ES2540501B1 - Food composition of honey bees and procedure to control infections in said bees by said food - Google Patents
Food composition of honey bees and procedure to control infections in said bees by said food Download PDFInfo
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- ES2540501B1 ES2540501B1 ES201331155A ES201331155A ES2540501B1 ES 2540501 B1 ES2540501 B1 ES 2540501B1 ES 201331155 A ES201331155 A ES 201331155A ES 201331155 A ES201331155 A ES 201331155A ES 2540501 B1 ES2540501 B1 ES 2540501B1
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K50/00—Feeding-stuffs specially adapted for particular animals
- A23K50/90—Feeding-stuffs specially adapted for particular animals for insects, e.g. bees or silkworms
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/111—Aromatic compounds
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/163—Sugars; Polysaccharides
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Abstract
Composición alimenticia de abejas melíferas y procedimiento para controlar infecciones en dichas abejas mediante dicho alimento.#La presente invención se refiere a una composición alimenticia azucarada de abejas melíferas que contiene en su formulación al menos una fitoalexina de calidad alimentaria, preferentemente trans-resveratrol, en una cantidad comprendida entre 0.1% y 2.8% en peso del total de composición, incluidos ambos límites. Dicha composición puede presentarse en forma de alimento líquido en solución azucarada (jarabe) o de alimento semisólido en forma de pasta azucarada con baja humedad -menor del 50%- (candy). Asimismo, la invención se dirige a un procedimiento de control de las infecciones de las colonias de abejas melíferas, especialmente por Nosema apis y Nosema ceranae, (Nosemosis A y C), que comprende introducir en las colmenas la composición alimenticia, en al menos una dosis. El procedimiento permite el tratamiento de la colmena mediante aplicaciones programadas de los alimentos asegurando niveles efectivos durante al menos cinco semanas, todo ello sin producir efectos de rechazo en la colonia de abejas.Food composition of honey bees and method for controlling infections in said bees by said food. # The present invention relates to a sugary food composition of honey bees containing in its formulation at least one food grade phytoalexin, preferably trans-resveratrol, in an amount between 0.1% and 2.8% by weight of the total composition, including both limits. Said composition may be presented in the form of liquid food in sugar solution (syrup) or semi-solid food in the form of sugary paste with low humidity - less than 50% - (candy). Likewise, the invention is directed to a method of controlling infections of honey bee colonies, especially by Nosema apis and Nosema ceranae, (Nosemosis A and C), which comprises introducing into the hives the food composition, in at least one dose. The procedure allows the treatment of the hive through programmed applications of food ensuring effective levels for at least five weeks, all without producing rejection effects in the bee colony.
Description
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DESCRIPCIONDESCRIPTION
COMPOSICION ALIMENTICIA DE ABEJAS MELIFERAS Y PROCEDIMIENTO PARA CONTROLAR INFECCIONES EN DICHAS ABEJAS MEDIANTE DICHO ALIMENTOHONEY BEES FOOD COMPOSITION AND PROCEDURE FOR CONTROLLING INFECTIONS IN SUCH BEES THROUGH SAID FOOD
Campo tecnico de la invencionTechnical field of the invention
La invencion se enmarca en el sector tecnico de la Agricultura, mas concretamente en el area de la Apicultura (crianza de abejas) y la Veterinaria (tratamiento de enfermedades en colmenas), aunque tambien esta relacionada con el sector de la Alimentacion y Consumo de productos provenientes de la crla de abejas.The invention is part of the technical sector of Agriculture, more specifically in the area of Beekeeping (bee breeding) and Veterinary (treatment of diseases in hives), although it is also related to the sector of Food and Product Consumption from beehive.
Objeto de la invencionObject of the invention
La presente invencion se refiere a una composition alimenticia para las colonias de abejas mellferas que contiene fitoalexinas de calidad alimentaria, principalmente resveratrol (y especialmente trans-resveratrol), compuestos que son capaces de favorecer el desarrollo normal de las colonias, y en especial combatir las nosemosis (A y C). Asimismo, la invencion esta relacionada con un procedimiento para combatir las infecciones de las colonias de dichas abejas mellferas mediante la alimentacion de las colonias de abejas con dicha composicion alimenticia que contiene fitoalexinas.The present invention relates to a food composition for honey bee colonies containing food grade phytoalexins, mainly resveratrol (and especially trans-resveratrol), compounds that are capable of promoting normal colony development, and especially combating nosemosis (A and C). Likewise, the invention is related to a method to combat the infections of the colonies of said honey bees by feeding the bee colonies with said food composition containing phytoalexins.
Antecedentes de la invencionBackground of the invention
Las colonias de abejas mellferas son susceptibles a sufrir infecciones e infestaciones por agentes tales como virus, acaros, lepidopteros, hongos, bacterias, etc., que afectan la salud de las abejas y en consecuencia disminuyen la productividad de las explotaciones aplcolas y las acciones polinizadoras de estos insectos, originando colateralmente graves perjuicios economicos y medioambientales. Una de las principales infestaciones sufridas en la ultima decada por las abejas a nivel mundial es atribuida a la action del microsporidio Nosema ceranae, considerado como uno de los factores claves del reciente fenomeno de colapso masivo de colonias de abejas. Efectivamente, Nosema ceranae es actualmente el patogeno de las abejas mas prevalente en Espana (Revisado por Higes M., Meana A., Bartolome C., Botlas C., Martln-Hernandez R. Nosema ceranae (Microsporidia), a controversial 21st century honey bee pathogen. Environmental Microbiology Reports 2013, 5 (1), 17-29)Colonies of honey bees are susceptible to infections and infestations by agents such as viruses, mites, lepidoptera, fungi, bacteria, etc., which affect the health of bees and consequently decrease the productivity of beekeeping farms and pollination actions of these insects, collaterally causing serious economic and environmental damage. One of the main infestations suffered in the last decade by bees worldwide is attributed to the action of microsporide Nosema ceranae, considered one of the key factors of the recent phenomenon of massive collapse of bee colonies. Indeed, Nosema ceranae is currently the most prevalent bee pathogen in Spain (Reviewed by Higes M., Meana A., Bartolome C., Botlas C., Martln-Hernandez R. Nosema ceranae (Microsporidia), a controversial 21st century honey bee pathogen. Environmental Microbiology Reports 2013, 5 (1), 17-29)
superando incluso la prevalencia de la Varroa destructor (Garrido-Bailon, Tesis doctoral, 2012) lo que sugiere la importancia sanitaria que presenta. Ademas esta misma situation ha sido detectada en diferentes palses europeos (Austria, Francia, Holanda, Suiza, Eslovenia,even surpassing the prevalence of the destructive Varroa (Garrido-Bailon, Doctoral Thesis, 2012) which suggests the sanitary importance it presents. In addition, this same situation has been detected in different European countries (Austria, France, Holland, Switzerland, Slovenia,
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Polonia, Italia, Grecia, Portugal, Alemania, etc.) y del continente Americano (Estados Unidos, Argentina, Chile, Uruguay). En todas estas zonas, se ha podido constatar cientlficamente el papel central de Nosema ceranae en el conocido como Slndrome de Despoblamiento de las Colmenas (SDC) o Desorden de Colapso de las Colonias (CCD) (Higes y col., 2008 Environmental Microbiology, Higes y col, 2009 Environmental Microbiology Reports), de ahl la necesidad de mejorar las estrategias de control. Estamos sin lugar a dudas ante el principal problema sanitario de la apicultura mundial en el siglo XXI. Tambien los resultados de investigaciones realizadas por los inventores nos permiten descartar como factor prioritario los pesticidas agricolas, fundamentalmente neonicotinoides, como responsables del problema generalizado de despoblamiento de las colmenas.Poland, Italy, Greece, Portugal, Germany, etc.) and of the American continent (United States, Argentina, Chile, Uruguay). In all these areas, the central role of Nosema ceranae in what is known as Hives Depopulation Syndrome (SDC) or Colony Collapse Disorder (CCD) (Higes et al., 2008 Environmental Microbiology, Higes) et al, 2009 Environmental Microbiology Reports), hence the need to improve control strategies. We are undoubtedly faced with the main health problem of world beekeeping in the 21st century. Also the results of investigations carried out by the inventors allow us to rule out as a priority factor agricultural pesticides, mainly neonicotinoids, as responsible for the widespread problem of depopulation of hives.
