The present invention relates to a pollen and/or pollen substitute comprising bee food composition and a method for preparing such bee food composition. The present invention further relates to the use of the bee food composition of the present invention.

In Europe, a decrease in managed honey bee colony numbers has been observed since 1965, but the pattern is diverse (Potts, S.G., et al.; 2010; Declines of managed honey bees and beekeepers in Europe; Journal of Apicultural Research 49(1): 15-22). Since 1998, individual beekeepers have been reporting unusual weakening and mortality in colonies, particularly in France, Belgium, Switzerland, Germany, the United Kingdom, the Netherlands, Italy and Spain. Mortality has been extremely high when activity is resumed at the end of winter and beginning of spring. In North America, a significant and constant decline in domestic honey bee colony numbers has been occurring during the past decades in this region (Ellis, J., et al.; 2009; Reviewing colony losses and Colony Collapse Disorder in the United States; Journal of Apicultural Research 49: 134-136 and Aizen, M.A.; 2009; The Global Stock of

Domesticated Honey Bees Is Growing Slower Than Agricultural Demand for

Pollination; Current Biology 19: 1-4). Losses of honey bee colonies since 2004 have left North America with fewer managed pollinators than at any time in the last 50 years. In this region, honey bees pollinate nearly 95 kinds of fruits such as almonds, avocados, cranberries and apples, as well as crops like soybeans (Stokstad, E.; 2007; The Case of the Empty Hives; Science 316: 970-972).

In Asia, beekeepers in Japan and China have faced several inexplicable and complex symptoms of colony losses in recent years (Natural News; Honey bee Collapse Strikes Japan, Up to Fifty Percent of Honey bees Gone; 28 April 2009). Certain losses are known to be caused by Varroa mites, foulbrood, viruses and Tropilaelaps mites including other factors. In Africa, Egyptian beekeepers based along the Nile River have reported symptoms of, what is called by scientists, the "Colony Collapse Disorder" (CCD) (Hassan 1, A.R.; 2009; Proceedings of the 4th COLOSS Conference). The term CCD is to define this multi-factor syndrome affecting beehives annually, particularly where low numbers of adult bees with food supplies such as honey and bee bread and immature or capped brood bees are present.

As it can be derived from the instabilities of pollinator populations, e.g. bee colonies, have become a worldwide phenomenon. Driving forces of the pollinator population instabilities may be the activities of humans which have impacted the landscape through fragmentation, degradation and destruction of natural habitats and the creation of new anthropogenic ones. Degradation and fragmentation of natural habitats are considered as key adverse changes for pollinator populations (Thomas, J.A., et al.; 2004; Comparative Losses of British Butterflies, Birds, and Plants and the Global Extinction Crisis;

Science 303(5665): 1879-1881). The degradation and fragmentation of natural habitats can lead to the reduction of food sources for all pollinator species. When large habitats are fragmented into small isolated patches, food sources become scarcer for resident animals.

The present invention aims to provide a solution to the extremely high mortality of bee colonies as it is observed in recent years.

In a first aspect, the invention thereto provides a bee food composition comprising a dough made of pollen and/or pollen substitute, and a mineral mixture comprising at least one mineral salt compound, wherein the amount of the mineral salt compound present in the bee food composition is at least 0,085 mmol per dose of bee food composition administered to 1 kg bees per week. It was found that by increasing the amount of minerals present in the bee food which is subsequently administered to the bee colony resulted in an increase of size of the colony and a drop in the mortality rate. It was found that after a period of 10 weeks after the first treatment of the bee colony with the bee food composition of the present invention the size of the bee colony was increased with 40% and a very low mortality rate was observed: less than or equal to 0,5%, a significant drop in mortality rate compared to the control group having a mortality rate of 8%, which control group received a bee food composition without an increased amount of minerals, i.e. a composition having an amount of mineral salt of less than 0,085 mmol per dose of bee food composition administered to 1 kg bees per week. It was further found that using a bee food composition according to the present invention comprising an increased amount of minerals, the activity and life expectancy of the bees increased also.

Furthermore, the bee food composition of the present invention also provides a better development of the larvae of the bee colony resulting in a healthier and stronger bee colony compared to other methods available nowadays.

