOFA) , which is available in a variety of compositions dif ^¬ fering in the fatty acids and resin acids content. Be- cause TOFA is an inexpensive source of fatty acids, it has previously been used in animal nutrition as an en ^¬ ergy source. For instance, GB 955316 discloses the use of alkali metal salts of tall oil fatty acids to im ^¬ prove weight gain and nitrogen retention in ruminant animals. PURPOSE OF THE INVENTION

The purpose of the invention is to provide a new type of tall oil fatty acid/feed supplement for use in the modulation of microbial population of the animal digestive tract.

The present inventors have surprisingly found that TOFA modulates microbial population of the animal digestive tract. SUMMARY

Use of a tall oil fatty acid according to the present invention is characterized by what is present ^¬ ed in claim 1.

Use of a feed supplement according to the present invention is characterized by what is present ^¬ ed in claim 9.

Use of a feed composition according to the present invention is characterized by what is present ^¬ ed in claim 13.

DETAILED DESCRIPTION OF THE INVENTION

FIG 1. Effect of TOFA on the total SCFA pro ^¬ duction in the ileal simulation.

FIG 2. Effect of TOFA on the acetic acid pro ^¬ duction in the ileal simulation.

FIG 3. Effect of TOFA on the propionic acid production in the ileal simulation.

FIG 4. Effect of TOFA on the lactic acid pro- duction in the ileal simulation.

The present invention is based on the reali ^¬ zation that tall oil fatty acid can be used in the modulation of microbial population of the animal di ^¬ gestive tract.

The modulation of microbial population of the animal digestive tract is carried from homofermenta- tive towards heterofermentative metabolical route and in one embodiment of the present invention it improves the feed utilization (improved nutritional value) . In another embodiment of the present invention, the modu- lation of microbial population of the animal digestive tract improves the feed conversion ratio.

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. TOFA typically com ^¬ prises 90-98% (w/w) fatty acids. Further, TOFA may comprise 1-10% (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, TOFA comprises 2-9 % (w/w) resin acids.

In one embodiment of the present invention, TOFA comprises 5-9% (w/w) resin acids.

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 TOFA varies e.g. according to the species of the trees the TOFA is obtained from and the pro ^¬ cessing 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, TOFA comprises 90-98% (w/w) of fatty acids.

The tall oil fatty acid (TOFA) is produced by refinement from distilled tall oil. Distilled tall oil (DTO) is produced by fractional distillation from crude tall oil, obtained as a by-product of the Kraft process of wood pulp manufacture.

In one embodiment of the present invention, the TOFA is dried. The TOFA can be dried by spray dry- ing, drum drying or by any other known suitable drying method .

The present invention also relates to use of a feed supplement comprising the tall oil fatty acid in the modulation of microbial population of the ani- mal digestive tract.

The feed supplement of the present invention is effective in the modulation of microbial population of the animal digestive tract.

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

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

In one embodiment of the present invention, the feed supplement comprises a tall oil fatty acid which comprises 5-9% (w/w) resin acids.

In this context, the term "feed supplement" 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 supplement may comprise different active ingredients. The feed supplement may be added in the feed in a concentration of 0.0001 - 5 kg//ton of dry weight, preferably 0.005 - 1 kg/ton of the dry weight of the total amount of the feed. The TOFA or the feed supplement comprising the TOFA ac ^¬ cording to the invention may be added to the feed or feed supplement as such, or it may in general be fur ^¬ ther processed as desired.

Further, the TOFA or the feed supplement com ^¬ prising the TOFA according to the invention may be added to the feed or feed supplement, or it may be ad- ministered 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 supplement comprises 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 supplement comprises TOFA which is dried.

The present invention also relates to use of a feed composition comprising the feed supplement comprising the tall oil fatty acid in the modulation of microbial population of the animal digestive tract.

In one embodiment of the present invention, the feed composition comprises the feed supplement in an amount of 0.00001 - 0.5 % (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 feed supplement in an amount of 0.0005 - 0.1 % (w/w) of the dry weight of the total amount of the feed.