Como se ha indicado anteriormente, en los ultimos anos se ha producido una elevada mortandad de abejas en diferentes zonas aplcolas tanto en Espana, como en palses vecinos con caracterlsticas productivas similares (Faucon y col., 2002; Chauzat y col., 2004; Marinelli y col., 2004; Higes y co., 2005; Stoktad, 2007; Plischuck y col., 2009) o en otras zonas geograficas (Finley y col., 1996; Mangum 1999). En todos los casos se denunciaron elevadas mortalidades de colonias de abejas, sobre todo durante el otono y el invierno, aunque durante 2005 tambien se detectaron a lo largo de todo el ano.As indicated above, in recent years there has been a high mortality of bees in different beekeeping areas both in Spain and in neighboring countries with similar productive characteristics (Faucon et al., 2002; Chauzat et al., 2004; Marinelli et al., 2004; Higes et al., 2005; Stoktad, 2007; Plischuck et al., 2009) or in other geographical areas (Finley et al., 1996; Mangum 1999). In all cases, high mortalities of bee colonies were reported, especially during the fall and winter, although during 2005 they were also detected throughout the year.
Uno de los principales problemas que ha planteado este hallazgo es el hecho de que la fumagilina, el tratamiento disponible y de eleccion actual para la nosemosis causada por N. apis (Katznelson and Jamieson, 1952; Hartwig and Przelecka, 1971; Higes et al., 2004), si bien resulta efectiva tras su aplicacion (Higes y col., 2008, 2009) actualmente no esta autorizada en apicultura en la mayorla de los palses de la Union Europea, por lo que resulta fundamental desarrollar nuevas moleculas que suplan con exito la ausencia de la fumagilina. Por lo tanto, la busqueda de compuestos efectivos frente a N. ceranae, debe ser tambien un objetivo prioritario teniendo en cuenta la situacion actual.One of the main problems that this finding has raised is the fact that fumagillin, the current available and choice treatment for nosemosis caused by N. apis (Katznelson and Jamieson, 1952; Hartwig and Przelecka, 1971; Higes et al. , 2004), although it is effective after its application (Higes et al., 2008, 2009) is currently not authorized in beekeeping in most of the countries of the European Union, so it is essential to develop new molecules that supplant successfully the absence of fumagiline. Therefore, the search for effective compounds against N. ceranae should also be a priority objective taking into account the current situation.
Por otra parte, y dado que muchos antifungicos son de uso cllnico limitado ya que frecuentemente conducen a varias toxicidades en pacientes, tales como efecto hemolltico frente a eritrocitos humanos, aparece otra opcion orientada hacia el empleo de algunos productos naturales, que parecen no tener ese efecto drastico. Uno de los actualmente considerados es el resveratrol, que entre otras propiedades muestra un atractivo potencial como antifungico en humanos, si bien todavla no se conoce el mecanismo real de su accion (Jung, 2005).On the other hand, and given that many antifungals are of limited clinical use since they frequently lead to various toxicities in patients, such as hemolytic effect against human erythrocytes, another option appears oriented towards the use of some natural products, which seem not to have that drastic effect One of those currently considered is resveratrol, which among other properties shows a potential appeal as an antifungal in humans, although the real mechanism of its action is still unknown (Jung, 2005).
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A este respecto, recientemente Maistrello y col. (2008) demostraron en pruebas realizadas unicamente en laboratorio y sin contrastar estos resultados con pruebas in situ que el resveratrol estandar analltico (no apto para consumo humano) adicionado a alimentos aumento la longevidad de las abejas y resulto eficaz para inhibir el desarrollo de Nosema ceranae en abejas infectadas. Sin embargo, no se han correlacionado aun estos datos con estudios de campo ni con alimentacion convencional a colmenas. Esta sustancia ya ha sido tambien testada con exito frente a otros microsporidios de humanos y mamlferos (Leiro y col., 2004; Jung y col., 2005; Didier y col., 2006).In this regard, recently Maistrello et al. (2008) demonstrated in tests carried out only in the laboratory and without contrasting these results with in situ tests that the analogous standard resveratrol (not suitable for human consumption) added to food increased the longevity of bees and was effective in inhibiting the development of Nosema ceranae In infected bees. However, these data have not yet been correlated with field studies or with conventional hive feeding. This substance has also been successfully tested against other microsporidia of humans and mammals (Leiro et al., 2004; Jung et al., 2005; Didier et al., 2006).
El resveratrol se ha detectado como el principal componente bioactivo en extractos de plantas usadas desde siglos pasados en la medicina tradicional para enfermedades humanas, de hecho se ha mostrado tan potente como la anfotericina B frente a S. cerevisae. Estudios recientes han mostrado que puede inhibir el desarrollo del microsporido Encephalitazoon cunicoli en experimentos in Vitro (Leiro, 2004).Resveratrol has been detected as the main bioactive component in plant extracts used since past centuries in traditional medicine for human diseases, in fact it has been shown to be as potent as amphotericin B against S. cerevisae. Recent studies have shown that it can inhibit the development of microsporide Encephalitazoon cunicoli in in vitro experiments (Leiro, 2004).
Trans-resveratrol es una fitoalexina producida por las angiospermas en respuesta a las infecciones microbianas, danos, radiacion UV, frlo, calor, ataque de microorganismos, estres abiotico, etc. La forma trans (t-r) del resveratrol aparece en la piel de uvas, cacahuetes, pistachos, soja (Langcage, 1976; Dercks, 1989; Jeandet, 1993). El isomero cis (c-r) no se ha encontrado en Vitis vinifera aunque ambos suelen aparecer en los vinos. El resveratrol es un precursor de las viniferinas, que son potentes agentes antifungicos (Palomino,2000; Loredana, 2004). Ambos isomeros aparecen de forma natural como dos isomeros geometricos, generalmente el trans es mas predominante (Filip, 2003) aunque tambien aparecen sus glicoxidos, piceidos, trans (t-p) y cis (c-p) que tambien poseen propiedades similares. In Vitro la forma trans es mas termoestable y fotoestable y la cis mas inestable, pasando rapidamente a la forma trans (Goldberg, 1995) aunque el trans tambien puede sufrir fotoisomerizacion, generando algo de cis (Hanzlikova, 2004), de modo que en experiencias prolongadas e intensas puede incrementar la cantidad de cis (Trela,1996).Trans-resveratrol is a phytoalexin produced by angiosperms in response to microbial infections, damage, UV radiation, cold, heat, attack of microorganisms, abiotic stress, etc. The trans (t-r) form of resveratrol appears in the skin of grapes, peanuts, pistachios, soybeans (Langcage, 1976; Dercks, 1989; Jeandet, 1993). The cis isomer (c-r) has not been found in Vitis vinifera although both usually appear in wines. Resveratrol is a precursor to viniferins, which are potent antifungal agents (Palomino, 2000; Loredana, 2004). Both isomers appear naturally as two geometric isomers, generally trans is more predominant (Filip, 2003) although their glycoxides, piceids, trans (t-p) and cis (c-p) also have similar properties. In Vitro the trans form is more thermostable and photostable and the cis is more unstable, moving quickly to the trans form (Goldberg, 1995) although the trans can also undergo photoisomerization, generating some cis (Hanzlikova, 2004), so that in experiences prolonged and intense can increase the amount of cis (Trela, 1996).
Descripcion de la invencionDescription of the invention
Un primer objeto de la presente invencion esta constituido por una composition alimenticia azucarada para abejas mellferas que contiene en su formulation al menos una fitoalexina, que es comunmente y de manera mas preferida resveratrol en su forma trans-, en unaA first object of the present invention is constituted by a sugary food composition for honey bees containing in its formulation at least one phytoalexin, which is commonly and more preferably resveratrol in its trans- form, in a
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cantidad comprendida entre 0.1% y 2.8% en peso del total de composition, incluidos ambos llmites.amount between 0.1% and 2.8% by weight of the total composition, including both limits.
La fitoalexina es de grado farmaceutico. Se entiende por grado farmaceutico una fitoalexina apta para uso y consumo humano con calidad de suplemento alimentario (food grade), que debe cumplir las normas USFDA, EFSA o AEMPS (U.S.Food and Drug Administration, European Food Safety Authority y Agencia Espanola de Medicamentos y Productos Sanitarios, respectivamente), ya que dicho alimento dejara rastro en la miel producida por las abejas y no puede ser una fitoalexina, por ejemplo resveratrol industrial, como las que se conocen en el mercado. En definitiva, no todas las fitoalexinas comercializadas, como el resveratrol, son de grado farmaceutico y aptas para consumo humano, por lo que la presente invention supone un avance en el campo al emplear fitoalexinas optimas para alimentation de abejas de production de miel, ceras y otros productos.Phytoalexin is pharmaceutical grade. A pharmaceutical grade is understood as a phytoalexin suitable for human use and consumption with food grade quality, which must comply with USFDA, EFSA or AEMPS (USFood and Drug Administration, European Food Safety Authority and Spanish Medicines Agency and Sanitary Products, respectively), since said food will leave a trace in the honey produced by the bees and cannot be a phytoalexin, for example industrial resveratrol, such as those known in the market. In short, not all of the phytoalexins marketed, such as resveratrol, are of pharmaceutical grade and suitable for human consumption, which is why the present invention represents an advance in the field by using optimal phytoalexins for feeding honey bees, waxes and other products.
