Propolis extract wi th enriched flavonoid content, its production, products containing this, and their use

Abstract

The invention relates to propolis extract with enriched flavonoid content, which contains raw propolis, one or more polyethylene glycols, one or more emulsifiers and water. The invention also relates to the production of the propolis extract with enriched flavonoid content, to a medicinal product, dietary supplement, vitamin product, feed or feed additive containing the propolis extract with enriched flavonoid content and to the use of the propolis extract with enriched flavonoid content.

Description

[0001] Propolis extract with enriched flavonoid content , its production , products containing this , and their use

[0002] The field of the invention

[0003] The invention relates to an extract containing propolis containing a high flavonoid content , to a method for the production of such an extract , to products produced from such extract and to their use .

[0004] The state of the art

[0005] Within the context of the present invention we are dealing with a propolis extract with enriched flavonoid content . Bees collect propolis in the interest of the protection of the hive , against bacteria and other pathogens . Propolis is a sticky yellowy-brown substance with a pleasant smell , which bees collect from the buds of trees ( such as black poplar, alder , oak, species of pine , etc . ) and their young branches and then transform. Bees use propolis mainly as a construction material , as insulation and adhesive . They fill gaps and cracks in the hive with propolis to keep the hive hermetically sealed .

[0006] The healing effect of propolis has been known for millennia , it contains wound-healing, anti-inflammatory and antibiotic components , in addition it also has an antifungal and immunostimulant effect . The maj ority of the advantageous effects of propolis are attributed to its flavonoid content [ I . Kosalec et al . , Flavonoid analysis and antimicrobial activity of commercially available propolis products , Acta Pharm . , 2005 , 4 , 423-30 ] .

[0007] Several solutions are known of according to the state of the art that deal with propolis extracts for use in animals .

[0008] A propolis extract with a high active ingredient content may be obtained with the method disclosed in patent document number CN113750120B , which involves a reduced extraction time . The method contains the following steps : production of propolis extract following pre-freezing of the propolis , dissolving of the propolis with foodquality alcohol and its dewaxing, then the propolis extract is subj ected to purification steps . The areas of use of the propolis extract produced in this way include use as an animal and poultry feed additive .

[0009] Patent application number CN114558035A relates to a propolis-f lavone extract and to its production and use . During the production method the propolis is mixed with an aqueous ethanol solution until the propolis is dissolved . The insoluble material is filtered out . The propolis extract is used for the treatment of necrotic bowel inflammation in poultry .

[0010] Patent application number CN108835388A relates to a feed additive and its production . The ingredients of the additive include , among others , propolis flavone . The application contains a method for the production of the propolis extract , which is as follows : The propolis is added to polyethylene glycol 6000 melted at 60 to 80 ° C and the stirring is continued for 10 minutes after the addition of 0 . 1 to 5 % of lecithin . This is followed by ultrasonic shaking for 30 minutes , then another 5 minutes of stirring . Following this the mixture is frozen ( -30 ° C ; 2 h ) , dried and the dried propolis is dissolved in 20 to 50 times its amount of water and filtered using membrane filtration through a 0 . 2 pm microfilter . The feed additive may be given to sheep , cattle , pigs and chickens .

[0011] The product according to patent application number CN107897558A is for increasing growth of poultry, the rate of laying eggs and egg weight , which is produced from propolis emulsion, aloe , earthworm, spirulina and probiotic . The propolis emulsion is produced in the following way : the solid propolis at room temperature is dissolved in polyethylene glycol in one of the embodiments at 37 ° C , for example . The wax-like , viscous consistency of the dispersion obtained is further improved with polyoxyethylene-ethers of fatty acids . Patent application number KR1020150083166A deals with improving the immunity of hens , improving the egg production rate and reducing the mortality rate . In the interest of this an additive containing propolis among others is mixed into the poultry feed . According to the specification the alcohol - aqueous extraction is used to produce the propolis extract .

[0012] Patent application number BRPI0604488A discloses the production of 20 to 35 % propolis extract . During the production method the frozen and ground propolis is dissolved in ethanol . Then this is centrifuged at a low temperature , the supernatant is separated and, finally, zinc oxide is added to it .

[0013] During the dilution with water of the propolis extracts ( alcoholic, glycol extracts ) produced today for human and animal use a significant amount of active ingredient is precipitated, which precipitation results in a significant reduction in the ratio of the useful components absorbed from the propolis . In other words , it is a general problem that during the industrial-scale production of propolis extracts low-content extract can be produced, or although the extract itself has a better content in view of useful substances , however when dissolved in water or other materials this content is precipitated, causing a significant loss in availability . In other words , no high flavonoid-content propolis extract and products made from it for human and animal use are known of according to the state of the art , the dissolution of which in water or other material would not involve significant precipitation, thereby causing a loss in availability .