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

The invention also relates to a method of modulating microbial population of the animal diges ^¬ tive tract, comprising the step of administering to an animal the tall oil fatty acid according to the inven ^¬ tion. In this context, the term "harmful bacteria" should be understood as referring to any bacteria that is capable of affecting the digestive tract or health of an animal in an adverse manner, including competi- tion for nutrients with the host animal. In this con ^¬ text, the term "microbial population" should be under ^¬ stood as referring to the microorganisms that inhabit the digestive tract, including the Bacteria and Ar- chaea domains and microscopic members of the Eukaryote domain and also intestinal parasites. The microbial population will vary for different animal species de ^¬ pending on e.g. the health of an animal and 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 TOFA is administered to an animal in an effective amount .

The present invention has a number of ad- vantages. TOFA is a readily available, natural, low- cost and environmentally friendly material. Further, it is non-toxic and well tolerated. The invention is effective in modulating the composition of the micro- biota in the animal digestive tract to a direction that is beneficial for animal performance. Subsequent ^¬ ly, other benefits of the invention are e.g. improved animal productivity, improved feed conversion ratio, higher product quality, uniformity, nutritional value and food and product safety and lower costs per pro- duction unit. The invention also allows the production of feed compositions and supplements at low cost. 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

This experiment was conducted to study the effect of TOFA with 5 % resin acids with or without Sugar Beet Pulp (SBP) carrier on the microbial popula ^¬ tion and fermentation of broiler chick ileum in vitro.

Experiment

Ileal contents of 40-days old broiler chicks were used for the simulation media and as inoculants in the simulation models. The trial treatments were prepared from a batch of TOFA.

Preparations of TOFA with 5% resin acids were produced:

1. TOFA with 20 % dry matter content An aliquot of the TOFA was heated to 90°C, mixed with finely ground SBP powder, and dried to con ^¬ tain 375 g dry TOFA /kg.

2. Digested TOFA

Gastrointestinal digestion of the TOFA: Part of the liquid TOFA and the carrier-absorbed TOFA was digested by a pepsin-HCl -treatment (pH 2.25) followed by a pancreatin bile-acid-NaOH treatment (pH 6.2) in a dilution series. The digestion was made to evaluate whether the products would resist the conditions of the upper gastrointestinal tract before they enter the distal intestine with higher microbial activity.

The simulation was conducted in a total of 160 2-ml plastic microcentrifuge vials, in 1.5 ml vol- ume, with 10 hours simulation time. Samples were tested at four concentrations of the dry matter of TOFA: 0%, 0.005%, 0.01%, 0.01% and 1%.

All the simulation samples were analysed for short chain fatty acids and the total number of mi- crobes. In addition, selected samples were analysed for a number of microbial species or groups by quanti ^¬ tative real-time PCR (qPCR) . Ileal simulation samples were analysed for lactobacilli , enterococci and strep ^¬ tococci .

Results

The results show that in the ileal simulation model, TOFA at 1 kg/ton level increased the concentra ^¬ tions of acetic and propionic acids and decreased the concentration of lactic acid. This suggests modulation of microbial metabolism from homofermentative towards heterofermentative metabolical route, which can be seen as a very positive change improving the feed con ^¬ version ratio. TOFA amendment at 0.1 kg/ton negatively affected the population numbers of lactobacilli, en- terococci and streptococci, all of which are lactate producers. The total bacterial numbers in the ileum were not affected by the TOFA, which may indicate that other bacterial populations were increased as a re ^¬ sponse to the TOFA amendment. Pre-digestion of the TOFA affected many of the studied parameters, while the sugar beet pulp carrier had little effect on the fermentation .

Example 2 This experiment was conducted to study the effect of TOFA with 5% resin acids on nutritional val ^¬ ue of feed and feed conversion ratio.

Experiment

240 newly-hatched, male Ross 508 broiler chicks were allocated into 40 open pens, six birds per pen and eight replicate pens per feeding treatment.