En una realization preferida, la fitoalexina es concretamente resveratrol, siendo mas preferentemente todavla su forma trans- (uno de los isomeros del resveratrol). Preferentemente, del total de fitoalexinas que comprende el alimento el 99% es trans- resveratrol. De manera mas preferida todavla, el 99,95% de la cantidad de fitoalexina que contiene la composicion (0.1%-2.8% en peso del total de composicion) es solo de trans- resveratrol.In a preferred embodiment, the phytoalexin is specifically resveratrol, its trans- form still being more preferably (one of the isomers of resveratrol). Preferably, of the total of phytoalexins that comprise the food 99% is trans- resveratrol. More preferably, 99.95% of the amount of phytoalexin contained in the composition (0.1% -2.8% by weight of the total composition) is only trans-resveratrol.
Basicamente, la composicion comprende una fitoalexina que es resveratrol en su forma trans- de forma mayoritaria, y al menos una segunda fitoalexina. Preferentemente, la segunda fitoalexina o fitoalexinas son seleccionadas entre cis-resveratrol, un isomero de piceido y cualquier combinacion de los mismos. Por isomero del resveratrol se entiende cualquiera de sus formas cis- y trans- (estando esta ultima siempre presente en esta realizacion); de igual forma, por isomero de piceido se entiende cualquiera de sus formas cis- y trans-. Esta alternativa es la mas preferida, por cuanto las composiciones de fitoalexinas comerciales y de calidad alimentaria que se emplean para preparar la composicion alimenticia de abejas siempre presentan trazas de estas formas: cis-resveratrol e isomeros de piceido.Basically, the composition comprises a phytoalexin which is resveratrol in its majority trans-form, and at least a second phytoalexin. Preferably, the second phytoalexin or phytoalexins are selected from cis-resveratrol, a piceid isomer and any combination thereof. The isomer of resveratrol means any of its cis- and trans- forms (the latter being always present in this embodiment); in the same way, by isomer of piceido is understood any of its cis- and trans- forms. This alternative is the most preferred, since the commercial and food grade phytoalexin compositions used to prepare the bee food composition always have traces of these forms: cis-resveratrol and piceid isomers.
La composicion alimenticia puede ser seleccionada entre un alimento llquido en solution azucarada y un alimento semisolido en forma de pasta azucarada con humedad inferior al 50%. En el primer caso, el llquido puede ser preferentemente un jarabe de alimentacion deThe food composition can be selected from a liquid food in a sugary solution and a semi-solid food in the form of a sugary paste with humidity below 50%. In the first case, the liquid may preferably be a feed syrup of
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abejas. En el segundo caso, puede ser preferentemente una pasta de azucares o miel (- alimento fabricado a base de miel, tambien llamado "candy”, muy seco y duro que se reblandece por la absorcion progresiva de la humedad de la colmena-). Por "azucarada se entiende que la composition alimenticia esta principalmente constituida por azucares, concretamente estando estos comprendidos de manera general entre 20%-85%, y mas preferentemente entre 40% y 80%, en peso del total de composicion.bees In the second case, it can preferably be a paste of sugars or honey (- food made from honey, also called "candy", very dry and hard that is softened by the progressive absorption of moisture from the hive-). "sugary is understood that the food composition is mainly constituted by sugars, specifically these being generally comprised between 20% -85%, and more preferably between 40% and 80%, by weight of the total composition.
Cuando la composicion alimenticia es un jarabe de alimentation de abejas, este puede contener preferentemente al menos una fitoalexina en una cantidad comprendida entre 0.4% y 2.8% en peso total de composicion, incluido ambos llmites. Asl por ejemplo, puede contener entre 1 y 7 gramos de fitoalexinas cada 250 gramos de jarabe. Cuando la composicion alimenticia es una pasta de azucares o miel, esta puede contener preferentemente entre 0.1% y 0.7% en peso total de composicion, incluido ambos llmites, de fitoalexinas. Asl por ejemplo, puede contener entre 1 y 7 gramos de fitoalexina por cada 1000 g de pasta. En un caso u otro, la composicion alimenticia comprende en su forma mas preferida 0.6% en peso del total de composicion de fitoalexinas.When the food composition is a bee feeding syrup, it may preferably contain at least one phytoalexin in an amount comprised between 0.4% and 2.8% by total composition weight, including both limits. Thus, for example, it can contain between 1 and 7 grams of phytoalexins per 250 grams of syrup. When the food composition is a paste of sugars or honey, it may preferably contain between 0.1% and 0.7% by weight of total composition, including both limits, of phytoalexins. Thus, for example, it can contain between 1 and 7 grams of phytoalexin per 1000 g of pasta. In one case or another, the food composition comprises in its most preferred form 0.6% by weight of the total phytoalexin composition.
En un caso particular mas preferido de la invention cuando la composicion alimenticia es un jarabe, en su forma mas preferida contiene entre 1 y 2 gramos, mas preferentemente entre 1.5 y 1.75 gramos (0.6%-0.7%), de fitoalexinas, cada 250 ml de jarabe de azucar. Dicho jarabe puede ser jarabe de azucar al 50% de agua.In a more preferred particular case of the invention when the food composition is a syrup, in its most preferred form it contains between 1 and 2 grams, more preferably between 1.5 and 1.75 grams (0.6% -0.7%), of phytoalexins, every 250 ml. of sugar syrup. Said syrup may be sugar syrup 50% water.
En otro caso particular de la invencion cuando la composicion alimenticia es una pasta semisolida azucarada, en su forma mas preferida contiene entre 5 y 7 gramos a, mas preferentemente 6 gramos (0.6%), de fitoalexinas, por cada 1 kg de pasta semisolida azucarada, que puede comprender preferiblemente en su formulation:In another particular case of the invention when the food composition is a semi-solid sugary paste, in its most preferred form it contains between 5 and 7 grams to, more preferably 6 grams (0.6%), of phytoalexins, for every 1 kg of semi-solid sugary pasta , which you can preferably understand in your formulation:
• 30% de glucosa• 30% glucose
• 50 % de fructosa• 50% fructose
• 18% agua; y• 18% water; Y
• 2% de componentes adicionales.• 2% additional components.
De manera preferida, la composicion alimenticia contiene tambien en cualquiera de sus formas vitaminas y/o aminoacidos que complementan el alimento.Preferably, the food composition also contains in any of its forms vitamins and / or amino acids that complement the food.
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La composition alimenticia descrita en cualquiera de sus variantes puede prepararse a partir de la combination de un alimento azucarado para abejas, preferentemente en forma de composition llquida (por ejemplo jarabe, preparado comunmente a partir de la mezcla de agua y azucar en partes iguales en peso) o semisolida (por ejemplo candy, preparado comunmente con miel y agua, aunque tambien puede prepararse con jarabe de glucosa y agua) con una composition de fitoalexinas de consumo humano, es decir de calidad alimentaria, segun los criterios antes especificados. Esta composition de fitoalexinas puede estar compuesta exclusivamente por resveratrol, concretamente por trans-resveratrol que es la forma mas comun, o puede contener otras fitoalexinas como las mencionadas anteriormente, que es el caso mas generalizado ya que suelen existir trazas de otras fitoalexinas conocidas.The food composition described in any of its variants can be prepared from the combination of a sugary food for bees, preferably in the form of a liquid composition (eg syrup, commonly prepared from the mixture of water and sugar in equal parts by weight ) or semi-solid (for example candy, commonly prepared with honey and water, although it can also be prepared with glucose and water syrup) with a composition of phytoalexins for human consumption, that is to say food grade, according to the criteria specified above. This phytoalexin composition may be composed exclusively of resveratrol, specifically trans-resveratrol which is the most common form, or it may contain other phytoalexins such as those mentioned above, which is the most widespread case since traces of other known phytoalexins usually exist.