[0014] On the basis of the above , in addition to the ethanol generally used during the production of the propolis extracts currently known of being flammable and volatile , meaning work performed with this should be avoided, it counts as an excise raw material , therefore its acquisition, storage and use are subj ect to very strict controls , which means its use is more costly . In addition to this an alcoholic propolis extract does not remain stable when diluted with water , the ethanol shifts over to the aqueous phase and the dissolved flavonoid propolis fraction becomes precipitated and / or crystalizes thereby damaging its availability . Furthermore , the materials costs of the currently available alcoholic propolis extracts currently available , their production duration and energy demand are also higher, in other words the known methods for the production of propolis extracts cannot be deemed to be optimal for the reasons explained above .

[0015] Therefore , it is necessary to produce an extract containing propolis that has a higher flavonoid content than those known of , during the production of which the use of ethanol is not involved, its dilution with water or dissolution in other materials is possible without the precipitation of propolis or flavonoids and which may be produced in a simpler , faster and more economical way than using the methods currently known of .

[0016] A brief description of the invention

[0017] During the economical industrial production of propolis extract with enriched flavonoid content , it was found in a way surprising for a person skilled in the art that using an extraction method performed with certain polyethylene glycol derivatives and by adding non-ionic emulsifiers , a high flavonoid content propolis extract may be produced that is soluble in water and even when dissolved in other substances the dissolved propolis and flavonoid fraction does not precipitate .

[0018] Therefore , in accordance with the above the obj ect of the present invention relates to a propolis extract with enriched flavonoid content that contains raw propolis , one or more polyethylene glycols , one or more emulsifiers and water .

[0019] According to a different preferable embodiment of the present invention the propolis extract with enriched flavonoid content contains 10 to 40 m / m% of raw propolis , 10 to 60 m / m% of one or more polyethylene glycols , 5 to 20 m / m% of one or more emulsifiers and 0 . 01 to 20 m / m% of water, preferably 30 m / m% of raw propolis , 50 m / m% of polyethylene glycol and 10 m / m% of one or more emulsifiers and 10 m / m% of water . According to a preferable embodiment of the present invention the one or more polyethylene glycols is / are polyethylene glycol ( s ) the melting point of which is a maximum of 62 ° C .

[0020] According to another preferable embodiment of the present invention the one or more emulsifiers is / are selected from the following list : polysorbate-20 , polysorbate-60 , polysorbate-80 , PEG derivatives of hydrogenated ricin oil , triton x-100 , polyglyceryl esters of fatty acids , sugar esters of fatty acids ; the emulsifiers are preferably polysorbate-20 and polysorbate-80 .

[0021] According to another preferable embodiment of the present invention the propolis extract with enriched flavonoid content contains 8 . 5 m / m% of polysorbate-20 and 1 . 5 m / m% of polysorbate-80 .

[0022] The present invention also relates to a method for the production of the propolis extract with enriched flavonoid content , which method contains the following steps : a ) the one or more polyethylene glycols and the one or more emulsifiers are added together and mixed until becoming homogenous while being continuously heated; b ) during this the raw propolis is pulverised in a knife chopper ; c ) then the pulverised raw propolis obtained in step b ) is added to the homogenous mixture of the one or more polyethylene glycols and the one or more emulsifiers heated in step a ) during continuous stirring , while care is taken to maintain the temperature in a constant range ; d ) the mixture is stirred for 20 to 40 minutes , preferably for 30 minutes whilst maintaining a given temperature range extraction; e ) following the extraction preheated, purified water is added to the mixture ; f ) after 30 minutes of stirring has elapsed the dark brown, thick, inhomogeneous mixture also containing solid supernatant is centrifuged; g ) after the centrifuging the inhomogeneous mixture separates into two phases , the lower dark brown liquid (propolis extract rich in flavonoid ) is separated from the light brown supernatant .

[0023] According to a preferable implementation of the method according to the present invention the temperature range used in steps a ) , c ) and d) is 47 to 62 ° C .

[0024] According to another preferable implementation of the method according to the present invention is that in step f ) the centrifuging is performed for 10 minutes at a rotational speed of 6000 rpm .

[0025] The present invention also relates to a dietary supplement , vitamin product and medicinal product containing the propolis extract with enriched flavonoid content , which is present as an aqueous solution, contains the propolis extract disclosed according to the above and its propolis content is at least 15 m / m% .

[0026] The present invention also relates to the use of the propolis extract with enriched flavonoid content according to the above in humans and animals as a dietary supplement , vitamin product , medicinal product and animal feed, and the use as a feed additive .

