Additive to food or drinking water containing botanical antioxidants together with probiotic microorganisms.

Technical field

The present invention relates to a feed additive with antioxidative properties based on natural or nature-identical substances combined with probiotic microorganisms and a feed mixture containing the additive, and to a method of use for domestic animals.

Technical background

The use of antibiotic or chemotherapeutic substances as so-called growth promoters in domestic animal production is well known. In recent years increasing attention has been directed to the fact that the widespread use of antibiotics or chemotherapeutical substances leads to problems. Larger and larger numbers of pathogenic microorganisms can develop resistance to the antibiotics concerned, which thus become less effective. There is a risk of residues of antibiotics in meat products, so that antibiotics are also consumed by humans, and consequently, microorganisms that cause disease in humans may also become resistant. Similarly, there is a risk that microorganisms that are resistant to antibiotics may be eaten with meat products. Finally, there is a risk that antibiotics may be returned to the groundwater and watercourses.

It is now considered that the continued use of various antibiotics as growth promoters will among other things increase the risk of the occurrence of an epidemic which cannot be controlled with antibiotics among animals and/or humans. A considerable number of people have already developed allergies to specific antibiotics. In addition, one of the chemotherapeutical growth promoters used up to now has been shown to cause cancer.

Consequently, the use of antibiotic growth promoters in pig breeding has been forbidden in the EU.

Because of the factors described above, and in order to safeguard the economy and sustainability of breeding domestic animals, alternative methods are now being sought to optimize the health and productivity of domestic animals without the use of antibiotic growth promoters and other synthetic products as antioxidants (TBHQ, ethoxyquin, BHT, BHA).

These measures can with advantage be based on natural or nature-identical active substances and probiotic microorganisms. They should also have a corresponding effect on the animals' growth and exploitation of feed to that of the classical growth promoters, to ensure that these new additives are accepted by the farmer on the one hand and by the animal feed industry on the other.

It is well known that many plants contain various functional substances, including saponins, flavonoids, tannins, hydroxycymenes and terpenes, and the use of such active substances from plant material in pharmaceutical products is also well known. Some of the positive properties of these active substances are due to their antioxidative properties. Antioxidants protect cells against breakdown and against harmful substances. They also reduce the risks of infection and have a special effect in periods of high production and stress.

The use of these natural substances as daily dietary supplements has also been proposed for both animals and humans. In many cases the claimed effects of such active plant components are not sufficiently documented, however, and many of these components are only present in small quantities in the plants concerned, while they are often too costly and/or difficult to obtain to be used daily in animal feed.

It is well known that olives have a number of health-promoting effects, such as reducing the risk of cardiovascular disease and cancer, which have been explained to some extent by the high content of natural antioxidants in olives. Among the antioxidants isolated from olive oil are phenol components such as simple phenols {hydroxytyrosol ((3,4- dihydroxyphenyl)ethanol), tyrosol (p-hydroxyphenylethanol)), secoiridoids (oleuropein) and lignans (pinoresinol) and other polyphenols (Owen et al., 2000: Olive-oil consumption and health: the possible role of antioxidants. The Lancet Oncology 1, 107-112). These active substances have been shown to be biologically active, for instance inhibiting cell growth in cancer cells, and actively antiviral or antioxidative (e.g. inhibiting oxidation of low-density lipoproteins (LDL) and inhibiting oxidative stress). The antioxidative activity of the active substances found in olives is far more effective than a synthetic form of vitamin E (Owen et al, 2000: Olive-oil consumption and health: the possible role of antioxidants. The Lancet Oncology 1, 107-112). Antifungal activity has also been demonstrated (Del Rio et al. 2003: Enhancement of phenolic compounds in olive plants and their influence on resistance against Phytophthora sp. Food Chemistry 83 (1): 75-78).

It is also well known that both wine and by-products from wine production have a number of health-promoting effects. Among other things, wine contains phenols such as pigments and tannins, in addition to organic acids and vitamins. Chromatographic studies have shown a number of flavan-3-ols and flavanols. Wine is known to have antiviral, antiallergic, antioxidative and cancer-inhibiting effects among others (Bombardelli & Morazzoni, 1995 : Vitis Vinifera L. Fitoterapia, LXVI (4)).

Other well known plants with antioxidative properties are green tea, berries and citrus fruits, but other plants with antioxidative properties are also included in this model for use.

