WO2016043610A1 - Nutritional supplements with prophylactic effect for bees and their preparation - Google Patents

Abstract

The invention relates to the formulation and the manner of obtaining nutritional supplements with prophylactic effect for bees, based on the conjugated action of iron (II) salts of the amino acids similar to the ones present in the honey and pollen, of volatile vegetable oils, preferably citronella oil alone or in admixture and p-coumaric acid, as concentrates, or as syrups or cakes on glucidis support, to which for the potentiation there can be added: essential vitamins, essential minerals, proteins and fats and which can be administered permanently or in certain periods.

Description

NUTRITIONAL SUPPLEMENTS WITH PROPHYLACTIC EFFECT FOR BEES

AND THEIR PREPARATION

The present invention relates to the formulation and the manner of obtaining nutritional supplements with prophylactic effect for bees, said effect being based on the conjugated action of the iron (II) salts obtained from amino acids similar to the ones present in the honey and pollen, of volatile vegetable oils, citronella oil alone or in admixture with p-coumaric acid, as concentrates or as syrups or cakes on a glucidic support, to which essential vitamins, essential minerals, proteins and fats are added for potentiation, and which can be administered permanently or in certain periods.

The sensitivity of the bees to the most frequent diseases: varroosis, nosemosis, European foulbrood, American foulbrood and so on, depends on many factors: insufficient food, lack or insufficiency in the food of some essential nutrition principles: proteins, essential vitamins, essential minerals; p-coumaric acid, action of the pesticides and extreme external factors such as excessive heat or cold. Generally, bees find all the components for a complete nutrition in the nectar, pollen and in the honey/bee bread reserves derived therefrom. But there may also exist situations when the local flora, soil or climatic conditions determine deficiencies of essential components even in the natural food for bees. Among these diseases, varroosis is one of the most destructive. There exist a series of medicaments such as: AMITRAZ; MAVIROL; API STAN, APITOL, BAYVAROL; PERIZIN, FOLBEX. There can also be used spraying with formic, lactic and oxalic acids. All these shall be applied cautiously, since they are also toxic for the bees, impurify the honey and do not eliminate the parasite completely. For the other diseases there can also be used antibiotics/bactericidal substances such as: fumagillin; oxytetracycline; terramycin; streptomycin; erythromycin and sulfathiazole. The antibiotics may be used only if strictly necessary, as the honey contamination / H C§iiite&s thereof imposes the elimination of this honey from the human consumption. The present tendency is the disposal as advanced as possible of the synthesis organic products, by replacing them with products extracted from plants, and as far as possible, the disposal of any products, even natural, which do not naturally occur in the nectar, honey or pollen. An example of natural product is API-LIFE-VAR, consisting of a mixture of thymol, camphor, menthol crystals extracted from plants and volatile eucalyptus oil, fixed on a vermiculite support (CHEMICALS LAIF). It is placed at the bottom of the hive, wherefrom, it releases by sublimation the active substances. Another product based on volatile oils, vitamins from the group B and essential minerals, is PROTOFIL (ICDA), efficient in the prophylaxis of nosemosis. The treatments based on these products containing volatile oils, vitamins and essential minerals do not eradicate the diseases of the bees either.

An efficient and ecological prophylaxis is based on strengthening the immunity of the bees to diseases and pesticides. First, there must be ensured a nutritional support sufficiently quantitatively and as complete as possible in essential nutrients. This is ensured first by the natural nutrition: nectar, honey and pollen, coming back with the observation that these cannot always guarantee the amounts of essential nutrients: glucides, proteins, fats, vitamins, minerals and immuno-fortifying compounds. The nutritional supplements added to the natural food shall complete the basic need and to correct possible deficiencies. The formulation of the nutritional supplements shall avoid products which can wear out the bees either due to the chemical composition, or due to exagerated concentrations.

The glucidic component shall avoid sugar, particularly beet sugar, as much as possible. It is accepted that in order to obtain the same amount of energy, the bee consumes an amount of sugar by 25% higher in relation to the one obtained from the same amount of glucose or fructose. Moreover, the beet sugar contains 0.5-2.5% raffinose, that is toxic for the bees. When using syrups with high fructose content, there shall be taken into accouoLifaat certain metals, such as Mn can catalyse the formation of 5-hydroxymethylfurfural (HMF), that is toxic for bees. Therefore, the concentration of metals in the syrups containing fructose shall not exceed the content level of these metals in the honey.

The proteins used in formulating the nutritional supplements, in order to be efficiently assimilated, have to contain ten essential amino acids in an optimal proportion or at least close to it (g/16N): threonine 3, valine 4; methionine 1.5; leucine 4.5; isoleucine 4.5; phenylalanine 2.5; lysine 3, histidinel .5; arginine 3, tryptophan 1 (DeGroot 1953). The major amino acid in honey (between 20 and 60% of the total amino acids) is proline [ ; 2].The identification of proline is also employed for attesting the authenticity of honey. Recent studies reassessed that proline has also a special role in bees physiology; no other amino acid can be metabolized so fast, immediately generating the enzymes necessary for the rapid release of high amounts of energy by "burning the glucose". Proline is essential for the bees flight and particularly for the "take off" [3]. Proteins, with the exception of proline, are not absolutely necessary for adult bees, but are necessary for raising bee brood and for preparing the bees for winter. Formation of the "fat body", namely the accumulation of a content of 60% of proteins in the body is a condition necessary for the survival of bees during the winter.

The source of proteins is also important. Soybean meal contains sufficient stachyose and raffinose, both toxic for bees, because, used in a too high amount, particularly with beet sugar syrup that also contains raffinose, it can cause the mortality of bees. Most protein additions: soy, barley, inactivated yeast derivatives etc have a very high potassium, natrium and chlorine content. Potassium at concentrations higher than 5000 ppm in the food is repellent for bees. Natrium chloride, at concentrations exceeding 0.1% in the food, drastically shortens the longevity of bees. Proteins extant in the integral egg or yolk powder contain between 40 and 60% lipids and 1.5 - 2.5% cholesterol. Cholesterol added to the bees food increases the longevity thereof, but a lipid content of 2- 4%, depending on the composition, reduces it drastically.