[0027] Furthermore , the present invention also relates to the use of the propolis extract with enriched flavonoid content according to the above in a procedure for the treatment of poultry infected with the Salmonella Enterl tldls serovar ; to achieve a weight increase in winged birds and / or poultry; and for supporting the general state of health of pets , where the medicinal product , dietary supplement or vitamin product is preferably in the form of an aqueous solution .

[0028] A detailed description of the invention If not mentioned to the contrary, m / m% in the present specification understood to mean mass percent (massi ) , and ratios are understood to mean the mass ratios of the individual components .

[0029] In the scope of the present specification if a numerical value is given, this is understood to mean that the last digit of the given number shows the precision of the given value according to the rounding-up rules . Therefore , for example , 1 . 5 m / m% is understood to mean all values falling within the range of 1 . 45 m / m% to 1 . 54 m / m% .

[0030] In the scope of the present specification, if a range between two values is defined, then the concrete numerical values indicated as the limit values of the range are viewed as constituting parts of the range .

[0031] Within the context of the present invention propolis extract with enriched flavonoid content is understood to mean an extract with a high flavonoid and propolis content that is obtained from raw propolis as the result of an extraction separation operation using an appropriate solvent . The propolis extract with enriched flavonoid content according to the present invention contains at least 15 m / m% propolis and at least 90 m / m% of the flavonoid content of the initial raw propolis .

[0032] As a result of literature research, it is recommended to use polyethylene glycol derivatives ( abbreviated as PEG, Macrogols ) or alcohol for the extraction of the raw propolis , the reason for this is that by using PEG the flavonoids in the raw propolis may be very efficiently extracted [ Elbe Langner . Phytochemische und mikrobiologische Untersuchungen von Propolis verschiedener Provenienzen als Beitrag zur Kenntnis der Wirkprinzipien in Propolis . Dissertation, Berlin 1995 ; Shaker Verlag ( BoD ) . Shimizu et al . : Antiinfluenza virus activity of propolis in vitro and its efficacy against influenza infection in mice . Antivir Chem Chemother . 2008 ; 19 ( 1 ) : 7 -13 ( PMID 18610553 ) . Kosalec et al . : Flavonoid analysis and antimicrobial activity of commercially available propolis products . In : Acta Pharm. Dez 2005 , Bd . 55 , Nr . 4 , S . 423-30 , ( PMID 16375832 ) ] . Emulsifiers or emulsifying agents are substances that facilitate the distribution of liquids that do not dissolve in each other ( such as water and oil ) , i . e . the creation of an emulsion .

[0033] Among emulsifiers we differentiate between emulsifiers that have hydrophilic and lipophilic properties , two groups of hydrophilic emulsifiers are ionic and non-ionic emulsifiers . Within the scope of the present invention emulsifiers are understood to be hydrophilic emulsifiers , specifically non-ionic emulsifiers . The water-solubility of the propolis extract with enriched flavonoid content according to the present invention is a consequence of the non-ionic emulsifier ( s ) used, furthermore , as is the fact that the propolis extract with enriched flavonoid content according to the present invention dissolved in water or another substance the dissolved propolis and flavonoid fraction is not precipitated out of the extract .

[0034] Within the scope of the present invention propolis extract with enriched flavonoid content is understood to mean an extract that contains raw propolis , one or more polyethylene glycols , one or more emulsifiers and water in a way not expected according to the current state of the art (water is an unexpected component in the dissolving of the propolis ) . As a consequence of the composition also containing water the efficiency of the absorption of the extract in the body increases significantly .

[0035] The propolis extract with enriched flavonoid content according to the present invention contains 10 to 40 m / m% of raw propolis , 10 to 60 m / m% of one or more polyethylene glycols , 5 to 20 m / m% of one or more emulsifiers and 0 . 01 to 20 m / m% of water , preferably 30 m / m% of raw propolis , 50 m / m% of one or more polyethylene glycol , 10 m / m% of one or more emulsifiers and 10 m / m% of water . It should be noted that the actual propolis content of the raw propolis used was an average of 76000 mg per kilogram . Raw propolis consists of several hundred different substances , primarily of wax, resin, essential oils and pollen . At the same time , it contains a significant amount of flavonoid and various trace elements , such as iron, copper, magnesium, calcium, silicon, manganese , zinc , aluminium, cobalt , etc . In order to extract the useful substances to be found in the raw propolis a solvent must be used during the extraction process that, as a result of its properties, is suitable for dissolving the useful substances to be found in the raw propolis . As it was explained in the section concerning the state of the art, the solvent frequently used for performing extraction of raw propolis is ethanol, however, the use of ethanol, as explained in detail above, involves numerous disadvantages. Ethanol is flammable, volatile, its use during extraction is costly, and, in addition, when diluted with water propolis extract dissolved in ethanol is unstable, and may cause the precipitation of the propolis and flavonoid fraction. As mentioned above, propolis and flavonoid may be very effectively extracted from the raw propolis using PEG. The reason PEGs are preferable is that they may be mixed with water or melted by heating. In order for it to be possible to produce a dispersion of the PEG propolis extract in water it is necessary to also use one or more emulsifiers, the daily intake limit value of which with respect to humans and animals is 75 mg / bodyweight kg.