Nowadays available methods for reducing the mortality rate of a bee colony are based on the principle that the mortality rate needs to be minimized in the wintertime period, i.e. the most critical period for a bee colony. Unlike other insects, the bee does not die off in the fall or hibernate, but is active all winter eating and metabolizing honey to keep warm. A, so called, "winter bee" is produced at the end of the summer. The winter bee is physiologically different than the summer bee, with a different hemolymph (blood) protein profile than the summer bee. Winter bees also have fatter bodies which they rely for nourishment during the non- foraging months. A winter bee will live much longer (4 to 6 months) than a summer bee (40 days). The sole purpose of the winter bee is to get the bee colony through the winter until the spring. Commonly used methods are based on the principle to prevent infection of the bee colony during winter period. For example miticides, e.g. oxalic acid and formic acid, are used to prevent the occurrence of varroatosis, i.e. the infection and subsequent parasitic disease caused by varroa mites.

Recently developed alternative methods reduces the mortality rate of a bee colony during the wintertime period by replacing the honey with a sugar syrup composition comprising additional vitamins and minerals in order to reduce the mortality of the winter bees.

Since the sugar syrup composition is consumed by the grown bee only, the above described method does not provide a solution to increase the health and strength of the bee colony during the year. The queen and larvae do not consume the sugar syrup composition and are therefore not supplied with additional minerals needed to develop in a healthy way. Additionally, in regions where the wintertime period is short or even not present at all, the above mentioned method cannot be used to reduce the mortality rate of a bee colony. The present invention provides a solution for the above identified problems of current available methods. By administering the bee food composition of the present invention to a bee colony outside the wintertime period, i.e. preferably at the end of the summer and beginning of the spring, the bee food composition is used to feed the bee colony and indirectly the larvae developing in the hive. By providing a bee food composition with an increased amount of minerals, the larvae do have access to the minerals, also during the first few days of the larva, which is of great importance in the development of the larva to bee.

The dough used in the bee food composition of the present invention may be made of (natural) pollen and/or a pollen substitute. The dough may be made by the beekeeper himself or can be bought as a ready- for-use pollen patty. There is no general recipe for such a pollen substitute composition, but most pollen substitute compositions comprise soy flour, sugar, yeast and sugar syrup and/or honey. Especially, the amount of sugars, e.g. maltose or honey, needs to be high in order to enhance the uptake of the dough by the bee. It is also possible that a mixture of natural pollen and a pollen substitute is administered to the bee colony.

Preferably the dough made of pollen and/or a pollen substitute may further comprise a vitamin and/or mineral supplement. Such a vitamin and/or mineral supplement are readily available and provide well-balanced pollen dough.

The mineral salt compound present in the bee food composition of the present invention may be selected from compounds comprising essential minerals. Preferably, the mineral salt compound is selected from copper salt compound, iron salt compound, zinc salt compound or combinations thereof.

In a further embodiment of the present invention, the mineral salt compound is an iron(II) salt compound. It was found that the presence of an iron(II) salt increases the health and strength of the larvae and bees present in the bee colony significantly. In order to provide iron(II) salt to the bee food composition of the present invention, the mineral salt compound is preferably selected from reduced iron, carbonylic iron, electrolytic iron, ferrous L-ascorbate, saccharated ferrous carbonate, ferrous chloride, ferrous chloride dihydrate, ferrous chloride tetrahydrate, ferrous citrate, ferrous fumarate, ferrous gluconate dihydrate, ferrous lactate dihydrate, ferrous lactate trihydrate, ferrous phosphate, ferrous pyrophosphate, ferrous sulfate, ferrous sulfate heptahydrate, ferrous sodium citrate or combinations thereof. In fact, all ferrous salts can be used, however the use of ferrous manganese salt is not preferred. More preferred the mineral salt compound is selected from ferrous fumarate, ferrous lactate dihydrate, ferrous gluconate dihydrate, ferrous sulfate, ferrous sulfate heptahydrate or