Vitamins are not essential for adult bees, but are important in the food of larvae and queen. The essential vitamins in the honey are those in the complex B and vitamin C. In the pollen, the majority is the entire complex o vitamins B, but there can also be found vitamins A, D and E, which have a favourable role in the diet of bees. loc pt prima fiqurj

The p-coumaric acid was emphasized in pollen and honey (0.3-7 ppm) [4; 5], but cannot be found in nectar. It was experimentally proved that the p-coumaric acid is the most efficient immuno-protector among all these similar compounds that can be found in the bees food. Unlike the other phenolic acids in the honey or pollen, the immunologic activity of the p-coumaric acid increases linearly with the increase of its concentration in the food, unlike other phenolic acids whose activity increases to a maximum, then decreases to zero. The sequence analysis of RNA demonstrated that the p-coumaric acid regulates a special suite of genes which act as defence against pesticides and pathogenic agents [6]. Since it cannot be found in the nectar, it is possible that, inside the hive, it is not accumulated in the sufficient amount for carrying out a complete immunologic reaction.

The major essential minerals: Na, K, Mg, Ca, Cu, Zn, Mn, Fe, P, S, CI expressed as elements, are to be found in the honey at a total content of 500-8oo mg/kg, and in the pollen they can reach 10000 mg/kg. A mineral content of more than 1- 1.5% in the food, has wearing out effects for bees. The role of the transition metals in the minerals is connected to metallo-enzymes, enzymes in the oxidative systems containing iron, manganese, zinc and copper have an important role in the energetic and immunolc¾0i£b^^tem; enzymes containing manganese or zinc (metalloproteases) have the role of cleaving the proteins. The metallo-enzymes also work in the immunity processes. Although it is desirable for the metals to be in stable and bioassimilable forms, the use of too strong chelating agents, such as EDTA, deactivates the metallo-enzymes. For many enzymes, iron, manganese, zinc and copper are interchangeable. Potassium and natrium take part in transmitting the neuronal impulses. Phosphorus and calcium participate in the processes of energetic conversion of glucides and in the formation of magnetite granules in the bees body. Calcium also has an essential role upon the olfactory memory of the bees. Sulphur has a role in the synthesis of some essential amino acids. Copper and zinc additionally have fungicidal and bactericidal effects.

Recent researches revealed that manganese in excess in the bees food, leads to the intensification of the reproduction processes, leading to an unbalance between the available food and the number of bees in the colony, the final being the colony collapse (CCD - Collapse Colony Disorder). As a matter of fact, manganese is a link which makes the connection with varroosis, T.J.Schuijt [7], in 2011 , formulated the so-called 'tick model" showing that, upon the infestation with mites/ticks, an important role is played by the bacterium Borellia. Borellia can be found in the mites body wherefrom it is transferred into the organism parasitized by them. Important is the fact that Borellia uses in the energetic metabolism Mn instead of Fe. Borellia replaces the iron with manganese also in the organism where it enters by means of the tick, thereby disturbing the metabolism thereof. In case of the bees, manganese does not hinder the transfer of oxygen, as the bees do not have respiratory pigment in hemolymph, but disturbs the iron metabolism. Researches performed by Chin-Yuan Hsu and Chia Wei Li [8; 9] showed that the iron is present in the bees body as magnetite granules, randomly distributed in the trophocytes of the worker bees and drones, while in the queen's body, they are distributed as peripherally located clusters. This differentiation suggests that the iron-containing granules have some essential biological functions, which are only partially deciphered. It is certainly known that the magnetite granules are the ones ensuring the bees orientation in the terrestrial magnetic field. The formation of the iron-containing granules in the bees body is made by means of the Fe²⁺ cations, there taking part the calcium and phosphorus compounds. The disturbance of iron metabolism, upon parasitizing with Varroa, does not directly kill the bees, but lowers their resistance to diseases, also adding the aggravating effect of excessive multiplication. Other effects pointed out in case of the iron are the ones connected to the stimulation of the bees and the ones connected to slowing down the reproduction thereof.

Volatile/essential vegetable oils are used in the prophylaxis of bees diseases, these having acaricidal, fungicidal and bactericidal properties. Of the volatile vegetable oils, the thyme, menthol, lemon, citronella oils have toxic action upon parasitizing with Varroa, Acarapis woodi and Paenibacillus larvae. Of these, the mint and thyme oils, though frequently used, are very toxic for bees, too. The lemon oil is the least toxic, but it may favour the multiplication of Varroa. Citronella oil is practically non toxic for bees even at relatively high concentrations.

There are some patents relating to the prophylactic use of the metal salts in the nutritional supplements for bees. Bounias and Dufour emphasized the prophylactic effect of the copper salts on Varroa [9]. The preferred compound was copper gluconate administered in the sugar syrup. The presence of copper gluconate in the syrup, unlike other salts which have repellent effect, increases the syrup attractivity for bees. There was also emphasized a stimulating effect of the copper gluconate on the bees. Copper passes from the bees hemolymph into the parasite hemolymph, affecting the respiratory pigment (hemocianine) thereof. However, it has no effect in case of acute infestation with Varroa. It usually ensures a reduction by 60-90% of the parasites population, the action stopping at an equilibrium level. Used excessively it may contaminate the honey. The main disadvantage of using the copper salts is the use of high copper concentrations in the syrup (by 50-200 mg/L), the maximum alowable limit in the fodder being of Cu 25 mg/L, according to the directive (EC) no. 1334/2003.

A prophylactic treatment with iron (II) salts: formate, lactate, gluconate or a mixture thereof, founded on the "tick model" was elaborated by Maarten Van Hoorn and Rik Ter Horst [11]. There is preferred the use of iron (II) formate in admixture with vinasse and various glucides, either as solutions, or as cakes administered to bees. There is also claimed the addition of proteins, amino acids, volatile oils and antioxidants, but without specifying them and the concentration thereof. Vinasse is a product resulting from the fabrication of sugar and in the distillation of alcohol from molasses or fermented cereals. These fabrication residues contain fulvic acids, sugars, proteins, vitamins and mineral salts. The vinasse-type products are used in a small quantity since they are toxic for bees. Their role would be, according to authors of the process, to facilitate the biochemical transfer of iron into the bees body. Gordon Wardell and Fabiana Ahumada-Segura use, in the food of bees, a product similar to vinasse, named SOLULAC, which is obtained by grinding the fermented cereals grains resulting from the distillation of alcohol [12].