[0036] One or more polyethylene glycols is / are used as solvent in the propolis extract with enriched flavonoid content according to the present invention, the melting point of which is a maximum of 62 °C or below. The reason for this is that in the method according to the present invention the temperature of the extraction is between 47 to 62 °C, in other words all of the polyethylene glycols defined according to the above will be in liquid phase during the extraction disclosed in detail later on. Such polyethylene glycols are, for example, PEG400, PEG200, PEG300, PEG600, PEG800, PEG1000, PEG1500, PEG2000, PEG3000, PEG4000, PEG6000, PEG8000 or a combination of these. According to a more preferable embodiment the polyethylene glycol is PEG400.

[0037] The one or more emulsifiers in the propolis extract with enriched flavonoid content according to the present invention is selected from the following list: polysorbate-20, polysorbate-60, PEG derivatives of hydrogenated ricin oil, triton x-100, polyglyceryl esters of fatty acid (e.g. polygliceryl-10 laurate, polyglyceryl-5 caprylate, etc. ) , sugar esters of fatty acid (e.g. sucrose stearate, sucrose palmitate, etc . ) , polysorbate-80 . Preferably two emulsifiers are used, which are polysorbate-20 and polysorbate-80 . Even more preferably 8 . 5 m / m% of polysorbate-20 and 1 . 5 m / m% of polysorbate-80 are used in the propolis extract with enriched flavonoid content according to the present invention .

[0038] The present invention also relates to a method for the production of the propolis extract with enriched flavonoid content , which method contains the following steps : a ) the one or more polyethylene glycols and the one or more emulsifiers are added together and mixed until homogenous while being continuously heated; b ) during this the raw propolis is pulverised; c ) then the pulverised raw propolis obtained in step b ) is added to the homogenous mixture of the one or more polyethylene glycols and the one or more emulsifiers heated in step a ) during continuous stirring , while care is taken to maintain the temperature in a constant range ; d ) the mixture is mixed for 20 to 40 minutes , preferably for 30 minutes whilst maintaining a given temperature range extraction; e ) following the extraction preheated, purified water is added to the mixture ; f ) after 30 minutes of stirring has elapsed the dark brown, thick, inhomogeneous mixture also containing solid supernatant is centrifuged; g ) after the centrifuging the inhomogeneous mixture separates into two phases , the lower dark brown liquid (propolis extract rich in flavonoid ) is separated from the light brown supernatant .

[0039] Essentially, in step a ) the non-ionic emulsifier or emulsifiers is / are dissolved in the one or more polyethylene glycols during stirring and continuous heating then, whilst maintaining a constant temperature , the obj ective is to obtain a homogenous liquid . The pulverised raw propolis ( for example , pulverised in a knife chopper ) is added to the homogenous mixture obtained in step a ) while stirring and heating continuously . The obj ective with the stirring and heating applied in steps c ) and d) is to facilitate the dissolving of the raw propolis and, essentially, the extraction taking place in step d) . The duration of the extraction stirring was determined empirically at 20 to 40 minutes , more preferably at 30 minutes . In step e ) purified water heated up to the temperature of the extraction mixture is added to the mixture , the purpose of which is to help the precipitation of the waxes from the mixture and increase the efficiency of the following separation step . The amount of purified water added is preferably one third of the mass of the initial raw propolis . In step f ) the inhomogeneous mixture is centrifuged, the purpose of this is to separate the undissolved parts of the raw propolis (primarily waxes that are concentrated in the supernatant phase ) from the dark brown liquid phase rich in flavonoid . As a result of the centrifuging the inhomogeneous mixture becomes separated into two phases that may be separated from each other, which are then separated from each other in step g ) . The lower phase is a thick, dark brown liquid with a characteristic odour , which in fact is the propolis extract with enriched flavonoid content .

[0040] According to a preferable implementation of the method for producing the propolis extract with enriched flavonoid content according to the invention, the temperature range used in steps a ) , c ) and d ) is 47 to 62 ° C . This temperature range was selected to be lower than the melting point of 63 ° C of the wax contained in the raw propolis , thereby the infiltration of waxes into the product that are obstructive in respect of the separation may be avoided .