TOFA with 5% resin acid content was absorbed into ground sugar beet pulp (SBP) carrier and added to the feeds. The feed was wheat-soy -based starter for ^¬ mula. The dietary treatments:

1. Control, no TOFA

2. Control + TOFA 0.1% (1 kg/ton)

3. Control + TOFA 0.05% (500 g/ton)

4. Control + TOFA 0.01% (100 g/ton)

Chicks were weighed on days 1, 11, 14, and

17. Feed consumption was measured and feed conversion ratio (FCR) was calculated for the same periods. Daily mortality was recorded.

After day 17, 105 ileal and 105 cecal digesta samples were analysed for short chain fatty acids (SCFAs) with gas chromatography and a number of microbial species or groups by qPCR.

Results

The results show that the dietary TOFA with 5% resin acids, fed at the level of 0.1-1 kg/ton, dose-dependently increased the body weight of broiler chicks on days 8, 11, and 14. TOFA at 0.1-1 kg/ton favourably and dose-dependently modulated the small intestinal microbial fermentation from homofermenta- tive to heterofermentative direction. Ileal and cecal numbers of CI. perfringens were not significantly af ^¬ fected by dietary TOFA amendment. TOFA at 0.5 kg/tn decreased the frequency of samples with more than 1*10 ⁹ cells of enterococci or streptococci, or more than 1*10 ¹² cells of lactobacilli . TOFA at 1 kg/ton de- creased the frequency of samples with high counts of streptococci or lactobacilli .

The results show that the TOFA modulates the microbial population of the digestive tract of broiler chicks or other species of poultry if given in the feed and improve the feed conversion ratio.

Example 3 This experiment was conducted to study the effect of TOFA with 9 % resin acids on the microbial population and fermentation of swine small-intestine in vitro

Experiment

The trial treatments were prepared from TOFA oil with 9 % resin acid content. To mimic the conditions prevailing in stomach and duodenum of live piglet, the tested product was initially treated by pepsin-HCl (pH 3- 4) for 1 hour and by bile acid+pancreatin+NaOH (pH 6.8- 7.2) for 3 hours) at 37°C prior to introducing them in the simulation vessels. Samples were tested at two concen ^¬ trations of the dry matter of TOFA: 0, 1.5 and 3.0 kg/tn i.e. 0%, 0.15% and 0.3%).

For the authentic anaerobic growth medium of the small-intestine simulation, distal ileal digesta of 5 piglets (25-30kg) was recovered and pooled. The digesta was subsequently centrifuged to remove the solid particles and combined with buffer solution (pH 6.5) . The final growth medium prepared was maintained anaerobic and treated with TOFA at two doses.

Inoculum from fresh pooled ileal digesta of two piglets was introduced into simulation vessels in an anaerobic glove box. After inoculation, the vessels were sealed with thick butyl rubber stoppers, trans- ferred to 37°C and continuously mixed in a gyratory shaker at 100 r.p.m. The simulations had 5 replicate vessels for each TOFA concentrations, and the inocula- tion was performed in a random order to avoid any po ^¬ tential systematic shifts. Incubation was continued for 10 hours prior to sampling of the vessels for microbial metabolic analyses.

The simulation was conducted in a total of 25

20 -ml glass vials, in 10 ml volume, with 10 hours sim ^¬ ulation time.

All the simulation samples were analysed for total short-chain fatty acids as well as acetic, propionic and lactic acids.

Results

The results are illustrated in Figures 1 - 4. In the ileal simulation model, inhibition of the total Short-chain fatty acid (SCFA) formation with TOFA treatments was observed (Figure 1) . The magnitude of inhibition was -11 to -14% from the control.

TOFA increased the concentrations of acetic (+23 to +31%) and propionic (+94% to +113%) acids and decreased the concentration of lactic acid (-64% to - 82%) in a dose-dependent manner from the control treatment (Figures 2 to 4) . This suggests modulation of microbial metabolism from homofermentative towards heterofermentative metabolical route, which can be seen as a very positive change.

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.