The main disadvantages of the formulations of Maarten Van Hoorn and Rik Ter Horst are: the use of a toxic component (vinasse) and of iron (II) formate that has a repellent effect, as well as the fact that they cannot be used all year round

The technical problem solved by the present invention is the formulation of nutritional supplements with prophylactic effects for bees, as concentrates, syrup or cakes, containing three basic components with conjugated prophylactic action: iron (II) salts of the amino acids present in the honey or pollen (according to the "tick model"), p-coumaric acid and essential oils having components commun with the pheromones released by the bees. These are administered on a glucidic support: honey, for any period of the year, other glucides for the periods with reduced activity of the bees. The formulations are completed with essential vitamins, essential minerals, amino acids and fats. The formulations use > 99.8% organic compounds of biological origin and between 97 and 99.8% only compounds to be found in honey and pollen. Supplements are obtained in homogenous and chemically and biologically stable forms without toxic products for bees, without synthesis preservatives and without repellent effect to the bees.

The formulation of compositions of nutritional supplements with prophylactic effect for bees, in compliance with the present invention, is based on the observation that there exists a correlation between the anti-Varroa effect which interferes upon the administration, in the food for bees, of metals (copper or iron) and the pheromones released by the Nasonov gland of the worker bees, Apis Melifera. Nasonov gland consists in several hundreds of cells situated under the sixth intertergal membrane, near the dorsal surface of the abdomen. When the bee flexes the terminal segments, this membrane is exposed and releases volatile secretions mainly consisting of cis-geraniol and trans-geraniol (nerol), citral and their oxidation products [13]. Bees always release these pheromones when they are stimulated: for example when they identify water or nectar. The Nasonov gland pheromones have a powerful repellent effect on Varroa. Therefore, when the bees are stimulated, the parasites detach therefrom. Consequently, upon the stimulation of the bees with copper or iron salts, the detaching of the parasites does not take place as a consequence of ingesting the same, but due to the stimulation of Nasonov gland. The iron (II) salt does not kill Varroa but acts upon Borellia. Borellia does not withstand a high iron concentration and it is destroyed at the level of the digestive system, interrupting the introduction of manganese into the hemolymph of the bees. This way the iron participation in the bees metabolism is reestablished, significantly reducing the effects of parasitizing with Varroa. The volatile citronella oil has a composition very similar to that of the pheromones released by the Nasonov gland. For these reasons, in the nutritional supplements with prophylactic effect for bees, formulated in compliance with the present invention, the Fe(ll) compounds are associated with the volatile citronella oil, following to reinforce the repellent effect on Varroa. Volatile citronella oil kills the Varroa parasite by direct contact, but this fact, when this is introduced into the nutritive supplements only occurs by accident. More important is the fact that, depending on the concentration in the bees food, the volatile citronella oil passing into the Varroa organism hinders the reproduction thereof or delays the development of eggs which are laid in the cells with larvae. By delaying the development of the parasite eggs, the bee larvae finish their development before the maturation of Varroa eggs and leave uninfested the cells. The citronella oil additionally has antifungal and bactericidal effects, ensuring both the preservation of the nutritional supplements up to the application and also a prophylactic action against the fungi and bacteria which parasitize the bees colony. The prophylactic effect of the iron (II) compounds and of the volatile citronella oil or of the mixtures of citronella oil with other volatile oils, is reinforced by adding the p-coumaric acid, as an immunity increase factor.

Unlike the products protected by patents by Maarten Van Hoorn and Rik Ter Horst, the iron (II) in the nutritional supplements with prophylactic effect, formulated in compliance with the present invention is administered as salts of the amino acids present in the honey and pollen. These are chemically stable, soluble and easily assimilable, without the need of adding a substance which facilitates their biochemical transfer. For the supplements which are administered more particularly in the active period of the bees, there is preferred the use of the iron II salt of the proline (H-Pro) and for the other periods, any other amino acid or mixture of amino acids, preferably glycine (H-Gly), alanine (H-Ala), lysine (H- Lys) and threonine (H-Thr). Unlike the products protected by patents by Maarten Van Hoorn and Rik Ter Horst, the formulation of the supplements according to the present invention is conceived for administering them permanently, for the purpose of maintaining the iron content, at a sufficient and necessary level in the organism of the bees, eliminating or attenuating the negative manganese effect. Since the iron content in the bees body vary depending on the activity period and consequently, on the availability and composition of the natural resources: nectar and pollen, a rigorous and efficient application can be made periodically monitoring the iron content in the beesyfcwijv The preparation of the iron (II) salts of the amino acids can be carried out by the reaction thereof with: metal iron in the presence of citric acid [13], ferrous carbonate in deaerated aqueous solution [14], ferrous sulfate in the presence of the calcium salts [15] or barium [16]; iron (II) salts in the presence of monosodic glutamate [17] or of the potassium hydroxide [18].

In the present invention there were used two methods for the preparation of iron (II) salts of the amino acids: by the reaction between the ferrous sulfate and amino acids in the presence of the potassium carbonate. As a medium for dissolving the reagents there may be used deionized ^"water or preferably an aqueous solution of 5-40% glucose in deionized water, with role of preventing the oxidation reactions. The ferrous sulfate is dissolved to a Fe concentration of 10- 50g/L, the potassium carbonate to K 10-80 g/L and the amino acids to 50-250 g/L.