Berries, and especially blackcurrants, blueberries, elderberries and cranberries, have a high content of polyphenols with antioxidative capacity (Kahkonen et al., 2001 : Berry pheno lies and their antioxidant activity. J. Agric. Food Chem 49, 4076-4082). Among other things, these berries contain anthocyanins, flavonol, rutin and catechin. Studies have shown the potential of berry fruits in preventing diseases like cancer and arteriosclerosis, and the mechanisms are assumed to be based on the way in which polyphenols counteract oxidative stress and inflammatory conditions (Neto, 2007: Cranberry and blueberry: Evidence for protective effects against cancer and vascular diseases. MoI. Nutr. Food Res. 51, 652-664). Besides these, cranberries contain procyanidins, which are believed to exert an effect by reducing linking of bacteria and toxins to the epithelium, so they are thus beneficial against urinary tract infections and others (Duthie, 2007: Berry Fruits. MoI. Nutr. Food Res. 51, 643). Elderberry has certain antiviral effect; it stimulates the immune system, and it has been suggested that it is effective against influenza (Wu et al. 2005: Aglycones and sugar moieties alter anthocyanin absorption and metabolism after berry consumption in weanling pigs. The Journal of Nutrition 135, 2417-2424).

A combination of diverse plants with antioxidative active elements may have particularly valuable properties, since the polyphenols with antioxidative properties found for instance in olives are partially soluble in the oil phase and partially soluble in the water phase (Rodis et al. 2002: Partitioning of olive oil antioxidants between oil and water phases. Journal of Agricultural and Food Chemistry 50, 596-601.). For example, hydroxytyrosol from olives is soluble in both the lipid and the water phase (Briante et al. 2002: Olea europaea L. leaf extract and derivates: Antioxidant properties. Journal of Agricultural and Food Chemistry 50, 4934-4990.). The antioxidants in for example vines and green tea are highly active, but are exclusively soluble in the water phase. A combination of products, for example from vines and olives, would thus result in a product that contained both fat- soluble and water-soluble antioxidants, which could thus function as a sort of transport from one phase to the other. Spreading the solubility and by that means the activity would thus increase the antioxidative activity, largely because the exploitation of the antioxidants would be increased. Synergic activity might thus result from a mixture of various plants with different antioxidative properties.

It is also well known that the use of probiotics is advantageous to the production and health of domestic animals (Alexopoulos et al. 2004. Field evaluation of the effect of a probiotic- containing Bacillus licheniformis and Bacillus subtilis spores on the health status, performance and carcass quality of grower and finisher pigs. J. Vet. Med. 51, 306-312) since among other things probiotics inhibit coliform bacteria and promote bacteria that form lactic acid. Up to the present, however, no one has considered the combination of probiotics and antioxidative plant elements.

A combination of antioxidative elements from plants with probiotics has now surprisingly been shown to have a more advantageous effect on production than the individual elements alone. Various patents describe methods for extracting antioxidants from material based on olives (US Nr. 467439). An International Patent Application (WO 03/086442) deals with natural extracts, including oleuropein, to protect health-promoting products for human use. Patents relating to vines deal with a feed additive made of a by-product purely from vines (EP 1 419 701 Al) or in combination with other named botanical elements containing tannins or flavonoids. Similarly, other patents deal with vine components as animal feed. A European patent deals with the combination of olives and vines (EP 1 790 234). Various patents deal with probiotics of various strains and methods, e.g. US 5073367 and US 4999193.

No patents describe a combination of antioxidative elements from plants with probiotics used as an additive for domestic animals.

In addition, many of the applications mentioned and others, which relate to antioxidative properties in products for domestic animals, are based on extracts that involve high costs. Thus there is a need for a cost-effective additive with such health-promoting antioxidative properties, which can be used in products for animals. A product in the form of a plant- based material with antioxidative properties combined with probiotics offers an obvious possibility of meeting the need.

Brief description of the invention

The present invention relates to a natural additive with probiotic and antioxidative properties for use as an additive to feed for domestic animals, and containing natural and/or nature-identical substances comprising a) A first active component, which contains antioxidative active substances and can be obtained from plant material primarily derived from olives (Olea europaea), vines (vitis vinifera), green tea {Camellia sinensis), citrus {citrus spp.) and various berries, primarily blackcurrants {Ribes nigrum), elderberries {Sambucus spp ), blueberries {Vaccinium myrtillus), cranberries {Vaccinium oxycoccus; V. microcaroum; V. macrocarpum), chokeberries {aronia spp., especially aronia arbutifolia; aronia melanocarpa), blackberries (Rubus spp., especially R. plicatus and R. fruticosus), cloudberries {Rubus Chamaemorus), cowberries {Vaccinium vitis idaea), crowberries (Empetrum nigrum), raspberries {Rubus idaeus), redcurrants {Ribes rubrum) b) a second active component, containing probiotic microorganisms, primarily Bacillus, Lactobacillus, Enter occocus, Lactococcus, Pediococcus, and if necessary conventional processing aids or additives, and which enhances productivity and the health status of animals yielding marketable produce.