[0041] According to another preferable implementation of the method according to the present invention, the centrifuging of the inhomogeneous mixture performed in step f ) is carried out for 10 minutes at a rotational speed of 6000 rpm .

[0042] The present invention also relates to a dietary supplement , vitamin product and medicinal product containing the propolis extract with enriched flavonoid content . The form of these products is an aqueous solution, with this facilitating its administration, thereby it is not necessary to dilute the propolis extract with enriched flavonoid content with water to the concentration required for administration, instead the users obtain it ready for use at the production facility . The product produced in this way is stable , may be stored at room temperature for at least 3 years and its propolis content is at least 15 m / m% .

[0043] According to the above the propolis extract with enriched flavonoid content according to the present invention may be used as a dietary supplement for humans and animals , as a vitamin product , a medicinal product , feed or feed additive . Medicinal product is understood to mean a product containing material of natural origin that has a beneficial biological effect , that may also be used without a doctor' s prescription and, in the case of use in accordance with the instructions , does not cause harm to health . [Website of the National Centre for Public Health and Pharmacy, Medicinal Products not classified as medicines [ in Hungarian] ,

[0044] <https : / / ogyei . gov . hu / gyogys zernek nem minosulo gyogyhatasu kes zitme nyek>] . Dietary supplements are foodstuffs serving as a supplement to a balanced, mixed diet that contain nutrients in concentrated form or other substances that have a dietary or physiological effect [Website of the National Centre for Public Health and Pharmacy , Public information in connection with dietary supplements [ in Hungarian] , <https : / / ogyei . gov . hu / etrend kiegeszitokkel kapcsolatos lakossagi tu dnivalok >] . Vitamin products , in addition to vitamins and minerals , as a supplement may contain propolis extract with enriched flavonoid content . The propolis extract with enriched flavonoid content according to the present invention may also be administered as animal feed or as an animal feed additive , where the feed additive is added to the feed or drinking water and exerts one or more beneficial effects on animals or on the quality of the animal product [ on the basis of Regulation ( EC ) No 1831 / 2003 of the European Union and of the Council ] . The medicinal product produced with the use of the propolis extract with enriched flavonoid content according to the present invention may be used for the following in animals : for the treatment of poultry infected with the Salmonella Enterl tldls serovar; to achieve weight increase in winged birds and / or poultry; and for supporting the general state of health of pets ; it may be used for the following in the case of humans : for strengthening the immune system; for pain relief ; for reducing inflammation, where in both cases the medicinal product is preferably in the form of an aqueous solution. The propolis extract according to the present invention contains more than 260 flavonoids, among which the most are polyphenols .

[0045] Examples

[0046] In the following the present invention is explained through examples . The examples facilitate better understanding of the invention, therefore under no account may they be interpreted as limitations to the scope of protection.

[0047] Example 1 : The production of propolis extract with enriched flavonoid content

[0048] 25.00 kg of PEG400, 4.25 kg of polysorbate-20 and 0.75 kg of polysorbate-80 purchased from MOLAR CHEMICALS Kft are added together, while performing continuous stirring the mixture is heated up to between 47 and 62 °C. In the meanwhile, 15.00 kg of raw propolis is pulverised in a knife chopper, which is then added to the homogenous mixture of PEG400 and the emulsifiers. The mixture was stirred at a temperature of between 47 and 62 °C for 30 minutes. After the mixture was stirred for 30 minutes, 5.00 kg of heated, purified water was added to the inhomogeneous mixture obtained and then the mixture was centrifuged while warm using the centrifuge purchased from Thomas Apiculture (10 minutes, 6000 rpm) . Following this the inhomogeneous mixture separates into two phases, and the lower dark brown liquid phase (propolis extract with enriched flavonoid content) is separated from the light brown supernatant .

[0049] The analytical test result of the finished propolis extract with enriched flavonoid content for total flavonoid content: 31688 mg / kg. According to our calculations the total flavonoid content of the raw propolis is 32828 mg / kg, in other words the efficiency of the flavonoid extraction is 31688 mg / kg / 32828 mg / kg x 100% = 96.5%. This value significantly exceeds the values given to date in the literature (approx. 75-80 %) . After increasing the scale , the experiment was repeated for a 100 -kg amount , from which 85 . 0 g water-soluble propolis could be extracted .