According to the first method, the ferrous sulfate solution is poured while stirring, at 30-40°C, in nitrogen atmosphere, onto the potassium carbonate solution, dosed so that finally there is obtained a pH = 7.2 - 7.4, when the ferrous carbonate is precipitated:

FeS0₄ + K2CO3 = FeC0₃ + K₂S0₄

Besides the ferrous carbonate there are also formed quantities of ferrous hydroxide, but this fact does not influence the process of obtaining the iron (II) aminocarboxylates. The suspension obtained is easily filtrable. The filtering is carried out in nitrogen atmosphere, then the precipitate is washed with deionized water or preferably with an aqueous solution of 5-40% glucose, there being obtained a precipitate with 18-22% Fe and 0.04 - 0.06% K. The ferrous carbonate is quantitatively taken from the filter and it is added, while stirring, and under nitrogen atmosphere, to 30-40°C onto the amino acid solution. The amount of amino acid is calculated so as to exist an ej¾ep¾ss6£3-10% depending on the amount of Fe(ll) present in solution. There is exemplified with the reaction of forming Fe(ll) chelate with the proline:

FeCOs + 2H -PRO = FePro₂ + CO₂ + HOH

The Fe(ll) aminocarboxylates have a good solubility in aqueous solutions.

According to the second method, the solution with amino acids is neutralized with potassium carbonate solution, so that 90-97% of the amino acid amount is converted into potassium salt:

K2CO3 + 2H -Gly = 2KGIy + H₂O + CO₂

To the potassium aminocarboxylate solution there is added, while stirring and under nitrogen atmosphere, the ferrous sulfate solution, dosed so that the molar ratio ferrous sulfate/potassium aminocarboxylate = 1 :

2KPGIy + FeSO₄ = FePro₂ + KSO₄

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Working at too high concentrations of potassium in solution, it is possible the crystallization of the potassium sulfate, but these crystals are easily filtrable and the ferrous aminocarboxylate solution is further used. The increase of the potassium in solution, in case of using the second method, generally does not matter, since the K content in the honey is of about 500 ppm and in the pollen it may reach to more than 5000 ppm.

The volatile citronella oil or the mixtures of volatile citronella oil with other volatile oils, preferably lavender, melissa and marjoram are introduced into the nutritional supplements formulated in compliance with the present invention, as emulsion in deionized water or preferably in aqueous glye^^olutions. The mixtures of volatile oils are used for increasing the prophylactic effect as well as of the attractivity of the supplements for bees. The volatile oils are emulsified by addition in water or glucose solution, 1-5% of liquid soy lecithin, at 40-60°C, while vigorously stirring, then there is added alimentary emulsifier (higher ethoxylated alcohols, fatty ethoxylated acids, ethoxylated monoglycerides) 8-30% related to the previous solution, while vigorously stirring and then, also while vigorously stirring, citronella oil or citronella oil in admixture with other 5-40% volatile oils as against the lecithin and emulsifier mixture. Then the emulsion of volatile oils is diluted, with deionized water or glucose solution, up to a concentration of 10-80 ml/L of volatile oil.

The p-coumaric acid is introduced into the nutritional supplements, either as a very finely ground solid, or as a potassium or ammonium salt solution containing 1g/L coumaric acid.

The nutritional supplements with prophylactic effects for bees, obtained according to the invention can be formulated as: a. Liquid concentrates containing 5-25 g/L iron, citronella oil 1-10 gt/L and p- coumaric acid 100-1000 mg/L to which essential vitamins may be added. Additionally there may be introduced Cu and Zn salts, preferably sulfates to achieve a Cu concentration of 10-50 mg/L and Zn 20-100 mg/L in the liquid concentrate. Cu and Zn additionally ensure the stability to biochemical degradation. Concentrates are destined to prepare cakes and syrups administered to bees, the dosage being 1/50-1/200 in relation with the mass thereof. At these dilutions, Cu and Zn used as preservation additions, have an insignificant influence on the composition of cakes or syrups.

b. Liquid or solid supplements contain Fe 20-300 mg/kg; citronella oil or a mixture of volatile oils 10 μΙ/kg-l mL/kg, p-coumaric acid 1-5 mg/kg and preferably essential vitamins, essential minerals, proteins and fats on a glucidic support. These are administered directly to the bees. The addition of vitamins has the role of reinforcing the nutritive and prophylactic effects of the supplements. The preferred proportion of vitamins, established in correlation with the average ratios in honey and nectar, is illustrated in Table 1.

Table 1. Proportion of vitamins in the nutritional supplements formulated according to the present invention

There is initially prepared a vitamins concentrate in deionized water or preferably in aqueous glucose solutions which is then dosed in formulations of the supplements. Vitamins B1, B3, B5, B6, B7 have a good solubility in aqueous solutions, while the others have low or negligible solubility and shall be brought in stable emulsion/suspension form. For the preparation of the vitamins concentrate 1-6% of liquid lecithin is dissolved, while vigorously stirring at 40-60°C in deionized water or preferably in aqueous glucose solution and then 10-25% emulsifier, related to the lecithin solution. It is cooled to the room temperature and there are added, while vigorously stirring, 10-40% vitamins related to the lecithin and emulsifier solution, the vitamins being in the proportions presented in Table 1. The solid vitamins are previously finely ground/milled. Then there are added gradually, in portions, while vigorously stirring, deionized water or glucose solution, up to obtaining a vitamins concentration of 10-40g/L.

The essential minerals, others than the iron, are another useful addition for sustaining the nutritional and prophylactic effects of the supplements. They are introduced into syrups or cakes as a concentrate of minerals in solution. The preferred proportion of the minerals, established mainly according to the average proportions thereof in the honey, is illustrated in Table 2.

Table 2 . Proportion between the minerals (except for iron) in the minerals concentrate formulated according to the present invention

In order to ensure the stability of metal cations in solution and for a better bio- absorption, these are complexed with citric, gluconic acids or with amino acids. The preparation of the mineral concentrate, using the citric acid (H Cit) is carried out in several stages. During the first stage there is prepared a solution of citric acid in deionized water or aqueous glucose solutions, the citric acid amount being calculated so as to form potassium dihydrogen citrates, magnesium and calcium in the established amounts. First, there are dissolved, while stirring, the oxides, preferably the magnesium and calcium carbonates in citric acid solution at 40-60°C:

CaC0₃ + 2H₃Cit = Ca(H₂Cit)₂ + C0₂ + HOH

MgC0₃ + 2H₃Cit = Mg(H₂Cit)₂ + C0₂ + HOH

Then there are added a solution of potassium carbonate dissolved in deionized water or glucose solutions, which react with the free citric acid left after the reaction with the magnesium and calcium carbonates:

K₂CO₃ + 2H₃Cit = KH₂Cit + CO₂ + HOH

The potassium, magnesium and calcium citrates solution is diluted with deionized water or glucose solutions, to a total metal content of 15-25 g/L.