In addition, the invention relates to a feed mix for domestic animals containing the additive together with conventional feed, and a method of use for domestic animals by which the animals are fed with the feed mix.

Moreover, the additive may be partially based on one or several waste products, which at present have no other practical use, and thus the active components are very cheap and cost effective, while the production of the product is simplified.

The extent of the usefulness of the invention will be shown in the following detailed description. It should be understood, however, that the detailed description and the specific examples, in which the preferred embodiments of the invention are indicated, are given only for illustration, since various alterations and modifications within the scope of the invention will be apparent to a person skilled in the art based on the detailed description.

Detailed description of the invention

The additive according to the invention can be obtained based on a by-product of olive-oil production, or based on olive leaves as a possible first active component. During the production of olive oil a by-product appears, also called "pulp", as well as waste water. In some processes only the pulp is left, as a semi-dry product, also called "alperujo". This latter product will also be called "pulp" hereafter. These products have a high antioxidant content, as is the case with the leaves of olive trees, which are not utilized to any great extent.

Olives are the fruit of the olive tree (Olea europaea L). Olive oil is produced by washing the olive fruits, after which they are crushed in a hammer mill. The resulting "pomace" is a mixture of olive stones and the pericarp of the olives (the actual fruit flesh). This is homogenized and transferred to pressing plates, where the oil is pressed out. The water is then separated from the clear oil. Several fractions of oil can be obtained by pressing the pomace repeatedly.

Another possible first component contained in the additive can be obtained based on a by- product of wine production. This by-product may consist of the entire residue from pressing or pomace - i.e. everything except the grape juice. In this description and the claims, this total residue or pomace will also be called "the primary pomace".

The primary pomace (also called grape pomace) from wine production is traditionally used as the raw ingredient for a number of different products. After each stage of the process it is possible to obtain a more or less suitable pomace product, which can be included as a larger or smaller proportion of the additive according to the invention.

When making red wine, the grape juice is fermented together with the skin and seeds. After three to seven days, the juice is pressed out, and the primary pomace may be one of several possible raw ingredients of the active component. In certain areas this primary pomace is used to produce different types of alcohol, Grappa and various acids.

The primary pomace can subsequently be processed further. Thus it is technically possible to separate the seeds from the skin in order to extract oil from the seeds. Both the residues from oil extraction and the discarded skin are suitable pomace products for use as raw ingredients for the active component.

In certain wine districts there is a tradition of processing the skin fraction further to obtain natural dyes and the like. In connection with most extraction processes, the residues left from this will have an increased content of the active substances of interest for the invention.

A preferred pomace product is based on red grapes. These may for instance be of the following types: Cabernet Sauvignon, Merlot, Syrah, Pinot Noir, Lemberger or Tempranillo. The pomace product used can with advantage be derived from red grapes which have been used to produce white wine, since the residues of the grapes are separated from the grape juice as soon as is practically possible after the grape juice is pressed out. This means that all the active substances relevant for the additive are preserved in the pomace product.

It is normal in many cases to make use of the primary pomace to extract a variety of products of fermentation, usually alcohol for technical use, citric acid, tartaric acid and the like. Before fermentation, a fraction of grape seeds is normally separated out. This fermentation process too leaves a residue, the secondary pomace, which can be used as the additive according to the invention or a part of it. In the secondary pomace, the high content of easily converted energy has been consumed in fermentation, while the concentration of the relevant active substances according to the invention is increased. Finally, the concentration of active substances relevant for the additive according to the invention can be increased even further, by means of conventional processes to extract other products - which are of no interest in relation to the additive according to the invention - in the form of grape seed oil and natural dyes such as enocianina, which has been produced commercially for over 100 years. (Mazza, G. (1995): "Anthocyanins in Grapes and Grape Products". Critical Reviews in Food Science and Nutrition, vol 35(4): p. 341-371) When these are extracted from the by-product of alcohol production, i.e. a secondary pomace, the active substances still remaining - relevant to the additive according to the invention - are found in a particularly high concentration in the final residual pomace product, a "tertiary pomace".