[0050] Example 2 : The innocuity ( toxicology profile ) of the aqueous solution of the finished propolis extract with enriched flavonoid content was tested at the lx normal dose level , and at 3x and 5x overdose levels in comparison with propolis powder , which was administered mixed in the feed of broiler chickens infected with the Salmonella Enterl tldls serovar . In addition to this , the experiment was also aimed at determining the effective dose of the propolis extract with enriched flavonoid content .

[0051] During the investigation, in the course of the rearing of the broiler chickens ( 33 days ) we monitored the weight increase of the chickens individually and compared this with the control group , which received no treatment at all .

[0052] In addition to this we measured the feed intake amount of the animals per group and compare this too with the data experienced from the control group .

[0053] Furthermore , we also examined whether the mixing of various doses of the propolis extract with enriched flavonoid content in the feed of chickens experimentally infected with the Salmonella Enterl tldl s serovar changed the probability of the infection taking hold, the severity and frequency of the appearance of the symptoms and the duration of the excretion of the infecting agent in comparison with the control group .

[0054] Finally, the broiler chickens were subj ected to a pathological autopsy in the interest of identifying the macroscopic changes and the microscopic changes were also observed in the course of a histopathology test .

[0055] The tests were performed with the obj ective of being able to determine whether the administration of various doses of the propolis extract with enriched flavonoid content influences the bodyweight increase of the broiler chickens , their feed utilisation, the probability of the development of salmonellosis , the severity of the symptoms and the appearance of pathological changes compared with these phenomena in the control group .

[0056] I The animal experiment condition were as follows : The animal testing level category was mild .

[0057] The propolis was administered by mixing it with the animals ' drinking water . The concentration in which the chickens receive the propolis every day is determined by multiple parameters , for example the weight of the chicken, its age , and even the season of the year . Determining this was the task of a specialist in animal nutrition , who determined that during rearing each individual animal should receive a total of 15 mg of propolis . Accordingly, during the rearing period each chicken received drinking water prepared with propolis extract with enriched flavonoid content equivalent to 15 mg of flavonoid . Chyme were collected during the investigation .

[0058] The day-old chicks were Ross 308 broiler chickens . In the incubator the chickens received the Hatchback AVINEW, Hatchback IBD120 and RISMAVAC + CA126 vaccines . The day-old chicks were received under the environmental parameters prescribed in the husbandry technology used ( 30 ° C , 60% relative humidity) , under infrared lighting , then the parameters were varied in accordance with the demands corresponding to the age of the chickens .

[0059] The chickens were individually marked with a serial number affixed to their legs , this was necessary for recording weight gain every week and for collecting the chyme .

[0060] The feeding of the animals took place ad libitum with broiler starter feed, in addition to this the animals received fresh drinking water every day in which the propolis extract with enriched flavonoid content according to the invention was dissolved . The intensive broiler starter crumb feed mixture without coccidiostat and without added copper was purchased from Farmer-Mix Kft . ( 23 )

[0061] I I The Salmonella infection process :

[0062] The Salmonella Enterl tldls bacteria strain previously frozen in a bacteria bank was removed from the -80 ° C store on the day of infection, a yellow loop of bacteria was placed into 30 ml of tryptone soy agar , which was then incubated at 41 ° C for 3 hours . Samples were taken from the suspension immediately following inoculation and then at the end of the incubation we prepared a tenfold dilution series in sterile tubes , from which 50 pl was spread out onto the tryptone soy agar for the purpose of determining the initial number of colony forming units (CFU) . In this way the initial number of bacteria grew from the initial value of 3 . 6 x 106CFU / ml to the value of 1 . 3 x 108CFU / ml following 3 hours of incubation .

[0063] The chicks were infected on their 4thday of life , which was repeated on the 5thday of life . 0 . 3 ml of bacteria suspension was inoculated into one chick using a feeding tube , which corresponded to 3 . 9 x 107CFU .

[0064] Following the second Salmonella infection, in order to monitor the success of the infection and to determine the excretion of Salmonella , we took cloacal swab samples on days 1 , 3 , 6 , 14 , and 21 from all the infected animals and from 5 animals each in the non-inf ected groups . The chyme samples were incubated for one hour at 41 ° C in tubes containing 3 ml of Rappaport-Vassiliadis broth . Following this , after vortex mixing the tubes , 100 pl of suspension was spread onto Rambach agar , which was then incubated for 18 to 24 hours at a 41 ° C thermostat . Positive results were viewed as the appearance of mauve coloured colonies , samples of which were transferred to XLD agar, which were then incubated for a further 18 to 24 hours at a constant temperature of 41 ° C .

[0065] The appearance of black colony forming units counted as positive samples , which samples were transferred once again to Chromagar Salmonella Plus selective agar and incubated for an additional 18 to 24 hours at a 41 ° C thermostat . The samples that also formed mauve coloured colonies on this agar were viewed as effectively positive .