The transition metals: Cu, Zn, Mn, Co are dissolved as sulphates in deionized water or glucose solutions. It is preferred that each of the sulphate solutions have a metal concentration so that the added volume is 5-20 mL of each of them, for the preparation of 1 L of minerals concentrate.

Upon mixing the minerals concentrate with the iron (II) aminocarboxylates it is possible for it to interfere with the transition metal citrates, a part of the iron (II) being able to pass to citrate, such as:

Fel_ys2 + Me(H₂Cit)₂ = Fe(H₂Cit)₂ + el_ys₂

In order to eliminate this reaction, the amount of amino acid necessary for the reaction with the citrates of the transition metals is additionally introduced into the solution of metals concentrate:

2HLys + Me(H₂Cit)₂ = MeLys₂ + H₃Cit

Sulphur is introduced into the minerals concentrate by means of the sulphates of transition metals and phosphorus as a solution of phosphoric acid or in aqueous glucose solutions, at a concentration P 50-150 g/L. Natrium and chlorine are added as alimentary natrium chloride.

At the end, the whole mixture is diluted with deionized water or with aqueous glucose solutions up to a content of minerals of 10-20 g/L, expressed as elements. There is obtained a clear and stable solution with the pH of 3-4.

In case of using the gluconic acid or amino acids it is proceeded similarly, with the observation that, in these situations neutral salts of the metals are formed.

The nutritional and prophylactic effect of the supplements can be also improved by the addition of proteins in syrups and cakes. In cakes, the preferred concentration of proteins ranges between 0.1 and 0.3%, comprising the average content of proteins extant in honey. In the cakes, the protein concentration ranges between 10 and 20%, close to the concentration of the proteins in pollen. In order to avoid introducing compounds that are toxic for bees, such as raffinose, stachyose and pectine and in order to avoid exagerately increasing the amount of minerals in cakes, it is preferred to use the soy protein isolate and the integral egg or yolk powder. The latter also ensures the fat and cholesterol intake. The ratio between the eggs powder and the soy protein isolate is preferably 1/10 - 1/ 5.

The glucidic support is important for the supplements as syrups and cakes. For the syrups administered to the bees during the harvesting period, natural honey is employed. For the syrups and cakes administered outside this period there are preferred the glucose and fructose syrups, such as for example the one prepared with AMIDEX™ 312 (TATE & LYLE) containing in (%): 59.5 glucose, 3.5 fructose; di- and polysaccharides 7 or the syrup ISOSUGAR 031™ (TATE & LYLE), containing in (%): 37.6 fructose; 31.4 glucose; 6.0 di- and polysaccharides.

The advantages of the present invention are:

• Supplements can be formulated both for being used in any period of the year, when they have honey as a glucide support, and for applications in the periods of reduced activity of the bees, when they have sugars of vegetal origin as glucide support.

• Supplements can be formulated both as concentrates which can be brought in the shape of cakes or syrups by the beekeepers, and as cakes and syrups which can be administered to bees as such.

^• There is administered the iron (II) as easily assimilable soluble salts of the natural amino acids and without repellent effects for bees.

^• The immunity of bees is increased by introducing into the composition the p- coumatic acid in association with iron (II) aminocarboxylates and essential/volatile vegetable oils, preferably citronella oil.

^• The formulations are perfectly compatible to the addition of vitamins, essential minerals, proteins, lipids which improve the nutrj^aT¾¾¾^ophylactic effects. • The preservation of the products against biochemical degradation is carried out with essential/volatile vegetable oils and with copper and zinc salts, the Cu and Zn concentration in the products administered directly to bees, being in compliance with the concentration thereof in the honey and pollen.

• The products do not have harmful effects on the bees, since they contain >90% non-toxic natural products.

• When used correctly, the supplements do not contaminate the honey produced by the bees, since they contain between 97 and 99.98% only components that can naturally be found in honey and pollen, at concentrations correlated to the average compositions thereof.

• The formulations are compatible with other prophylactic products or medicaments administered to the bees.

There are given hereinafter 9 examples of formulations of nutritional supplements with prophylactic effects for bees, in compliance with the present invention:

Example 1

Formulation A of the concentrated nutritional supplement with prophylactic effect for bees is prepared in the following sequences: a. There is prepared a 25% glucose solution by dissolving, while stirring, at the room temperature, 420 g of AMIDEX™ 312 in 580 g of deionized water, for each kilogram of solution prepared, solution hereinafter named glucose solution. b. 200 g of iron sulphate heptahydrate (p. a. SIGMA - ALDRICH) are dissolved while stirring, at the room temperature, under nitrogen atmosphere, in 750 mL of glucose solution, then it is brought to the volume of 1 L, with the same solution, to result in a Fe(ll) solution of 40 g/L, hereinafter named ferrous sulphate Fe 40 g/L in glucose solution.

c. There is dissolved, while stirring, at the room temperature, 84 g of potassium carbonate 1.5 hydrate (p.a MERCK) in 800 mL of glucose solution, thereafter it is brought to the volume of 1L with the same solution. There is obtained a solution k 40 g/L, hereinafter named potassium carbonate K 40 g/L in glucose solution.

d. 400 mL of ferrous sulphate Fe 40g/l_ in glucose solution, is poured slowly, at the temperature of 40°C under nitrogen atmosphere and while stirring, onto 565 mL of potassium carbonate K 40g/L in glucose solution. There is obtained a precipitate of cream-coloured ferrous carbonate which is filtered under nitrogen and then it is washed on filter with 300 mL of glucose solution.