Other possible components of the first component of the additive according to the invention are obtainable based on a by-product from berries or citrus fruit used in the production of foods, especially jellies or juices and the like, derived from the berries mentioned above or obtained from the actual berries or the leaves.

A by-product, known as pomace, results from the production of jelly or juice. This pomace consists of the skins of berries, seeds, and sometimes fruit pulp. The antioxidant content of the pomace is high, and it is similarly high in the leaves of blueberry, blackcurrant and cranberry bushes and elderberry trees. Furthermore, components of the first component may be obtainable based on dried and ground parts of olive leaves, citrus fruit peel and similar elements from the plants or fruits mentioned.

The component can be exploited either as whole elements or as extracts of the whole elements or of the by-products. Extraction can be carried out with the aid of water, alcohol or a combination of these.

The active part of the second component consists of probiotic microorganisms produced by fermentation and subsequent drying. They may originate from microbiota, soil, plants etc. The second active component contains probiotic microorganisms, predominantly Bacillus (e.g. B. subtilus, B. lichniformis) , Lactobacillus (e.g. L. acidophilus, L. pentosus, L. plantarum, L. brevis, L. paracasei, L. casei), Enteroccocus (e.g. E. faecium), Lactococcus (e.g. L. lactis), Pediococcus (e.g. P. pentosaceus, P. acidilactici) .

Immediately after the juice is pressed out to produce wine or juice, the resulting pomace products and the waste water from olive oil can be reduced until the water content is not more than 8-15%, normally to a maximum water content of 10%; or it can be preserved, for instance by adding 3 to 10% by weight of NaCl or some other salt; and it can then be stored for drying later, or it can be preserved. To ensure the keeping qualities further, it is advantageous to add 0 to 5% of a type of acid that is acceptable in feed, such as citric acid, tartaric acid, propionic acid, ascorbic acid or lactic acid or a mixture of these, the addition of which will at the same time positively influence the effectiveness of the product. Other appropriate preservatives might include calcium propionate, potassium sorbate or sodium benzoate.

The waste water, pomace products and dried and powdered olive leaves can be further concentrated to the extent necessary by the removal of some of the water from the product. This can be done by filtration, extraction, centrifugation and other conventional methods.

The invention contains a number of active substances which are of interest. Among the most essential are primarily polyphenols with antioxidative properties and the anthocyanine and oligomeric procyanidine sub-groups.

The additive according to the invention has the following advantageous effects among others:

1) When used as a feed additive, it has an antioxidative effect in the animal through

* protection against cell breakdown

* reducing the consumption of other antioxidants such as vitamins E and C and selenium

2) It improves the animal's productivity and health because

* it has antiviral properties

* improves feed exploitation

* improves exploitation of nutrients generally * improves health by protecting the epithelial cells in the gastro-intestinal tract

* modulates microbiota

3) It improves product quality by

* improving the water-retention capacity (reduction of drip loss), through protection of cell membranes

* improving meat color by retarding pigment oxidation from red oxymyoglobin to brown metmyoglobin

* improving fatty acid stability by retarding lipid oxidation through the product's antioxidative activeness * greater muscle volume

The additive according to the invention can be used with great advantage for both monogastric domestic animals and ruminants, fish and shellfish, and also for pets. Formulation of Commercial Products with the Additive According to the Invention

Depending on the concentration in the raw product, and the variation of the concentration of the active substances relevant to the additive according to the invention, it may be necessary to mix the raw product with a suitable carrier, so that a final product appears which on the one hand will be reasonably standardized, and on the other hand is not too concentrated to allow it to be mixed directly into the feed either on the premises of ready- feed manufacturers or by breeders who mix feed at home.

Suitable carriers in order to obtain the properties mentioned in the commercial feed additive are primarily mineral carriers in the form of clay minerals and zeoliths - especially sepiolite and clinoptilolite and diatomic earth - and secondarily vegetable carriers such as wheat flour, wheat bran, ground soya husk, etc. Calcium carbonate or calcium magnesium carbonate (dolomite chalk) may also be usable as a carrier, not so much because of its technical properties, but because of its reasonable price. The same applies to grape seed flour, a known carrier that is frequently used in animal feed.

Vegetable matter and the pomace from the production of olive oil, wine and juice can to a greater or lesser extent be pre-ground before being mixed with the carrier in question.