Las composiciones alimenticias de colonias de abejas mellferas como las que se describen aqul, contribuyen de forma significativa al control de la infection, manteniendo los niveles de parasitacion dentro de llmites que permiten a las colonias ser productivas gracias a la presencia de las fitoalexinas, que se muestra como una alternativa eficaz, economica y ambientalmente mas sostenible y sana para el consumidor que otros compuestos empleados en la actualidad, como la fumagilina (ver Ejemplos). Estas composiciones alimenticias se muestran especialmente eficaces frente a la nosemosis, y particularmente la nosemosis Tipo A y C. En el ambito de la presente memoria se entiende por control de una infection a la reduction en la parasitacion sin eliminar completamente la infection.The food compositions of honey bee colonies such as those described here contribute significantly to infection control, keeping parasitization levels within limits that allow colonies to be productive thanks to the presence of phytoalexins, which are It shows as an effective, economically and environmentally more sustainable and healthy alternative for the consumer than other compounds currently used, such as fumagillin (see Examples). These food compositions are especially effective against nosemosis, and particularly nosemosis Type A and C. In the scope of the present specification it is understood as control of an infection to the reduction in parasitization without completely eliminating the infection.
La presente invencion se refiere tambien al uso de la composicion alimenticia descrita, en cualquiera de sus variantes para el control de infecciones por microorganismos patogenos en colmenas de abejas mellferas, y especialmente de infecciones por nosemosis, siendo mas preferible aun la nosemosis tipo A y C.The present invention also relates to the use of the described food composition, in any of its variants for the control of infections by pathogenic microorganisms in hives of honey bees, and especially of infections by nosemosis, with nosemosis type A and C being even more preferable. .
De esta forma, un tercer objeto de la presente invention lo constituye un metodo para controlar infecciones por microorganismos patogenos en colmenas de abejas mellferas, que comprende administrar a la colmena la composition alimenticia antes descrita en cualquiera de sus variantes, que contiene en su formulation al menos una fitoalexina en una cantidad comprendida entre 0.1% y 2.8% en peso del total de composition, incluidos ambos llmites, en al menos una dosis.Thus, a third object of the present invention constitutes a method for controlling infections by pathogenic microorganisms in honey bee hives, which comprises administering to the hive the food composition described above in any of its variants, which contains in its formulation the less a phytoalexin in an amount between 0.1% and 2.8% by weight of the total composition, including both limits, in at least one dose.
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La infeccion a tratar es en el caso mas preferido la nosemosis, siendo mas preferible aun la nosemosis tipo A y C.The infection to be treated is in the most preferred case nosemosis, with nosemosis type A and C being even more preferable.
Como se ha dicho anteriormente en relacion al producto, el resveratrol y mas preferentemente el trans-resveratrol es la fitoalexina de grado farmaceutico de eleccion preferida, aunque tambien puede combinarse con otras fitoalexinas, siendo asl como normalmente se presenta en las composiciones de fitoalexinas de consumo humano o de calidad alimentaria que se utilizan para preparar la composition. Ademas, la composition alimenticia puede ser administrada en forma de alimento llquido o de alimento semisolido como se describe en las variantes anteriores, que son preferidas. En el primer caso, el llquido azucarado puede ser preferentemente un jarabe de alimentation de abejas. En el segundo caso, puede ser preferentemente una pasta semisolida azucarada de alimentacion de abejas. En ambas variantes de presentation del alimento, el metodo puede llevarse a cabo de forma mas preferida con las composiciones especlficas descritas anteriormente para el jarabe y para la pasta semisolida azucarada.As stated above in relation to the product, resveratrol and more preferably trans-resveratrol is the pharmaceutical grade phytoalexin of preferred choice, although it can also be combined with other phytoalexins, being as usual in the phytoalexin compositions of consumption human or food grade that are used to prepare the composition. In addition, the food composition can be administered in the form of liquid food or semi-solid food as described in the above variants, which are preferred. In the first case, the sugary liquid may preferably be a bee-feeding syrup. In the second case, it may preferably be a semi-solid sugar-sweetened bee-feeding paste. In both variants of food presentation, the method can be carried out more preferably with the specific compositions described above for syrup and for semi-solid sugary pasta.
Preferentemente, la composicion alimenticia se administra a la colmena repartiendola entre 1 y 4 dosis (es decir, la dosis total de composicion alimenticia se administra en una, dos, tres o cuatro veces). Cuando la composicion es un llquido azucarado, se administra preferentemente en cuatro dosis, mientras que cuando es una pasta semisolida se administra de una vez; esto se debe a que las composiciones llquidas azucaradas, como el jarabe, se consumen mas rapido, no pudiendose administrar en cantidades elevadas de producto porque si se liberara por accidente podrla matar a las abejas. Por el contrario, cuando el producto es semi-solido, este problema no se da y por tanto puede administrase toda la dosis de una vez. Asl, por ejemplo, se puede administrar la composicion alimenticia a una colmena forma llquida (jarabe) o en forma semisolida (candy), conteniendo una cantidad de 7 gramos de fitoalexina, concretamente trans-resveratrol. En el primer caso, se administrarlan cuatro dosis de 250 gramos de jarabe contiendo cada una 1,75 gramos de fitoalexina (25% de la dosis total de fitoalexinas por colmena). En el segundo caso, se darla una unica dosis del candy, siendo de 1 kilogramo de candy que contiene los 7 gramos de fitoalexinas (100% de la dosis de fitoalexinas por colmena, de una vez). En definitiva, independientemente de la forma de presentacion, la dosis a administrar de composicion alimenticia y de fitoalexinas que contiene es la misma.Preferably, the food composition is administered to the hive by distributing it between 1 and 4 doses (ie, the total dose of food composition is administered in one, two, three or four times). When the composition is a sugary liquid, it is preferably administered in four doses, while when it is a semi-solid paste it is administered at once; This is because sugary liquid compositions, such as syrup, are consumed faster, and can not be administered in large quantities of product because if it were released by accident it could kill the bees. On the contrary, when the product is semi-solid, this problem does not occur and therefore the entire dose can be administered at once. Thus, for example, the food composition can be administered to a hive in liquid form (syrup) or in semi-solid form (candy), containing an amount of 7 grams of phytoalexin, specifically trans-resveratrol. In the first case, four doses of 250 grams of syrup each containing 1.75 grams of phytoalexin (25% of the total dose of phytoalexins per hive) will be administered. In the second case, a single dose of candy will be given, being 1 kilogram of candy containing 7 grams of phytoalexins (100% of the dose of phytoalexins per hive, at once). In short, regardless of the form of presentation, the dose to be administered of food composition and phytoalexins it contains is the same.
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Tambien de manera preferida, cuando la composition se administra en mas de una dosis, cada dosis se administra con 7 dias de intervalo entre ellas.Also preferably, when the composition is administered in more than one dose, each dose is administered with a 7-day interval between them.
Se ha comprobado que es mas conveniente que la administration de la composicion alimenticia en primavera se realice en forma de alimento liquido (jarabe), mientras que en otono es mas eficaz la administracion como alimento semisolido (candy).It has been proven that it is more convenient for the administration of the food composition in spring to be carried out in the form of liquid food (syrup), while in autumn it is more efficient to administer it as a semi-solid food (candy).
Asi, se ha constatado un efecto significativo del metodo descrito en el control de la infection de abejas infectadas por nosemosis, observandose una reduction en la parasitacion, es decir, en el numero de abejas infectadas por colmena, y en la production de esporas en dichas abejas infectadas tras la administracion de la composicion alimenticia.Thus, there has been a significant effect of the method described in the control of the infection of bees infected by nosemosis, observing a reduction in parasitization, that is, in the number of bees infected by hive, and in the production of spores in these infected bees after the administration of the food composition.
Al poder estar la composicion alimenticia constituida por la combination de un alimento azucarado de abejas comun y conocido y una composicion de fitoalexinas preparada para su uso humano y con calidad alimentaria, la memoria cubre asimismo un metodo de control de las infecciones por microorganismos patogenos en colmenas de abejas meKferas, y especialmente de infecciones por nosemosis, siendo mas preferible aun la nosemosis tipo A y C, que comprende administrar a la colmena de forma separada pero simultaneamente ambos componentes, alimento azucarado de abejas y composicion de fitoalexinas. Dicha administracion simultanea de alimento y fitoalexinas se puede realizar siguiendo todas las alternativas y especificaciones planteadas en esta memoria para la composicion alimenticia en cuanto a proporciones, tipos de alimentosa y fitoalexinas, etc. y en cuanto al metodo (formas de administracion: cantidades, frecuencias, formas de administration...).Being able to be the food composition constituted by the combination of a common and known sugary bee food and a phytoalexin composition prepared for human use and food quality, the memory also covers a method of controlling infections by pathogenic microorganisms in hives of honey bees, and especially of infections by nosemosis, being even more preferable nosemosis type A and C, which comprises administering to the hive separately but simultaneously both components, sugary food of bees and composition of phytoalexins. Said simultaneous administration of food and phytoalexins can be performed following all the alternatives and specifications set forth herein for the food composition in terms of proportions, types of food and phytoalexins, etc. and as for the method (forms of administration: quantities, frequencies, forms of administration ...).