e. 102 grams of L-proline (>98.5% MERCK) are dissolved, while stirring at the room temperature, in 800 mL of glucose solution and are brought to 1 L with the same solution, there resulting in a solution with the concentration of L-proline 100 g/L.

f. The ferrous carbonate cake obtained according to letter d of this example, is taken over quantitatively from the filter and added, while stirring in nitrogen atmosphere, in 690 mL of L-proline solution 100 g/L, obtained according to letter e of this example, the molar ratio L-proline/Fe(ll) being 1.05 . There is obtained a solution of the L-proline salt cu Fe(ll).

g. 1 g of liquid soy lecithin (GMO free, STERA CHEMICALS) is added in 100 mL of glucose solution and it is mixed vigorously for 1 minute at 45°C, using a stirrer T-25 ULTRA - TURRAX®. Onto the obtained solution there are added 12 g of alimentary emulsifier CETARETH 20 (STERA CHEMICALS). The emulsion formed this way is brought to 500 mL cu glucose solution, to result in a stable emulsion, containing essential citronella oil 40 ml/L, hereinafter named citronella oil emulssion 40 ml/L in glucose solution.

h. 5 g of copper sulphate pentahydrate (>98.5% SIGMA ALDRICH) and 1 1 g of zinc sulphate heptahydrate (>99% SIGMA ALDRICH) are dissolved while stirring at the room temperature, in glucose solution and it is brought to 250 mL, there being obtained a solution (g/L): Cu 5, Zn 10, hereinafter named glucose solution Cu 5g/L; Zn 10 g/L.

i. Onto the L-proline salt solution with Fe(ll), obtained according to letter f of this example, there are introduced, while stirring, at the room temperature 100 mL of citronella oil emulsion 40 ml/L in glucose sok&on, 10 mL of glucose solution Cu 5g/L; Zn 10g/L and 500 mg of p-coumaric acid (98.34%NANJING ZELANG MEDICAL TECHNOLOGY) very finely milled. It is left for one hour to homogenize, while stirring, then it is brought to 1 L with glucose solution, there being obtained the formulation A as a clear and stable solution, having the composition:

The product kept in brown-coloured bottles at room remperature for 6 months does not show any signs of chemical or biochemical degradation.

The product obtained according to formulation A is destined first to prepare the honey syrups administered to bees, during the harvesting time, but it can also be used for the preparation of syrups or cakes obtained with syrups of vegetable extraction glucides. The recommended doses are 1/100-1/200, related to the total amount of syrup or cake.

Example 2

Formulation B of the concentrated nutritional supplement, with prophylactic effect for bees is prepared as follows: 115 g of potassium carbonate 1.5 hydrate (p.a MERCK) is dissolved, while stirring, in 700 mL of deionized water at 50°C. After dissolving, 150 g amino acids are added, while stirring at 50°C in the quantities presented in Table 3, when the potassium aminocarboxylates are formed. Table 3. Quantities of amino acids used in Example 2

The solution obtained is cooled to the room tmperature and it is brought to 1 L with a glucose solution, to result in a solution of potassium aminocarboxylates containing K 54 g/L. Onto the 445 mL of the solution obtained this way there is gradually added, while stirring at 40°C, 400 ml of ferrous sulphate Fe 40 g/L in glucose solution, the ratio between the moles of amino acids/moles of Fe(ll) being of 1.07. In the iron (II) aminocarboxylates and potassium sulphate solution obtained there are added 100 mL of emulsion of citronella oil 40 ml/L in glucose solution; 10 mL of glucose solution Cu 5g/L; Zn 10 g/L, 15 g of citric acid monohydrate (99% SIGMA- ALDRICH) and 500 mg of finely milled p- coumaric acid. It is stirred for 40 minutes for homognization, cooled to the room temperatrure and brought to 1 L with glucose solution, there being obtained formulation B which is presented as a clear solution, having the composition:

The product kept in brown bottles at the room temperature, for 6 months, does not show any signs of chemical or biochemical degradation. The product obtained according to formulation B is intended first for the preparation of syrups with honey, administered to bees during the harvesting period, but, it can be also used for the preparation of cakes based on vegetal glucides. The recommended doses are 1/100 - 1/200, related to the total amount of syrup or cake.

Example 3

Formulation C of the concentrated vitaminized nutritional supplement with prophylactic effect for bees is prepared in the following sequences:

a. In 100 ml_ of 25% glucose solution prepared as in Example 1 letter a there are introduced 3 g of lecithin while vigorously stirring with a stirrer T-25 ULTRA - TURRAX®, at 40°C, for 1 minute and then there are added 15 g of emulsifier, further stirring for 1 minute at the same temperature. The solution thus obtained is cooled to 25°C, then the amountrs of vitamins presented in table 4 are added, the solid ones being previously finely ground.

Table 4.The quantities of vitamins used in example 3

It is vigorously stirred with a stirrer T-25 ULTRA - TURRAX®, for 5 minutes, thereafter 800 mL of glucose solution are gradually added, in portions of 100 mL, vigorously stirring for 1 minute, after eacbf^S¾)¾0. Then it is brought to 1 L with glucose solution, to obtain an emulsion/stable suspension of vitamins, hereinafter named vitamins concentrate.

b. 250 mL of vitamins concentrate are added in thin jet, while stirring at the room temperature, onto 500 mL of product formulation A. Then there are added 5 mL of glucose solution Cu 5g/L; Zn 10 g/L and it is left for 30 minutes for homogenization, then it is brought to 1 L with glucose solution, to obtain formulation C which is presented as a clear solution having the following composition:

The product kept in brown bottles at the room temperature, for 6 months, does not show any signs of chemical and biochemical degradation.

The product obtained according to formulation C is indicated first for preparation of syrups or cakes based on vegetable glucides, which do not contain vitamins in composition. It can be also used for preparation of syrups with honey for supplementing the doses of vitamins and p-coumaric acid already extant in the honey. The recommended doses are 1/50 - 1/100, related to the total amount of syrup or cake.