combinations thereof. Most preferred the mineral salt is selected from ferrous fumarate, ferrous sulfate heptahydrate or combinations thereof, since those mineral salts are easily available and exhibit good solubility, which increases the availability of iron(II) to the bees and larvae present in the bee colony. As already stated above, the amount of the mineral salt compound present in the bee food composition of the present invention is at least 0,085 mmol per dose of bee food administered to 1 kg bees per week. It was found that the amount of mineral salt compound present in the bee food composition of the present invention is preferably in the range of 0,085 to 25 mmol. In bee food compositions having an amount of mineral salt above 25 mmol per dose no further positive effect was observed in the mortality rate, health and strength of the bee colony. Possible negative effects may be observed by administering an amount of more than 25 mmol mineral salt compound per dose of bee food per kg bees per week, however, those possible negative effects have not been observed by the inventors so far. It was found that a bee food composition wherein the mineral salt compound is present in an amount of less than 0,085 mmol no positive effects have been observed compared to the control group.

The mineral salt compound present in the bee food composition of the present invention may be present in an amount in the range of 0,085 to 25 mmol per dose of bee food administered to 1 kg bees per week. In a further embodiment the mineral salt compound may be present in the bee food composition of the present invention in an amount in the range of 0, 1 to 20 mmol per dose of bee food administered to 1 kg bees per week, preferably in the range of 0,2 to 15 mmol per dose of bee food administered to 1 kg bees per week. In an even further embodiment of the present invention the bee food composition may comprise an amount of mineral salt compound in the range of 0,3 to 10 mmol per dose of bee food administered to 1 kg bees per week, preferably the amount of mineral salt compound is present in the range of 0,5 to 7,5 mmol, more preferably the amount of mineral salt compound is present in the range of 0,6 to 5 mmol and even more preferably the amount of mineral salt compound is present in the range of 0,7 to 2,5 mmol per dose of bee food administered to 1 kg bees per week. It was found that using a bee food composition comprising an amount of mineral salt compound in the range of 0,085 to 1 mmol per dose of bee food administered to 1 kg bees per week optimum results are obtained.

In an embodiment of the present invention a bee food composition is provided comprising wherein the amount of the mineral salt compound present in the bee food composition is about 0,3 mmol per dose of bee food administered to 1 kg bees per week or about 0,8 mmol per dose of bee food administered to 1 kg bees per week.

The mineral mixture of the present invention may further comprise at least one chelating agent. In order to provide biological active mineral salt to the bees and larvae, the mineral salt needs to be present in its biological active form, e.g. Fe ²⁺ in case of the mineral salt being iron(II). By using a highly soluble mineral salt compound, e.g.

ferrous fumarate and/or ferrous sulfate heptahydrate, the mineral salt is present in its biological active form. In order to improve the presence of the biological active form of the mineral salt even further, a chelating agent may be used. The chelating agent not only improves the presence of the biological active form of the mineral salt (even for less soluble mineral salt compounds), but also ensures mineral salts remaining biologically active in the extreme temperature conditions in the hive of the bee colony.

In an embodiment of the present invention, the chelating agent is selected from natural chelating agent, chelators used in nutritional supplements, etheric oils having chelating groups, food preservatives or combinations thereof. Examples of such chelating agents and suitable for the mineral mixture of the present invention are vinasse, molasses, aetheric oils, ethylenediaminetetraacetic acid (i.e. EDTA), and the like, either used alone or in combination. Many different forms of chelating agents may be used. For example, commercially available vinasse is sold after a partial dehydration and usually has a viscosity comparable to molasses. However, the bee food composition of the present invention may comprise a substantially dried form of vinasse produced by a method such as described in US 2012/0210759 Al .

The mineral mixture of the present invention may further comprise zinc, chrome, molybdenum and/or copper. In a second aspect, the present invention provides the use of the bee food composition of the present invention, wherein the bee food composition is administered to a bee colony at least every 2 weeks. In a further embodiment, the bee food composition of the present invention may be administered on a continuous basis using a feed system adapted to release a predetermined amount of bee food composition for a certain period of time. The bee food composition of the present invention may be administered more frequently as well as less frequently as long as the amount of the mineral salt compound is at least 0,085 mmol per kg bees per week. Furthermore, the bee food composition of the present invention may be administered in the form of a powder or a patty.