Breve description de las FigurasBrief description of the Figures
Figura 1. Comparacion del numero de esporas contabilizado en colmenas de abejas infectadas por N. ceranae tratadas con o sin resveratrol de acuerdo con el Ejemplo 1. La figura en gris claro representa el grupo R, y la figura de gris oscuro representa al grupo NR.Figure 1. Comparison of the number of spores counted in beehives infected with N. ceranae treated with or without resveratrol according to Example 1. The light gray figure represents the R group, and the dark gray figure represents the NR group .
Figura 2. Resultados del primer ensayo comentado en el Ejemplo 2.2 de comparacion de colmenas tratadas con la composicion alimenticia objeto de la presente invention y alimentos que comprenden fumagilina. RJ: resveratrol en jarabe; RC: resveratrol en candy; AF: acido formico; FUMA: fumagilina; CJ: control del tratamiento con jarabe; CC: control del tratamiento con candy; NI: sin intervention.Figure 2. Results of the first test commented on in Example 2.2 of comparison of hives treated with the food composition object of the present invention and foods comprising fumagillin. RJ: resveratrol in syrup; RC: resveratrol in candy; AF: formic acid; SMOKE: fumagiline; CJ: control of syrup treatment; CC: control of candy treatment; NI: without intervention.
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Figura 3. Porcentaje de parasitacion medio en cada grupo tratado en el estudio (CR= grupo ensayo con resveratrol adicionado a candy; JR= grupo ensayo con resveratrol adicionado a jarabe; FUM= grupo testigo positivo al que se administro fumagilina; C= grupo testigo solo candy; J= grupo testigo solo jarabe; ST= grupo testigo sin intervention). M1: antes del tratamiento en otono 2011; M2: despues del tratamiento en otono 2011; M3: antes del tratamiento en primavera 2012; M4: despues del tratamiento en primavera 2012; M5: antes del tratamiento en otono 2012; M2: despues del tratamiento en otono 2012.Figure 3. Percentage of average parasitization in each group treated in the study (CR = test group with resveratrol added to candy; JR = test group with resveratrol added to syrup; FUM = positive control group to which fumagiline was administered; C = control group only candy; J = control group only syrup; ST = control group without intervention). M1: before treatment in autumn 2011; M2: after treatment in autumn 2011; M3: before treatment in spring 2012; M4: after treatment in spring 2012; M5: before treatment in autumn 2012; M2: after treatment in autumn 2012.
Figura 4. Porcentaje de viabilidad medio en los grupos estudiados en abril de 2013. CR= grupo ensayo con resveratrol adicionado a candy; JR= grupo ensayo con resveratrol adicionado a jarabe; FUM= grupo testigo positivo al que se administro fumagilina; C= grupo testigo solo candy; J= grupo testigo solo jarabe; ST= grupo testigo sin intervencion.Figure 4. Average viability percentage in the groups studied in April 2013. CR = trial group with resveratrol added to candy; JR = test group with syrup added resveratrol; FUM = positive control group to which fumagiline was administered; C = witness group only candy; J = control group only syrup; ST = control group without intervention.
Figura 5. Production de miel media en cada grupo en verano 2012. CR= grupo ensayo con resveratrol adicionado a candy; JR= grupo ensayo con resveratrol adicionado a jarabe; FUM= grupo testigo positivo al que se administro fumagilina; C= grupo testigo solo candy; J= grupo testigo solo jarabe; ST= grupo testigo sin intervencion.Figure 5. Average honey production in each group in summer 2012. CR = test group with resveratrol added to candy; JR = test group with syrup added resveratrol; FUM = positive control group to which fumagiline was administered; C = witness group only candy; J = control group only syrup; ST = control group without intervention.
EjemplosExamples
Ejemplo 1. Evaluation en laboratorio de la actividad de resveratrol frente a Nosema spp La evaluacion de la eficacia del resveratrol requirio realizar previamente ensayos de toxicidad del producto en laboratorio, y para ello se debieron realizar estudios preliminares de solubilidad y valoracion de aceptacion del resveratrol para determinar la concentration maxima que se puede administrar a las abejas. Dado que los ensayos se deben realizar con abejas de edad controlada, fue necesario tomar cuadros de crla de las colmenas que se introdujeron en incubadores en el laboratorio hasta el nacimiento de las abejas. Por ello estos ensayos solo se pueden realizar en los meses desde mediados de primavera e inicio del otono.Example 1. Laboratory evaluation of the activity of resveratrol against Nosema spp The evaluation of the efficacy of resveratrol required to previously carry out toxicity tests of the product in the laboratory, and for this, preliminary studies of solubility and acceptance assessment of resveratrol had to be carried out. Determine the maximum concentration that can be given to bees. Since the tests must be carried out with bees of controlled age, it was necessary to take pictures of the hives from the hives that were introduced in incubators in the laboratory until the bees were born. Therefore, these tests can only be carried out in the months from mid-spring to the beginning of the fall.
Se adapto la metodologla siguiendo las recomendaciones de la OECD 213 (GUIDELINES FOR THE TESTING OF CHEMICALS. Honeybees, Acute Oral Toxicity Test) para la determination de la toxicidad via oral del resveratrol. Esta gula indica que para las sustancias de baja toxicidad esperada (como es el resveratrol), se pueden realizar test limites con 100 pg principio activo/abeja. Si la toxicidad es superior a ese valor no se requieren pruebas adicionales de toxicidad.The methodology was adapted following the recommendations of OECD 213 (GUIDELINES FOR THE TESTING OF CHEMICALS. Honeybees, Acute Oral Toxicity Test) for the determination of oral toxicity of resveratrol. This guideline indicates that for substances with low expected toxicity (such as resveratrol), limit tests can be carried out with 100 pg active ingredient / bee. If the toxicity is higher than that value, no additional toxicity tests are required.
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Tras la realization de varios test, se pudo determinar que la DL50 del resveratrol es superior a 100 pg/abeja, abordando posteriormente los ensayos de eficacia en el laboratorio. En estos estudios, se establecieron 2 grupos con 4 replicados por grupo utilizando abejas nacidas en el laboratorio. Un primer grupo (R) de abejas infectadas individualmente con 160.000 esporas de N. ceranae, utilizando la metodologla desarrollada y puesta a punto. A los 7 dlas post-infection, se les administro una dosis (oral) de 6mg/ml que se mantuvo ad limitum durante 24 horas. El segundo grupo (NR), estaba formado por abejas infectadas (a la misma dosis que el grupo anterior) a las que no se les suministro resveratrol sino una solution de agua y azucar. Se tomaron 5 abejas por replicado a los dlas 9, 10 y 11 dlas post-infection, que en el grupo R correspondla a 24, 48 y 72 horas despues de la administration del resveratrol. Se observo que 72 horas despues de la administration se produce una reduction estadlsticamente significativa (p<0.05) en los recuentos de esporas en abejas infectadas por N. ceranae.After performing several tests, it was possible to determine that the LD50 of the resveratrol is greater than 100 pg / bee, subsequently addressing the efficacy tests in the laboratory. In these studies, 2 groups were established with 4 replicates per group using bees born in the laboratory. A first group (R) of bees individually infected with 160,000 spores of N. ceranae, using the methodology developed and tuned. At 7 days post-infection, they were given a dose (oral) of 6mg / ml that was maintained ad limitum for 24 hours. The second group (NR) was made up of infected bees (at the same dose as the previous group) that were not given resveratrol but a solution of water and sugar. Five bees were taken per replicate on days 9, 10 and 11 days post-infection, which in group R corresponds to 24, 48 and 72 hours after administration of resveratrol. It was observed that 72 hours after administration there is a statistically significant reduction (p <0.05) in spore counts in bees infected with N. ceranae.
Tabla 1. Comparacion del numero de esporas contabilizado en colmenas de abejas infectadas por N. ceranae tratadas con o sin resveratrol.Table 1. Comparison of the number of spores counted in beehives infected by N. ceranae treated with or without resveratrol.