Example 4

Formulation D of the nutritional supplement with prophylactic effect for bees as honey syrup is prepared as follows: 100 mL of product obtained according to formulation A, is added gradually while stirring, at 30-35°C, using a stirrer for viscous liquids IKA EUROSTAR 200 in 1 kg of polYfloral honey and it is left to be homogenized for 1 hour. Further 8.9 kg of polyfloral honey is gradually added to obtain 10 kg of product of formulation D with the composition:

* additional content added to the honey by formulation D

The increase of the mineral content, first Cu and Zn in the honey used as dispersion medium is insignificant and does not affect the quality of the one produced by the bees fed with this formulation.

Formulation D can be administered to bees all year round, but mostly during the harvesting period without the risk of honey contamination. Its role is to maintain, at the necessary level, the iron concentration in the hive, to complete the deficiency in p-coumaric acid necessary to increase the bees immunity, to create a repellent medium for Varroa, due to the citronella oil and to supplementing the amount of proline necessary for the worker bees. Administration is made depending on the seasonal activity of bees, with doses for one month of 20-100 mg Fe in syrup/kg of bees. The recommended doses for one hive which contains in March 2 kg of bees for an average climatic season, are presented in Table 5.

Table 5. Doses recommended of nutritional supplement with prophylactic effect for bees, as honey syrup; formulation C, depending on the bees acyivity

Doses are less from May till August, since, the bees can get the necessary iron from pollen in that period. The syrup administration is recommended to be carried out in 5-10 equal portions/month.

The prophylactic treatment can also be completed by application of other additional treatments.

Example 5

Formulation E of vitaminized nutritional supplement with prophylactic effect for bees - fructose - glucose syrup is prepared as follows: 200 ml_ of product of formulation G, prepared according to example 3 of the present invention is added gradually, at 30-35°C in 1 kg of syrup ISOSUGAR 031™, stirring with a stirrer for viscous liquids IKA EUROSTAR 200, being stirred for 1 hour, thereafter 8.8 kg of ISOSUGAR 031™ is added gradually, while stirring to obtain 10 kg of syrup representing the formulation E, having the following composition:

Formulation E is administered to bees, preferably outside the honey collecting period, in September - November and March - May, respectively, depending on the climatic conditions. The recommended dosage in a month is Fe 20-100 mg/kg of bees; 60-80 mg/kg of bees in September - October and in March-May, depending on the bees activity, determined by the climatic conditions. Example 6

Formulation F of the nutritional supplement with prophylactic effect for bees, as glucose-fructose syrup with a content of vitamins, minerals and amino acids is carried out according to the following sequences: a. 62 g of citric acid monohydrate is solubilized while stirring, at 40°C, in 170 mL of glucose solution, thereafter 6.4 g of calcium carbonate (>99% SIGMA ALDRICH) and 3.35 g of magnesium carbonate (>99% SIGMA ALDRICH) are added. After completely passing the Ca and Mg into dihydrogen citrates, there are added in a thin jet, while stirring, 250 mL of potassium carbonate K 40gL in glucose solution.

b. There is prepared 1 L of solution for each component, with the concentrations presented in Table 6.

Table 6. Quantities of components dissolved in 25% glucose solution, prepared according to paragraph a of example 1 , necessary to obtain 1 L of each one

Each of these components is dissolved, while stirring, at room temperature, in glucose solution, then bringing it to 1 L with the same solution. There are obtained 5 solutions having the concentrations (g/L: Cu 40; Zn 100; Mn 2.5; Co 0.05; P 100.

c. In Ca, Mg and K solution prepared according to letter a of this example, there are added, while stirring, at the room temperature, 400 mL of glucose solution, then by means of a pipette there are added: 10 mL of Cu lOg/L solution, 15 ml of Zn 15g/L solution; 8 mL of Mn 2.5 g/L solution and 8 mL of Co 0.05% solution, these solutions being obtained according to letter b of this example. Stirring is continued and then there are added 2 g of glycine for chelation with Cu, Zn, Mn and Co as aminocarboxylates. It is homogenized for 15 minutes and then there are added 2.34 g of alimentary natrium chloride and 8 mL of phosphoric acid P 100 g/L solution, obtained according to letter b of this example. Stirring is continued for 40 minutes for homogenization and then it is brought to 1 L with glucose solution. There is obtained a clear and stable solution with pH of 3.7, having the composition (mg/L): K 10000; Na 800; Mg 400; Ca 1200; Cu 200; Zn 600; Zn 600; Co 0.4; Mn 20; P 800; S 454; CI 1540, hereinafter named minerals concentrate,

d. 50 mL of minerals concentrate is mixed, while stirring, at room temperature, in 335 g of syrup ISOSUGAR 031™ for 20 minutes, using a stirrer IKA EUROSTAR 200, adding 5 mL of product of formulation A and 5 mL of vitamins concentrate. Finally, while stirring, there are added in three equal portions, 1 g of soy protein isolate (composition in Table 7) and 200 g of syrup ISOSUGAR 031™, stirring for 15 minutes after each portion.

Table 7. Composition of soy protein isolate; 91% proteins (REDIS NUTRITIE)

used in the formulations accordin to the invention

There is obtained 1 Kg of liquid clear and homogenous product representing formulation F with the composition:

2.5

Vitami Vitami Vitamin Vitamin Vitami Lecithin Emulsifie Protein

n B9 n C D E n A mg/Kg r s

mg/Kg mg/Kg : mg/Kg Mg/Kg Ul/Kg 16 mg/Kg %

8 10 4 4 1600 87 0.27

Glucose Fructos Total

% e glucids

30 % %

35.7 71.4

Formulation F is administered to bees similarly to formulation E.