[0066] I I I Measurement of the bodyweight of the animals and their feed intake , sampling

[0067] The weight increase of the animals was measured once a week, individually, on scales . The measurement of feed intake took place per group , by remeasuring the amount of feed given the previous day .

[0068] Sterile cloacal swabs were used for collecting the chyme samples , which swabs were washed into tubes containing Rappaport Vassiliadis broth immediately after the samples were taken, then the tubes were sealed .

[0069] Monitoring of the clinical symptoms : the following were observed and recorded every week on the basis of yes-no answers a ) whether there is any change in the consistency of the chyme of the animals ( examined per group ) ; b ) uric acid deposits accumulated around the cloaca ( examined individually) ; c ) crouching ( examined individually) ; d ) wing drooping ( examined individually) ; e ) limping ( examined individually) .

[0070] IV Autopsy and histopathology test

[0071] An autopsy was performed on each animal ( according to the international standard ) , which was performed according to the rules relating to the diagnostic post-mortem of birds ( external examination, internal examination per organ system) .

[0072] The histopathology tests were performed on 5 animals from each group from the j ej unum and ileum intestine sections , with three parallel measurements performed in the case of each sample .

[0073] The depth of the intestinal crypts and the height of the intestinal villi were measured . We calculated the average from three parallel measurements and examined the villi height / crypt depth ratio , which we compared with the values from the control groups .

[0074] In the case of the group receiving the drinking water solution, by day 12 it was observed that less feed intake resulted in a better bodyweight increase compared to the control group . Table 1 contains the comparison of the effect of the aqueous propolis extract with enriched flavonoid content on feed intake and weight gain with respect to the control group .

[0075] Table 1

[0076] The feed intake of those chickens that received the propolis extract with enriched flavonoid content in their drinking water was significantly lower by day 12 compared to the control group . When viewing weight gain in this respect it was observed that , compared to the control group , all the treated groups had a much better weight gain with a lower feed intake . In the case of every group, between days 12 and 24 the difference with respect to feed intake dropped to a degree nearly the same as that of the control group then by the end of the experiment feed intake became better once again, however weight gain only exceeded that of the control group in the case of triple the amount of administered propolis extract . It should be noted that the administration of the triple and quintuple doses was performed for the purpose of the overdose test . Feed intake was better than that of the control group during 86% of the whole investigation period ( 33 days ) in the case of the single dose , and better during 91% of the duration of the study in the case of the triple and quintuple doses , it was better than that of the control group in 80% of the duration of the investigation in the case of the drinking water solution .

[0077] After calculating specific feed intake , it may be determined that each of the propolis extract treatments significantly reduced the amount of feed required to product one kilogram of bodyweight from day 25 compared to the control group .

[0078] In summary the conclusion drawn is that the use of various doses of the propolis extract with enriched flavonoid content according to the invention ( single , triple , quintuple ) in animals has a safe and positive effect on bodyweight gain, compared with the control group . Histopathology shows that the crypt depth measured in the j ej unum in the case of propolis extract with enriched flavonoid content administered at a quintuple dose ( group 7 ) displayed a significantly better result compared to the negative control group . In a significant part of the rearing time of the chickens , as a result of the administration of the propolis extract with enriched flavonoid content , feed conversion was better , which is verified by the specific feed intake measured from day 25 of the investigation period . On the basis of the present investigation results the propolis extract with enriched flavonoid content may be brought into consideration as a product with medicinal effect in the interest of preventing salmonellosis in broiler chickens , and also has beneficial biological effects on weight gain and feed conversion .

[0079] I I After the in vivo investigations , we started large-scale experiments , in the course of which we performed the large-scale testing of the propolis extract with enriched flavonoid content according to the present invention in chickens .

[0080] Table 2 summarises the large-scale experiment and its results .

[0081]

[0082] BTI means the Broiler Production index, which is understood to mean the broiler chicken rearing index, a BTI value of over 400 is exceptionally good.

[0083] The OMMI index is the chicken clone identifier used for rearing.

[0084] Our objective with the large-scale experiment was to prove that the propolis extract with enriched flavonoid content according to the present invention reduces mortality by 20-30% (see table 2: there were 415 fewer mortalities) and, furthermore, reduces feed use (see the value in the first row of table 2 compared with the control group) , the reduction in feed use during rearing may be as much as 6 to 9%.

[0085] From table 2 it may be seen that in the case of the animals that received drinking water made with the propolis extract with enriched flavonoid content mortality dropped to 8.67% compared to the value of 11.26% measured in the case of the control group.