In a third aspect, the present invention provides a method of preparing a bee food composition of the present invention, comprising the following steps:

a) providing a dough comprising pollen and/or pollen substitute;

b) providing a mineral mixture comprising at least one mineral salt; and c) mixing the dough and the mineral mixture.

The amount of the mineral salt compound in the bee food obtained in step c) is at least 0,085 mmol per dose of bee food administered to 1 kg bees per week. The amount of the mineral salt compound may be varied as identified above.

In an embodiment of the present invention, the dough may be first mixed with a vitamin and/or mineral supplement prior to step c).

The mineral mixture may be first mixed with a chelating agent prior to step c).

Furthermore, the mineral mixture may be in the form of a solution or powder.

In a further embodiment of the present invention, the dough and the mineral mixture may be mixed wherein the weight ratio of the dough to the mineral mixture may be in the range of about 40: 1 to about 1 : 10, preferably about 10: 1.

In an even further embodiment of the present invention, the amount of mineral salt added to the dough is at least 0,085 mmol per dose of bee food administered to 1 kg bees per week. Preferably, the amount of mineral salt added to the dough is in the range of 0,085 to 1,0 mmol per dos of bee food administered to 1 kg bees per week. More preferred, the amount of mineral salt added to the dough is at least about 0,3 mmol per dose of bee food administered to 1 kg bees per week. Even more preferred, the amount of mineral salt added to the dough is at least about 0,7 mmol per dose of bee food administered to 1 kg bees per week.

Example

A bee food composition was prepared by mixing the ingredients mentioned in table 1 to form a pollen patty.

Table 1. Bee food composition

The Vitamins and Electrolytes composition (AgriLabs®) comprises the following ingredients: vitamin A supplement, vitamin D supplement, vitamin E supplement, menadione sodium bisulfite complex (source of vitamin K activity), niacin supplement, riboflavin supplement, calcium pantothenate, pyridoxine hydrochloride, folic acid, thiamine mononitrate, vitamin B12 supplement, citric acid, ascorbic acid, magnesium sulfate, calcium chloride, potassium chloride, sodium chloride, iron sulfate

monohydrate, dried Lactobacillus Acidophilus, dried Streptococcus Faecium and dextrose. The amount of ingredients is further provided in table 2 below. Table 2. Specification Vitamins & Electrolytes "Plus" (AgriLabs®)

The Pro Health FD-350 composition (Mann Lake Ltd.) comprises sucrose, water, spearmint oil, lemongrass oil, thymol, lecithin and emulsifier.

The Ultra Bee Dry composition (Mann Lake Ltd.) comprises plant protein products, high oleic canola oil, lemongrass oil, potassium chloride, sodium chloride, niacinamide, vitamin E supplement, stabilized ascorbic acid, sodium citrate, d-calcium pantothenic acid, vitamin A supplement, vitamin D3 Supplement, riboflavin, botin supplement, vitamin B12 supplement, pyridoxine hydrochloride, thiamine hydrochloride, magnesium sulfate, folic acid, ferrous sulfate, copper sulfate, zinc sulfate, manganese sulfate, cobalt sulfate, calcium lactate, potassium sulfate, magnesium carbonate and ethylenediamine dihydroiodide. The Ultra Bee Dry composition comprises several amino acids of which the concentrations are shown in table 3. Table 3. Ultra Bee Dry amino acid concentrations

Furthermore, 1 bottle BEEingAlive.com comprises 45 g ferrous fumarate and 375 ml vinasse.

The formed pollen patty was divided into equal portions of 325 g. 180 bee hives (average amount of 25.000 bees which corresponds to around 1,8 kg) were treated two times with 325 g pollen patty. The second treatment took place two weeks after treating the bee hive with the first dose of pollen patty.

The control group (250 bee hives with an average amount of 25.000 bees) received a pollen patty having a composition according to the one disclosed in table 1, excluding BEEingAlive.com. Ten weeks after the first treatment 2 of the 400 bee hives, i.e. 0,5%, treated with the bee food composition of the present invention died, whereas in the control group 32 bee hives, i.e. 8%, died. It was further noted that the bee hives treated with the composition of the present invention were 30-40 % stronger.