- 24 h 48 h 72 h 24 h 48 h 72 h
- Grupo R R group
- 40.106.200 35.106.500 33.253.400* 40,106,200 35,106,500 33,253,400 *
- (Resveratrol) (Resveratrol)
- (± 16.535.833) (±12.739.944) (± 16.203.105) (± 16,535,833) (± 12,739,944) (± 16,203,105)
- Grupo NR NR Group
- 40.000.933 34.710.800 52.490.000* 40,000,933 34,710,800 52,490,000 *
- (No Resveratrol) (Not Resveratrol)
- (± 11.425.964) (± 15.008.312) (± 24.177.759) (± 11,425,964) (± 15,008,312) (± 24,177,759)
Ejemplo 2. Analisis y valoracion del metodo de tratamiento de infestaciones (nosenoma) en colonias de abejas mellferas.Example 2. Analysis and evaluation of the method of treatment of infestations (nosenoma) in colonies of honey bees.
2.1 Desarrollo de metodologla analltica para analisis de trans-resveratrol y compuestos relacionados en materia prima, miel, polen cera y alimentos para abejas Con el fin de poder conocer tanto la composition de la materia prima como establecer correctamente la cantidad de resveratrol que debla anadirse al alimento, se han desarrollado metodos basados en el empleo de cromatografla llquida de alta eficacia (HPLC) con detection ultravioleta y fluorescencia.2.1 Development of analytical methodology for analysis of trans-resveratrol and related compounds in raw material, honey, pollen wax and food for bees In order to be able to know both the composition of the raw material and correctly establish the amount of resveratrol that should be added to the For food, methods based on the use of high-performance liquid chromatography (HPLC) with ultraviolet detection and fluorescence have been developed.
Los ensayos han sido realizados sobre trans-resveratrol, riqueza minima del 99%, grado farmaceutico de Megaresveratrol® (Danbury,CT,USA); tambien se ha ensayado con trans-The tests have been carried out on trans-resveratrol, minimum wealth of 99%, pharmaceutical grade of Megaresveratrol® (Danbury, CT, USA); It has also been tested with trans-
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resveratrol al 50% de pureza de la misma firma comercial. Para conocer la riqueza del producto comercial expedido como suplemento alimenticio aprobado por FDA como fuente de trans-resveratrol y otras fitoalexinas, y puesto que se partio de un etiquetado de riqueza minima del 99%, se aplico Cromatografla llquida con detection diodos en fila y fluorescente (HPLC- DAD-FLD) y se encontro que dentro de la variation prevista el producto cumplla la especificacion; empleando cromatografla llquida con deteccion espectrometrica de masas(HPLC-MS) se detecto la presencia de trans-piceido y trazas de cis-resveratrol.50% purity resveratrol from the same commercial firm. To know the richness of the commercial product issued as an FDA-approved food supplement as a source of trans-resveratrol and other phytoalexins, and since it was based on a labeling of minimum wealth of 99%, liquid chromatography was applied with row and fluorescent diode detection (HPLC-DAD-FLD) and it was found that within the expected variation the product meets the specification; using liquid chromatography with mass spectrometric detection (HPLC-MS) the presence of trans-piceid and traces of cis-resveratrol was detected.
Se hicieron posteriormente analisis de los alimentos suministrados a las abejas, y preparados como se piensa pueden hacer los apicultores en campo, sobre diversas preparaciones siguiendo la dosis recomendada; se encontraron para candy valores comprendidos entre 0,17 y 0,32 mg/L de trans-piceido y entre 7,94 y 8,55 mg/L para trans- resveratrol. En el caso del jarabe los valores oscilaron entre 0,24 y 0,18 mg/L para t-piceido y 6,94 a 8,03 mg/L para trans-resveratrol.Subsequently, analyzes of the food supplied to the bees were made, and prepared as is thought by beekeepers in the field, on various preparations following the recommended dose; values between 0.17 and 0.32 mg / L of trans-piceido and between 7.94 and 8.55 mg / L were found for trans- resveratrol. In the case of syrup, the values ranged between 0.24 and 0.18 mg / L for t-piceid and 6.94 to 8.03 mg / L for trans-resveratrol.
Examinada la variacion de los preparados alimenticios con el tiempo se aprecio que eran estables por al menos un mes.Examined the variation of food preparations over time it was appreciated that they were stable for at least one month.
Para la evaluation de las cantidades residuales, despues de efectuados los tratamientos, se ha aplicado el mismo sistema pero incorporando un detector espectrometrico de masas lo cual no solo ha permitido confirmar la identidad de los analitos sino tambien rebajar notablemente los llmites de deteccion. La metodologla analltica ha sido publicada en Liquid chromatographic determination of resveratrol and piceid isomers in honey M. E. Soto, J. Bernal, Ma. T. Martin, M. Higes, J. L. Bernal, M. J. Nozal, Food Analytical Methods, 5 (2011) 162-171, que complementa el estudio sobre la utilization de la fumagilina, sobre cuya eficacia se ha comparado la de la presente invention: The stability and efectiveness of fumagillin in controlling Nosema ceranae (Microsporidia) infection in honey bees (Apis Mellifera) under laboratory and field conditions. M. Higes, M. J. Nozal, A. Alvaro, L. Barrios, A. Meana, R. Martin, J. L. Bernal, J. Bernal. Apidologie, 42 (2011) 364-377.For the evaluation of the residual quantities, after the treatments have been carried out, the same system has been applied but incorporating a mass spectrometric detector which has not only allowed to confirm the identity of the analytes but also significantly reduce the detection limits. The analytical methodology has been published in Liquid chromatographic determination of resveratrol and piceid isomers in honey ME Soto, J. Bernal, Ma. T. Martin, M. Higes, JL Bernal, MJ Nozal, Food Analytical Methods, 5 (2011) 162- 171, which complements the study on the use of fumagillin, on which efficacy has been compared to that of the present invention: The stability and effectiveness of fumagillin in controlling Nosema ceranae (Microsporidia) infection in honey bees (Apis Mellifera) under laboratory and field conditions. M. Higes, M. J. Nozal, A. Alvaro, L. Barrios, A. Meana, R. Martin, J. L. Bernal, J. Bernal. Apidologie, 42 (2011) 364-377.
En todas las muestras se han evaluado trans- y cis-resveratrol (t-r y c-r) as! como trans- y cis-piceido (t-p y c-p).Trans- and cis-resveratrol (t-r and c-r) as! as trans- and cis-piceido (t-p and c-p).
En los ensayos realizados con la cantidad teorica maxima de 7 gramos de trans-resveratrol por colmena se analizaron por HPLC-MS las muestras de miel, polen y cera recogidas aIn the tests carried out with the maximum theoretical quantity of 7 grams of trans-resveratrol per hive, samples of honey, pollen and wax collected at HPLC-MS were analyzed by HPLC-MS
distintos intervalos de tiempo en la colmena asl como al momento de la cosecha, los valores encontrados fueron muy variables como sucede siempre en el caso de este tipo de muestras y muestreo.Different time intervals in the hive as well as at the time of harvest, the values found were very variable as always happens in the case of this type of samples and sampling.