The additive according to the invention has proved to have a significant effect on domestic animals' intake of feed, their daily growth and exploitation of feed, and product quality. The reasons for the effects referred to are not fully clarified, since there may still be further effects that have not been discovered up to the present. The reasons already confirmed include the following.

The Additive according to the invention

* reduces the consumption of other antioxidants such as vitamin E and selenium in animals yielding marketable products and in pet animals

* has antiviral properties * improves nitrogen exploitation

* improves product quality, e.g. in meat and milk, through improved fatty acid stability among other things, through its antioxidative activeness Examples

A number of tests have been carried out to verify the additive on selected Danish farms. Some preliminary test results with weanling pigs are described in examples 1 to 3.

Example 1

Trials on a herd of pigs on Zealand (S). The trial started after weaning on 12 February 2007. The trial lasted three weeks. The weaned pigs were divided into four groups (control, product B, probiotics and "botanic element") balanced for weight and gender. All the pigs were fed ad libitum with the same standard feed. A premix with 2000 ppm of the test products was added to the feed of the test groups. The pigs were weighed at the start, after 10 days and after 21 days. Feed intake was measured on the same days, so that exploitation of the feed could be calculated.

To produce lkg of feed additive - formulated as a premix - the following materials must be mixed in the dry state, referred to in the following as "Probiotic":

Probiotic (Lactobacillus): 150 g

Sepiolite 850 g

To produce lkg of feed additive - formulated as a premix - the following materials must be mixed in the dry state, referred to in the following as "Product B":

Probiotic (Lactobacillus): 75 g

Water extract of grape pomace 7.5 g

Water/ethanol extract of olive leaves: 7.5 g Sepiolite 910 g

To produce lkg of feed additive - formulated as a premix - the following materials must be mixed in the dry state, referred to in the following as "Botanic element":

Water extract of grape pomace 7.5 g

Water/ethanol extract of olive leaves: 7.5 g

Sepiolite 985 g To produce lkg of feed additive - formulated as a premix - the following materials must be mixed in the dry state, referred to in the following as "Product C":

Probiotic {Bacillus): 200 Blackcurrant pomace 50 Sepiolite 750

Table 1. The effect of a combination of probiotics and botanic antioxidative elements on the growth and feed exploitation. Trial 1

Example 2

Trials on a herd of pigs on Zealand (G). The trial started after weaning on 8 March 2007. The trial lasted three weeks. The weaned pigs were divided into two groups (control, product B) balanced for weight and gender. All the pigs were fed αd libitum with the same standard feed. 2000 ppm of the given test product were added to the feed of the test groups. The pigs were weighed at the start, after 10 days and after 21 days. Feed intake was measured on the same days, so that exploitation of the feed could be calculated. Table 2. The effect of a combination of probiotics and botanic antioxidative elements on the growth and feed exploitation. Trial 2

Example 3

Trials on a herd of pigs on Zealand (S). The trial started after weaning on 28 July 2008. The trial lasted six weeks. The weaned pigs were divided into four groups (control, product B and "botanic element") balanced for weight and gender. All the pigs were fed αd libitum with the same standard feed. 2000 ppm of the given test product were added to the feed of the test groups. The pigs were weighed at the start, after 10 days, after 21 days, after 31 days and after 42 days. Feed intake was measured on the same days, so that exploitation of the feed could be calculated.

Table 3. The effect of a combination of probiotics and botanic antioxidative elements on the growth and feed exploitation. Trial 3

Example 4

The trial was carried out on a commercial herd of pigs on Zealand (S). The trial started after weaning on 9 November 2009. The trial lasted three weeks. The weaned pigs were divided into two groups, the control group and the trial group, balanced for weight and gender. All the pigs were fed αd libitum with the same standard feed, to which 2500 ppm zinc oxide had been added. A premix with 2000 ppm of the test product was added to the feed of the test group. The pigs were weighed at the start, after 10 days and after 21 days. Feed intake was measured on the same days, so that exploitation of the feed could be calculated.

Table 4 - The effect of Product C on production parameters in weanling pigs.

As is clearly shown in the trial results, the addition of "Product B" or "Product C" resulted in improved growth, ad in most trials an improvement in feed exploitation compared with the control group and with the addition of probiotics or the botanic element alone.

Since the product has now been described, it will become apparent that it can be varied in many ways. Such variations shall not be regarded as deviations from the scope of the invention, and it will be apparent to those skilled in the art that all such modifications should also be considered to be comprised in the scope of the claims that follow.