Example 7

Formulation G a nutritional supplement with prophylactic effect for bees, as fructose-glucose syrup, with increased content of essential oils, is carried out in the following sequences:

a. 1 g of alimentary lecithin is added to 100 mL of glucose solution. It is vigorously mixed for 1 minute, at 45°C, using a stirrer T-25 ULTRA - TURRAX®. There are added onto the solution obtained 12 g of emulsifier and 20 mL of essential oil mixture (% by volume): citronella 50; melissa 10; lavender 20 (COZACPLANT) and marjoram 20 (COZACPLANT) and are vigorously mixed for further 1 minute. The emulsion formed this way is brought to 500 mL with glucose solution. There is obtained a stable emulsion containing 40 ml/L of a mixture of four essential oils, hereinafter named within the invention emulsion of mixture of essential oils 40 ml/L.

b. 25 mL of emulsion of mixture of esential oils 40 ml/L is mixed, while stirring at the room temperature in 100 mL of syrup formulation E. It is left to be homogenized for 30 minutes and then further 875 mL of syrup formulation E are gradually added, there being obtained about 1 L of syrup with Fe 154 mg/L and essential oils 1 ml/L.

Formulation E combines the effect of Fe(ll) aminocarboxylates of p-coumaric acid and of the oils essential in the prophylaxi&afsiiarroosis and of other diseases of the bees. Limited use is recommend^! 0.5 - loJfcg/kg bees in the periods: August -November and March-May, respectively, only once, maximum two times within a given period.

Example 8

Formulation H of the nutritional supplement with prophylactic effect for bees, as a glucidic cake is carried out as follows: 120 g of alimentary sugar is dissolved in 210 g of deionized water, while stirring at 50°C, thereafter 650 g of AMIDEX™ 312 are added, while stirring up to total dissolution. The solution thus obtained is cooled at the room temperature and then there are added 30 g of glicerine while stirring (> 99.5 SIGMA ALDRICH) and 20 mL of product of formulation C. It is stirred for further 45 minutes for homogenization, then it is left at room temperature. Within 24-48 h, there occurs the solidification, there being obtained about 1 kg of formulation H, as a white paste, having the composition:

The product obtained according to formulation H is used for feeding bees during winter, preferably after performing the first cleansing flight. The recommended dose in a month is 0.5-1 kg/kg of bees.

Example 9

Formulation J of the nutritional supplement with prophylactic effect for bees, as cake with protein content is prepared as follows: 450 mL of deionized water is gradually added into 3850 g ISOSUGAR 031™ and it is mixed for 20 minutes, raising the temperature to 40°C. Heating is sie∞^ and there are further added 250 mL of minerals concentrate, 25 rrjjssof pFpdu¾¾f formulation B obtained according to example 2; 25 ml_ of vitamins concentrate and 10 mL of emulsion of essential oils 40 ml/L. Stirring is continued for 30 minutes and then it is passed into a mixer UTG-0130 with the volume of 10 L, where there are added 750 g of soy protein isolate and 125 g of yolk powder (OVOPROT) having the average composition: organic materials (%): fats 56, proteins 37; hydrocarbons 2.2; cholesterol 2.2; minerals (mg/g): Ca 2.3; P 9.2; K 2.4; Na 1.3. These are mixed in a mixer for 40 minutes at 35°C and there are obtained about 5 kg of paste representing formulation J with the composition:

Formulation J is administred to bees similarly to formulation H.

Claims

1. Formulation and process for obtaining nutritional supplements with prophylactic effect for bees, characterized in that they have in the composition iron (II) salts of the amino acids similar to the ones present in the honey and pollen, at iron concentrations ranging from 10 mg/L - 25 mg/L.

2. Concentrated nutritional supplements with prophylactic effect for bees, obtained in compliance with claim 1 , characterized in that, in them volatile vegetable oil, preferably citronella oil, is added, at concentrations ranging from 10 pL/kg - 50 mL/kg and p-coumaric acid, at concentrations ranging from 1 mg to 5 mg/kg.

3. Concentrated nutritional supplements with prophylactic effect for bees, obtained in compliance with claims 1 and 2, characterized in that they have in the composition: 5-25 g/L of Fe as aminocarboxylates, 1 -10 ml/L volatile citronella oil or mixtures of volatile oils, preferably citronella, lavender, melissa and marjoram oils, 50-1000 mg/L p-coumaric acid, 10-50 mg/L Cu and 20 -100 mg/L Zn.

4. Concentrated nutritional supplements with prophylactic effects for bees, in compliance with claim 3, characterized in that, vitamins, preferably vitamins B1 , B2, B3, B5, B6, B7, B9, A, C, D, E are added.

5. Nutritional supplement with prophylactic effect for bees, as syrup obtained according to claims 1 and 2, characterized in that it has the natural honey as glucidic support and comprises: 10-200 mg/kg of Fe as iron (II) aminocarboxylates, preferably the iron (II) salt of proline, 10 -1000 μΙ/kg of volatile citronella oil or mixtures of volatile oils and 1 - 5 mg/kg of p-coumatic acid.

6. Nutritional supplements with prophylactic effect for bees, as syrup or cake, obtained according to claims 1 and 2, characterized in that, they have as a support vegetable origin glucides, preferably glucose or glucose-fructose syrups and they have the composition: Fe 10 -200 mg/kg as iron (ii) aminocarboxylates, preferably iron (ii) salt of proline, 10 -1000 μΙ/kg of volatile citronella oil or mixtures of volatile oils and 1 - 5 mg/kg of p-coumaric acid.

7. Nutritional supplements with prophylactic effect for bees, as syrup or cake, obtained according to claim 6, characterized in that vitamins, preferably B1 , B2, B3, B5, B6, B7, B9, A, C, D, E are added into the composition.

8. Nutritional supplements with prophylactic effect for bees, as syrup or cake, obtained in compliance with claim 6, characterized in that in the composition there are added essential minerals, preferably K, Mg, Ca, Cu, Zn, Mn, Co, P, S, CI, said metals being complexed as citrates, gluconates or aminocarboxylates.

9. Nutritional supplements with prophylactic effects for bees, as syrup, obtained in compliance with claim 6, characterized in that, in the composition there are added 0.1-0.3% proteins, preferably soy protein isolate.

10. Nutritional supplements with prophylactic effect for bees, as cake, obtained in compliance with claim 6, characterized in that, in the composition there are added proteins in concentration of 10-20%, resulting preferably from soy protein isolate and yolk powder in a yolk powder/soy protein isolate ratio of 1/10 / 1/15.

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[18] EP2717713

[19] US4216144