5 Tabla 2. Contenido total de los diferentes isomeros en miel en funcion de tipo de alimento5 Table 2. Total content of the different isomers in honey depending on the type of food
en que se ha administrado el resveratrol y el muestreoin which resveratrol and sampling have been administered
Concentration total (mgkg-1)Total concentration (mgkg-1)
Alimento Muestreo ________________________________Food Sampling ________________________________
- t-p c-pa t-r c-r t-p c-pa t-r c-r
- Candy R Candy R
- 1° 4.4 55.3 206.9 8.9 1st 4.4 55.3 206.9 8.9
- Candy R Candy R
- 2° 1.6 50.2 209.2 4.5 2nd 1.6 50.2 209.2 4.5
- Jarabe R R syrup
- 1° 2.9 10.5 200.4 4.1 1 2.9 10.5 200.4 4.1
- Jarabe R R syrup
- 2° 1.7 36.0 362.3 41.2 2nd 1.7 36.0 362.3 41.2
a ygkg1to ygkg1
Tabla 3. Concentraciones (mgkg-1) encontradas de los diferentes isomeros en muestras de 10 polen de colmenas tratadas con resveratrol anadido en candy o jarabeTable 3. Concentrations (mgkg-1) found of the different isomers in 10 pollen samples of hives treated with resveratrol added in candy or syrup
- Tratamiento Treatment
- Muestra t-p c-p t-r c-r Sample t-p c-p t-r c-r
- Candy R Candy R
- TN-03 ND ND 0.44 ND TN-03 ND ND 0.44 ND
- Candy R Candy R
- TN-09 ND ND 0.07 ND TN-09 ND ND 0.07 ND
- Candy R Candy R
- TN-19 ND ND 5.38 0.11 TN-19 ND ND 5.38 0.11
- Candy R Candy R
- TN-23 ND ND 0.91 ND TN-23 ND ND 0.91 ND
- Candy R Candy R
- TN-27 ND ND 10.29 0.05 TN-27 ND ND 10.29 0.05
- Jarabe R R syrup
- TN-08 ND ND 0.43 ND TN-08 ND ND 0.43 ND
- Jarabe R R syrup
- TN-11 ND ND 0.23 ND TN-11 ND ND 0.23 ND
- Jarabe R R syrup
- TN-14 ND ND 0.03 ND TN-14 ND ND 0.03 ND
- Jarabe R R syrup
- TN-17 ND ND 1.91 0.05 TN-17 ND ND 1.91 0.05
- Jarabe R R syrup
- TN-29 ND ND 1.82 0.09 TN-29 ND ND 1.82 0.09
Candy R: candy con resveratrol Jarabe R: jarabe con resveratrolCandy R: candy with resveratrol Syrup R: syrup with resveratrol
En cuanto a la action contra la nosemosis, mostraron una eficacia notable con una 15 reduction en la parasitacion que mantuvo a las colmenas en niveles de viabilidad y productivos similares a los grupos que recibieron fumagilina y muy por encima de los grupos testigo que recibieron solo placebo.Regarding the action against nosemosis, they showed a remarkable efficacy with a reduction in parasitization that kept the hives in viability and productive levels similar to the groups that received fumagiline and well above the control groups that received only placebo .
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2.2. Evaluation en campo de la actividad frente a Nosema spp. del trans-resveratrol y sus isomeros. Determination de la posolog'ia adecuada.2.2. Evaluation in the field of activity against Nosema spp. of trans-resveratrol and its isomers. Determination of the appropriate dosage.
Se partio siempre de trans-resveratrol, bien al 99% o al 50% de la firma Megaresveratrol®.It always started with trans-resveratrol, either 99% or 50% of the Megaresveratrol® firm.
Se iniciaron ensayos en Otono 2011 con 65 colmenas, divididas en los siguientes grupos: RJ (Resveratrol en jarabe, n=10), RC (Resveratrol en candy, n=10), AF (Acido formico, n=5), FUMA (Grupo testigo positivo, tratado con Fumagilina, n=10), CJ (Grupo testigo solo con jarabe, n=10), CC (Grupo testigo solo con candy, n=10), ST (Grupo testigo sin intervention, n=10). El grupo RJ recibio 1,75g MegaResveratrol® en 250 ml de jarabe de azucar al 50% (4 tratamientos, 1 por semana, lo que supone 6 g en total del producto activo resveratrol) y el grupo RC recibio 7g MegaResveratrol® (6 g en total del producto activo) en 1 kg de candy (glucosa 30%, fructosa 50%, agua 18%). Estos ensayos (excepto el grupo AF y ST) se han continuado en el ano 2012, realizandose tratamientos en primavera y otono. En total se han realizado 6 muestreos, recogidos antes y despues de las intervenciones en otono 2011, primavera de 2012 y otono de 2012. De esta manera , se dispone de datos de un gran grupo de colmenas que se ha mantenido durante un largo periodo de tiempo, pudiendo asl conocer el efecto de los tratamientos (aplicacion del resveratrol en diferentes formas y los controles) a largo plazo, obteniendo datos de production, viabilidad y evolution de las colmenas. Tambien sirvio para determinar el momento mas adecuado de aplicacion de los tratamientos asl como para elegir la mejor forma de aplicacion.Trials began in Otono 2011 with 65 hives, divided into the following groups: RJ (Resveratrol in syrup, n = 10), RC (Resveratrol in candy, n = 10), AF (Formic acid, n = 5), FUMA ( Positive control group, treated with Fumagilina, n = 10), CJ (Control group only with syrup, n = 10), CC (Control group only with candy, n = 10), ST (Control group without intervention, n = 10) . The RJ group received 1.75g MegaResveratrol® in 250 ml of 50% sugar syrup (4 treatments, 1 per week, which is 6 g in total of the active product resveratrol) and the RC group received 7g MegaResveratrol® (6 g in total of the active product) in 1 kg of candy (30% glucose, 50% fructose, 18% water). These trials (except the AF and ST group) have been continued in 2012, with spring and autumn treatments. In total, 6 samples were taken, collected before and after the interventions in autumn 2011, spring 2012 and autumn 2012. In this way, data are available from a large group of hives that have been maintained for a long period of time, being able to know the effect of treatments (application of resveratrol in different forms and controls) in the long term, obtaining data on production, viability and evolution of hives. It also served to determine the most appropriate time of application of treatments as well as to choose the best form of application.
Los resultados del primer ensayo de otono 2011 mostraron que en los grupos que recibieron resveratrol se produjo una reduction en la parasitacion (reduction en el numero de abejas infectadas por colmena) comparables al grupo que recibio el tratamiento con fumagilina (Figura 2).The results of the first fall test 2011 showed that in the groups that received resveratrol there was a reduction in parasitacion (reduction in the number of bees infected by hive) comparable to the group that received the treatment with fumagiline (Figure 2).
Continuando con el estudio de las colmenas durante un ano posterior (excepto el grupo AF que se saco del estudio), se pudo observar que durante la primavera de 2012, el porcentaje de parasitacion bajo en todas las colmenas como efecto del aumento de la poblacion que se produce de forma natural en las colmenas durante este periodo. Debido a esto no se pudo observar el efecto de los tratamientos en este periodo. Por el contrario, en el muestreo realizado antes de los tratamientos de otono 2012, se pudo observar claramente que los porcentajes de parasitacion son mucho mas elevados en aquellos grupos que no recibieron ningun tratamiento en primavera (C, J) cuando se comparan con los grupos tratados con resveratrol o fumagilina (CR, JR y FUM). El efecto de los tratamientos en otono tambienContinuing with the study of the hives during a subsequent year (except the AF group that was removed from the study), it was observed that during the spring of 2012, the percentage of parasitization low in all the hives as an effect of the population increase that It occurs naturally in hives during this period. Due to this the effect of the treatments in this period could not be observed. On the contrary, in the sampling carried out before the 2012 autumn treatments, it was clearly observed that the parasitacion percentages are much higher in those groups that did not receive any treatment in spring (C, J) when compared with the groups treated with resveratrol or fumagiline (CR, JR and FUM). The effect of autumn treatments also
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produjo una reduction en el porcentaje de parasitacion. La aceptacion del resveratrol en otono es mas adecuada en candy que en jarabe, si bien se reduce en primavera, cuando por el contrario las abejas consumen mejor la administration en jarabe. La administration del CR es un sistema comodo que no requiere de muchas visitas al colmenar, facilitando la labor del apicultor (Figura 3).produced a reduction in the percentage of parasitization. The acceptance of resveratrol in autumn is more appropriate in candy than in syrup, although it is reduced in spring, when on the contrary bees consume syrup administration better. The administration of the CR is a comfortable system that does not require many visits to the apiary, facilitating the work of the beekeeper (Figure 3).
La viabilidad de las colonias al final del estudio en los grupos tratados con resveratrol (JR y CR) fue similar a la del grupo que se trato con fumagilina (Figura 4). Aunque los resultados de production de miel solo fueron significativamente mayores en el grupo FUM (Figura 5).The viability of the colonies at the end of the study in the groups treated with resveratrol (JR and CR) was similar to that of the group treated with fumagiline (Figure 4). Although the honey production results were only significantly higher in the FUM group (Figure 5).
Por todo ello, el resveratrol muestra ser eficaz para el tratamiento y control de la Nosemosis. Si bien el efecto en la reduccion de la parasitacion es mas evidente en otono, la administracion del producto en primavera facilita que las colmenas lleguen al otono en con unos niveles de parasitacion menores.For all these reasons, resveratrol is shown to be effective for the treatment and control of Nosemosis. Although the effect on the reduction of parasitization is more evident in autumn, the administration of the product in spring facilitates that the hives reach the autumn in with lower levels of parasitization.