[0086] The average weight of the animals in the test group at the end of the cycle was 3.15 kg / chicken, while the average weight of the animals in the control group was 3.11 kg / chicken. This, in itself, does not count as a deviation, however the amount of feed used to produce 1 kg of bodyweight differs significantly. In the case of the test group this was 1.42 kg of f eed / bodyweight kilogram (58718 kg feed / 41262 kg bodyweight) , while in the case of the control group this was 1.59 kg f eed / bodyweight kilogram (65721 kg feed / 41329 kg bodyweight) .

[0087] The total feed use in the test group was 3.15 kg * 1.42 kg / bwkg = 4.47 kg, and in the control group 3.11 kg * 1.59 kg / bwkg = 4.94 kg. In other words, in order to achieve the same weight in the test group ( 4.94-4.74 ) / 4.94 = 4.04% less feed was required.

[0088] This is also underpinned by the change in the BTI value in the test group. The higher BTI value compared with the control group is proof of more efficient, more economical rearing.

Claims

Claims1. Propolis extract with enriched flavonoid content, characterised by that it contains raw propolis, one or more polyethylene glycols, one or more emulsifiers and water.

2. Propolis extract with enriched flavonoid content according to claim 1 wherein it contains 10 to 40 m / m% raw propolis, 10 to 60 m / m% of one or more polyethylene glycols, 5 to 20 m / m% of one or more emulsifiers and 0.01 to 20 m / m% of water, preferably 30 m / m% of raw propolis, 50 m / m% of polyethylene glycol and 10 m / m% of one or more emulsifiers and 10 m / m% of water.

3. Propolis extract with enriched flavonoid content according to any of claims 1 to 2, wherein the one or more polyethylene glycols is / are polyethylene glycol (s) the melting point of which is a maximum of 62 °C.

4. Propolis extract with enriched flavonoid content according to any of claims 1 to 3, wherein the one or more emulsifiers is / are selected from the following list: polysorbate-20, polysorbate- 60, polysorbate-80, PEG derivatives of hydrogenated ricin oil, triton x-100, polyglycerin esters of fatty acids, sugar esters of fatty acids; the emulsifiers are preferably polysorbate-20 and polysorbate-80.

5. Propolis extract with enriched flavonoid content according to any of claims 1 to 4 wherein it contains 8.5 m / m% of polysorbate- 20 and 1.5 m / m% of polysorbate-80.

6. Method for the production of the propolis extract with enriched flavonoid content according to any of claims 1 to 5, characterised by that it contains the following steps : a) the one or more polyethylene glycols and the one or more emulsifiers are added together and mixed until homogenous while being continuously heated; b) during this the raw propolis is pulverised in a knife chopper;c ) then the pulverised raw propolis obtained in step b ) is added to the homogenous mixture of the one or more polyethylene glycols and the one or more emulsifiers heated in step a ) during continuous stirring , while care is taken to maintain the temperature in a constant range ; d ) the mixture is stirred for 20 to 40 minutes , preferably for 30 minutes whilst maintaining a given temperature range extraction; e ) following the extraction preheated, purified water is added to the mixture ; f ) after 30 minutes of stirring has elapsed the thick, dark brown inhomogeneous mixture also containing solid supernatant is centrifuged; g ) after the centrifuging the inhomogeneous mixture separates into two phases , the lower dark brown liquid (propolis extract rich in flavonoid) is separated from the light brown supernatant .7 . Method for the production of the propolis extract with enriched flavonoid content according to claim 6 , wherein the temperature range used in steps a ) , c ) and d ) is 47 to 62 ° C .8 . Method for the production of the propolis extract with enriched flavonoid content according to any of claims 6 to 7 , wherein in step f ) the centrifuging is performed for 10 minutes at a rotational speed of 6000 rpm .9 . Product with medicinal effect , dietary supplement , or vitamin product containing propolis extract with enriched flavonoid content , characterised by that the product with medicinal effect , dietary supplement , or vitamin product is an aqueous solution, it contains the propolis extract with enriched flavonoid content according to any of claims 1 to 5 and its propolis content is at least 15 m / m% .10 . The use of the propolis extract with enriched flavonoid content according to any of claims 1 to 5 in humans and animalsas a dietary supplement , vitamin product , product with medicinal effect and animal feed, and as a feed additive product .11 . The use of the propolis extract with enriched flavonoid content according to any of claims 1 to 5 in a procedure for the treatment of winged birds infected with the Salmonella Enteritidis serovar ; to achieve a weight increase in winged birds and / or poultry; for supporting the general state of health of pets , wherein the product with medicinal effect , dietary supplement or vitamin product is preferably in the form of an aqueous solution .

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