En cuanto a los residuos, podemos observar que la miel puede presentar resveratrol de forma natural ya que se ha detectado en los grupos no tratados (C y J) aunque en cantidades mucho menores que en los grupos tratados (CR y JR). En estos ultimos se puede encontrar trans-resveratrol en cantidades hasta 1000 veces superiores. Las otras formas isomericas que se detectan en mayor proportion son el trans-piceido y el cis- resveratrol.Regarding the residues, we can observe that honey can present resveratrol naturally since it has been detected in the untreated groups (C and J) although in much smaller quantities than in the treated groups (CR and JR). In the latter, trans-resveratrol can be found in amounts up to 1000 times higher. The other isomeric forms that are detected in greater proportion are trans-piceid and cis-resveratrol.
Tabla 4: Concentraciones en microgramo por kilogramo, obtenidas por HPLC-MS, de los distintos isomeros encontrados en mieles procedentes de colmenas tratadas. PrimerTable 4: Microgram concentrations per kilogram, obtained by HPLC-MS, of the different isomers found in honeys from treated hives. First
muestreo (1) entre 2-6 diciembre de 2011. Segundo muestreo (2) entre 9-10 enero de 2012.sampling (1) between 2-6 December 2011. Second sampling (2) between 9-10 January 2012.
- _____t-p_____ c- p -r c-r _____ t-p _____ c- p -r c-r
- 1 one
- 2 1 2 1 2 1 2 2 1 2 1 2 1 2
- TN-03 TN-03
- CR 874.3 127.7 6.8 ND 32.3a 24.3a 1.3a 135.0 CR 874.3 127.7 6.8 ND 32.3a 24.3a 1.3a 135.0
- TN-07 TN-07
- CR 19.3 9.3 NQ NQ 730.9 389.7 363.8 9.5 CR 19.3 9.3 NQ NQ 730.9 389.7 363.8 9.5
- TN-09 TN-09
- CR 537.4 NQ 17.8 NQ 21.6a 29.2 1.9a NQ CR 537.4 NQ 17.8 NQ 21.6a 29.2 1.9a NQ
- TN-13 TN-13
- CR NQ ND NQ ND 318.5 NQ 21.2 ND CR NQ ND NQ ND 318.5 NQ 21.2 ND
- TN-16 TN-16
- CR 4.9 NQ NQ NQ 211.7 535.2 23.4 NQ CR 4.9 NQ NQ NQ 211.7 535.2 23.4 NQ
- TN-19 TN-19
- CR 250.9 878.1 NQ 15.4 8.6a 93.0a 518.1 2.0a CR 250.9 878.1 NQ 15.4 8.6a 93.0a 518.1 2.0a
- TN-23 TN-23
- CR 4.6 ND NQ ND 738.2 93.0 367.3 NQ CR 4.6 ND NQ ND 738.2 93.0 367.3 NQ
- TN-27 TN-27
- CR 657.0 31.9 9.2 NQ 28.3a 11.3a 1.3a 15.0 CR 657.0 31.9 9.2 NQ 28.3a 11.3a 1.3a 15.0
- TN-31 TN-31
- CR 2.1a 365.2 21.5 14.7 114.1a 34.0a 3.2a 403.6 CR 2.1a 365.2 21.5 14.7 114.1a 34.0a 3.2a 403.6
- TN-35 TN-35
- CR ND 213.1 ND 20.1 12.6 16.5a 10.5 2.0a CR ND 213.1 ND 20.1 12.6 16.5a 10.5 2.0a
- TN-01 TN-01
- JR 380.1 16.7 NQ 6.2 28.9a 70.8a 431.4 1.0a JR 380.1 16.7 NQ 6.2 28.9a 70.8a 431.4 1.0a
- TN-04 TN-04
- JR ND 21.5 ND 5.8 12.0 47.5 ND NQ JR ND 21.5 ND 5.8 12.0 47.5 ND NQ
- TN-08 TN-08
- JR NQ ND NQ ND 35.7 7.7 11.2 ND JR NQ ND NQ ND 35.7 7.7 11.2 ND
- TN-11 TN-11
- JR 9.7 77.1 NQ 5.4 348.7 3.7a 41.1 36.6 JR 9.7 77.1 NQ 5.4 348.7 3.7a 41.1 36.6
- TN-14 TN-14
- JR NQ 27.3 NQ 6.9 132.9 62.8a 15.1 1.7a JR NQ 27.3 NQ 6.9 132.9 62.8a 15.1 1.7a
- TN-17 TN-17
- JR NQ 509.6 NQ 8.5 75.3 151.6a 12.9 1.2a JR NQ 509.6 NQ 8.5 75.3 151.6a 12.9 1.2a
- TN-21 TN-21
- JR NQ 7.3 NQ NQ 38.9 209.4 12.4 8.4 JR NQ 7.3 NQ NQ 38.9 209.4 12.4 8.4
- TN-25 TN-25
- JR NQ ND ND ND 30.4 10.1 10.9 ND JR NQ ND ND ND 30.4 10.1 10.9 ND
- TN-29 TN-29
- JR 1.8a 99.0 10.5 NQ 124.7a 12.2a 3.0 90.7 JR 1.8a 99.0 10.5 NQ 124.7a 12.2a 3.0 90.7
- TN-33 TN-33
- JR 725.5 935.7 NQ 3.2 46.2a 60.9a 488.7 690.8 JR 725.5 935.7 NQ 3.2 46.2a 60.9a 488.7 690.8
- PR-11-03 PR-11-03
- ST NQ NQ NQ NQ 10.9 5.1 ND ND ST NQ NQ NQ NQ 10.9 5.1 ND ND
- PR-11-10 PR-11-10
- ST ND ND NQ NQ 11.5 NQ ND ND ST ND ND NQ NQ 11.5 NQ ND ND
- PR-11-20 PR-11-20
- ST ND ND ND ND 11.3 36.1 ND ND ST ND ND ND ND 11.3 36.1 ND ND
- PR-11-22 PR-11-22
- ST ND ND ND ND 11.1 ND ND ND ST ND ND ND ND 11.1 ND ND ND
- PR-11-31 PR-11-31
- ST ND ND ND ND 11.1 NQ ND ND ST ND ND ND ND 11.1 NQ ND ND
- PR-11-34 PR-11-34
- ST ND ND ND ND 11.0 NQ ND NQ ST ND ND ND ND 11.0 NQ ND NQ
- PR-11-01 PR-11-01
- C ND NQ ND NQ 48.2 47.1 14.5 NQ C ND NQ ND NQ 48.2 47.1 14.5 NQ
- PR-11-04 PR-11-04
- C NQ NQ ND NQ ND 6.4 ND ND C NQ NQ ND NQ ND 6.4 ND ND
- PR-11-07 PR-11-07
- C ND ND NQ NQ 16.0 NQ ND ND C ND ND NQ NQ 16.0 NQ ND ND
- PR-11-14 PR-11-14
- C ND ND ND ND 10.0 ND ND ND C ND ND ND ND 10.0 ND ND ND
- PR-10 PR-10
- C ND NQ NQ NQ 10.9 NQ ND ND C ND NQ NQ NQ 10.9 NQ ND ND
- PR-11-28 PR-11-28
- C ND ND ND ND 61.7 NQ 14.2 ND C ND ND ND ND 61.7 NQ 14.2 ND
- PR-11-09 PR-11-09
- J ND NQ ND NQ 45.3 NQ ND ND J ND NQ ND NQ 45.3 NQ ND ND
- PR-11-12 PR-11-12
- J ND ND ND ND ND 10.7 ND NQ J ND ND ND ND ND 10.7 ND NQ
- PR-11-15 PR-11-15
- J NQ NQ NQ NQ 16.0 NQ ND ND J NQ NQ NQ NQ 16.0 NQ ND ND
- PR-11-17 PR-11-17
- J NQ NQ ND ND 11.4 NQ ND ND J NQ NQ ND ND 11.4 NQ ND ND
- PR-11-33 PR-11-33
- J ND ND ND ND 10.8 NQ 10.7 ND J ND ND ND ND 10.8 NQ 10.7 ND
a: mg/kga: mg / kg
Tabla 5. Limites de detection (LOD) y Cuantificacion (LOQ) en ug/kg obtenidos enTable 5. Limits of detection (LOD) and Quantification (LOQ) in ug / kg obtained in
LC-MS para los distintos isomerosLC-MS for the different isomers
- Analito Analyte
- LOD LOQ Analito LOD LOQ LOD LOQ Analyte LOD LOQ
- t-p t-p
- 0.51 1.71 c-r 0.98 3.25 0.51 1.71 c-r 0.98 3.25
- c-p c-p
- 0.98 3.25 t-r 1.84 6.13 0.98 3.25 t-r 1.84 6.13
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